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The Negative Effect of Corticosterone on Murine Bone
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THE NEGATIVE EFFECT OF CORTICOSTERONE ON MURINE BONE MARROW
B LYMPHOCYTES: MODULATION BY IL-7 AND STROMAL CELLS

By

Tonya S. Laakko

A Dissertation
Submitted to Michigan State University
in partial fulﬁllment of the requirements
for the degree of
Doctorate of Philosophy
The Department of Biochemistry and Molecular Biology

2000

ABSTRACT

THE NEGATIVE EFFECT OF CORTICOSTERONE ON MURINE BONE MARROW
B LYMPHOCYTES: MODULATION BY IL-7 AND STROMAL CELLS

By

Tonya S. Laakko

B cells are critical to many facets of host defense making their development and
production in the marrow of vital importance. Glucocorticoids (Ge), especially those
produced endogenously in trauma patients, burn victims, the malnourished, etc., where
the neuroendocrine stress axis is activated, can greatly impair lymphopoiesis. This
reduces the numbers of circulating B cells and compromises immune defense.
Considering the importance of B cell development, surprisingly little was known about
the mechanisms involved in the downregulation of lymphopoiesis by Go. The
experiments performed in this thesis were designed to address the following questions: 1)
How do increased concentrations of the natural glucocorticoid, corticosterone (Cs), effect
the various stages of development of cells of the B lineage in murine bone marrow? 2)
Can stromal cells that support hematopoiesis modulate the in vitro response of precursor
B cells to corticosterone? 3) Can the cytokines interleukin-7 and/or stem cell factor,
which are essential to B cell development, modulate the adverse effects of corticosterone
on precursor B cells in vitro? Using a Cs implantation system, circulating Cs was
elevated to concentrations analogous to that seen during normal physiological stress in
the mouse model system. Flow cytometric identiﬁcation of the subpopulations of
developing B lymphocytes indicate that Cs had adverse effects within 12 hours especially

on pre B cells. By 36 hours nearly all (70-90%) of early-pro. late-pro, pre and immature

cells were lost from the bone marrow. Only mature B cells and the earliest deﬁned
progenitor, the pre-pro B cell, showed resistance to Cs. Phenotypic and DNA analysis
via ﬂow cytometry showed that Cs induced apoptosis in pro, pre and IgM+ B cells ﬁom
murine bone marrow and decreased cell cycling in pro and pre B cells in vitro.
Surprisingly, interleukin-7 which promotes lymphopoiesis, but not stem cell factor,
completely inhibited Cs—induced apoptosis and cell cycle arrest in the earliest B
lymphocytes, the pro B cells. IL-7 also had a modest protective effect on pre B cells,
reducing apoptosis by 30%, but it did not protect them from the Cs-induced decrease in
cell cycling. It provided only limited protection to immature IgM+ bearing cells. Stromal
cells also signiﬁcantly reduced Cs—induced apoptosis (30-50%) for all stages of B
lymphocyte development in vitro although they did not restore cell cycling to normal
levels. Clearly these experiments have shown that Cs can have a rapid and adverse effect
on the development of cells of the B lineage in mouse BM in vivo and in vitro.
Nevertheless, substantial protection afforded the precursor B cells by interleukin-7 and
stromal cells in vitro suggest that with further investigations this cytokine and other
factors produced by stromal cells could potentially promote immune recovery from stress

and Cs induced damage.

TABLE OF CONTENTS

LIST OF TABLES ............................................................................ vi
LIST OF FIGURES ........................................................................... vii
ABBREVIATIONS ........................................................................... ix
INTRODUCTION ........................................................................... 1
CHAPTER I: LITERATURE REVIEW ................................................... 4
B Cell Lymphopoiesis ............................................................... 5
Background .................................................................. 5
Model Systems ............................................................... 6
Hematopoiesis ................................................................ 8
Flow cytometry: Phenotypic Markers for Monitoring
B Cell Lymphopoiesis ............................................. 8
Irnmunoglobulin Gene Rearrangement .................................. 11
B Lymphocyte Homeostasis and Selection .............................. 12
Stage Speciﬁc Gene Expression .......................................... 15
Microenvironmental Effects on B Lymphopoiesis ..................... 16
Cell-Cell and Cell-Extracellular Matrix Contact ....................... 20
Stromal Cell Derived Cytokine Support ................................. 22
Summary ..................................................................... 28
Glucocorticoids and the Immune System ........................................ 30
Background .................................................................. 3O
Glucocorticoid Biochemistry .............................................. 33
The Glucocorticoid Receptor .............................................. 34
Glucocorticoid Receptor Signaling ....................................... 36
Summary ..................................................................... 4O
Apoptosis .............................................................................. 41
History ........................................................................ 41
Apoptotic Morphology and Detection .................................... 42
Apoptosis Activation (Caspases and Mitochondria) ................... 44
Apoptosis Inhibition (Bel-2 Family of Proteins) ........................ 47
Bel-2 Family Members in B Cell Lymphopoiesis ...................... 50
Cytokine-Withdrawal Induced Apoptosis ................................ 51
Glucocorticoid Induced Apoptosis ....................................... 53
Summary ..................................................................... 55

CHAPTER II: THE IN V1 V0 EFFECT OF CORTICOSTERONE ON
DEVELOPING B LYMPHOCYTES: FROM COMMITTED

PROGENITOR TO MATURITY .................................................. 64
Abstract ................................................................................ 65
Introduction ........................................................................... 67

iv

Materials and Methods ............................................................... 72
Results ................................................................................. 77
Discussion ............................................................................. 83

CHAPTER III: STROMAL CELL PROTECTION AGAINST CS-INDUCED

APOPTOSIS IN BONE MARROW B LYMPHOCYTES ..................... 100
Abstract ................................................................................ 101
Introduction ........................................................................... 103
Materials and Methods ............................................................... 107
Results ................................................................................. 111
Discussion ............................................................................. 1 17

CHAPTER IV: INTERLEUKIN-7 PROTECTS EARLY STAGES IN E
LYMPHOYCTEDEVELOPMENT FROM CORTICOSTERONE-INDUCED

APOPTOSIS AND CELL CYCLE ARREST .................................... 132
Abstract ................................................................................ 133
Introduction ........................................................................... 135
Materials and Methods ............................................................... 138
Results ................................................................................. 141
Discussion ............................................................................. 148
REFERENCES ................................................................................ 164

Table 2.1

Table 3.1

Table 4.1

Table 4.2

LIST OF TABLES

Bone marrow cellularity of Cs or sham implanted mice ............... 91
Bone marrow B cells in S/Gz/M phases of the cell cycle .............. 125
Recovery and viability of bone marrow cultured for

15 to 16 Hours ............................................................... 152
Effects of IL-7 and SCF on apoptosis in pro B cells ................... 163

vi

Figure l .1

Figure 1.2

Figure 1.3

Figure 1.4
Figure 1.5
Figure 1.6
Figure 1.7
Figure 2.1
Figure 2.2

Figure 2.3

Figure 2.4

Figure 2.5

Figure 2.6

Figure 3.1

Figure 3.2

Figure 3.3

Figure 3.4

LIST OF FIGURES

Differentiation of various blood cell lineages from a
pluripotent stem cell ......................................................... 57

Bone marrow B lymphocyte development scheme ..................... 58

Developing B lymphocyte association with stromal cells in

the bone marrow ............................................................ 59
Corticosteroid biosynthesis ................................................ 60
The glucocorticoid receptor ............................................... 61
Apoptotic pathways ........................................................ 62
Cytokine survival via Bad inactivation .................................. 63
Plasma corticosterone concentrations .................................... 88
The effect of Cs or cholesterol implants on thymic weights .......... 90
Flow cytometric proﬁle of developing bone marrow B

lymphocytes .................................................................. 93
Cs-induced changes in bone marrow B lymphocyte

populations over time ....................................................... 95
Cs-induced cell cycle changes in pro and pre B cells .................. 97

Cs effect on major hematopoietic lineages in the bone
marrow ........................................................................ 99

Spontaneous and Cs-induced apoptosis in pro, pre and IgW B cells
in vitro ........................................................................ 122

Stromal cell modulation of spontaneous and Cs-induced

apoptosis ..................................................................... 124
Flow cytometry of S10 and/or IL—7 effects on apoptosis

and cycling of Pro B cells .................................................. 127
Relative expression of IL-7 and SCF message .......................... 129

vii

Figure 3.5
Figure 4.1

Figure 4.2

Figure 4.3

Figure 4.4

Figure 4.5

Cs effect on stromal cell expression of SCF and IL-7 RNA ........... 131
IL-7 modulation of spontaneous and Cs—induced apoptosis ........... 154

IL-7 and/or Cs effect on cell cycle distribution among pro and

pre B cells .................................................................... 156
Flow cytometry of IL-7 and/or Cs effect on pro B cell

apoptosis and cell cycle .................................................... 158
IL-7 effect on apoptosis induced by varying concentrations of

