0": - nl‘womwnw-uuwwu-u v...— .

u".- .10.

 

 
 

 

 

 
 

 

 

 

 

, r '

 

 

 

 

.

 

Hahn; .im

0 “Vat." O

 

 

 

 

 

fink?“ ,3 ‘3 cm IGAN snreu

 _, _. ‘ II'IIII IIIII llllllllIllllllllllllll

5'! ."f I" l 301564 4655

 

 

 

 

 

 

 

 

 

 

 

 

This is to certify that the
dissertation entitled

PERTURBATION OF LYMPHOPOIESIS BY DIETARY
ZINC DEFICIENCY IN YOUNG ADULT A/J MICE

presented by

Farzaneh Osati—Ashtiani

has been accepted towards fulfillment
of the requirements for

Ph.D. degree in Eachglflg1—

 

fwatw MM”

jor professor

Date 12/19/96

MS U is an Affirmative Action/Equal Opportunity Institution 0- 12771

 

 

LiBRARY
Michigan State
University

 

 

 

PLACE III RETURN BOX to remove thie checkout from your record.
TO AVOID FlNES return on or before date due.
I:_

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

-—-l I,—
—— —___*T ——
fil‘F—l—a

MSU ie An Ai'i‘imetive Actior'MEquel Opportunity lnetituion
WWI

 

 

 

 

 

 

7 _ _. W. .__—.. _.

PERTURBATION OF LYMPHOPOIESIS BY DIETARY ZINC DEFICIENCY
IN YOUNG ADULT A/J MICE

By

Farzaneh Osati-Ashtiani

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Pathology

1996

ABSTRACT

PERTURBATION OF LYNIPHOPOIESIS BY DIETARY ZINC
DEFICIENCY IN YOUNG ADULT A/J MICE

By

Farzaneh Osati-Ashtiani

Deficiencies in dietary zinc represent a world wide nutritional problem which
compromises the host immune defense capabilities, and leads to increased susceptibility to
disease and infection. Dietary zinc deprivation in mice, a compatible model for human zinc
deficiency, causes rapid thymic atrophy and decreases cell and antibody mediated responses
all of which correlates with the losses in the number of peripheral lymphocytes. This
nutritional deficiency also results in chronic elevation of endogenous glucocorticoid
concentration, which itself is irnmunosuppressant. Thus, disruption of lymphopoietic
processes which could lead to lymphopenia and diminished host defense capacity during zinc
deficiency was hypothesized. The primary objective of this research was to evaluate the
effects of suboptimal dietary zinc intake on developing B-cells, particularly progenitor and
precursor compartments of the B-lineage in the marrow of young adult mice. This

investigation was also extended to the examination of the status of T-cells at different stages

‘Wt‘

ofdevdopmentinthethynmsofdietarya'ncmice, asthesecond objective ofthis dissertation.
Since glucocorticoids are known to suppress the immune system, the third objective of this
research was to correlate glucocorticoid elevation during zinc deficiency with the suppression
of lymphopoiesis through apoptotic cell death of susceptible lymphocytes. The results
strongly indicate a selective and stage specific alteration in B-lymphopoiesis in the marrow
of fine deficient mice. Precursor (B220‘CD43‘IgM‘) and immature (BZZO*IgM*IgD‘) B-cells
exhibited significant sensitivity to the efi‘ects of zinc deficiency, whereas progenitor
(B220‘CD43*IgM') and mature (BZZO‘IgM‘IgD‘) B-cells were substantially resistant.
Similarly, significant depletion of immature thymocytes (CD4*CD8*) with concomitant
resistance of progenitor (CD4'CD8‘) and mature T-cells (CD4‘CD8‘/CD4‘CD8*) in the
thymus indicated a stage specific sensitivity of residual thymocytes to the effects of zinc
deficiency. Thus, disruption of lymphopoiesis particularly of early developing immature
lymphocytes by zinc deficiency was indeed the underlying cause of impaired immunity. To
investigate the role of glucocorticoids in the observed immunological alterations in zinc
deprivation, adrenalectomies were performed. Removal of corticosterone via adrenalectomy
completely protected thymus integrity and bone marrow cellularity while thymus weights and
phenotypic distribution of early developing and immature B-cells were analogous to those
found in the control groups. Furthermore in vivo and in vitro verification of apoptosis in the
thymus of zinc deficient mice indicated that the elimination of immature double positive
thymocytes (CD4‘CD8‘) occurred via apoptosis. Taken together, the results strongly indicate
the disruption of lymphopoiesis due to dietary zinc deprivation. Furthermore, a potential role

for GC-induced suppression of lymphopoiesis and apoptotic elimination of susceptible

lymphocytes in zinc deprived mice are suggested.

Dedicated to:
my husband, Eraj Poureslami
my daughters, Ghazaleh and Bahareh
and

my parents, Ali and Pari Ashtiani.

iii

ACKNOWLEDGMENTS

I am truly grateful to my thesis advisor, Dr. Pamela Fraker, for her guidance,
cncouragements, and support in the past few years. Pam has shown me in many ways how
to be a strong, descent, and honest scientist and I believe my experience as her student will
be beneficial for my future career. I am very thankful to Dr. J on Patterson who served as my
academic advisor, for his unconditional time, guidance, and support throughout the years.
I greatly acknowledge my other committee members Dr. Richard Schwartz, and Dr. Cheryl
Swenson for their guidance, advice and encouragement along the way.

I am also indebted to Dr. Louis King for his significant contribution to this project.
Without his guidance, thankless teachings in the use of flow cytometer and suggestions I
would not have reached this point. Special appreciation goes to the past and present members
of our lab, Dr. Deborah Elghanian, Dr. Bill Telford and my buddy Tonya Laakko for their
friendship, advice, and encouragement throughout the years. I would also like to express my
appreciation to Teresa Vollmer for her technical assistance that was always available for me.

This section of my dissertation would not be complete without acknowledging my
parents. Their continuing and unconditional love, support, and encouragement throughout

the years that I have been away fiom home have been and always will be remembered and

iv

appreciated Iwould also like to thank mydear brother and his wife and my lovely niece and
nephew for their love and support.

_ Last but not least, I must thank my dear husband, Eraj, and my beautifiil, lovely
children, Ghazaleh and Bahareh. Without their love, understanding, sacrifice and
unconditional arpport, completion of this journey would not have been possible. For this and

countless other things, I will be always indebted to them.

Thanks for everything.

God bless you all.

EST I
m
[\TR

CHI!

CHII

CHiI

TABLE OF CONTENTS

LIST OF TABLES ................................................. viii
LIST OF FIGURES ................................................. ix
LIST OF ABBREVIATIONS ......................................... xiii
INTRODUCTION .................................................... 1
CHAPTER ONE: LITERATURE REVIEW .............................. 5
SECTION 1: Biochemical and Physiological Significance of Zinc ........... 5
SECTION 11: Zinc Deficiency and its Effects on Immune System .......... 35
' SECTION III: Phenotypic Characterization of B and T Lymphocytes

at Different stages of development in Murine System ....... 53

SECTION IV: Efi'ects of Zinc Deficiency on Stress Axis; Role of
Glucocorticoids on Immune System ..................... 78

CHAPTER TWO: Depletion of Cells of the B-lineage in the Bone

Marrow of Zinc Deficient Mice ........................ 112
Abstract ..................................................... 1 13
Introduction .................................................. 1 14
Materials and Methods .......................................... 116
Results ...................................................... 120
Discussion ................................................... 123

CHAPTER THREE: Effect of Deficiencies in Zinc on the Status of Progenitor

and Precursor-B cells in the Marrow of Mice .......... 135
Abstract ..................................................... 136
Introduction .................................................. 138
Materials and Methods ..................... ' ..................... 140
Results ...................................................... 146
Discussion ................................................... 150

CHAPTER FOUR: Role of Glucocorticoids in the Suppression of B-Lymphopoiesis

During Zinc Deficiency ............................. 170

Abstract ..................................................... 171
~ Introduction .................................................. 173
Materials and Methods .......................................... 176
Results ...................................................... 182
Discussions .................................................. 185

CHAPTER FIVE: Evaluation of the Effects of Zinc Deficiency on T-cell Maturation
in the Thymus of Young Adult A/J Mice: A Possible Role for

Apoptosis in Zinc Deficiency .......................... 199

Abstract ..................................................... 200
Introduction .................................................. 202
Materials and Methods .......................................... 206
Results ...................................................... 213
Discussion ................................................... 217
SUMMARY AND SUGGESTIONS .................................... 233
LITERATURE CITED .............................................. 239

vii

LIST OF TABLES

CHAPTER THREE
Table 1: Composition of the Diet ................................. 155

CHAPTER FOUR
Table 1: Body weights of sham operated and adrenalectomized mice
maintained on zinc adequate or zinc deficient diet during eight

weeks of diet study ..................................... 190
CHAPTE FIVE
Table 1: Body and thymus weights, thymus cellularity and plasma CS
levels of mice after 27 days on zinc dietary study ............... 224

viii

SECTION I:

Figure 1:

Figure 2:

SECTION III:

Figure 1:

Figure 2:

Figure 3:

Figure 4:

SECTION IV:

Figure 1:

Figure 2:

Figure 3:

. Figure 4:

LIST OF FIGURES

CHAPTER ONE
A schematic view of DNA binding of a transcription factor via
zinc finger domains. ................................ 31
Schematic view of the classical zinc finger domain of TFIIIA. . . 33
Differentiation and maturation pathways of all major blood
cell types fi'om a common hematopoietic stem cell ........... 70
A schematic view of the events required for heavy
chain gene rearrangements and generation of u heavy

chain protein ...................................... 72

Cell surface and molecular marker expression during the stages
of B-cell development in murine bone marrow. ............. 74

Osmond’s scheme of B-lymphocyte differentiation and dynamics
of B-cell production in murine bone marrow. .............. 76
Biosynthetic pathways of adrenal steroid hormones ......... 104

Hypothalamus-pituitary—adrenal axis; Regulation of
glucocorticoid production. ........................... 106

The functional domains of the steroid/nuclear (glucocorticoid)
receptor. ......................................... 108

Schematic representation of the glucocorticoid receptor
heterocomplex in untransformed state. .................. 110

CHAPTER TWO

Figure l: The efi‘ect of zinc deficiency on body weights, thymus weights,
and zinc content of serum ............................ 127

' Figure 2: Assessment of efl'ects ofa'nc deficiency on nucleated cells of the
bone marrow by flow cytometry of mice fi'om ZA, RZA, MZD
and SZD groups afier a 28 day dietary period .............. 129

Figure 3: Flow cytometric light scatter profiles of nucleated cells of bone
marrow prepared fiom normal and severely zinc deficient mouse
after a 28 day dietary period. .......................... 131

Figure 4: Phenotypic distribution of early, immature, and mature cells of
the B-lineage in marrow of ZA, RZA, MZD and SZD mice at

day 28 by flow cytometry. ............................ 133
CHAPTER THREE
Figure 1: Effect of ZD on body and thymus weights for the 27 day
dietary study. ..................................... 156
Figure 2: Analysis of the zinc content of sera and tissues of mice fiom the
ZA, RZA, MZD, and SZD groups at day 27. ............. 158
Figure 3: Schematic representation of stages of B-cell development in the
BM ofmurine system as proposed by Hardy et al., 1991. . . . . 160
Figure 4: Effects of ZD on phenotypic distribution of early B-cells
of the marrow ..................................... 162
Figure 5: Three-dimensional presentation of flow cytometric scatter
profiles of 3220+ gated nucleated BM cells from a ZA and a
SZD mice after a 27-day dietary period. ................. 164

Figure 6:

Figure 7:

Figure 1:

Figure 2:

Figure 3:

Figure 4:

Figure 1:

Figure 2:

Expression of precursor (Pro-B) and progenitor (Pro-B) B-cells
as a proportion of BM B-cell compartment for ZA, RZA, MZD,

and SZD mice at day 27. .............................

Evaluation of BM cellularity for 27 day of a dietary zinc study.

CHAPTER FOUR
Comparison of thymic weights of adrenalectomized and sham
operated mice maintained on zinc adequate or zinc deficient
diet, obtained at the terminal point of the diet study (day 56).

Plasma corticosterone levels of adrenalectomized and sham

operated mice fed zinc adequate or zinc deficient diet ........

Comparison of plasma zinc levels of adrenalectomized or sham
operated mice maintained on zinc adequate or deficient diet for

eight weeks. .....................................

Proportion of early developing (B220‘Ig‘), immature
@220*IgMIgD') and mature (B220‘IgM‘IgD”) B-cells in the
marrow of mice that were sham operated and maintained on zinc
deficient diet (ZD sham) or zinc adequate diet (ZA sham) or
which were adrenalectomized and maintained on zinc deficient

(ZD Adr) or zinc adequate diet (ZA Adr). ................

CHAPTERFIVE

Evaluation of the phenotypic distribution of thymic T-
lymphocytes in ZA, MZD, and SZD groups after 27 days of

166

168

. 191

193

195

197

diet. ............................................ 225

Absolute number (total numbers) of immature thymocytes in the

thymus onA, MZD, and SZD mice. .................. 227

Figure 3: Apoptosisinimmaturethymocytesofmice fromZA, MZD,and
SZD groups at day 27 . ............................. 229

Figure 4: Apoptosis in thymic T-cells in in vitro culture system. ....... 231

AC

Adi

AI

AK

AP

AP

AV

BL

Bl

Co

D.‘

ACTH_

Adr

AN OVA

APA

AV-PE

BL

BM

CS

DAPI

DN

LIST OF ABBREVIATIONS

adrenocorticotropin hormone
adrenalectomized
Acrodennatitis entereopathica
analysis of variance

amino peptidase A

amino peptidase N
avidin-phycoerythrin

basal

bone marrow

Chinese hamster ovary cells
concanavalin A
corticotropin-releasing factor
cysteine-rich intestinal protein
corticosterone
4',6—diamidine-2-phenylindole
dexamethasone

double negative

Di

Oil

Oil

il‘

fl

DP

DTAF

DTH

dUTP

EDTA

FACS

GR

HBSS

double positive

dichlorotriazinyl amino fluorescein
delayed type hypersensitivity reactions
deoxyuridil triphosphate
ethylenediarninetetracetic acid
fluorescently activated cell sorter

fetal bovine serum

facteur timique serique

forward scatter

glucocorticoid

glucocorticoid receptor

Hank’s balanced salt solution

high

hypothalarnus-pituitary-adrenal axis
hormone responsive element

heat stable antigen

heat shock protein

inductively coupled plasma-atomic emission spectroscopy

irrununoglobulin

xiv

NaCI

NaOH

NIST'

PBMC

its

interleukin

leukocyte common antigen
low-density lipoprotein

low

monoclonal antibodies

major histocompatibility complex
mononuclear phagocytic cells
metal responsive element
metallothionein

moderately zinc deficient

sodium chloride

sodium hydroxide

national institute of standards and technology
natural killer

nuclear localization signal
peripheral blood mononuclear cells
phosphate bufi‘er saline
programmed cell death

protein calorie malnutrition

 

PD
ilC
PHI

iliii

Pi“

N.‘

Iii

RD

RH

PD

PD
PFC
PHA
PHSCs
Pl)n
pro-B

PTPase

SD

SERPIN

prednisolone

protein deficient

plaque forming cells
phytohemagglutinin

pluripotent hematopoietic stem cells
part per million

progenitor B cells

protein tyrosine phosphatase
pokeweed mitogen

recombination activating gene

red blood cell

recommended daily allowance
rheumatic heart disease

restricted zinc adequate

severely combined immunodeficient
standard deviation

serine protease inhibitor

superoxide dismutase

single positive

TrlT
TEC

TFIHA

TNP-LPS

sheep red blood cells

side scatter

severely zinc deficient

T-cell receptor

T-cell dependent

terminal deoxynucleotidyl transferase
thymic epithelial cell

transcription Factor III A or Factor A
T-cell independent

trinitrophenyl conjugated lipopolysaccharide
undetectable

zinc adequate

zinc deficiency/zinc deficient

Introduction

The importance of zinc as a nricronutrierrt in the growth and development of mammals
was first recognized by Todd etal in 1934. Since then, a growing body ofliterature has been
devoted to the importance of zinc as well as deleterious effects of its deficiencies in biological
systems, particularly in immune system (Prasad, 1985; Fraker et al., 1986; 1993; Endre et al.,
1990; Walsh etaI., 1994; Hadden, 1995; Cousins, 1996). Being one of the largest tissues in
the body and a dynamic system with constant development, differentiation, and proliferation,
the immune system requires a continuous flow of nutrients including zinc for its pr0per
function. The frequency of zinc deficiency (ZD) in humans and its adverse efi‘ects on the
imnmne components emphasizes the necessity for understanding the role of zinc in immunity.
The high reliability of mice as an immunological model for humans has greatly enhanced our
knowledge about the interrelationship between zinc deficiency and impairment of the immune
system. Information gathered to date indicates that a period of 30-day insufficient zinc intake
in young adult mice is suflicient to cause: 1) weight loss, 2) rapid thymic atrophy; and 3)
reduced numbers in lymphocytes and macrophages in the peripheral immune system (eg.,
spleen) (Fraker et al., 1986; 1993). Furthermore, the deficiency caused severe depressions
in antibody and cell mediated immune responses (DePasquale—Iardieu and Fraker, 1984;

Femandes et al., 1984; Jardieu and Fraker, 1990). Further investigations revealed that in

2

spite of severe depletion oflymphocytes in the spleen, the proportion of residual splenocytes
('1‘ -cells to B-cells) were normal (King et al., 1991). Similarly, the fimctional capacity of
residual splenocytes in terms of their proliferation capacity and antibody and cytoldnes
production in response to different mitogenic stimuli was normal (Cook-Mills and Fraker,
1993a). Taken together, it appeared that lymphopenia was the most probable cause for
reduced host defense capacity. Thus, alteration in lymphopoiesis in the bone marrow (BM),
the site oflymphopoiesis in adult mammals, appeared to be a possibility causing reduction in
lymphocyte numbers and impaired immunity in ZD. This hypothesis was used as the main
frame for investigations on this dissertation. The primary objective of this dissertation was
to identify the status of B-lymphopoiesis in the marrow and T-cell maturation in the thymus
of ZD mice. It was of interest to determine if suboptimal levels of zinc result in a uniform
reduction in lymphocyte subsets or if selective alterations occur in certain subclasses.
Experiments reported in Chapters two and three will extensively examine different stages of
B-cell development through irnmunophenotyping the marrow of mice on a 27-day zinc
deficient dietary regime. Likewise, Chapter five will address the status of T-cell maturation
in the thymus of dietary zinc mice, as the second objective of this research.

Chronic elevation of glucocorticoid concentrations (GCs) in the later stages of ZD has
been established (Quarterman, 1972; 1974; Quarterrnan and Humphries, 1979; DePasquale-
Jardieu and Fraker, 1979; Prasad, 1985). DePasquale—Jardieu and Fraker demonstrated that
ZD constitutes a stress that activates the hypothalamus-pituitary-adrenal cortex axis (stress
axis) and subsequently leads to the chronic production of corticosterone (CS, the predominant

form of GCs in murine system) (DePasquale-Jardieu and Fraker, 1979). Furthermore,

3

removal of CS via adrenalectomy protected against involution of the cortex in which
inunature thymocytes reside (DePasquale-Iardieu and Fraker, 1980). Thus, it appeared that
GC had profound efi‘ects on thymic integrity, in particular on immature thymocytes, as has
been also shown by others (Quarterman and Humphries, 1979; Miller et 01., 1991; 1994;
Flaherty et 01., 1993). Recent literature has extensively demonstrated the sensitivity of
'nnmsturethymccytesto GCs followedbyinduction ofapoptosis asa mean for the elimination
of vulnerable lymphocyte populations (Cohen and Duck, 1992; Sun et 01., 1992; Brown et
01., 1993; Cidlowski et 01., 1996). In fact, several recent studies from this lab have indicated
the susceptibility and apoptotic elimination of thymocytes as well as developing B-cells to
short exposure of low levels of synthetic (dexamethasone) and natural (corticosterone,
cortisol) GCs in vitro (Garvy et 01., 1991; 1993b; Voetberg et 01., 1994). These findings
provided a foundation for the two other objectives in this dissertation. Thus, the third
objective of this research was to identify the role of endogenously elevated levels of CS in B-
lymphopoiesis in the marrow of ZD mice. This objective was met by the removal of GCs
M the circulation via adrenalectomy. It was of interest to investigate whether removal of
the GC would protect BM cellularity in ZD mice as it did for the thymus (DePasquale-Jardieu
and Fraker, 1980). This will be addressed in Chapter Four. The final objective was to verify
whether apoptosis played a role in elimination of GC-susceptible lymphocyte populations in
ZD mice. Use of a homogenous tissue such as thymus provided an easier tool for this
investigation. Chapter Five, along with evaluation of T-cell subsets in ZD, concomitantly
examines the presence of apoptosis among immature thymocytes in the thymus of ZD mice.

Due to rapid in viva phagocytosis of apoptotic cells by phagocytic cells (Savill et 01., 1989a;

 

Be

aHV.
A. I

O

4

1989b; Cohen, 1991; Evan et 01., 1992), the verification of apoptosis in ZD was attempted
though two approaches. In the first approach (in viva system), fi'eshly isolated thymocytes
fi'om difi‘erent dietary groups were shortly incubated (6 hrs) in regular media (RPM-1640)
to allow for the completion of apoptotic processes in cells that received the death signal in
viva, but escaped phagocytosis. In the second approach (in vitra system), fieshly prepared
thymocytes from regular mice were incubated (8 hrs) in culture media (RPMI-l640) in which
known levels of CS and zinc relevant to the levels detected in zinc dietary mice were
supplemented. This approach allowed for the identification of the role of zinc and CS or their
synergistic action in thymocyte survival or death and allowed detection of the apoptotic
elimination in immature thymocytes with higher magnitude compared to the in viva system.
Identification of apoptotic cells were made possible by a rapid and highly quantitatively
method recently introduced by this lab (I'elford et 01., 1991). Using multicolor FACS analysis
for the identification of different subpopulations along with their simultaneous cell cycle
analysis via fluorescent DNA dye, the precise proportion of each subpopulation undergoing
apoptosis was rapidly detected.

Collectively, these studies strongly indicate a selective disruption in lyrnphopoietic
processes in zinc deprived mice. Further, they indicate a potential role for GC in alteration

in lymphopoiesis leading to apoptotic elimination of susceptible lymphocytes.

SECTION I: Biochemical and Physiological

Significance of Zinc

"'3

 

it:

is 1
Tries

.975.

 

6

Introduction: It was not until 1934 that zinc was shown to be necessary for the
growth of mammals (Todd et 01., 1934). Later in 1963, the importance of zinc for human
health was first documented by Prasad and his colleagues. This documentation was based on
clinical studies on patients from Iran (Prasad et 01., 1961) and Egypt (Prasad et 01., 1963)
who demonstrated severe growth retardation, hypogonadism, hepatosplenomegaly, rough
skin, geophagia (in male Iranian subjects) and Schisiosamiasis (in Egyptian patients). In
addition the zinc content of plasma, red blood cells and hair was dramatically decreased in
dwarfs compared to normal subjects. The diet of these patients consisted of bread made of
wheat flour which is known to contain high phytate, which decreases the availability of zinc.
These clinical manifestations and laboratory observations indicated zinc deficiency as a
principal feature in these patients. Since then investigations related to metabolic functions and
esseruiality of one has expanded rapidly. Tire essential role of zinc in the survival, growth and
irnegrity of cellular components of different organisms is due in part to the utilization of this
element by more than 200 metalloenzymes for their fiinction (Coleman, 1992; Vallee and
Auld, 1990). In these enzymes, zinc participates in catalytic, cocatalytic (coactive) or
structural activities (V slice and F alchuk, 1993). Thus, if the metal is removed by chelating
agents or if it is not available at optimal levels (eg., dietary zinc deficiency) the catalytic
fimetions and structural stability of these zinc-dependent enzymes will be abolished, thereby
leading to profound physiological alterations. The biochemical features of zinc have lead to
the recognition of zinc in wide variety of biological systems. In the last few decades many
investigators have specified the significant role of zinc in biomembrane integrity, wound

healing, reproduction, the nervous system, carcinogenesis, the endocrine system, and as an

at”.

 

db

~I

it?

7
antioxidant and therapeutic agent (Prasad, 1988; Sharnbaugh, 1989; Endre et 01., 1990;
Watanabe et 01., 1995). However, this chapter will review the characteristics and the
importance of zinc in biological systems and the impact of its deficiencies on some
components of the immune system.

Chemical Properties: Zinc is widely known as one of the most ubiquitous elements
inthenatureandoneofthemostessentialtracemetalsinthe human body. This element with
an atomic number of 30 and atomic weight of 65.37 is placed in Group II B transition
elements in the periodic table (\Vrlliams, 1989; Walsh et 01., 1994). Zinc is generally present
as a divalem cation (Zn2*) with a completely filled d shell (10 d electrons) which allows zinc
to play its unique structural roles. First, it readily undergoes ligand exchange reactions which
explains its catalytic role in metalloenzymes. Second, as an electron acceptor molecule, it can
strongly interact with ligands such as thiolate fiom cysteine, amine fi'om histidine, oxygen
fi'om aspartate, glutamate and water. Third, it shows no tendency for oxidation and reduction
activities which highlights its biological functions, since redox activity results in the fi'ee
radical formation (Williams, 1989; Da Silva and Williams, 1991). In fact zinc is known to act
as fiee radical scavenger by displacing redox-active transition metals (eg., iron and copper)
from their specific site and preventing hydroxyl radical (OH') formation at the active site
(Hovering and Dean, 1992; Powell et 01., 1990). The scavenging mechanism of zinc was
demonstrated in a recent study by Powell et 01., (1994) in which they showed that the
perfusion of isolated rat hearts with zinc significantly improved the cardiac postischenric
recovery. When they measured the copper concentration in hearts treated with zinc (30uM),

they noticed 27% less copper than the control hearts. Thus it seems that by competing with

8

copper and displacing it, zinc could suppress the OH' formation and improve postischemic
recovery.

The other mechanisms by which zinc may fiinction as an antioxidant is the protection
ofailfirydryl groups against oxidation This is achieved by direct binding of zinc to sulfirydryl
groups, thus preventing intrarnolecular disulfide formation (Bray and Bettger, 1990).
Furthermore, the presence of zinc at the active site of superoxide dismutase (SOD)
(eliminates superoxide anions, 02°, formation), and its association with metallothionein (a fi'ee
radical scavenger) indicates the indirect contribution of zinc in antioxidation activities and
'urtensifies the importance of zinc in biological systems (Bray and Bettger, 1990). In fact a
study by Richard et 01., (1991) reported high lipid peroxidation and tissue injury in patients
with clu'onic renal failure due to decreased enzymatic activity of SOD. The decrease in SOD
activity and subsequent hyperlipoperoxidation in these patients were explained by a
disturbance in the status of zinc as decrease in serum zinc level was also noted. SOD
converts superoxide to molecular oxygen and hydrogen peroxide which is subsequently
degraded to oxygen and water by catalases and proxidases. Thus, by removing these highly
reactive radicals via scavenging capacity of SOD, the tissue damage is prevented (Borg et 01.,
1992).

Taken together, these data suggest that zinc is involved in antioxidative defense
systems; thus, its deficiency could result in an increase in tissue oxidative damage. Recent
studies fiom different laboratories have in fact shown the oxidative damage to proteins and
lipids in rats fed a low zinc diet due to the low zinc metalloenzymes activities and increased

reactive oxygen species (Coudray et 01., 1991; Disilvestro and Carlson, 1994; Oteiza et 01.,

9

1995). This, some of the observed physical and biological alterations in zinc deficient mice
studied in this dissertation could be related to the disturbance in the antioxidative mechanisms.
Zinc Metabolism:

Absorption: Zinc commonly enters the body through ingestion of food and drink and
its absorption is primarily by the small intestine including duodenum, jejunum and ileum
(Solomons and Cousins, 1984; Cousins, 1985; Cousins, 1989; Lennerdal, 1989). Most
recently rat colon was identified as another site of absorption (14%) for this nutrient (Naveh
et 01., 1993). Although zinc absorption has been investigated widely, the mechanism (8)
involved in this process has not been clearly identified. However, most of the investigators
think that zinc absorption occurs via difi‘usion (nonmediated) and carrier-mediated
mechanisms (Cousins, 1989; Lennerdal, 1989). At high zinc concentrations, the brush border
membrane of the intestinal mucosal cells absorb zinc by passive difiiision (Tacnet et 01., 1990;
Rafi'am'ello et 01, 1992) and under normal dietary conditions zinc is absorbed by the carrier-
mediated mechanism which is most active at low luminal zinc concentration (Rafi‘aniello and
Wapnir, 1989; Hempe and Cousins, 1991). Thus, one would expect that at suboptimal
dietary zinc intake the intestinal mucosa would have higher zinc uptake. In fact in an earlier
study by Steel and Cousins (1985) they demonstrated that the intestinal absorption rate in the
zinc deficient rats was eight fold higher than in the control rats, suggesting an increase in
membrane transport mechanism when the zinc supply decreases. However, there was no
indication of whether or not the higher zinc uptake in these patients was associated with zinc
mobilization from other tissues (eg., muscles, bone) which can affect the tissue zinc

distribution. This will be addressed in Chapter Three of this dissertation.

10

The carrier-mediated zinc absorption is regulated via two proteins, namely,
metallothionein (MT) and cysteine-rich intestinal protein (CRIP) (Rafi‘aniello and Wapnir,
1991; Hempe and Cousins, 1992). This mechanism was primarily identified as a low
molecular weight (6500 daltons) cysteine rich protein, namely metallothionein (MT) (Hoadley
and Cousins, 1987; Seal and Heaten, 1987). This protein which binds many divalent cations
(eg., Zn, 0i, Cd, etc.) has been suggested as a storage protein for zinc (Chesters, 1991). MT
synthesis is directly correlated to dietary zinc intake and inversely related to zinc absorption
(Hoadley etal, 1988; Cousins and Lee-Ambrose, 1992). The interrelationship between zinc
and MT and its relevancy to this dissertation will be addressed in this section.

The cysteine-rich intestinal protein (CRIP) has been identified as a 77-amino acid, 8.6
KDa protein with seven cysteine residues (Birkenmeier and Gordon, 1986). This protein is
mainly expressed in small and large intestine with minimal to no expression in other tissues
(Hempe and Cousins, 1991). A recent study by Hempe and Cousins (1992) was specifically
aimed at the identification of a role for CRIP in zinc absorption and the interrelationship
between CRIP and MT in the regulation of zinc transport. Using isolated intestinal loops
fiorn rats fed either low (1 ug zinc/g) or high (180 ug zinc/g) zinc diet, an inverse relationship
in zinc absorption was shown between these two proteins suggesting a competitive binding
interactions between MT and CRIP for intracellular zinc transport.

Many agents afi‘ect zinc absorption. Agents that form insoluble complexes with zinc
are the primary source ofmalabsorption. For example, phytate (a phosphate rich compound
found in cereal grains, legumes and soy-based infant formulas) histidine and cysteine (high

afinity binding amino acids), have all been shown to reduce or inhibit zinc absorption (Han

11

et 01, 1994; Cousins, 1996). Thus, due to high amnity binding of zinc to many ligands (eg.,
phosphate groups, sulfirydryl groups) and the possible formation of insoluble complexes with
fine, malabwptionofzincwithasubsequentzincdeficiency is expected. In fact, human zinc
deficiency in the Middle East due to the high consumption of plant proteins which contain
large amounts of phytate (inositol hexaphosphate) is a classical example of zinc malabsorption
and zinc deficiency (Prasad, 1963; Prasad, 1991). Furthermore, clinical problems of
gastrointestinal absorption, kidney disorders, alcohol, and inborn errors of metabolism also
contrilnrte to the zinc malabsorption (Cousins, 1985; Walsh e101, 1994). This dissertation,
however, will specifically address the adverse efi‘ects of suboptimal dietary zinc intake and its
outcomes on some immune components of the murine system.

Transport: Plasma is the major route of zinc transport throughout the body once it
has been absorbed. The total zinc content of plasma varies from 95-130 mg/dl in a healthy
adult. However, it comprises only 0.1% of the total body zinc (Cousins, 1989; Bremner and
May, 1989). Within the plasma, zinc appears to be bound to two main carrier proteins,
namely macromolecular and micromolecular zinc ligands. The macromolecular zinc ligands
which comprise more than 98% of the circulating zinc include transferrin, albumin and a2-
macroglobulin (Cousins, 1989; Walsh et 01., 1994; Cousins, 1996). Approximately 66% of
the plasma zinc is bound to albumin presumably to one of the histidine moieties of this
molecule. Albumin is considered as the major and the more dynamic source of zinc binding
protein and zinc carrier within the circulation. Approximately one-third of the zinc carried in
theplasmaisboundto armaeroglobulinwhich comprisesatightlybound pool ofplasma zinc.

The incorporation and metabolism of this complex only occurs in the liver. The physiological

In

 

12
sigru'ficsnce ofzinc binding az-macroglobulin has not been identified yet. The micromolecular

portion of the zinc binding carriers which comprise only 1-2% of the circulating zinc is
composed of amino acids such as histidine, cysteine, glutamine, and threonine. The amino
acid-zinc complexes, due to their low molecular weights, are able to transport to all tissues
and body organs including brain. This small zinc pool may act as a zinc donor for a high
afinity zinc uptake system within cells (Cousins, 1989; Cousins, 1996).

Distribution: Zinc is found in almost all organs, tissues and body fluids, however,
at different concentrations. The highest tissue concentrations of zinc have been found in
human prostate gland (520 mg/kg dry weight), muscle (197-226 mg/kg dry weight), bone
(218 mg/kg dry weight), kidney (184-230 mg/kg dry weight), liver (141-205 mg/kg dry
weight), and pancreas (115-135 mg/kg dry weight) (Walsh et 01., 1994). The rest of the
tissues contain relatively trace amounts of zinc. The zinc content in extracellular fluids is
relatively low (plasma contains only 0.1% of the total body zinc) compared to intracellular
stores (about 99%) (V slice and F alchuk, 1993). However, plasma zinc serves as a primary
source of the element available to all cells. Thus, small variations in the zinc content of
tissues would have dramatic effects on plasma zinc. Furthermore, any fluctuations in the
dietary zinc intake could have the same consequences. A decrease in plasma zinc levels has
been repeatedly reported in both humans and animals following zinc deprivation where
introduction of a zinc deficient diet was followed by rapid depression of the plasma zinc
(Endre et 01., 1990; Prasad, 1991; Cook-Mills and Fraker, 1993a; King et 01., 1995).

The distribution of zinc between extracellular fluid pool and intracellular pools has

been shown to be sensitive to several factors. These include variations in hormonal balance

13

(eg, glucagon, insulin and glucocorticoids), inherited disorders of zinc metabolism(eg.,
Acrodennatitis enterapathica) and diets either low in zinc or with zinc chelating agents, in
all of which the plasma zinc level is depressed (Cousins, 1985; Bunce, 1989; Jackson, 1989).
This is of significant interest to this dissertation where a combination of zinc deprivation and
chronic elevation of glucocorticoids which accompanies zinc deficiency could be evaluated
for their role in zinc mobilization. This subject will be addressed in Chapter Three of this
dissertation

Excretion: Following zinc absorption, the unabsorbed portion of the ingested zinc
which is in the form of insoluble complexes is carried in the intestine and excreted in the feces.
This fecal fine is composed in large part of unabsorbed zinc. A portion of the zinc in feces
also reflects the secreted zinc fiom gastrointestinal tract and the bile. Zinc in feces represents
the major source of zinc lost fi'om the body (70-80% of the total ingested zinc). Fecal zinc
excretion ranges from 5-10 mg/day and depends on the dietary zinc intake and physiological
condition (Cousins, 1985; Jackson, 1989).

A small portion of zinc is also excreted in the urine of healthy subjects. This portion
is composed of zinc primarily bound to amino acids. The zinc-amino acid complexes would
drerrreadilypassfliererialglonmdusuidareerrcretedintheurine. The urinary zinc excretion
(approximately 500-800 rig/day) reflects changes in nutrition, disease and physiology of both
launans and animals. For example, urinary zinc loss is a common feature of acute and chronic
liverdiseaseandrenaldiseaseinwhich, duetothedamagetc thecells and theirfunction, n'nc
cannotbehandledproperlyandisexcreted intheurine(Barry etal., 1990). Asrnall amount

ofzinc is also excreted in sweat (115 ug/dl) which under extreme heat would increase to as

14

. ligh as 2-3 mg daily. There is no known body store for zinc with the exception of zinc stored
in the bone. Therefore, homeostasis of zinc mostly depends on the balance between
absorption and excretion where an adequate nutritional diet is provided.

Role of Zinc in Metalloenzymes: The essential role of zinc in metabolism,
transmission of genetic messages, growth and development is mainly due to association of
zincasanirnegralcomponent ofoverZOOzincmetalloenzymes (Walsh 2101., 1994; Coleman,
1992). By incorporation ofn'nc into enzymes such as carbonic anhydrase, which dehydrates
bicarbonate in the lungs and hydrates carbon dioxide fi'om other tissues; alcohol
dehydrogenases that catalyze the oxidation of alcohols and certain steroids; carboxypeptidases
which catalyze carboxyl-terminal peptide bond hydrolysis in peptides and proteins; alkaline
phosphatases that catalyze the hydrolysis of a variety of phosphate esters; nucleotide
polymerases which are involved in DNA and RNA synthesis; superoxide dismutase that
catalyzes the production of hydrogen peroxide; and many other zinc-dependent enzymes, the
structural stability and the biological activities of these enzymes are maintained (V allee and
Glades, 1984; Vallee and Auld, 1990; Vallee and Auld, 1993).

Carbonic anhydrase is an enzyme in which the catalytic activity could be impaired by
insufieient zinc. In patients with sickle-cell disease in wlu'ch a conditioned zinc-deficient state
is observed, the content of carbonic anhydrase in the erythrocytes was decreased in
correlation with the zinc content of the erythrocytes (Vallee and Auld, 1990).

Another interesting case is the liver alcohol dehydrogenase in which both catalytic and
structrn'alrolesofthezinc ion canbefound. This enzymehas two active sites and four ions

ofzinc per molecule. Two zinc ions are essential for catalytic activity and are bound to the

15

enzyme via two cysteinyl-SH groups and the imidazole ring. The other two zinc ions appear
to have a structural function and are each bound to four cysteinyl-SH groups (V allee and
Auld, 1990).

In some cases zinc plays a non-catalytic role in the zinc-dependent enzymes. For
instance, in case of superoxide dismutase, There are two atoms of copper and two atoms of
zinc present per molecule of protein and all four atoms are required for optimal superoxide
dismutase activity. Zinc is thought to be needed for the stability of the enzyme (Bremner and
Beattie, 1990). This enzyme which is a member of oxidoreductases, zinc metalloenzymes,
catalyzes the production of hydrogen peroxide in aerobic cells which in turn protect the cells
floor the oxygen fi'ee radicals formation. The protective activity of SOD against free radical
formation could be either tlu'ough disrnutation of superoxide anion (02’) or by preventing iron
(Fe’*) reacting with hydrogen peroxide, thereby generating fi'ee hydroxyl radical (OH') in the
well-known Fenton reaction (Willson, 1989; Bray and Bettger, 1990; Bremner and Beattie,
1990; Borg et 01., 1992).

In addition to the cytosolic copper and zinc containing SOD (Cu-ZnSOD), a
mitochondrial manganese-containing SOD (MnSOD) has been also identified in mammalian
tissues (Borg e101, 1992; Giglio et 01., 1994). The efl‘ects of zinc deficiency on antioxidant
activities of Cu-ZnSOD and MnSOD was investigated by Taylor and colleagues (1988). The
Ctr-ZnSOD activity was shown to be unchanged in the liver and elevated in the lungs of the
zinc deficient rats. The MnSOD activity were unchanged in the liver and lung of zinc
deficient rats compared to zinc adequate controls. In general, even though the animals were

severely zinc deficient, the changes in the free radical defense system were small. Thus, it

16

seems that the antioxidant defense system may be so important to the survival of the zinc
deficient animal that the growth of the animal is halted in order to maintain tissue zinc
concentrations and other components of the antioxidant defense mechanism.

Furthermore, zinc has been recognized as a structural regulator of many transcription
factors that are necessary for gene activation. The structural role for zinc in transcription
factors was first recognized in transcription factor IIIA (TFIIIA) from fi'og Xenopus leavis
(Miller et 01., 1985). Most of these transcription factors including TFHIA include small zinc-
based domains called zinc fingers that are necessary for DNA recognition and subsequent
gene transcription (Rhodes and Klug, 1993). Over the past decade, more than ten classes of
arch zinc finger domains have been discovered and characterized (Schwabe and Klug, 1994).
This subject will be discussed in more detail in upcoming section under zinc and gene
transcription.

Metallothionein: Metallothionein (MT) is a significant macromolecular ligand for
zinc with a unique structural features including low molecular weight (<10,000 daltons), a
high content of heavy metals (4-12 atoms/mole) and high cysteine content (30% of the
residues). Mammalian MT is a 61-62 amino acid peptide containing 20 cysteines, 6-8 lysines,
7-10 serines, one acetylated methionine at the amino terminus and no aromatic and
hydrophobic amino acids. The majority of cysteine residues are present in cys-x-cys and cys-
cys sequences where x represents any other amino acid (Harrier, 1986; Bremner and Beattie,
1990; Cousins, 1985; Chester’s, 1992). MT binds the essential metals copper and zinc under
physiological conditions and the toxic metals cadmium and mercury under pathological

conditions (Nagel and Vallee, 1995). This protein (MT) has higher binding affinity for copper

17

relative to zinc, whereas its induction is more responsive to dietary zinc than to copper
(Cousins, 1985). In fact, the low stability constant of zinc-MT would serve as labile source
of zinc that could be utilized for the activation of many zinc-dependent enzymes. All the
cysteine reddues are involved in metal binding with some of the cysteine residues sharing the
metal ion. Metals are associated with MT through thiolate bonds to all 20 cysteine residues
(tetrahedrally coordinated to four cysteine thiolate ligands) and are contained in two distinct
clusters. The A cluster contains eleven cysteines, binds four atoms of zinc or cadmium or five
to six atoms ofcopper, and is contained within the carboxyl-terminal ct domain. The B cluster
contains nine cysteines, binds three atoms of zinc or cadmium or six atoms of copper and is
contained in the amino-terminal B domain(Hamer, 1986; Vallee and Auld, 1990; Kay et 01.,
1991).

MT has been isolated from a variety of species and a wide range of tissues, including
liver, kidney, pancreas, intestine, brain, thymus, bone marrow and reproductive organs.
However, the concentration of the protein in tissues is highly variable and is influenced by
many nutritional (dietary), physiologic and developmental factors (Bremner, 1987; Bremner
and Beattie, 1990; Huckle et 01., 1993). For example, the concentration of MT is greatly
reduced in tissues of zinc-deficient animals (Bremner et 01., 1987; Vruwink et 01., 1988;
Cousins and Lee-Ambrose, 1992) and are increased after induction of many types of stress
or metal administration (Cousins, 1985). Its ubiquity, and inducibility by a wide range of
stimuli, including zinc, copper, infections, and stress suggest that it plays a vital role in the

regulation of metabolic processes that utilize this essential trace element.

18
IthasbeenshcwnthatMTperfomrsanimportantregulatory roleinthe cells by being

involved in the intracellular phase of zinc absorption, in cellular detoxification or storage of
zinc, and in direct activation ofzinc dependent enzymes by donating its metal to apoenzymes
and converting them into fully active enzymes (Hamer, 1986; Bremner, 1987; Vallee and
Falchuk, 1993). In vivo and in vitro studies have suggested a role as a free radical scavenger
asithasbeen showntobeapowerful binder ofhydroxyl radicals (Thornally and Vasak, 1985;
Bremner and Beattie, 1990; Schwarz, 1994; Powell et 01., 1994; Palmiter, 1994).
Furthermore, rapid induction of MT synthesis in response to increased intracellular zinc
concentrations is consistent with its action as a scavenger of metal ions. Thus in zinc
deficiency where fine is at suboptimal levels, MT synthesis would be diminished, thereby the
removal of toxic heavy metals such as cadmium and to some extent copper will be impaired.
This could result in fiee radical formation, lipid peroxidations, and subsequent tissue damage.

Synthesis: The Synthesis of the major storage-regulating metalloprotein
(metallothionein) is triggered by increasing levels of the fi'ee metal ions (eg., Zn,Cd,Cu) in the
cell (Hamer, 1986; Palmiter, 1994). In the liver, metallothionein synthesis functions in uptake
and storage of zinc in hepatocytes. In the intestinal mucosal cells, this protein competes with
CRIP,a zinc-binding ligand involved in zinc absorption, regulating the amount of zinc
available for transfer to the plasma as discussed (Cousins, 1983; 1989; 1996). Thus, the MT
synthesis controlled by the plasma zinc concentration demonstrates a homeostatic mechanism
in zinc metabolism.

In an early investigation by Richards and Cousins (197 5), they demonstrated zinc

regulation of an actinomycin-D sensitive mechanism that involves the synthesis of hepatic and

19

intestinal metallotlu'onein Their data clearly showed that plasma zinc uptake by hepatocytes
mist involve metallothionein synthesis since this is the only zinc binding protein that has the
dynamic capacity to respond to changes in zinc status. So they proposed that when plasma
zinc levels are high, liver metallothionein synthesis is stimulated which facilitates the uptake
of zinc into hepatocytes. Zinc remains bound to this protein until needed to meet cellular
requirements. Furthermore, The control of metal-induced MT synthesis at the transcription
level has bun shown by inhibitory efi'ects of cycloheximide, actinomycin D, and puromycin
on the process (Dumam and Pahniter, 1987; Raffaniello and Wapnir, 1991). This effect has
been confirmed by measurement of mRNA levels using cell-free translation systems and by
direct assay using cDNA probes. The rate of protein synthesis closely parallels the production
ofrnetallothionein mRNA (Dumarn and Palmiter 1987), and a high rate of transcription can
be detected within one hour of stimulation by metals. The mRNA levels reach a maximum
of at about 6-8 hours after exposure to an inducer, although the maximal levels of
metallothionein occur after 1-2 days (Palmiter, 1987). Cadmium is a particularly potent
inducer of metallothionein synthesis (Webb, 1986). The dynamics of metallothionein
synthesis in relation to zinc and copper metabolism has been comprehensively reviewed
(Cousins, 1985). Generally, a close relationship exists between zinc status and the levels of
metallothionein in tissues.

In a recent study by Cousins and Lee-Ambrose (1992), the influence of dietary zinc
levels in the regulation of MT gene expression in rats was demonstrated. Utilizing different
dietary levels ofzinc (5,30,130 mg Zn/kg), they showed that the intestine and liver took up

more zinc than other tissues. Nuclei purified from liver, kidney, and spleen accumulated

20

substantial amounts of zinc which was directly related to the amount of dietary zinc intake.
Furthermore, northern analysis demonstrated that MT expression was also proportional to
dietary zinc intake, where it was greatest in kidney followed by liver, intestine, spleen and
heart. These data strongly suggest that the induction ofMT synthesis is directly correlated
with dietary zinc supply as well as the bioavailability of zinc. Although not specifically
addressed in this dissertation, the possibility of reduced MT synthesis due to suboptimal zinc
intake should be considered as one of the underlying causes of observed manifestations of
zinc deficiency.

There is also a number of nonmetal factors which induce metallothionein synthesis.
Many of the stress inducers which raise circulating levels of glucocorticoids (GCs) stimulate
metallothionein synthesis in the liver and to a lesser extent in the heart, kidney, skeletal
muscle, and spleen of the mouse (Hager and Palmiter, 1981). Glucocorticoids were therefore
thought to mediate some of the efi‘ects on MT synthesis. Other steroid hormones, such as
estrogens and progesterone, can also induce MT synthesis (Kagi and Schafi‘er, 1988).
Endotoxin is another potent inducer of MT synthesis in the liver and other tissues. This
process is mediated by the release of cytokines (De et 01., 1990). Rats injected with
endotoxin showed reduced serum zinc levels only 3 hrs after treatment and greatly elevated
hepatic metallothionein after 18 hrs (Abe e101, 1987). Furthermore, hepatic metallothionein
mRNA levels in Syrian hamsters increased fourfold, 6 hrs after administration of endotoxin
(Etzel et 01., 1982).

In fact, in a recent study, a combination of regulatory factors such as zinc, copper,

endotoxin and glucocorticoids on MT synthesis in different tissues, specially brain, was

21

investigated (Gasull er al., 1994). Administration of zinc, glucocorticoids (dexamethasone,
corticosterone) and endotoxins significantly increased the level of MT concentrations in the
liver and kidney, with non-uniform distribution of MT in different areas of the brain. When
they examined the efi‘ect of zinc deficiency on MT synthesis, by feeding rats a zinc deficient
diet, they observed lower liver and serum MT and zinc concentrations compared to the
control group, with no significant change in the MT levels in different parts of the brain.
Once again these data confirm the multi-factor regulation of MT synthesis.

Bacterial infection also induces a marked increase in hepatic metallothionein levels and
a decrease in serum a'nc concentrations (Sobocinski et at, 1978), both characteristic of acute-
phase response. Several mechanisms have been suggested for the induction of
metallothionein synthesis during infection. Interleukin-l (IL- l), which is produced and
released from monocytes and activated macrophages in response to infection, stimulates the
synthesis of metallothionein and uptake of zinc by the liver of male (Cousins and Leinart,
1988) and pregnant female (Held and Hoekstra, 1984) rats. This finding suggests that GCs
mediate some of the cytokine-stimulated induction of metallothionein, since IL-l causes the
release of these hormones via stimulation of adrenocorticotropin hormone (ACTH) release
(Held and Hoekstra, 1984). More recently Schroeder and Cousins (1990) showed the effects
of IL-6 as a major cytokine mediator of MT gene expression and zinc metabolism in rat
hepatocytes However, bone marrow MT gene expression was shown to be solely dependent
on dietary zinc and not on IL-1 or IL-6 (Huber and Cousins, 1993).

Functions of MT: Unique features of the MT include its expression in most tissues,

its high content of heavy metals (eg., zinc, copper, cadmium, mercury, etc.) and its being a

22
highly conserved protein in evolutionary terms. As was mentioned earlier, MT has been

shown to act in the detoxification of heavy metals especially cadmium (Webb, 1987), and in
the control in zinc and copper homeostasis via regulating their intestinal absorption in
difiierent physiological conditions (Cousins, 1985; Menrad et al., 1981; Richards and Cousins,
1976). MT also acts as a metal transfer protein, a metal-storage protein, a sulfidr storage
protein, an acute phase protein, and a free radical scavenger. The antioxidant activity of MT
could be viewed as a mechanism in cells resistance to cancer chemotherapy. Cells with
acquired resistance to antineoplastic agents have shown overexpression of MT which tends
to bind these alkylating agents to a higher extent than the non-resistant cells (Ebadi and
Iverson, 1994). The mechanisms underlying this observation have not been clearly identified.
However, the results could be partly explained by the detoxification and free radical
scavenging mechanisms of MT. Increased accumulation of MT occurs in the liver and to a
lesser extent in bone, thymus and other tissues in animals subjected to different types of stress
(Bremner and Beattie, 1990). These stresses include restriction of food intake, bacterial
infection, and inflammatory and physical stress such as exposure to high or low temperatures.
The increased synthesis of metallothionein is accompanied by increased tissue zinc
concentrations, and the incorporation of zinc into the MT structure (Karin and Herschmann,
1981). The hypozincemiathat usually occurs in stressed animals appears to be a consequence
of the induction of hepatic metallothionein synthesis via increased zinc uptake (from the
circdation (Cousins, 1989). The influx of zinc into the cells under these circumstances plays

key roles in DNA synthesis and gene expression. Moreover, the induction of MT gene

23
expression and synthesis which provide flee-radical scavenging mechanism is another
beneficial aspects of increased cellular zinc uptake in stressed subjects.