Cs among B cells ........................................................... 160
SCF effect on spontaneous and Cs-induced apoptosis ................. 162

viii

BM
Cs
Gc
Dex
IL
SCF

BCR
TCR
HSA
SCID
RAG
tdt

, BSAP-S
NFKB
IKB
LTBMC
VCAM
ECM
SDF-l
TSLP
CSF
VLA
PI3K
yc
ACTH
CRH
HPA
LDL
HDL
CBG
GcR
SHR

GRE
SCR
GRIP
HAT
AIDS
PS
TNFR
ICE

LIST OF ABBREVIATIONS

bone marrow

corticosterone

glucocorticoid

dexamethasone

interleukin

stem cell factor

immunoglobulin

B cell receptor

T cell receptor

heat stable antigen

severe combined immunodeﬁciency
recombination activation gene
terminal deoxynucleotidal transferase
B-cell-speciﬁc activator protein—5
nuclear factor kappa B

inhibitor of nuclear factor kappa B
long term bone marrow culture
vascular cellular adhesion molecule
extracellular matrix

stromal derived factor-1

thymic stromal lymphopoietin

colony stimulated factor

very late antigen
phosphatidylinositol 3-kinase
common gamma chain

adrenal corticotrophin hormone
corticotrophin releasing hormone
hypothalamic-pituitary—adrenal
low density lipoprotein

high density lipoprotein
corticosterone binding globulin
glucocorticoid receptor

steroid hormone receptor

heat shock protein

glucocorticoid response element
steroid receptor coactivator
glucocorticoid receptor interacting protein
histone acetyltransferase

aquired immunodeﬁciency syndrome
phosphatidylserine

tumor necrosis factor receptor
interleukin l-B converting enzyme

ix

APAF
DD
STAT
Jak
Smase
PI-PLC

apoptotic protease activating factor

death domain

signal transducers and activators of transcription
janus kinase

sphingomyelinase

phosphatidylinositol phospholipase C

INTRODUCTION

The successful generation of mature B cells (B cell lymphopoiesis) is critical to
maintenance of the immune system. Aberrations in lymphopoiesis can result in a variety
of diseases and/or failure to adequately respond to pathogens. Research in this area of
immunology has been limited due to the complexity of the development process, the
difﬁculty in identiﬁcation of developing B lymphocytes and the very heterogeneous
nature of the bone marrow where B cells develop in adult mammals. Therefore much of
the research on B lymphocytes has been performed using immortalized cell lines, rather
than natural cells. However, over the past decade methodology has been developed to
successfully identify B lymphocytes from the other cell types that exist in the bone
marrow (BM) (Cofﬁnan and Weissman, 1981). In addition, distinct stages in B
lymphocyte development have been deﬁned based on the differential expression of
surface proteins (Hardy et al., 1991). These stages have also been deﬁned functionally
based on Ig gene rearrangement. This thesis utilizes this sophisticated methodology to
study, in depth, the effect of glucocorticoid (Go) elevation on different substages of cells
of the B lineage.

Chronic elevation of Ge, due to either physiological stress or pharmacological
administration, can result in immunosuppression. In viva research from our lab has
shown that Go elevation reduces the development of B cells in mice (Garvy et al., 1993).
It is likely that induction of apoptosis in these developing cells is at least one mechanism
whereby Gc elevation adversely affects lymphopoiesis. However in viva studies of

apoptosis are severely limited due to the rapid phagocytosis of cells early in the apoptotic

process. Evidence that Go can induce signiﬁcant amounts of apoptosis in these
populations have come from in vitra studies; the synthetic Gc, dexamethasone (dex), has
been used for decades as a classic inducer of apoptosis in thymocytes (developing T
lymphocytes). Although fewer studies have been performed using normal developing B
lymphocytes, some research has shown that glucocorticoids can also induce apoptosis in
these cells, mostly IgM' B cells, in vitra (Garvy et al., 1993; Grifﬁths et al., 1994; Merino
et al., 1994). Very few in vitra studies of the effect of Gc on B or T lymphocytes have
been performed using the natural Ge (corticosterone [Cs] in rodents). Natural and
synthetic Gc have been reported to have different afﬁnities for the Ge receptor and
different biological efﬁcacies. To focus on physiological rather than pharmacological
effects of Go, the effect of the natural steroid hormone, Cs, rather than synthetic Gc has
been investigated here. In viva studies were performed herein to determine the effect of
increased concentrations of circulating Cs on each stage in B lymphocyte development to
determine if losses induced by Cs correspond with populations thought to be more
susceptible to apoptosis. In vitra studies were also performed to thoroughly determine
the apoptotic effect of Cs on developing B lymphocytes in culture.

Additionally, this thesis investigates potential modulation of Cs-induced apoptosis
in developing B lymphocytes in vitra by cells and factors that would normally be found
in the BM microenvironment. B cell lymphopoiesis depends on contact with, and factors
secreted from, large ﬁbroblast-like cells found in the bone marrow called stromal cells.
Stromal cell lines have been utilized here to determine if, in addition to promoting B cell
lymphopoiesis, these cells could modulate the B lymphocyte response to Cs. A cytokine

derived ﬁ'om stromal cells, interleukin-7 (IL-7), has been shown to support B cell

lymphopoiesis, when added to B lymphocyte cultures not containing stromal cells
(Namen et al., 1988). This cytokine has been shown to be speciﬁc for early stages of B
and T cell lymphopoiesis and clinical interest in this cytokine for immunotherapy has
recently developed (Maeurer et al., 2000; Westermann et al., 1998). Therefore we have
investigated the direct effect of IL-7 addition on B lymphocyte response to Cs in vitra.
The cytokine stem cell factor (SCF) promotes the commitment of early blood cell
progenitors to the B lineage in combination with IL-7 (McNiece et al., 1991). SCF also
ampliﬁes the lymphopoietic effect of IL-7 on early stages during B cell development, so
the affect of this cytokine on B lymphocyte induced apoptosis, alone and in combination
with lL-7 has also been investigated.

In Chapter 1 of this document the literature on B cell lymphopoiesis,
glucocorticoids and apoptosis will be reviewed. This will be followed by three data
chapters covering 1) the in viva effect of Cs elevation on developing B lymphocytes 2)
stromal cell mediated modulation of the affect of Go on B lymphocytes in vitra and 3) the
in vitra effect of IL-7 and/or SCF on Gc-induced apoptosis and cell cycle arrest in bone

marrow B lymphocytes.

CHAPTER 1: LITERATURE REVIEW

B CELL LYMPHOPOIESIS

Backgron

B cell lymphopoiesis occurs in the bone marrow of adult marmnals and is a highly
complex, multi-stage process that results in the generation of mature B cells that are
involved in host defense. Membrane bound immunoglobulins (Ig) are the molecules
responsible for B cell recognition of antigen and are composed of two heavy chains and
two light chains. The generation of the immense diversity required for antigen
recognition is via the recombination of the heavy and light chain genes (Rast and Litman,
1998). Brieﬂy, recombination is achieved by rearrangement of several variable gene
segments to the constant domain. This occurs for both the heavy and light chains. In
addition to diversity derived from recombination, there are ﬁve heavy chain isotypes that
result in various effector functions. The genes are mu, delta, gamma, alpha and epsilon
which result in ﬁve isoforrns, IgM, IgD, IgG, IgA and IgE, respectively. On the cell
surface Ig molecules are associated with Igor and IgB coreceptors that are part of the B
cell receptor (BCR) complex (Hombach et al., 1990; Kashiwamura et al., 1990;
Wienands, 2000). Ligation of Ig causes an intracellular phosphorylation cascade through
the cytoplasmic domains of [go and IgB that results in increased proliferation and
differentiation to plasma cells that produce large quantities of soluble lg (antibodies)
(Justernent, 2000; Matsuo et al., 1993; Nomura et al., 1991). Antibodies mediate
neutralization of toxins, initiate the complement cascade and elicit T cell responses in

defense of foreign pathogens.

The dysregulation of B cell lymphopoiesis can result in several diseases. Multiple
disorders such as systemic lupus erythematosis, asthma and rheumatoid arthritis can
result from B cells with faulty or “anti-self” Igs produced via dysregulated
lymphopoiesis. This thesis is concerned with the down-regulation of lymphopoiesis,
which can occur during physiological stress and can result in compromised immune
responses. In fact, this lab has extensively studied on the affects of zinc deﬁciency (a
common example of physiological stress) on lymphopoiesis (DePasquale—Jardieu and
Fraker, 1984; Fraker et al., 1977; King etal., 1995; Osati-Ashtiani et al., 1998). Zinc
deﬁciency results in massive losses of precursor (3220+IgM') B lymphocytes in the bone
marrow, suggesting that downregulation of B lymphopoiesis may be the major
mechanism whereby peripheral B lymphocytes are decreased, leading to a compromised
immune system. During zinc deﬁciency and other conditions of chronic stress, a steroid
hormone, glucocorticoid (Go), is believed to induce many of the physiological responses.
In fact this lab has also shown that chronic elevation of Go decreased BM B cell in mice
(Garvy et al., 1993). The mechanism by which lymphopoiesis is downregulated by

glucocorticoids is not well deﬁned and this is the phenomenon that this thesis will focus

011.

Model Systems

There are currently few treatments available for the devastating, sometimes fatal,
health scenarios presented above and there are often no reliable cures. Lack of progress
in this ﬁeld can, at least in part, be attributed to a lack of understanding of the

mechanisms whereby B lymphocytes develop into functional mature cells and how

homeostasis is maintained. A multi-stage, complex maturation process in the highly
heterogeneous bone marrow (BM) compartment can be concluded to be one main reason
for our lack of advancement. The majority of our knowledge about B cell lymphopoiesis
has been attained from cell lines, short-term whole bone marrow cultures or long-term
bone marrow cultures. The murine (mouse) model has proven to be very useful in
studying lymphopoiesis, and while not identical to the human system has shown
fundamental similarities (Akashi et al., 2000; LeBien, 1998). The use of immortalized
cell lines to study B lymphocytes has proven to be useful for determining important
intracellular processes such as transcription and signaling in speciﬁc stages of B cell
development. However, a limitation of cell lines is that they are not always a reliable
model for determining cellular functions such as differentiation, cell cycle and cell death
responses, since these are the systems often modiﬁed from normal by irnmortalization.
Short-term bone marrow cultures have been valuable for studying some responses of
primary developing B cells, although within a few days the majority of B cells will die
due to the absence of microenvironmental support factors. The advent of a long term
culture system developed by Whitlock and Witte (Whitlock et al., 1984), which will be
revisited later, has resulted in a reliable in vitra system to speciﬁcally grow and
regenerate B lymphocytes for long periods of time. This review addresses the murine
system and the experiments presented in this thesis were performed on primary mouse

cells or stromal cell lines unless otherwise speciﬁed.

Hematopoiesis

Figure 1.1 is a diagram of the generation of the various lineages of blood cells
from a hematopoietic stem cell. In the BM of adult mammals all lineages of blood cells
are generated and, for the most part, go through multiple stages of development there. T
cells are a notable exception; they are generated in the BM and then migrate to the
thymus for maturation. Overall, there are at least eight different blood cell lineages
present in various stages of maturation in the BM. Developing and mature B
lymphocytes compose approximately 30% of the total cells of the marrow. The B cells
are a branch of the lymphoid lineage along with T and natural killer (NK) cells. In fact
many parallels are seen between the development of B and T cells, including
differentiation, proliferation and selection based on the generation of a ﬁrnctional BCR or
T cell receptor (TCR), respectively. The study of T cell lymphopoiesis has been aided by
the nearly homogeneous tissue that the majority of their development takes place in, the
thymus. On the other hand, to study primary B lymphocytes there was a vital need for
methodology to identify speciﬁc subsets of cells within the very heterogeneous

population of cells found in the marrow.

Flow Cytometry: Phenotypic Markers for Monitoring B Cell Lymphopoiesis

F low cytometry has proven to be of great use in analysis of heterogeneous tissues,
such as the BM. F luorochrome conjugated antibodies to surface proteins can be used to
identify various cell types. Multi-parameter ﬂow cytometry can allow for the analysis of
several ﬂuorochromes simultaneously, thus the expression of several cellular proteins can

be analyzed simultaneously. This “multi-color” labeling has been useful in not only

identifying the various developmental stages of B lymphocytes in BM, but also in
identifying distinct developmental subsets within the population. For many years mature
B lymphocytes were identiﬁed and puriﬁed based on surface Ig expression. Precursor
cells, committed to the B lineage but lacking surface lg, could not be identiﬁed from
other cell types found in the marrow. By immunizing rats with murine B cells and
neoplastic pre B cell lines, a monoclonal antibody was produced which could identify lg
expressing B cells and precursor B cells (Cofﬁnan and Weissman, 1981). The antibody
recognized a 220,000 MW glycoprotein (B220, CD45RA); it was determined that this
antibody was speciﬁc for the B lineage. 3220 is a phosphotyrosine phosphatase that
dephosphorylates and inactivates src tyrosine kinases such as 1yn (Katagiri et al., 1999;
Satterthwaite and Witte, 1996). The precursor cells have been further differentiated since
then based on stage-speciﬁc expression of various proteins.

Several labs have developed murine B lymphocyte identiﬁcation schemes
independently and the incongruent nomenclature and differing phenotypic markers used
in identiﬁcation has added confusion to the ﬁeld, although each supports the same order
of development based on the progression of Ig recombination (Lu et al., 1998; Osmond et
al., 1998). This lab has adopted the established phenotypic nomenclature developed by
Hardy et a1 (Hardy et al., 1991). Figure 1.2 is a detailed representation of the ordered
development of B lymphocytes showing surface proteins, relative size and expression of
signiﬁcant proteins in each deﬁned stage in development. It incorporates information
that will be presented throughout this review on B lymphopoiesis. Pro B cells can be
identiﬁed by the surface expression of the molecule recognized by the S7 monoclonal

antibody (leukosialin, CD43) (Hardy et al., 1989). S7 is the clone designation and it is

also used as the nomenclature for the antigen it recognizes. Although it is expressed on
many cell types in the marrow, it is speciﬁc for the pro B cell stage of development in
8220+ B lymphocytes of the bone marrow. S7 is a highly sialylated integral membrane
protein which has been shown to play a role in adhesion (Laferte and Dennis, 1988,
Walker, 1999 #195). The pro B cells can be subdivided further based on expression of
heat stable antigen (HSA) (CD24) and a membrane bound aminopeptidase, BP—l (Ly-51,
6C3) (Wu et al., 1991). HSA (CD24) is a highly glycosylated protein which is involved
in adhesion and in the regulation of lymphopoiesis. In transgenic mice that overexpress
HSA and in HSA knockout mice, the generation of early B lymphocyte precursors is
downregulated (Hough et al., 1996; Nielsen et al., 1997). Although BP-l expression is
tightly regulated in B cell development a functional role has not been assigned, and
lymphopoiesis is normal in BP-l deﬁcient mice (Lin et al., 1998; Wang et al., 1998).
The earliest pro B cells, pre-pro, do not express either HSA or BP-l (BZ20+S7+HSA'BP-
1'). The expression of HSA on the surface identiﬁes the next stage of development, the
early-pro B cells (3220+S7IHSA+BP-l'). Late-pro B cells are identiﬁed by the
expression of BP-l (B220+S7+HSA+BP-l+) as development proceeds toward the pre B
cell stage the expression of S7 is lost (8220+S7‘HSA+BP-l+). Irmnature B cells have
lgM on their surface, and maturity of this lineage is marked by the coexpression of
surface lgM and IgD. The orderly progression through these stages to maturity is based
on the rearrangement of Ig heavy and light chain genes and the expression of various
proteins that play a role in the rearrangements, differentiation, proliferation and
apoptosis. This will be discussed more in-depth below and is addressed throughout the

experimental sections.

Immunoglobulin Gene Rearrangement

In general pro B cells are deﬁned as cells committed to the B lineage prior to
expression of cytoplasmic 1; heavy chain. The identiﬁcation of a variety of other
distinctive features of these cells has led to a better functional understanding of their
development and maturation. Using reverse transcriptase polymerase chain reaction (RT-
PCR) ampliﬁcation techniques, the ordered progression of heavy chain gene
rearrangements was elucidated (Hardy et al., 1991). The earliest pro B cells (pre-pro as
deﬁned by Hardy) have Ig genes in a gerrnline conﬁguration and the cells are small and
noncycling. Although evidence exists which suggests that this population may not be
entirely composed of cells committed to the B lineage, a substantial ﬁ'action of these cells
retain the ability to differentiate into mature B cells. The next deﬁned stage, the early-
pro B cells, are a large, proliferating cell type undergoing recombination of the p. heavy
chain diversity regions (DH) to joining (JH) regions. In the late pro B cells, DJH
recombination is completed and joined to one of several variable gene segments (V H).
These cells are also large and proliferating. Upon completion of [.1 heavy chain
rearrangement, and subsequent cytoplasmic expression, the cells are considered pre B
cells. There exists a stage where the pre B cells are large and actively cycling followed
by a stage of non-proliferation and smaller size (Coffrnan, 1982, Osmond, 1986 #132)
(see Figure 2). At this stage the pre B cells undergo recombination of the joining region
to either a kappa or lambda light chain variable region (VK or VA, respectively).

Completed lgM rearrangement results in export to the cell surface, deﬁning the cells as

11

immature B lymphocytes. Maturity is accomplished following production and surface

expression of IgD.

B Lymphocyte Homeostasis and Selection

The complexity of this recombination process suggests a high potential for faulty
rearrangements, yet its importance suggests a need for quality control. In fact studies on
the population dynamics of B lymphopoiesis indicated that the production of BM B
lymphocytes far exceeds the number of mature cells generated (Osmond, 1990; Osmond,
1986). Using tritiated thymidine incorporation to study in viva proliferation it was
discovered that certain precursor B cells actively cycle. These were identiﬁed as being
large cellsgin the late pro B cell stage and in the pre B cell stages of development. Further
experiments using vincristine to block cells in metaphase were performed to determine
the rate of proliferation of the various B cell subsets (Opstelten and Osmond, 1983).
Together, these experiments led to the estimation that approximately ﬁfty million B cells
are generated per day in the bone marrow, but only around ten percent of those are
exported ﬁom the marrow. The major losses appear to occur in transition from the pro B
to pre B and from pre B to immature B cells (Lu and Osmond, 1997). This suggested
that, indeed, many B lymphocytes were somehow being eliminated and this was
consistent with the probability of the generation of faulty and anti-self Ig gene
rearrangements. Therefore, it is widely accepted that B lymphocytes with nonsense and
anti-self lg gene rearrangements are eliminated before reaching maturity.