In zinc deficiency, where a combination of low zinc availability and increased stress
hormones (GCs) is present, a more complex phenomenon is expected. It is possible that
elevation ofglucocorticoids in zinc deficient subjects induces the MT gene expression which
in turn is accompanied by nuclear zinc uptake from the circulation and active MT synthesis.
However, the observed low MT concentration in zinc deficient subjects could be the result
of the low availability of zinc for the synthesis of the active protein (MT). Although this
subject ins not been specifically addressed in this dissertation, it is possible to speculate that
at early stages of zinc deficiency the tissue MT concentration is increased, but as zinc
deficiency persists, a reduction in MT levels as well as circulating zinc levels would evolve.

A study by Nagel and Vallee (1995) proposed an additional function for MT: cell
cycle regulation in actively proliferating cells (human colonic cancer cells). They observed
the oscillation of MT during the mitotic cell cycle of HT-29 cells with its maximum near the
61/8 transition of the cells cycle, at the onset of DNA synthesis. They also claimed that the
elevated levels of MT in actively proliferating cells can serve as a marker for proliferation.

Studies on the regulation of MT indicate that much of the regulation occurs at the
level of transcription by heavy metals including zinc (Thiele, 1992). The promoter structure
of mouse MT-I (one of the MT isoforms) has been most thoroughly investigated and is
known to contain multiple metal responsive elements (MRE) responsible for the
transcriptional sensitivity of the gene to divalent metal ions (Thiele, 1992). The underlying

theory for the role of MRE in MT gene transcription indicates that an interaction occurs

24
between the inducing metal (eg., zinc) and a specific nuclear factor that through altered

confomntion recognises the MRE sequence and participates in the initiation of transcription.
Altermtively tln's factor could bind fine in the cytoplasm and then enter the nucleus for DNA
bindings (Cousins and Lee-Ambrose, 1992). Thus dietary zinc could control MT genes by
influencing the intracellular zinc concentration and interaction with metal responsive
transcription factors which in turn bind to MRE and initiate transcription.

Role of Zinc in DNA and RNA Synthesis: The impaired rate of grth that
accompanies zinc deficiency in mammals could also be a consequence of impairment in the
synthesis of DNA, RNA and proteins. Studies were conducted in which whole animals or
isolated cells were used to qualify DNA, RNA and/or to measure the incorporation of the
radioactive nucleotide precursors (thymidine and uridine) into DNA or RNA under conditions
of zinc deficiency (Auld etal., 1975; Duncan and Dreosti, 1976; Dreosti et 01., 1981; Duncan
and Dreosti, 1976). In these studies a consistent observation was that both total DNA and
incorporation rates were reduced significantly as a consequence of the zinc deficiency.
However, RNA synthesis showed less effects. Many nucleic acid polymerases, as we know,
are zinc metalloenzymes. Thus the reduction of uridine incorporation into RNA and
tlrymidine incorporation into DNA of zinc deprived animals or cell cultures could explain the
reduced activities of RNA and DNA polymerases.

RNA polymerase contains more than one mole of zinc/mole protein. Hence, it is likely
that zinc has both structural and catalytic roles in this enzyme. In an early study by Falchuk
et 01., (1978), they reported abnormal synthesis of RNA polymerase and altered base

composition of the synthesized RNA in a zinc deficient Euglena gracilis model. Impaired

25
mRNAsynthesis would lead to altered protein synthesis. In fact, Hicks and Wallwork (1987)

inve shown a significant depression of protein synthesis in rat liver and rat hepatocytes fi'om
zinc deficient rats. They concluded that the defect probably occurred in the tRNA synthetase
fiinction suggesting a zinc-dependent activity for this enzyme.

Recent studies on growth requirements for 3T3 cells clearly demonstrated the role of
Zn" for initiation of DNA synthesis. In 1990 Chesters et al., showed impaired thymidine
incorporation associated with decreased thymidine kinase activity and a comparable decrease
in mRNA by inadequate supply of Zn“. One year later, in another study by Chesters and
Boyne (1991), they further demonstrated the requirement of adequate zinc supply for
tramition of 3T3 cells fi'om quiescence to S phase. This implies the requirement of zinc for
mRNA and protein synthesis involved in the progression of 3T3 cells into S phase. The
combination of low Zn2+ availability with inhibition of mRN A synthesis or of protein synthesis
almost completely returned the cells to a quiescent state. Thus it seems that the lack of zinc
primarily restricted gene expression rather than enzyme activation. In fact, Chesters et al.,
(1995) in their recent investigation on this subject, showed that the inhibition of thymidine
kinase activity due to the lack of zinc was associated with increased binding of a specific
protein to the gene's promoter in the region between -55 and -83 bp 5' to the transcription
initiation site, and inhibition of transcription. This protein whose nature is not known yet,
apparently competes with the transcription factors responsible for thymidine kinase expression
for the same binding site and by displacing them inhibits the transcription of the gene. Thus
inthiscasetheirnpact ofthe lack ofzinc on thymidine kinase occurred at apretranslational

step. These observations strongly suggest the impairment of DNA, RNA, and protein

26

ynthesis in zinc deficiency which may subsequently result in some of the observed
physiological and immunological alterations presented in this dissertation.

Role of Zinc in Gene Expression: Structural and functional roles of the intrinsic zinc
in many DNA and RNA polymerases have been investigated. In this regard, direct zinc
removal, and replacement or substitution with other divalent metals have revealed multiple
roles for zinc in gene transcription (Chatterji and Wu, 1982).

In an early study by Falchuck et 01., (1975), they demonstrated the specific zinc
requirement for transition of Euglena gracilis from G] to S, S to G2 and G2 to M phases of
the cell cycle where the gradual depletion of the zinc content of the medium lead to the
growth arrest. Similarly, a recent study correlated low thymidine kinase mRNA levels in zinc-
depleted cells to the presence of two zinc dependent steps during G1 to S phase transition
(Chesters et al., 1993). The role of zinc in gene expression has been also identified at the
level of chromatin structure. An early study by Subiranal (1973) demonstrated that the
presence of zinc ions facilitated the first phase of chromatin denaturation consisted of the
loosely condensed chromatin in the inter-nucleosomal regions. This process is closely related
to gene unmasking which is required for synthesis of the enzymes for DNA synthesis. The
major change in genetic expression and chromatin structure within the cell cycle occurred in
62 phase immediately before mitosis. Thus the role of zinc in alteration of chromatin
structure could explain the arrest of E. gracilis cells in 62 as their zinc supply diminished.

In a most recent study conducted by Kimball and his coworkers (1995), the effects
of fine deficiency on hepatic protein synthesis and gene expression of retinol-binding protein

and transthyretin (plasma thyroxine—binding protein) in weanling rats was investigated. Their

27

results showed the inhibition of protein synthesis in livers from zinc deficient rats. This
inhibition was accompanied by altered expression of mRNAs in the liver, suggesting that zinc
has a vital role in regulating gene expression and protein synthesis.

The role of zinc in gene expression could also explain one of the characteristic signs
of zinc deficiency, namely, parakeratosis. This condition, which is the result of poor cell
difi‘erentiation, could be due to insufficient zinc levels for DNA synthesis and expression of
molecules required for normal difl‘erentiation of these cells. Several possible mechanisms
could underlie the impaired gene expression in zinc deprived animals. First of all, many
enzymes involved in nucleic acid synthesis are zinc dependent enzymes, such as DNA
polymerase and RNA polymerase. These enzymes have shown to have lower activities in
tiswes fi'om zinc-deficient animals than from their controls. Second, zinc has been implicated
in the stabilization of nucleic acid conformation. Therefore, alteration in tissue zinc
concernration could modify the DNA template used for transcription or translation. Finally,
zinc-dependent changes in the concentration of other ions, known to have interactions with
zinc, such as iron and copper might also alter nucleic acid structure or activity of enzymes
involved in their metabolism. For instance, in an eariy study ofFalchuk et a1., (1977) on zinc-
deficient E. gracilis, they observed the accumulation of iron, manganese and copper, and
subsequent effect on template specificity and products generated by RNA polymerase
specifically due to manganese accumulation.

Another contribution of zinc to gene expression is the zinc requirement of many
transcriptional factors for their structural stabilities and their functional activities (Klug and

Schwabe, 1995). Many transcription factors include small projections called ”zinc fingers"

28

that are needed for DNA recognition These structural domains connect transcription factors
to their target gene mainly by binding to specific sequences of DNA base pairs (Figure 1).
In 1985, Rhodes' lab at the Medical Research Council in Cambridge, England was the first
to identify zinc finger structures in a transcription factor of the immature oocytes fi'om the
fiochnaprLs ms, namely TFIIIA or factor A (Klug and Rhodes, 1987). Since then more
tlarn 200 proteins, many of them transcription factors, have been shown to incorporate such
zinc fingers.

The amino acid sequence of TFIIIA was found to be rather unusual, because it
contains nine repeat units of about 30 amino acids with conserved cysteine, histidine, and
hydrophobic residues (eg., Tyr,Phe,Leu). These sequences are arranged as: Y-X-Cys-XM-
Cys-X,-Y-X,—Y-X¢-His-X,,,-His, where Y represents a hydrophobic residue, X any amino
acids, Cys cysteine, and His histidine (Miller et al., 1985; King and Schwabe, 1995). The
cysteine and histidine side chains coordinate the zinc and the other hydrophobic residues pack
to form a hydmphobic core to somehow help stabilize the arrangement (Berg and Shi, 1996),
thus making a Cys, His, type zinc finger domain. The two tightly bound zinc ions in TFIIIA
are involved in the multiple roles in the regulation of ribosomal SSRNA synthesis (Klug and
Rhodes, 1987). The structure of each of these zinc finger domains consists of two antiparallel
Banandsfollowedbyanahelixwhichinteractwiththemajor groove ofDNA(Berg and Shi,
1996). A schematic view of a Cys2 His2 zinc finger domain is presented in Figure 2.

Over the past decade more than 10 classes of such zinc based domains have been
identified and biochemically characterized (Schwabe and Klug, 1994). It has been estimated

that between 300 and 700 human genes (about 1% of human genome) encode zinc finger-

29

containing proteins (Hoovers et al., 1992). A recent study by Bianchi et a1., (1992)
dammtratedflreerdstaneofaCys,Ifis,zincfingamofifinP2protmnines, one ofthe sperm
rudear proteins. Zinc is abundant in human sperm nuclei and is presumed to stabilize sperm
chromatin through a reversible binding to the thiol groups of mature spermatozoa. Thus the
presence of zinc fingers in P2 protamines could contribute to sperrnatogenesis and fertility,
since zinc deficiency leads to infertility.

More relevant to the studies presented in this dissertation is the presence of zinc finger
structures in the steroid-thyroid hormone receptor superfamily (Evans, 1988; Carson-Jurica
er al., 1990). Cloning and sequencing of the glucocorticoid receptor (GR) has revealed a
cysteine-rich region in the DNA binding domain. This region was found to contain two zinc
finger structures ( Archer et 01., 1990; Vallee et a1., 1991). While the zinc fingers reviewed
so far utilize two cysteines and two histidines to bind zinc in a tetrahedral structure, steroid
receptors including GR utilize four cysteines to coordinate zinc (Freedman, 1992; Dahlman-
Wright et al., 1992). The requirement of zinc occupancy in the zinc fingers for the DNA
binding is absolute, since chelation of incorporated zinc in GR, reduced receptor aflinity for
DNA binding by over 20 fold (Freeman et al., 1988).

Collectively, the unique characteristics of zinc including its small size, lack of redox
activity and its relatively rapid ligand exchange reactions appear to be at least partially
responsible for its role as a structural element in nuclear acid-binding or other gene regulatory
proteins. Thus, the physiological and immunological alterations observed in zinc deficiency
could be partly due to the impaired zinc-dependent molecular activities which are the keys to

the biological functions. Alternatively, the reduced zinc availability could exert its effects

30

indirectly via induction of other molecules (eg., GCs) that are known to be

irnmunosuppressive. The latter will be addressed in Chapter Four of this dissertation.

31

Figural; A schematic view of DNA binding of a transcription factor via zinc finger
domains. Three zinc finger structures have facilitated the binding of the
transcription factor to the major groove of a DNA molecule (Rhodes and

Klug,1993).

32

 

33

W Schematic view of the classical zinc finger domain of TFIIIA. (Top)
Tetrahedral binding of zinc to cysteine (C) and histidine (H) residues along with
other amino acid sequences involved in zinc finger domain formation in TFIIIA
(Rhodes and Klug, 1993). (Bottom) Three dimensional view of the structure

of a Cys2 I-Iis2 zinc finger domain (Berg and Shi, 1996).

34

ZINC-FINGER MODULE LINKER ZINC-FINGER MODULE

 

J l l
I if IF I
HYDROPHOBIC
AMINO ACID

.4 / r -
.-‘.> "' ”R ‘J
U _.' “t I

CYSTE'NE HISTIOINE

G (:1 3 it:
\@/ \@/
@//\\¥i ? ©// \\H
a; (3,.

ZINC

1 $ICSFADGGAAQNKwaflo-A0Lc
2166K-PGPCKEEGCEKGOTSLHHurraoSL

K6
T0

3TGEKoNGTCDSOGCOLROTTKANMK-KOFNRFG

4NIKrcvmVCHFENCGKAGKKHNOGK~v00F
STOOL-PQECPHEGCDKROSLPSRBK-995K

50
V0

GAG----@PoKKooscstcxrwrfivLKGVAECO

7ODs-rLAVC--DVCNRKORHKOYflR-OOOK

T0

aEkearmeCPaoccoaserrAPuma-sorosso

9550a.PevceuAcchcoAMKKsee-aOsv

 

vo

W

SECTION II: Zinc Deficiency and its Effects on Immune

System

35

36

Zinc Deficiency: As mentioned earlier, zinc is an essential trace element for normal
growth and development in both humans and animals (Endre et at, 1990; Walsh et al., 1994;
Cousins, 1996). In this regard, the body has developed a very sophisticated control
mechanism for the correct delivery and utilization of this trace element to maintain the
biological activities eficiartly. Therefore, inadequate dietary zinc intake as well as any defect
or abnormalities in the utilization, absorption, transportation, and delivery of this molecule
to the appropriate tissues could impair homeostasis. One of the most important consequences
of this imbalance is the state of zinc deficiency (ZD). It is well established that ZD is a world
wide nutritional deficiency which affects both men and women of all ages and socioeconomic
status (Prasad, 1991; Walsh et al., 1994). However, the deficiency is prevalent among
mahiourished patients and children, pregnant teenagers, and elderly with low socioeconomic
status (Prasad, 1988). This deficiency predisposes patients to increased risk of infection, and
threatens their recovery or even their survival (Prasad, 198 8), indicating impaired immune
function.

Theimportanceofzincinhumanhealthandthe occurrence ofdietaryZD in manwas
first recognized in 1963 when Prasad et 01., described a syndrome of dwarfism and
hypogonadisrn in cases fi'om Iran and Egypt (Prasad et al., 1961; 1963). The syndrome
described in male Iranian subjects consisted of severe growth retardation, severe anemia,
hypogonadism, hepatosplenomegaly, and rough, dry skin (Prasad et a1., 1961). Similar
clinical manifestations were noted in subjects fiom the Cairo area in Egypt (Prasad et 01.,
1963). The onlydifi‘erencebetweenthesetwo groupswasthat Iranian patients had geophagia

whereas almost all the Egyptian cases exhibited Schistosomr‘asis or hookorrn infections. It

37
was hypothesized that lack of zinc was primarily responsible for the observed manifestations

in these patients. This was supported by the low concentrations of zinc in plasma, red blood
cells (RBC) and hair of these individuals. Furthermore, successful treatment of this syndrome
withzinc supplementation confirmed the presence onD. The primary cause onD in these
subjects was related to their poor diet. The main food consumed by these patients consisted
of bread urd beans which are high in phytate, therefore making insoluble complexes with zinc
and inhibiting zinc absorption (Prasad et al., 1961; 1963). In addition to the poor diet,
excessive blood loss due to infections and loss of zinc by sweating in hot climates, were
several other causes of ZD in these human subjects.

Zinc deficiency is characterized by anorexia and growth retardation as the primary
signs in both humans and animals (Chesters and Quarterrnan, 1970; O’Dell and Reeves,
1989). In humans the manifestations of ZD include poor appetite, growth retardation, male
hypogonadism, mental disturbances, delayed wound healing, and skin disorders. If ZD
persists, the symptoms become more pronounced with the addition of recurrent infections due
to the depressed immune system. Ifuntreated, the ZD becomes fatal (Prasad, 1988; Mills,
1989; Aggett, 1989; Harnbidge, 1989). In experimental animals the manifestations include
anorexia, growth cessation, gastrointestinal malfunction, diarrhea, dermatitis (eg.,
parakeratosis) and impaired irnmunocompetence (Carlomagno and McMurray, 1983;
Hambidge et at, 1986).

The importance of zinc in numerous biological functions and the deleterious efi'ects
of its deficiency have initiated a growing body of literature on the etiologies of this nutritional

deficiency which are summarized below.

38

EtioloyonincDeficiency: Sincedietisthemajorsourceofzinc intake, a common
cause of mammalian ZD is underlined by improper diet and insuficient food intake (O’Dell
and Reeves, 1989, Walsh et at, 1994). In spite of the ubiquity of zinc in almost every tissue,
there is no lmown body store for zinc, as there is for iron; therefore zinc must be supplied in
the diet continuously. The recommended daily allowance (RDA) of zinc in human is about
10-15 mg (Walsh et al., 1994).

In addition to adequate dietary zinc intake, dietary composition contributes to the
bioavailability of zinc for intestinal absorption. For instance, severe ZD is prevalent in
populations whose diet is mainly consisted of large quantities of plant proteins (eg.,
unleavened grain products) containing phytate which is known to form insoluble complexes
with zinc and decrease the absorption of zinc (Solomons and Cousins, 1984; Sandstrbm and
Lennredal, 1989). As described previously, early studies in southern parts of Iran and in
Egypt docrnnented the excessive intake of plant proteins mainly in form of unleavened bread
as the principal cause onD in these areas (Prasad, 1991; 1995). Furthermore a recent study
on peri-urban Guatemalan school children with low socioeconomic status and poor diet
revealed a state of ZD in these subjects (Cavan et al., 1993). The main food consumed by
these children were corn tortillas and black beans which contain high amounts of phytate,
dietary fiber and calcium, all of which are known to inhibit zinc absorption. Selected zinc
indices such as hair, plasma zinc concentrations and the activity of alkaline phosphatase in
plasma and red blood cell membranes were examined and were shown to be diminished in
both sexes, being more pronounced in boys. Upon zinc supplementation, these conditions

were corrected to the normal levels.

39

Acrodernrafirr's enteropathr’ca (AB), a genetic disorder in zinc assimilation, also
rearlts in ZD. AB is a rare childhood disease of high morbidity and mortality and is inherited
as an autosomal recessive trait, afi‘ecting the sexes equally (Dillaha et al., 1953; Moynahan,
1974). The role of zinc in this genetic disorder was recognized when Moynahan and Barnes
(1973) used zinc supplementation on a case with this disorder. The patient showed a
remarkable recovery after administration of zinc. This observation was rapidly confirmed by
Moynahan in 1974. The possible mechanism responsible for ZD in AB has been related to
impaired intestinal uptake (77% decrease) and transfer of zinc (Aggett et al., 1981; Weismann
et al., 1979; Atherton et 01., 1979). In a most recent study by Grider and Young (1996), it
was noted that the impairment in zinc uptake in AB patients is not restricted to intestinal
mucosal cells. Using fibroblasts fiom AE patients, they demonstrated significant decrease in
zinc content, S’rarcleotidase activity ( a zinc metalloenzyme), and altered zinc uptake kinetics
cmpuedwifirnumdfibrobhfls.11esedatawggefledeainpstheAEmumfionafi‘ecu
the number of functional zinc transport proteins by either reducing their total number or
afl‘ecting their function. This condition is associated with low plasma zinc, growth
retardation, hypogonadism, skin lesions, bowl disorders, central nervous system malfunction
and fi'equent immunodeficiency (Moynahan, 1974; Van Wouwe, 1989). The close
resemblance between AE manifestations in human subjects and symptoms of ZD in animals
(eg., adult mice) support the use of animalmodels for the study of ZD in humans.

ZD has also been reported in patients with sickle cell anemia, an inborn error of
harroglobin metabolism which results in red cell sickling and continuous hemolysis, with an

accompanying high rate of infections (Ballester and Prasad, 1983 ; Prasad, 1993). An early

40
study by Prasad and colleagues (1978) reported decreased zinc in plasma erythrocytes and
hair of these patients with concomitant increased urinary zinc excretion. The continued
hyperzinairia and hernolysis in these patients and the existence of zinc as an important
constituent of erythrocytes may have been responsible for the state of ZD.

In addition to dietary ZD and inherited disorders in zinc absorption, there are several
disease states which may lead to ZD. The diseases and the possible explanations for the
presence of ZD (indicated in parenthesis) in each case are summarized as: chronic renal
disease (proteimria and failure of tubular reabsorption); chronic alcoholism (increase in renal
clearance of zinc and hyperzincuria); gastrointestinal disorders (insoluble zinc-protein and
zinc-fat complex formation); cirrhosis of the liver (abnormal zinc assimilation and
hyperzincuria); diabetes mellitus (increased urinary losses of zinc); various neoplastic diseases
(anorexia and starvation); parasitic infections (blood loss); burns (losses in exudates);
psoriasis (loss of skin epithelial cells and the massive formation of new cells); and AIDS
(reamed appetite, decreased dietary intake, gastrointestinal malabsorption, and fiequent acute
and chronic infections) (Prasad, 1979; Pai and Prasad, 1988; Endre et 01., 1990; Shippee et
01., 1992; Odeh, 1992; Walsh et al., 1994).

Collectively, this information supports the critical role of zinc in many biological
fimctions, with zinc deficiency resulting in numerous physiological complications including
impairment of immune fiinction. The studies provided herein have utilized the induction of
dietary ZD in mice as a general model to emphasize the importance of zinc in immune
integrity and to provide significant information on the status of various immune components

inZD.

41

fine Deficiency and the Immune System: The interrelationship between nutritional
deficiencies and the immune response has been well established in both human and animal
studies (Good andLorenz, 1992; Sherman, 1992; Fraker et al., 1993; Prasad, 1995). Being
a dynamic system with constant development, differentiation, and proliferation, the immune
system requires a continuous flow of nutrients for its proper function. Among nutritional
elements zinc has been recognized as an essential trace element for the development and
integrity of the immune system, as discussed previously (Endre et 01., 1990; Sherman, 1992;
Aggett and Comerford, 1995). In fact numerous clinical and experimental observations have
documented the deleterious effects of ZD on immune fiinction (Keen and Gershwin, 1990;
Prasad, 1991; Dardenne, 1993; Fraker et 01., 1993; Walsh et al., 1994).

Asmarfiawdpreviwdyflwmrponmeofa'minhmnumywasfirstrecogruzedwith
the discovery of human ZD due to a poor diet (Prasad et aL, 1963). Dwarfisrn, testicular
atrophy, low n‘nc concentrations in plasma, hair, and erythrocytes, and increased susceptibility
to infections were characteristic complications in these patients. The reversibility of these
clinical manifestations upon zinc supplementations established the presence of ZD in these
individuals.

Further evidence relating zinc deficiency to impaired immunity have come fi'om
patients with Acrodennatitis enteropathica, a classical model for zinc deficiency. Early
studies by Moynahan and Barnes (1973) described a 2-year-old giri with severe AE and
several immunological complications such as skin lesions, gastrointestinal disorders,
decreased delayed hypersensitivity reactions, and reduced numbers of peripheral T and B

cells. This lymphopenia is a condition accompanying ZD. Upon zinc supplementation all the

42
symptoms were corrected. The immunological abnormalities generally described in patients
with AE are thymic atrophy, lymphopenia, reduced proliferative response to mitogens,
decreased natural killer (NK) cell activity, decreased neutrophils and monocytes chemotaxis
activity, deficient thymic hormone activity and increased susceptibility to infections (Chandra
1980; Van Wouwe, 1989; Endre et al., 1990). The presence of ZD in these patients is thought
to be due to congenital malabsorption of zinc.

The development of experimental dietary ZD in humans was an advancement in the
characterization of specific physiological and immunological alterations caused by mild ZD
(Abbasi et 01., 1980; Prasad, 1991; Rabbani er a1., 1987). Consumption of a semisynthetic
soy-protein based diet (3-5 ngn/day) by male volunteers for 28 weeks resulted in
oligospermia, decreased serum testosterone and thymulin activity, decreased lean-body mass,
and decreased interleukin II (IL-2) production. In addition, diminished NK activity, and
alterations in T-lymphocytes subpopulations with selective decrease in T-helper cells were
noted. Zinc supplementation of 30 mg/day for 20 weeks reversed all the above symptoms
(Rabbani et al., 1987).

The incidence of ZD and immunological abnormalities have been also reported in
children with severe protein calorie malnutrition (PCM). Indeed lower serum zinc levels are
often noted in PCM. The common features observed in human ZD and PCM include:
anorexia, diarrhea, growth retardation, thymic atrophy, lymphopenia, reduction in lymphoid
tissues, impaired cell-mediated and immoral-mediated immunities, and increased susceptibility
to infections. These manifestations as well as reduction in plasma, muscle, and liver zinc

levels led to the suggestion of the role of ZD in the development of PCM. As expected, zinc

43

therapy in children with PCM reversed the manifestations and resulted in thymus growth in
these subjects (Golden et 01., 1977; Kuvibidila et aI., 1993).

Children with Down's syndrome, trisomy of chromosome 21, also demonstrate a
muginal zinc deficiency associated with impairment of thyroid functions, reduced thymulin
activity, reduced neutrophil chemotaxis function and subsequent increased susceptibility to
infections (Licastro et 01., 1992). Improved immunological efficiency observed in these
subjects after zinc supplementation confirms the role of ZD in the pathogenesis of some of
these immune defects (Licastro et 01., 1992).

The role of low bioavailability of zinc on the impairment of immune system, has also
been reported in patients with rheumatic heart disease (rum), cystic fibrosis, leukemia, and
chronic uremia. The impaired cell-mediated immunity, decreased delayed hypersensitivity
responses, decreased thymulin activity, and significant low serum zinc levels are the common
immunological defects in these patients (Gorodestky et aI., 1985; Consolini et 01., 1986;
Gupta et aI., 1992; Bonomini et aI., 1993; Mocchegiani et aI., 1994; Mocchegiani et aI.,
1995).

The role of zinc in cellular immunity, or the thymus-dependent immune firnctions, has
bear recently related, in part, to the regulatory role of thymulin in T-cell differentiation and
matrn'ation (Prasad et al., 1988). Thymulin (previously known as facteur timique serique, or
FTS), one of the best known thymic hormones, is a zinc dependent hormone synthesized by
the epithelial cells of the thymus and is adversely afl‘ected by ZD. Zinc is required to confer
biological activity to thymulin, which has a modulating action on cell-mediated immunity.

The inc-unbound form is inactive and can inhibit the active form possibly via competing for

44
the thynarlin receptor (Fabris et aI., 1984; Prasad et aI., 1988). It has been shown that with

ZD the thymic peptide, thymulin, is synthesized and secreted in normal amounts, but only a
fiaction of it binds zinc ions and becomes active. However, in vitro addition of zinc ions
causes an increase in the active form of thymulin (zinc-bound thymulin) which in turn
edunces thymocyte responses to mitogens (eg., phytohemagglutinin, PHA; concanavalin A,
ConA) (Saha et aI., 1995).

In addition to disease states, many physiological conditions in which the state of zinc
deficiency is observed are accompanied by altered immunological functions. For instance,
during aging there is a considerable loss in both zinc status, possibly due to reduced intestinal
zinc absorption, and immunological fiinction particularly cell-mediated immunity (Chandra,
1990; Ripa and Ripa, 1995). Several investigators have shown improvement of immune
function-specially delayed type hypersensitivity reactions (DTH) and significant restoration
of serum thymic hormones-activities in elderly subjects after low dose zinc supplementation
(20-30 ngn/day) (Bunker et 01., 1987; Prasad, et aI., 1993; Boukaiba et 01., 1993; Bogden
er‘ aI., 1994; Bogden, 1995). This indicates that zinc supplementation in elderly populations
provides significant immunological benefits. Furthermore, it should be noted that the zinc
requirement is higher during periods of rapid growth such as infancy, childhood, adolescence,
and pregnancy, thereby putting these populations at a greater risk of ZD (Moore et aI., 1984;
Harnbidge et aI., 1986; Prasad 1995; Ripa and Ripa, 1995).

Parallel to human studies, dietary studies in animals have also shown similar patterns
of impaired cellular and humoral-mediated immunity as well as thymic atrophy and

lymphoperfia due to zinc deprivation and the restoration of immune function by zinc repletion.

45
Using a murine model in a 30-day dietary a'nc studies, Fraker and colleagues initiated a series
of investigations on the efi'ects of suboptimal dietary intake on immunity. The following
information is a review of these accomplishments as well as some studies by other
investigators which in parallel explain the immune capacity of the murine system in zinc
deficiency.

In 1977, Fraker etaI. demonstrated that 4-week dietary ZD (0.5 uan/g diet) caused
rapid thymic involution (>60%) in young adult (6-week old) All mice. In 6 weeks, mice fed
ZD diet became athymic. When the antibody-mediated response of these mice to sheep red
blood cells (SRBC) was evaluated, ZD mice produced only 10% of the number of anti—SRBC
antibody producing cells (PFC; plaque forming cells) generated by zinc adequate (ZA) control
mice. Reconstitution of the ZD mice with thymocytes from normal mice restored their
response and generated 61% as many plaques as the control mice. These data clearly
indicated that ZD greatly affected T-cell helper function. In a similar dietary zinc study,
Fernandes and coworkers ( 1979) investigated the immune response of difl‘erent strains of
young adult mice (A/J, C57BI/Ks, and CBA/H) to deficiencies in zinc. When placed on ZD
diet, these mice showed loss of body weight, significant involution of thymus, and low serum
zinc levels within 4-8 weeks afier initiation of diet. Almost 50% of the mice on ZD diet
developed severe skin lesions on tail and paws and diarrhea. Furthermore, defective
development of both direct and indirect PFC after in viva immunization with SRBC,
depressed T—killer cell activity against in viva immunization with tumor cells, and low NK cell

activity were observed in ZD group. These data confirmed the previous findings on the

46

impairment of T-cell dependent immune firnctions in ZD and demonstrated similar
immunological response fi'om different strains of mice to this nutritional deficiency.

To further characterize the effects of ZD on antibody-mediated responses of young
achrlt mice, the capacity of ZD mice to respond to T-cell independent (TI) antigens were also
evaluated (Jardieu and Fraker, 1990). The TI antigens primarily elicit responses from
macrophages and B-cells with less involvement of T-helper cells. Furthermore, response to
TI antigens develops at difi‘erent stages of B-cell development. Trinitrophenyl conjugated
lipopolysaccharide (TNP-LPS), a TI-class 1 antigen, stimulates immature B-cells; whereas,
TNP-Ficoll, a class 2 TI antigen, stimulates more mature B-cells (F raker et al., 1984). After
a 28-day period of suboptimal intake of zinc, the deficient mice exhibited only 32% as many
total splenic anti-TNP PFC to TNP-LPS and 50% as many PFC in response to TNP-Ficoll
as compared to ZA fed mice at day four afier immunization. By day 5, the deficient mice
could only produce 28,000 anti-TNP PFC per spleen compared to 69,000 PFC produced by
ZA mice. These data represented a significant reduction in host defense capacity. The
kinetics of the response of the ZD mice to each antigen was delayed by two days fiom the
optimal time of response to the antigen (day 3). In evaluation of the proportion of
splenocytes responding to antigenic challenge (PFC/10‘ viable splenocytes) over time (3, 4,
5 days post immunization), the optimal response of the deficient mice was again delayed by
two days compared to theresponse onAgroup. However by day 5, the number ofPFC/lO‘
splenocytes had increased in the ZD group, being equivalent to ZA group at their optimum
day (day 3). This suggested that the residual B-cells of ZD mice were functional, with

perhaps slower rate of activation and proliferation in response to antigenic stimuli. In a

47
sirm'lar dietary a'nc study conducted by Moulder and Steward (1989), mice fed ZD diets had

reduced numbers of T-cells and T-cell subsets, decreased antibody and cell-mediated
responses to T-cell dependent (TD) and TI antigens as well as decreased IL-2 production.
Overall, the significant reduction in antibody-mediated responses to either TD or T1 antigens
indicate a substantial loss in host defense capacity in zinc deprived mice.

As indicated earlier susceptibility to infection is also pronounced in ZD. In this
regard, Fraker et 01., (1982) investigated the effects of this deficiency on host resistance to
infection by T opanosoma cruzi, an intracellular parasite causing Chagas’ disease in South
America. The extreme vulnerability of ZD mice to this pathogen was demonstrated by a
substantial increase in blood parasites in ZD mice (50 fold) compared to controls (infected
mice in restricted or zinc adequate fed groups). Furthermore, by 22 days post-infection,
almost 80% of the infected ZD mice died, whereas there was no mortality among either the
uninfected ZD or the infected ZA mice. These data clearly demonstrated the synergistic
interaction between ZD and T. cmzi on the observed death rate.

Since mononuclear phagocytic cells (MNPs) are the first line of defence against T.
cruzi, the follow-up experiments were designed to evaluate MNP function. In this case, it
wasshownthattheinability oftheZD mice to defend against infection by T cmziwas most
likely due to the defective phagocytic and microbicidal activities of resident macrophages
(Wirth et 01., 1989). At time zero after infection, the percentage of macrophages engaged
with T crust and the number of T. mm’ per 100 macrophages was substantially reduced in
the ZD group. Twenty four hours after infection, macrophages from the ZD group were

unable to destroy as many parasites as macrophages from the ZA group. In other words,

48

there was a reduction (20-50%) in the number of parasites per 100 macrophages in the ZA
group but not in the ZD group. Interestingly, pro-incubation of MNPs from the ZD mice for
one hour with zinc chloride (10 ug/ml) restored all the functions (Wuth et 01., 1989). Thus
it was evidernt that the macrophages fiom ZD mice had some functional processes that were
impaired due to the low availability of zinc. This defective function was later related to the
oxygern burst activity which is thought to be heavily dependent on metals such as zinc (Cook-
Mills and Fraker, 1993b).

The profound efl‘ects of zinc deficiency on immune integrity against pathogens has
beern further emphasized in recent murine studies in which animals fed difl‘erent dietary zinc
levels were challenged with a variety of intestinal parasitic worms (F enwick et aI., 1990a;
1990b; Nawar et 01., 1992). In all cases zinc deficiency depressed both humoral and T-cell
mediated immunity, enhanced establishment of the parasites and impaired expulsion of the
parasite from the intestine. Zinc supplementation, however, restored rapidly the ability to
expel the infection by few days postinfection (F enwick et al., 1990a; 1990b).

In contrast to these studies, when Minkus and coworkers (1992) challenged the mice
with Helignsosmroidespoblgmas, (an intestinal nematode), their results showed no sigrnificant
differences between zinc restricted fed (5 mykg) mice and control mice with respect to cell
mediated immune response, worm proliferation (worm numbers), and egg production.
However, plasma a'nc corncerntrations were sigrnificantly lower in the zinc restricted fed mice.
These outcomes were reversed when the same examinations were evaluated in mice made
severely zinc deficienct (<0.75 mg/kg in the diet) (Shi et aI., 1995). These findings clearly

irndicate that marginal intake of dietary zinc (5 mg/kg) is not sufficient to affect the survival

49

of tlne intestiml rnernatodes. Furthermore, the low plasma zinc cannot be used by itself as an
index for zinc deficiency, since many physiological alterations (including infections) causes
plasma zinc depletion (Walsh et aI., 1994).

As with ZD human subjects, delayed type hypersensitivity reactions (DTH) in which
macrophages and T-helper cells are primarily involved are also afi‘ected in ZD mice. When
DTH response was nneasured in mice, ZD animals gave a very poor response (50% less than
ZA corntrols). However, after 21 days of nutritional repletion, re-feeding the ZD group with
ZA diet (55 uan/g diet) reanlted in normal DTH response in previously ZD mice (Fraker et
al., 1982).

With regard to these observations, it appeared that in spite of multiple defects in
immune system due to ZD, nutritionally deficient animals had the capacity to repair their
immune system when re-fed nutritionally adequate diets. To confirm this, 5-week old All
mice were fed ZD diet (<1 uan/g diet) for 31 days (Fraker et aI., 1978). Thymuses fiom ZD
mice were one-third of nomnal size with preferential involution of the cortex. The direct PFC
(IgM response) and the indirect PFC (IgG response) produced per mouse spleen to SRBC
immuniution were 34% arnd 18% of the normal, respectively. The deficient mice were then
re-fed ZA diet (50 uan/g diet) and the degree of restoration of the thymus as well as
regeneration of T-cell helper function were evaluated at 1, 2, and 4 weeks. By 4 weeks, the
thymus weights and antibody-mediated responses to SRBC were normal. Thus it was
obvious that ZD young adult mice had the capacity to restore T-cell dependent antibody-

mediated responses after dietary zinc repletion.

50

In a subsequent study in which the functional capacity of residual lymphocytes from
ZD mice was investigated, cultured splenic T-cells in autologous sera from ZD mice gave
normal proliferation and adequate IL-2 activity when stimulated with Con A Furthermore,
splenic B-cells from ZD mice which were stimulated in vivo with SRBC produced
significantly lower rannbu' of PFC (50%-70% depression based on the degree of deficiency)
compared to the ZA group. However, the proportion of B-cells responding as determined
by the number of PFC/ 10‘ viable splenocytes remained unchanged in ZD mice. It was also
demonstrated that similar amounts of IgM and IgG antibodies per activated B-cell were
produced by all cells fi'om all dietary groups. In addition, the average number of splenocytes
irn MZD and SZD was reduced by 43% to 47%, respectively, compared to ZA control. These
firndirngs emphasized that many of residual lymphocytes of tine deficient mouse were firnctional
and correlated the impaired immunity in ZD to lymphocytopenia ( Cook-Mills and Fraker,
1993 a).

The presense of lymphopenia and defective host defense in ZD prompted the
investigations on the degree of sensitivity of lymphoid subsets to the effects of this deficiency.
In this regard, the spleern, which contains predominantly mature lymphocytes of difi‘erent
subsets, was used to evaluate the proportion or the phynotypic distribution of T and B cells
(King et 01., 1991). In spite of sigrnificant reduction in the total numbers of splenic
lymphocytes (~50% loss), it seemed that ZD had no sigrnificant efi‘ect in the distribution of
the more mature lyrrnphoid subsets residing in the spleen. Marginally ZD mice showed a
rnormal ratio of T cells to B cells, and severely ZD mice demonstrated a 20% increase in the

overallratioofThelpertoTsuppressorcells. Thesedata, aswellasotherhumanandanimal

5 1

studies in which thynnic atrophy and lymphopenia were predominantly observed in ZD
arbjects (Moulder and Steward, 1989; Fraker et 01., 1993; Kuvibidila et al., 1993), suggested
that reduction in absolute number of lymphocytes involved in immune response was the
principal cause of reduced host defense. Knowing that zinc plays a sigrnificant role in the
firnctioml activities of many enzymes involved in cell proliferation and growth, alteration of
lymphopoiesis in ZD was possible. Thus, extensive studies on the BM, as the site of
lymphopoiesis in adult mammals, were initiated. In fact, Chapter Two represents the first
detailed study on the status of developing B-lymphocytes in the marrow of ZD mice. This
was acollaborativeworkwithDr. Louis King, a sernior investigator in our laboratory. As will
be shown, this study demonstrates a significant loss (40-90%) in the proportion of nucleated
marrow B-cells with substantial sensitivity of more immature B-cells (3 5%-80%) depending
on the severity of the deficiency in ZD animals. These observations mediated more close
examination of B-lymphopoiesis in earlier stages of development (eg., progenitor and
precursor B-cells) to further specify the pattern of alterations in these populations. This will
be addressed in Chapter Three.

Thus, the overall pattern of immunological changes in ZD arnimals shows a high
correlation with alterations observed in zinc deprived human subjects. Furthermore, both
arnimal and human studies once again emphasize the multiple roles of zinc in the developing
immune response. The summary of above findings mainly indicate that a substantial portion
of the impairment of immune response was due to the lymphopernia and decreased number of
lymphocytes engaged in defense rather than functional disruption. This reduction in

lymphocytes rnurnbers could be either due to susceptibility of these cells to the less availability

52

of zirnc, as zinc is critical to cellular and metabolic activities and survival of the cells, or due
to alterations irn lymphopoietic processes responsible for generation of these cells. In addition
to the direct efl‘ects of zinc deficiency by itself, the elevation of glucocorticoids in zinc
deficiency, which is krnown to be irnmunosuppressive, could be an additional sigrnificant factor
in depressed cellularity of the immune system. These are some important possibilities that
unfortunately have been neglected in the study of nutritional-immunology and
immurnopathology. However, this study has tried to focus and to evaluate the aforementioned
possibilities in the suppression of lymphopoiesis in zinc deficiency. Since BM is the primary
site of lymphopoiesis in adult mammals, and thymus is the site of T-cell maturation, these
tissues were utilized in this work. Chapters Two and Three will exclusively demonstrate the
efi‘ects of ZD on B-lymphopoiesis in the marrow and the extent of the susceptibility of each
subpopulation of B-lymphocytes to the efl‘ects of this deficiency. Furthermore, the status of

T-cell maturation in the thymus of dietary ZD mice will be addressed in Chapter Five.

SECTION III: Ph enotypic Characterization of B and T
Lymphocytes at Different stages of development in

Murine System

53

54

Introduction: During mammalian ontogeny, the lymphohernopoietic lineages are
generated seqnerntiallyinyolksac, fetalliver, fetal spleen, and bone marrow (BM) whichthen
becomes the major site of lymphohemopoiesis during postnatal life (Kincade, 1981; Ikuta et
aI., 1992; Marcos et aI., 1994). The process of lymphohemopoiesis is believed to be
regulated by the BM microenvironment. The hematopoietic microenvironment in the bone
marrow, which consists of adherent stromal cells such as endothelial cells, fibroblasts, and
dendritic cells as well as various cytokines and adhesion molecules, supports a continuous
proliferation and differentiation of lymphohematopoietic cells of multilineages, in addition to
the pluripoternt hematopoietic stem cells (PHSCs) (Dorshkind, 1990; Ikuta et aI., 1992).
These stem cells, with an estimated fiequency of 0.01-0.005% of all nucleated cells in the BM
(T sai et aI., 1994), have the capacity for extensive self-renewal, and the ability to rescue
lethally irradiated arnimals by giving rise to all difi‘erent blood cell types which are the cellular
components of the immune system (Figure l) (Heimfeld and Weissman, 1992; Ikuta et al.,
1992).

Although, the question of whether T and B lymphocytes arise fiom a common
lymphoid progenitor or directly fi'om multipotent hematopoietic stem cells is still unclear, it
is certain tlnat their site of nnaturation is different. Progernitor T-cells are generated in the BM,
arnd tlnern migrate irnto the thymus, where they proliferate and difl‘erentiate into mature T-cells.

Mature Tocells which reside in the thymic medulla leave this tissue through veins and
lymphatic vessels and join the circulating lymphocyte pool (von Boehmer, 1992; Kruisbeck,
1993; Pawlowski and Staerz, 1994). In contrast, B progenitors in mammals are generated

and remain in the BM, where they differentiate into mature immunoglobulin (Ig) bearing

55

B—cells via a complex series of steps. These maturation steps involve lymphocyte-stromal cell
irnteractiorn, Ig gerne rearrangement, and surface expression of Ig molecules (Osmond, 1993;
Melclners etal., 1995). The mature B-cells then enter the blood stream and migrate into the
secondary lymphoid organs (eg., spleen, lymph nodes) where they participate in immune
responses. Tlnus, since all development and maturation of B-lymphocytes occurs in the EM,
it is easy to assess the status of difi‘erent stages of B-cell development and the firnction of BM
lyrrnphopoiesis irn ZD in which lymphopenia is predominantly observed. Thus to comprehend
the status of lymphopoiesis in ZD, it is necessary to review the stages of lymphocytes
development as well as associated cellular and molecular events, all of which will be discussed
in the following sections.

B-cell development in the BM:

As mentioned earlier, adult mammalian B-cells, which comprise 23-25% of the
heterogernous BM population, are predominantly generated and mature in the primary
lymphoid orgarn, BM (Osmond et aI., 1994). Although early stages of B-cell development
are not well defined, a combination of cellular and molecular events such as Ig gene
rearrangements and gene expression, the expression of specific surface and molecular
markers, as well as growth requirements have been utilized to characterize the sequential
steps irn B-lymphopoiesis in the BM. In this regard a few models have been proposed, some
of which will be discussed here. However, with the use of Hardy's scheme of B-cell
development (1991) in this dissertation, the main emphasis will be given to this system.

The most primitive cells of tire B-lineage are called progenitor B (pro-B) cells. These

B lineage restricted cells, which comprise 4-6% of the total nucleated bone marrow cells,

56
retain Ig genes irn the germlirne configuration arnd lnave the capacity to differentiate into mature
B cells expressing diverse antigen receptors (Hardy et 01., 1991). These progenitor B-cells
that are believed to start gene rearrangement by joirning the D (diversity) and I (joint)
segrnernts of the Ig heavy chain (D-JH), subsequently become large precursor B-cells (pre-B)
in which the variable(V) segment of the Ig heavy chain (IgH) is joined to the previous D-JH
complex (V -D-J“) (T arlinton, 1994). This recombination generates a complete IgH chain
gene (u), shown in Figure 2, which is expressed in the cytoplasm of pre-B cells (Cu) and is
the characteristic of tlne pre-B stage of development. This population accounts for 8-12% of
the total BM cells. Furthermore, studies on early B-cell development have identified the
association of a pseudo light chain, known as surrogate light chain (nlrL), with u heavy chain
(pH) of Ig irn place of the conventional light chain (KL or AL) of Ig on the surface of pre-B
cells (Karasuyanna et aI., 1994). This protein is composed of two polypeptide chains and
encoded by the Vpre-B arnd A5 genes (Rolink et al., 1994). the Vpre-B gene has sequence
honnology to the V region of the IgH and IgL chain genes and the A5 gene has homology to
the J and C regions of Ig AL chain gene (Rolink et aI., 1994). The expression of these two
genes is largely restricted to developmental stages before light chain gene rearrangement,
namely the pro-B cell stages and the large pre-B (Melchers et 01., 1993; Li et 01., 1993;
Karasuyarna et aI., 1994). In vitro studies on the sigrnificance of the uH-nlrL complex
formation have assigrned sigrnal transduction activities to this complex, since its expression was
accompanied by an increase in intracellular Ca++ levels as well as tyrosine phosphorylation of

irmacellular proteins (Misener et aI., 1991). Moreover, using AS-deficient mice generated by

gene targeting technique, it was shown that B-cell development of early stages (pro and pre-B

57
cells) was disrupted in these mice (Kitarnura et 01., 1991). In the next step of Ig gene
rearrarngemerls, the liglnt chain gene rearrangement (V-JL) occurs in small pre-B cells carrying
cytoplasrra'cuhesvychain Therearrangementoflightchainbeginswiththexgene segment.
Ifthe rearrarngernernt is successive, the light chain rearrangement would stop with It being the
predominant light chain of the Ig molecule. If the rearrangement of both I: alleles is
nonproductive, the A gene segment undergoes rearrangement. Successful light chain
rearrangement together with it heavy chairn results in the expression of a complete Ig molecule
(IgM) on the surface of immature B-cells. Further differentiation of immature B-cells will
lead to the coexpression of IgM and IgD on the surface of mature B-cells (Rolink and
Melclners, 1991; 1993). The mature B-cells will then leave the BM and migrate to the spleen
and lymph nodes where they could be stimulated by foreign antigens (Picker and Butcher,
1992; Butcher and Picker, 1996). Upon activation of B-cells by antigens, IgD is down
regulated arnd the cells develop into memory cells and antibody secreting plasma cells with
IgM secretion as the early primary response (Roes and Rajewsky, 1993; Desiderio, 1994).
Recent studies on the structure of the B-cell membrane bound Ig molecules have
demonstrated that tlnese Ig receptors are non-covalently associated with a heterodirner of two
transmembrane proteins, namely Ig-a and Ig-B (Hombach et 01., 1988; 1990; Campbell and
Cambier, 1990). In fact, several studies have concluded that the IgM molecules will be
expressed on the surface only when associated with Ig-a/Ig-B chains, becoming a complex
receptor analogous to T-cell receptor complex (CD3 -ctB/TCR) (Sakaguchi et 01., 1988;
Hombach et aI., 1988; Kaslniwamura et aI., 1990). The Ig-a and Ig-B proteins are the

product of mb-l arnd 829 genes, respectively (Hombach etal., 1990; Matsuuchi et aI., 1992).

These

Norm

these

58
Tlnese transmennbrarne glycoproteins seem to be involved in sigrnal transduction since

modies agairnst mb-l causes Ca++ influx in pre-B lymphoma cells (Y amarnishi et al., 1991;
Normrra et aI., 1991; Matsuo et 01., 1993). The heavy chain of IgD is also associated with
these heterodimer glycoproteins, however, with slightly different Ig-a size (Campbell and
Cambier 1990; Chen et aI., 1990).

Besides Ig gene expressiorn, the progression of early immature B-cells to mature Ig
bearirng B-cells is also marked by acquisition or loss of B-cell differentiation markers as well
as expression of specific genes and their products required for B-cell development. Hardy
et 01., (1991) in their recent investigations on determination of early stages of B-cell
development were able to subdivide B-lineage cells of the marrow into several distinct
fi'actiorns or subpopulations. This investigation was based on flow cytometric analysis of cell
surface markers such as CD43 (S7) or leukosialin, BP-l, heat stable antigen (HSA), IgM,
IgD, immunoglobulin gene rearrangement arnd growth requirements. Thus, they proposed an
ordered difl'erentiation pattern of B lineage cells as: Pre-pro B @220*CD43 ‘HSA'BP-l';
Fraction A), early Pro-B (B220*CD43*HSA*BP1'; Fraction B), late Pro-B plus to precursor
B-cells (8220+ CD43”HSA‘BP-l"; Fraction C), small pre-B (B220‘CD43'IgM‘; Fraction D),
immature B (B220’CD43'IgM‘IgD'; Fraction E), and mature B (B220‘CD43'IgM’IgD’3
Fraction F) (see Figure 3). The modified version of this scheme was utilized in tlnis
dissetation to identify the subpopulations of pro and pre-B cells, as demonstrated in Figure
3. When the growth requirement of each isolated subset was evaluated, they found that the
growth arnd proliferation of cells from the earliest fi'action (Pre-Pro B) was absolutely

dependent on contact with a stromal layer whereas later fractions (early Pro-B, late Pro-B,

59

large Pre-B) could proliferate irn the presence of soluble mediator interleukine—7 (IL-7) alone.
This data is in agreement with earlier studies from Dorshkind Laboratory (1985; 1990) which
slnowed the absolute dependence of earliest B lineage progenitors on direct connection with
stromal layer arnd the proliferation of large pre-B cells on r1L-7 alone (without the presence
of stronnal cells). Furthermore, using a deletions! PCR assay, which permits the amplification
of DNA sequences that are normally deleted upon gene rearrangement, they noticed that cells
of the earliest fiaction (Pre-Pro B) showed no Ig rearrangement, and as cells progressed
toward irntermediate (eariy Pro-B) arnd late fractions (late Pro-B, large Pre-B) they possessed
D-Jfl rearrangements (Hardy et 01., 1991).