The mechanism by which cells identify faulty or functional heavy chains began to

be elucidated by the identiﬁcation of the expression of a surrogate light chain in early

stages of B cell development. The surrogate light chain is composed of two non-variable
molecules, termed A5 and VpreB (Kudo and Melchers, 1987; Sakaguchi and Melchers,
1986). Surrogate light chain associates with rearranged )1 heavy chains via a disulﬁde
bond and allows for its transient surface expression (Karasuyama et al., 1990). These
associated molecules along with Igor and IgB are termed the pre-B cell receptor (pre-
BCR). The pre-BCR appears to be used as a checkpoint molecule for either ﬁrrther
differentiation or elimination of the cell (Karasuyama et al., 1994; Rolink et al., 2000).
Transgenic mice with a defective pre-BCR, due to a disrupted A5 gene, show a block in
the progression of pro B cells into pre B cells (Kitarnura et al., 1992; Rolink et al., 1993).
In addition, the SCID defect in mice results in the inability to rearrange Ig genes and
therefore B lymphopoiesis is halted early in the pro B cell stage (Bosma et al., 1999;
Bosma et al., 1983; Schuler et al., 1986). Insertion of a completed it heavy chain and
pre-BCR expression in SCID mice allows the progression to the pre B cell stage (Chang
et al., 1995). Another suggested checkpoint in B lymphopoiesis occurs immediately
following surface expression of the completed lgM molecule. This can again be
demonstrated using the SCID model. After insertion of a completed 11 and progression
to the pre B cell stage, maturity only occurs with insertion of a recombined light chain
(Reichman—Fried et al., 1990). The transgenic expression of a kappa light chain allowed
pre B cells to differentiate into mature B cells, although at a two to three fold lower
number of cells. These experiments have given strong evidence for the stages in B cell
development where cells with functional lg gene rearrangements are positively selected.
It is now widely accepted that elimination of deve10ping B lymphocytes during

lymphopoiesis occurs via apoptosis. Several lines of evidence support this hypothesis.

In viva detection of apoptotic cells is minimal due to rapid phagocytosis of cells at early
stages of death (F adok and Henson, 1998; F adok et al., 1992), but in vitra culturing
techniques have given some insight into death in developing B lymphocytes. Isolation of
BM and short term culture have shown that B lymphocytes undergo apoptosis. Pre, late
pro and immature B cells undergo a higher rate of apoptosis, relative to the early pro and
mature B cells, when removed from the BM microenvironment (Lu and Osmond, 1997).
In addition, it has been shown that early pro B cells and mature B cells have elevated
levels of Bel—2, a protoonco gene with anti-apoptotic potential (Li et al., 1993; Merino et
al., 1994). ‘Cursory studies have also shown that precursor B cells, not expressing surface
IgM, are more susceptible to apoptosis induced by the synthetic glucocorticoid,
dexamethasone (Garvy et al., 1993; Lu and Osmond, 1997; Merino et al., 1994). In
transgenic mice with elevated expression of Bel-2 in B lymphocytes, cell survival is
heightened (Strasser et al., 1991). In Bel-2 deﬁcient mice, B lymphopoiesis is normal
through birth, but soon thereafter B lymphocytes are absent in the bone marrow, spleen
and periphery (Nakayama et al., 1993; Veis et al., 1993). These observations are
fundamental to this thesis, since we are thoroughly studying Gc induced apoptosis in B
cell lymphopoiesis. An in depth review of the mechanisms of apoptosis and

glucocorticoid affects on immune cells will be covered later.

Stage Specific Gene Expression
The expression of some important genes, whose products are involved in B
lymphopoiesis, have also been determined. RT-PCR was used to detect these genes from

sorted primary cell populations (Li et al., 1993). The recombinase activating genes,

14

recombination activating genes 1 and 2 (RAG-l and RAG-2), code for proteins involved
in the recombination machinery (Schatz et al., 1989); their gene expression pattern
correlates speciﬁcally with the B cell populations undergoing active rearrangement.
They are upregulated in pro B cells, transiently down regulated in large, proliferating pre
B cells and again are expressed in small pre B cells, undergoing light chain
rearrangements. Mice lacking functional RAG-1 or 2 are unable to undergo I g gene
rearrangements and B lymphopoiesis is blocked in the pro B cell stage (Mombaerts et al.,
1992; Shinkai et al., 1992). Terminal deoxynucleotidal transferase (tdt) is an enzyme
involved in adding random nucleotides to the heavy chain joining regions to increase
diversity (Desiderio et al., 1984; Landau et al., 1987). It is expressed in the pro B cell
populations, but it is not detectable in the pre B cells that have completed heavy chain
rearrangements (Opstelten et al., 1986). These expression patterns correlate with the
stages in which their functions are critical, therefore in addition to surface phenotype and
status of Ig rearrangement, the presence of certain intracellular proteins can deﬁne the
various stages of development.

Recently several transcription factors have been identiﬁed which appear to be
critical in the differentiation of developing B lymphocytes. PU.1 and Ikaros are two such
factors. Transgenic PU.1 -/- or Ikaros -/- mice, have arrested B cell development at a
stage prior to the generation of pro B cells (McKercher et al., 1996; Scott et al., 1994;
Wang et al., 1996). Another transcription factor indicated in development of B
lymphocytes are the E2A gene products E12 and E47. Mice with a targeted null E2A
mutation fail to develop B cells early in development, before DJ rearrangements (Bain et

al., 1994; Bain and Murre, 1998). B-cell-speciﬁc activator protein (BSAP-S) is a

transcription factor that has been shown to be involved in heavy chain recombination
(Hagman et al., 2000). It is expressed in B cells through the pro B cell stage of
development and its elimination blocks the development of cells after DJ H
rearrangements (Nutt et al., 1997; Urbanek et al., 1994). Nuclear factor kappa B (N FKB)
is a molecule that was originally identiﬁed as a transcription factor that binds to an
enhancer element for the K light chain (Sen and Baltimore, 1986). Several family
members have been identiﬁed and appear to have redundant function in B lymphopoiesis,
since eliminating one of these factors does not halt lymphopoiesis (Burkly et al., 1995;
Sha et al., 1995). Although, the constitutive expression of a dominant negative form of
the NFKB inhibitor (IKB) does result in inhibition of VKJ K rearrangement and
transcription of the K light chain (Scherer et al., 1996). These ﬁndings demonstrate that
speciﬁc transcription factors are involved in stage-speciﬁc differentiation during B
lymphopoiesis. This underscores the complex and tightly regulated nature of the

development of B lymphocytes through various differentiation stages.

Microenvironmental Effects on B Lymphopoiesis

The BM microenvironment is critical for B cell lymphopoiesis. In the BM,
precursor B lymphocytes develop in close association with the cytoplasmic projections of
reticular-like cells. Figure 1.3 is a diagram depicting the development of B cells within
the context of the matrix of the microenvironment. In viva injection of radiolabeled
antibodies to B lymphocytes demonstrated that the earliest progenitors are found near the
bone cortex and as the cells mature they progress towards the central core of the marrow

for exportation to the periphery (Jacobsen and Osmond, 1990; Jacobsen et al., 1990;

16

Osmond, 1990; Osmond et al., 1992). The microenvironment plays an integral role in
lymphopoiesis as is demonstrated by high losses of B lymphocytes following their
removal from the BM. It was believed that the reticular cells that the lymphocytes were
in contact with were supporting lymphopoiesis by providing direct contact and soluble
factors for the developing cells. Cells that provide support during the development of
other cell types are called stromal or nurse cells. Here we use the term stroma as the
nomenclature for the cells of the bone marrow that support B cell lymphopoiesis

Long term bone marrow cultures (LTBMC) developed by Whitlock and Witte
(Whitlock et al., 1984) provided a system by which B lymphopoiesis could be generated
and sustained for months or even years. Whole bone marrow is placed in culture and
after approximately 2 weeks an adherent layer of cells, that contains stromal cells, is
established and following 3-4 weeks after initiation lymphoid cells are constitutively
generated. In young cultures, through 8 weeks, the predominant lymphocytes in culture
are IgM' B cells with few lgM cells. These precursor cells retain the ability to fully
recombine Ig genes and differentiate into mature B cells. In older cultures the
predominant cell type is lgMI. This culture system has provided a means to reliably
elucidate many key players in B lymphopoiesis. Prior to the development of the B
lymphopoietic culture system a culture system that promoted myelopoiesis was
developed (Dexter et al., 197 7). Interestingly, a major difference between these culture
systems was the addition of glucocorticoids to the medium, which promoted the
development of myeloid cells. This suggested that perhaps glucocorticoids might
suppress lymphopoiesis and/or promote myelopoiesis. The work presented in this thesis

addresses the negative effect of glucocorticoids on B lymphopoiesis, thereby providing

17

insight into the mechanism whereby these two different culture systems can lead to the
development of two different types of blood cells. Additionally, the affect of increased
concentrations of Cs on myelopoiesis will be addressed in the experimental portion of
this dissertation.

Morphological analysis of the adherent layer of LTBMC showed two distinct cell
types, a large, cytoplasmically spread, ﬁbroblast-like cell and a macrophage-like cell
(Witte et al., 1987). The ﬁbroblast-like cells were termed stromal cells, since the B
precursors developed in close association speciﬁcally with this cell type (W itte et al.,
1987). Although stromal cells have not been shown to be identical in nature, some
general characteristics of the cell type were established. For example, stromal cells ﬁom
LTBMC express surface vascular cellular adhesion molecule (VCAM l) and CD44, and
various components of the extracellular matrix (ECM) such as collagen IV, ﬁbronectin
and laminin are also expressed (Witte et al., 1993; Witte et al., 1987). More importantly,
stromal cells are responsible for the production of various cytokines that support
lymphopoiesis (Funk et al., 1995). In fact, two cytokines that will be discussed
thoroughly later in this review, interleukin 7 (IL-7) and stem cell factor (SCF, kit ligand,
steel factor), are produced speciﬁcally by stromal cells in LTBMC. In addition, stromal
cells produce a pre B cell growth stimulating factor (stromal derived factor-1, SDF-l) and
thymic stromal lymphopoietin (TSLP) (Levin et al., 1999; Nagasawa et al., 1994). The
production of macrophage colony stimulating factor (M-CSF) is also speciﬁc to stromal
cells, whereas the macrophages produce the interleukin 1 beta (IL-113) cytokine. Stromal
cell lines have also been established from LTBMC, many of which can support both

myelopoiesis and lymphopoiesis depending on the culture conditions used (Collins and

Dorshkind, 1987; Dorshkind et al., 1986). Some variation in expression of proteins can
be observed with different clones and the degree of support provided to B lymphocytes
can also vary (Henderson et al., 1990). In fact, some BM stromal cell lines have been
shown to have a negative affect on lymphopoiesis (Borghesi et al., 1997). This suggests
that stromal cells have the ability to both support or inhibit B cell development, yet little
is known about the mechanism by which these cells can affect the lymphocyte so
differently.

It has also been noted that stromal cell function can be modulated by exogenous
factors that in turn can affect lymphopoiesis. For example, the addition of the aryl
hydrocarbon 7,12-dimethylbenzanthracene to cultures containing B lymphocytes and
bone marrow stromal cells resulted in apoptosis of the B cells (Yarnaguchi et al., 1997).
This induction of death was due to either the stromal cells or stromal cell derived factors
since the B lymphocytes were shown to contain very few aryl hydrocarbon receptors and,
in cultures without stromal cells, the B lymphocytes did not undergo apoptosis. Estrogen
also appears to down regulate B cell lymphopoiesis in long term cultures via modulating
stromal cell function rather than by directly affecting the B cells (Smithson et al., 1995).
Treatment of B lymphocytes alone, did not induce cell death or inhibit proliferation,
whereas a reduction in B cell lymphopoiesis occurred when B cells were cultured on
stromal cells that were pretreated with estrogen. Speciﬁc cytokines have also been
shown to modulate the expression of surface proteins on stromal cells. IL-l B addition to
stromal cell clones resulted in the upregulation of both CD54 and VCAM-1 on the cell
surface, whereas IL-4 upregulated only VCAM-l and TGF-B downregulated expression

of VCAM-1 on stromal cells (Dittel et al., 1993). Both CD54 and VCAM-1 have been

19

indicated in providing direct cell-cell contacts with B lymphocytes that promote B cell
lymphopoiesis. Clearly various molecules that would normally be found in the bone
marrow microenvironment can cause modiﬁcation in stromal cell ﬁrnction, thereby
potentially modifying their affect on the development of blood cells. Although the
normal effects of stromal cells on B cell development have been studied in depth, little is
known about the effect that Cs might have on these cells. Considering what is known of
Cs negative long-term effects on B lymphocyte development this thesis addressed the
potential of stromal cells to modify B cell responses to Cs and the potential for Cs to

directly modify the production of cytokines known to be involved in lymphopoiesis.

Cell-Cell and Cell-Extracellular Matrix Contact

Considering the close association of the precursor B lymphocytes with stromal
cells both in LTBMC and in situ, the role of direct contact appeared to be important in
lymphopoiesis. One hypothesis regarding the close association between B lymphocytes
and stroma was simply the physical retention of developing cells in the marrow.
However, it was observed that suspending lymphocytes over stromal cells by using a
porous membrane resulted in decreased cell viability and loss of cell differentiation, thus
arguing for a more intricate role for cell-cell and cell-extracellular matrix (ECM)
adhesion in lymphopoiesis (Borghesi et al., 1997; Kiemey and Dorshkind, 1987; Manabe
et al., 1994). Contact between pro B cells and stromal cells enhanced survival of the pro
B cells and resulted in an upregulation in the anti-apoptotic Bel-2 protein and a
concomitant decrease in pro-apoptotic Bax protein levels (Gibson et al., 1996). In

addition to adhesion playing a role in supporting B cell lymphopoiesis it has also been

20

suggested that lymphocytes can initiate signaling cascades in stromal cells (Jarvis and
LeBien, 1995; Jarvis et al., 1997). The incubation of B lymphocytes directly on a stromal
cell line, resulted in tyrosine phosphorylation at focal adhesion points in stromal cells,
indicating the initiation of adhesion mediated signaling. Therefore the interactions
between stromal cells and lymphocytes appear to be more complex than originally
thought with each cell type potentially able to affect the other in a positive or negative
manner.

There are multiple potential adhesive interactions that could play a role in stromal
cell supported B lymphopoiesis. Miyake et a1 (Miyake et al., 1991) attempted to identify
if adhesion molecules on bone marrow stromal cells could directly affect B cell
development. This was done by creating monoclonal antibodies against cell surface
proteins. Two antibodies, M/K-l and M/K-2, had speciﬁc afﬁnity to a surface molecule
on stromal cells present in LTBMC and in the bone marrow. Addition of the antibodies
to newly initiated LTBMC resulted in the ablation of lymphopoiesis in preestablished
cultures and also resulted in the rapid detachment of lymphoid cells. This antibody
recognized a molecule with sequence homology to human VCAM-1. One ligand of
VCAM-1 is the 01481 integrin (VLA-4), therefore an antibody to the or subunit of VLA-4
was made to determine if it would have similar effects on lymphopoiesis (Miyake et al.,
1991). In fact, it did cause rapid detachment of lymphocytes in LTBMC and its addition
to newly established cultures completely blocked lymphopoiesis. This indicated that
VCAM-l on stroma and VLA-4 on lymphocytes interact and facilitate lymphopoiesis.
VLA-4 is also known to use ﬁbronectin as a ligand, but blocking peptides that disrupted

this interaction did not disrupt adhesion or lymphopoiesis. Additionally, irnmunostaining

21

showed that VCAM-l and VLA-4 are located together in areas of contact between
stromal cells and precursor B lymphocytes (Murti et al., 1996, Jacobsen, 1996 #78). It
has also been demonstrated that adhesion of human precursor B cells to stromal cells is
mediated to a great extent by the interactions of VLA-4 and VCAM-l (Dittel et al.,
1993). Thus, direct cell-cell interactions between stromal cells and B lymphocytes are

important in the normal development of B lymphocytes in the bone marrow.

Stromal Cell Derived Cytokine Support
In addition to the important role of contact in stromal cell support of
lymphopoiesis another critical role of stromal cells is the production of cytokines,
although cytokine effects and close association between stromal cells and lymphocytes
can be interrelated. Many cytokines are produced in membrane bound forms as well as
soluble forms. Although close association with the stroma may augment these effects it
has also been shown that soluble support via the stroma can also increase B lymphocyte
survival and proliferation, although not to the extent of direct association (Borghesi et al.,
1997). In 1988 a stromal cell derived factor, IL-7, was identiﬁed and puriﬁed, that
supports proliferation of pre B lymphocytes (N amen et al., 1988; Namen et al., 1988).
Biochemical analysis identiﬁed it as a 25-kilodalton glycoprotein and its structure of four
’ alpha helical bundles placed it in the Type-I class of cytokines. In culture, without
stromal cell support, recombinant IL-7 can support prolonged proliferation of IgM' cells
(Lee et al., 1988). Initially the responding cells are consistent with late pro B cells and

pre B cells: cytoplasmic if, BP-l+ and IgM' (Lee et al., 1989). Extended culture periods

with IL-7 ultimately resulted in an accumulation of cytoplasmic u” B lymphocytes,

22

corresponding with the early pro B cell stage. Subsequently it was discovered that IL-7
also played a similar role in thyrnocyte development (Okazaki et al., 1989; Watson et al.,
1989). It induced proliferation in early CD8'CD4' thymocytes. Since then, T cells and
thymocytes have been used extensively in the study of IL-7 and the IL—7 receptor (IL-7R)
and relatively little work has been done using B lymphocytes. It can be surmised that, at
least in part, the complexity of the B cell system has hindered research efforts in this area.
In addition to inducing proliferation, IL—7 could promote survival of early B and T
cells. One mechanism whereby IL-7 might promote survival is by the activation of the
phosphatidyl inositol 3-kinase (PI3K) pathway (Dadi et al., 1993; Pallard et al., 1999).
This survival pathway will be discussed in greater detail later in this thesis, but brieﬂy
PI3K phosphorylates AKT (protein kinase B) thereby inactivating the pro-apoptotic Bad
protein. It has also been shown that IL-7 can promote survival by maintaining or
upregulating anti-apoptotic Bcl-2 levels and decreasing pro-apoptotic Bax levels (Lu et
al., 1999). IL-7 upregulated Bel-2 protein levels and increased cell survival in early
precursor thymocytes and in a T lymphoma cell line (Kim et al., 1998; Lee et al., 1996;
von Freeden-Jeffry et al., 1997). Although Bel-2 may play a role in IL-7 induced
survival it may not be the exclusive survival mechanism. In mice deﬁcient for the IL-7
receptor, overexpression of Bel-2 did not rescue B lymphopoiesis, but did promote the
survival of circulating (mature) B cells (Maraskovsky et al., 1998). It should be noted
that IL-7 receptor deﬁciency also resulted in a lack of proliferation in the developing B
lymphocytes, therefore Bel-2 could potentially promote the survival of B lymphocytes
but this effect would not be seen due to the lack of expansion of the cells. Therefore, the

role of Bel-2 in IL-7 mediated survival has not been completely elucidated. This thesis

23

will provide further insight into the effect of IL-7 on apoptosis and cell cycle arrest in B
lymphocytes induced by Cs.