Several years later (1993) Li and his colleagues from Hardy's laboratory confirmed
and extended the aforernerntiorned observations by identifying the stage-specific expression of
genes involved in Ig gene rearrangements such as terminal deoxynucleotidyl transferase
(T dT), recombirnation activating genes (RAGl , RAG2) (Ferguson et 01., 1994) and genes that
associate with heavy chain, eg., A5, and Vpre-B (Melchers et aI., 1993). In this
complementary study they detected high expression of TdT in early Pro-B, late Pro-B, and
large Pro-B, and its absence in small pre-B cells. TdT, an intranuclear zinc metalloenzyme,
plays a role in generating immunological diversity by addition of non-germline encoded
nucleotides (N regions) at the V-D and DJ junctions of IgH chain genes (Landau et aI.,
1987). PresenceofnrcthegionsinIgI-Ichaingeneanditsabsencein light chain gene (V-J.)
correlates with the detection of TdT early in B cell difl‘erentiation and its absence in later
stages of development reported by Li et al., (1993) findings. The other two genes, A5 and

Vpre-B, which form the surrogate light chain (Karasuyarna, et 01., 1994), showed the same

60

espruionpattern, beingdetected inearlyPro-B, latePro-B, and largePre—B cells and absent
when light chain gene rearrangement occurs. In contrast with these three genes, the co-
expression of RAG-1 and RAG-2 enzymes responsible for heavy and light chains gene
rearrangements (Ferguson et 01., 1994) was significant in small pre-B , where much of the
light chain gene rearrangement takes place. Moreover, these enzymes were also detected in
early and late pro-B cells (Li et aI., 1993).

Parallel to Hardy's scheme of B-cell development, Osmond and his colleagues in a
series of studies have defined stages of B-cell differentiation using a combination of B-lineage
associated surface and molecular markers as well as mitotic arrest techniques (Park and
Osmond 1987; 1989a; Osmond 1990; 1991). As demonstrated in Figure 4, the B-cell
compartment of the bone marrow were divided as: Pro-B cells subdivided to early pro-B
(TdT*B220’), intermediate pro-B (TdT”B220*) and late pro-B (TdT‘B220*); pre-B cells
exhibiting cytoplasmic u heavy chain and lacking TdT subdivided to large pre-B and small
pre-B cells; and finally B lymphocytes expressing surface IgM.

Osmond (1990, 1991) in his kinetic studies demonstrated that the early B-cell
compartment including pro-B and pre-B cells undergo a series of mitoses, at least once at
each phenotypic stage of development with the exception of small pre-B cells that show much
lower turnover rate (Figure 4). As shown in Figure 4, the turn over rate of large pre-B cells
followed by smaller turn over rate of small pre-B cells indicated a dramatic change in this
transition state. Ifthis significant change is translated into less survival of small pre-B cells
,asitissuggestedbyOsmond(1993), then, thereisasignificantcell loss at this stage ofB-cell

development. This substantial cell death has been also reported by other investigators

61

(Jacobean etal, 1994; Osmond et 01., 1994). It is suggested that tlnis cell loss is mainly due
to defective lg gene rearrangements (Jacobsen et 01., 1994), which has also been suggested
to occur among cells of late pro-B cell subset (Td'I‘B220‘p') which may have undergone
nonproductive gerne rearrangements (Rolink arnd Melclners 1993; Rolink et 01., 1994). In vivo
studies have suggested that much of the loss occurs via apoptotic cell death followed by
mophage mediated phagocytic elimination of these cells (Jacobson et 01., 1994; Osmond
et a1., 1994). This cell loss provides a control mechanism to eliminate cells with
nonfunctional gene rearrangement and to regulate cell numbers entering the circulation. It
is estimated that out of 5x107 cells generated from B-cell progenitors in the BM of adult
mouse, only 2-Sx10‘ cells are released to the peripheral pool each day (Rolink and Melchers,
1993). Indeed, this dramatic cell loss at the pre-B stage of development is highly sigrnificant
for the study presented in this dissertation since it suggests the high susceptibility of these
cells to apoptotic cell death which might play a regulatory role in lymphopoietic processes.
The question of whether or not zinc deficiency and the subsequent increased endogenous
glucocorticoids would adversely afi‘ect this population that is highly programmed to die will
be addressed in Chapters two and three of tlnis dissertation.

As previously nnentiorned, early stages of B-cell development have been identified via
surface expression or elimination of maturation markers, gene recombination and expression,
and growth requirernernts. However, the studies herein have utilized only one aspect of these
developmental markers That is, the surface expression of B-cell maturation markers. The
availability arnd the specificity of nnonoclonal antibodies (mAbs) against stage specific surface

markers on B-lymphocytes and the flow cytometric determination of difl‘erent lymphocyte

62
subpopulations were efliciernt tools for the evaluation of the BM B-cell genesis. Thus, a brief

reviewonthestnncturalandfiunctional properties ofmajorearin-cells surface markers, most
of which were used in this dissertation, will be given below.
Structural and Functional Properties of Key B-Cell Maturation Markers:

B-lineage cells can be recogrnized by exclusive expression of tlne high molecular weight
fornn (220 KDa) of the leukocyte common antigen (L-CA) designated as B220 (CD45R)
tlnrouglnout the stages of B-cell maturation on the surface of all B committed and mature B-
cells (Cofi‘man and Weissmarn, 1981). This transmembrane glycoprotein is composed of
composed of elongated, heavily glycosylated exterior domain (~ 538 residues and less
conserved) arnd a large globular cytoplasmic domain (705 residues and highly conserved) with
a 22 amino acids membrane spanning region. Within the cytoplasmic domain there is an
internal duplication of about 300 amino acid residues with 33% homology within each
duplication (Thomas and Lefrancois, 1988). These regions of duplications has been shown
to have protein tyrosine phosphatase (PTPase) activity (Koretzky et al., 1992). It is
suggested that this portion of the molecule is involved in the signal transduction pathway by
acting on P593, arnd PS6“, of the Src-farnily protein-tyrosine kinases, in T-cells and P21m in
B-cells, activating the signal transduction complex (Guttinger et al., 1992; Rothstein et aI.,
1993; Kawauchi et aI., 1994). This is the molecule that has been selected in this dissertation
to identify BM B-lymphocytes, by using the mAb, RAB-6B2. There is no known cross
reactivity of this mAb with the L-CA found on T-cells (T-200) (Thomas and Lefrancois,

1988) (see Figure 3).

63

HSA, Heat stable antigern, is a molecule widely distributed on many cell types with
heterogeneous forms of 30.60 KDa depending on the cell type (Wenger et aI., 1991).
Molecular cloning arnd sequencing analysis of HSA showed a very short 27-amino acid
polypeptide that is anchored to the cell surface through a glycosyl-phosphatidylinositol
linkage with extensive N—and O-linked glycosylation (Wenger et aI., 1991; 1993). This
molecule slnows a fluctuating pattern of expression during B-cell differentiation indicating its
possible role in B-cell development. HSA is first detectable at low levels on the DJ
rearranged (BZ20‘CD43 +) cells, then reaches the highest levels at the large cycling pro-B to
late pre-B stage (Hardy et aI., 1991; Ehlish et aI., 1993). Immature B cells and newly
generated B cells in the spleen all express high levels of HSA. The expression of this
molecule is down regulated on most peripheral B-cells, increases upon B-cell activation and
becomes low or non-detectable in memory B cells and plasma cells (Allman et aI., 1992;
1993) (see Figure 3). HSA is recogrnized by several antibodies such as 30Fl, 111d, M1/69,
and 79, all of which have shown similar specificity and staining patterns for HSA molecule V
(Hardy et 01., 1991; Hough et aI., 1996; Hahne et aI., 1994).

In terms of its finnction, HSA was first identified as a costimulatory molecule on
activated B-cells, dendritic cells and epidermal langerhans cells being essential for the
irnduction of proliferative response in CD4+ T-helper cells (Hubbe and Altevogt, 1994; Liu
et a1., 1992; Enk and Katz, 1994). The second function assigned to HSA is as an adhesion
nnolecule (Sammar et 01., 1994). It has been shown that mAb 79 against HSA inhibited the
aggregation of lipopolysaccharide (LPS)-activated spleen B cells and induced an increase in

intracellular Ca++ levels (Hahne etal, 1994). Hough and colleagues in their two most recent

64
studies evaluated the role of murine HSA in lymphocyte maturation. Using transgenic mice

with overexpressed HSA, their data clearly showed perturbation of T and B lymphocyte
developrnernt. This was indicated by major reductions in both double-positive (CD4+CD8")
arnd single-positive (CD4*CD8'; CD4' CD8”) thymocytes, profound depletion of pro and pre
B-cells especially at the level of IL7-responsive B-cells, and reduced mature peripheral and
splenic B-cells with impaired response to LPS stimulation (Hough et 01., 1994; 1996). Thus,
these observations suggested a regulatory function for HSA tlnroughout the early stages of
T and B cell development. Being a general marker for B-lymphocytes and being detected on
more than 90% of nucleated BM cells (personal observation), the HSA molecule was not
evaluated in phenotypic determination of B-lymhpocytes presented in Chapter four.
NevethdesetlnisstnrdywasabletoidentilytlnesameB-cell subsets aswhen HSAwould have
been irncluded.

C1143 or leukosialin (also known as sialophorin in humans, LY48 or mouse CD43
(S7) in mice, and W3/ 13 in rats) is a major sialoglycoprotein present on granulocytes,
macrophages, T cells, erythroid and B cells at specific stages of development (Gulley et al.,
1988; Fukuda, 1991). In mice, this molecule is recogrnized by rat mAb S7 and has been
detected on early B cell progenitors arnd is be as these cells differentiate to pre-B and mature
B-cells but is upregulated on te'minally difi‘erentiated plasma cells (Hardy et aI., 1991). More
recently an additional site of expression for this molecule was found on both mice and human
pluripotent lnematopoietic stem cells (PHSC) by Moore and his coworkers (1994). These
cells (PHSC) upon transfer into SCID (severely combined immunodeficient) mice caused

rapid population of BM, spleen and thymus and repopulation of lyrnphohemopoietic cells in

65

secondary recipients The expression of CD43 on only a subset of B-lymphocytes in the BM
was significant to tlis study where the combirnation of this marker with a predominant B-cell

determinarnt such as B220 could exclusively identify the population of interest, namely
progenitor B-cells. This will be addressed in Chapter three of this dissertation.

CD43 molecule has a highly O-glycosylated extracellular domain and a highly
consevedtrannnenbraneandinuacelhrhrdomainswithinnufineand human species (Pallant
et 01., 1989). The conserved intracellular/transmembrane domains in CD43 have been
postulated to play a role in intracellular events such as signal transduction or interaction with
cytoskeletal structures (Y onemura et 01., 1993). Furthermore, the heavy glycosylation of
CD43 extracelhnlar domain is thought to interact with lectin-like receptors on cells (Greaves
et (11., 1992). In terms of its function, human CD43 has been shown to act as cell adhesion
molecule on T cells via birndirng to intracellular cell adhesion molecule 1 (ICAM-l) on stromal
cells (Rosenstein et al., 1991), therefore, suggesting its involvement in cell adhesion and cell
proliferation. Most recently, a group of investigators studied the in-vivo efl‘ects of
disregulated expression of CD43 in B-cell lineage of transgenic mice (generated by
microirnjection of tlne infused nnouse CD43-IgH chain enhancer gene) (Dragone et al., 1995).
These transgeru'c mice exhibited splenomegaly due to increased number of B-cells, and
prolonged survival of their B-cells in culture with decreased apoptosis. Based on these
obse'vations they suggested a role for CD43 in delivering signals by itself or by its
conjurnction with other molecules in the adhesion cascade (i.e., lectin-like receptor on stromal
cells) to rescue B-cells from apoptotic death arnd to promote B-cell expansion and

development. Indeed the suggested antiapoptotic role of CD43 molecule in B-lineage cells

66
would give better understanding for the observed distribution pattern of CD43 bearing early

B-lineage cells in the zinc deficient mice which will be presented in Chapter three of this
dissertation.

BP-1/6C3 molecule is a 140 KDa cell surface homodimer glycoprotein formed by
disulfidelinked chainswlnich isidentified innnicebytlne mAb BP-l and rat mAb 6C3 (Cooper
et aI., 1986; Wu et al., 1989). This molecule is expressed on early B-lineage cells in BM and
irn relatively lnigh levels on nnost neoplastic pre-B cells; pre-B cells in long term bone marrow
cultures; certain stromal cell lines; brush borders of the proximal renal tubules and small
intestinal enteocytes; arnd a subpopulation of tlnymus cortical epithelial cells (Wu et 01., 1989;
Wlnitlock et aI., 1987; Welch et al., 1990). The broad tissue distribution and the transitional
extinction of BP 1/6C3 on early B-cells support the diverse biological function and the highly
ordeed regulation of this molecule in B-cell development.

In a sequencing study, BP- l/6C3 was shown to be a member of zinc-metalloprotease
farme with a highest homology to aminopeptidase N (APN; microsomal aminopeptidase)
which acts on peptides with an N-terminal neutral amino acid (Kenny et aI., 1987). A few
years later, however, another group of investigators demonstrated anninopeptidase A (APA)
activity of BP-1/6C3 (Wu et aI., 1991). This enzyme catalyzes the removal of N-terminal
acidic arrnirno acid (i.e., glutarnic arnd aspartic) residues fi'om peptides. This peptidase activity
of BP-l molecule may be sigrnificant in activating or inhibiting the activity of molecules that
are involved in cell progression through differentiation pathway. Collectively, the limited
expression of BP-l molecule on early stages of B-lymphopoiesis; its expression on BM-

deived stronnal cell lines; and the IL-7 (a BM stromal cell cytokine) induced proliferation of

67
B-cell precursors expressing BP-l, all suggest the important role of this molecule in growth
and difi‘erewation of early B-cells. Thus, the study presented in Chapter three utilized this
molecule to specifically identify and investigate a small subset of bone marrow early B-cells
(late Pro-B) that would have not been possible, otherwise.
T Cell Development in the Thymus:

In contrast to B-lymphocytes that are produced arnd mature in the BM, the cells
cormnitted to differentiate into T-lymphocytes are generated in the BM, but then migrate to
the thymus to mature. By analogy to B-cell development in the BM, differentiation of
progenitor T-cells is associated with expression of difi'erent surface markers as well as
rearrangements of the germ-line T-cell receptor (T CR) genes (Pawlowslci and Staterz, 1994).
The early precursors of T-lymphocytes within the thymus express neither of the major T-cell
accessory molecules CD4 and CD8 so are referred to as double-negative (DN) cells. These
DN (CD4’CD8’) tlnymocytes difi‘erentiate irnto early double-positive (DP) cells, which express
low levels of CD4 and CD8 (CD4*CD8*). This transition in phenotype is associated with
extensive proliferation and rearrangement of germ-line TCR a and [5 gene segments which
is very similar to lg gene rearrangement in B-cells (von Boehmer, 1992; Kruisbeck, 1993;
Godfi’ey et aI., 1994). DP thymocytes with non-productive TCR-gene rearrangement will be
programmed to die but the productively rearranged all heterodimer of TCR will then be
associatedwithCD3 andwillbeexpressedontlnecell surfaceofDP thymocytes (Hedrickand
Eidelman, 1993).

The fate of most developing thymocytes is intrathymic death, either early in the

development due to the failure of the TCR molecule to be engaged by self-MHC, or later in

68

maturation due to self-reactive thymocytes. Through the combination of positive and
negative selections, these cells (>95%) will be eliminated via programmed cell death
(apoptosis), and the remaining thymocytes (~5°/o) that are capable of interacting with self-
MHC/arntigen will survive (Sprent et 01., 1988; von Boehmer and Kisielow, 1990; Tough and
Sprent, 1994). Differentiation and maturity of thymocytes are thought to be supported by
thymic epithelial cells (TECs) through direct cell contact and secretion of thynnic hormones
(von Boehmer, 1992; Anderson et aI., 1994; Coto et 01., 1992). A number of thymic
hormone-like peptides that influence this maturity have been isolated from the thymus,
irncluding thymulin (Bach et 01., 1975). This zinc-dependent hormone is thought to play an
important role in T-cell difl'erentiation and maturation and subsequent cell-mediated immunity
(Coto et 01., 1992; Okamato et 01., 1993), as in vitro addition .of thymulin to cultured
thymocytes enhanced their response to mitogens (i.e., PHA; ConA) (Saha et al., 1995).

The irnrrnature CD4"CD8‘TCR'° thymocytes that survive the thyrnnic selection develop
into TCR'll single-positive CD4‘ or CD8+ mature T-cells representing 10-14% or 5-8%.of
total thynnic T-lymphocytes, respectively (Sprent and Webb, 1987; Fowlkers and Pardoll,
1988; van Ewijk, 1991). These mature single positive T-cells (CD4‘CD8'TCR‘i/CD4'
CD8‘TCR“) leave the medulla through veins and lymphatic vessels to join the recirculating
lymphocyte pool arnd home to peripheral lymphoid organs (Shortman et 01., 1990). In terms
of cell numbers, it has been estimated that about 1x10‘ mature T-cells per day leave the
thymus and enter the peripheral blood in young mice (Tough and Sprent, 1994).

Due to the fact that immature thymocytes are highly susceptible to apoptotic death,

the presence of thymic atrophy in ZD with preferential involution of the cortex, where

69

immature thynnocytes reside, could suggest of a similar phenomenon. Thus, it was of interest
to evaluate the effects of ZD on phenotypic distribution of thymic T-cells, particularly
innrnature DP tlnyrrnocytes as well as the verification of apoptosis as a mean for elimination of
cortical tlnynnocytes marnifested as thymic atrophy in ZD. These will be addressed in Chapter

Five of this dissertation.

70

.AN8_ :3 E 338 :8 see. 828998:

cos—Eco a 89¢ 89: :8 woo—n 3.88 =a me ”$353 5:853: 93 eouaweobba g

71

 

 

 

 

Gonzaeog

@n

 

 

 

 

 

 

 

 

 

.00 «635 .

 

 

Esau...

 

 

 

 

 

 

 

 

 

 

 

72

W A schenatic view of tlne events required for heavy chain gene rearrangements and

generation of u heavy chain protein (Kuby, 1992).

73

     
 
 

          

'— V"! L Va” DH! Du.) Jul JH3 C" C5 C13 C11 CTZb C723 CC CC

c,n

     

      

L van LVHZIZ c,3 qzb cyza

 

 

 

DNA
Primary RNA transcript
lRNA processing
1. V D] C“
“m I IEE- A)-
lTranslation
l. V D] C"
Nascent polypeptide I It-
v D] (In

u-lneavy chain

74

W Cell surface and molecular marker expression during the stages of B-cell

development in murine bone marrow.

7S

 

 

 

    

. 3220 3220 HSA
c043 3220 CD43 HSA CD43 /
33-1
/
D'JH .
l I \ \
\ \ \
\h. \> ‘5
Pre Pro-B Early Pro-B Late-Pro-B
+ +
4-6%
”SA 31 HSA H A IM "5 7 'M
322° ’ 3220 3P" 3220 322° I90

V'D'JH
__> ——>
\ (50%) ——"
\
\‘ non-eyeing non-cycling non-cycling
Large are-a small Pre-B Immature B-cell stature B—cell
¢ + ‘___, <——>

 

842% 742% 3-6%

76

W Osmond’s scheme of B-lymphocyte differentiation and dynamics of B-cell

production in murine bone marrow.

77

 

 

 

 

Pro-B cells Pro-8 cells B lymphocytes
Early Intermediate Late Large Small Immature
8220
Incidence (°/o) 0.9 0. 9 43. 3 s 8
Turnover 6 2.5 S 36 36 17 16
(cells it 10 Id) ‘ '
o( 021‘ @@—~
< Proliferation

 

 

————————— < Death / Survian

WEE

SECTION IV: Effects of Zinc Deficiency on Stress Axis;

Role of Glucocorticoids on Immune System

78

79

Effects of zinc deficiency on stress axis:

Selye in his book, The Stress of Life (1956), clearly demonstrated that stress fi'om a
variety of sources caused adrenal enlargement, increased serum glucocorticoids (GCs) and
thymus atrophy and that stress-induced atrophy was much diminished in adrenalectomized
rats. These obse'vatiorns led to the specnrlation that the activation of hypothalamus-pituitary-
adrernal axis (HPA axis; stress axis) played as an important link between the neuroendocrine
and immune systems during stress. Since then, chronic elevation of endogenous GCs
followed by downsizing of the immune system has been documented in various stress related
conditions including: burn and trauma (Maldonado et aI., 1991); infection (Hermann et aI.,
1994); poa surgery; arnd malnutrition (Barone et 01., 1993; Fraker et aI., 1995). It has been
noted that zinc deficiency (ZD) activates the stress axis and leads to the chronic elevation of
GCs irn the circulation. In fact, there are studies suggesting the role of elevated endogenous
GCs irn the induction of immunological alterations such as thymic atrOphy, and lymphopernia
observed in ZD (Quarterman and Humphries, 1979; DePasquale-Jardieu and Fraker, 1979;
1980; Fraker etal, 1995). In addition to trace element nutritional deficiency, other forms of
malnutrition, such as protein-enegy-malnutrition, which resenble ZD in terms of the impaired
immune system also result in elevation of GCs followed by profound thymic atrophy, and
lymphopenia (Becker, 1983; Barone et aI., 1993; Kuvibidila et al., 1993). Thus it appears
that the elevation of glucocorticoids is common to all the conditions in which downsizing of
the immune system has been reported.

A series of early studies by Quarterman (1972; 1974; Quarterman and Humphries,

1979) denonstrated thynnic atrophy and enlargement of the adrenal glands with concomitant

80
increase irn cholesterol (precursor ofglucocorticoids) content in ZD rats. These observations

were followed by a significant increase in plasma corticosterone (CS) level (about 2-fold) in
ZD rats compared to the control group. Interestingly, when ZD rats were given a large
arnournts ofzirnc (~10 mg zirnc sulfate per day irn drinltingwater), an increase in thymus weight
and a decrease in plasma CS concentration to levels detected in control zinc adequate (ZA)
rats were obseved. In a similar study, increased pituitary and adrenal weights, accompanied
by hypersecretion of adrenocorticotropin hormone (ACTH; released fi'om pituitary gland),
arnd adrenal corticoid hormones were reported in ZD albino rats (Macapinlac et aI., 1966).
These obsevations led to speculation that stress-induced elevation of GCs was playing a
major role in suppression of irmnunity. A few years later this hypothesis was investigated by
DePasquale-Jardieu and Fraker (1979; 1980). They noted that ZD mice exhibited thymic
atrophy with a prefeential invohrtion of the thynnic cortex as well as sigrnificantly elevated GC
levels (3-fold lnighe' than control mice). Furthemore, a concomitant reduction in T-dependent
antibody rrnediated response and rise in CS concentration suggested a role for GCs in the
suppression of immune response (DePasquale-Jardieu and Fraker, 1979). IfCS played a key
role in impaired immunity, specifically thymic involution in ZD, then the elimination of this
hormone should be able to block such immunological defects. This was tested by
adrenalectomizing mice and removing CS fi'om the circulation (DePasquale-Jardieu and
Fraker, 1980). The results indicated complete thynnic protection in adrenalectonnized ZD
mice, whereas the sham-operated ZD mice showed a significant thymic involution. Similar
results were also obseved irn adrenalectonnized ZD rats by another investigator (Quarterman

and Humphries, 1979). When DePasquale-Jardieu and F raker evaluated the T-cell helper-

8 1

mediated arntibody response of zinc dietary mice, a significant drop (50%) in the response in
the ZD nnice was noted before any elevation of serum CS, possibly due to other factors (eg.,
nutritional deficiency). However, another reduction (20%) in antibody response was noted
irn just sham-operated ZD mice who exhibited significant elevation of GCs (~7 fold increase)
irn their serum (DePasquale—Jardieu arnd Fraker, 1980). These findings indicated the impaired
T-lnelper finnctiorn arnd the sensitivity of cortical thymocytes (immature T-cells) to the elevated
levels of GCs marnifested as thymic involution since adrenalectomized ZD mice showed
complete thymic protection. Furthemore, it suggested a combination of both suboptimal zinc
intake arnd elevated CS in the suppression of immune response.

Thus the use of adrenalectomy was able to begin to clarify the role of elevated GC
levels during ZD in tlnynnic involution arnd elimination of immature cortical thymocytes as well
as the impairment of cell-mediated immunity due to T-helper cell dysfunctions. In fact the
sensitivity of immature T-cells to GCs followed by glucocorticoid-induced apoptotic death
irn tln's population is a well established phenomenon (McConkey et a]. 1989; Cohen and Duck,
1992; Sun et aI., 1992; Brown et 01., 1993). The GC sensitivity was not just restricted to T-
cells, as B-cells wee also shown to be affected. In a CS pellet implantation system in young
adult mice delivering CS analogous to levels detected in ZD, a significant depletion in BM
total B—cells and immature B-cells (75% and 25% respectively) were noted within five days
(Garvy et al., 1993a). However, mature B-cells were modeately increased. This suggested

the prefe'erntial elimination of immature B-cells by CS and the resistance of more mature B-

82
cell population. Detailed studies on distribution of BM B-lymphocytes in ZD mice will be

presented irn Chapters two and three.

Knowing that both immature T and B cells are highly sensitive to the elevated GCs
arnd adrenalectomy provides protection against thymic involution, it was of interest to examine
the status of B-lymphocytes in adrenalectomized ZD mice. Chapter four will thoroughly
address the role of chronic elevation of GCs in ZD on BM B-cell development, thus,
identifying a more clear role for GCs in nutritional deficiencies.

General Characteristics of Glucocorticoids:

Glucocorticoids are in a class of major steroid hormones released from zona
fasciculata of the adrenal cortex. These hormones have been shown to have a wide range of
effects on components of the immune system and inflammatory responses in both humans and
animals (DePasquale-Jardieu and Fraker, 1979; 1980; Garvy et aI., 1993a; 1993b; Flaherty
et aI., 1993; Adcock etal, 1995; Marx, 1995). In terms of their synthesis, cholesterol is the
known biosynthetic source of all steroid hormones, including GCs (Miller, 1988).
Adrenocortical cells have large numbers of receptors that mediate the uptake of low-density
lipoprotein (LDL), the predorm'nant form of cholesterol, into the cells. The cholesterol is then
enzymatically converted to pregnenolone via cholesterol desmolase. Dehydrogenation of
pregnenolone yields progesterone which is the precursor to all steroid hormones (Bolander,
1994; We, 1988; Hadley, 1992; Rudney and Sexton, 1986). The biosynthelic pathways of
steroid hormones are summarized in Figure 1.

Production and release of GCs by the adrenal cortex is primarily under control of

ACTH released fiom the anterior pituitary gland. In turn, the release of ACTH is regulated

83

by a variety of neurohypophysical peptides produced by hypotlnalarnic neurons. One of the
mairn regulating peptides is corticotropirn-releasing factor (CRF) which is released at neuronal
endings and transported via the portal vein to the anterior pituitary where it stimulates the
synthesis and secretion of ACTH (Figure 2) (Berczi, 1994; Bolander, 1994; Hadley, 1992).
The efl‘ects of CRF arnd ACTH are mediated by activation of adenylate cyclase and the cyclic
AMP-dependent protein kinase (Hadley, 1992). Briefly, ACTH interacts with plasma
membrane receptors of the zona fasciculata cells of adrenal cortex and activates adenylate
cyclase. This activation results in increased cAMP levels and subsequent activation of one
or more protein kinases. The active phosphorylated protein will activate a cholesterol ester
hydrolase to convet sequesteed cholesterol into free cholesteol available for steroidogenesis
(Hadley, 1992).

Cortisol (hydrocortisone; 119, 17a, 21-trihydroxy-pregn-4-ene 3, 20, dione) arnd
corticosterone (17a, 2l-dihydroxy-pregn-4-ene 3, ll, 21 trione) are the main GCs secreted
by the adrenal cortex. However, the relative amounts of these hormones that are generated
depend on the species. For example, in man, dog, and monkey, cortisol secretion
predominates, whereas irn rats, mice and rabbits, corticosterone is the main secretory GC
(Simpson and Waterman, 1988). The rate of secretion of cortisol in normal human subject
under optimal conditions is about 20-30 mg/day. However, the rate is not steady and exhibits
circadian rhythmic, be'ng relatively high in the early morning hours, declining during the day,
and reaching a minimum during the evening (Smith et al., 1981; Hadley, 1992). Being
nocturnal, the rodents exhibit the reverse diurnal cycle, with the highest CS concentrations

in the evening (20 mg/dl) and the lowest in the early morning. \Vrth this in mind and due to

84
thefinctthatcla'onicelevationofGCs accompaniesZD, carewastaken in this work to collect

all blood samples required for CS assay during early morning, 8-9 AM, at the lowest
concentration time point in the diurnal cycle for rodents.

In terns of GC transport system, plasma is the main route of GC transport to target
tissues. In the plasma, 90% or more of the cortisol and CS is reversibly bound to two main
proteins, namely corticosteroid-binding globan (CBG, transcortin) and albumin (Ballard,
1979; Siiteri et aI., 1982; Baxter and Tyrell, 1987). The CBG has been identified as a
member of the serine protease inhibitor (SEPRIN) superfannily, with a molecular weight
between 50,000-60,000 daltons in most species (Hammond et aI., 1987; Nyberg et aI., 1990).
Tlnis protein has lnigh afinity but low total binding capacity for GCs, whereas albumin has low
affinity, but relatively large binding capacity. The remaining fraction of GCs in plasma
comprises the pool of nonprotein bound or "fies” steroid that is generally assumed to be
biologically active (Brien, 1981; Siiteri et 01., 1982; Vermeulen, 1986). The fi'ee hormone
model of steroid action has been defined by Lan et 01., (1984) as: ”only those steroids which
are not bound (eg., flee) to CBG in plasma are available to difl‘use out of the capillary bed
irnto the interstitial space, arnd go across cell membranes to initiate hormonal effects". In fact
in an early study by Slaunwhite et 01., (1962), they showed that injection of cortisol alone
increased liver glycogen in adrenalectomized mice, whereas injection of cortisol-CBG
complex caused no increase, thus demonstrating that CBG-bound cortisol is biologically
inactive.

The free hormone model was further supported by the work of Faict et aI., (1985).

They investigated wlnether trarnscortin (CBG) modulates the in vitro efl‘ects of cortisol on the

85
prolifeation of human peripheral blood mononuclear cells (PBMC) stimulated by different

mitogens. Doses of cortisol in Physiological range (10-1000 nM) strongly inhibited the
prolifeation of PBMC (80% lymphocytes, 20% monocytes) stimulated by the mAb OKT3,
a nnitogen specific for T-lymphocytes, and by phytohaemagglutinin (PHA). However, a
smaller degree of inhibition was obseved with pokeweed nnitogen (PWM). As expected,
CBG alorne lad no influence on the proliferation of stimulated PBMC. However, addition of
pure cortisol-flee transcortin to the cultures sigrificantly reduced the effects of cortisol. It
was, thus, concluded that when evaluating the effects of GCs on lymphoid tissues, only the
free steroid level rather than the total steroid concentrations should be considered. In fact,
this was considered in the study presented in Chapter five in which the amount equall to the
estimated biologically active free CS level as opposed to total concentration of CS detected
in ZD mice was added to the culture system.

Structure and Function of Glucocorticoid Receptor:

The action of steroid hormones including GC is primarily mediated via binding to a
cytoplasmic receptor followed by nucleus translocation and binding of the hormone-receptor
complex to the specific DNA sequence of the target cell genome. Briefly, the hormone
initially interacts with the cytoplasmic receptor in the target cell, thereby inducing a change
in the receptor which is followed by dissociation of the receptor-associated protein complex
and exposure of the DNA binding domain. The activated receptor-ligand complex
translocates toward the nucleus via the cytoskeleton transport system, and into the nucleus
via nuclear pores, where it binds to a consensus binding sequence of target genes. The

bindirng of the receptor-steroid complexes to DNA initiates changes in gene expression that

lie

Mei

sit

bi

8i

 

86

are translated into the cellular response to GC (Gustafsson et aI., 1987; Distelhorst, 1989;
Meisfield, 1990).

The glucocorticoid receptor (GR) is a member of the steroid receptor superfamily,
which is fournd irn all mammalian tissues but at difl‘erent levels (Pratt, 1993). The firnction of
GR as transcriptional activator is uniquely hormone dependent, since its nuclear translocation
and transcriptional activation only occurs upon ligand binding (Gustafsson et aI., 1987;
Burnstein and Cidlowski, 1989; Godowski and Picard, 1989; Smith and Tell, 1993). The
structure of GR is composed of a ligand binding domain at the C-terminal, a DNA binding
domain in the center and a modulating domain at the N-terminal (Figure 3). The DNA
binding domain is composed of two zinc-finger structures which have been proposed to
interact with the hormone responsive elements (HRE; GRE in case of glucocorticoids) on
DNA arnd regulate transcription of the gene downstream. These zinc atoms are located in a
cysteine-rich region of the DNA binding domain, and are each tetrahedrally bound to 4
cysteine residues, forming zinc finger structures (Gustafsson et 01., 1987; Luisi et aI., 1991;
Hutchison et aI., 1992). Using point mutations, the presence of zinc fingers in the DNA
binding domain has been shown to confe the specificity of receptor binding to GREs (Archer
et al., 1990). The GRE is a 15 base-pair, partially palindromic sequence that consist of two
hexameic half-sites separated by three bases (Beato, 1989). Based on sequence analysis, a
consensus binding sequence, 5 ’-AGAACAnnnTGTTCT-3 ’ (where n can be any nucleotide)
located within the 5 ’-flanking regions of the promoter of the targeted genes was proposed
(Beato, 1989; Gustafl‘son et at, 1990). In nature, the sequence of the half-sites of GREs may

vary considerably, but the spacing between the half-sites is always three bases. The

 

 

87

palindromic nature of GREs suggest that the receptor binds its targets as dimers. The
crystallographic analysis (Luisi et al., 1991) or the electrophoretic mobility studies (Alroy and
Freedman, 1992) have shown that two monomers of the GR DNA-binding domain bind to
the DNA target site face to face, making extensive protein-protein contacts. The binding of
the first monomer increases the afinity of the second monomer by two orders of magnitude
(Hard et 01., 1990). Regions required for dimeization are located in both the hormone
‘ binding and DNA binding domains (Dahlman-Wright et 01., 1993) (see Figure 3).

Early studies on isolation arnd characterization of steroid receptors led to the isolation
of two ste'oid birnding forms: a large 8-9S form and a smaller 4S form (Pratt, 1987). The 8-
9S receptor form was found predominantly in the cytoplasm, whereas the 48 form was
detected irn the nucleus. Furthemore, stimulation of hormone decreased the amount of 8-98
form in the cytoplasm and increased the amount of 4S form in the nucleus, indicating
horrnnone irnduced transformation and nuclear translocation of the receptor (LaFond et al.,
1988). The untransformed cytosolic receptor is predominantly associated with a 90KDa heat
slnock protein (hsp90) which is dissociated upon ligand binding and receptor transformation
to the 48 form( Sanchez et 01., 1986; Housley etal., 1990). Further studies have established
flntlnp90assodafionismqunedforeeoidbhndingmflnekaheeasdissociafion ofhsp90
precedes the DNA birnding of receptor-steroid complex (Meshinchi et aI., 1990; Hutchiso et
aI., 1992). Furthermore, hsp90 is thought to stabilize the receptor, and prevent it from
birnding to DNA by masking the donnairns required for receptor localization and DNA binding
(Housley et 01., 1990; Pratt, 1993). Sequence studies and insertional/deletional mutations of

GR lave identified a 20 amino acid sequence at the N-terminal region of the steroid binding

dc

of

dc

pf

 

 

 

88
domain for hsp90 association (Pratt et 01., 1988; Housley et aI., 1990). Thus the proximity
of hsp90 birnding domain to the steroid binding, DNA binding, and nuclear localization
domains support the hsp90 dissociation upon ligand binding and disposing the domains
necessary for DNA birnding.

In addition to the association of hsp90 as the predominarnt non-hormone binding
protein with GIL thee are also a number of proteins associated with the untransformed GR
Among tlnen is hsp70 that is present in chinese hamster ovary cells (CHO) that overexpress
the mouse GR arnd for some unknown reasons is entirely nuclear in hormone free CHO cells
(Sanchez et aI., 1990a; l990b). It is important to note that as opposed to hsp70 association
arnd localization of mouse GR in CHO cells, the mouse GR in L cells is cytoplasmic and not
associated with hsp70 (Snachez et aI., 1990b). This has caused an speculation that the
presence of receptor-associated hsp70 is in some way related to the arrival of steroid
receptors in the nucleus, thereby serving as a molecular chaperon (Sanchez et aI., 1990b;
Pratt arnd Scherer, 1994). It is thought that hsp70 binds to hydrophobic regions of proteins
to facilitate their unfolding to cross organelle’s menbranes (Rotlnrnan, 1989). The association
of GR with a complex containing hsp70 could play a role in maintaining steroid receptors in
an unfolded state for passage through the nuclear membrane (Sanchez et aI., l990b; Pratt et
01., 1992). In this respect Shi and Thomas (1992) have shown an hsp70 requirement for
transport of nucleoplasrnin into Hela cell nuclei.

Besides the association of hsp90 arnd hsp70 with GR, a number of other proteins such
as hsp59 (also krnown as hsp56, hsp60), p50, p23, and p14 has been reported to occur as a

part of heteomeric structure of untransformed GR (Pratt, 1992; Pratt, 1993; Lebeau et aI.,

89

1994) (Figure 4). In fact, coirnmunoadsorption studies with GR (Bresnick et aI., 1990;
Sanchez et aI., 1990b; Pratt, 1990) suggest that the receptor-hsp90 complex is a core unit
deived flour a larger heteromeric complex that also contains hsp70, hsp56, and some other
proteins. The heat shock protein heterocomplex exists in cytosol independent of steroid
receptors (Sanchez et aI., l990a; Tai et 01., 1992) and the three heat shock proteins in the
complex are thought to be involved in protein folding/unfolding and protein traflicking in the
cell (Pratt et aI., 1992). This heterocomplex has been suggested to function as a transport
particle, tlars temed a 'Transportosome" to which the steroid receptor remain attached while
they undergo trafficking via cytoskeleton-mediated transport within the cell (Pratt, 1992).
Following steroid binding, receptor dissociation fiom hsp90 with its simultaneous
transformation to DNA binding state, and cytoskeletal-mediated transport, the receptor must
translocate across the nuclear nnenbrarne to access to the nuclear chromatin via nuclear pores.
Nuclear proteins larger than relative molecular mass of ~40,000 appear to require a nuclear
localization signal (NLS) for passage through the nuclear pores (Pratt and Scherrer, 1994).
Picard arnd Yamamoto (1987) have identified two nuclear localization signals, NLl and NL2,
in the COOH-teminal half of the GR. NLl maps to a short segnent at the COOH-terminal
side of the DNA binding domain, whereas NL2 is located within the hormone binding of the
receptor. Both NLS are hormone dependent, accounting for cytoplasmic location of the GR
irn the absence of hormone (Picard and Yamamoto, 1987). Thus, it appears that hsp90 caps
both DNA binding arnd rnuclear localization domains, which are uncapped upon ligand binding,
tlnus allowing the progression of both processes. The transformed receptor would then move

along the cytoskeletal (nnicrotubules, nnicrofilarnents) pathway toward the nuclear envelope

90

and translocate across the nuclear membrane through nuclear pores using NLS to reach the
rarclearchromatin Thesereceptorscanbeexported outofthe rnucleus to be recycled (Madan
arnd DeFranco, 1993). Subsequent birnding of the transformed active receptor to the GRE on
DNA, would then either stimulate or inhibits transcription of the targeted gene (Pratt et aI.,
1989; Meisfield, 1990; Pratt, 1993).

Two receptor subtypes for adrenal steoids have been characterized: type I receptors,
or mineralocorticoid receptors, and type H receptors known as glucocorticoid receptors.
Type I receptors lnave a higher afiinity for naturally occurring adrenal steroids (cortisol in men
and corticosterone in rodents) (Beaumont and Fanestil, 1983; Spencer et 01., 1991). On the
contrary, type 11 receptors have a highe' affinity for synthetic glucocorticoid, dexamethasone
(Reul and deKloet, 1985; Sutanto and deKloet, 1987; Reul et al., 1987). Both of these
receptors have been identified in immune cells, however, with considerable expression
variation among irrnmune tissues (Lowy, 1989; Miller et 01., 1990; Spencer et 01., 1991).
Thymus has been shown to express the highest type II receptor concentration in the body,
whereas spleen exhibited both typeI and type 11 receptors (Lowy, 1989; Miller et aI., 1990).
This pattern of receptor expression is suggestive of the difl‘erent responses or different
sensitivity of various immune compartments to the glucocorticoid hormones (Miller et aI.,
1990; Miller et aI., 1994; Spencer et 01., 1993).

Effects of Glucocorticoids on Lymphocytes:

Glucocorticoid hormones have wide-ranging efi‘ects on the immune system (Cupps

and Fauci, 1982). At pharmacological levels, these hormones exert anti-inflammatory and

imrrrunosuppressive efi‘ects, whereas at physiological levels they play important

91
inmarnoregulatory roles (Munck etal, 1984; Flahety et aI., 1993; Adcok et al., 1995; Marx,
1995; Auphan et aI., 1995).

In an early study by Fauci and Dale (1974), the in viva efl‘ects of hydrocortisone
(cortisol) on ubpopulations of lymphoid cells in human peripheral blood were investigated.
A single intravenous injection of either 100 mg or 400 mg of hydrocortisone showed a
profound decrease in absolute numbers of circulating lymphocytes and monocytes between
4-6 hours after either concentration of hydrocortisone. However, the counts returned to
rnorrnal by 24 hours. The depletion of lymphocytes fi'om the circulation was selective in that
there was a greater decrease in the number of thymus derived T-cells than B-cells, which
returned to baselirne by 24 hours after hydrocortisone iry'ection. The in vitro response to PHA
was relatively unafl‘ected, while responses to concanavalin A (Con A), PWM (at high
hydrocortisone concentrations=400 mg), and in vitro responses to antigens (streptokinase-
streptodorrnase arnd tetarnnrs toxoid) were significantly diminished. This selective depletion of
nnonocytes and lymphocytes was suggested to be the result of redistribution of these cells out
of tlne circulation irnto the other body compartments. However, this proved not to be the case
in studies hereirn, which will demonstrate the selective sensitivity of difl‘erent immune
components to the GCs resulting in their elimination or survival rather than their
redistribution.

The susceptibility of lymphocytes to GCs was further demonstrated by the study of
Mille’etal, (1991). In this study they showed that dexamethasone (Dex) treatment (0.3 to
10 ug/hr) had a significarnt efl‘ect on T-cell proliferation where a stepwise increase in the Dex

concentration added to the drinking water was associated with a stepwise decrease in the

92
Con-A 'mduced splenocyte prolifeative response. To ascertain that the observed effects were

irndeed due to the GCs, these investigators initiated a multidimentionals study. This study
examined the effects of GCs and the GR agonist on the number and percentage of immune
cells irn the peripheral blood arnd spleen of rats (Mille et 01., 1994). Irnplanting corticosterone
irn adrenalectomized and sham-operated rats for seven days, they demonstrated a significant
increase (>50%) in the neutroplnil population and a substantial depletion of all lymphocyte
subsets irncluding T-cells (helper and suppressor), B-cells, natural killer cells, as well as
monocytes in the peripheral blood and the spleen. In addition, the effects of RU283 62, a
potent GR agonist, on immune cell distribution in the peripheral blood was examined.
Implanting osmotic nninipumps which delivered 1, 4, and 10 uglinr of RU283 62 resulted in
a significant decrease in lymphocyte numbers with concomitant increase in neutrophil
population in a dose-dependent manner. Similarly, the high concentration of RU28362 (10
rig/hr) significantly reduced the spleen cellularity, decreased the absolute numbers and the
percentage of all lynrphocyte subsets, with B—cells exhibiting the greatest decline (>80%), and
a substantial increase in the percentage of neutrophils. Consistent with the depletion of B-
cells irn RU28362 treated rats, a significant increase in B-cell population of peripheral blood
in non-treated adrenalectomized animals compared to sham adrenalectonnized and RU283 62
treated rats was observed. These firndirngs denonstrated the potent efi‘ects of chronic delivery
of both naturally occurring GCs, corticosterone, and the GR agonist, RU28362, on the
distribution of immune cells mainly manifested as lymphopenia and neutrophilia, as
adrenalectomy blocked the efl‘ects. In this regard, studies presented in Chapter Two and

Four will demonstrate the pattern of BM B-lymphocyte development in the presence of

93
elevated endogenous CS (ZD mice) and in the absence of CS (ZD adrenalectomized mice)
in zinc dietary studies, respectively.

The sensitivity of lymphocytes, particularly T-cells, to exposure to GCs was
emphasized in a recent study by Flaherty et 01., (1993). In this investigation they evaluated
the efl‘ects of corntinuous CS adnninistration on peipheral blood lymphocytes of Fischer 344
rats. This was accomplished by subcutaneous pellet implantation releasing CS (0.07, 0.48,
arnd 4.8 mg/day) over a 21-day period. As expected, a significant decrease in total
lymphocytes with reductions in the absolute numbes of the T-helper, T-cytotoxic and B~cells
was observed. Histopathological examination of animals treated with the high dose CS, for
seven days, revealed involution of the thymus with diminished thymic lymphocytes. Due to
the lu'gh sensitivity of T-cells to GCs, it was important to relate the distribution and the
susceptibility of thymic T-lymphocyte subpopulations to the effects of ZD, which is
accompanied by tlnynnic atrophy and elevated levels of GCs. This will be addressed in Chapter
Five.

Besides the irnmunosuppressive effects of pharmacological doses of synthetic GCs,
stress induced alteration of immune cell distribution due to endogenous GCs has been also
reported recently. Dhablnar et aI., (1995) denonstrated that restraining rats for up to 2 hours
caused a rapid and significant increase in plasma corticosterone levels. This elevation was
accomparnied by a significant decrease in numbers and percentages of lymphocytes (T -cells,
B-cells, NK cells) and monocytes and an increase in numbe’s and percentages of neutrophils
in the peripheral blood. The observed alteration in immune cells was related to the high

concentration of corticosterone, since adrenalectomy significantly reduced the depletion of

94

immune cells. Furthermore, administration of corticosterone to adrenalectonnized rats
resulted irn the same irnnnnurnological alteration as those observed irn stress-induced intact rats.
In thermal irnjury which results in secretion of elevated levels of GCs, depression of
host defense systen was observed (Organ et. 01., 1989; Calvano et aI., 1987; 1988). In this
regard, Calvano and his co-workers studied 10 thermally injured human subjects over time
for both percentages arnd absolute numbers of peripheral blood lymphocytes. Their results
showed a significarnt reduction irn CD3+ lymphocytes percentage in the early post burn period,
with a concomitant decline in CD4+ T-cell subset. The percentage of CD8+ T-cells did not
change significantly at any time post burn. The change in T-lymphocyte subsets caused a
general lyrnphopernia on day four following the injury. A previous work from the same
laboratory (Calvano et 01., 1987) clearly demonstrated the role of GCs in the change of
lymphocyte distribution irn burn patients, by comparing these patients with healthy individuals
infused with hydrocortisone for 6 hours. Both groups showed significant lymphopenia,
monocytopenia and granulocytosis. Additionally, there was a substantial decrease in
percentage of CD4+ T-cells with no significant change in the percentage of CD8+ cells.
Parallel to tlnese studies, the irnmunosuppressive efi‘ects of GCs on cells of the immune
systen 'm protein-calofie-malrnutrition (Becker, 1983; Barone et at, 1993) have revealed close
similarity to those observed in other stress induced conditions (eg., ZD, trauma and burn
patients) (DePasquale-Jardieu arnd Fraker, 1980; Organ et 01., 1989; Maldonado et aI., 1991;
Fraker et aI., 1995). Recent studies on the evaluation of the role of elevated serum CS
observed in protein malnutrition demonstrated a substantial sensitivity of T-lymphocytes,

particularly immature CD4*CD8+ and mature CD4”CD8'subsets along with impaired

95

nnacroplnge finnction in nnice fed protein deficient (PD) diet (Barone et 01., 1993; Hill et al.,
1995). Blocking the stress CS response with adrenalectomy or using RU486 to block CS
receptor prevented the impairment of nnacrophage function (Hill et al., 1995). Furthermore,
administration of CS via a subcutaneous pellet implantation reproduced macrophage
impairmernt (Hill et 01., 1995) and resulted in severe thymic atrophy and lymphopenia
analogous to thou of PD nnice (Barone et at, 1993). Collectively, These results, once again,
strongly support the critical role of glucocorticoids in lymphopenia and selective alteration
in immune cell distribution which are observed in ZD.

Interestingly, endogenous levels of GC in the absence of any stimuli has also been
shown to control the connponents of tlne immune system (DelRey et 01., 1984). In this study,
normal mice and adrenalectonnized mice were evaluated. In normal mice, there was an
inverse correlation between endogenous levels of GCs (4-26 mg/dl) and splenic nnass, splenic
cellularity, arnd numbers of Ig secreting cells. These observations were reversed in
adrenalectonnized mice, which showed trace amounts of CS (3-4 mg/dl), confirming the
contribution of GCs to the irnmunosuppression.

Recent studies on the evaluation of the efi‘ects of GCs on B-cells indicate that B-
lymphocytes in the early stages of their development bear the same sensitivity to GCs as
irnnnature T-cells do. A recent study by Voetberg et 01., (1994) demonstrated the significant
susceptibility of murine lymphocytes, specifically BM B-cells, to the GCs, in this case
predrnisolone (PD). Ten days of exposure to 2-5 mg PD/ml of plasma via pellet implant in
mice resulted in a dramatic depletion (50%) of the circulating lymphocytes. Early B-cells

(B220*IgM') arnd immature B-cells (3220*IgM’IgD') showed a significant susceptibility to

96
PD (3-fold decrease), whereas the nnature B-cells (B220”IgM*IgD*) were resistant.

Furthermore, exposure to PD both in viva and in vitro also afl‘ected the finnctional capacity
of BM-B cells to the T-cell-independent antigen TNP-LPS (trinitrophenylated-
lipopolysacclnaride). This was denonstrated by a significant inhibition of plaque-forming cells
(PFC) production. However, as expected, the GR antagonist, RU3 8486, caused 50-70%
greater PFC production in PD treated cultures.

Garvy and Fraker (1991) showed a substantial decrease (50%) in in vitro responses
of BM innrnature B-cells to TNP-LPS with physiological levels of GCs. This inhibition was
nnore significant (SO-80% decrease in plaque fornning cells) when dexamethasone, the more
_ potent synthetic GC, was utilized. The specificity of the observed effects by GCs was
confirmed when RU3 8486 counteracted the inhibitory efl‘ects of the glucocorticoids.
Interestingly, the marked inhibition of plaque formation by B-cells was accompanied by a
significam depletion in the proportion of B-cells present in Dex-treated culture. Thus the data
clearly irndicate the inhibitory efl‘ects of glucocorticoid hormones on BM B-cell function via
significant reduction in the proportion of immune B-cell population.

A significarnt study recently presented by Garvy et al, (1993a) revealed a clear picture
of tire role of GCs on BM B-cell component of the immune system in mice. In viva delivery
of levels of GCs analogous to that detected during ZD was shown to down regulate the
immune system. Implantation of CS tablets in mice resulted in chrornic exposure to the
hormone levels normally detected during stress, including ZD (DePasquale-Jardieu and
Fraker, 1979). The imnarnosuppressive effects of the chronic exposure to CS was primarily

indicated by the seve'e tlnynnic atrophy within the first day of pellet implantation. Analysis of

ht.

dcl

97

BM B-lirneage cells indicated a 70% decrease in total B-cells as well as 40% decrease in Ig“
B-cells. The early B-cells were completely depleted by day 5 and the cells remaining were
shown to be mature B-cells. The results were also accompanied by a decrease in the
proportion of B-cells in the S phase of the cell cycle. These observations once again
emphasize the down regulation of the immune system by the chronic elevation of GCs as
shown by thynnic atrophy and depletion of immature lymphocytes. Furthermore, the
evaluation of the status of B-cell subsets in the presence of GCs enriches the literature which
is heavily focused on T-cell susceptibility to GCs.

Although a substantial investigation has been devoted to the immunosuppressive
effects of GCs, the effects of chronic elevation of GCs accompanying ZD on the immune
systen, particularly early developing B—cells, is still unclear. In this regard, it was of interest
to evaluate arnd identify the role of elevated corticosterone accompanying ZD on the
distribution of developing BM B-cells. Use of adrenalectomized ZD mice in which CS is
eliminated clarified the role of tlnis hormone in distribution of BM B-lineage cells. This study
will be presented in Chapter Four.