In 1990 the receptor for human and murine IL-7 was cloned (Goodwin et al.,
1990). Binding studies revealed that the receptor exists in both a high and low afﬁnity
form. The receptor is observed on many murine cell types, both myeloid and lymphoid.
The high afﬁnity form of the receptor is found on IgM' B lymphocytes and in CD4'CD8'
or single positive thymocytes. Injection of a blocking antibody to the high afﬁnity form
of the IL-7R resulted in a dramatic decrease in B cell precursors and thymocytes,
although peripheral lymphocytes remained for two weeks (Sudo et al., 1993). These data
supported an important role for IL-7 in lymphopoiesis. Later studies, utilizing transgenic
mice, showed that elimination of IL-7 or IL-7R resulted in ablation of B lymphopoiesis,
except for an odd population of immature B cells found in the spleen (Corcoran et al.,
1998; von Freeden-Jeffry et al., 1995). The exact role of IL-7 in B lymphopoiesis is still
being investigated. It was originally thought that the cytokine was involved strictly in
proliferation and survival of precursor B cells. More recently support is growing for a
role of IL—7 in differentiation as well. It was shown that IL—7Ror knockout mice have
precursor B lymphocytes with normal D-J recombination of the heavy chain but there is a
marked reduction in V gene recombination, correlating with the physical distance of the
V gene from the recombination site (Corcoran et al., 1998). In addition, the expression of
the pax-5 gene, whose product, BSAP-S, binds the heavy chain and stimulates
recombination is signiﬁcantly reduced. To determine if signaling through the IL7R could
result in lg gene rearrangement, a mutation was made on the cytoplasmic domain of the

receptor (Corcoran et al., 1996). This mutation caused elimination of the proliferative

effects of IL-7 but did not affect Ig heavy chain rearrangement. The IL-7R, therefore, has
been shown to promote several distinct intracellular responses through its cytoplasmic
domain.

Crosslinking studies showed that the IL-7Ror subunit was associated with the
common y chain (yo) of the IL-2 receptor (Noguchi et al., 1993). Association with yo
enhanced the binding affinity of IL-7Ror and increased receptor internalization upon
ligand binding. In addition, blocking antibodies to yo inhibited IL-7 induced
proliferation of a B cell line (Kondo et al., 1994). IL-4, IL-9 and IL-15 also contain the
yc as part of their receptors. The cytoplasmic tail yc has been shown to associate with
janus kinase family member Jak3 and stimulate STAT signaling resulting in proliferation
(Malabarba et al., 1995; Miyazaki et al., 1994). Src kinasesgand phosphatidyl inositol 3-
kinases (PI3K) have also been shown to be activated in response to IL-7.

Another cytokine that appears to be involved in B cell lymphopoiesis is stem cell
factor (SCF). SCF is a glycosylated homodimer that plays a broad role in aiding the
commitment of hematopoietic progenitors to the various lineages when present in
combination with speciﬁc grth factors for those speciﬁc cell types (W itte, 1990). To
date, the only cytokine that has been shown to be critical in B lymphopoiesis is IL-7, but
SCF synergizes with IL-7 to increase proliferation of pro and pre B cells (McNiece et al.,
1991). In addition, this combination of cytokines results in differentiation of 3220'
murine bone marrow cells into B220+ B lymphocytes, whereas neither cytokine, alone,
can promote the commitment to the B lineage. SCF can be produced in both soluble and

membrane bound forms. Experiments whereby only the soluble isoforrn of SCF was

25

produced indicated that the membrane bound form likely is the active form of the
cytokine (Miyazawa et al., 1995).

The receptor for SCF is c-kit which is a receptor tyrosine kinase expressed on the
surface of many hematopoietic cells; on B cells its expression is restricted to the pro B
cell stage (Rico-Vargas et al., 1994). Like IL-7, SCF can initiate multiple signaling
cascades in the cell and it has shown some ability to promote survival. It may, in part,
promote survival through the PI3K induced activation of AKT (Blume-Jensen et al.,
1998). Other activating cell signaling pathways that have been shown to be activated by
SCF binding of c-kit are the JAK/ STAT, MAPK and Src family transduction pathways
(Linnekin, 1999). Another tyrosine kinase receptor, ﬂt-3, appears to parallel c-kit in its
function on B lymphocytes. Like c-kit, it is expressed on pro B lymphocytes and its
ligand, FL, can induce proliferation in pre-pro B cells (Hirayama et al., 1995; Matthews
et al., 1991). Considering the similarities and the differences between the biological
effects and signaling pathways of IL-7 and SCF this thesis investigates the effect of both
of these cytokines on background and Cs-induced apoptosis to determine whether they
have similar, different or additive effects on B lymphocytes.

Since the discovery of the above-mentioned factors, other stromal cell derived
molecules that may play a role in lymphopoiesis have been discovered. Two such factors
are thymic stromal lymphopoietin (TSLP) and stromal derived factor-l (SDF-l). TSLP
has been shown to act as a costimulatory factor with IL-7 and promotes proliferation
through the immature stage of development (Friend et al., 1994). Interestingly, it was
shown that the receptor for TSLP is composed of the IL-7Ror chain and the novel TSLP

receptor (Levin et al., 1999). Whereas TSLP appears to affect later stages in B cell

26

development, SDF-l appears to elicit its effect on earlier B cells. SDF-l can induce
proliferation in pre B cells and has also been shown to be a chemoattractant factor for
various progenitor populations (Aiuti et al., 1997; Nagasawa et al., 1994). Mice deﬁcient
in SDF-1 or its receptor, CXCR4, have a block in fetal lymphopoiesis before the pro B
cell stage. It is becoming clear that many factors are involved in the successful
generation of mature B lymphocytes and that they may elicit their effects at different

developmental stages.

27

Summary

The generation and development of B lymphocytes in adult mammals is a tightly
regulated, multi-stage process that occurs in the highly heterogeneous bone marrow. It is
the complex and critical rearrangement of Ig genes that appears to drive the development
of B cells. Two advances have aided in progress in this ﬁeld, ﬂow cytometry and a long
term culturing technique that promotes B cell lymphopoiesis. Flow cytometry has
provided a powerful tool for analysis of the B lymphocytes in their various stages of
development and LTBMC has allowed for the discovery of several microenvironmental
support factor critical in lymphopoiesis. lL-7 is a stromal cell derived factor that, in
particular, has been shown to be critical in early stages of B lymphocyte development in
mice. Without IL-7 pro and pre B lymphocytes cannot survive or proliferate long-term in
culture. In addition to LTBMC, transgenic mice have provided a system for deﬁning the
in viva effects of many of these lymphopoietic molecules. Taken together, it is becoming
clear that during the development of B lymphocytes a myriad of factors work together to
promote cell survival, proliferation and differentiation and that response to these factors
depends on the stage of development. Each developmental stage can be deﬁned by
selective expression of relevant proteins and surface markers and proper recombination
of lg is critical for advancement to maturity. Even with the experimental advances that
have been made in the last two decades, much work remains in eliciting the mechanism
of B lymphopoiesis, especially in determining how normal physiological changes may
modulate this process. This lab has a unique advantage in studying normal bone marrow
B lymphocytes in various stages of development in part due to access to a multi-

pararneter ﬂow cytometer. Considering this advantage we have chosen to investigate the

28

effect of Go on normal developing B lymphocytes from progenitor through maturity. As
will be further discussed in the next portion of this literature review, Gc can have
dramatic negative effects on B cells, yet little is known of this phenomenon. Here we
will determine in viva and in vitra responses of normal B lymphocytes to Go, as well as

the potential modiﬁcation of these responses by stromal cells, IL-7 or SCF.

GLUCOCORTICOIDS AND THE IMMUNE SYSTEM

Background

Glucocorticoids (Gc) are steroid hormones that are produced in the cortex of the
adrenal gland. Their basal production is critical to mammals, since the lack of Ge can
result in death. Natural variations in serum levels of Ge occur in a diurnal fashion with
levels in humans being highest in the morning hours and in nocturnal animals, such as
rodents, levels are highest at night (Nichols and Tyler, 1967). Levels of Go are regulated
by adrenal corticotrophin hormone (ACTH; corticotrophin) produced in the anterior
pituitary gland. ACTH is upregulated by vasopressin and corticotrophin releasing
hormone (CRH), whereas glucocorticoids cause its down-regulation (Engler et al., 1999).
Together this cascade of hormones link together neurobiology and endocrinology and is
referred to as the hypothalamic-pituitary—adrenal (HPA) axis. The major external effector
of the HPA axis is stress. Stress can be deﬁned as anything that disturbs homeostasis.
Some stressors, such as exercise, are thought of as positive and elicit a transient increase
in Go. Other stressors induce a chronic increase in the production and circulation of Ge.
Classic examples of negative stress are seen in burn patients, trauma victims,
malnourishment and depression. It is chronic exposure to Gc that can have a variety of
negative effects including compromised immune function. Therefore it is this
phenomenon that is of interest here.

As early as 1947 the relationship between stress and Ge elevation was suggested
(Selye, 1947). Just 2 years later, it was proposed by Hench (Hench, 1949) that Go

mediate anti-inﬂammatory action. In fact, for decades synthetic Gc such as prednisone

30

and dexamethasone have been used as anti-inﬂammatory drugs in diseases such as
arthritis, autoimmunity and asthma. This provides both pharmacological as well as
physiological reasons for studying the effects of glucocorticoids. Along with the noted
anti-inﬂammatory effects of glucocorticoids, immunosuppression is evident. In patients
with the aforementioned stressors, there is often a marked decrease in peripheral
lymphocytes (lymphopenia) and ultimately compromised immune defense against
infection.

This lab became interested in glucocorticoid induced irrnnunosuppression while
studying zinc deﬁciency in mice. It was observed that zinc deﬁciency resulted in chronic
elevation of the naturally occurring Gc, Corticosterone (Cs) (in humans the major
circulating glucocorticoid is cortisol) (DePasquale-Jardieu and Fraker, 1980).
Adrenalectomy of these mice resulted in protection of the immune system during zinc
deﬁciency (DePasquale-Jardieu and Fraker, 1980). There is evidence that suppression of
lymphopoiesis is at least partially the mechanism whereby Gc induces lyrnphopenia.
Thyrnic atrophy has been a hallmark of elevated Gc, indicating that it may decrease the
numbers of developing T lymphocytes. It was later determined that indeed developing T
lymphocytes were being lost, most notably the double positive (CD4+CD8+) precursor
thymocytes (Cohen, 1992). The effect of elevated Go on B cell lymphopoiesis was not
investigated until relatively recently, when our lab and others analyzed the BM
compartment in mice with elevated levels of the steroid. Garvy et a1 (Garvy et al., 1993)
demonstrated that chronic elevation of Cs in mice resulted in selective depletion of the
developing B lymphocytes in the BM. It was also noted that IgM‘ precursor B cells were

more sensitive to these Cs induced losses and suggested that the mechanism may be due

31

to Go mediated cell cycle arrest and apoptosis in these cells. The role of glucocorticoid
induced apoptosis and cell cycle arrest will be thoroughly discussed in the apoptosis
section of this review.

In addition to the negative regulatory effect of elevated Go on developing B and T
lymphocytes, it appears that Go can also play a role in normal lymphocyte selection and
even survival under some conditions. In the thymus it was recently shown that
corticosterone could be produced by epithelial cells (Vacchio et al., 1994). In
experiments using radiolabeled steroid precursors, thymic tissue was capable of complete
glucocorticoid metabolism, suggesting that the thymus contains all of the necessary
enzymes for steroid biosynthesis (Lechner et al., 2000). In thymic organ cultures, the
elimination of Cs production resulted in the loss of cells that would normally be
positively selected by crosslinkage of the TCR (V acchio and Ashwell, 1997; Vacchio et
al., 1998). In addition to promoting survival of “selected” T cells, it was observed that
glucocorticoids could inhibit apoptosis induced by serum depletion in a Bel-2 expressing
T lymphoma cell line (Huang and Cidlowski, 1999). To date, there does not appear to be
any evidence for the role of Go in the positive selection of B lymphocytes, but given the
parallels between the two processes it seems likely that Go may play a similar role in B
lymphopoiesis. These observations underscore the complex role that Go plays in the
immune system and suggest that the steroid may act as an effector for cell survival or cell
death depending on the context of the cell. This thesis addresses Gc induced
downregulation of B lymphopoiesis, induction of apoptosis and cell cycle arrest on
normal B lymphocytes, thus deﬁning conditions whereby Gc has a negative effect on B

cell development.

32

Glucocorticoid Biochemistry

As previously stated the synthesis of glucocorticoids occurs in the cortex of the
adrenal gland. The precursor for all steroid hormones is cholesterol and the majority of
cholesterol used in steroid hormone biosynthesis is trafﬁcked into the adrenal gland by
either low density lipoprotein (LDL) in humans or high density lipoprotein (HDL) in
nrice. In the adrenal the majority of cholesterol is stored in lipid vesicles until stimulation
of synthesis (Vinson et al., 1992). Figure 1.4 is a diagram of the pathways of steroid
biosynthesis and the enzymes involved. The ﬁrst step is the generation of pregrrenalone
by the cytochrome P450 side chain cleavage enzyme. This is followed by 3 B-
hydroxysteroid dehydrogenase A 4,5-isomerase reaction resulting in progesterone. In
humans a Nor-hydroxylase followed by a 21- and 113- hydroxylase reaction generates
cortisol. It has been long believed that the Nor-hydroxylase is absent in rodents,
therefore the 21-hydroxylase and 1 1 B-hydroxylase reactions result in the production of
corticosterone, which differs from cortisol only in lacking a hydroxyl group at carbon 17.
More recently though, evidence for l l B-hydroxylase activity in murine in vitra adrenal
cultures has been demonstrated (Touitou et al., 1990). In addition to synthesis of
glucocorticoids, mineral corticoids and sex hormones are also derived from cholesterol in
the adrenal gland.

Following synthesis and release from the adrenal gland, Gc are transported
through the system bound to the corticosteroid binding globulin (CBG, transcortin)
(W estphal and Devenuto, 1966). However, it is believed that the biologically active form
of Go is not bound to CBG, but exists in a ﬁ'ee state (Bright, 1995; Mendel et al., 1989).

A dynamic equilibrium between free and bound Go is apparent, therefore suggesting that

33

regulation of Go binding to CBG might result in partial control of Go activity. In fact,
CBG levels have been shown to decrease in response to stress such as surgery in humans
(V ogeser et al., 1999). These decreases have also been observed in rodents exposed to
classical stressors (Tannenbaum et al., 1997). It is believed that the downregulation of
CBG results in an upregulation of free Go. In addition, at sites of inﬂammation it has
been shown that elastase can cleave CBG, drastically reducing its afﬁnity for Go
(Pemberton et al., 1988). These results suggest that not only are Go actions controlled by

upregulated production of the steroid but also by modulation of its availability.

The Glucocorticoid Receptor
Following transport throughout the system, Gc elicit their effects by binding to
the glucocorticoid receptor (GcR). The GcR belongs to the steroid hormone receptor
(SHR) superfamily of proteins and is present in nearly all mammalian tissues at
thousands of molecules per cell (Ballard et al., 1974). This family includes receptors for
the sex hormones, thyroid hormones and arylhydrocarbons (such as dioxin). In 1985 the
human GcR was cloned, demonstrating the existence of both or and B isoforrns
(Hollenberg et al., 1985). The or isoform was shown to be the ligand binding form
composed of 777 amino acids whereas the non-ligand binding B form was missing 15
amino acids at the carboxyl terminus. It was recently shown that the GcRB is expressed
at modest yet varying levels and may act as a ligand independent negative regulator of
GcR (Bamberger et al., 1995). The or form of GcR has several functional domains, which
are consistent throughout the SHR family. The protein contains a ligand binding domain,

a DNA binding domain and two transactivating domains (AF-l and AF -2). A diagram of

34

the GcR is shown in Figure 1.5. Sequence analysis and crystallography of the DNA
binding domain revealed the presence of two zinc ﬁnger motifs (Freedman and Luisi,
1993). The loop structure formed by zinc binding between four cysteine residues is a
common feature of DNA binding proteins. The ligand binding domain is located at the
carboxyl terminal end of the protein. Radiolabeled ligand afﬁnity studies have shown
that dexamethasone binds the receptor with high afﬁnity. The natural steroids, cortisol or
corticosterone, bind with modest afﬁnity and sex hormones bind with only low afﬁnity.
The transcription activating domain, AF- 1 , is located at the amino-terminal end of the
receptor and its activity is independent of ligand binding (Almlof et al., 1997). In
contrast the AF -2 transactivating domain is located in the ligand binding region and is
dependent on steroid binding (Danielian et al., 1992). Steroid binding is thought to cause
a transformation of the AF-2 region, allowing coactivating proteins, which mediate
transcription, to bind the SHR (Feng et al., 1998; Wagner et al., 1995).

Early studies showed that under hormone free conditions a large form of GcR was
located in the cytosolic fraction of cell extracts (Baxter and Tomkins, 1971).
Interestingly, the addition of steroid resulted in a lower mass receptor that could be
isolated from nuclear fractions (Higgins et al., 1973; Schmidt et al., 1982). This led to
the hypothesis that Go binds to a large inactive receptor complex in the cytosolic
compartment resulting in release of associated factors and translocation to the nucleus. It
was later determined by irnmunohistochernical studies that indeed Gc binding to the GcR
resulted in translocation of the receptor from the cytosol to the nucleus. In pursuit of
identiﬁcation of the putative associated proteins of the inactive GcR complex, cytosolic

receptor was isolated and puriﬁed (Gustafsson et al., 1989; Rexin et al., 1992). A 90-

35

kilodalton protein was isolated and ﬁrrther immunolabeling and sequence analysis
showed the protein to be heat shock protein 90 (hsp90). Point mutations in the ligand
binding domain of the GcR resulted in loss of hsp90 binding. This suggested that hsp90
bound the ligand binding domain of the OCR (Chakraborti et al., 1992). Further
coprecipitation studies of the GcR suggested that there were a variety of associated
proteins. To date the cytosolic form of the OCR has been shown to have two hsp90
molecules, hsp70, hsp56 and ‘a small immunophillin p23 molecule associated with it.
Upon binding of steroid to the receptor, this protein complex dissociates allowing bound
receptor to translocate to the nucleus. GcR are phosphorylated proteins that undergo
hyperphosphorylation upon agonist ligand binding (Bodwell et al., 1998). The
hyperphosphorylation appears to be critical in GcR regulation of transcription, since
receptors mutated at certain phosphorylation sites have severely impaired transcriptional
transactivation. Additionally, phosphorylation occurs in regions of association with cell
cycle dependent kinases. Interestingly, phosphorylation of the OCR appears to be
dependent on the cell cycle status. In the experiments performed thus far, ligand binding
induced hyperphosphorylation in the S phase of the cycle, but not during szM (Hu et al.,
1997). Collectively these data suggest that the regulation of GcR activity is modulated

by more than just ligand binding.