Glucocorticoid-Induced Apoptosis:

The association of elevated levels of endogenous GCs with thynnic atrophy and the
subsequent suppression of immature thymocytes have been extensively documented in both
lnumans arnd arnimals (Weissman, 1973; DePasquale-Jardieu and Fraker, 1979; Becker, 1983;
Barone et aI., 1993). The cells affected in this system are predominantly the immature
double-positive (CD4+ CD8+) thymocytes. Early studies considered this type of cell loss in

thymocytes as cytolysis (Clarnan et al., 1971). However, it is now clear that exposure of cells

98

to GCs innitiates a range of events that leads the cells to “commit suicide.” This type of cell
death is termed progarnnmed cell death (PCD) or apoptosis (Wyllie, 1980; Cohen, 1992).

Apoptosis (A,) or PCD is a process responsible for selective deletion of cells during
morphogenesis, enbryogenesis, tumor regessiorn, and tissue involution in response to
chemical or physical stimuli (Wyllie et 01., 1980; Wyllie, 1987; Walker et aI., 1988). This
form of cell death was first applied to cells dying physiologically without swelling, necrosis
or inflammation by Kerr et 01., (1972). Since then many investigators have evaluated the
morphological arnd molecular events in apoptotic cells in a wide range of tissues. Compared
to necrosis, in which cells swelling is followed by nnpture of plasma and organelle membranes
snnd an inflammatory response, apoptotic death is unique and different. Based on histological
examinations and electron nnicrogaphs, apoptosis is distinct from necrosis in various
morphologic features: reduction in cell volume; condensation and margination of nuclear
clnromatin; maintenance of organelle integity; blebbing of the cell surface and packaging of
cytoplasmic organelles into membrane-bound fragnents called apoptotic bodies; and lack of
an innflarnmatory response. These alterations are accompanied by endonucleosomal cleavage
of chromatin DNA into 180-200 base pair multinners also known as DNA ladders (Wyllie,
1987; Ker et 01., 1987; Cohen, 1992). The DNA ladders are extensively used as a primary
arnd predonnirnarnt marker to identify apoptotic death (Telford et aI., 1991; Cohen and Duke,
1992; Schwartzrnan and Cidlowski, 1993).

Apoptosis is triggeed by divese signals including abnormal expression of oncogenes
sucln as bcl-2, and c-myc, or tumor suppressor genes such as p53, and intracellular elevation

of Ca‘ concentration which activates nuclear Ca‘lMgtdependent endonuclease activity

99

(Bissonnette et 01., 1992; Caelles et 01., 1994; Herrneking and Rick, 1994; Nicotera and
Rossi, 1994). Besides the morphological features of apoptotic cells, the formation of the
DNA ladders was primarily used to identify apoptotic populations. This was primarily
achieved by determination of total and fragnented DNA in whole cell lysates either
colorimetrically or by electrophoretic separation of low-molecular—weight DNA fiagments on
agarose gels (Wyllie, 1980). However, these techniques have the disadvantages of not
quantifying the apoptotic proportion of a population and substantial time consumption (2-3
days). The use of flow cytometry in conjunction with surface and DNA irnmunofluorescent
labeling provides a rapid, reliable and quantitative method of identifying snnbpopulations of
cells within heterogeneous tissues such as BM that are apoptotic based on forward light
scatteing (size); and fluorescent intensity. This method was recently developed in our
laboratory by William Telford and Louis King (Telford et 01., 1991). Using cell cycle
analysis, they demonstrated that cells undergoing apoptosis accumulated in the so called
hypodiploid region to the left of GolG, phase of the cell cycle, also termed A,. This region
(A) was also correlated with cells with fragnented DNA (DNA ladder) on agarose gels and
morphological characteristics of apoptotic cells identified by electron nnicrographs (Telford
etal, 1991). Furthemore, using apoptotic inhibitors (eg., zinc, GR antagonist) the apoptotic
peak (A) was elinnirnatcd, all of which confirmed the detection of true apoptotic cells by this
rapid arnd highly quantitative method. This technique was extensively used in quantitation of
apoptotic cultured thymocytes presented in Chapter Seven. In addition to the use of DNA
birndirng dye for detecu'on of A, recently more sophisticated techniques have been introduced

for deection of this population. Among them, in situ nick translation of nucleotide analogs

100
into the DNA strand breaks and TdT labling of DNA breaks at their 3’-OH terminal with

dUTP have shown to be more sensitive and nnore eficient methods compared to those
previously described (reviewed by Telford et 01., 1994).

Among many inducers of apoptosis, glucocorticoid-induced apoptosis in mouse
thymocytes has become a classical model in understanding and characterization of
biochenical events in this type of cell death (Cohen, 1992). Apoptosis of immature
tlnynnocytes can be induced not only by GCs but also by anti-CD3 antibodies (Smith et aI.,
1989; Slni et 01., 1991). Furthermore, removal of autoreactive T-cells and cells with
nonfunctional gene rearrangement during thynnic T-cell development have been shown to
occur via apoptosis (Murphy et 01., 1990; Kisielow, 1995). Thus it seems that the sensitivity
of difi‘erent subsets of T-lineage cells to GCs is based on the level of differentiation and
maturation, sinnce the nnore mature single positive cells are much less sensitive (Telford et al.,
1991). This was further proved by Telford et al. (1994). Their in vitro studies of
glucocorticoid-induced apoptosis in thymocytes denonstrated geater sensitivity of cells
exhibiting CD4*CD8" phenotype with low expression of TCR. However, single positive T-
cells with high expression of TCR showed resistance to apoptotic death. The stage-
dependernt sensitivity ofthymocytes has been also demonstrated in viva (Sun etaL, 1992).
Intrapeitoneal injection of rats with dexamethasone (1 mgkg), caused 50% thynnic atrophy.
A loss inn thymocytes occurred within 2-8 hours after exposure to Dex primarily in only one
fiction of the two main fi'actions isolated by percoll gradients. This fi'action was shown to
be innnnature thymocytes. This loss was accompanied by the appearance of small dense cells

with characteistics of apoptosis falling in the hypodiploid peak on flow cytometric analysis.

101
Fmthermore, cells eliminated in this fractionwere presumed to be immature thymocytes (Sun
etal, 1992) These results suggested that the widely used in vitro model of glucocorticoid-
induced thymocyte apoptosis closely mimics the in viva events.

Although studies on glucocorticoid-induced apoptosis in B-lineage cells are limited,
evidence indicate that B-cells show similar sensitivity to GCs as T-cells. In fact the
elinnination of autoreactive B-cells, B-cells with non-functional gene rearrangement at the
transition state from pre-B cells to immature B-cells, and positive selection of B-cells with
high afinity surface Ig for foreign antigens have been suggested to occur via apoptosis (Liu
et 01., 1989; Rolink et 01., 1991; Scott, 1995).

Voetberg arnd colleagues demonstrated that the delivery of prednisolone (PD) to nnice ‘
at a rate of a few nanograms per milliliter of plasma significantly reduced the proportion of
early B-cells (BZZO‘IgM’) and immature B-cells (B220”IgM). To ascertain whether
apoptosis played a role in elimination of these developing B-cells, BM cells were cultured in
low levels of PD (0.1 nM) for 16 hours. Approximately 40% of cells expressing B220
(B220‘) and IgM (IgM') were located in the sub GJG, or A, region of the cell cycle
representing the apoptotic population. Further use of RU3 8486 elinninated the cells irn A,
region confirming the GC-induced apoptotic death of tlnis population (Voetberg et 01., 1994).
Tln'sstudydemonstratedtlnatbothinvivoandinvitrochronic exposure ofBMB-cells to low
levels of PD dirnirnslned the population of early and immature B-cells via apoptosis.

A similar study by Garvy and co-workers demonstrated a large proportion of murine
BM 8220+ and IgM (45-65%) B-cells underwent apoptosis when exposed to physiological

levels of GCs for 12 hours inn-vitro. Apoptosis was inhibited by high levels of zinc (500 pM),

102

a known inhibitor of apoptosis, and RU38486 (Garvy et aI., 1993b). Furthermore, in viva
CS pellet implantation in nnice mimicking plasma CS levels detected during chronic stress (30-
100 ml/dl) also indicated apoptotic loss of BM 3220* and IgM“ B-cells (Garvy et aI., 1993a).
However, the apoptotic proportion of B-cells from CS treated mice was considerably lower
than in vitro exposure of cells to the same apoptotic cue. This study demonstrated the rapid
clearance of apoptotic cells over the first 24 hours alter pellet implantation, since depletion
of 8220+ cells at 50% of the controls diminished to 4% in the A0 region. This is expected
since the rapid clearance of apoptotic cells by macrophages in viva has been well documented
(Wyllie et 01., 1980; Cohern, 1991). In fact, The rapid clearance of apoptotic cells by
macroplnages substantially prevents the leakage of toxic cell contents and local tissue injury.
The degradation of ingested apoptotic cells by macrophages is remarkably fast as this process
has been reported as short as 10-20 minutes (Savill et al., 1989a; 1989b) or even shorter
(Evan et 01., 1992) after the occurance of histological changes of apoptosis. This significant
phenomenon has been considered in Chapter Five of this dissertation.

In addition to normal developing B-cells, some transformed B-cells also exhibit
glucocorticoid-induced apoptosis. In vitro exposure of neoplastic B-cell lineage (B-chronic
lynnplnocytic leukenu'a, B-cell) to methylprednisolone caused influx of Ca+ followed by DNA
fragmentation which are the characteristics of apoptosis. Furthermore, addition of protein
synthesis inhibitor (cycloheximide) and RU3 8486 elinninated apoptosis in this population
(McConkey et al., 1991). This observation indicates the pharmacological importance of GCs

in the suppression of leukemic cells in leukemia.

103

Knowing the sensitivity of developing B-cells to GCs in both in viva and in vitro and
their elimination via apoptosis upon exposure to GCs (Voetberg et aI., 1994; Garvy et aI.,
1993 a; 1993b), it was of interest to exannine whether or not the same phenomenon would
occur in zinc deficiency where chronic elevation of GCs is present. This study will be

presented in Chapter Five.

104

mm; Biosynthetic pathways of adrenal steroid hormones (Hadley, 1992).

1135

 

 

 

 

 

Cholesterol
Desmotose —-——) (EH3 CH 3
C=O C =0 0
.. .. OH ll
l7a-hydr0sylose
1, .______)
H0 HO HO
Preqnenolone War-Hydroxypregnenolone Dehydroeplond rosterone
w'oH°de"Yd'oqenase . : 3' a
A5 A4 isomerase —’
§”3 9“:
C=O C=O ('3'
— — H
17a - hydmxylose 0
A——> ‘ ——-——->
Oé O4 0’,

Progesterone

2| - hydrOxylose _____.)'

I7a-Hydroxyproqesterone

 

 

CHon CHZOH
C=O C=O
;|: --OH
0* 0”
Il- Deoxycortocosterone II-Deoxycornisol
lip-hydroxylose ..__—) ’>
CHZOH CHZOH
6:0 c=o
(3?: HO
0’ 0
Corticosterone Cortisol
lB‘hydroxylose
and Liver __—)
IB-OH-dehydroqenose
QHZOH (EHZOH
g :0 C:O
HO og -0H
04 0‘

Aldosterone

C0! tisone

Androstenedione

H o.

04

TeSloslerone
OH

HO

Estrodiol

106

W Hypotlnalarms—pituitary—adrenal axis; Regulation of glucocorticoid production.

107

Higher ‘.
Brain Centers (-)

HYPOllIalamus ‘- --------------------

(-)
CRH

Pitu tary " """"""""""""

(-)

ACTH

 

t
Adrenal
Cortex

r"‘ ‘

‘OQ‘ ‘. k” ~\

V
I
\--O’

Target Tissues

)

\b-.-.-.-..._.-.-l—.—-»----- -‘- -4—.—‘—._—£-.-.-

108

c998. gearboxes—3 32083803 05 mo mean—av gauge 25. g

109

 

 

was

new new OS

I :ozmufioo; 320:2

9.65m 8%..

 

0 59:00 9.65m 20.85

 

59:00
4.20

 

 

 

EeEoo mess—522

 

+

.853.
was:

 

110

m Schematic representation of the glucocorticoid receptor heterocomplex in

untransformed state.

111

 

 

 

 

 

. Depletion of Cells of the B-lineage in the Bone

Marrow afZinc Deficient Mice

"A collaborative study with Dr. Louis E. King"

112

113

Abstract

Though lymphopenia is often noted in malnourished humans and rodents, little is known
about tlne efl‘ects of suboptinnal nutriture on lymphopoietic processes. Focusing primarily on
cells of the B-lineage in the marrow of young adult mice, this study demonstrated that a
moderate degree of zinc deficiency (MZD) caused a 43% decline in the proportion of
molested cells bearing B220 with a 91% decline noted among more severely zinc deficient
nnice (SZD). Early B-cells (B220‘Ig') were lniglnly sensitive to the deficiency, being barely
detectable in SZD mice and reduced by almost 60% in MZD mice. Immature B-cells
(3220‘IgM‘IgD’) were similarly afl‘ected, declining 35% to 80% depending on the degree of
the deficiency. In MZD mice, mature B-cells (IgM‘IgD‘) exhibited moderate losses, being
somewhat resistant. A nnore profound loss in this population was noted for SZD mice. Flow
cytometric (FACS) scatter profiles indicated that zinc deficiency caused a sharp decline in the
proportion of small nucleated cells which in the marrow are thought to contain a high
proportion of developirng lymphoid cells. There was a concomitant increase in large granular
cells which paralleled a substantial increase in the proportion ofnucleated cells bearing Mac-1
for both MZD and SZD mice. Given the dramatic depletion of cells of the B-lineage in the
marrow created by a deficiency in zinc, it is probable that disruptions in lymphopoietic
processes 'm the marrow play a key role in the resulting lymphopenia observed in many types
of malnutrition.

114

Introduction

Thyrm'c atrophy and lymphopenia are alien noted irn malnourished humarns and rodents (Endre
etal, 1990; Kuvibidila et 01., 1993; Fraker er al, 1993; Cook-Mills and Fraker, 1993a). The
resulting reduction in leukocytes, especially lymphocytes, suggests that some nutritional
deficiencies might be altering bone marrow firnction and reducing its ability to produce
lymphocytes. However, there is little infornnation in the literature on this important topic.
Therefore, the present study focuses on the effects of zinc deficiency, because it is a well
characterized mtritional-immunological paradigm, on lymphopoietic processes in the marrow
of young adult mice (Endre, et al., 1990; Fraker et al., 1993). Dietary deficiencies in zinc
(ZD) are frequently noted in underdeveloped countries and occasionally in Western nations
(Endre et 01., 1990; Walsh et at, 1994). However, the deficiency also accompanies a variety
of common disease states such as renal disease, alcoholism, chronic gastrointestinal disorders,
sickle cell anemia, AIDS, certain cancers, etc. (Endre et al., 1990; Keen and Gershwin, 1990;
Walsh et al., 1994; Prasad, 1995). Increased incidences of sepsis, respiratory infections, and
various other secondary infections are seen in individuals with suboptimal intake of zinc,
indicating the immune system is compromised (Endre, et 01., 1990; Keen and Gershwin, 1990;
Fraker et aI., 1993; Hadden, 1995). The prevalence of this deficiency in the human
population spawned interest in the development of rodent models for the study of zinc
deficiency. In the case of the mouse, thirty days of an inadequate intake of zinc by young
adult mice reduced thymic weights and splenocyte numbers 50% to 75% depending on

whether the deficiency was moderate or severe (Cook-Mills and Fraker, 1993 a).

115

Nevertheless, the phenotypic distribution or proportion of various subsets of mature T and
B-cells were nearly normal even in the spleens of severely zinc deficient mice (King and
Fraker, 1991). The residual mature splenic T and B-cells in the deficient mice responded
appropriately to mitogenic and antigenic challenges producing normal levels of cytokines,
antibodies, plaque forming cells, etc., when considered on a per cell basis (Cook-Mills and
Fraker, 1993a). Indeed, reductiorns in the capacity of zinc deficient mice to respond to in viva
antigenic challenges directly correlated with reductions in the number of mature peripheral
lymphocytes available to participate in such responses (Cook-Mills and F raker, 1993 a).

. Because the reduction in mature lymphocytes noted in zinc deficiency is substantial,
and because zinc is krnown to play a key role in cell division and replication (Chesters, 1992;
Vallee arnd Falchuk, 1993; Cousins, 1996), it seemed probable that the deficiency had altered
lymplnopoiesis in the borne marrow arnd thymus. The study herein represents the first detailed
study of the efi‘ects of a nutritional deficiency on lymphopoietic processes, focusing on
developing cells of the B-lineage. It shows that zinc deficiency causes significant losses in the
proportion of cells of tine B-lirneage in the marrow that correlate with the degree of deficiency.
This depletion of developing B-cells probably represents a seminal event in the eventual
reduction of mature B—lymphocytes observed in the peripheral immune system as zinc
deficiency advances. This is a collaborative work with Dr. Louis King who initiated this

investigation and remained as a potential investigator throughout the study.

116

Materials and Methods

11' III 12' l I"

Six week old All female mice weighing 17.1:t:0.1g (Jackson Labs, Bar Harbor, ME) were
distributed irnto three dietary groups. The zinc adequate group (ZA) received diet containing
30 ug zinc/g diet, and the deficient group (ZD) received diet containing less than 0.7 pg
zinc/g diet ad Iibitum. A third group, received zinc adequate diet restricted to the average
quantity of food consumed the previous day by the ZD mice to control for inanition that
accomparnies the deficiency (RZA, restricted zinc adequate) (King and Fraker, 1991; Cook-
Mills arnd Fraker, 1993a). All three dietary groups were housed in stainless steel cages with
mesh bottonns to reduce recycling of zinc for a period of 28 days . Feed jars and bottles were
washed in 4N HC1 and the drinking water was acidified to reduce Pseudamanas infections.
Mice in all tlnree dietary groups were weighed weekly to establish the mean weight 3; standard
deviation After a 28 day dietary period, ZD mice were subdivided into moderately afi‘ected
mice by zinc deficiency (MZD), weighing 72%-76% of ZA mice and bearing a modest degree
of parakeratosis of the eyes, ears, and tails; and severely affected mice by zinc deficiency
(SZD), weighing 64%-68% of ZA nnice and bearing extensive parakeratosis (King and Fraker,
1991; Cook-Mills and Fraker, 1993a). Zinc adequate and restricted zinc adequate rnnice
selected for experimental analysis were randomly chosen to approximate the mean weight 1

standard deviation for their respective group.

1 17

BI lClIIi IS 2' l I"

Bloodcollectedfiomthesubclavianarteiesofanesthetized mice fromall fourdietary
groups was processed individually in acid washed microtubes. For zinc analysis, serum
sanples were diluted 1:10 in 1% HCl and analyzed immediately by flame atomic absorption
spectrophotometry (V arian AA-20 Plus, Mulgrave, Victoria, Australia) at 213.9 nm with
WWW Astarndard curvewasestablishedusingconcentrationsof
zinc ranging fi'om 0.1 to 1 ppm (ug Zn/ml) prepared fiom an ultra-pure standard (Sigma, St.
Louis, MO). Addition of known concentrations of Zn to serum samples (standard addition)

resulted in greater than 90% recovery of zinc by this method.

WW1;

Bone marrow fiom five to seven mice fi'om each of the four dietary groups was
fiushedfiomfirnanrandtibiaandindividuallyprocessed using harvest buffer (Harnk's balanced
salt solution contairning 10 mM Hepes, pH 7.4 and 4% heat inactivated fetal bovine serum
absorbed with mouse cells) (Garvy et al., 1993a). Red blood cells were removed by
centrifirgation over a 3 ml Histopaq gradient (1.083 g/ml) (Sigma Chemical Co, St Louis,
MO) except where noted. Alter washing, cells were resuspended in harvest buffer containing
0.15% sodium azide and kept at 4°C for phenotypic labeling and FACS analysis (Garvy et
al., 1993a). Cell viability at the completion of processing was greater than 95% by trypan

blue dye exclusion.

 

Bone marrow fi'om each mouse was processed separately and phenotyped in single
arnd two color protocols as desenbed below. Antibodies used were: 3220 (RA3-632 isolated
fi'om ascites fluid by protein G column chromatography arnd biotinylated), IgM (u chain
specific and afinity purified, Tago, Burlingame, CA) or IgD (Fe specific, absorbed against
a chain, Nordic, CA). Single antibody phenotyping was used to identify changes in the
proportion of all 3-cells of the marrow (3220*), immature-mature cells (IgM*), and mature
3-cells (IgD*). Two color antibody combirnations were used to identify specific 3-cell
subpopulation changes in the marrow such as early 3-cells (3220*Ig'), immature 3-cells
(IgMIgD'), mature 3—cells (IgM*IgD"), etc. Antibody combinations were: 1) biotinyl-anti-
3220/Strep-Avidin-phycoerythrin (AV-PE) vs anti-Ingichlorotriazinyl amino fluorescein
(DTAF) for the detection of early 3-cells versus immature-mature 3-cells; 2) biotinyl-anti-
IgD/Strep-AV-PE vesus anti-IgM DTAF for the detection of immature and mature 3-cells;
3) anti-3220-PE versus biotinyl-anti-Mac- l/Avidin-fluorescein-isothiocyanate (AV-FITC)
for the detection of 3-cells versus myeloid derived cells. One million marrow cells were
labeled irn the presence of harvest buffer plus 0.15% azide for 30 nninutes at 40 with each
reagent. The cells were analyzed immediately by flow cytometry.

- One and two color flow cytometric arnalyses were done using an Ortho 50H
Cytofiuorograph/803 86 computer system using Acqcyte software. Cells were selected for
arnalysis based on presence irn a scatter cytogram region consisting of low angle forward light
scatter (channels 12-90, y-axis) vs orthogonal light scatter (channel 2-98, x-axis) which

excluded cell debris and cell aggregates. Cells included in the scatter gate were examined for

51'

re

1 19
the presence of antibody in single phenotype experiments or simultaneously examined for
green. (DTAF) and orange (PE) fluorescence in two color phenotypic experiments.
Thymocytes were labelled in parallel, saving as negative controls. Background fluorescence
found within positive fluorescence gates was subtracted from all data. In two color
phenotyping less than 10% electronic color compensation was required to correct for spectral
ove'lap irn the detection of Wm. FITC, DTAF, and PE were excited using the 488
nm line of an argon laser. FITC and DTAF ennission was detected at 525 i 10 nm and PE
enission was detected at 570 i 5 nm using band pass interference filters. The proportion of
small nucleated cells in the marrow was determined by light scatter based an inclusion of

greater than 80% of a thymocyte population within the small nucleated cell scatter cytogram

region.

SI l' l' I l I . .

Nornpararnetric statistical analysis was used for all data presented. The nonparametric
Kruskal-Wallis test was used to determine whether significant differences existed between
treatrnnent groups. Tukey's test, which does not presume normal data distribution, was used
in posthoc arnalysis to determirne which experimental groups were sigrnificantly different (Steel

and Torrie, 1980). The mean : standard deviation is presented in all cases.

120

Results

'l—nl' ‘ l. ’ l» .. :.. .. I. .. -. .-,. .. .. .. ,. .a . -

The reuniting body weights for a typical 28 day dietary study are shown in Figure 1A
wheethe average ZD and RZA mouse weighed 76% and 83%, respectively of ZA controls.
The ZD mice were then subdivided into MZD and SZD based on body weights (~75% and
~65% respectively) and external signs of zinc deficiency as described in the Methods.
Accordingly, tlnynnic weiglnts were nearly rnormal for RZA mice but exhibited extensive weight
loss of 47% and 72%, respectively for MZD and SZD mice (Figure 13), all of which is
analogous to past dietary experiments. The zinc content of the serum was reduced 56% for
ZD mice arnd were nearly normal among RZA mice (Figure 1C). As observed here, the zinc
content of fluids and tissues often can not distinguish between degrees of deficiency though
irmrnurne paranneters often do (Endre et 01., 1990; Fraker et al., 1993; Cook-Mills and Fraker,
1993 a). MZD and SZD mice also exhibited more than a two-fold elevation in serum
corticosterone levels compared to ZA mice (data not shown), which is also characteristic for

zinc deficiency (DePasqualeJardieu and Fraker, 1980; Fraker et aI., 1993).
l~l\ ‘l:\[ 'tl't 'l'lrsr l. It ”"0 I U'II".
With regard to the bone marrow, scatter analysis of the nucleated cells by FACS

showed statistically sigrnificarnt draps of 24% and 57%, respectively, for MZD and SZD mice

in the proportion of small nucleated cells thought to contain large numbers of developing

12 1
lynnplnoid cells (Figure 2A) (Osmond, 1986). A sample scatter profile of the nucleated cells

fi'omthemarrowofaZAandaSZDmouseisprovided (Figure 3). It clearly shows a marked
depressioninsmallnucleatedcellswithaconcomitant increase ingranular cells in SZD nnice.
A brief exploratory experiment indicated that cells of the myeloid lineage bearing Mac-1
(3220MAC-1*) increased substantially. However, it correlated with the loss of 3-lineage
cells inMZD and SZD mice (Figure 23). This fits with the scatter profiles (Figure 3) where
an irneease in the proportion of more granqu cells (side scatter) was clearly observed in the
marrow of SZD nnice. Thus, the myeloid compartment is probably more resistant to the
efl‘ects of zinc deficiency than differentiating marrow 3-cells.

From Figure 4, it is evident that zinc deficiency had a profound effect on cells of the
B-lineage. The proportion of cells of the 3-lineage among nucleated cells of the marrow
(total 3220*) declined 43% in MZD and 91% in SZD mice. Using dual color analysis, the
following stages of 3-cell development were also analyzed: (1) 3220*Ig‘, early 3-cells
corntairnirng pro arnd pre-B cells; (2) 3220*IgM*IgD', immature B-cells; (3) IgM*IgD*, mature
3—cells (Osmond, 1986; Hardy et al., 1991). The early B-cell (3220*Ig‘) population was
most severely affected by ZD. MZD mice exhibited a 56% reduction in the proportion of this
population, whereas the early B-cells were almost completely eliminated by SZD (4% of
controls). The profound efl'ect that nutritional deficiencies may have on early B-cells is
further evident in the RZA mice, which exhibited a 40% drop in the proportion of this
populafioneventhouglntheirreductionincaloricirntakewasmodest. It serves to demonstrate

that reduced food intake that accomparnies deficiencies in zinc in humans or rodents, also

122

contributes to changes in the immune system as previously shown (Prasad, 1991; Fraker et
aI., 1993; Cook-Mills and Fraker, 1993a).

Immature B-cells (3220*IgM‘IgD') were somewhat more resistarnt with no statistically
significant decline in this population noted among RZD mice. Nevertheless, the immature
B-cells declined about 35% in MZD mice. The severe form of the deficiency took a greater
toll with a loss of 80% of immature cells among SZD mice (Figure 4). Mature 3-cells
normally account for only 3-5% of the marrow as is the finding here and thus, can be more
diflicult to measure quantitatively (Osmond, 1986). A small decline in this population of cells
among MZD mice was repeatedly observed, though it was never statistically different from

ZA mice (Figure 4). However, SZD mice experienced more than a 70% drop in the

proportion of IgM‘IgD* bearing cells.

123

Discussion

The data presented herein indicate that 28 days of a suboptimal intake of a single essential
rutrient, zirnc, has a profound efi'ect on lymphopoietic processes of the marrow. Both MZD
and SZD caused sigrnificant depletions in the proportion of small nucleated cells of the
marrow and of cells bearing 3220* in particular. Early 3-cells (3220 1g) were especially
sensitive to suboptimal zinc with immature B-cells (IgMIgD‘) following closely in sensitivity.
The more mature IgM’IgD* cells, while harder to measure accurately, appeared to be
somewhat nnore resistant to moderate levels of zinc deficiency. Thus, some resistance to the
deficiency seemed to be acquired with lineage maturity. Experiments presented in Chapter
Three will extensively examine the effects of the deficiency on progenitor (pro-3) and
precursor (pre-3) 3-cells in the marrow of mice in dietary zinc study. Whether or not the
nanltipotent sten cells are affected by the deficiency is also of interest, though it is krnown at
least some stem cells and/or early progenitor cells must survive since the immune system is
almost completely rejuvenated within a two weeks of rnutritional repletion in the case of MZD
mice (Fraker et aI., 1978).

It seems clear that the ability of tlne marrow to produce and/or maintain lymphoid cells
was severely compromised by ZD with gradations in magrnitude of effects noted between the
MZD arnd SZD mice. The rapid atrophy of thymus and reduction in thynnic hormone activity
previously observed during ZD would probably greatly hamper, if not prevent, the maturation
of any early precursor T-cells that were generated in the marrow of the deficient mouse

(Dardenne et at, 1982). "Thus, it is higlnly probable tlnat production and/or maturation of both

124

3 arnd T-cells are altered by ZD. This further suggests that changes in bone marrow function
trust at least partly accournt for the lymphopenia commonly observed in zinc deficient rodents
and people (Frake et aI., 1993; Prasad, 1995).
Shncelynnphopeuaunddnynficatrophyuefiequendynotedinbothdeficiencies ofzinc
and protein-calories (Endre et aI., 1990; Prasad, 1991;Kuvibidi1a et al., 1993; Fraker et al.,
1993; Cook-Mills and Fraker, 1993a), one wonders if a purposeful down-sizing of this part
of the immune system is set in motion as the nutritional deficiencies advance. The large
number of lymphocytes that must be produced each day by the marrow would require high
arnraunts of rartrients. As the body shifls from a well fed to a starved state, nutrients must be
saved for truly vital tissues such as the brain, heart, liver, kidney, etc, so perhaps production
of new lymphocytes must be reduced. In this light, it is interesting to point out that blood
concentrations of the glucocorticoids cease their normal circadian rhythm and become
chronically elevated albeit at modest levels during the course of zinc and protein calorie
deficiencies in both lnunnarns arnd rodents (DePasquale-Jardieu and Fraker, 1980; Smith et aI.,
1981; Fraker et al., 1993; Kuvibidila et al., 1993). Recently it was demonstrated that
nnodeately elevated levels of corticosterone delivered over a ten day period analogous to the
concentrations observed during ZD, caused substantial thynnic atrophy (by day 3), and
depletedthennarrowofcellsoftheB-lineage in nnice (by day 5) (Garvy eral.,1993a). Early
and immature 3-cells were preferentially affected with some resistance noted among
IgM’IgD*, as was observed for ZD mice (Garvy et al., 1993a). Furthermore, such levels of
steroid increased three to four fold the proportion of 3220* cells in the marrow undergoing

apoptosis (Garvy et al, 1993a). In vitro 10"M cortisol induced 30% of 3220* marrow cells

125
to undergo apoptosis in 8 in, further demonstrating that early 3-cells are highly sensitive to

glucocorticoids, arnd very prone to undergo steroid induced apoptosis (Garvy et aI., 1993b).
In orde to renove glucocorticoids from the equation, mice were adrenalectomized prior to
placingthemonZAorZD diets. Itwasfound that in the adrenalectomized nnice thymocytes
weeprovided anbstantial protection duringthe course ofzinc deficiency (DePasqualeJardieu
and Frake, 1980). This finding prompted the question of whether adrenalectomy also
provides protection for the cells of the 3-lineage in ZD. This will be addressed in Chapter
Foun
Though apoptosis appears to be one likely cause of depletion of 3-cells during zinc
deficiency, at least two other possrbilities must also be considered: 1) reduction in the rate of
B-celliproductiorn, and 2) disruption of the cell cycle of precursor B-cells. In first case, zinc
deficiency could alter the activities of one or more zinc dependent enzymes (V allee and
Falchuk, 1993; Cousins, 1996) such that cell cycle progression would be lengthened. In the
secornd case, zinc deficiency irn connbirnation with the initial rise in serum corticosterone could
block the cell cycle. In support of the latter, this lab has previously shown corticosterone
treatment significantly reduces the percentage of 3220* B-cells in the S phase of the cycle
(Garvy et al., 1993a). In addition, the effects of zinc deficiency and corticosterone on 3-cell
cycle status arnd rate of lymphopoiesis are other important parts of the puzzle, and questions
for upcoming projects in this laboratory.
- Finally, theincreaseingraranlarcellsnotedintheenclosed scatter profiles onD mice,
along with the greate proportion of 3220' Mac-1* cells, fits with past observations that cells

of the myeloid lineage are more prevalent in marrow exposed to glucocorticoid than are

126
lymphoid cells (Dexter et al., 1977; Dexter arnd Testa, 1980). Sirnce cells of the myeloid series

provide substantial immune protection, being part of our first line of defense, their
preservation may represent a sort of immunological “fail-safe” mechanism. Collectively, the
data indicate a substantial sensitivity of early developing B-cells to the efi‘ects of ZD.
Furthermore, they suggest that induction of the hypothalamus-pituitary-adrenocortical axis
during zirnc deficiency may play an important irnmunoregulating role by reducing production

of new lymphocytes which require substantial amounts of nutrients.

127

$258598 29: ca 025 .«o gauge. 98 3.5 9.9% <N 05 55 @8383
35 w a. goesmnmaqsaaaasewssas .Ewiesaaeesfig
:08 mo 88:3 0:“ 880m ADV in": ”macaw 88335 .3 3.303 man—Eu an .95 A5
2 as 8 is sera .a 3323. 8:: mum as .32 {Na 4N 5.. gave 9.2»
assesses €82 goose BE .8338 See .2. 43": seas base 5
an 2a 3.3 £85 See an as {Na {N 8a same; 38 2C .82. .3833
can .3 8? Bases snags .aNm seems 8e B 8a. Bee... 5885 .3:
.088 3333 can we» 38%. {Ne A82. .8288 02:. Be 8333 2i (N

Seacoaoasofiesgesggeaessgfiaflofifia:

128

QNm ONE <Nm

<N

 

QNm ONE <Nm

(N

 

 

 

 

 

§

(6w) nqblaM Sl’lLU/(qi

“K \\\\\\\
- ’7/ /////7/

\

 

 

 

 

 

 

o a
OF M
om w
on Z
oe m.
cm a
cm W
on /
om mu
om {\
65 :0 980
Fm e—

 

 

q

A

 

O—

(6) WWW K908

129

:32: mm Om H 5.0:. 80:3
mafiseessies 8... w a. .asasaseilgeseasageeo
3820:: 90:8 .500 Eo_0.»8 + _ .082 :5 £00m Emma 30.5.8 0:3 .«0 :oegtamv
0:50:25 A3 $58598 .8030... 8... 88m 3:830 9.03 83 8:85 8.5:»
808803 be 9m": “£00 380—03: :88. :8 9.38:8 we vote... 8388.55 9.2.3
8305 8:08 9%: £00 38.88 0:3 vogue—0:: .«o :oesntfie one .8 58.33% A3
8.5.. .98... 3.. 8 a a... as... cam a... :82 42 {N :5... 8.8.... 5.8%

3oz .3 38.88 0:3 05 .«o ”:00 v0.8.0.8 :o 3:333 0:8 .«o 83b... mo «gong

ad:

-0 r. 6.

 

 

 

 

 

~\\\\\\\\\\\‘
~7///////////A

monomomomo
vwnnNNr-c-

 

 

 

 

 

 

 

snag MOJJDW euog

pemalonN IIOUUS %

RZA MZD SZD

ZA

130

  
  
 
  
  
 
 
  

 
      

 
 
  
  
   
 

'zzzzzzzzzzzz‘
3 393039 0 0-
30303939 0 o o 0 o

oooooooo~
. 3o . 3 3 3 3 3 303930393-

   
  
  
  

 

 

 

 

8220+Moc1

 

 

(ED

 

 

 

BZZO-Mocl

 

ZA
MZD
SZD

 

 

 

I§I

[111llljllllllJlllllllllllllllllllllJ

O C) O O O O O O O

(D l\ LO LO fi’ ”7 (\l '—
snag MOJJDW euog

pamapnN JO 0/0

‘Efi

 

131

W Flow cytometric light scatter profiles of nucleated cells of bone marrow
prepared from normal and severely zinc deficient mouse after a 28 day dietary
period. (A) ZA mouse. (B) SZD mouse weighing 68.5% of ZA controls and
exhibiting extensive parakeratosis. The peaks at low forward and side scatter
identify the heterogeneous small nucleated cell population of the bone marrow.
The peaks at intermediate forward and high side scatter identify cells of the‘

granular myeloid series in bone marrow.

'61
35"].

(A)
l

46

2A Mouse
” 3.

it“

   

55°70

i'r
'3. c

,‘ ‘3 ”Mr J?
all! . _utl,,«..4;v.,
'1' 93$ a1l,t‘,‘l'f;)“5 pl? {a ‘.|-' iii} ‘( .
....;tzge.e;:tge’;%:m,},-e),}¢,’va,._
433" ‘ififiu‘ii‘fi'fm‘hfirg’rh
«.‘wfié‘.’

..

 

      

 

    

   
 
 
 

 

r 12
0 9a
2
128 1
91'

Forward scatte'

(B)

SZD Mouse

 

 

 

 

 

133

:00...“ 8. 2868.2. 8.8 28.. .8. .23 80.25. 620...... 0...... 2828....»
8.00 03:... x... .3 08.2.9.0 8.0 0.0 2.80.. 2.... 0.3.0:: .300 03. w:.2. 2:2...898
:3. mo 03.088080. 0.0 0.0m. .:0>.w m. Gm H :02: 0.2.3 9.2m <N 0... 5.3 3.09:8
3... w .7 2.8.0 .2. 0.0": 2.8.: b02228 8.2.88 2 8.2.2.2. .8... 232..
2... 22.282. :8... 232...... 2.. 82...... 8.2.2.2. 00:2... 2:. 3.8 0.2.. .2...
a... 8.2.2.2. :8... 2:8 2.. 8:02. 88.2.8 20800: 2.... .8000. ......2.o..<....U
=00.m ho 8&0: 2.. .«o 80.0%. ommm w:...00.. 0:0?m :0 .0 30.83 5.3.2.2.
=8... .52 2.: 8.28...U 8.. 2. 8 .8 .a 8.... an .22 :02 £5. 40 .8

39:08 :. 0w8:=-m 0... .0 0:00 0:32: 0:0 6.3088. «€00 .0 5.25.20... 085..

134

 

 

 

ZA

E
m RZA

     

m MZD

 

 

* \\\\
WMW

 

 

*-

*——.\\\\\\\\\\\\\\\\\\\\\\\\V
W/W/WM

*

 

 

 

 

 

0 LO 0 LO Q LO Q

+

+

lgM+|gD

+
E3220 igM

IgD

IgM

TotolB Cells

Effect of Deficiencies in Zinc on the Status of Progenitor

and Precursor-B cells in the Marrow of Mice

135

136

Abstract

Short periods of zinc deficiency (ZD) have adverse efl‘ects on immune system, causing thymic
atrophy and lymphopenia in both humans and rodents. The previous chapter demonstrated
that early B-cells (B2201g’) exhibited high sensitivity to the efi‘ects of ZD, with 60%-90%
depletion depending on the severity of deficiency. Conversely, mature B-cells
(B220”IgM‘IgD") showed moderate losses, being somewhat resistant to the suboptimal
dietary zinc. Whether the high sensitivity of different stages of early B-cells to ZD was stage
specific was not clear. To investigate the status of early developing B-cells in the marrow
of ZD mice, BM cells were immunofluorescently labeled with monoclonal antibodies (mAbs)
directed to the key B-cell maturation surface markers (13220, CD43/S7, 6C3, and IgM)
followed by flow cytometric analysis. Three color immunofluorescence staining of BM B-
lymphocytes indicated 25% to 50% decline in B220 bearing cells, 50% to 70% decline in pre-
B cells (BZZO‘STIgM') and 30 % to 60% drop in immature/mature B-cells (B220+STIgM")
(data not shown) of moderately and severely affected mice by zinc deficiency (MZD,SZD),
respectively. However, early progenitor B-cells (early pro-B) (B220*S7*6C3') and late pro-B
cells (3220*S7*6C3*) showed only moderate losses in some cases being otherwise resistant
depending on the severity of ZD in individual mice. Total cellularity of BM was also
examined in all dietary groups and there were no significant differences among groups as the
results of zinc deficiency. Evaluation of the zinc content of serum as well as some tissues
detected 55% depression in the zinc content of serum in both MZD and SZD, and 32% to

39% decline in zinc level in leg bones of the MZD and SZD, respectively, compared to the

131

mc

is:

137
ZA nn'ce. Interestingly, there was no detectable change in the zinc levels of marrow or liver

cells of ZD mice. The pattern of tissue zinc distribution observed in this study suggest the
redistribution or mobilization of zinc in the body to maintain the survival and the function of
the remaining cells. Taken together, the results indicate that the sensitivity of developing B-
lymphocytes to ZD is stage specific and may depend on the degree of ongoing cellular and
molecular activities which are zinc dependent. Furthermore, the role of other
irnrmmoregulatory factors (eg., GCs), secondarily to ZD, in the alteration of B-lymphopoiesis

is suggested.

fed
Sul

will

Sub

the

 

138

Introduction

Lymphopenia and thymic atrophy are hallmarks of many forms of malnutrition including zinc
deficiency (ZD) (Endre et 01., 1990; Fraker et aI., 1993; Kuvibidila et 01., 1993). In previous
studies, a strong correlation was noted between the loss in host defense capacity and losses
in the number of lymphocytes populating the secondary immune tissues in zinc deficient adult
mice. This suggested that alterations in the production of lymphocytes in the marrow might
be an underlying cause of the lymphopenia so commonly observed in malnourished animals
and people (King and Fraker, 1991). It seemed likely that insuflicient zinc would alter
lymphopoiesis since this essential trace element is critical to cell division, maturation, and
developmental processes, all of which are integral to the production of lymphocytes by the
bone marrow (Chesters, 1992; Vallee and Falchuk, 1993; Cousins, 1996).

The previous study presented in Chapter 2 examined the efl‘ects of a 30 day period of
mboptimal intake of dietary zinc on young adult mice focusing on B-cell development in the
marrow, which is the prime site for production and maturation of B-cells in all higher animals
(King et aI., 1995). Using flow cytometric analysis it was evident that ZD substantially
reduced the B-cell compartment by 40% to 90% depending on the degree of zinc deficiency.
Substantial losses of 56% to 95% were noted among the so-called early B-cells (B220‘Ig’)
with losses of 35%-80% noted for immature B-cells (B220*IgM*IgD'). The mature B-cells
(IgM‘IgD‘) showed greater resistance to ZD with more moderate losses observed in this
subset during the course of the deficiency. Clearly, suboptimal intake of zinc substantially

altered the B-cell compartment of the marrow.

139

" The current study expands upon the earlier work by employing three color flow
cytometric analysis to specifically examine the efl‘ects of ZD on early and late pro-B cells as
well as pre-B cells. It was of .significance to know how ZD afi‘ected pro-B cells, with their
immunoglobulin (1g) genes still in the gerrnline configuration, versus pre-B cells, which are
actively engaged in rearranging the Ig genes (Rolink and Melchers, 1991; Li et aI., 1993).
The serum a'nc content and the intracellular zinc concentration of marrow, several lymphoid,
and non-lymphoid organs along with analysis of the effects of ZD on the overall cellularity

of the marrow also were investigated.

PE

w:

140

Materials and Methods

M' ”1' l'

. Six week old female All mice (Jackson Laboratory Bar Harbor, ME) weighing
11110.7 gm were provided a biotin fortified egg white based diet using ADI-936 vitamins
and minerals (Reeves etal., 1993) to which was added 30 pg Zn/g diet (zinc adequate group;
ZA) or ~ 0.5 pg Zn/g diet (zinc deficient group; ZD) (Table l). A restricted-fed group which
was a control (RZA) for monitoring inanition that accompanies ZD, were fed zinc adequate
diet (30 pg Zn/g diet) being limited to the average amount of food consumed by the zinc
deficient group the previous day (Cook-Mills and Fraker, 1993 a). All three dietary groups
were maintained in stainless steel cages with mesh bottoms to reduce recycling of zinc for a
period of 27 days. Feed jars and water bottles were washed in 4 N HCl and the drinking
waterwas acidified to reduce Pseudomonas infections. Food consumption was recorded
dailyandbodyweightsweremeasuredmiceaweek. At the end ofthe dietary study, theZD
group was subdivided into moderately affected mice by zinc deficiency (MZD) weighing
72%-76% of the ZA group with moderate signs of parakeratosis of the ears and tail or
severely afl‘ected mice by zinc deficiency (SZD) weighing 66%-68% of the ZA group which
exhibited extensive parakeratosis per previous studies (King and Fraker, 1991; King et 01.,

1995). The specific contents of the diet is presented in Table l.

Wabash:
Blood collected floor the subclavian arteries of anesthetized mice fi'om all four dietary

141
groups was processed individually in acid washed microtubes. For zinc analysis, serum
sampleswa'e diluted 1:10 in 1% BC] and analyzed immediately by flame atomic absorption
spectrophotometry (V arian AA-20 Plus, Mulgrave, Victoria, Australia) at 213.9 nm with
deuterium background correction A standard curve was established using concentrations of
fine ranging from 0.1 to 1 ppm (pg Zn/ml) prepared from an ultra-pure stande (Sigma, St.
Louis, MO). Addition of known concentrations of Zn to serum samples (standard addition)

resulted in greater than 90% recovery of zinc by this method.

What:

The following procedures were used to avoid zinc contamination from the
environment. Throughout sample preparation acid washed disposable plastic pipers, tubes
and containers and EDTA washed surgical tools were used to reduce zinc contamination.
Isotonic saline (0.9% NaCl) prepared from trace metal grade sodium chloride
(SigmaUltra, Zn <0.0005%, Sigma chemical co.,St.Louis,MO) dissolved in milli Q
water was used for cell suspensions. Tissues, including bone marrow, bones (devoid of
marrow) and liver from mice in all dietary groups were processed and assessed
individually. Marrow was flushed from femurs and tibias of 5-8 mice from each dietary
group and suspended in isotonic saline. After removal of red blood cells (RBC) from
extmded marrow by lysis (Coligan et aI., 1991a), the nucleated marrow cells were washed
twice, and counted using Turk's nuclear staining solution (Whitlock et al. , 1984). They
were then aliquoted at 1x107 cells in acid washed tubes to be centrifuged at 400 xg for 5

min. The upper left liver lobe from each mouse was removed, rinsed in isotonic saline,

142

blotted, weighed and placed in acid washed tubes. Bones devoid of marrow followed the
same procedure. An inductively coupled plasma-atomic emission spectroscopy
(ICP/AIE), a polyscan 61E simultaneous/sequential instrument (Thermo Jarnell Ash Corp,
Franklin, MS) interfaced to a U-SOOO ultrasonic nebulizer (Cetac Technologies Inc,
Omaha, NB) was used for tissue zinc analysis (Nixon et al., 1986). Marrow cells
(lxlO’lsample) and whole tissues (liver and bones) were digested in 250 pl and 500 pl of
Baker analyzed Ultrex II ultrapure nitric acid (Zn <20 pg/g) (J.T.Baker Inc, Phillipsburg,
NJ), respectively, for 6 hrs in 60-65°C waterbath. The samples were then cooled to room
temperature and brought to a final concentration of 5 % nitric acid by addition of Ultrex
II ultrapure water (Zn < 100 pg/g) (J.T.Baker Inc, Phillipsburg, NJ).

The accuracy of the analysis was verified by comparing the readings obtained with
sundards from National Institute of Standards and Technology (NIST) multielement mix
A-1 and multielement mix-B diluted to 1 ppm with 5% nitric acid. Quality control was
achieved by using NIST bovine liver standardized for the ICP/AES method (Rajaram ct

01.1995). TheICP/AFS valuesobtained from standards were within 97% to98% ofthe

NIST established range.

 

‘Bonemarrow(BM)samplesfromallmicewereprocessedandassessed
individually. Marrow was flushed from femurs and tibias of 6—9 mice from each dietary
group and suwended by gentle aspiration into Hepes buffered modified Hank's balanced

salt solution (HBSS) supplemented with 4% fetal bovine serum (FBS). Red blood cells

143
were removed by density gradient centrifugation (Histopaq 1083, Sigma Chemical, St.
Louis, MO). The nucleated cells at the interface were washed twice, counted, and tested
for viability by trypan blue exclusion (>90% viable) (Coligan et al. , 1991b). To evaluate
the absolute number of BM nucleated cells, marrow cell suspensions were prepared by the
same investigator to provide consistency. In this case cells were immediately counted
using Turk's nuclear staining solution (Whitlock et al., 1984) without the use of any

separation methods. All marrow cell suspension were kept at 4°C throughout processing.

 

R-phycoerythrin (R-PE) conjugated rat anti-mouse CD45R (3220); fluorescein
isothiocyanate (FITC) conjugated rat anti-mouse leukosialin (CD43/S7); biotin-conjugated
rat anti-mouse 6C3 (Ly-6C) as well as the isotype-matched controls (biotin, PE or FITC
conjugated rat 1gG,,,x) were all obtained from Pharmingen (San Diego, CA). Biotin—
conjugated affinity-purified F(ab’)2 goat anti-mouse IgM which was p-chain specific was
purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA) while
streptavidin Red 670 (R-670) was obtained from the Gibco BRL (Grand Island, NY). All
the antibodies were used at their saturating concentration subsequent to titration.

For labeling with antibodies 1x10“ nucleated BM cells were resuspended in label
buffer containing Hepes buffered modified HBSS containing 0.1% NaN3 with 2% FBS
which was supplemented with 5% rat serum and 5% goat serum to reduce nonspecific
binding. Cells were incubated in this mixture at 4°C for 20 min. After washing cells

were then incubated in 150 pl of label buffer containing either anti-BZZO—PB, anti-CD43-

144
FITC, anti-IgM—biofin or anti-BZZO—PE, anti-CD43-FITC, anti-6C3-biotin for 30 min at
4°C and washed twice. To detect the biotinylated antibodies, an additional ‘15 hr
incubation at 4°C with 100 pl of R-670 was required. Samples were then washed and the
cells were suspended in 1 ml of label buffer, kept at 4°C being immediately analyzed on
a Becton Dickinson Vantage equipped with a Consort 32 HP computer system with LYSIS
II software. Fluorochrome excitation was accomplished using an 1LT model RCP-SO
argon laser tuned to 488 nm for FITC, PE, and R-670. Fluorochrome emission for FITC,
PE, and R-670 was detected at 5301.15 nm, 575:1;13 nm, and 670i14 nm, respectively.
Negative controls included unstained cells to define the negative population and detect
autofluorescence. Cells exposed to R-670 alone or labeled with isotype-matched non-
specific antibodies were used to detect background fluorescence. Single and dual color
controls were used to define the negative as well as the positive population for each
antibody and to set color compensation. To calculate the background correction for each
gated phenotypic population in the BM, a three color negative control composed of
isotype-matched non-specific antibodies was used for each corresponding primary antibody
that was used. Multistep gating was used to limit phenotypic analysis to lymphocyte
populations using the following gates: cytogram gated BM cells which excluded debris and
doublets (gate 1); 3220 histogram gated selected for B220” cells (gate 2); and the
cytogram scatter gated selected for lymphocytes only (gate 3). Cells which met these

criteria were used for the phenotypic analysis.

145
W
All data were analyzed by the Kruskal-Wallis nonparametric test and the analysis
of variance (ANOVA) for parametric data in order to identify significant differences
between dietary groups (Daniel, 1987). A Tukey's post-hoe test was then applied to locate
differences among the means of the dietary groups (Daniel, 1987). Differences were
considered statistically significant at P<0.05. All data are presented as the mean :1; SD

where n is 6 to 9 mice unless otherwise indicated.

146

Results

WWW

Figure 1 illustrates the changes in body weight and thymus size for all dietary
groups. By day 27, the average ZD mice weighed 70% of the ZA controls while RZA
mice showed no statistieally signifieant decrease in body weight (Figure 1A). Prior to the
termination of diet study, mice in the ZD group were subdivided into MZD and SZD
groups based on the degree of body weight (7296-7696 and 66%-68% of the ZA group,
respectively) (Figure 1A) and the severity of skin parakeratosis as described in Materials
and Methods. With this subdivision, MZD mice exhibited an almost 50% decline in
thymus weight, while SZD mice exhibited severe thymic atrophy or a 70% decline in

weight (Figure 113). RZA mice had nearly normal thymic weights (Figure 18).

Winner

The zinc content of sera (2A), nucleated BM cells (2B), liver and bones devoid of
marrow (2C) of mice from all dietary groups on a 27 day of the diet study are shown in
Figure 2. The serum zinc in both MZD and SZD groups declined similarly to about 41%
of that in the control ZA group, whereas RZA group showed no statistically significant
change in their serum zinc content (Figure 2A). The lack of distinction in serum zinc
levels between MZD and SZD groups has been noted in previous studies (King and
Fraker, 1991; Cook-Mills and Fraker, 1993a; King et aI., 1995). By contrast, mice from

all four dietary groups showed the same zinc concentration for nucleated cells from the

147

marrow (Figure 28). Likewise, the liver zinc content showed no significant difference
among mice from the four dietary groups (Figure 2C). However, the zinc level in the
bonedeclined signifieantlyintheZDgroups, showinga32% declineinMZD, anda39%

in SZDcompared to thatonA group (Figure 2C).

Wm

To evaluate the effects of ZD on early stages of B-cell development, Hardy’s
scheme of B-cell development in murine system ,illustrated in Figure 3, was extensively
used. Multiple three color phenotyping of BM cells using monoclonal antibodies against
B-cell differentiation antigens (eg., 3220, S7, 6C3, IgM) were carried out and analyzed
usingaduallaserflowcytometer. Inthefirst setofthreecolorphenotyping, B220+ gated
BM B-lymphocytes were screened for the expression of CD43/S7 and/or IgM molecules.
This approach enabled us to identify multiple stages of B-cell development to include:
early B-cells (B220‘IgM'), pro-B cells (B220"S7+IgM’), pre-B cells (B220"STIgM‘),
immture and nature B-cells (B220*STIgM") (Hardy et al., 1991). As shown in Figure
4A, the proportion of all cells of the B-lineage (3220*) in the marrow was reduced by
approximately25% inMZDmiceand50% in SZD mice. Thepr compartrnentofthe
BM normally comprising 12-15% of the nucleated BM cells was affected the most by ZD
declining almost 50% in MZD mice, and 70% in SZD mice. Interestingly, in contrast to
the pre-B cells, the pro-B cell population comprising 4%-6% of the nucleated BM cells
exhibited significant resistance to the deficiency remaining at near normal levels in the

marrow of both MZD and SZD animals (Figure 4A). In agreement with these findings,

148

the flow cytometric scatter profile of total B220+ cells from the marrow of a ZA and a
SZD mouse is presented in Figure 5. It clearly demonstrates a significant depression in
8220* gated lymphocyte population (right panels), as well as precursor B-cells with
relative resistance of progenitor B—lymphocytes in the marrow of SZD mouse (left panels).

Since the pro-B cells could be further divided into subcompartments, another set
of three color phenotyping was used, replacing the biotinylated-anti—IgM with biotinylated
anti-6C3 (Hardy et al., 1991) (Figure 4B). The 6C3 (BPl) molecule is a cell surface
glycoprotein whose expression is limited to early B-lineage cells in hematopoietic tissues
and is not found on mature B-lymphocytes. This new combination of antibodies
subdivided pro—B cells into early pro-B cells (8220*S7*6C3') and late pro-B cells
(B220‘S7‘6C3‘) as well as pre-B cells (8220+ST6C3*) and immature and mature B-cells
@220*ST6C3'). Pre—B cells, as expected, were significantly affected by ZD showing
50% to 70% decline in both MZD and SZD mice, respectively (Figure 4B). As
demonstrated in Figure 4B, evaluation of total pro-B cells and late pro-B compartments
indicated no significant decline in these populations in either the MZD and SZD mice.
There was, however, a moderately significant loss of early pro-B cells in SZD mice, but
not MZD mice. Clearly, pre-B cell precursors were much more affected by ZD than
pro-Bcells.

The phenotypic data presented above was expressed as a percentage of the total
population of nucleated cells found in the BM. To better demonstrate overall degree of
the change in the early B-cell compartment during ZD, the pro and pre-B cell populations

oftheBMwereexpressedasaproporfionofdreB—cellcompamnentitself. Ascanbeseen

149
in Figure 6, the differences in sensitivity of pre-B cells and pro-B cells to ZD are
highlighted using this type of analysis. The signifieant depletion of pre-B cells detected
in MZD and SZD groups (26% and 47 %, respectively) was concomitant with a moderate
accumulation of pro-B cells for MZD (+10%) and SZD (+43%) groups. This more
clearly shows the severity of depletion of pre-B cells by ZD and the much higher resistance

of the pro-B cell population to ZD.

W

Given the signifieant decline in the number of cells in the B-cell compartment, we
were also interested in the effects of the deficiency on the overall cellularity of the BM.
To investigate this parameter, marrow suspensions were prepared from mice in all dietary
groups in order to determine the total number of nucleated BM cells per two legs.
Interestingly, the data showed almost no change in the overall cellularity of the BM of
mice in any of the dietary groups. Thus, in spite of depletion of the B-cell compartment
in the marrow during ZD, the overall BM cellularity was not significantly altered (Figure

7).

150

Discussion

The results herein confirm results from the previous study (Chapter 2) in which depletion
of total nucleated BM B-cells as well as early B-lineage cells in the course of 28 days of
dietary zinc deprivation in mice were demonstrated (King er al. , 1995). It furthermore
identifies the effects of ZD on the status of a small subpopulation (4-6%) of early B-
lineage cells, namely, progenitor B-cells that are the earliest committed cells in the B-
lineage and therefore the key to the B-cell development. The results of this study indicate
a selective and stage-specific sensitivity of B—lymphopoiesis to the effects of zinc
deficiency in the marrow of young adult mice. The BM cells bearing B220 surface antigen
(total B-cells) demonstrated a 25 % to 50% decline (based on the severity of deficiency)
in zinc deficient mice. Utilizing Hardy's scheme of B-cell development (Hardy et al. ,
1991) in subdividing the early B-lineage cells into subpopulations (Figure 3), a significant
depletion of precursor B—cells (pre-B) (B220*STIgM') was noted in both MZD (50%) and
SZD (70%) groups indicating their high sensitivity to the effects of suboptimal zinc intake.
The progenitor B—cells (pro-B) (B220‘S7 *IgM ), on the other hand, demonstrated a
substantial resistance to the effects of ZD with the exception of the early pro-B cells
(3220*S7*6C3‘) in the SZD group which showed a moderate but statistically signifieant
loss. These data fit the scatter profiles (Figure 5) where a remarkable depletion in the total
lymphocyte population due to significant alteration in the phenotypic distribution of pre-B
and immature/mature—B cells and only a moderate decline in pro-B cells, in SZD mice

were noted. Furthermore, the extensive loss of pre-B cells and the protection of pro-B

151

cells in the course of zinc deficiency were even more magnified when these populations
were expressed as proportions of the B-cell compartment.

This unique stage specific effect of ZD on BM B-cell development could be
evalum from different prospective views. It is well documented that zinc is an essential
element involved in DNA, RNA and protein synthesis, and gene expression. Zinc also is
astructmalorfimctionalcomponentofmany metalloenzymes (Endreetal.,1990;Vallee
and Falchuk, 1993; Cousins, 1996). With this in mind, this study evaluated two early B—
cell populations which exhibit different levels of biochemical activities. Progenitor B-
cells, ononelnnd, arethemostimmatureB—lineagecommitted cells whichretainlg genes
in the germline configuration; they exhibit low turnover rate with an average of 25le
cells/day (Hardy et 01., 1991; Li et al. , 1993; Osmond et aI., 1994). Thus the survival
ofthispopulationisakeytotheB—celldcvelopment. Precursor B-cells, on theother hand,
are actively cycling cells with an average turnover rate of 28x106 cells/day (Osmond et al. ,
1994). This pool is heavily involved in the Ig gene rearrangement, gene expression and
protein synthesis, all of which are required for successive progression of cells from this
stage to the next stage ofB-cell development (Rolinkand Melchers, 1991; Li et al., 1993).
Therefore, the bioavailability of zinc for these biological activities and survival of this
population may be more crucial.

Furthermore, the notion of stage-specific expression of Bel-2 oncogene in
developing B-cells and its correlation with glucocorticoid-induced death (Merino et al. ,
1994; Nunez et 01., 1994; Cory, 1995) could also play a role in the observed pattern of

B-cell development in the course of zinc deficiency. Previous studies from our lab have

152

shown the modulation of the stress axis (hypothalamus-pituitary-adrenocortical axis) via
zinc deficiency and the subsequent chronic elevation of glucocorticoids in the circulation
in mice (DePasquale-Jardieu and Fraker, 1979; 1980). A lO—day in viva delivery of
corticosterone analogous to the concentrations detected during zinc deficiency caused
substantial depletion of B-cells, preferentially early and immature B-cells (Garvy et al.,
1993a). In addition, this chronic elevation of corticosterone caused a three to four fold
increase in the proportion of B-cells in the apoptotic region (Garvy et a1. , 1993a).
Moreover, the recent study by Merino et al. (1994) regulation of Bel-2 in B-cell
development, demonstrated high expression of the product of the Bcl-2 protooncogene, a
known inhibitor ofapoptosis, in progenitor B-cells and its downregulation in precursor and
immature B—cells. They further showed the sensitivity of pre-B cells and the resistence of
pro-B cells to dexamethasone induced apoptosis and their correlation with the Bel-2 levels.
Thus, chronic elevation of glucocorticoids during zinc deficiency along with changes in
expression of Bcl-2 at different stages of B-cell development could be considered as
anotherpoasibility for depletion in one case (pre-B) and protection in another (pro-B). In
this regard, the significant depletion of pre—B cells and the substantial resistance of pro—B
cells observed in ZD mice could be a reflection of their sensitivity to the elevated level of
GCs induced by insufficient dietary zinc intalne along with thdr level of Bcl-2 expression.

Inthecaseofserum zinc determination, thedata confirms theprevious reports in
which serum zinc depletion has been repeatedly observed in both humans and rodents
suffering from 21) (Endre er 01.1990; Walsh et 01., 1994; King et 01., 1995). However,

the same level of depletion in serum zinc concentrations in both MZD and SZD as

153

observed in this study is indicative of problems observed in both human and rodent
research where plasma or serum zinc content give only a relative idea of zinc status
without indicating the severity of the deficiency (Cook-Mills and Fraker, 1993a; Walsh
et 01., 1994; King et aI., 1995). In regard to tissue zinc analysis, bone which is known
as one of the largest mobilizable zinc pools (Jackson, 1989), showed significant drop (32%
to 39%) in its zinc content in both MZD and SZD mice. This is possibly due to the
redistribution of zinc from this tissue to other vital tissues such as the liver in the course
of zinc deficiency (Brown et al., 1978; Masters et al., 1983; Guigliano and Millward,
1984), as there is no bodily store for zinc (Golder, 1989). Soft tissues such as marrow and
liver of ZD mice, on the other hand, did not demonstrate any change in their zinc content
and still maintained the same levels of zinc detected in ZA mice. This suggests that the
survival and function of the cell machinery particularly in vital tissues, is based upon a
continuuous inflow of nutrients. Thus, in situations where zinc becomes suboptimal,
extracellular sources such as serum and mobilieable sources such as bone would be the
available sources of zinc redistribution for vital tissues to survive (Jackson, 1989).
Alternatively, the similar zinc content in soft tissues of all dietary groups could suggest the
contribution of other factors in the disruption of lymphopoiesis; factors such as elevated
levels of GC secondary to the ZD as well as overexpression or downregulation of some
oncogenes (e.g. Bel-2) which have been shown to regulate cell survival (Merino et al. ,
1994; Cory, 1995; Fraker et 01., 1995).

Fimlly, the unchanged cellularity of BM in all dietary groups indicates the survival

of other cell types such as myeloid lineage in the BM of zinc deficient mice, as was

154
demonstratedinChapter2(Kingetal., 1995). Ithasbeenshownthatthein vitro survival

of myeloid lineage (Dexter culture condition) is based upon GCs (hydrocortisone)
supplementation to the culture system (Dexter and Tests, 1980). Thus, in ZD, where
chronic elevation of GCs is reported (DePasqualeJardieu and Fraker, 1979; 1980; Fraker
et 01., 1995; King et 01., 1995), survival of cells of myeloid lineage (King et aI., 1995)
is expected.

Taken together, these observations strongly indicate that the effects of ZD upon B-
cell development is stage specific. The sensitivity of pre-B cells and the resistant of pro-B
cells to the effects of ZD could be due to the degree of biological activity and related zinc
requirements. Furthermore, the stage specific expression of molecules such as Bel-2 in
B-lymphocytes could regulate B-cell survival by protecting their elimination via GC-
induced apoptosis which is thought to happen in ZD. Finally, the pattern of zinc
distribution in different tissues of ZD mice suggest the redistribution of zinc in the body
to maintain homeostasis and biochemical activities in vital tissues which are key to the

survival.

155

Table 1: Composition of the Diet

 

 

 

 

 

g/kg Percent

_I_§gredients diet diet Source
Glucose monohydrate 609 60.9 Harlan/Teklad, Madison, WI
Egg white solids 200 20.0 Harlan/Teklad, Madison, WI
Corn oil 100 10.0 Michigan State Uni., Food Services
Salt mix‘ 40 4.0 Bioserv Inc., Frenchtown, NJ I
Fiber2 30 3.0 Bioserv Inc., Frenchtown, NJ
Vitamin mix3 10 1.0 Bioserv Inc., Frenchtown, NJ
Biotin-premix‘ 10 1.0 Made in house
Ethoxyquin’ 1 0.1 Monsanto Chemical Co. St.Louis, MO
Total 1000 100

 

 

 

 

‘ AIN-93G mineral mix without zinc carbonate, supplemented with appropriate amount
of zinc carbonate added in house to the zinc adequate diet to make it ~30 ngn/g
diet.

2 Cellulose-type fiber.

3 AIN—93 vitamin mix

‘ One part d-biotin mixed with five parts glucose monohydrate to offset the avidin found
in the egg white protein.

5 Ethoxyquin or santoquin for prevention of oxidation of polyunsaturated fatty acids.

156

Emmi; Effect of ZD on body and thymus weights for the 27 day dietary study. (A)
Changes in body weights of ZA (O—O), RZA (Au-A) and 2D (Du-Cl) dietary
groups at various time points during the dietary study. Filled symbols
represent final mean body weights of mice selected for the ZA (O), RZA (A),
MZD (I), and SZD (6) groups at day 27. (B) Average thymus weights for
each dietary group at day 27. ZA, n=7; RZA, n=5; MZD, n=9; SZD, n=8.
Data are meaniSD being representative of 3 separate experiments. * Denotes

data significantly different from ZA group at p<0.05.

Body Weight (g)

Thymus Weight (mg)

157

 

26
24 -
22 -
20 -

14-
12-

—
~
—_-

18S:i
16:

 

 

10

T
T -
--T~*””:i:-i
$:::——-$-”"’” 1 l
\i\l.;
1 l
i 114 21

Days on Diet

 

35

30-

25-

20-

 

 

 

 

  

 

 

 

 

 

ZA RZA MZD SZD

 

 

 

158

Emmi: Analysis of the zinc content of sera and tissues of mice from the ZA, RZA,
MZD, and SZD groups at day 27. (A) Serum from individual mice was
evaluated for zinc content using atomic absorption spectroscopy. (B)
Aliquots (1x107 cells) of nucleated BM cells from individual mice in each of
the four dietary groups were digested and analyzed for zinc content via the
ICP/AES method. (C) Tibia and femurs devoid of marrow, and the upper
left liver lobe from mice were digested and analyzed by ICP/AES analysis.
ZA, n=8; RZA, n=5; MZD, n=7; SZD, n=7. Data are expressed as
meaniSD. "‘ Denotes data significantly different from control (ZA) at

P<0.05.

159

H t-- H
I# O) o: O N #-
O O O O O 0

Serum Zinc ( ,ug/dl )
N
C

 

O

ZA RZA MZD SZD

HNNNOD
OOOnFCDN

( ,ug Zn/109 cells )
ES

 

Nucleated Bone Marrow Cells
0 uh co

ZA RZA MZD SZD

 

 

 

 

3,... c
g roo~ ‘
“53 80- §
36‘ a
[3, 401 §
.30 20 §
0

 

 

 

 

 

 

ZA RZA MZD SZD
BONE LIVER

160

W Schematic representation of stages of B-cell development in the BM of
murine system as proposed by Hardy et 01., 1991. Acquisition and/or loss
of surface maturation markers, progression through Ig gene rearrangement,
and the growth requirement (not shown) were utilized to identify different

subpopulations in the B—cell compartment of the murine BM in this diagram.

161

 

 

 

CD43 CD43 HSA CD43 /
BP-1
D'JH I ___>
1 I \ l
\ \ \
\ \b ‘5
Pre Pro-B Early Pro-B Late-Pro-B
« >
4-6%

   

HSA H A | M HS 7 I M
. 3"" 3220 3220 IgD
—> ——> ——>
* non-cvdlng non-cycling non-cycling
Large Pre-B small Pro-B Immature B-cell Mature B-cell
< 5 <———> <——>

 

8-1 2 % 7-1 2 % 3-6 96

162

W Effects of ZD on phenotypic distribution of early B-cells of the marrow. (A)
A three color phenotyping of BM B—cells using anti-B220-PE, anti-CD43/S7-
FITC, anti-IgM-biotin was used to identify precursor and pro-B cells. Total
B-cells consist of all the B220+ cells, pro-B cells are defined as
(B220*S7*IgM') and pre-B cells are B220*STIgM'). (B) The phenotypic
distribution of early pro-B cells (B220"S7*6C3') and late pro-B cells
(B220*S7*6C3”) using a second set of three color phenotyping. For these
experiments ZA, n=7; RZA, n=5; MZD, n=9; SZD, n=8. Data are shown
as meaniSD being representative of 3 separate experiments. * Denotes data

significantly different from ZA group at p < 0.05.

 

163

 

 

 

 

 

emwa
2.
.NNM
*IL é
mmm- .TNH D. E
gm

 

 

 

 

  

 

   

  

 

 

 

 

 

 

 

 

 

 

 

 

+ TE
0
a. 2
2
a $\\\\\\\ B *
eeeeeeee§e_n .ilttxxxx
Tor. "0H0".HOHOHOHOHOHOHOMONOHONOHOMO"Queue”. ,
T [E VA VA VA VA VA VA VA VA VA VA
5 0 5 0 5 0 5 0 5 O 5 O
3 3 2 2 1. 1.. 1 1.
D Em UQHGUHOSZ HO figmnvhmnm WHHUU 2m U0ade05z HO .«HHwOHmnH

LATE

EARLY
PRO—B PRO—B PRO—B

TOTAL

PRE—B

164

Emmi: Three-dimensional presentation of flow cytometric scatter profiles of B220+
gated nucleated BM cells from a ZA (Top panels) and a SZD (Bottom
panels) mice after a 27-day dietary period. Left panels demonstrate the 2-
color (IgM vs CD43) cytogram scatter profiles of three major subpopulations
of the B-cell compartment (Pro, Pre, and immature/mature B-cells) in the
marrow of a ZA or a SZD mouse. Right panels illustrate the light scatter
profiles of lymphocytes gated B220+ population in the BM of a ZA and a
SZD mice, respectively. Forward scatter (FSC) is indicative of cell size
while side scatter (SSC) indicates cell granularity. Data are from a ZA

(control) and a SZD mouse representative of seven to eight mice in each

dietary group, respectively.

165

3 S

L mphocuta ,
ngulatton-> 3

5 ZA.

 

  
  

Immature 8.
Mature
8 Cells

 

 

 

Pro B Cells

 

'8
L h
“ nguficct’foert.
6 SZD
4

Immature 81.
Mature Pro B

 

 

 

166

an: dam an: .3: Wu: £5— 2.": .<N .5. 23E s 3885 .8555
2: 52. e 58 .8 5. a 8?. 3m 93 .52 £5. (N E .ficeaeB =8.m

2m .8 acetone:— 5 8 «=3 5.9:: 858? v5 3.2% 3 no 899.5— g

167

 

 

 

 

 

 

 

 

 

 

  

 

 

up "9

a: 8

n. a.

D E

é é é é é é é.
luawuedwog “33-8 ;0 %

 

SZD

RZA

ZA

168

figural: Evaluation of BM cellularity for 27 day of a dietary zinc study. Marrow from
mice in ZA, MZD, and SZD dietary groups were extruded from femurs and
tibias and individually processed in order to determine the total number of
nucleated cells by staining with Turk's solution. ZA, n=7; MZD, n=9; SZD,

n=9. Data are expressed as mean :1; SD.

169

 

 

I
IW\\\\\\\\

 

 

 

 

 

 

 

 

 

‘ d 1 d - 1 di ‘ I ‘ 1 d ‘ d‘ - I l I‘ J

bod M £30 Em Umpemfiodz Bach.

MZD SZD

ZA

Role of Glucocorticoids in the Suppression of B-

Lymphopoiesis During Zinc Deficiency

170

171
Abstract

Previous investigations fi'om this laboratory have provided strong evidence that the rapid
thymic atrophy and bone marrow (BM) B-lineage lymphopenia associated with zinc
deficiency in mice correlated with chronically elevated levels of plasma corticosterone (CS).
Furthermore, removal of endogenous C8 by adrenalectomy provided significant thymic
protection against zinc deficiency in adrenalectomized mice (ZD Adr) as compared to zinc
deficient non-adrenalectomized controls (ZD sham). However, it was not clear whether
adrenalectomy in ZD mice would also protect BM cellularity, particularly the B-cell compart-
ment. To address the role of endogenous glucocorticoid (GC) elevation on B-lymphopoiesis
in 21) mice, a zinc dietary study using adrenalectomized and non-adrenalectomized mice was
conducted. However, due to the special care and handling required of adrenalectomized
mice, a typical 30—day dietary study was lengthened to 8 weeks. As a result, mice in the ZD
group were only moderately deficient. Plasma CS levels showed a six-fold increase in ZD
shams compared to ZD Adr mice, indicating the induction of the stress axis by ZD and
successful removal of GCs via adrenalectomy. Consistent with the earlier report from this
laboratory, removal of adrenal glands provided substantial protection of the thymus of ZD
mice, whereas ZD non-adrenalectomized group exhibited a 28% thymic atrophy compared
to ZA sham. Interestingly, two color flow cytometric analysis of BM B-lymphocytes in ZD
Adr indicated a complete protection of early developing (B220*s1g') and immature
(BZZO‘IgM‘IgD‘) B-lineage populations, as their proportions were analogous to those of ZA

sham group. As expected, ZD sham operated mice exhibited significant depletion in early

 

 

172
(57%) and immature (50%) B-cells comparing with ZA shams. However, there was no

significant change in the mature (BZZO‘IgM‘IgD‘) B-cell compartment in either of ZD Adr
or ZD sham operated mice. Taken together, the data confirms the selective sensitivity of
developmental stages of BM B-lymphocytes to the efl‘ects of ZD, as was demonstrated in
previous chapters. More importantly, it suggests a role for chronic elevation of CS during

ZD in the alteration of B-lineage lymphopoiesis in the murine system.

173

Introduction

Malnutrition including zinc deficiency has been shown to exert a rapid and deleterious efl‘ect
on host defense in both humans and animals (Fraker et aI., 1986; Fraker et aI., 1993;
Kuvibidila etal, 1993; Prasad, 1995). Earlier studies from this lab have shown a significant
alte'ationinbothantibodyandcellmediatedreaponseaduringthe course onD (Frakeretal.,
1986; Fraker et aI., 1993). Nevertheless, splenic T and B-cells from ZD mice were
responsive to different mitogenic stimuli indicating normal firnctional capacity (Cook-Mills
and Fraker, 1993 a). Further studies indicated rapid thymic atrophy as well as lymphopenia
in zinc deficient mice (DePasquale-Jardieu and Fraker, 1979; Fraker et 01., 1982; King and
Fraker, 1991). Thus, it was clear that part of the reduction of host defense in ZD was due
to the reduced number of leukocytes actively participating in immune responses. Data
presented in previous chapters (2,3) clearly indicated that ZD profoundly afl‘ected
lymphopoiesis, in particular B-lyrnphopoiesis, in the marrow of mice. The deleterious effects
on B-cell development were mainly observed in precursor and immature B-cells, leaving the
progenitor and mature B-cells relatively unaffected. However, the mechanism(s) underlying
these alteration was still unknown.

It is well documented that physiological stresses such as surgery, burns, infections,
and malnutrition are associated with a two to five fold increase in circulating plasma GC
levels (Maldonado et aI., 1991; Barone et aI., 1993; Hermann et 01., 1994; Fraker et 01.,
1995). In fact, there are studies in which the role of elevated levels of endogenous GCs in

thymic atrophy and lymphopenia in ZD has been suggested (Quarterman, 1973; Quarterman

174
and Hamphires, 1979; DePasquale—Jardieu and Fraker, 1979; 1980). A few years ago, this

laboratory demonstrated that ZD, possibly through the activation of the stress axis, led to the
chronic elevation of GC from a basal level (IO-20 ug/dl) to a higher level (100-120 ug/dl)
which was shown to be irnmunosuppressive (DePasquale-Jardieu and Fraker, 1979). In
addition, a series of in vivo studies were performed using implanted CS pellets in mice to
chronically deliver levels of CS (32 to 94 uydl) similar to the levels detected in ZD (Garvy
et al, 1993a). Interestingly, a short exposure (3 days) to these levels of CS resulted in thymic
atrophy and significant reduction (~70%) of BM early developing B-cells (B220‘, sIg')
(Garvy et aI., 1993a). In separate studies, this lab also demonstrated that removal of
endogenous CS via adrenalectomy provided complete thymic protection against atrophy in
ZD Adr compared with ZD sham (DePasquale-Jardieu and Fraker, 1980). Considering the
above observations, one might hypothesize that if immature B-cells are as sensitive as
immature thymocytes to chronically elevated levels of GC, removal of circulating
corticosteroid by adrenalectomy should also provide BM protection against ZD as it did to
the thymus. To investigate this question, mice were either adrenalectomized or sham
operated and after 7-10 days of recovery they were started on a dietary zinc study. In a
typical diet study, mice are normally housed in hanging stainless steel cages with open-wire
mesh bottoms to prevent zinc contamination fiom their feces (Cook-Mills and Fraker, 1993a).
However, to keep these vulnerable mice as warm and protected as possible, the metal cages
were replaced with EDTA washed polycarbomte cages with hardwood chips bedding. While
these conditions protected the mice fi'om additional stress, they increased the possibility of

zinc recirculation between the mice and their surrounding environment. For these reasons,

175
the diet study was lengthened to eight weeks resulting in only moderate degree of deficiency
among mice in the ZD group. Results presented herein confirm the role of adrenalectomy in
protection of the thymus against atrophy in ZD, as was demonstrated in an early study by
DePasquale-Jardique and Fraker (1980). More significantly, removal of the CS provided

substantial protection of B-lymphopoiesis in the marrow of ZD mice as well.

176

Materials and Methods

B I' I D . |'

Young adult A/J female mice (4-5 weeks old) obtained from the Jackson Laboratory
(Bar Harbor, Maine) were used in this study. Mice were housed in transparent polycarbonate
cages with woodchip bedding and maintained in a temperature (72-74°F) and humidity (45-
50%) controlled facility on a 12 hr/ 12 hr light/dark cycle (lights on 7 am.) Mouse chow and
acidified water were fieely available and 5-7 days of climate adjustment was allowed prior to
the surgery. Following surgery, at least 7-10 days were allowed for healing before starting
the dietary study. In addition, to maintain the mice as warm and comfortable as possible,
hanging stainless steel cages were not used. Instead, the mice were housed in pairs in EDTA
washed polycarbonate cages provided with 4N HCl washed feed jars and water bottles.
These conditions lengthened the period of dietary study to about 8 weeks resulting in only
a moderate degree of deficiency. To initiate the dietary study, mice were subdivided into four
dietary groups: zinc adequate adrenalectomized (ZA Adr), zinc adequate sham operated
(ZA sham), zinc deficient adrenalectomized (ZD Adr) and zinc deficient sham operated
(ZD sham). Mice in each group were fed ad libitum a biotin-fortified egg white containing
either deficient (~05 ppm) or adequate (~30 ppm) levels of zinc (see Table l in Chapter 3).
Adrenalectomized and non-adrenalectomized mice were maintained on physiological saline
(0.9% NaCl) and deionized distilled drinking water, respectively. The diet study was
terminated using criteria for moderately zinc deficient mice such as moderate changes in coat

color, texture, eye, tail and skin along with diminished food consumption and body weights.

177

MW

' Mice that were to be adrenalectomized were anesthetized with methoxyfiurane
(Metafane, Pitman-Moore, Inc., Mundelein, IL) using a nose cone and received bilateral
dorsal incisions through the skin and muscle wall at the level of the last rib. To maintain
consistency in the operational procedure, the adremrl gland on the left side was always
removed first and the right adrenal gland was removed subsequently. The incision was closed
with 6-0 ethilon sutures (Ethicon Inc., Somerville, NJ). Sham operated mice (controls) were
exposed to the same surgical procedure except the adrenal glands were left intact. All
surgical procedures were done using sterile techniques. Following the surgery, all ofthe mice
received 0.3 ml subcutaneous injections ofBuprenex (2.5 nyml) in Lactated Ringers solution
followed by two to three more injections at 12-hr intervals to minimize post-surgical pain and
dehydration Adrenalectomized and sham operated (non-adrenalectomized) mice were then
placed individually in cages with sterile, autoclaved bedding material. A recovery period of
7-10 days was allowed before the initiation of the zinc dietary study. The success of the
operation was verified via visual inspection for any trace amount of adrenal glands and by

measurement of the plasma CS levels at the time of sacrifice.

BI ICII I' III C |° | IZ' ! .

To prevent elevation of endogenous corticosterone and obtain it at the lowest levels
during the diurnal cycle, mice were bled within 90 seconds of disturbing the cages between
8-9 am. Blood samples collected fi'om the subclavian arteries of anesthetized mice fi'om all

four dietary groups were processed individually in acid washed heparinized microtubes. A

178

spectrofluorometric method for measuring corticosterone in small volume of plasma (Garvy
etal, 1993a) was utilized. Briefly, CS was extracted fiom 30 ul of plasma by addition of 600
pl dichlorornethane (Aldrich,Milwaukee,Wl). Alter vigorous shaking and centrifugation, the
aqueous layer (top phase) was discarded. Then the solvent layer (bottom phase) was treated
with 0.1N NaOH, mixed well and centrifuged. The organic phase (bottom layer) was then
used for the fluorescence development. A volume of 200 pl of a pre-fluorescence mixture
consisting of three parts concentrated sulfuric acid (Aldrich,Milwaukee,WI) and one part
absolute ethanol (200 proof) was added to the organic phase. To develop the fluorescence,
samples were then vigorously shaken for 2 min, centrifuged and the solvent layer (top phase)
was discarded by aspiration. After 30 min. incubation of the acid layer at room temperature,
the fluorescence intensity was determined on a spectrofluorometer (Perkin Elmer, model 650-
40) at 475 nm excitation wavelength and 525 nm anission wavelength. Standards containing
10-75 ng CS (Sigma, St.Louis, M0) were carried through the same procedure resulting in a
linear relationship between the fluorescence intensity and the standard concentrations used
in this study. Addition of known amount of standards to the plasma samples of known
concentrations resulted in 85% to 95% recovery by this method.

For zinc analysis, plasma samples were diluted 1:10 in 1% HCl and analyzed
immediately by flame atomic absorption spectrophotometry (V arian AA-20 Plus, Mulgrave,
Victoria, Australia) at 213.9 nm with deuterium background correction. A standard curve
was established using concentrations of zinc ranging from 0.1 to 1 ppm (pg Zn/ml) prepared

fiom an ultra-pure standard (Sigma, St. Louis, MO). Addition of known concentrations of

179
zinc to plasma samples (standard addition) resulted in greater than 90% recovery of zinc by

this method.

2 |° [C II S . .

BM cell suspensions were prepared by flushing the femoral and tibial shafts with
Hepes buffered modified Hank’s balanced salt solution (HBSS) (Sigma, St. Louis, MO)
supplemented with 4% FBS. After removal of erythrocytes using density gradient
centrifugation over Histopaque (1083) (Sigma, St. Louis, M0), the interphase was removed,
washed two times and resuspended at 1x10‘ cells/ml in cold HBSS with 2% FBS and 0.1%

sodium azide and kept on ice for phenotypic analysis.

! H l' .

Biotin-conjugated rat anti-mouse B220 (RAB-6B2) prepared in house, sheep anti-
mouse biotinylated IgD (7 chain specific) (The Binding Site, San Diego, CA), and
dichlorotriazinyl amino fluorescein (DTAF) conjugated goat anti-mouse IgM (u chain
specific) fi'om the Jackson Labs (West Grove, PA) were used in this study. For biotinylated
antibodies, streptavidin conjugated phycoerythrin (AV-PE) fluorochrome, purchased from

Vector (Burlingame, CA), was used to detect the biotin.

I II SI . . .
Iabelingofcellsurfaceantigenswasperformed by incubating one million cells for 30

min. on ice with 150 pl of appropriately diluted antibody combinations. The antibody

180
combirnations were either biotin-anti-B220 and DTAF-anti-IgM to detect early B-cells versus

immature/mature B-Cells, or DTAF-anti-IgM and biotin-anti-IgD for the detection of
immature and mature B-cells. Cells stained with biotinylated antibodies were washed once
and irncubated for another 30 min with streptavidin conjugated phycoerythrin for developing
fluorescence. The negative controls were unstained cells, cells stained with secondary

antibody (AV-PE), and cells fi'om tissue negative in expression of cell surface antigens (eg.,

thymocytes).

mama:

Two color FACS analysis of stained cells was performed on a Becton Dickinson
Vantage flow cytometer linked to a HP consort 32 computer system on which multiparameter
data were collected and analyzed using LYSIS II software. The flow cytometer was equipped
with an 1LT laser exciting at 488 nm. Electronic compensation was used for color correction
of DTAF arnd PE Fluorochromes. DTAF emission was collected at 530115 and PE emission
was collected at 575:13. Singlet cells were gated by light scatter (forward vs side scatter)

to exclude debris and cell aggregates. Data fi'om 10,000 cells were collected in list mode.

SI |° |° I l I . .
All data were analyzed by the Kruskal-Wallis nonparametric test and the analysis of

variance (ANOVA) for parametric data in order to identify sigrnificant difl‘erences between

dietary groups (Daniel, 1987). A Tukey's post-hoe comparisons was performed when

181
appropriate (Daniel, 1987). Differences were considered statistically significant at P<0.05.

All data are presented as the mean i SD where n is 6 to 8 mice unless otherwise indicated.

182

Results

6 .Ie'al‘ufil'H ..:.. it. .in... 'vr'JI.\' 'I '
Mia:

The change in body weights of the four difl‘erent dietary groups obtained at difl‘erent
time points during the diet study are shown in Table 1. As indicated in Table 1, the body
weights in ZD Adr mice were not sigrnificantly different from their corresponding control
group, ZD sham, throughout the diet study. However, by the end of the dietary study ZD
adrenalectomized and sham operated mice weighed significantly less than adrenalectomized
and slnarn operated ZA fed groups. In this case, ZD Adr mice weighed 86% and ZD sham
79% of their corresponding controls, ZA Adr and ZA sham mice.

In terms of thymus integrity, the thymus weight (Figure 1) of the ZD sham mice
decreased significantly, being 72% of control sham operated ZA fed mice. The degree of
atrophy was in accordance with the values obtained fiom mice moderately affected by zinc
deficiency in the previous chapters (2,3). More importantly, the thymus weights of the ZD
Adrgroupremainedatabout90% oftheZAAdr and 1.5 fold greaterthanthat inZD shams,

indicating significant protection against the effects of ZD (Figure l).

jar-1r: ;rg,i',.,qnu in '1 ll *4! ,l , ,.tU. ll .1. .l 1‘
upon termination of the dietary study (day 56), mice fi'om each of the four dietary
groups were bled arnd their plasma CS was determined by a fluorometric assay (Garvy et aI.,

1993a). As shown irn Figure 2, the mean plasma CS level in ZD sham operated mice was 2.6

183
and 6 fold greater than that in the control ZA sham and ZD Adr groups, respectively.

Further, the plasmaCS levels inZD AdrandZAAdrgroups remained at 2.8-4.1ug/dl, while
the levels in ZA sham operated mice was elevated slightly to 6.6 ug/dl (Figure 2).
Theresultsoftlneplasmazinclevelsofthemiceineachgroup obtainedatthe
termination of diet study are illustrated in Figure 3. Adrernalectonnies appeared to have no
efl‘ect on the plasma zinc levels, as no sigrnificant difference in plasma zinc level was noted
between ZD Adr and ZD sham nnice. Likewise, the zinc level in ZA Adr mice showed no
significam change compared to the levels detected in ZA sham operated group. By contrast,
plasma zinc levels in mice fi'om ZD fed adrenalectonnized and sham operated groups were

moderately declined (21-28%) compared to the control ZA fed adrenalectomized and sham

operated mice, respectively (Figure 3).

HI I i"Z°Dfi°|!I II'IM"
To determine the effects of adrenalectomy or removal of GC on B-cell development

in ZD mice, fluorescently labeled antibodies directed against major B-cell surface markers,
B220, IgM and IgD, were used, and flow cytometric analysis was performed. Using this
approach, the proportion of early (B220‘Ig'), immature (B220‘IgM’IgD'), and mature
(B200”IgM‘IgD‘) B-cell subcompartments in the marrow of mice in each of the four dietary
groups were determined (Figure 4). In Figure 4, it is clear that ZD had a profound efl‘ect on
cells of the B-lineage at the early and immature stage of development as shown in previous
studies (Chapters 2,3). The proportion of early developing B-cells (B220"Ig') in ZD sham

mice indicated 57-70% depletion compared to that of ZA sham and ZD Adr groups,

184
respectively. Similarly, immature (B220‘IgM‘IgD‘) B-cell declined significantly (5 7-65%)
inflnenmrowonDshmnope'atedgroupwhencomparedtoflneZAshamandZD Adrnnice.
However, the more mature (B220‘IgM*IgD*) B-cell subset showed marginal but not
statistically sigm'ficant reduction in ZD sham operated mice. Interestingly, adrenalectomized
mice maintained on the ZD diet (ZD Adr) demonstrated complete protection in their B-cell
subpopulations. The immature (BZZO’TgM‘IgD') arnd mature (B220‘IgM*IgD”) B-cell subsets
in the ZD Adr mice exhibited no depletion, as their proportions were analogous to those
fournd in ZA Adr and sham operated nnice. Likewise, the early B-cells in the marrow onD
Adr group were not afl‘ected by the deficierncy, showing a moderate accumulation (+23% to

+3 9%) compared to that in ZA Adr and ZA slnam controls, respectively (Figure 4).

185

Discussion

The disruption of B-cell development in the marrow of ZD mice resulting in lynnphopenia,
along with chronic release of known immunosupressor, GCs, during zinc deprivation
suggested a role for GC for these observations. In the light of an early study (DePasquale-
Iardieu arnd Fraker, 1980), in which removal of GC via adrenalectomy provided complete
protection of the thymus of ZD mice against atrophy, speculation on the role of GC with
respect to B-cells was intensified. To investigate the role of C8 (the predominant form of
GCs in narrine system) in lymphopoiesis, adrenalectomy was performed to essentially remove
GC from the circulation. Using this approach, it was possible to ascertain how much of the
alteration in lymphopoiesis was due to suboptimal zinc levels versus the chronic levels of CS
that are produced in later stages of the deficiency in mice. Adrenalectomy has been
previously shown by this laboratory as a convenient way for removal of GC (DePasquale-
Jardieu and Fraker, 1980). Furthermore, the use of tlnis technique by many other investigators
for the characterization of the potential role of difl‘erent adrenal hormones including GCs
(Dunn, 1988; Sloviter et aI., 1989; Jerrells et 01., 1990; Mitchell and Meaney, 1991; Rinner
et aI., 1992; Kitson et aI., 1994; Dhabhar et 01., 1995) added more reasonings for the
selection of this technique. It should be noted that although adrenalectomy also removes
mineralocorticoids, the substitution of isotonic sodium chloride for drinking water has been
slnown to compensate for the loss of this hormone (Saksi and Epsteirn, 1990). An alternative

to adrenalectomy would be drugs which block glucocorticoid receptors or release of GCs.

186
Such treatment would require continuous delivery to the already stressed dietary mice

throughout the experiment (at least four weeks), which is impractical.

Just aszincinfluencesthehormonal secretion, particularlyfromtheadrenalglands,
changesinthe concentrations ofadrernal hormones (eg., GCs) have been shown to afl‘ect zinc
concentrations. Early studies have indicated that adrenalectomy or adrenocortical
insufliciency (eg., hypopituitarisrn) have been accompanied by increased serum zinc
concentrations, decreased urinary zinc excretion, and increased retention of zinc in several
tissues (Flynn et at, 1973; Hernkin, 1974). Thus, it was of interest to measure zinc levels of
all mice, particularly those in adrenalectomized groups, to monitor any possible variation in
zinclcivelsduetoadrenalectomyaswellassuboptimalzinc intake. As shown inFigure 3, the
results indicated a moderate but statistically significant decline in the zinc levels of ZD Adr
andZD slnamgoupsindicatingtheexpected depletion ofzincin moderate deficiency. Onthe
other hand, the plasma zinc levels in ZD Adr group showed no significant variations
censured to ZD sham operated goup. Likewise, no significant change in plasma zinc levels
of ZA Adr compared to that in ZA sham group was observed. These data suggest that
adrenlectomy or removal of CS from the circulation had no apparent efi'ects on serum zinc
distribution as opposed to what was indicated by earlier studies (Flynn et aI., 1973; Henkirn,
1974).

‘ Although moderately zinc deficient, the mice in ZD sham operated group had 6 fold
higherplasrmCS levelscomparedtothelevelsdetected inZD Adrgroup. This indicatesthe
activation of the hypothalaIms-pimitary-adrenocortical axis (stress axis) in ZD sham mice as

reported in the literature (DePasquale-Jardieu and Fraker, 1979; 1980; Quarterman and

187
Hunpll'ies, 1979; Prasad, 1985;) at even moderate stages of the deficiency. Furthermore, the

low levels of CS in adrenalectomized mice compared to non-adrenalectomized mice (sham
operated) wee irndicative of proper adrenalectomy or nearly complete elinnination of CS via
adrenlectorny, andthelackofdetectibleadrenaltissueatthetimeofsacrifice supported this.

ExanninationofthednyrmsesonDgouprevealedthatinspiteofmodeate induction
of ZD in this study, the thymus of ZD sham operated mice were involuted 28% to 33%,
comparedtothethyrmsweightsoftheZAshamsand ZD Adr mice. This observation, along
with the similarity between the thynnic weights of ZD Adr and ZA Adr groups, strongly
suggested a role for GC in thynnic atrophy, as the elinnination of this hormone significantly
protected the thymus from atrophy (DePasquale-Jardieu and Fraker, 1980; Jerrells et al.,
1990).

The major question to be addressed in this study was the degee to which
adrenalectomy had protected the development of cells of B-lineage in the ZD Adr mice. As
shown in figure 4, B—cell subpopulations whose susceptibility to the effects of ZD has been
shown herein and in the previous chapters (2 and 3) were now completely protected with
adrenalectomy. InZDshamopeatedmicewheeCSwaselevated,earlyandimmatureB-cell
arbcompartments exlnibited significant depletion in accordance to the previous findings (King
et aI., 1995). However, adrenalectomy appeared to provide substantial protection to the
developirng B-cells irn ZD mice. This was demonstrated by the presence of all the BM B-cell
arbpopulatiomevaluatedintlu’sstudyinproportions similarto those detected inZAAdr and
ZA slnarn operated mice. Thus, suboptimal zinc may not be directly affecting the marrow's

ability to produce new lymphocytes during moderate deprivation. Secondary efl‘ects of zinc

188
deficiency, especially the elevation in production of glucocorticoids, may be significantly
reducins lymphopoiesiS-

As stated earlier, in viva exposure ofnnice to the levels ofCS irnduced by ZD were
shown to be suficient to alter B-lymphopoiesis, particularly in early and immature stages of
B-cell development (Garvy et aI., 1993 a). Furthermore, sensitivity of these populations to
slnort exposure (8 Inns) of synthetic (dexamethasone) and natural (cortisol, corticosterone) GC
invitrowasalsodenonsflatedbytheirsubstantialdepletioninflneBM andtheiraccumulation
irn the apoptotic region of DNA histogam evaluated by flow cytometry (Garvy et aI., 1993b;
Voetberg et 01., 1994). These findings are in accordance with the more recent literature in
which adrenalectomy protected the suppressive effects of GC on cells of the immune system
(Kitson et 01., 1994; Dhabhar et aI., 1995). The results fi'om this study clearly demonstrated
the neuroendocrine (stress-induced CS release) regulation of the immune system suggested
in the literature (Bateman et aI., 1989; Dantzer and Kelley, 1989; Homo-Delarche and
Dardenns, 1993; Berczi, 1994).

It appears that although zinc is an essential element in many biochemical activities
inside cells (Vallee arnd Falchuk, 1993; Cousins, 1996), the deficiency ofzinc by itselfmay not
be directly exerting suppressive efl‘ects on the cells of immune system. Instead, it is the
stimulation of the stress axis with the production of GCs at elevated levels, which leads to
irrnrranrnosuppression in ZD (DePasquale-Jardieu and Fraker, 1979; Prasad, 1985; Hambidge
etal, 1986; Bunce, 1989). Tlnus, it seem that ZD purposely downsizes the immune system,
which is one of the largest tissues and major users of nutrients in the body, to provide this

limited nutrient to other vital tissues for their protection. This is achieved apparently via

189

activation of the stress axis and subsequent chronic release of GC in later stages of the
deficiency. 0n the other hand, the survival of nnature lymphocytes (T and B) in the course
of ZD provides a fully functional defense system (Cook-Mills and Fraker, 1993 a), but with
lower magnitude in the periphery. Taken together, these observations identified the
significant role of CS in the alteration of murine BM B-lymphopoiesis in the course of
suboptimal dietary zirnc intake. Howeve, the possibility of other factors (eg., oncogenes) that
might play additional mechanistic roles (eg., blocking or facilitating GC actions) can not be

ruled out and needs to be investigated.

190
In“ Body weights of sham operated and adrenalectomized mice maintained on zinc

adequate or zinc deficient diet during eight weeks of diet study.

 

 

16.48 :1: 0.31 16.84 :1: 0.68 17.01 :l: 1.22' 16.74 :I: 1.14

 

17.18:]: 1.14 17.143: 1.67 17.08:1:0.61 16.293: 1.25
18.04 :1: 0.83 18.32 :1: 1.2 17.50 :h 0.72 16.53 :1: 1.36
18.66 :1: 0.50 19.41 :1: 1.0 17.35 :1: 0.78 16.58 :I: 1.96
19.70 :t: 0.56 20.44 :1: 1.22 16.90 :l: 0.85 16.14 :1: 1.63‘=

 

 

 

 

«Os-BN6

 

 

 

 

 

a Data are expressed as mean i: SD of 6 to 8 mice per dietary group, and
. represent 2 separate experiments.
b Significantly different from ZA Adr at P< 0.05.

c Significantly different from ZA sham at P< 0.05.

191

Bun-J; Comparison of thymic weights of adrenalectonnized and sham operated mice
mairntairned on zinc adequate or zinc deficient diet, obtained at the terminal point
of the diet study (day 56). ZA Adr, n=6; ZA shann, n=8; ZD Adr, n=7; ZD
sham, n=8. Data are mean i SD and represent two separate diet studies.
‘Denotes significantly different fiom ZA sham operated and ZD Adr mice at

P<0.05, where comparisons were made separately .

192

 

 

 

 

 

 

 

is\\\\\\§

 

 

 

 

 

 

 

5
3

q q _ u q u A
O 5 O 5 0 5 O
3 2 2 1.. 1..

REV gamma? manage

ZA sham ZD Adr ZD sham

ZA Adr

193

W Plasma corticosterone levels of adrenalectonnized and sham operated mice fed
zinc adequate or zinc deficient diet. Upon termination of diet study (day 56),
mice from each of the four dietary goups were bled and their plasma was
collected individually for a fluoromfic CS assay. ZA Adr, n=6; ZA shann, n=8;
ZD Adr, n=7; ZD sham, n=8. Data are expressed as mean :1: SD and are
representative of two separate diet studies. * Denotes significantly different

from ZA slnam and ZD Adr groups at P< 0.05 in separate comparisons.

Plasma CS (,ug / d1)

194

 

 

 

 

ZA Adr

 

 

 

 

 

 

 

 

 

 

 

l
l
T
s\\\
ZA sham ZD Adr ZD sham

 

195

m Comparison of plasma zinc levels of adrenalectonnized or sham operated mice
maintained on zinc adequate or deficient diet for eight weeks. Plasma zinc
concentration of individual mice from each of the four dietary groups were
determined via flame atomic absorption spectroscopy on the last day of diet
study(day56). DatarepresentmeaniSD,wheen=6to 8 miceineach dietary
goup. "' Indicates significantly difl‘erent flom ZA sham at P< 0.05. " Indicate

significantly different fi'om ZA Adr at P<0.05.

Plasma Zn (Mg/d1)

196

 

110
100-
90-
80-
70-
601
50-
410--
30‘
20-
‘10-

 

 

ZA Adr

 

 

 

**

 

V//////

 

 

    

 
    

vvvzyvvv
szozozegezozozq
0099090

0 O O O O O O
0 O...0.0.0.0.Q

      
      

     

AAAAAAAA

 

ZA sham

ZD Adr

ZD sham

 

197

m Proportion of early developing (B220‘Ig‘), immature (B220’IgM’IgD') and
mature (B220*IgM*IgD*) B-cells in the marrow of mice that were sham
operated and maintained on zinc deficient diet (ZD sham) or zinc adequate diet
(ZA sham) or which were adrenalectomized and maintained on zinc deficient
(ZD Adr) or zinc adequate diet (ZA Adr). Fluorescently labelled antibodies
were used to quantitate the proportion of B220‘, Iglvf and IgD” cells in the
marrow of each dietary goup as dete'mined by flow cytometry where data from
10,000 cells were collected for each analysis. Data are expressed as the mean
values of the percentage of all nucleated cells of tlne marrow for each population
:|: SD, and represent two separate studies. The number of mice per treatment
goup ranged from n = 6 to 8. * Denotes numbers significantly different from
ZA sham and ZD Adr mice at P< 0.05, where separate comparisons were

performed.

198

 

 

 

- ZA Adr
l: ZA sham
m ZD sham

g 11% was

*

 

1%
.IJrll.

 

 

 

O
.4.

. . _ . _ _ .
5 O 5 O 5 O 5 O
3 3 2 2 1 1

230 Sm veaeefiodz no omeucooaem

Immature B Mature B

Early B

CHABIEREIKE

Evaluation of the Effects of Zinc Deficiency on T-cell
Maturation in the Thymus of Young Adult A/J Mice: A

Possible Role for Apoptosis in Zinc Deficiency

199

200
Abstract

The presence of thynnic atrophy and lymphopenia and the disruption of B-lymphopoiesis in
zinc deprived nnice led to the investigation of two main questions in this study. The first
question was to detemine how suboptimal zirnc irntake afl‘ects phenotypic distribution of major
thynnic T-cell subsets including progenitor (CD4'CD8'), immature (CD4”CD8*), and mature
(CD4*CD8'/CD4‘CD8*) T-cells. The second question was to deternnine if apoptosis was the
means for the elirnnination of susceptible lymphocytes in the course of dietary zinc deficiency.
To irnvestigate the first objective, young adult All mice were provided zinc adequate (ZA) or
zinc deficient (ZD) diet for 27 days. This was followed by flow cytometric evaluation of the
phenotypic distribution of T-lymphocyte subsets in the thymus of mice in zinc dietary study.
The results fiom this investigation indicate that T-lymphocyte subsets are selectively afl‘ected
by the deficiency. Phenotypic analysis of thynnic T-lymphocytes revealed a significant
resistance of double negative (DN) progenitor (CD4'CD8‘) as well as single positive (SP)
mature (CD4*CDB‘/ CD4‘CD8‘) T-cells to the effects of ZD. By contrast, the proportion of
double positive (DP) immature T-cells (CD4‘CD8”) showed marginal decline in moderately
afl‘ected (MZD) and geater depletion (15%) in severely affected mice by zinc deficiency
(SZD). However, when the absolute number of this population was evaluated a substantial
decrease (3 8%) in MZD mice and a significant reduction (86%) in SZD group were noted.
Furthermore, moderate levels of zinc deficiency (MZD) caused a 41% depletion in thynnic

cellularity, with substantial depletion (83%) in SZD mice. The suppression in T-cell numbers

201
was directly correlated to a 36 and 67% involution of the thymus in MZD and SZD goups,
respectively.

Detemimtion of apoptosis in zinc deficiency, the second objective, was investigated
botln in vivo and in vitro. However, due to rapid clearance of apoptotic cells by phagocytic
cells in viva, detection of apoptosis after 27 days of diet study was hardly possible. Thus, the
in vivo approach utilized a short incubation (6 hrs) of thymic T—cells from mice in a 27-day
dietary zirnc study in regular culture media (RPMI-l640), to reveal apoptosis in cells that had
received the death signal in viva, but had escaped detection by phagocytic cells. This
approach was able to detect a 23% level of apoptosis in immature T-cells of SZD mice,
whereas MZD nnice showed almost the same level of apoptosis in this population (17%) as
did control ZA fed nnice (16%). This result indicated that perhaps most of the apoptotic
thymocyteshadalreadybeenclearedinvivobyphagocytic cells and onlyavery small portion
ofapoptotic cells had the chance to escape in vivo phagocytosis and to be captured by this
system. To bypass this problem, simulation of ZD - though for a short time- in an in vitro
culture systen was investigated. This approach utilized a short incubation (8 hrs) of regular
mouse thymic T-cells in culture media (RPMI-1640) to which known amounts of zinc (Zn)
and corticosterone (CS) at the estimated levels in ZA and SZD mice, were added. This
system detected a substantial percentage of apoptotic T-cells particularly among immature
T-cells (2.8 fold) in the SZD simulated culture media compared to the control ZA simulated
culture condition Taken togethe, the data presented herein indicate the selective sensitivity
of thynnic T-lineage subsets to the efl‘ects of zinc deficiency and verifies the occurrence of

apoptosis in the thymus of SZD mice.

202

Introduction

Previous investigations on the evaluation of the efi‘ects of suboptimal zinc intake on the
immune system has strongly documented the immune impairment, manifested as thymic
atrophy, lymphopenia and impaired antibody and cell mediated immunity (Fraker et 01., 1986;
1987; Keen and Gershwin, 1990; Zeng et 01., 1991; King et aI., 1995). In addition, the
results from previous chapters (2, 3, and 4) indicated that ZD resulted in stage-specific
alteration in BM B-lymphocytes which were otherwise protected in adrenalectomized mice.
However, the status of phenotypic distribution of developing T-cells in the thymus of ZD
mice, and the mechanism by which thymocytes were eliminated remained unclear. Thus, the
objectives of the study presented herein were two fold. The primary objective was to
investigate the relative sensitivity or resistance of thymic T-lineage cells (progenitor,
immature, and mature T-cells) to the effect of ZD. It was of interest to see whether the
results would show a pattern of alterations similar to that observed in BM B-lymphopoiesis
in ZD mice. In this regard, thymocytes prepared from the three dietary groups (ZA, MZD,
SZD) were phenotyped with monoclonal antibodies against T-cell maturation markers (CD4
and CD8) followed by flow cytometric analysis. This is the first detailed examination of cells
of the T-lineage in the thymus of zinc dietary mice.
. The second objective of this study was to investigate whether apoptosis played a role
in the elimination of susceptible lymphocytes in zinc deficiency. One of the earliest
investigations which linked ZD to apoptosis, was reported by Elmes (1977). He observed

significant increase in the number of apoptotic bodies in the mucosa of small intestine of ZD

203

rats compared to ZA fed rats, suggesting the induction of apoptosis via deficiency in zinc.
Martin and colleagues (1991) examined the survival ofthree cell lines ofhuman lymphoid
(Molt-3 and Raji) or myeloid (EL-60) origin in vitro under zinc sufficient or zinc deficient
mirrors. Zinc deficiency resulted in diminished proliferative capacity and viability ofall the
cell types. Cell death occurred mainly via apoptosis in the HL-60 and Raji cells and via
necrosis in the Molt-3 cells. These studies suggested that ZD can induce apoptosis both in
viva and in vitro. A more recent study by Treves et aI., (1994) suggested a role for zinc as
an intracellular regulator of apoptosis, since the chelation of zinc resulted in induction of
DNA fi'agmentation into 200 bp in peripheral blood lymphocytes.

Parallel to the above studies, GC hormones are also thought to induce thymic atrophy
in viva through the enhancement of apoptosis (Munck and Grabtree, 1981; Compton et al.,
1987). It has been long recognized that mouse thymocytes undergo apoptosis in response to
a variety of apoptotic cues including GCs (Wyllie, 1980; Morris et aI., 1984; Telford et 01.,
1991; Nieto et aI., 1992; Cohen, 1992). These findings prompted the question of whether
the sensitivity ofirnmature thymocytes to the effects onD might be relevant to the in viva
and in vitro elimination of thymocytes via GC-induced apoptosis. Thymocytes have been
extensively used over the past decade as an easy and reliable model for the study and
characterization of GC-induced apoptosis (Wyllie, 1980; Cohen, 1992; Sun et 01., 1992;
Gruber et aI., 1994; Korsmeyer, 1995). The homogeneity of the thymus (populates >90%
thymocytes) not only makes it easier to assess apoptosis but also brings the opportunity to

determine the efi‘ects of ZD on T—cell development.

204
Thus, the occurrence of apoptosis in ZD, as the second objective of this study, was

investigated in the thymus both in viva and in vitro. However, based on a small pilot study,
we knew that the detection of apoptotic thymocytes afier a period of four weeks diet study
would not be successful, perhaps due to rapid clearance of apoptotic cells by macrophages
in viva (Wyllie et 01., 1980; Cohen 1991). Short incubation of thymocytes (6 hours),
however, might allow those cells that had just received the death signal in viva, to complete
the apoptotic phase in vitro without detection by macrophages. To investigate, thymocytes
prepared fiom difl‘erent dietary groups were incubated in RPMI-1640 medium at 37°C for
6 hours, followed by irnmunophenotyping and DNA staining. Not to our surprise, this
approach could only detect a small portion of the apoptotic lymphocytes occurring in a ZD
mouse. Therefore, a second approach consisting of an in vitro culture system designed to
simulatetheinvivaCS andanevelsofaZAand aSZDmouse, was investigated. Although,
the establishment of in viva conditions of 28-day dietary mice in an s hrs-in vitro culture
system is absolutely impossible, the in vitro system presented herein was an attempt to test
the survivability of thymocytes in culture conditions where Zn and CS levels were analogous
to the levels observed in ZA and SZD mice. Since addition of CS to the culture media at
levels detected in zinc dietary mice (ZA and SZD) resulted in >70% cell death in thymocytes
(preliminary data), the estimated fi'ee CS (unbound CS in plasma) levels which is believed to
exert the biological activity of GCs (Faict et 01., 1985; Vermeulen , 1986), were utilized. In
this system thymocytes prepared from regular mice were incubated for 8 hours in RPM-1640
culture media supplemented with estimated levels of Zn (50 or 100 uydl) and fi'ee CS (2 or

6 ug/dl) found in ZA and SZD mice, respectively. Subsequent to incubation, thymocytes

205

were subjected to two color immunofluorescent phenotyping against major T-cell surface
markers (CD4 and CD8) followed by DNA staining to detect apoptosis in distinct T-cell
subsets.

The data presented in this study indicates the high susceptibility of immature
thymocytes and the relative resistance of progenitor and mature T-cells to the efi‘ects of ZD.
Furthermore, the high incidence of apoptosis in thymocytes, particularly in immature
subpopulation, indicates the presence of apoptosis in ZD, and suggests that it plays a
significant role in the elimination of vulnerable lymphocytes (lymphopenia) in the course of

the deficiency.

206
Materials and Methods

Six week old female All inbred mice (Jackson laboratory Bar Harbor, ME) weighing
17. 1+ 0.7 g were used throughout the study. All mice were fed ad libitum a biotin fortified
egg white based diet which contained either 30pg Zn/g diet (zinc adequate group; ZA) or
~0.5ug Zn/g diet (zinc deficient group; ZD). The composition of the diet has been described
in Chapter 3. All dietary groups were maintained in stainless steel cages with mesh bottoms
to reduce recycling of zinc for a period of 27 days. Feed jars and water bottles were washed
in 4N HCl and the drinking water was acidified to reduce Pseudamanas infections. Food
consumption was recorded daily and body weights were measured twice a week. At the end
of the dietary study, the ZD group was subdivided into moderately affected mice by zinc
deficiency (MZD) weighing an average of 78% of the ZA group with moderate signs of
parakeratosis of the ears and tail or severely afi‘ected mice by zinc deficiency (SZD) weighing

an average of 71% of the ZA group which exhibited more severe parakeratosis.

Wall:

To prevent elevation of endogenous corticosterone and maintain it at the lowest levels
during the diurnal cycle, mice were bled within 90 seconds of disturbing the cages between
8-9 in the morning. Blood collected fi'om the subclavian arteries of anesthetized mice fi'om
all three dietary groups, was processed individually in acid washed heparinized microtubes.

A spectrofluorometric method for measuring corticosterone in small volume of plasma (Garvy

207
eral, 1993a) was utilized. Briefly CS was extracted fiom 30 ul ofplasma by addition of600

ul dichloromethane (Aldrich, Milwaukee, WI). After vigorous shaking and centrifugation,
the aqueous layer (top phase) was discarded. Then the solvent layer (bottom phase) was
treated with 0.1N NaOI-i, mixed very well and centrifirged. The organic phase (bottom layer)
was then used for the fluorescence development. A volume of 200 pl pre-fluorescence
nirdureconsistingofflueepaflsconcentratedsulfirficacid(Aldrich, Milwaukee, WI) and one
part absolute ethanol (200 proof) was added to the organic phase. To develop the
fluorescence, samples were then vigorously shaken for 2 min., centrifirged and the solvent
layer (top phase) was discarded by aspiration. After 30 min. incubation of the acid layer at
room temperature, the fluorescence intensity was determined on a spectrofluorometer (Perkin
Elmer, model 650-40) at 475 nm excitation and 525 nm emission. Standards containing 10-
75 ng CS (Sigma, St. Louis, M0) were followed through the same procedure as plasma
samples. Emciency of the recovery of CS fi'om plasma was determined by adding known
concentration ofstandards to the plasma samples ofknown concentration and ranged between

85% to 95%.

int. i I. Hustle .... I. a u .. a . i 14’ i a ..
atAmmm

Thymuses fiom mice in difl‘erent dietary groups (6-8 mice per group) were removed,
weighed, minced and passed individually through sterile 100 micron mesh stainless steel
screen into sterile Hepes bufl‘ered Hank's balanced salt solution (HBSS) supplemented with

4% heat imctivated fetal bovine serum (FBS) (Hyclone,Logan,UT). Single cell suspensions

208
were washed twice, counted for total T-cell numbers (thymus cellularity) and tested for

viability using trypan blue exclusion dye (>95% viability). The cells were then resuspended
in RPM-1640 supplemented with 5% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 100
IU/ml penicillin, 100 rig/nu streptomycin, 1% non-essential amino acids, 50 jig/ml gentamicin
and 5x10" M 2-mercaptoethanol (2-ME). Aliquots of cells (10‘ cells/ml/well) were plated
in24-welltisare cultureplates (triplicates ofeach sample) and incubated for 6 hours at 37°C,
5% C02 atmosphere. Cultured thymocytes were then irnmunophenotyped and stained with
DNA dye to evaluate the phenotypic distribution as well as apoptosis in distinct T-cell

subsets.

I . .0
'. I '.',' ' " "‘Jhlie ll" \9‘4 ' U . ' r ..’l’ . '1 .l' l 6 la" '

Thymrses fi'orn young adult (6 to 10 weeks old) A/J female mice were removed and
extruded through 100 micron stainless steel screen into modified HBSS supplemented with
2% FBS. Single cells were washed twice by centrifirgation at 400 xg for 5 min., resuspended

in HBSS/FB S, and counted using trypan blue-exclusion viability dye (>97% viable).

WWW

Since the core of the in vitro culture system was based upon the supplementation of
the system with known amounts of CS and zinc, it was necessary to minimize the possible
interference of the basal levels of GCs and zinc normally present in the FBS, with the in vitro
assay described below. To do this, the FBS was treated with dextran coated charcoal which

as reported removes GC fi'om serum (Hayashi et al., 1984). Subsequently, the charcoal

209
treated FBS was treated with Chelex-lOO (Bio-Rad laboratories, Hercules, CA) using the

batch method (Bio-Rad instructional manual) to remove zinc. Briefly, FBS
(Hyclone,Logan,UI') was mixed with 1 mg/ml dextran (Sigma Chemical Co., St. Louis, MO)
and 10 mg/ml Norit A activated charcoal (Matheson Clernan and Bell, Norwood,OH) and
incubated for 30 min. in a 50°C waterbath subjected to fi'equent shaking. Dextran coated
charcoal was removed from FBS by centrifilgation at 4000 xg for 10 min. at 4°C and
subsequent filtration through a 0.22 pm filter. These steps resulted in CS deficient FBS. To
remove a'nc fiom this serum, Chelex-IOO was added to the FBS at 5 g/ml followed by gentle
stirring on an electronic mixer for one hour. The resin was separated from FBS by filtration
through a 0.22 pm filter and the FBS was stored at -20°C. The FBS obtained fiom these
treatments (Zn‘ CS') was periodically tested for Zn and CS levels both of which were below
the detection limit of the assays (< 0.01 rig/d1). This serum was used to supplement the
culture media to which known amounts of CS (2 or 6 ug/dl)(4-Preg'lene-11B,21-diol-3,20—
dione, Signs Chemical Co., St. Louis, MO) and/or Zn (0, 50 or 100 ug/lezinc sulfate
heptahydrate, J.T.Baker, Phillipsburg, NJ) were added depending on the culture condition
described below.

[III [I ll EEIZIDII' [l |°‘

RPMI-1640 medium (Signs Chemical Co., St. Louis, MO) supplemented with 2 mM
glutamine, 1 mM sodium pyruvate, 100 rig/ml of streptomycin, 100 IU/ml of penicillin, 50
rig/ml gentarnicin, 1% non-essential amino acids, and 5x10" M 2-mercaptoethanol was used
in this assay. This medium was then supplemented with one of the combinations illustrated

in the chart below:

210

 

 

 

 

 

_ CM“ W, C___ded Zd
1) Basal CS/basal Zn 20% 0.06 uM 3.5 uM
(BL CS/BL Zn) (2 pydl) (100 Jydl)
2) High CS/low Zn 20% 0.17 pM 1.25 uM
(HI CS/LO Zn) (6 pg/dl) (50 1 d1
3) High CS/high Zn 20% 0.17 uM 500 uM
' (HI CS/HI Zn) (6 pg/dlL (14.3 1 ill
4) Basal CS/undetectable Zn 20% 0.06 uM no addition
(BL CS/UN Zn) (2 ugldl) undetectable

 

 

 

 

 

 

Thyrnic T-lymphocytes prepared from regular mice were added at 2x10‘ cells/ml/well
to the various culture media in 24-well plates. The cultured cells were incubated for 8 hours

at 37°C in 5% CO2 atmosphere. Each culture condition was tested in quadruplicate.

I 'II III |' ISI" [5 II'CII'

Afler incubation of thymic T-cells fiom dietary mice in regular culture media (6 hrs),
or thymic T-cells fi'om regular mice in CS/Zn supplemented culture conditions (8 hrs), the
percent recovery (100%) and the viability (>90%) of the cultured cells were examined.
Aliquots of 1x10‘ cells/ml were then resuspended in cold label buffer (HBSS, 2% FBS, 0.1%
sodium azide), washed at least once, and immunophenotyped using antibodies to T-lineage
surface markers. CD4 and CD8 surface markers on mouse T-cells were simultaneously

labeled using 150 pl of phycoerythrin (PE) conjugated rat anti-mouse CD4 and fluorescein

21 1
isothiocyanate (FIT C) conjugated rat anti-mouse CD8 monoclonal antibodies (Pharmingen,
SanDiego, CA). All labeling was performed at 4°C for 1/2 hour followed by two washes in
cold label bufifer. Furthermore, all the antibodies were used at their saturating concentration
subsequent to titration.

To evaluate the presence of apoptosis in thymic T-lymphocytes, phenotyped samples were
resuspended in one part (0.4 ml) cold phosphate buffer saline (PB S) supplemented with 50%
FBS, and fixed by dropwise addition of three parts (1.2 ml) of cold ethanol followed by gentle
mixing. Cells were kept overnight at 4°C, washed twice with cold label bufl‘er to remove
fixative and resuspended in DNA staining dye 4',6-diamidino-2-phenylindole (DAPI) at 1

rig/ml. Stained samples were kept on ice prior to flow cytometric analysis.

W111

Irmunofluorescently labded samples were analyzed on a Bacton Dickinson Vantage
equipped with a consort 32 HP computer with LYSYS‘“ II and MultiPlusTM sofiwares.
Fluorochrome excitation for one and two color analysis (PE, FITC and PE/FITC) was
accomplished using an ILT model RCP-SO argon laser at 488 nm. Fluorochrome emission for
FITC and PE was detected at 530:15 nm and 575113 nm, respectively. For three color
analysis of samples stained with DAPI, PE and FITC, simultaneous use of a Krypton laser
exciting at 350 nm for DAPI and the argon laser was required. Negative controls included
unstained thymocytes to define the negative population and detect autofluorescence. Cells
labeled with isotype-matched non-specific antibodies were used to detect background

fluorescence. Single and dual color controls were used to define the negative as well as the

212
positive population for each antibody and to set color compensation. Phenotypic distribution

of T-cells as well as quantitation of apoptotic thymocytes were determined by initial gating
through DAPI DNA width vs ares fluorescence, excluding debris and doublets and including
apoptotic cells. The gated population was then used for cytogram scatter phenotypic and cell

cycle histogram analysis.

In order to identify any significant differences among dietary groups or different cell
culture conditions, all the data were analyzed by the Kruskal-Wallis nonparametric test and
the analysis of variance (ANOVA) for parametric data (Daniel, 1987). A Tukey's post-hoc
comparisons was then applied where appropriate (Daniel, 1987). Differences were
considered statistically significant at P<0.05. All the data are presented as the meantSD of
6 to 8 mice for the dietary study, and quadruplicates of each culture condition for the in vitro

culture samples.

213

Results

' The effect of ZD on growth, thynurs weight, thymic cellularity, and plasma CS levels
ofmiceona27-daydietarystudyarepresented inTable1.0nthe last day ofdiet study, ZD
mice were divided into MZD and SZD groups based on body weight and degree of
parakeratosis. As shown in table 1, consumption of ZD diet for a period of about four weeks
caused 22% to 29% drop in body weight in ZD group based on the degree of deficiency. The
thymus weights in ZD mice declined significantly, with 36% to 67% depletion in the thymus
weight of MZD and SZD groups, respectively, compared to that in ZA controls. Thymus
cellularity was also affected by ZD. Mice in the MZD group lost 41% of their total
thymocytes with more severe depletion of thymocytes (83%) observed in SZD group. The
plasma CS levels exhibited significant elevation in ZD group, showing about 2.4 to 3 .4 fold
increase in MZD and SZD mice, respectively, compared to ZA mice. The presence of growth
retardation, thymic involution as well as elevation of GC in this study resembles the previous
diet studies (DePasquale-Jardieu and Fraker, 1980; King and Fraker, 1991; King et 01., 1995;

Fraker et al., 1995).

EflillZD IIIMI l' 'IIII .
To evaluate the efl‘ects of ZD on phenotypic distribution of thymic T-cells, 6 hrs

cultured thymocytes prepared fi'om individual mice in each dietary group were

214

immunofiuorescently labeled with monoclonal antibodies against major T-cell surface
markers. The labeled cells were then fixed and stained with DAPI DNA binding dye for in
viva detection of apoptotic thymocytes which will be explained in the next section. The
irnmunophenotyping of cells resulted in detection of four distinct thymic T-cell subsets,
including progenitor (CD4'CD8'), immature (CD4*CD8*), and mature (CD4*CD8')/(CD4'
CD8”) thymocytes. Figure 1 demonstrates the distribution of CD4/CD8 T-lymphocyte
subsets in all dietary groups. As seen in figure 1A, the proportion of progenitor T-cells (CD4‘
CD8).wlu'ch are the earliest committed T-cells in the thymus was increased to +20% in MZD
and +48% in SZD compared to the ZA controls. Likewise, the proportion of mature T-cells
(CD4‘CD8'ICD4’CD8‘) exhibited +36% to +69% (Figure 1B) and +33% to +82%
accumulation (Figure 1C), respectively, depending on the level of the deficiency. The
immature T-cells (CD4*CD8*), however, showed moderate decline in MZD and a greater
depletion (15%) in SZD group compared to ZA control mice (Figure 1D). This cell loss was
more significant when the absolute number of CD4“CD8+ cells were examined for each
dietary group (Figure 2). As Figure 2 demonstrates, the total number of immature T-cells in
SZD nice dropped significantly to about 14% of ZA control group. Likewise, the immature
population in MZD mice declined to a lesser degree, being 62% of that in ZA mice. When
the absolute number of progenitor and mature T-cell compartments were evaluated, a

moderate decline in each population was noted (data not shown).

215

WWW

‘ The irnmunofiuorescently labeled/DNA stained cultured thymocytes fiom mice in
difi‘erent dietary groups were evaluated for in viva apoptosis. Two color phenotype-gated
DAPI cell cycle analysis was applied to specifically investigate the degree of apoptosis in
double positive thymocytes. As shown in Figure 3, a small percentage of CD4“CD8” subset
(16%) 'm ZA nice appeared in the apoptotic region of DNA histogram (Bottom panel). This
may indicate the normal level of apoptotic death among self-reactive and non-fianctional T-
cells in the thymus (Kisielow et 01., 1988; Murphy et aI., 1990). In the evaluation of ZD
group, it was noted that while moderate levels of deficiency exhibited no significant change
in the levels of apoptosis, severe deficiency in zinc resulted in 23% apoptosis in immature
thymocytes (Figure 3). Progenitor and mature T-lymphocytes in ZD groups also showed
some low degree of apoptosis which was not difi‘erent fi'om that of ZA controls (data not
shown). These results indicated that perhaps most of the apoptotic cells had been eliminated
by macrophages in viva, leaving only a very small portion of apoptotic cells undetected by
phagocytic cells. To overcome this problem, an in vitro culture system simulating ZD-though
short-wasdesigned. Itwasofinterestto determinewhetherinasystemwherelnandCS
are added at the estimated levels observed in zinc dietary mice, and without the presence of

macrophages, apoptosis could be detected.

1! | |° E l | . fl [2! .
Thymic T-cells prepared from regular mice were incubated in difl‘erent culture

conditions ( see the chart in materials and methods) for 8 hrs and were subsequently

216
immunophenotyped against CD4/CD8 T-cell surface markers with simultaneous DNA

staining. As Figure 4 illustrates, total thymic T-cells in control ZA simulated culture
condition (BL CS/BL Zn) demonstrated a low level of apoptosis (16%) similar to the in viva
level detected in ZA mice shown in Figure 3. By contrast, T-lymphocytes incubated in
HI CS/LO Zn culture media (simulated culture condition of a SZD mouse) showed almost
three fold increase in their apoptotic population (45% apoptosis) compared to the control
(BL CS/BL Zn). Furthermore, among difl‘erent thymic T-cell subpopulations, immature
thymocytes showed the most sensitivity, comprising almost 80% of the total apoptotic events
in mouse thymic T-cells (Figure 4). Moreover, addition of high concentrations of zinc (500
nM), which is known to inhibit apoptosis, to the SZD simulated culture media (HI CS/HI Zn)
prior to incubation, suppressed apoptosis close to the background levels observed in control
ZA sirmlated culture condition (Figure 4). It was also important to investigate how absence
of zinc would affect T-cells survival. As it can be seen fiom Figure 4, short term incubation
of cells in culture condition deficient in zinc (BL CS/UN Zn) showed significant apoptosis (~2
fold increase) in total T—cells fiom which 80% were immature thymocytes. Thus, this
approach could demonstrate the significant increase in apoptosis in immature thymocytes
treated in conditions in which CS and Zn levels closely resembled the in viva levels in SZD

mice.

217

Discussion

Earlier chapters (2 and 3) demonstrated that ZD exerts deleterious efl‘ects on B-cell genesis,
mainly on early precursor and immature B-cells with no substantial effects on early progenitor
or mature B4ymphocytes. Furthermore, it was shown (Chapter 4) that chronic elevation of
GCs in ZD played a major role in thymic atrophy and alteration in B-cell development as
elimination of this hormone via adrenalectomy protected thymic atrophy and lymphopenia and
resulted in normal distribution of B-cell subcompartrnents in adrenalectomized ZD mice. The
study presented herein extended our knowledge on lymphopoietic processes in ZD by
providing the first detailed study on the status of T-cell development in zinc deficient mice.
Furthermore it showed that apoptosis was a possible mechanism by which lymphocytes
susceptible to suboptimal zinc intake and elevated GC were eliminated.

The availability of a panel of mAb directed to T-cell associated surface antigens of
mice (CD4 and CD8) as well as DNA fluorochrome (DAPI) in conjunction with
multiparameter FACS analysis allowed for the analysis of thymic T-cells carrying these
antigens and their cell cycle status for detection of apoptotic population. The first objective
of this study which was to evaluate the phenotypic distribution of thymic T—cells in mice in
a zinc dietary study, clearly demonstrated that T-cell maturation was adversely affected by
ZD similar to the observed alteration in B-lymphopoiesis (Chapters 2 and 3). The highest
susceptibility among different thymic T-cell subpopulations was observed in DP immature
(CD4*CD8‘) thymocytes, in particular in the SZD group. This is analogous to the alteration

in B-cell development where precursor B-cells exhibited the most sensitivity and losses in zinc

218

deprived mice (Chapter 3). Interestingly, both of these populations are heavily involved in
Ig or TCR gene rearrangements which require many enzymatic and molecular activities most
of which are Zn dependent (Vallee and Auld 1990; 1993; Coleman, 1992; Li et 01., 1993;
Osmond et al, 1994; W at at, 1994). Sensitivity of the immature population was even
more magnified when their absolute number was evaluated. This analysis demonstrated that
as the level of ZD intensified it created greater losses in immature thymocyte population
where in SZD almost 85% of the DP population were lost. By contrast, the proportion of
progenitor and mature T-cells showed significant accumulation, specifically in SZD mice,
suggesting the less sensitivity or to some degree the resistance of these populations to zinc
deprivation. As was shown in earlier Chapters (2,3) progenitor and mature IgD” B-cells also
demonstrated more resistance to the effects of ZD. These are the populations that their
antigen receptor genes (Iy'l‘ CR) are either in a germline configuration or have been
niecesafiilly rearranged (Hardyetal., 1991; Li et 01., 1993; Godfrey et aI., 1994; Pawlowski
and Staterz, 1994), thereby, are not actively involved in enzymatic and molecular activities.
These cells might be, thus, less susceptible to nutritional deficiency and subsequent cell loss.

The second objective of this study was to investigate the presence of apoptosis in ZD
mice as a mean of elimination of lymphocytes susceptible to zinc deprivation. As was
described earlier in this chapter, this investigation was examined via two approaches. In the
first approach (in viva), in which thymic T-cells fi'om dietary mice were shortly incubated in
regular media, moderate levels of apoptosis, specifically in immature thymocytes of SZD
mice (23%) was noted. MZD mice, however, did not show a detectable change in their

apoptotic T-cells compared to the control ZA mice. With regard to the significant depletion

219
in immature thymocytes of ZD mice, it was clear that this approach was only capturing a

small portion of apoptotic cells that had survived the in viva elimination by macrophages.
Thus apparently the rapid clearance of apoptotic cells by macrophages in viva was still a
barrier in the detection of actual apoptotic events in immature thymocytes during ZD. It is
well documented that apoptotic cells are rapidly recognized and cleared by macrophages
through the expression of specific surface changes (eg., phosphatidyl serine expression )
(Fadok eral., 1992a; 1992b; 1993) before their membrane integrity is lost (Ran et aI., 1995).

To overcome this problem the second approach including the in vitro culture system
was investigated. As shown in Figure 4, short time incubation of normal T-lymphocytes in
media supplemented with basal levels onn and CS was accompanied with a low level (16%)
ofapoptotic T-cells in dis culture condition This level reflects the normal ongoing apoptotic
events in thymocytes that will not survive during thymic education (positive and negative
selections) (Hedriclt and Eidelman, 1993; Tough and Sprent, 1994). However, as expected,
a significara number of T—cells, in particular DP population, died apoptotically when Zn and
CSinculturewere adjusted tothelevels ofa SZD mouse. Infact, anearly studybyElmes
described the presence of apoptotic bodies in the small intestine of severely deficient rat
(Elmes, 1977). This phenomenon was, however, explained to be due to decreased DNA
synthesis. Furthermore, addition of high levels of Zn (500 nM) to the system suppressed
apoptosis to the background levels observed in the control culture condition. This is in
agreementwiththe literature in which zinc at high levels (500-1000 uM) has been shown to
act as an apoptotic inhibitor of GC-iriduced apoptosis (Cohen and Duke, 1984). It has been

suggested that the inhibitory action of zinc at high levels on GC-induced apoptosis might

220
occur at any steps during GC binding to its cytoplasmic GR, GR transformation or

translocation in target cells (Fraker and Telford, 1995). When the deficiency of Zn by itself
inthepresenceofbasal levels ofCS was examined (BL CS/UNZn), a significant increase in
apoptosis, specifically in DP population was observed. Similarly, culture media deprived of
zinc also resulted in apoptotic death in certain lymphoid and myeloid cell lines, indicating the
induction ofapoptosis by zinc deprivation (Martin et al., 1991). Thus it appears that zinc at
suboptimal levels can by itself eliminate susceptible cells by triggering apoptosis (T elford and
Fraker, 1995).

The pattern of alteration in T-cell maturation and the detection of apoptosis both in
viva and in vitro in immature thymocytes, which are the core of sensitivity in the thymus of
ZD mice, are all indications of a synergistic actions of zinc and GC in down sizing of the
immune system. As mentioned in earlier chapters, the crucial role of zinc for cell growth,
development, anddifi‘ererrtiationaswellasanessential component ofmorethan200enzymes
has been well documented (Vallee and Auld, 1993; Walsh etal., 1994; Cousins, 1996).
Considering these facts, one would expect that cells that are more metabolically or
enzymatically active would be in more need for this nutritional element. Thus in situations
where zinc is in suboptimal levels, cells such as immature thymocytes that are actively
involved in TCR gene rearrangement and require zinc for their zinc-dependent biochemical
activities would be sensitive to this deficiency and are programmed to die. On the contrary,
cells of progenitor or mature T-cells that are either prior to gene rearrangement (TCR) or

have successfully rearranged their gene tolerate this deficiency and are less prone to die.

221
In addition, chronic elevation of GCs in later stages of ZD and its induction of

apoptoa'sspeciallyinimmaturelymphocytesis anotherimportant phenomenonthatmustbe
considered in apoptotic depletion of sensitive lymphocytes in zinc deprivation. GC-induced
apoptosis of lymphoid cells, in particular the immature lymphocytes of the thymus gland, is
perhaps the most widely studied model of programmed cell death (Compton and Cidlowski,
1992; Cohen, 1992). Furthermore, in vitro exposure of lymphocytes to low levels of synthetic
or naturallyoccuningGCs resultedinaccunuilationofimmatureTandearlyB cellsin
apoptotic region of the cell cycle (Telford et aI., 1991; Garvy et aI., 1991; 1993a; 1993b;
Voetberg et al., 1994). These observations are strong indications of the suppressive effects
of GCs an immature lymphocytes via induction of apoptosis.

Another consideration in observed alteration in T-cell development and its correlation
to the apoptotic elnnination of immature thymocytes is the expression of protooncogenes such
as Bel-2 and its related gene Bel-x oncogene. The protooncogene Bcl-2 (apoptosis-
suppressing gene) was the first gene studied in the context of program cell death (PCD)
regulation (Reed, 1994; Korsmeyer, 1995). Over expression of Bcl-2 in DP thymocytes of
transgenic mice enhances the resistance of these cells to y-radiation and GC-induced PCD
(Sentman etal, 1991; Strasser et al., 1994). Elimination of Bcl-2 by gene targeting in mice
results in progressive apoptosis of B and T lymphocytes beginning at 3 to 4 weeks of age
(Nakayams et 01., 1993; Veis etal, 1993). Based on these experiments it appears that Bel-2
regulates the survival of lymphocytes in response to a variety of stimuli that induce PCD.

As in the case of B—cell development explained in Chapter 3, the pattern of Bcl-2

expression in T-lymphocytes closely resembles the stage specific sensitivity of T—cells to

222

ZD/GC-induced apoptosis. Murine Bel-2 is expressed in the developmentally early CD4'CD8'
progern'tor T-cdls, but diminishes as T-cell difi‘erentiation progresses into the CD4“ CD8* DP
stage such that little or no Bel-2 is detected in immature thymocytes (Veis et 01., 1993) - the
stage at which tersncn-seir screening and negative selection occurs. This is followed by
upregulation of Bel-2 in mature T- cells. Making the T -cell development scenario potentially
more complex is the recent obwation that expression of apoptotic-promoting Bcl-x, peaks
during the immature stage (Boise et 01., 1993), and it diminishes as cells progress to
CD4"CD8'/CD4‘CD8* mature T-cells. The stage specific upregulation or downregulation
of Bcl-2 and its related gene Bel-x, in thymocytes overlaps the stage specific resistance or
susceptibility of thymocytes to the effects of ZD. Thus it could be suggested that in addition
to deficiency of zinc and chronic elevation of CS as the main core underlying apoptotic
elimination of immature thymocytes in ZD, the presence of other factors such as expression
or absence of some oncogenes in the target cells could also contribute to the overall fate of
the cells.

Collectively, it may be possible to suggest that at early stages of ZD prior to the
elevation of GCs there might be some degree of apoptotic death among cells in need for this
raitrient, largely due to deficiency in zinc. However, as ZD progresses, specifically in severe
zinc deprivation, it activates stress axis and subsequent release of GC hormones that remains
chronically elevated through the later stages of deficiency (DePasquale-Jardieu and Fraker,
1979; 1980; Fraker et aI., 1995). At this point GCs will possibly take over the situation by
triggering cells that are susceptible to ZD (in this case immature thymocytes) to die

apoptotically. This would limit the pool of zinc mostly available to those tissues that their

223
function is vital to the survival of the system. In fact high levels of GC hormones has been

shown to redistribute zinc in the body (demonstrated in Chapter 3) and possibly increases zinc
uptake by cellsthat are in need forthis essential nutrient (Henkin, 1974). Thus,it appears that
mture has set a series of events to protect vital tissues fi'om adverse effects of this nutritional
deficiericyandinairethesurvivalbydownsizingthe immune systemvia increased GC release

and subsequent induction of apoptosis in susceptible cell populations.

224

m Body and thynnisweights, thymus cellularity and plasma CS levels ofmice after
27 days on zinc dietary study‘.

noun Groups ”In“

 

 

 

Initial body weight” (g) 17.2 i 0.7 17.0 i 0.5 17.0 i 0.5
Final body weight‘ (g) 20.3 s: 1.4 _ 15.8 3: 0.3’ 14.4 i 0.6’

‘
Thymus weight (mg) 25.1 i 1.1 16.2 i 0.9‘ 8.4 i 1.9‘
Thymus cellularity (x107) 5.1 i 1.7 3.0 i 1.1‘ 0.85 i 05‘
Plasma cs (pg/d1) f f 17.6 i 50 , f 414 i583 59.8 i 2.4‘

 

a = Data represent two separate diet studies.

b = Mean :1; SD of 18 mice on ZA and 24 mice on ZD diet on day Q ofdiet study.
c = Mean :1; SD of6 to 7 mice in each dietary group assayed on day 27.

* = p < 0.05 or greater as compared to ZA mice.

225

.8... ve a aoeeoc <N ace usage anaemia 8.82 a see... an
3939... 9.3 n=9.—82? deacw 4933.. :03 E 025 w 3 30 Om Hg»: v9.8.9.8
on sea coeecaa as use See can 5.5% 83:? 2e a 852.3 232::
$8.38 A8 c5 .e=co..y ease S8 .38 g 38.9 seas £8.38 8.
£89 cocaomoa £8.58 a: co aoeaem «353 eggs so: .8583
as, names cocoa «no e5 30 a as: see 323 seeoceocoéoceaé
so; 8? 333 see acacia»: case 8335 cacao a... a be. a8.»

an ce. .3: {N a 8522.5 omega consume caeoecfi 23o Sufism

O
N

<

(OI‘CDDV'C'JN

CO
H

N a)
H

_aaonaa z

_QGD_i7(IO %

 

H

 

O

MZD SZD

ZA

MZD SZD

ZA

226

100

ODCDNCOIOfi'GJNr-‘O

O
Q

 

O O
CO V'

+8CIO+i7CIO z

 

+8CIDJ7CIC) z

MZD SZD

ZA

MZD SZD

ZA

227

Elam}; Absolute number (total numbers) of immature thymocytes in the thymus of ZA,
MZD, and SZD mice. The total number ofimrnature CD4*CD8+ T-cells in each
dietary group was determined as: % CD4*CD8+ thymocytes in each mouse
multiplied by the total number of T-cells in that corresponding mouse. Data are
mean i SD of 6 to 8 mice in each dietary groups and represent two separate
zinc dietary study. " denotes significantly different at P< 0.05 as compared to

ZA mice.

228

 

 

 

 

 

 

 

 

 

 

 

 

6

* h
\
Till]
‘ 11‘ ‘ ‘JJ ‘ ‘1‘ 1 ‘1‘ ‘ d 4 A. ‘ ‘ ~ ‘ fl ‘ C d 11 ‘ ‘ q
5 4 3 2 1.. 0

pots +80 +30
«0 confidz 330m“:

MZD SZD

ZA

229

«case some cannon
93 «c 26839: 2a 23 3:on become :08 5 3:: w 3 o .«o Gm u... 508 2:
a cases a. see .aoem cum as am: {Nee one :8 22o cone canoes
e< 22%—cog: 05 5 393255 cacao? mo caged—38:08 2: no @295 .23 .82—
93 05 E 83 05 mo max—«5 Banana 0.96 :3 05 «:80? Joe!— Eeuem case»
See use a a 858a... $8.38 .5 ceases a ceases ac eoecoeoa
05 36% .2...— aeh ease—.88“. 83 anew >386 :08 5 naive—FE“ 033.3%
mo cow—cache 05 tacos—camps» v5 0% <75 .23 3&8» v5 comboeoanoeafig
one a8 6373.2 5 agcgé can... no couscous a: c .32

.R .3. a seam em as .32 {N ace 88.3 8.8%.: ceases a cacao?

230

QNm

 

 

 

 

eeoeeoo <75

 

 

 

DMd

 

 

 

 

 

 

mNm QNE «N

o

-m

10H

. I“: I“. .2
lml tom
H ®5HG> Gwmz Ill smm
o2: 33365, .8
.mm

 

ouv

 

+8CID+i7CID U! 0V z

00.

JequmN 119;)

231

W Apoptosis in thymic T-cells in in vitro culture system. Thymic T-lymphocytes
fi'om regular mice were incubated in RPMI-1640 culture media with 20% CS‘
Zn' FBS supplemented with various CS and Zn concentrations for the period of
8 hrs. Cells were then immunophenotyped and stained with DNA dye and were
subsequently analyzed by flow cytometry as described in the text. Basal
CS/basal Zn (BL CS/BL Zn) (control) represents CS at 2 ug/dl and Zn at 100
ug/dl; high CS/Low Zn (HI CS/LO Zn) represents CS at 6 pg/dl and Zn at 50
rig/d1; high CS/high Zn (HI CS/HI Zn) indicates CS at 6 rig/d1 and Zn at 500
M (14.3 mg/dl); and basal CS/undetectable Zn (BL CS/UN Zn) indicates CS
at 2 ug/dl with no addition of zinc to the culture. Data are expressed as mean
3; SD of quadruplicates of each treatment and representative of three separate
experiments. " indicates significantly different (P< 0.05) fi'om total thymocytes
in control group (BL CS/BL Zn). ” Denote significantly difi‘erent (P< 0.05)

compared to CD4"CD8+ thymocytes in the control group.

232

 

60
55-4 Total Thymic T—cells

504 [Z] CD4+CD8+,,
45 .
40 -
35 ~
30 4

 

*-
*

 

 

25-1
20~
15-
101

% Apoptosis

 

 

 

 

 

////////////////// k1
t \\\\\\\\e.—.

 

 

 

 

 

 

7//////~
\5

 

 

 

 

 

 

 

 

BL CS
UN Zn

BL CS
BL Zn

Hm

a a m
Q4.
\\\\\ 3

SE
5‘8
N0

233

Summary and Suggestions

The world wide prevalence of zinc deficiency with deleterious effects on the immune system
demanded investigation on the specific efi‘ects of this deficiency on lymphocyte
subpopulations. The occurrence of lymphopenia in zinc deficiency prompted the question of
whether the deficiencies in zinc interfered with lymphopoietic processes. Furthermore, it was
important to determine if elevation of glucocorticoids associated with ZD was playing a role
in the alteration of lymphopoiesis, as immunosuppressive effects of GCs are well established.
Finally, the possibility of apoptotic death as a mean for elimination of vulnerable lymphocytes
in ZD was investigated.

The results presented in this dissertation clearly demonstrate for the first time that
B-cell development is adversely affected by ZD. Examination of the marrow of ZD mice
revealed that small nucleated BM cells representing the lymphocyte population in the marrow
were significantly diminished, whereas a significant accumulation in the myeloid lineage was
noted. Precursor B-cells were the most severely affected population, followed by a significant
depression in the proportion of immature B-cells in the BM of zinc deprived mice. By
contrast, progenitor B-cells which are the earliest identifiable committed cells in the B-lineage
exhibited substantial resistance to the deficiency. Likewise, the mature B-cell subpopulation
demonstrated much less sensitivity, being less affected by the deficiency. In spite of
significant depletion in some B-cell subcompartments, the marrow cellularity was unafi‘ected,

showing the analogous number of BM nucleated cells in all dietary groups. This finding

234
indicated the resistance of other lineages (eg., myeloid) in the marrow of ZD mice as was
identified earlier.

Evaluation of the efi‘ects of ZD on T-cell maturation in the thymus revealed alteration
in T-lymphocyte subsets in a pattern analogous to the B-lymphocyte subpopulations in the
marrow. That is, the progenitor and mature T-cells showed substantial resistance to the
effects of ZD, whereas immature thymocytes were depleted significantly. Together, these
observations clearly indicated a selective and stage specific alterations in B-lymphopoiesis as
well as in T-cell maturation in the course of ZD.

In investigation of the zinc content of several lymphoid and non-lymphoid tissues, a
sigriificantdepletioninthebonezincwithno changeinthezincconcentrationintheBMand
liver cells was noted. This pattern suggested a possible mobilization of n'nc fi'om bone to
other tissues such as BM and liver as has been also indicated by other investigators.
However, dietary studies terminated at different time points in which the zinc content of
larger lumber of tissues are evaluated would better identify the kinetics of zinc depletion and
the possibility of zinc redistribution in the course of the deficiency. Furthermore, the
assessment of the zinc content of sorted populations (eg., B220‘Ig', B220”Ig*, CD4*CD8*)
along with the determination of their phenotypic distribution at various time points in the
course of dietary study would be able to better correlate the intracellular zinc levels to the
survivability of each population. However, in order to specifically identify the role of
suboptimal zinc levels by itself, the use of adrenalectomized mice in which there is no

interference of GC hormones is suggested.

235

Alterationinlymphopoieaisalongwiththeuncharigedzinclevels in the marrow onD
mice also suggest the contribution of other factors secondary to ZD. As it is documented,
progression of ZD activates the stress axis and results in chronic release of glucocorticoids
which remain elevated throughout the deficiency. Due to the immunosuppressive efi‘ects of
GCs on lymphocytes, particularly an immature populations, their role in the alteration of
lymphopoiesis during ZD was speculated. In this regard, removal of CS via adrenalectomy
clearly showed that ZD adrenalectomized mice exhibited complete thymic protection and
normal phenotypic distribution in early developing, immature and mature B-cell
subpopulations compared to ZD sham control mice. This data identified a significant role for
CS in the suppression of lymphopoiesis in fine deprived mice. Furthermore, the accumulation
of glucocorticoid resistance cells of the myeloid lineage in the marrow of ZD mice
emphasized the role of GC as one possible mechanism in the suppression of vulnerable
lymphocytes. Nevertheless, other possibilities such as alteration in the rate of lymphopoiesis
and cell cycling can not be ruled out. The necessity of zinc for cell division and proliferation
along with the presence of elevated GC during ZD, increases the possibility of reduced rates
of lymphopoiesis and cycling in early developing B-lymphocytes. Using the FACS
methodology developed in this laboratory along with metaphase arrest techniques (eg., use
of vincristine to block cells at GM phase) in a time point diet study would identify the
contribution of these parameters to the observed lymphopenia in zinc deprived mice.
Furthermore, interrelating these parameters to the plasma zinc and CS levels, as well as zinc

content of lymphoid tissues would provide a more detailed picture of how gradual changes

236
in the (zinc and CS levels in the course of ZD would play a role iii lymphopoietic processes
and determine the eventual fate of lymphocytes.

Over expression or down regulation of some of the oncogenes particularly Bel-2 and
its family members (eg., Bel-x, A1, Bax, Bak, etc.) at difi‘erent stages of B and T—cell
development could also determine cell survival or cell death to maintain the potentially
fiinctional cells and also homeostasis of the system . As a clear example, Bel-2, an anti-
apoptotic oncogene has been shown to be expressed in progenitor and mature B-cells and
down regulated in precursor and immature B-cells. The pattern of on and off expression of
dis oncogene in these lymphocytes overlaps with the resistance or the sensitivity of different
B-cell subpopulations to the efl'ects of ZD, namely GCs. Thus, the expression or down
regulafionofoncoguiescmddraideracdleiflia'sensifiveorresistance to deficienciesinzinc
or its secondary outcomes (eg., GC hormones).

Along with these factors, there are other possibilities that might contribute to the
selective sensitivity of lymphocytes in zinc deprivation, one of which is the BM
microenvironment in which lymphopoiesis occurs. Stromal cells, as one of the key
components of this microenvironment, provide a substantial support for lymphohemopoietic
processes. This is achieved via production of different cytokines and expression of various
cell adhesion molecules that are involved in growth, maturation and differentiation of difi‘erent
cell lineages. It is possible that in the course of zinc deficiency the organization of this
microenvironment is altered in such a way that cells that are greatly dependent on cell
contacts or specific cytokines for their survival would somehow lose these supportive

elements and, therefore, become eliminated in the course of ZD. Our insuficient knowledge

237

about the status of the microenvironment of the BM or thymus during zinc deprivation,
demands arterisive investigations which will likely reveal some explanations for the observed
immunological defects due to this nutritional deficiency.

The results from the in viva verification of apoptosis in ZD mice showed a moderate
level of apoptosis in immature thymocytes detected by flow cytometric cell cycle analysis.
However, due to rapid elimination of apoptotic cells by macrophages in viva, it was
speculated that the data was not a true representative of apoptotic events occurring in viva.
Therefore, an in vitro culture system with supplementation of different levels of zinc and CS
analogous to their levels in dietary zinc mice was established. Data fiom these experiments
clearly showed that in culture conditions where zinc was as low and CS was as high as their
levels in ZD mice, a significant accumulation in apoptotic thymocytes, particularly, in
immature thymocytes were evident. Furthermore, addition of high amounts of zinc to this
culture condition substantially inhibited apoptosis, maintaining it at basal levels detected in
the control media. These data suggested that apoptosis apparently occurs in ZD mice and
might be a mechanism for the elimination of susceptible lymphocytes in ZD, thus creating
lymphopenia Nevertheless, more thorough investigations employing time point diet studies
would greatly enhance the possibility of capturing apoptotic lymphocytes in viva before their
rapid clearance by macrophages.

In conclusion, the work described here has shed considerable light on the status of
lymphopoietic processes in zinc deprived mice and has provided some insights into the
possible roles of glucocorticoids and apoptosis in the observed lymphopenia. Although,

future studies examining the role of the afi‘ormentioned factors in the impaired immunity in

238
zinc deficiency are highly required, the importance of zinc in the integrity of immune system
and the its deleterious efi‘ects on immune components were substantially provided by this
project. More importantly, this work has laid a foundation for further investigations which
are likely to provide considerable insight into the treatment of human subjects, particularly
children, that are either malnourished due to improper diet or are sufi‘ering from difi‘erent

diseases that would lead them to malnourished state.

LITERATURE CITED

Abbasi, AA, Prasad, AS., Rabbani, P., et a]. (1980) Experimental zinc deficiency in man:
efi‘ect of testicular function. J. Lab. Clin. Med. 96, 544-550.

Abdulwajid, AW, and Wu. F.Y-H. (1986) Chemical modification of Escherichia 0011' RNA
polymerase by diethyl pyrocarbonate: evidence of histidine requirement for enzyme activity
and intrinsic zinc binding. Biochemistry 25, 8167-8172.

Abe, S., Matsumi, M., Tsukioki, M., et a1. (1987) Metallothionein and zinc metabolism in
endotoxin shock rats. Exs. 52, 587-594.

Adcock, IM, Lane, S.J., Brown, C.R. er a1. (1995) Differences in binding of glucocorticoid
receptor to DNA in steroid-resistant asthma. J. Immunol. 154, 3500-3505.

Aggett, P.J., Delves, H.T., Thorn, I.H., er a1. (1981) The therapeutic efi‘ect of amphotericin
in Actadernratitis enterapathica. Eur. J. Pediatr. 137, 23-25.

Aggett, P.J., and Comerford, JG. (1995) Zinc and human health. Nutr. Rev. 53, 516-522.

Aggett, PI, and Harries, IR. (1979) The current status of zinc in health and disease states.
Arch. Dis. Child. 54, 909-917.

Aggett, PI. (1989) Severe zinc deficiency. In C.F. Mills (ed), Human Nutrition Reviews.
Zinc in Human Biology, Springer-Veflag, London, pp 259-279.

Allman, D.M., Ferguson, SE, and Cancro, MP. (1992) Peripheral B cell maturation. I.
Irmnature peripheral B cells in adults are heat-stable antigen" and exhibit unique signaling
characteristics. J. Immunol. 149, 2533-2540.

Allman, D.M., Ferguson, S.E., Lentz, V.M., and Cancro, MP. (1993) Peripheral B cell
maturation. II. Heat-stable antigen“ splenic B cells are an immature developmental
intermediate in the production of long-lived marrow-derived B cells. J. Immunol. 151,
4431-4444.

Alroy, 1., and Freedman, LP. (1992) DNA binding analysis of glucocorticoid receptor
specificity mutants. Nucl. Acids Res. 20, 1045-1052.

239

240

American Institute of Nutrition (1993) AIN-93 purified diets for laboratory rodents: Final
Report of the American Institute of Nutrition Ad Hoc Writing Committee on the
Reforrinrlation of the AIN-76A Rodent Diet. J. Nutr. 123, 1939.

Anderson, G., Jerikinson, E.J., Moore, NC, and Owen, J.J.T. (1993) MHC class II+ thymic
epithelium and mesenchyme cells are both required for T-cell development in the thymus.
Nature 362, 70-73.

Anderson, G., Owen, J.J.T., Moore, NC, and Jenkinson, E.J. (1994) Thymic epithelial cells
provide unique signals for positive selection of CD4*CD8” thymocytes in vitro. J. Exp. Med.
179, 2027-2031.

Archer, T.K., Hager, G.L., and Omichinski, J.G. (1990) Sequence-specific DNA binding by
glucocorticoid receptor “zinc finger peptides”. Proc. Natl. Acad. USA 87, 7560-7564.

Atherton, D.J., Muller, D.P.R, Aggett, P.J., and Harries, J .T. (1979) A defect in zinc uptake
by jejunal biopsies in Acradennatiris enterapathica. Clin. Sci. 56, 505-507.

Auld, D.S., Kawaguchi, H., Liningston, D.M., and Vallee, BL. (1975) Zinc reverse
transcriptase from mammalian RNA type C viruses. Biochem. Biophys. Res. Commun. 62,
296-302.

Auphan, N., DiDonato, J.A, Rosette, C., er a1. (1995) Immunosuppression by
glucocorticoids: inhibition of NF-itB activity through induction of I-KB synthesis. Science
270, 286-290.

Bach, J.F., Dardenne, M., Pleau, J .M., and Bach, AM. (1975) Isolation biochemical
chmacteristics, and biological activity of a circulating thymic hormone in the mouse and in the
human. Ann. NY. Acad. Sci. 249, 186-210.

Ballard, P.L., Ballard, RA, Gonzales, I.K., et al. (1984) Corticosteroid binding by fetal rat
and rabbit lung in organ culture. J. Steroid Biochem. 21, 117-126.

Ballard, PL. (1979) Delivery and transport of glucocorticoids to target cells. In 1D. Baxter,
and G.G. Rousseau (eds), Glucacarticaid hormone receptors, Springer-Verlag, Berlin and

New York, pp 25-48.

Ballester, O.F., and Prasad, AS. (1983) Energy, zinc deficiency, and decreased nucleoside
phosphorylase activity in patients with sickle cell anemia. Ann. Intern. Med. 98, 180-182.

Bands, N..,K Bernier, J., Kwahara, D.K. er a1. (1992) Crosslinking CD4 by human
iirlrainodeficiency virus gp 120 primes T cells for activation induced apoptosis. J. Exp. Med.
176,1099-1106.

241

Barone, KS, O’Brien, PCM, and Stevenson, JR (1993) Characterization and mechanisms
of thynn'c atrophy in protein malnourished mice: role of corticosterone. Cell. Immunol. 148,
226-233.

Barry, M., Keeling, P. W. N., and Feely, J. (1990) Tissue zinc status and drug elimination in
patients with chronic liver disease. Clin. Sci. 78, 547-549.

Bateman, A, Singh, A, Kral, T., and Solomon, S. (1989) The immune hypothalamic-
pituitary-adrenal axis. Endocr. Rev. 10, 92-112.

Baum, M., and Quigley, R (1991) Parental glucocorticoids stimulate neonatal
juxtamedullary prominal convoluted tubule acidification. Am. J. Physiol. 261, F746-F752.

Baxter, J.D., and Tyrrell, J.B. (1987) The adrenal cortex. In P. Felig, J.D. Baxter, AE.
Broadus, and L.A.Frohman (eds), Endocrinology andMetabalism, 2nd ed, McGraw-Hill,
New York, pp 511-650.

Baxter, J .D., and Rousseau, CG. (1979) Glucocorticoid hormone action: An overview. In
J .D. Baxter, C.G. Rousseau (eds), GlucocorticaidHarmone Action, Springer-Vedag, Berlin,
Heidelberg, and New York, pp 1-24.

Beato, M. (1989) Gene regulation by steroid hormones. Cell 56, 335-344.

Beaumont, K, and Fanestil, DD. (1983) Characterization of rat brain aldosterone receptors
reveals high amnity for corticosterone. Endocrinology 113, 2043-2051.

Becker, DJ. (1983) The endocrine responses to protein calorie malnutrition. Ann. Rev. Nutr.
3, 187-212.

Berczi, I. (1994) Hormonal interactions between the pituitary and immune systems. In C.
J. Grossman (ed), Bilateral Communication Between the Endocrine and Immune system,
Endocrinology and Metabolism: Progress in Research and Clinical Practice, Springer-Veflag,
New York, pp 96-144.

Berg, 1M, and Shi, Y. (1996) The galvanization of biology: a growing appreciation for the
roles ofa'nc. Science 271, 1081-1085.

Bettger, W.J., and O’Dell, B.L. (1981) A critical physiological role of zinc in the structure
and function of biomembranes. Life Sciences 28, 1425-1438.

Bianchi, F., Rousseaux-Prevost, R., Sautiere, P., and Rousseaux, J. (1992) P2 protarnines
from human sperm are zinc-finger proteins with one CYSIIHIS2 motif. Biochem. Biophys.
Res. Comm. 182, 540-547.

242

Billingsley, G.D., Walter, M.A, Hammond, G.L., and Cox, D.W. (1993) Physical mapping

of four serpin genes: alpha-antitrypsin, alpha-antichymotrysin, corticosteroid-binding globulin,
and protein C inhibitor, within a 280-kb region on chromosome 14 q 32.1. Am. J. Hum.
Genet. 52, 343-353.

Birkerimeier, ER and Gordon, J .I. (1986) Developmental regulation of a gene that encodes
a cysteine-rich intestinal protein and maps near the murine immunoglobulin heavy chain locus.
Proc. Natl. Acad. Sci. USA 83, 2516-2520.

Bissonnette, RP. ,Echeverii, F. ,Mahboubi, A, and Green, D. R (1992) Apoptotitic cell death
induced by c-myc is inhibited by bcl-2. Nature 359, 552-554.

Bogden, JD. (1995) Studies on micronutrient supplements and immunity in older people.
Nutr. Rev. 53, 859-865.

Bogden, J .D., Bendich, A, Kemp, F.W., et al (1994) Daily micronutrient supplements
enhance delayed-hypersensitivity skin test responses in older people. Am. J. Clin. Nutr. 60,
437-447.

Boise, L.H., Gonzalez-Garcia, M., Postema, C.E., et al. (1993) Bel-x, a bcl-2-related gene
that functions as a dominant regulator of apoptotic cell death. Cell 74, 597-608.

Bolander, RF. (1994) Molecular Endocrinology. Academic Press, 2nd edition.

Bonomini, M., Manfiini, V., Cappelli, P., and Albertazzi, A. (1993) Zinc and cell-mediated
immunity in chronic uremia. Nephron 65, 1-4.

Bonomini, M., Palnn’eri, P.F., Evangelista, M., etal (1991) Zinc-mediated lymphocyte energy
charge modification in dialysis patients. ASAIO Trans. 37, 387-389.

Borg, L.A., Cagliero, E., Sandler, S., et al. (1992) Interleukin-1|} increases the activity of
superoxide dismutase in rat pancreatic islets. Endocrinology 130, 2851-2857.

Boukaiba, N., Flarnent, C., Archers, S., et al. (1993) A physiological amount of zinc

Implementation: effects on nutritional, lipid, and thymic status in an elderly population. Am.
J. Clin. Nutr. 57, 566-572.

Bovering, K.E., and Dean, RT. (1992) Restriction of the participation of copper in radical-
generating systems by zinc. Free Radical Res. Commun. 14, 217-225.

Bray. TM. and Bettger, W. J. (1990) The physiological role of zinc as an antioxidant. Free
Rad. Biol. Med. 8, 281-291.

243

Bremner, I. (1987) Interactions between metallothionein and trace elements. Prog. Food
Nutr. Sci. 11, 1-37.

Bremner, I., and Beattie, J.H. (1990) Metallothionein and the trace minerals. Annu. Rev.
Nutr. 10, 63-83.

Bremner, 1., Morrison, J.M., Wood, AM., and Arthur, JR (1987) Effects of changes in
dietary zinc, copper and selenium and of endotoxin administration on metallothionein 1
concentrations in blood cells and urine in the rat. J. Nutr. 117, 1595-1602.

Bremner, I., and May, RM. (1989) Systematic interactions of zinc. In C.F. Mills (ed),
Human Nutrition Reviews. Zinc in Human Biology, Springer-Verlag, London, pp 95-108.

Bresrn'ck, EH, Dalman, EC, and Pratt, W.B. (1990) Direct stoichiometric evidence that the
untransformed Mr 300,000, 98 glucocorticoid receptor is a core unit derived from a larger
heteromeric complex. Biochemistry 29, 520-527.

Brien, T.G. (1981) Human corticosteroid binding globulin. Clin. Endocrinol. 14, 193-212.

Brown, D.G., Sun, X.M., and Cohen, GM. (1993) Dexamethasone-induced apoptosis
involves cleavage of DNA to large fragments prior to intemucleosomal fiagrnentation. J.
Biol. Chem. 268, 3037-3039.

Brown, E.D., Chm, W., and Smith, J.C. Jr. (1978) Bone mineralization during a developing
zinc deficiency. Proc. Soc. Exp. Biol. Med. 157, 211-214.

Buchingliam, J. C., Safieh, B., Singh, S., et a1. (1992) Interaction between the hypothalamo-
pituitary adrenal axis and the thymus in the rat: a role for corticotropin in the control of
thyman release. J. Neuroendocrin. 4, 295-301.

Bunce, GE. (1989) Zinc in endocrine function. In C.F. Mills (ed), Human Nutrition
Reviews. Zinc in Human Bialag, Springer-Variag, London, pp 249-258.

Biirker, V. W., Hinks, L. J., Stansfield, M E., etal. (1987) Metabolic balance studies for zinc
and copper in housebound elderly people and the relationship between zinc balance and
leukocyte zinc concentrations. Am. J. Clin. Nutr. 46, 353-359.

Burnstein, K.L., and Cidlowski, J.A (1989) Regulation of gene expression by
glucocorticoids. Ann. Rev. Physiol. 51, 683-699.

Burr, RG. (1973) Blood Zinc in the spinal patient. J. Clin. Path. 26, 773-775.

Butcher, EC, and Picker, L]. (1996) Lymphocyte homing and homeostasis. Science 272,
60-66.

244

Caelles, C., Helrnberg, A, and Karin, M. (1994) p53-Dependent apoptosis in the absence of
transcriptional activation of p53-target genes. Nature 370, 220-223.

Calvano, S.E., Albert, J.D., Legaspi, A, et al. (1987) Comparison of numerical and

phaiotypic leukcyte changes during constant hydrocortisone infusion in normal humans with
those in thermally injured patients. Surg. Gynecol. Obstet. 164, 509-520.

Calvano, S.E., de—Riesthal, H.F., Marano, M.A, and Antonacci, AC. (1988) The decrease
inperipheralbloodCD4+Tcellsfollowingthermal injuryinhumans canbeaccounted forby
a concomitant decrease in suppressor-inducer CD4+ T cells as assessed using anti-CD45R
Clin. Immunol. Irnmunopathol. 47, 164-173.

Campbell, KS, and Cambier, JG (1990) B-lymphocyte antigen receptors (mIg) are non-
covalently associated with a disulfide-linked, inducibly phosphorylated glycoprotein complex.
EMBO J. 9, 441-448.

Carlomagna, MA, and McMurray, D.N. (1983) Chronic zinc deficiency in rats: its influence
on some parameters of humoral and cell-mediated immunity. Nutr. Res. 3, 69-78.

Carson-Jaiica, M.A, Schrader, WT, and O’Malley, B.W. (1990) Steroid receptor family:
structure and functions. Endocrine Rev. 11, 201-220.

Cavan, KR, Gibson, RS, Grazioso, C.F., et al. (1993) Growth and body composition of
periurbanGuatemalanchildren inrelationto zinc status: across-sectional study. Am. J. Clin.
Nutr. 57, 334-343.

Chandra, RR (1980) Acrademratitis enterapathica. Zinc levels and cell-mediated immunity.
Pediatrics 66, 789-791.

Clutterji, D., and Wu, F.Y. (1982) Selective substitution in vitro of an intrinsic zinc of
Escherichia aali RNA polymerase with various divalent metals. Biochemistry 21, 4651-4656.
Dreosti, I.E., Record, 1R, Manuel, 8.1., and Buckley, RA (1981) High plasma zinc levels
following oral dosing in rats and the incorporation of 3H-thymidine into deoxyribonucleic acid
in rat fetuses. Res. Commun. Chem. Pathol. Pharmacol. 31, 503-513.

Chen, J., Stall, AM., Herzenberg, LA, and Herzenberg, LA (1990) Differences in

glycoprotein complexes associated with IgM and IgD on normal murine B cells potentially
enable transduction of different signals. EMBO J. 9, 2117-2124.

Chesters, J .K., and Quarterman, J. (1970) Efi‘ects of zinc deficiency on food intake and
feeding patterns ofrats. Br. J. Nutr. 24, 1061-1069.

Chesters, J .K., and Boyne, R. (1991) Nature of the Zn2+ requirement for DNA synthesis by
3T3 cells. Exp. Cell. Res. 192, 631-634.

245

Chesters, J.K. (1978) Biochemical functions of zinc in animals. Wld. Rev. Nutr. Diet. 32,
135-164.

Chesters, J.K., Boyne, R, Petrie, L., and Lipson, K.E. (1995) Role of the promoter in the
sensitivity of human thymidine kinase to lack of Zn”. J. Biochem. 308, 659-664.

Chesters, J.K. (1989) Biochemistry of zinc in cell division and tissue growth. In C.F. Mills
(ed), Human Nutrition Reviews. Zinc in Human Biology, Springer-Vedag, London, pp
109-128.

Chesters, J.K., Petrie, L., and Lipson, K.E. (1993) Two zinc-dependent steps during G1 to
S phase transition. J. Cell. Physiol. 155, 445-451.

Chesters, J .K., Petrie, L. and Travis, J. (1990) A requirement for Zn” for the induction of
thymidine kinase but not ornithine decarboxylase in 3T3 cells stimulated from quiescence.
Biochem. J. 272, 525-527.

Chesters, J .K (1972) The role of zinc ions in the transformation of lymphocytes by
phytohaemagglutinin. Biochem. J. 130, 133-139.

Chesters, J .K. (1991) Trace element-gene interactions with particular reference to zinc.
Proc. Nutr. Soc. 50, 123-129.

Chesters, J .K. (1975) Comparison of the efl'ects of zinc deprivation and actinomycin D on
ribonucleic acid synthesis by stimulated lymphocytes. Biochem. J. 150, 211-218.

Chesters, J.K., Petrie, L., and Lipson, K.E. (1993) Two zinc-dependent steps during G1 to
S phase transition. J. Cell. Physiol. 155, 445-451.

Chesters, J.K. (1992) Trace element-gene interactions. Nutr. Rev. 8, 217-223.

Cidlowski, J.A, King, K.L., Evans-Storms, RB., et al. (1996) The biochemistry and
molecular biology of glucocorticoid-induced apoptosis in the immune system. Recent Prog.
Horm. Res. 51, 457-491.

Claman, HN., Moorhead, J .W., and Benner, W.H (1971) Corticosteroids and lymphoid cells
in vitro. 1. Hydrocortisone lysis of human, guinea pig, and mouse thymus cells. J. Lab. Clin.
Med. 78, 499-507.

Clarnan, H. (1972) Corticosteroids and lymphoid cells. New Engl. J. Med. 287, 388-398.
Clegg, M.S., Keen, C.L., and Hurley, LS. (1989) Biochemical pathologies of zinc deficiency.

In C.F. Mills (ed), Human Nutrition Reviews. Zinc in Human Biology, Springer-Verlag,
London, pp 129-145.

246

Coffman, RL., and Weissman, LL. (1981) 3220: A B cell specific marker at the T200
glycoprotein family. Nature 289, 681-683.

Cohen, J.J., and Duke, RC. (1992) Apoptosis and programmed cell death in immunity.
Annu. Rev. Immunol. 10, 267-293.

Cohen, J .J ., and Crnic, LS. (1982) Glucocorticoids, stress, and the immune response. In
DR Webb (ed), Immrmapharmacolagy and the Regulation of Leukocyte Function, Dekker,
New York, pp 61-91.

Cohen, 1.]. (1992) Glucocorticoid-induced apoptosis in the thymus. Sern. Immunol. 4,
363-369.

Cohen, J .J ., and Duke, RC. (1984) Glucocorticoid activation of a calcium-dependent
endonuclease in thymocyte nuclei leads to cell death. J. Immunol. 132, 38-42.

Cohen, H. (1991) Programmed cell death in the immune system. Adv. Immunol. 50, 55-85.

Coleman, J .E. (1992) Zinc proteins: enzymes, storage proteins, transcription factors, and
replication proteins. Annu. Rev. Biochem. 61, 897-946.

Coligan, J.E., Krusibeek, AM., Margulies, D.H., et al. (19918) ACK lysing bufi‘er. In:
Crm'entPratocals in Immunology. National Institutes of Health. Vol. 1, Greene Publishing
Associates and Wiley, Interscience, pp 3.1.5.

Coligan, J.B., Krusibeek, AM., Margulies, D.H., et al. (1991b) Trypan blue exclusion test
of viability. In Current Protocols in Immunology, National Institute of Health, Vol. 1,
Greene Publishing Associates and Wiley, Interscience, pp A3 .3.

Compton, M M., and Cidlowski, J. A (1992) Tliymocyte apoptosis: 8 model of programmed
cell death. Trends. Endocrinol. Metab. 3, 17-23.

Compton, M M, Caron, L-A M, and Cidlowski, J. A (1987) Glucocorticoid action on the
immune system. J. Steroid. Biochem. 27, 201-208.

Consolini, R, Cei, B., Cini, P., et al. (1986) Circulating thymic hormone activity in young
cancer patients. Clin. Exp. Immunol. 66, 173-180.

Cook-Mills, J ., and Fraker, RI. (1993 a) Functional capacity of residual lymphocytes fiom
zinc deficient adult mice. Brit. J. Nutr. 69, 83 5-848.

247

Cook-Mills, J .M, and Fraker, P.J. (1993b) Optimization of peroxide production by resident
macrophages. J. Leukoc. Biol. 53, 205-207.

Cooper, M.D., Mulvariey, D., Coutinho, A, and Cazenave, P.A (1986) A novel cell surface
molecule on early B-lineage cells. Nature 321, 616-618.

Cory, S. (1995) Regulation of lymphocyte survival by the bcl-2 gene family. Annu. Rev.
Immunol. 13, 513-543.

Coto, J.A, Hadden, EM, Sauro, M., et al. (1992) Interleukin 1 regulates secretion of zinc-
thymulin by human thymic epithelial cells and its action on T-lymphocute proliferation and
nuclear protein kinase C. Proc. Natl. Acad. Sci. 89, 7752-7756.

Coudray, C., Boucher, E, Richard, M.J., et al. (1991) Zinc deficiency, ethanol, and
myocardial ischemia affect lipoperoxidation in rats. Biol. Trace Elem. Res. 30, 103-118.

Cousins, RJ. (1985) Toward a molecular understanding of zinc metabolism. Clin. Physiol.
Biochem. 4, 20-30.

Cousins, R]. (1996) Zinc. Present Knowledge in Nutrition, 7th edition, pp 293 -306.
Cousins, RJ. (1989) Theoretical and practical aspects of zinc uptake and absorption. In J.A
Laszlo, and FR Dinta's (eds), Alineral Absorption in the Manogastric GI Tract: Chemical,
Nutritional and Physiological Aspects, Plenum, New York, pp 3-12.

Cousins, RJ., and Lee-Ambrose, L.M. (1992) Nuclear zinc uptake and interactions and
matallothionein gene expression are influenced by dietary zinc in rats. J. Nutr. 122, 56-64.

Cousins, RJ. (1989) Systemic transport of zinc. In C.F. Mills (ed) Human Nutrition Reviews.
Zinc in Human Biology, Springer-Vedag, London, pp 79-93.

Cousins, RJ., (1985) Absorption, transport, and hepatic metabolism of copper and zinc:
special reference to metallothionein and ceruloplasmin. Phys. Rev. 65, 23 8-309.

Cousins, RI. (1983) Metallothionein - aspects related to copper and zinc metabolism. J.
Inherit. Metab. Dis. 1, 15-21.

Cousins, RJ. and Leinart, AS. (1988) Tissue-specific regulation of zinc metabolism and
metallothionein genes by interleukin 1. FASEB J. 2, 2884-2890.

Cupps, TR, and Fauci, AS. (1982) Corticosteroid-mediated irnmunoregulation in man.
Immunological Rev. 65, 133-155.

248

da Silva, J. J. R, and Williams, R J. P. (eds) (1991) The biological chemistry of the elements:
the inorganic chemistry of life, Clarendon Press, Oxford.

Daeaclner, C.W., Carpentieri, U., Goldman, AS., and Haggard, ME. (1985) Zinc deficiency
and blood lymphocyte function with sickle cell disease. Scand. J. Haematol. 35, 186-190.

Dahlman-Wright, K., Wright, A, Carlstedt-Duke, J., and Gustafsson, IA (1992) DNA-
binding by the glucocorticoid receptor: a structural and functional analysis. J. Steroid
Biochem. Mol. Biol. 41, 249-272.

Dahlman-Wright, K., Grandien, K., Nilsson, S., et al. (1993) Protein-protein interactions
between the DNA-binding domains of nuclear receptors influence DNA-binding. J. Steroid
Biochem. Mol. Biol. 45, 239-250.

Daniel W.W. (1987) Biostatistics: A foundation for analysis in the health sciences. Fourth
edition. John Wiley & Sons, Inc., New York.

Dmtzer, R, and Kelley, KW. (1989) Stress and immunity: an integral view of relationship
between the brain and the immune system. Life Sci. 44, 1995-2008.

Dardenne, M, Pleau, J.M., Nabarra, B., et al. (1982) Contribution of zinc and other metals
to the biological activity of serum thymic factor. Proc. Natl. Acad. Sci. 79, 5370-5373.

De, S.K., McMaster, M.T., and Andrews, GK. (1990) Endotoxin induction of murine
metallothionein gene expression. J. Biol. Chem. 265, 15267-15274.

DelRey, A, Besedovsky, H., and Sorkin, E. (1984) Endogenous blood levels of
corticosterone control the immunologic cell mass and B cell activity in mice. J. Immunol.
133, 572-575.

DePasquale-Jardieu, P., and Fraker, P.J. (1979) The role of corticosterone in the loss in
immune function in the zinc-deficient A/J mouse. J. Nutr. 109, 1847-1855.

DePasquale-Jardieu, P., and Fraker, P.J. (1980) Further characterization of the role of
corticosterone in the loss of humoral immunity in zinc-deficient All mice as detennined by
adrenalectomy. J. Immunol. 124, 2650-2655.

DePasquale-Jardieu, P., and F raker, P.J. (1984) Interference in the development of a
secondary irmme response in mice by zinc deprivation: persistence of efi‘ects. J. Nutr. 114,
1762-1769.

Desiderio, S. (1994) The B cell antigen receptor in B-cell developement. Curr. Opin.
Immunol. 6, 248-256.

249

Dexter, T.M., Allen, TD., and Lajtha, LG. (1977) Conditions controlling the proliferation
of haemopoietic stem wlls in vitro. J. Cell Physiol. 91, 335-344.

Dexter, T.M., and Testa, N.G. (1980) In vitro methods in haemopoiesis and lymphopoiesis.
J. Immunol. Methods 38, 177-190.

Dhabhar, F.S., Miller, AH., McEwen, BS, and Spencer, RL. (1995) Efl'ects of stress on
immune cell diaribution Dynamics and hormonal mechanisms. J. Immunol. 154, 5511-5527.

Dhabhar, F.S., Miller, AH, McEwen, BS, and Spencer, RL. (1995) Differential activation
of adrenal steroid receptors in neural and immune tissues of Sprague Dawley, Fischer 344,
and Lewis rats. J. Neuroirnmunol. 56, 77-90.

Dillaha, C. J., Lorincz, A L., and Auvik, O. R (1953) Acrodermatitis enteropathica: review
of the literature and report of a case successfiilly treated with diodoquin. J. Am. Med. Assoc.
152, 509-512.

DiSilvestro, RA, and Carlson, GP. (1994) Effects of mild zinc deficiency, plus or minus
acute phase response, on CC], hepatotoxicity. Free Radic. Biol. Med. 16, 57-61.

Distelhorst, CW. (1989) Recent insight into the structure and function of the glucocorticoid
receptor. J. Lab. Clin. Med. 113, 404-412.

Dorshkind, K. (1990) Regulation of hemopoiesis by bone marrow stromal cells and their
products. Annu. Rev. Immunol. 8, 111-137.

Dorshkind, K, Schoueat, L., Fletcher, W.H. (1985) Morphologic analysis of long-tenn bone
marrow cultures that support B-lymphopoiesis or myelopoiesis. Cell Tissue Res. 23 9,
375-3 82.

Dragone, L.L., Barth, RK., Sitar, K.L., et al. (1995) Disregulation of leukosialin (CD43,
Ly48, sialophorin) expression in the B-cell lineage of transgenic mice increases splenic B-cell
number and survival. Proc. Natl. Acad. Sci. USA 92, 626-630.

Dtmcan, JR, and Dreosti, LB. (1976) A proposed site of action for zinc in DNA synthesis.
J. Comp. Pathol. 86, 81-85.

Dunn, AJ. (1988) Stress-related changes in cerebral catecholarnine and indoleamine
metabolism: lack of efi‘ect of adrenalectomy and corticosterone. J. Neurochem. 51, 406-412.

Dumam, D.M, and Palmiter, RD. (1987) Analysis of the detoxification of heavy metal ions
by mouse metallothionein. Exs. 52, 457-463.

Duvall, E., and Wyllie, A H. (1986) Death and the cell. Immunol. Today 7, 115-119.

250

Ebadi, M., and Iversen, RL. (1994) Mettalotionein in carcinogenesis and cancer
chemotherapy. Gen. Pharmac. 25, 1297-1310.

Ehlich, A, Schaal, 8., Cu, 11., et al. (1993) Immunoglobulin heavy and light chain genes
rearrange independently at early stages of B cell development. Cell 72, 695-704.

Elmea, M (1977) Apoptosis in the small intestine ofzinc-deficient and fasted rats. J. Pathol.
123, 219-223.

Endre,L.,Beck, P., andPrasad, AS. (1990)Theroleofa’ncinhuman health. J. TraceElem.
Exp. Med. 3, 337-375.

Enk, AH, and Katz, 8.1. (1994) Heat-stable antigen is an important costimulatory molecule
on epidermal Langerhans cells. J. Immunol. 152, 3264-3270.

Etzel, KR, Swerdel, MR, Swerdel, J .N., and Cousins, RJ. (1982) Endotoxin-induced
changes in copper and zinc metabolism in the Syrian hamster. J. Nutr. 112, 2363-2373.

Evan, G.I., Wyllie, AH., Gilbert, C.S., et al. (1992) Induction of apoptosis in fibroblasts by
c-myc protein. Cell 69, 119-128.

Evans, RM. (1988) The steroid and thyroid hormone recptor superfamily. Science 240,
889-895.

Fabris, N., Mocchegiani, E., and Amadio, L., et al. (1984) Thymic hormone deficiency in
normal aging and Down’s syndrome: is there a primary failure of the thymus? Lancet 1,
983-986.

Fadok, V.A, Savill, J.S., Haslett, C., etal. (1992b) Different populations of macrophages use
either the vitronectin receptor or the phosphatidylserine receptor to recognize and remove
apoptotic cells. J. Immunol. 149, 4029-35.

Fadok, V.A, Voelker, DR, Campbell, P.A, et al. (1992a) Exposure of phosphatidylserine
on the surface of apoptotic lymphocytes triggers specific recognition and removal by
macrophages. J. Immunol. 148, 2207-2216.

Fadok, V.A, Laszlo, D.J., Noble, F.W., et al. (1993) Particle digestibility is required for
induction of the phosphatidylserine recognition mechanism used by murine macrophages to
phagocytose apoptotic cells. J. Immunol. 151, 4278-4285.

Faict, D., Ceupens, J ., and DeMoor, P. (1985) Transcortin modulates the effects of cortisol
on mitogen-induced lymphocyte proliferation and immunoglobulin production. J. Steroid
Biochem. 23, 553-555.

251

Fairea, L.M. (1982) Inductively Coupled plasma: principles and horizons. Am. Lab. Nov.
18-22.

Falcluck, KR, Ulpino, L., Mzus, B., and Vallee, B.L. (1977) E grocilis RNA polymerase
I: a zinc metalloenzyme. Biochem. Biophys. Res. Commun. 74, 1206-1210.

Falcluk, K.H., Fawcett, D., and Vallee, B.L. ( 1975) Role of zinc in cell division of Euglena
grocilis. J. Cell. Sci. 17, 57-68.

Falclatk, K11, Hardy, C., Ulpino, L., and Vallee, B.L. (1978) RNA metabolism, manganese,
and RNA polymerases of zinc sufiicient and zinc deficient Euglena gracilis. Proc. Natl.
Acad. Sci. USA 75, 4175-4179.

Fauci, AS., and Dale, DC. (1974) The efi'ect of in viva hydrocortisone on subpopulations
of human lymphocytes. J. Clin. Invest. 53, 240-246.

Feldman, D., Funder, J.W., and Edelman, 1.8. (1973) Evidence for a new class of
carticosterone receptors in the rat kidney. Endocrinology 92, 1429-1441.

Felicia, Y.H., Wu, C.W., and Wu, W. (1987) Zinc in DNA replication and transcription. Ann.
Rev. Nutr. 7, 251-272.

Fenwick, PK, Aggett, P.J., Macdomld, D., et al (1990a) Zinc deficiency and zinc repletion:
effect on the response of rats to infection with T richinella spiralis. Am. J. Clin. Nutr. 52,
166-172.

Fenwick, P.K., Aggett, P.J., Macdonald, D., et al. (1990b) Zinc deprivation and zinc
repletion: effect on the response of rats to infection with Strongyloides ratti. Am. J. Clin.
Nutr. 52, 173-177.

Ferguson, S.E., Accavitti, M.A, Wang, D.D., et al. (1994) Regulation of RAG-2 protein
expression in avian thymocytes. Mol. Cell. Biol. 14, 7298-7305.

Femandes, G., Nair, M., Onoe, K., et al. (1979) Impairment of cell-mediated immunity
functions by dietary zinc deficiency. Proc. Natl. Acad. Sci. USA 76, 457-461.

Flaherty, D.K., McGarity, K.L., Sinzenburger, P., and Panyik, M. (1993) The efl‘ect of
continuous corticosterone administration on lymphocyte subpopulations in the peripheral
blood of the Fischer 344 rat as determined by two color flow cytometric analysis.
Immunophannacol. Immunotoxicol. 15, 583-604.

Flynn, A, Pories, W.J., Strain, W.H., and Hill Jr., VA (1973) Zinc deficiency with altered
adrenacortical function and its relation to delayed healing. Lancet 1, 789-791.

252

Fowlkes, RJ., and Pardoll, D.M (1989) Molecular and cellular events of T-cell development.
Adv. Immunol. 44, 207-264.

Fraker, P.J., and Telford, W. (1996) Regulation of apoptotic events by zinc. In C. Berdanier
(ed), Nutrition and Gene Expression, CRC Press, Boca Raton, FL, pp 189-208.

Fraker, P.J., Zwickl, CM, and Luecke, RW. (1982) Delayed type hypersensitivity in the zinc
deficient achilt mice: impairment and restoration of responsitivity to dinitrofluorobenzene. J.
Nutr. 112, 309-313.

Fraker, P.J., Gershwin, M.E., Good, RA, and Prasad, AS. (1986) Interrelationships
between zinc and immune function. Feder. Proc. 45, 1474-1479.

Fraker, P.J., King, L.E., Garvy, BA, and Medina, CA (1993) The immunopathology of zinc
deficiency in humans and rodents. In D.M. Klurfeld (ed), Human Nutrition - A
CW Treatise, Nutrition and Immunology, Vol. 8, Plenum Press, New Yorlg pp
267-284.

Fraker, P.J., DePasquale-Jardieu, P., Zwickl, CM, and Leucke, RW. (1978) Regeneration
of T-cell helper function in zinc-deficient adult mice. Proc. Natl. Acad. Sci. USA 75,
5660-5664.

Fraker, P.J., Haas, S.M., and Leucke, RW. (1977) Effect of zinc deficiency on the immune
response of the young adult A/J mouse. J. Nutr. 107, 1889-1895.

Fraker, P.J., Osati-Ashtiani, F., Wagner, M.A, and King, LE. (1995) Possible roles for
glucocorticoids and apoptosis in the suppression of lymphopoiesis during zinc deficiency: a
review. J. Am. Coll. Nutr. 14, 11-17.

Fraker, P.J., Caruso, R, and Kierszenbaum, F. (1982) Alteration of the immune and
mtritional status of mice by synergy between zinc deficiency and infection with Topanoswna
cmzi. J. Nutr. 112, 1224-1229.

Fraker, P.J., Hildebrandt, K., and Luecke, RW. (1984) Alteration of antibody-mediated
responses of suckling mice to T-cell-dependent and independent antigens by maternal
marginal zinc deficiency: restoration of responsivity by nutritional repletion. J. Nutr. 114,
170-179.

Freedman, LP. (1992) Anatomy of the steroid receptor zinc finger region. Endocrine Rev.
13, 129-145.

Freedman, L.P., Luisi, B.F., Korszvn, ZR, et al. (1988) The function and structure of the
metal coordination sites within the glucocorticoid receptor DNA binding domain. Nature
334, 543-546.

253

Fukuda, M. (1991) Leukosialin, a major O-glycan-containing sialoglycoprotein defining
leukocyte difi‘erentation and malignancy. Glycobiology 1, 347-356.

Garvy, R, King, L., Telford, W., etal. (1993a) Chronic levels of corticosterone reduces the
number of cycling cells of the B-lineage in murine bone marrow and induces apoptosis.
Immunology 80, 587-592.

Garvy, B.A, Telford, W.G., King, L.E., and Fraker, P.J. (1993b) Glucocorticoids and
irradiation-induced apoptosis in normal murine bone marrow B-lineage lymphocytes as
determined by flow cytometry. Immunology 79, 270-277.

Garvy, BA, and Fraker, P.J. (1991) Suppression of the antigenic response of murine bone
marrow B cells by physiological concentrations of glucocorticoids. Immunology 74, 519-523.

Gasull, T., Giralt, M., Hernandez, J ., et al. (1994) Regulation of metallothionein
concentrations in rat brain: efl‘ect of glucorticoids, zinc, copper, and endotoxin. Am. J.
Physiol. Soc. 266, E760-E767.

Giglio, MP, Hunter, T., Bannister, J .V., et al. (1994) The manganese superoxide dismutase
gene of Caenorhabditis elegans. Biochem. Mol. Biol. Int. 33, 37-40.

Giroux, B.L., and Henkin, RI. ( 1972) Competition for zinc among serum albumin and amino
acids. Biochem. Bi0phys. Acta. 273, 64-72.

Giugliano, R, and Millward, DJ. (1984) Growth and zinc homeostasis in the severely
Zn-deficient rat. Br. J. Nutr. 52, 545-560.

Godfiey, D.I., Kennedy, J ., Mombaerts, P., et al. (1994) Onset of TCRB rearrangement and
role of TCRB expression during CD3CD4‘CD8‘ thymocyte differentiation. J. Immunol. 152,
4783 -4792.

Godowski, P.J., and Picard, D. (1989) Steroid receptors: How to be both a receptor and a
transcription factor. Biochem. Pharmacol. 38, 3135-3143.

Godowski, P.J., Picard, D., and Yamamoto, KR (1988) Signal transduction and
transcriptional regulation by glucocortoid receptor. Lex A fusion proteins. Science 241,
812-816.

Golden, BE. (1989) Zinc in cell division and tissue growth: physiological aspects. In C.F.
Mills (ed), Human Nutrition Reviews. Zinc in Human Biology, Spring-Vedag, London, pp
1 19-128.

Golden, M.H.N., Jackson, AA, and Golden, BE. (1977) Effect of zinc on thymus of
recently malnurished children. Lancet H, 1057-1059.

254

Good, RA, and Lorenz, E. (1992) Nutrition and cellular immunity. Int. J.Immunopharmacol.
14, 361-366.

Gorodetsky, R, Fuks, Z., Sulkes, A, et al. (1985) Correlation of erythrocyte and plasma
levels ofzinc, copper and iron with evidence ofmetastatic spread in cancer patients. Cancer
55, 779-787.

Greaves, M.F., Brown, J., Molgaard, H.V., et al. (1992) Molecular features of CD34: a
hemopoietic progenitor cell-associated molecule. Leukemia 6, 31-36.

Grids, A, and Young, EM. (1996) The Aa'odermatitzs enteropahthica mutation transiently
afi‘ects zinc metabolism in human fibroblasts. J. Nutr. 126, 219-224.

Grubs, J., Sgonc, R, Hu, Y. H., Beng, H., et al. (1994) Thyrnocyte apoptosis induced by
elevated endogenous corticosterone levels. Eur. J. Immunol. 24, 1115-1121.

Guidos, C.J., Williams, C.J., Wu, G.E., et al. (1995) Development of CD4“CD8+ thymocytes
in RAG-deficient mice through a T cell receptor [3 chain-independent pathway. J. Exp. Med.
181, 1187-1195.

Gulley, ML, Ogata, L.C., Thorson, J .A., et al. (1988) Identification of a murine pan-T cell
antigen which is also expressed during the terminal phases of B cell difi‘erentiation. J.
Immunol. 140, 3751-3757.

Gupta, RK, Bhattacharya, S.K., Sundar, S., et al. (1992) A correlative study of serum zinc
and in vitro cell mediated immune status in rheumatic heart disease. Acta Cardiologica. 157,
297-304.

Gustafsson, J.A, Carlstedt-Duke, J ., Stromstedt, RB, et al. (1990) Structure, function and
regulation of the glucocorticoid receptor. In G.H. Sato, and J .L. Stevens (eds), Molecular
Endocrinology and Steroid Action, Alan R Liss, Inc., New York, pp 65-80.

Gustafsson, J.A, Carlstedt-Duke, J., Poellinger, L., et al. (1987) Biochemistry, molecular
biology, and physiology of the glucocorticoid receptor. Endocr. Rev. 8, 185-234.

Guttinger, M., Gassman, M., Amrein, K.E., and Burn, P. (1992) CD45 phosphotyrosine
phosphatase and p56lck protein tyrosine kinase: a functional complex crucial in T cell signal
transduction. Int. Immunol. 4, 1325-1330.

Hadden, J.W. (1995) The treatment of zinc deficiency is an immunotherapy. Int. J.
Immunopharmac. 17, 697-701.

Hadley, MC. (1992) Chapter 15: Adrenal steriod Hormones. Endocrinology, 3rd edition,
Prentice-Hall, Inc., New Jersey, pp 391-429.

255

Hager, L.J., and Palmits, RD. (1981) Transcriptional regulation of mouse liver
metallothionein-I gene by glucocorticoids. Nature 291, 340-342.

Hager, L.J., and Palmiter, RD. (1981) Transcriptional regulation of mouse liver
metallothionein-I gene by glucocorticoids. Nature 291, 340-342.

Halme, M., Wenger, RH., Vestweber, D., and Nielsen, P.J. (1994) The heat-stable antigen
can alter vsy later antigen 4-mediated adhesion. J. Exp. Med. 179, 1391-1395.

Hambidge, KM. (1989) Mild zinc deficiency in human subjects. In C.F. Mills (ed), Human
Nutrition Reviews. Zinc in Human Biology, Springer-Verlag , London, pp 281-296.

Hambidge, K.M., Casey, CE, and Krebs, NP. (1986) line. In W. Mertz (ed), Trace
Elements in Human and Animal Nutrition, Vol. 2, 5th ed, Academic Press, New York, pp
1-137.

Hams, DH. (1986) Metallothionein. Ann. Rev. Biochem. 55, 913-951.

Hammond, G. L. (1990) Molecular properties of coritcosteroid binding globulin and the sex-
steroid binding proteins. Endo.Rev. 1165-79.

Hammond, G.L., Smith, C.L., Goping, L.S., et al. (1987) Primary structure of human
corticosteroid binding globulin, deduced fi'om hepatic and pulmonary cDNAs, exhibits
homology with serine protease inhibitors. Proc. Natl. Acad. Sci. USA 84, 5153-5157.

Hammond, G.L., Smith, C.L., and Underhill, DA. (1991) Molecular Studies of corticosteroid
binding globulin structure, biosynthesis and function. J. Steroid Biochem. Mol. Biol. 40,
755-762.

Hart 0., Failla, ML, Hill, AD., et al (1994) Inositol phosphates inhibit uptake and transport
of iron and zinc by a human intestinal cell line. J. Nutr. 124, 580-587.

Hard, T., Dahlman, K., Carstedt-Duke, J ., et al. (1990) Cooperativity and specificity in the
interactions between DNA and the glucocorticoid receptor DNA-binding domain.
Biochemistry 29, 5358-5364.

Hardy, RR, Carmack, C.E., Shinton, S.A., et al. (1991) Resolution and characterization of
pro-B and pre-pro-B cell stages in normal mouse bone marrow. J. Exp. Med. 173,
1213-1225.

Hayashi, J ., Medlock, ES, and Goldschneider, I. (1984) A selective culture system for
generating terminal deoxynucleotidyl transferase-positive (T dT‘) lymphoid precursor cells
in vitro. J. Exp. Med. 160, 1622-1639.

256

Hedrick, SM, and Eidelman, F.J. (1993) T lymphocyte antigen receptors. Fund. Immun. 3rd
edition, 383-413.

Heimfeld, S., and Weissman, LL. (1992) Characterization of several classes of mouse
hematopoietic progenitor cells. Curr. Top. Microbiol. Immunol. 177, 95-105.

Held, DD. and Hoekstra, W.G. (1984) The efi‘ects of zinc deficiency on turnover of
cadmium-metallothionein in rat liver. J. Nutr. 114, 2274-2282.

Hempe, J.M. and Cousins, RJ. (1991) Cysteine-rich intestinal protein binds zinc during
transmucosal zinc transport. Proc. Natl. Acad. Sci. USA 88, 9671-9674.

Hempe, J.M., and Cousins, RJ. (1992) Cysteine-rich intestinal protein and intestinal
metallothionein: an inverse relationship as a conceptual model for zinc absorption in rats. J.
N. 122, 89-95.

Henkin, RI. (1974) On the role of adrenocorticosteroids in the control of zinc and copper
metabolism. In W.G. Hoekstra, J .W. Suttie, HE. Ganther, and W. Mertz (eds), Trace
ElementMetabolism in Animals-II. Proceedings of the second International symposium on
Trace Element Metabolism in Animals held in Madison, Wisconsin, University Park Press,
Baltimore, pp 647-651.

Hermann, G., Tovar, C.A, Beck, F.M., and Sheridan, JP. (1994) Kinetics of glucocorticoid
respome to restraint stress and/or experimental influenza viral infection in two inbred strains
of mice. J. Neuroimmunol. 49, 25-33.

Hermeking, H, and Eick, D. (1994) Mediation of c-myc -induced apoptosis by p53. Science,
265, 2091-2093.

Hicks, S.E., and Wallwork, JG (1987) Efi‘ect of dietary zinc deficiency on protein synthesis
in cell-free systems isolated from rat liver. J. Nutr. 117, 1234-1240.

Hill, AD., Naama, HA, Gallaghs, H.J., et at (1995) Glucocorticoids mediate macrophage
dysfirnction in protein calorie malnurtion. Sugery 118, 130-137.

Hoadley, J.B., Leinart, AS, and Cousins, RJ. (1988) Relationship of "Zn absorption kinetics
to intestinal metallothionein in rats: effects of zinc depletion and fasting. J. Nutr. 118,
497-502.

Hoadley, J .E., and Cousins, RJ. (1987) Cellular mechanisms for intestinal zinc absorption.
Fed. Proc. 46, 598-604.

Hombach, J ., Leclercq, L., Radbnrch, A, et al. (1988) A novel 34 kD protein co-isolated
with IgM molecule in surface IgM expressing cells. EMBO J. 7, 3451-3456.

257

Hombach, J., Lottspeich, F., and Reth, M (1990) Identification of the genes encoding the Iga
and IgB components of the IgM antigen receptor complex by amino-terminal sequencing.
Eur. J. Immunol. 20, 2795-2799.

Homo-Delarche, F ., and Dardenne, M. (1993) The neuroendocrine-irnmune axis. Springer
Sernin. Immunopathol. 14, 221-238.

Hoovss, J.MN., Mannens, MJR, Bliek, J., et al. (1992) High-resolution localization of 69
potential human zinc finger protein genes: a numbs are clustered. Genomics 12, 254-263.

Hough, M.R, Takei, F., Humphries, RK., and Kay, R (1994) Defective development of

thymocytes ovsexpressing the costimulatory molemle, heat-stable antigen. J. Exp. Med. 179,
177-184.

Hough, MR, Chappel, MS, Sauvageau, G., et al. ( 1996) Reduction of early B lymphocyte
precursors in transgenic mice overexpressing the murine heat-stable antigen. J. Immunol. 156,
479-488.

Housely, PR, Sanchez, ER, Danielson, M., et al. (1990) Evidence that the conserved
region in the steroid binding domain of the glucocorticoid receptor is required for both
optimal binding of hsp90 and protection from proteolytic cleavage. J. Biol. Chem. 265,
12778-12781.

Hsu, BR 8., Siiteri, P.K., and Kuhn, RW. (1986) Ineractions between corticosteroid-binding
globulin (CBG) and target tissues. Binding proteins of steroid hormones. Colloque INSERM/
John Libbey Evortext Ltd. 149, 577-591.

Hubbe, M., and Altevogt, P. (1994) Heat-stable antigen-CD24 on mouse T lymphocytes:
evidence for a costimulatory firnction. Eur. J. Immunol. 24, 731-737.

Huber, K.L., and Cousins, RJ. (1993) Metallothionein expression in rat bone marrow is
dependent on dietary zinc but not dependent on interleukin-l or interleukin-6. J. Nutr. 123,
642-648.

Huckle, J.W., Morby, AP., Turner, 18., and Robinson, NJ. (1993) Isolation of a prokaryotic
metallothionein locus and analysis of transcriptional control by trace metal ions. Molec.
Microbiol. 7, 177-187.

Hutchison, RA, Matic, G., Czar, M.J., et al. (1992) DNA-binding and non-DNA-binding
forms of the transformed glucocorticoid receptor. J. Steroid Biochem. Mol. Biol. 41,
715-718.

258

Hutchison, K.A, Czar, M.J., and Pratt, W.B. (1992) Evidence that the hormone-binding
domain of the mouse glucocorticoid receptor directly represses DNA binding activity in a
major portion of receptors that are “misfold” alter removal of hquo. J. Biol. Chem. 267,
3 190-3 195.

Ikuta, K., Uchida, N., Friedman, J ., and Weissman, LL. (1992) Lymphocyte development
from stem cells. Annu. Rev. Immunol. 10, 759-783.

Iwata, M., Hanaloka, S., and Sato, K. (1991) Rescue of thymocytes and T-cell hybridomas
fi'om glucocorticoid induced apoptosis by stimulation via the T cell receptor/CD3 complex:
Possible in vitro model for positive selection of the T cell repertoire. Eur. J. Immunol. 21,
643 -648.

Jackson, M.J. (1989) Physiology of Zinc: General aspects. In C.F. Mills (ed), Human
Nutrition Reviews, Zinc in Human Biolog, Springer-Verlag, London, pp 1-14.

Jacobsen, K., Prasad, V.S., Sidman, C.L., and Osmond, D.G. (1994) Apoptosis and
macrophage-mediated deletion of precursor B cells in the bone marrow of Eu-myc transgenic.
Blood 84, 2784-2794.

Jardieu, P., and Fraker, P.J. (1990) Influence of zinc deficiency on the magnitude kinetics, and
aflinity of the response to trinitrophenylated (TNP) lipopolysaccharide in TNP-ficol in adult
mice. J. Trace Elements Exp. Med. 3, 1-11.

Jays Rao, K.S., Srikantia, 8.6., and Gopalan, C. (1968) Plasma cortical levels in protein-
calorie malnutrition. Arch. Dis. Child. 43, 365-367.

Jenkinson, E.J., Anderson, G., and Owen, J.J.T. (1992) Studies on T cell maturation on
defined thymic stromal cell populations in vitro. J. Exp. Med. 176, 845-853.

Jerrells, TR, Marietta, GA, Weight, FF, and Eckardt, M.J. (1990) Effects of
adrenalectomy on ethanol-associated immunosuppression. Int. J. Irnmunopharrnacol. 12,
43 5-442.

Kagi, J.H., and Schafi‘er, A. (1988) Biochemistry of metallothionein. Biochemistry 27,
8509-8515.

Karasuyama, H, Rolirut, A, Shinkai, Y., et al. (1994) The expression of Vina/15 surrogate
light chain in early bone marrow precursor B cells of normal and B cell-deficient mutant mice.
Cell 77, 133-143.

Karin, M., and Herschmann, HR. (1981) Induction of metallothionein in Hela cells by
dexamethasone and zinc. Eur. J. Biochem. 113, 267-272.

259 4

Kasls'warmra, S.I., Koyama, T., Mataro, T., etal. (1990) Structure of the murine mb-l gene
encoding a putative surface IgM-associated molecule. J. Immunol. 145, 337-343.

Kawauchi, K., Lazarus, AH, Rapoport, M.J., et al. (1994) Tyrosine kinase and CD45
tyrosine phosphatase activity mediate p21ras activation in B cells stimulated through the
antigen receptor. J. Immunol. 152, 3306-3316.

Kay, J., Cryer, A, Darke, B.M., et al. (1991) Naturally occurring and recombinant
metallothioneins: structure, irnmunoreactivity and metal-binding firnctions. Int. J. Biochem.
23, 1-5.

Keen, C.L., and Gershwin, ME. (1990) Zinc deficiency and immune firnction. Annu. Rev.
Nutr. 10, 415-431.

Keller, S.E., Weiss, J.M., Schleifer, S.J., et al. (1981) Suppression of immunity by stress:
efi‘ect. of a graded series of stressors on lymphocyte stimulation in the rat. Science 213,
1397-1400.

Kenny, AJ., Stephenson, S.L., and Turner, AJ. (1987) In AJ. Kenny, and AJ. Turner (eds),
Mammalian Ectoenzymes, Elsevier, Amsterdam, pp 169-210.

Kerr, J.F.R, Searie, J., Harmon, B.V., and Bishop, C. J. (1987) Apoptosis. In C. S. Protter
(ed), Perwectives onMammalian Cell Death, Oxford University Press, Oxford, pp 93-128.

Kerr, J.F.R, Wyllie, AH, and Currie, AR (1972) Apoptosis: a basic biological phenomenon
with wide-ranging implications in tissue kinetics. Br. J. Cancer. 26, 23 9-257.

Kimball, SR, Chen, S.J., Risica, R, et al. (1995) Efi‘ects of zinc deficiency on protein
synthesis and expression of specific mRNAs in rat liver. Metabolism 44, 126-133.

Kincade, RW. (1981) Formation of B lymphocytes in fetal and adult life. Adv. Immunol. 31,
177-245.

King, L.E., Osati-Ashtiani, F ., and Fraker, P.J. (1995) Depletion of cells of the B lineage in
the bone marrow of zinc-deficient mice. Immunology 85, 69-73.

King, L.E., and Fraker, P.J. (1991) Flow cytometric analysis of the phenotypic distribution
of splenic lymphocytes in zinc-deficient adult mice. J. Nutr. 121, 1433-1438.

Kisielow, P., Bluthmann, H, Staerz, V.D., et al. (1988) Tolerance in T-cell-receptor
transgenic mice involves deletion of nonmature CD4*CD8+ thymocytes. Nature 333,
742-746.

260

Kisielow, P. (1995) Apoptosis in intrathymic T-cell developement. In C.D. Gregory (ed),
Apoptosis and the Immune Response, erley-Liss, Inc., New York, pp 13-53.

KiMura, D., Roes, J., Kuhn, R, and Rajewsky, K. (1991) A B-cell deficient mouse by
targeteddisruptionofthememlxaneexon ofthe imnmnoglobulinrm chain gene. Nature 350,
423-426.

Kitson, RR, Brunson, K.W., Miller, CA, and Goldfarb, RH. (1994) Neuroendocrine
modulation of tumor metastasis. In-Vivo. 8, 803-806.

Klug, A, and Schwabe, J.W.R (1995) Zinc fingers. FASEB J. 9, 597-604.

Klug, A, and Rhodes, D. (1987) Zinc fingers: A novel protein motif for nucleic acid
recognition. Trends Biochem. Sci. 12, 464-469.

Koch, B., Lute B., Briand, B., and Mialhe, C. (1976) Heterogeneity of pituitary
glucocorticoid binding evidence for a transcortia-like compound. Biochem. Biophys. Acta.
44, 497-507.

Koretzky, G.A, Kohmetscher, M.A, Kkadleck, T., and Weiss, A (1992) Restoration of T
cell receptor-mediated signal transduction by transfection of CD45 cDNA into a CD45-
deficient variant of the Jurkat T cell line. J. Immunol. 149, 1138-1142.

Korsmeyer, SJ. (1995) Regulators of cell death. Trends Genet. 11, 101-105.

Krafi, D.L., Weissman, LL, and Waller, ER (1993) Differentiation of CD3'4‘8' human fetal
thymocytes in viva: clumcterization of a CD3’4*8' intermediate. J. Exp. Med. 178, 265-277.

Kraujelis, K., and Desrius, A (1989) Transcortin biosynthesis and intracellular distribution
of rat liver polyribosomes synthesizing transcortin. J. Steriod. Biochem. 32, 121-125.

Kruisbeck, AM. (1993) Development of all T-cells. Curr. Opin. Immunol. 5, 227-234.

Kuby, J. (1992) Organization and expression of immunoglobulin genes. In Immunology,
W.H. Freeman and Company, New York, pp 165.

Kuvibidila, 8., Yu, L., Ode, D., and Warrier, RP. (1993) The immune response in protein-
energy malrnrtrition and single nutrient deficiencies. In D.M. Klurfeld (ed), Human Nutrition.
A Comprehensiw Treatise, Vol. 8., Nutrition and Immunology, Plenum Press, New York,
pp 121-155.

LaFond, RE, Kennedy, S.W., Harrison, RW., and “flea, CA (1988) Irnmunocyte chemical
localization of glucocorticoid receptors in cells, cytoplasts and nucleoplasts. Exp. Cell Res.
175, 52-62.

261

Laherty, C.D., Hu, H.M., Opipari, AW., et al. (1992) The epstein-barr virus LMPl gene
product induces A20 zinc finger protein expression by activating nuclear factor xB. J. Bio.
Chem. 267, 24157-24160.

Lan, N.C., Karin, M., Nguyen, T., et al. (1984) Mechanisms of glucocorticoid hormone
action. J. Steroid Biochem. 20, 77-88.

Landau, NR, Schatz, D.G., Rosa, M., and Baltimore, D. (1987) Increased fi'equency of N-
region insertion in a murine pre-B-cell line infected with a terminal deoxynucleotidyl
transferase retroviral expression vector. Mol. Cell. Biol. 7, 3237-3243.

Lapointe, MC, and Baxter, JD. (1989) Molecular biology of glucocarticoid hormone action.
In RP. Scheirns, H.N. Claman, and A Oronsky (eds), Anti-inflammatory Steroid Action -
Basic and Clinical Aspect, Academic Press, Inc., pp 3-29.

Lebeau, M.C., Binart, N., Cadepond, F., et al. (1994) Steroid receptor associated proteins:
heat shock protein 90 and p59 irnmunophilin. In V.K. Moudgil (ed), Steroid Hormone
Receptors: Basic and Clinical Amects, Birkhauser, Boston, pp 261-280.

Li, Y-S., Hayakawa, K., and Hardy, RR (1993) The regulated expression of B lineage
associated genes during B cell differentiation in bone marrow and fetal liver. J. Exp. Med.
178, 951-960.

Licastro, F., Mocchegiani, E., Zannotti, M., et al. (1992) line affects the metabolism of
thyroid hormones in children with Downs syndrome: normalization of thyroid stimulating
hormone and of reversal triiodotheronine plasmic levels by deitary zinc supplementation.
Intern. J. Neuroscience 56, 259-268.

Lifschitz, M.D., and Henkin, RI. (1971) Circadian variation in copper and zinc in man. J.
Appl. Physiol. 31, 88-92.

Liu, Y., Jones, R, Brady, W., et al. (1992) Co-stirnulation of murine CD4 T cell growth:
cooperation between B7 and heat-stable antigen. Eur. J. Immunol. 22, 2855-2859.

Liu, Y.J., Joshua, D.E., Williams, G.T., et al. (1989) Mechanism of antigen-driven selection
in germinal centers. Nature 342, 929-931.

Lennerdal, B. (1989) Intestinal absorption of zinc. In C.F. Mills (ed), Human Nutrition
Reviews. Zinc in Human Biology, Springer-Vedag , London, pp 33-35.

Lotem, J., and Sachs, L. (1995) Reguulation of bcl-2, bcl-XL and bax in the control of
apoptosis by hematopoietic cytokines and dexamethasone. Cell Growth Differ. 6, 645-653.

262

Lowy, M.T. ( 1989) Quantification of type I and II adrenal steroid receptors in neuronal,
lymphoid and pituitary tissues. Brain Res. 503, 191-197.

Luisi, B.F., Xu, W.X., Otwinowski, 2., et al. (1991) Crystallographic analysis of the
interaction of the glucocorticoid receptor with DNA Nature 352, 497-505.

Ma, A, Pena, J.C., Chang, B. et al. (1995) Bel-x regulates the survival of double-positive
thymocytes. Proc. Natl. Acad. Sci. USA 92, 4763-4767.

Macapinlac, MP. (1966) Some characteristics of zinc deficiency in the albino rat. In AS.
Prasad (ed), Zinc Metabolism, Thomas, Illinois, pp 142-165.

Madan, AP. and DeFranco, DB. (1993) Bidirectional transport of glucocorticoid receptors
across the nuclear envelope. Proc. Natl. Acad. Sci. USA 90, 3588-3 592.

Mahajan, s. K. (1988) Zinc metabolism in uremia. Int. J. Artif. Organs, 11, 223-228.

Maldonado, M.D., Venturali, A, Franco, A, and Nunez-Roldan, A. (1991) Specific changes
in peripheral blood lymphocyte phenotype fiom probable origin of the thermal injury-related
lymphocytopenia. Burns 17, 188-192.

Marcos, M.AR, Godin, 1., Cumano, A, et al. (1994) Developmental events fi'om
hsnopoieticstemcellstoBcellpopulationsandlgrepertoires. Immunol. Rev. 137,155-171.

Martin, S.J., Mazdai, 6., Strain, J.J., et al. (1991) Programmed cell death (apoptosis) in
lymphoid and myeloid cell lines during zinc deficiency. Clin. Exp. Immunol. 83, 338-343.

Marx, J. (1995) How the glucocorticoids suppress immunity. Science 270, 232-233.

Masters, D.G., Keen, C.L., Lonnerdal, B., and Hurley, LS. (1983) Zinc deficiency
teratogenicity: the protective role of maternal tissue catabolism. J. Nutr. 113, 905-912.

Matsuo, T., Nornura, J., Kuwahara, K., et al. (1993) Cross-linking of B cell receptor-related
MB-l molecule induces protein tyrosine phosphorylation in early B lineage cells. J. Immunol.
150, 3766-3775.

Matsuucli, L., Gold, MR, Travis, A, et al. (1992) The membrane IgM-associated proteins
MB-l and Ig-B are mflicient to promote surface expression of a particularly firnctional B-cell
antigen receptor in a nonlymphoid cell line. Proc. Natl. Acad. Sci. USA 89, 3404-3408.

McConkey, D. J., Nicotera, P. ,,Hartzell P., et al. (1989) Glucocorticoids activate a suicide
process in thymocytes through an elevation of cytosolic Ca concentration. Arch. Biochem.
Biophys. 269, 365-3 70.

263

McConkey, D.J., Aguilar-Santelises, M., Hartzell, P., et al. (1991) Induction of DNA
fiagmentationin chronic B-lymphocytic leukemia cells. J. Immunol. 146, 1072-1076.

Meisfield, RL. (1990) Molecular genetics of corticosteroid action. Annu. Rev. Respir. Dis.
141, 11-17.

Melchers, F., Rolink, A, Grawunder, U., et al. (1995) Positive and negative selection events
during B lymphopoiesis. Curr. Opin. Immunol. 7, 214-227.

Melchers, F., Karasuyama, H, Haasner, D., et al. (1993) The surrogate light chain in B-cell
developement. Immunol. Today 14, 60-68 .

Menard, P., McCormick, CC, and Cousins, RJ. (1981) Regulation of intestinal
metallothionein biosynthesisinrats by dietary zinc. J. Nutr. 111, 1358-1361.

Merino, R, Ding, L., Veis, D.J., et al. (1994) Developmental regulation of the Bel-2 protein
and susceptibility to cell death in B lymphocytes. EMBO J. 13, 683-691.

Mills, AH, Spencer, RL., and Trestman, RL. (1991) Adrenal steroid receptor activation
in vivo and immune function. Am. J. Physiol. 261, E126-E131.

Miller, AH, Spencer, RL., Stein, M., and McEwen, BS. (1990) Adrenal steroid receptor
binding in spleen and thymus after stress or dexamethasone. Am J. Physiol. 259, E405-E412.

Miller, W.L. (1988) Molecular biology of steroid hormone synthesis. Endocr. Rev. 9,
295-318. '

Miller, J., McLachlan, AD., and Klug, A (1985) Repetitive zinc-binding domains in the
protein transcription factor 111 A from Xenopus oocytes. EMBO J. 4, 1609-1614.

Mills, AH, Spencer, RL., Hassett, J., etal (1994) Effects of selective type I and II adrenal
steroid against an immune cell distribution. Endocrinology 135, 1934-1944.

Mills, CF. (1989) The biological significance of zinc for man: problems and prospects. In
C.F. Mills (ed), Human Nutrition Reviews. Zinc in Human Biology, Springer-Verlag,
London, pp 371-381.

Minkus, T.M., Koski, K.G., and Scott, ME. (1992) Marginal zinc deficiency has no efl‘ect
on primary or challenge infections in mice with Heligmosomoides polygyrus (Nematode). J.
Nutr. 122, 570-579.

Misener, V., Downey, GP, and Jongstra, J. (1991) The immunoglobulin light chain related
protein A, is expressed on the surface ofmouse pre-B cell lines and can firnction as a signal
transducing molecules. Int. Immunol. 3, 1129-1136.

264

Mitchell, J .B., and Meaney, M.J. (1991) Efi‘ects of corticosterone on response consolidation
and retrieval in the forced swim test. Behav. Neurosci. 105, 798-803.

Mocchegiani, E., Paolucci, P., Granchi, D. et al. (1994) Plasma zinc level thymic hormone
activity in young cancer patients. Blood 83, 749-757.

Mocchegiani, E., Provinciali, M., DiStefano, G., et al. (1995) Role of the low zinc
bioavailability on cellular immune effectiveness in cystic fibrosis. Clin. Immunol.,
Irnmunopathol. 75, 214-224.

Moore, T., Huang, 8., Tsstappen, L.W.M.M., et al. (1994) Expression of CD43 on murine
and human pluripotent hematopoietic stem cells. J. Immunol. 153, 4978-4987.

Moore, M.E.C., Moran, JR, and Greene, HL. (1984) line supplementation in lactating
women-evidence for mammary control of zinc secretion. J. Pediatr. 105, 660-672.

Morris, RG., Hargreaves, AD., Duvall, E., and Wyllie, AJ. (1984) Hormone-induced cell
death: surface changes in thymocytes undergoing apoptosis. Am. J. Pathol. 115, 426-436.

Moulder, K., and Steward, M.W. (1989) Experimental zinc deficiency: efi‘ects on cellular
responses and the affinity of humoral antibody. Clin. Exp. Immunol. 77, 269-274.

Moynahan, El. (1974) Acrodennatitis enteropathica: a lethal inherited human zinc deficiency
disorder. Lancet 11, 399-400.

Moynahan, E.J., and Barnes, PM. (1973) line deficiency and a synthetic diet for lactose
tolerance. Lancet I, 676-677.

Munck, A, Guyre, F.M., and Holbrook, NJ. (1984) Physiological firnctions of
glucocorticoids in stress and their relation to pharmacological actions. Endocr. Rev. 5,
25-44.

Munck, A, and Crabtree, GR (1981) Glucocorticoid-induced lymphocyte death. In I. D.
Bowen and R A Lockshin (eds), Cell Death in Biology and Pathology, Chapman and Hall,
London, pp 329-359.

Murphy, KM, Heimbergs, AB., and Loh, D.Y. (1990) Induction by antigen of intrathymic
apoptoxsis of CD4+ CD8+ TCR thymocytes in vivo. Science 250, 1720-1723.

Muza'oli, M, Mocchegiani, E., Bressani, N., et al. (1992) In vitro restoration by thymulin of
NK activity of cells from old mice. Int. J. Immunopharmacol. 14, 57-61.

Nagel, W.W., and Vallee, B.L. (1995) Cell cycle regulation of metallothionein in human
colonic cancer cells. Proc. Natl. Acad. Sci. USA 92, 579-583.

265

Nagel, W.W., and Vallee, B.L. (1995) Cell cycle regulation of metallothionein in human
colonic canes cells. Proc. Natl. Acad. Sci. USA 92, 579-583.

Nakayama, K., Nakayama, K., Negishi, I., et al. (1993) Disappearance of the lymphoid
system in Bel-2 homozygous mutant chimeric mice. Science 261, 1584-1588.

Naveh, Y., Lee-Ambrose, L.M., Samuelson, DA, and Cousins, RJ. (1993) Malabsorption
of zinc in rats with acetic acid-induced enteritis and colitis. J. Nutr. 123, 1389-1395.

Nawar, 0., Akridge, RE., Hassan, E., et al. (1992) The efi‘ect of zinc deficiency on
gramrloma formation, liver fibrosis, and antibody responses in experimental schistosomiasis.
Am. J. Trop. Med. Hyg. 47, 383-389.

Neggers, Y.H., Cutter, GR, Acton, RT., et al. (1990) A posistive association between
maternal ssum zinc concentration and birth weight. Am. J. Clin. Nutr. 51, 678-684.

Nicotsa, P., and Ross, AD. (1994) Nuclear Ca2+: physiological regulation and role in
apoptosis. Mol. Cell. Biochem. 135, 89-98.

Nieto, M.A, Gonzalez, F., Garnban, F., et al. (1992) Apoptosis in human thymocytes afier
treatement with glucocorticoids. Clin. Exp. Immunol. 88, 341-344.

Nixon D.E., Moyer T.P., Johnson P., et al. (1986) Routine measurement of calcium,
nngnesium, copper, zinc and iron in urine and serum by inductively coupled plasma emission
spectroscopy. Clin. Chem. 32, 1660-1665.

Nomura, J., Matsuo, T., Kubota, E., et al. (1991) Signal transmission through the B cell
specific MB-l molecule at the pre-B cell stage. In. Immunol. 3, 117-126.

Nunez, G., Merino, R, Grillot, D., and Gonzalez-Garcia, M. (1994) Bel-2 and Bcl-x:
regulatory switches for lymphoid death and survival. Immunol. Today 15, 582-588.

Nyberg, L., Marekeov, L.N., Jones, 1., et al. (1990) Characterization of the murine
corticosteroid binding globulin: variations between mammalian forms. J. Steroid Biochem.
35, 61-65.

Odeh, M (1992) The role of zinc in acquired immunodeficiency syndrome. J. Inter. Med.
231, 463-469.

Okamoto, M., Morishita, M., Setoguchi, C., and Nakata, K. (1993) Restorative efi‘ect of
short term administration of thymulin on thymus-dependent antibody production in restraint-
stressed mice. Int. J. Immunophannacol. 15, 757-762.

266

Orava, M., Zhao, X.F., Leiter, E., and Hammond, G.L. (1994) Structure and chromosomal
location of the gene encoding mouse corticosteroid-binding globulin: strain difl‘erences in
coding sequence and steroid-binding activity. Gene 144, 259-264.

Organ, B.C., Antonach AC., Chiao, J., et al. (1989) Changes in lymphocyte number and
phenotype in seven lymphoid compartments after thermal injury. Ann. Surg. 210, 78-89.

Osmond, D.G. (1986) Powhtion dynamics of bone marrow B-lymphocytes. Immunol. Rev.
93, 103-124.

Osmond, D.G. (1990) B cell developernent in the bone marrow. Semin. Immunol. 2,
173-180.

Osmond, D.G. (1991) Proliferation kinetics and lifespan of B cells in central and peripheral
lymphiod organs. Curr. Opin. Immunol. 3, 179-185.

Osmond, D.G. (1993) Production and selection of B-lymphocytes in bone marrow:
lymphostromal interactions and apoptosis in normal, mutant and transgenic mice. Adv. Exp.
Med. Biol. 355, 15-20.

Osmond, D. G., Rico-Vargas, S. ,Valenzona, H, et al. (1994) Apoptosis and macrophage-
mediated cell deletion in the regulation of B-lymphaporesrs in mouse bone marrow. Immunol.
Rev. 142, 209-230.

Oteiza, P.I., Olin, KL, Fraga, G.G., and Keen, CL. (1995) Zinc deficiency causes oxidative
damage to proteins, lipids and DNA in rat testes. J. Nutr. 125, 823-829.

O’Dell, B.L., and Reeves, P.G. (1989) Zinc status and food intake. In C.F. Mills (ed),
Human Nutrition Reviews. Zinc in Human Biology, Springer-Vedag, London, pp 173-181.

Pai, L.H, and Prasad, AS. ( 1988) Cellular zinc in patients with diabetes mellitus. Nutr. Res.
8, 889-897.

Pallant, A, Eskenazi, A, Mattei, M.G., et al. (1989) Characterization of cDNAs encoding
human leukosialin and localization of the leukosialin gene to chromosome 16. Proc. Natl.
Acad. Sci. USA 86, 1328-1332.

Palmiter, RD, and Findley, SD. (1995) Cloning and Functional characterization of a
mammalian zinc trasporter that confers resistance to zinc. EMBO J. 14, 689-649.

Palmiter, RD. (1987) Molecular biology of metallothionein gene expression. Exs. 63-80.
Palmiter, RD. (1994) Regulation of metallothionein genes by heavy metals appears to be

mediated by a zinc-sensitive inhibitor that interacts with a constitutively active transcription
factor, MTF-l. Proc. Natl. Acad. Sci. USA 91, 1219-1223.

267

Park, Y.H., and Osmond, D.G. (1987) Phenotype and proliferation of early B lymphocyte
precursor cells in mouse bone marrow. J. Exp. Med. 165, 444-458.

Park Y.H, and Osmond, D.G. (1989a) Dynamics of B lymphocyte precursor cells in mouse
bone marrow. prolifsation of cells containing terminal deoxynucleotidyl transferase. Eur. J.
Immunol. 19, 2139-2144.

Pawlowski, T.J., and Staerz, U.D. (1994) Thymic education- T cells do it for themselves.
Immun. Today 15, 205-209.

Perkins, DJ. (1964) Zinc binding to poly-L-glutamic acid and human serum albumin.
Biochem. Biophys. Acta. 86, 635-636.

Picard, D., and Yamamoto, KR (1987) Two signals mediate horrnone-dependent nuclear
localization of the glucocorticoid receptor. EMBO J. 6, 3333-3340.

Picker, L.J., and Butcher, EC. (1992) Physiological and molecular mechanisms of
lymphocyte homing. Annu. Rev. Immunol. 10, 561-591.

Powell, 8., Saltman, P., Uretzky, G, and Chevion, M. (1990) The efi‘ect of zinc on
repsfusion arrhythmias in the isolated pcrfirsed rat heart. Free Radic. Biol. Med. 8, 33-46.

Powell, SR, Hall, D., Aiuto, L., et al. (1994) Zinc improves postischemic recovery of
isolated rat hearts through inhibition of oxidative stress. Am. J. Physiol. 266, H2497-I-12507.

Powell, SR, and Tortalani, AJ. (1992) Recent advances in the role of reactive oxygen
intermediates in ischemic injury. J. Surg. Res. 53, 417-429.

Prasad, AS., Rabbani, P., Abbasi, A, et al. (1978) Experimental zinc deficiency in humans.
Ann. Intern. Med. 89, 483-490.

Prasad, AS. (1985) Clinical, endocrinological and biochemical effects of zinc deficiency. In
M.P. Cohen and RP. Fodk (eds), Special Topics in Endocrinology and Metabolism, Vol. 7,
Alan R Liss, New York, pp 45-76.

Prasad, AS. (1979) Clinical, biochemical and pharmacological role of zinc. Ann. Rev.
Pharmacol. Toxicol. 20, 393-426.

Prasad, AS. (1988) Clinical spectrum and diognostic aspects of human zinc deficiency.
Essential and Toxic Trace Elements in Human Health and Disease. Aspects of Zinc
Deficiency, Alan R Liss, Inc., pp 3-53.

Prasad, AS. (1995) Zinc: an overview. Nutrition 11, 93-99.

268

Prasad, AS., Miale, A, Farid, Z., etal. (1963) Zinc metabolism in patients with the syndrome
of iron deficiency anemia, hypogonadism, and dwarfism. J. Lab. Clin. Med. 61, 537-549.

Prasad, AS., Halsted, J.A, and Nadirni, M. (1961) Syndrome of iron deficiency, anemia,
hepatosplenomegaly, hypogonadism, dwarfism, and geophagia. Am. J. Med. 31, 532-546.

Prasad, AS. (1991) Discovery of human zinc deficiency and studies in experimental human
model. Am. J. Clin. Nutr. 53, 403-412.

Prasad, AS. (1993) Acquired zinc deficiency and immune dysfirnction in sickle cell anemia.
In S. Ctmningham-Rundles (ed), Nutrient Modulation of the Imune Response, Dekker, New
York, pp 393-410.

Prasad, AS., Meftah, S., Abdallah, J., et al. (1988) Serum thymulin in human deficiency. J.
Clin. Invest. 82, 1202-1210.

Prasad, A8,, Fitzgerald, J.T., Hess, J.W., et al. (1993) Zinc deficiency in the elderly.
Nutrition 9, 218-224.

Pratt, W.B., Scherrer, L.C., Hutchinson, KA, and Dalman, EC. (1992) A model of
glucocorticoid receptor unfolding and stabilization by a heat shock protein. J. Steroid
Biochem. Mol. Biol. 41, 223-229.

Pratt, W.B., and Scherrer, LC. (1994) Heat shock proteins and the cytoplasmic nuclear
traficking of steroid receptors. In V.K. Moudgil (ed), Steroid Hormone Receptors: Basic
and Clinical Aspects, Birkhttuser, Boston, pp 215-246.

Pratt, W.B., Sanchez, E., Bresnick, EH, et al. (1989) Interaction of the glucocorticoid
receptor with the Mr 90,000 heat shock protein: an evolving model of ligand-mediated
receptor transformation and translocation. Cancer Res. 49, 22223-22293. _

Pratt, W.B. (1987) Transformation of glucocorticoid and progesterone receptors to the DNA-
binding state. J. Cell. Biochem. 35, 51-68.

Pratt, W.B. (1993) The role of heat shock proteins in regulating the function, folding, and
traficing of the glucocorticoid receptor. J. Biol. Chem. 268, 21455-21458.

Pratt, W.B. (1990) Interaction of hsp90 with steroid receptors: organizing some diverse
observations and presenting the newest concepts. Mol. Cell Endocrinol. 74, C69-C76.

Pratt, W.B. (1992) Control of steroid receptor function and cytoplasmic nuclear transport by
heat shock proteins. BioEssays 14, 841-848.

269

Pratt,iW.B., Jolly, D.J., Pratt, D.V., et al. (1988) A region of the steroid binding domain
determines formation of the non-DNA-binding of 9S glucocorticoid receptor complex. J.
Biol. Chem. 263, 267-273.

QuartermanJ.(1974)Theefl‘ectsofzincdeficiencyorexcessontheadrenalglandsandthe
thymus of the rat. In W.G. Hoekstra, J .W. Suttie, HE. Ganther, and W. Mertz (eds), Trace
Element Metabolism in Animals, University Park, Baltimore, MD, pp 742-748.

Quarterman, J. ( 1972) The efi‘ects of zinc deficiency on the activity of the adrenal glands.
Proc. Nutr. Soc. 31, 74A-75A

Quarterman, J., and Humphries, W.R. (1979) Efi‘ect of zinc-deficiency and zinc
supplementation on adrenals, plasma steroids and thymus in rats. Life Sci. 24, 177-183.

Rabbani, P.I., Prasad, AS., Tsai, R, et al. (1987) Dietary model for production of
experimental zinc deficiency in man. Am. J. Clin. Nutr. 45, 1514-1525.

Rafl‘aniello, RD., and Wapnir, RA (1991) Zinc-induced metallothionein synthesis by Caco-2
cells. Biochem. Med. & Met. Biol. 45, 101-107.

Rafl‘aniello, RD., and Wapnir, RA. (1989) line uptake by isolated rat enterocytes: efi‘ect
of low molecular weight ligands. Proc. Soc. Exp. Biol. Med. 192, 219-224.

Rafi’aniello, RD., Lee, S-Y., Teichberg, 8., and Wapnir, RA. (1992) Distinct mechanisms
of zinc uptake at the apical and basolateral membranes of Caco-2 cells. J. Cell. Physiol. 152,
356-361.

Rajaram, S., Carlson, S.E., Koo, W., and Braselton, W.B. (1995) Plasma mineral
concentrations in preterm infants fed a nutrient-enriched formula after hospital discharge. J.
Pediat. 126, 791-796.

Reed, JG. (1994) Bel-2 and the regulation of programmed cell death. J. Cell Biol. 124, 1-6.
Reeves, P.G., Nielsen, RH, and Fahey, G.C. Jr. (1993) A1N-93 purified diets for laboratory
rodents: final report of the American Institute of Nutrition ad hoc writing committee on the
reformulation of the AIN-76Z rodent diet. J. Nutr. 123, 1939-1951.

Ren, Y., Silvsstein, RL., Allen, J., and Savill, J. (1995) CD36 gene transfer confers capacity
for phagocytosis of cells undergoing apoptosis. J. Exp. Med. 181, 1857-1862.

Raul, J.M, and de Kloet, ER (1985) Two receptor systems for corticosterone in rat brain:
microdistribution and difi‘erential occupation. Endocrinology 117, 2505-21511.

270

RarLJWMHM vandenBosch,F.,R anddeKloet,ER (1987) Difi‘erential response oftype
I and type II corticosteroid receptors to changes in plasma steroid level and circadian
rhythmicity. Neuroendocrinology 45, 407-412.

Revs; J., Brown, KH, Santizo, MC., et al. (1995) Efi‘ects of zinc supplementation on the
growth of young Guatemalan children (abstract). FASEB J. 9, A164.

Rhodes, D., and Klug, A (1993) Zinc fingers. Scientific Am. Feb., 56-65.

Riclurd, M.J., Arnold, J ., Jurkovitr, C., et al. (1991) Trace elements and lipid peroxidation
abnormalities in patients with chronic renal failure. Nephron 57, 10-15.

Richards, M.P., and Cousins, RJ. (1976) Metallothionein and its relationship to the
metabolism ofdietalyzincrats. J. Nutr. 106, 1591-1599.

Richards, M.P., and Cousins, RJ. (1975) Mammalian zinc homeostasis: requirement for RNA
and metallothionein synthesis. Biochem. Biophys. Res. Comm. 64, 1215-1223.

Rims, L., Schauenstein, K, Mangge, H, et al, (1992) Opposite effects of mild and severe
stress on in vitro activation of rat peripheral blood lymphocytes. Brain Behav. Immun. 6,
130-140.

Ripa, S., and Ripa, R (1995) Zinc in immune function. Minerva Med. 86, 315-318.

Robinson, P.A, Langley, M.S., and Hammond, G.L. (1985) Identification and
characterization of a 1mm corticosteroid binding globulin variant with a reduced afinity for
cortisol. J. Endocrinol. 104, 269-277.

Roes, J., and Rajewsky, K (1993) Irmnunoglobulin D (IgD)-deficient mice reveal an auxiliary
receptor function for IgD in antigen-mediated recruitment of B cells. J. Exp. Med. 177,
45-55.

Rolink, A, and Melchers, F. (1991) Molecular and cellular origins of B lymphocyte diversity.
Cell 66, 1081-1094.

Rolink, A, Karasuyama, H, Haassner, D., et al. (1994) Two pathways of B-lymphocyte
developernent in mouse bone marrow and the roles of surrogate L chain in this developement.
Immunol. Rev. 137, 185-201.

Rolink, A, Kudo, A, Karasuyama, H, et al. (1991) Long-term proliferating early pre b-cell
lines and clones with the potential to develope to surface Ig-positive, mitogen reactive B-cell
in vita and in viva. EMBO J. 10, 327-336.

271

Rolink, A, and Melchers, F. (1993) Generation and regeneration of cells of the B-lymphocyte
lineage. Curr. Opin. Immunol. 5, 207-217.

Roacnstein, Y., Park, J.K, Hahn, W.C., etal. (1991) CD43, a molecule defective in Wiskott-
Aldrich syndrome, binds ICAM-l. Nature 354, 233-235.

Rosner, W. (1990) The filnctions of corticosteroid-binding globulin and sex hormone-binding
globulin: Recent advances. Endocrine Rev. 11, 80-91.

Rothman, J.B. (1989) Polypeptide chain binding proteins: catalysts of protein folding and
related processes in cells. Cell 59, 591-601.

Rothstein, D.M., da Silva, A, Sugita, K, et al. (1993) Human CD4/CD45RA+ and
CD4/CD45RA' T cell subsets express CD4-p561ck complexes, CD4-associated lipid kinases,
TCR/CD3-p59fyn complexes, and share similar tyrosine kinase substrates. Int. Immunol. 5,
409-418.

Rudney, H, and Sexton, RC. (1986) Regulation of cholesterol biosynthesis. Ann. Rev. Nutr.
6, 245-272.

Saha, AP., Hadden, B.M., and Hadden, J .W. (1995) Zinc induces thymulin secretion fiom
human thymic epithelial cells in vitro and augments splenocyte and thymocyte responses in
viva. Int. J. Immunopharmacol. 17, 729-733.

Sakaguchi, N., Kashiwamura, S.I., Kirnoto, M, et al. (1988) B lyrnphocyte-lineage-restricted
expression of mb-l, a gene with CD3-like structural properties. EMBO J. 7, 3457-3464.

Sakai, RR, and Epstein, AN. (1990) Dependence of adrenalectomy-induced sodium appetite
on the action of angiotensin II in the brain of the rat. Behav. Neurosci. 104, 167-176.

Sarnrnar, M, Aigner, S., Hubbe, M., et al. (1994) Heat-stable antigen (CD24) as ligand for
mouse P-selectin. Int. Immunol. 6, 1027-1036.

Sanchez, ER, Hirst, M, Scherrer, L.C., et al. (1990a) Hormone-fies mouse glucocorticoid
receptors overexpressed in chinese hamster ovary cells are localized to the nucleus and are
associated with both hsp70 and hsp90. J. Biol Chem. 265, 20123-20130.

Sanchez, E.R, Faber, L.E., Henzel, W.J., and Pratt, W.B. (1990b) The 56-59 kilodalton
protein idsltified in untransformed steroid receptor complexes is a unique protein that exist
in cytosol in a complex with both the 70- and 90-kilodalton heat shock proteins.
Biochemistry 29, 5145-5152.

272

Sanchez, ER, Hausley, PR, and Pratt, W.B. (1986) The molybdate-stabilized
glucocorticoid binding complex of L-cells contains a 98-100 KDa steroid binding
pluplnpradnandsmmamnsoid-bindingphosphopmtdnthmispMofmemufine
heat-shock complex. J. Steroid Biochem. 24, 9-18.

Sandstrbm, B., and Lannerdal, B. (1989) Promoters and antagonists of zinc absorption. In
C.F. Mills (ed), Human Nutrition Reviews. Zinc in Human Biology, Springer-Vedag,
London, pp 57-78.

Savill, IS. (1995) The innate immune system: Recognition of apoptotic cells. In C.D.
Gregory (ed), Apaptaalsandthelmmrme Reparrse, Willy-Liss, Inc., New York, pp. 152-171.

Savill, J.S., Wyllie, AH, Henson, J.B., et al. (1989a) Macrophage phagacytosis of aging
neutrophils in inflammation Programmed cell death in the neutrophil leads to recognition by
macrophages. J. Clin. Invest. 83, 865-895.

Savill, J.S., Henson, RM, and Haslett, C. (1989b) Phagocytosis of aged human neutrophils
by macrophages is mediated by a novel “charge sensitive” recognition mechanism. J. Clin.
Invest. 84, 1518-1527.

Schroeder, J.J., and Cousins, RJ. (1990) Interleukin 6 regulates metallothionein gene
expression and zinc metabolism in hepatocyte monolayer cultures. Proc. Natl. Acad. USA
87, 3137-3141.

Schroeder, J .J ., and Cousins, RJ. (1991) Maintenance of zinc-dependent hepatic firnctions
in rat hepatocytes cultured in medium without added zinc. J. Nutr. 121, 844-853.

Schwabe, J.W., and Klug, A (1994) Zinc mining for protein domains. Nat. Struct. Biol. 1,
345-349.

Schwartzman, RA, and Cidlowski, J.A (1993) Mechanism of tissue-specific induction of
internucleosomal deoxyribonucleic acid cleavage activity and apoptosis by glucocorticoids.
Endocrinology 133, 591-599.

Schwarz, MA. (1994) Cytoplasmic metallothionein overexpression protects NIH 3T3 cells
fiom tert-butyl hydroperoxide toxicity. J. Biol. Chem. 269, 15238-15243.

Scott, D.W. (1995) Apoptosis in immature B-lymphocytes. In C.D. Gregory (ed), Apoptosis
and the Immune Remorse, Willey-Liss, New York, pp 187-215.

Scrocchi, L.A, Hearn, S.A, Han, V.K.M, and Hammond, G.L. (1993a) Corticosteroid-
binding globulin biosynthesis in the mouse liver and kidney during postnatal development.
Endocrinology 132, 910-916.

273

Scrocchi, L.A, Orava, M., Smith, C.L., et al. (1993b) Spatial and temporal distribution of
carticostsoid-binding and its messenger ribomlcleic acid in embryonic and fetal mice.
Endocrinology 132, 903-909.

Seal, C.J., and Heaton, RW. (1987) Zinc transfer among proteins in rat duodenum mucosa.
Ann. Nutr. Metab. 31, 55-60.

Sellins, KS, and Cohen, 1.]. (1987) Gene induction by y-irradiation leads to DNA
fi’agmentationin lymphocytes. J. Immunol. 139, 3199-3206.

Selye, H. (1955) The stress of life. McGraw-Hill, New York.

Sentrnan, C.L., Shutts, J.R., Hockenbery, D., et al. (1991) Bcl-2 inhibits multiple forms of
apoptosis but not negative selection in thymocytes. Cell 67, 879-888.

Ssaliru', G.E., Berube, D., Gagne, R, and Hammond, G.L. (1990) The human corticosteroid
binding globulin gene is located on chromosome 14q31-q32.1 near two other serine protease
inhibitor genes. Hum. Genet. 86, 73-75.

Shambaugh, GE. Jr. (1989) line: the neglected nutrient. Am. J. Otol. 10, 156-160.
Sherman, AR (1992) Zinc, copps, and iron nutriture and immunity. J. Nutr. 122, 604-609.

Shi, Y., Bissonnettern RP, Parfiey, N., et al. (1991) In viva administration of antibodies to
the CD3 T-cell receptor complex induces cell death (apoptosis) in immature thymocytes. J.
Immunol. 146, 3340-3346.

Shi, H.N., Scott, M.E., Koski, K.G., et al. (1995) Energy restriction and severe zinc
deficiency influence growth, survival and reproduction of Heligmasamoides palygrus
(Nernatoda) during primary and challenge infections of mice. Parasitology 110, 599-609.

Sl'n', S., and Thomas, J .O. (1992) The transport of proteins into the nucleus requires the 70-
kilodalton heat shock protein or its cytosolic cognate. Mol. Cell. Biol. 12, 2186-2192.

Shippee, RL., Koppenhefi‘er, T., Watiwat, SR, et al. (1992) The interaction of burn injury
and fine nutriture as assessed by the humoral response to sheep red blood cells in a burn rat
model. Burns 1, 45-48.

Shortrnan, K, Egerton, M, Spanglude, 6.1, and Scollay, R (1990) The generation and fate
of thymocytes. Semin. Immunol. 1, 3-12.

Siiteri, P.K., Murai, J.T., Hammond, G.L., et al. (1982) The serum transport of steroid
hormones. Rec. Prog. Horm. Res. 38, 457-510.

274

Simpson, ER, and Waterman, MR (1988) Regulation of the synthesis of steroidogenic
enzymes in adrenal cotical cells by ACTH. Annu. Rev. Physiol. 50, 427-440.

Slaunwhite, WR Jr., Lockie, G.N., Back, N, and Sandbsg, AA (1962) Inactivity in viva
oftranacortin-bound cortisol. Science 135, 1062-1063.

Sloviter, RS, Valiquette, G, Abrams, G.M., et al. Selective loss of hippocampal granaule
cells in the mature rat brain afier adrenalectomy. Science 243, 535-538.

Smith, H.F ,Latham, M. C. Azaburke, J. A, et al. (1981) Blood plasma levels ofcortisol,
insulin, growth hormone, and somatomedin in children with marasrnus, kwashiorkor and
intermediateformsofprotein-srsgymalmtrition Proc. Soc. Exp. Biol. Med. 167, 607-611.

Smith, GA, Williams, G.T., Kingston, R, et al.(1989) Antibodies to CD3 T-cell receptor
complex induce death by apoptosis in immature T-cells in thymic culture. Nature 337,
181-84.

Smith, DR, and Toft, DO. (1993) Steroid receptors and their associated proteins. Mol.
Endocrinal. 7, 4-11.

Sobocinski, P.Z., Canterbury, W.J., Mapes, CA, and Dinterman, RE. (1978) Involvement
of hepatic metallothionein in hypozincernia associated with bacterial infection. Am. J. Physiol.
234, E399-406.

Solomons, NW, and Cousins, RJ. (1984) Zinc. In N.W. Solomons and 1H. Rosenberg
(eds), Absorption and malabsorption of mineral nutrients, Alan R. Liss, New York, pp
125-197.

Spencs, RL., Miller, AH, Moday, H, et al. (1993) Diurnal difi‘erences in basal and acute
stress levels of type I and type II adrenal steroid receptor activation in neural and immune
tissues. Endocrinology 133, 1941-1950.

Spencer, RL., Mills, AH, Stein, M., and McEwen, BS. (1991) Corticosterone regulation
oftypelandtypellasenalstsoidreceptorsinbrainpituitaryand irnmunetissue. BrainRes.
549, 236-246.

Sprent, J. ( 1989) T lymphocytes and the thymus. Fund. Immun., 2nd edition, 69-93.

Sprent, J., Lo, D, Gao, E.-K, and Ron, Y. (1988) T cell selection in the thymus. Immunol.
Rev. 101, 173-190.

Sprent, J., and Webb, SR (1987) Function and specificity of T cell subsets in the mouse.
Adv. Immunol. 41, 39-133.

275

Steel, L., and Cousins, RJ. (1985) Kinetics of zinc absorption by luminally and vascularly
perfirsed rat intestine. Am. J. Physiol. 248, G46-G53.

Steel, RGD. and Torrie, J.H (1980) Principles and Procedures of Statistics: A Biametrical
Approach, McGraw Hill, New York, pp 185-186 and pp 544-545.

Strasser, A, Harris, AW., Jacks, T., and Cory, S. (1994) DNA damage can induce apoptosis
in proliferating lymphoid cells via p53-independent mechanisms inhibitable by Bel-2. Cell 79,
329-339.

Sun, K.M., Dinsdale, D, Snowden, RT, et al. (1992) Characterization of apoptosis in
thymocytes isolated from dexamethasone-treated rats. Biochem. Phannacol. 44, 2131-2137.

Sundennan, F.W., Jr., (1995) Influence of line on apoptosis. Ann. Clin. Lab. Science 25,
134-142.

Sutanto, W., and deKloet, ER (1987) Species-specificity of corticosteroid receptors in
hamster and rat brains. Endocrinology 121, 1405-1411.

Symington, F.W., and Hakomori, S. (1984) Hematopoietic subpopulations express cross
reactive, lineage-specific molecules detected by monoclonal antibody. Mol. Immunol. 21,
507-514.

Tacnet, R, Watkins, D.W., and Ripoche, P. (1990) Studies of zinc transport into brush-
bords membrane vesicles isolated from pig small intestine. Biochim. Biophys. Acta. 1024,
323-330.

Tai, P.K.K, Albers, M.W., Chang, H, et al. (1992) Association of a 59-kilodalton
immunophilin with the glucocorticoid receptor complex. Science 256, 1315-1318.

Tarlinton, D. (1994) B-cell difi‘erentiation in the bone marrow and the periphery. Immunol.
Rev. 137, 203-229.

Taylor, C.G., Bettger, W.J., and Bray, TM. (1988) Efi‘ects of dietary zinc or copper
deficiency on the primary fi'ee radical defense system in rats. J'. Nutr. 118, 613-621.

Telford, W.G., and Fraker, P.J. (1995) Preferential induction of apoptosis in mouse
CD4’CD8” aliTCR"CD3e'° thymocytes by zinc. J. Cell. Physiol. 164, 259-270.

Telford, W.G., King, L.E., and Fraker, P.J. (1994) Rapid quantitation of apoptosis in pure
and heterogeneous cell populations using flow cytometry. J. Immunol. Meth. 172, 1-16.

Telford, W., King, L, and Fraker, P.J. (1991) Evaluation of glucocorticoid induced DNA
fragmentation in mouse thymocytes by flow cytometry. Cell Prolif. 24, 447-459.

276

Thiele, DJ. (1992) Metal-regulated transcription in eukaryotes. Nucleic Acid Res. 20,
1183-1191.

Ihomas, ML, and Lefiancois, L. (1988) Difi‘erential expression of the leukocyte-common
antigen family. Immunol. Today 9, 320-326.

Thompson, EB. (1994) Apoptosis and steroid hormones. Molec. Endoc. 8, 665-673.

Thomalley, P. J., and Vasalg M (1985) Possible role for metallothionein in protection against
radiation-induced oxidative stress. Kinetics and mechanisms of its reaction with superoxide
and hydroxyl radicals. Biochim. Acta 827, 36-44.

Todd, W.R, Elvehjen, CA, and Hart, EB. (1934) Zinc in the nutrition of the rat. Am. J.
Physiol. 107, 146-156.

Tough, DE, and Sprent, J. (1994) Turnover of naive- and memory-phenotype T-cells. J.
Exp. Med. 179, 1127-1135.

Treves, S, Trentini, P.L., Ascanelli, M., et al. (1994) Apoptosis is dependent on intracellular
zinc and independslt of intracellular calcium in lymphocytes. Exp. Cell. Res. 211, 339-343.

Tsai, S. ,Bartelmer, S, Sitnicka, E, and Collins, S. (1994) Lymphohematopoietic progenitors
immortalized by a retroviral vector harboring a dominant-negative retinoic acid receptor can
recapitulate lymphoid, myeloid, and sythroid development. Genes Dev. 8, 2831-2841.

Vacchio, M.S., Papadopoulos, V., and Ashwell, ID. (1994) Steroid production in the
thymus: implications for thymocytes selection. J. Exp. Med. 179, 1835-1846.

Vallee, B.L., and Falchuk, KH. (1993) The biochemical basis of zinc physiology. Physiol.
Rev. 73, 79-118.

Vallee, B.L., Coleman, J.B., and Auld, D.S. (1991) Zinc fingers, zinc clusters, and zinc twists
in DNA-binding protein domains. Proc. Natl. Acad. Sci. USA 88, 999-1003.

Vallee, B.L., and Auld, HS. (1993) New Perspective on line Biochemistry: Cocatalytic Sites
in Multi-Zinc Enzymes. Amer. Chem. Soc. 23, 6494-6500.

Vallee, B.L., and Galdes, A (1984) The metallobiochemistry of zinc enzymes. Adv.
Enzymol. Relat. Areas Mol. Biol. 56, 283-430.

Vallee, B.L., and Auld, BS. (1990) line corrdination, function, and structure of zinc
enzymes and other proteins. Biochemistry 19, 5647-5659.

277

van Ewijk, W. (1991) T cell differentiation is influenced by thymic microenvironments. Annu.
Rev. Immunol. 9, 591-615.

Van Wouwe, J. P. (1989) Clinical and laboratory diagnosis of Acradermatitis enterapathica.
Eur. J. Pediatr. 149, 2-8.

Veis, D.J., Sentman, C.L., Bach, EA, and Korsrneyer, SJ. (1993) Expression of the Bel-2
protein in nalrine and launan thymocytes and in peripheral T lymphocytes. J. Immunol. 151,
2546-2554.

Vermeulsl, A (1986) TeBG and CBG as an index of endocrine filnction. Binding Proteins
of Steroid Hormones 149, 383-396.

Voetbsg, B.J., Garvy, B.A, Mayer, HK, et al. (1994) Apoptosis accompanies a change in
the phenotypic distribution and filnctional capacity of murine bone marrow B-cells chronically
exposed to prednisalone. Clin. Immunol. ImmunOpath. 71, 190-198.

von Boehmer, H. (1992) Thymic selection: a matter of life and death. Immunol. Today 13,
454-458.

von Boehmer, H, and Kisielow, P. (1990) Self-nonself discrimination by T cells. Science
248, 1369-1373.

Vruwink, KG, Hurley, L.S., Gershwin, M.E., and Keen, CL. (1988) Gestational zinc
deficiency amplifies the regulation of metallothionein induction in adult mice. Proc Soc. Exp.
Biol. Med. 188, 30-34.

Walker, N.I., Harmon, B.V., Gobe, GC, and Kerr, J.F. (1988) Patterns of cell death.
Methods Achiev. Exp. Pathol. 13, 18-54.

Walsh, C.T., Sandstead, HH, Prasad, AS, et al. (1994) Zinc: health effects and research
priorities for the 1990s. Environ. Health Perspect. 102, 5-46.

Watanabe, T., Arakawa, T., Fukuda, T., et al. (1995) Zinc deficiency delays gastric ulcer
healing in rats. Dig. Dis. Sci. 40, 1340-1344.

Webb, M (1986) Role of metallothionein in cadmium metabolism. Handb. Exp. Pharmacol.
80, 281-337.

Weiss, J .N, Do, Y.S., and Feldman, D. (1979) Synthesis and secretion of corticosteroid-
binding globulin by rat liver. J. Clin. Invest. 63, 461-467.

278

Weissman, K, Hoe, S, Knudsen, L, and Sorensen, SS. (1979) Zinc absorption in patients
suffering fiorn Acradennatitis enterapatlrica and in normal adults assessed by whole-body
counting technique. Br. J. Dermatol. 1010, 573-579. '

Weissman, I.L. ( 1973) thymus cell maturation: studies on the origin of cortisone-resistant
thymic lymphocytes. J. Exp. Med. 137, 504-510.

Welch, P.A, Burrows, P.D., Narnen, A, et al. (1990) Bone marrow stromal cells and
interleukin-7 induce coordinate expression of the BP-1/6C3 antigen and pre-B cell growth.
Inter. Immunol. 2, 697-705.

Wenger, RH, Rochelle, J .M, Seldin, M.F., et al. (1993) The heat-stable antigen (mouse
CD24) gene is difl‘sentially regulated but has a housekeeping promoter. J. Biol. Chem. 268,
23345-23352.

Wenger, RH, Ayane, M., Bose, R, et al. (1991) The genes for a mouse hemopoietic
difi‘erentiation marker called the heat-stable antigen. Eur. J. Immunol. 21, 1039-1046.

Wertharner, S, Govindaraj, S, and Amaral, L. (1976) Placenta, trascortin, and localized
immunoresponse. J. Clin. Invest. 57, 1000-1008.

Werthamer, S, Samuels, AJ., and Amnral, L. (1973) Identification and partial purification
of “transcortin” like protein within human lymphocytes. J. Biochem. 248, 6398-6407.

Whitehead, RG., Coward, WA, and Lunn, P.G. (1973) Serum-albumin concentration and
the onset of Kwashiorkor. Lancet 1, 63-66.

Whitlock, C.A, Robertson, D, and “fine, ON. (1984) Murine B cell lymphopoiesis in long
term culture. J. Immunol. Meth. 67, 353-369.

Whitlock, C.A, Tidmarsh, G.F., Muller-Sieburg, C, and Weissman, LL. (1987) Bone
marrow stromal cell lines with lymphopoietic activity express high levels of a pre-B neoplasia-
associated molecule. Cell 48, 1009-1021.

Wilder, RL. (1995) Neuroendocrine-irnmune system interactions and autoinununity. Annu.
Rev. Immunol. 13, 307-338.

Williams, GT. (1994) Apoptosis in the immune system. J. Pathology. 173, 1-4.

Williams, R.J.P. (1989) An introduction to the biochemistry of zinc. In C.F. Mills (ed),
Human Nutrition Reviews. Zinc in Human Biology, Springer-Veflag, London, pp 15-31.

'a.

279

Wilson, RL. (1989) Zinc and iron in he radical pathology and cellular control. In C.F. Mills
(ed) Hanna: Nrdrition Reviews, Zinc in Human Biology, Springer-Verlag, London, pp 147-
172. .

Wirth, J.J., Fraker, P.J., and Kierszenbaum, F. (1989) Zinc requirement for macrophage
fiinction: efi‘ect of zinc deficiency on uptake and killing of a protozoan parasite. Immunology
68, 114.119.

Woodward, B. (1991) Zinc, a pharmacologically potent essential nutrient: focus on
immunity. Can. Med. Assoc. J. 145, 1469.

Wu, Q, Li, L, Cooper, M., et al. (1991) Aminopeptidase A activity of the murine B-
lymphocyte differentiation antigen BP-1/6C3. Proc. Natl. Acad. Sci. USA 88, 676—680.

Wu, Q, Tidmarsh, GI“, Welch, P.A, etal. (1989) The early B lineage antigen BP-l and the
transfonnation-associated antigen 6C3 are on the same molecule. J. Immunol. 143,
3303-3308.

Wyllie, AH. (1987) Apoptosis: cell death under homeostatic control. Arch. T oxicol. 11,
3-10.

Wyllie, AH, Kerr, JFK, and Currie, AR. (1980) Cell death: the significance of apoptosis.
Int. Rev. Cytol. 68, 251-305.

Wyllie, AH. (1980) Glucocorticoid-induced thymocyte apoptosis is associated with
endogenous endonuclease activation. Nature 284, 555-5 56.

Yamanashi, Y., Kakuichi, T., Mizuguchi, J ., er al. (1991) Association of B-cell antigen
receptor with protein tyrosine kinase Lyn. Science 251, 192-194.

Yonemura, S, Nagafuchi, A, Sato, N, and Tsukita, S. (1993) Concentration of an integral
membrane protein, CD43 (leukosialin, sialophorin), in the cleavage firrrow through the
interaction of its cytoplasmic domain with actin-based cytoskeletons. J. Cell. Biol. 120,
43 7-449.

Yu, D.T.Y., Clements, P.J., Paulua, et al. (1974) Human lymphocyte subpopulations. J. Clin.
Invest. 53, 565-571.

Zeng, B, Qian, Y., Zheng, D, et al. (1991) Change of T lymphocyte subsets in peripheral
blood of clu'ldren with malnutrition and zinc deficiency. Hua. Hsi. 1. K0. Ta. Hsueh. Hsueh.
Pao. 22(3), 337-339.