Glucocorticoid Receptor Signaling
In 1974 it was observed that the insect glucocorticoid, ecdysteroid, caused pufﬁng
on polytene chromosomes of Drasaphila melanagaster, suggesting that Go may elicit its

effect by modulating transcription (Ashbumer et al., 1974). It was later determined that,

36

in fact, GcR was affecting transcription of certain genes via binding to a DNA consensus
sequence, 5’-GGTACAnnnTGTTCT-3’ (Beato et al., 1989; Chandler et al., 1983; Karin
et al., 1984). The GcR DNA binding site was termed the glucocorticoid response
element (GRE). Exactly how receptor binding to DNA regulates the transcription
machinery has not been determined. There is evidence for direct interactions between
GcR and the transcription initiation complex and for association of the GcRs with
coactivator proteins (Tsai and O'Malley, 1994). Members of the p160 family of proteins
(steroid receptor coactivator 1 [SCRl ], GcR-interacting protein 1 [GRIP1]) were found to
associate with the AF -2 region of nuclear receptors and act as coactivators of
transcription following ligand binding (Hong et al., 1996; Onate et al., 1995). Recently
these coactivators have been found to promote transcription activation by binding the
histone acetyltransferases (HAT) CBP or p300 (Torchia et al., 1997). The HAT activity
promotes transcription by acetylating histone H3 of chromatin, thus modifying chromatin
structure to allow transcription machinery access to the DNA (Kuo and Allis, 1998). In
addition to HAT activity CBP and p300 have been shown to directly bind the basal
transcription machinery. Therefore, GcR, may play a direct role in stimulating
transcription by controlling chromatin structure and perhaps by directly interacting with
the transcription initiation complex.

Active ligand-bound GcR has also been implicated in cross-talk with other
signaling cascades. Interestingly, it was long believed that steroid hormones mediated
transcription activation, but recently it was shown that SHR can also negatively regulate
transcription of certain genes. Gc appears to negatively regulate transcription of AP-l

and NF -kB regulated genes. AP-l promotes transcription of immediate early genes

37

stimulated by growth factors and NF -kB stimulates transcription of genes involved in
immune cell activation and inﬂammatory cytokines. In vitra experiments suggested that
GcR negatively regulates AP-l transcription by directly interacting with the fos/jun
heterodimer (Heck et al., 1994; Lucibello et al., 1990). This interaction is ligand
dependent, but does not require GcR binding to the DNA. In contrast, the repressive
effects of GcR on NF-kB activities do not appear to be mediated by direct interactions
between the two proteins. NF -kB is bound to 1ch in its inactive cytoplasmic form
[reviewed in Hatada, 2000 #323]. Activation of NF-kB requires the degradation of 1ch
followed by nuclear translocation and transcription initiation. It appears that GcR
inhibits NF-kB activity by upregulating 1ch gene transcription, thus rendering NF-kB to
its inactive form (Auphan et al., 1995). Cross-talk between nuclear receptors and
transcription factors is an active area of study and is proving to be important in the
understanding of how Gc affects cells.

In addition to the direct effect of GcR on cellular processes, it is also becoming
apparent that hsp90 may also mediate signaling following its dissociation ﬁ'om the GcR.
The creation of a fusion protein containing the GcR hormone binding domain (also the
hsp90 binding domain) and the adenovirus ElA protein, resulted in steroid
responsiveness by the otherwise innocuous ElA (Picard et al., 1988). The results
suggested that release of the inhibitory protein complex may result in at least a partial
steroid effect. In addition, hsp90 also interacts with pp60"'src kinase and has been shown
to diminish its kinase activity (Xu and Lindquist, 1993). It is becoming apparent that
signal transduction via the GcR is a multi-faceted process that can inﬂuence or be

inﬂuenced by various other cellular signals.

38

Another mechanism for regulating GcR mediated signaling may be via the direct
transcriptional upregulation or downregulation of the receptor. It has been shown that Go
itself can downregulate the transcription of GcR, ultimately resulting in decreased levels
of the receptor protein (Bumstein et al., 1994; Cidlowski and Cidlowski, 1981). In
addition to the negative effect on transcription, Gc binding to the OCR results in
destabilization of the receptor. Recently, the phosphorylation state of the GcR had been
implicated in the stability of the receptor, with hyperphosphorylation induced by ligand
binding correlating with decreased receptor stability (Bodwell et al., 1998). Abolishment
of the eight phosphorylation sites, either individually or in combination, has shown that
decreased levels of phosphorylation correlates with decreased transactivation of
responsive genes and increased receptor half-life (Webster et al., 1997). The exact
mechanism of GcR stability and modiﬁcation is not well understood, although it is

becoming clear that GcR mediated pathways are tightly regulated.

39

Summary

Glucocorticoids are hormones that have a diverse range of effects on many
physiological functions in mammals. Here we have focussed on the effect of Go on the
immune system, speciﬁcally the development of B lymphocytes. At basal concentrations
Gc appear to play a positive role in normal homeostasis during thymocyte development,
yet the effect of basal levels of Go on B lymphocyte development has not been conﬁrmed
but can be surmised to likely play a similar role. A chronic increase in Ge production and
circulation, due to the chronic activation of the HPA stress axis, clearly has a negative
affect on the immune system. This lab has had an integral role in determining how the
induction of the stress axis via zinc deﬁciency has had a negative affect on the immune
system and B lymphocyte development and the speciﬁc downregulation of BM B
lymphocytes by chronic Cs elevation. Since then identiﬁcation techniques and
methodologies have advanced dramatically in this ﬁeld, allowing for further investigation
into the effects that Go have on developing B lymphocytes in viva and in vitra. The
downregulation of developing B lymphocytes by Go likely occurs by both a decrease in
cell survival (apoptosis) and a downregulation in proliferation. These phenomenon will
be discussed in the next section of this review. Additionally, many in vitra experiments
studying B lymphocyte responses to Go have utilized the synthetic Gc, dex, yet little is
known of the effects of the naturally occurring Cs on B lymphocytes. This thesis will
thoroughly investigate the effects of Cs on normal BM B lymphocytes from mice to
provide new insights into the mechanism of Ge negative actions on these developing

immune cells.

40

APOPTOSIS

History

In 1972 Kerr and Wyllie (Kerr et al., 1972) observed that in normal tissue
development, certain cells underwent cell death in an ordered manner with distinct
morphological characteristics. This form of death was subsequently termed apoptosis
and varied ﬁom necrotic cell death in that cytoplasmic contents were not released to the
extracellular environment, thus avoiding an uncontrolled immune response. Nearly a
decade passed before a modest interest in apoptosis was scientiﬁcally revived. It wasn’t
until the 1990’s that the fundamental importance of apoptosis became widely recognized
and sparked intensive research in this area. It is now clear that cell death is germane to
the homeostasis of multicellular organisms and dysregulation of it can have serious health
consequences.

Excessive apoptosis has been indicated as playing a central role in several
diseases. A classic example is apparent in the etiology of acquired immunodeﬁciency
syndrome (AIDS). Central to the manifestation of AIDS is the excessive elimination of
helper T lymphocytes by apoptosis (Meyaard et al., 1992; Wang et al., 1999). The faulty
induction of apoptosis is also apparent in neurodegenerative diseases, such as Parkinson’s
and Alzheimer’s (Waggie et al., 1999). Along with excessive apoptosis, the failure to
undergo appropriate cell death also can contribute to disease. In B cell follicular
lymphoma, a genetic translocation results in the upregulation of the anti-apoptotic protein
Bel-2 (this protein will be discussed in detail later in this chapter) (Bakhshi et al., 1985;

Cleary et al., 1986). The malignant cells in this type of cancer did not exhibit increased

41

proliferation as was thought to be a hallmark of malignancies, rather they failed to die
normally. Apoptosis is also thought to play a signiﬁcant role in the proper development
of B and T lymphocytes with death being induced in cells with faulty Ig or T cell receptor
rearrangements, respectively. The failure of B or T cells with anti-self antigen receptors
to undergo proper elimination can lead to autoimmune diseases such as Systemic Lupus
Erythematosis, asthma and rheumatoid arthritis (O'Reilly and Strasser, 1999). Relative to
this thesis, our lab has implicated apoptosis in the mechanism of downregulation of
developing B lymphocytes in response to Go elevation (Garvy et al., 1993). A
downregulation in the development of B and/or T lymphocyte can result in decreased
circulating cells (lymphopenia) that ultimately results in a compromised immune
response. Clearly the past ten years have brought apoptosis to the foreﬁont in the study
of the etiology of many diseases and thus ﬁrrther study of this process could signiﬁcantly
contribute in the understanding, the control, and potentially the cure of sometimes fatal

diseases.

Apoptotic Morphology and Detection

Morphologically most apoptotic cells undergo a decrease in cell size and volume,
nuclear condensation, blebbing of the plasma membrane and eventual formation of
membrane bound apoptotic bodies. During early research on apoptosis a seminal
observation was made in thymocytes induced to undergo apoptosis with dexamethasone
(Wyllie, 1980). It was shown that in the apoptotic nucleus an endonuclease is activated
that caused DNA fragmentation, consistent with cleavage at the intemucleosomal linker

regions of chromatin. As previously stated, apoptotic cell death varies from necrotic cell

42

death in that the cytosolic contents remain membrane bound whereas during necrosis the
dying cell swell and ultimately releases its contents to the surrounding milieu. During
apoptosis the plasma membrane undergoes changes in ﬂuidity, resulting in a transition of
phosphatidylserine (PS) from the inner to the outer leaﬂet of the phospholipid bilayer
(Fadok et al., 1992). The exposure of PS then triggers rapid engulﬁnent by phagocytic
cells. Recently, a protein kinase C delta mediated scramblase has been proposed as the
mechanism whereby PS is exposed on the outer leaﬂet (Frasch et al., 2000). Another
ﬁrndamental difference between apoptosis and necrosis is the lack of intemucleosomal
DNA cleavage in necrotic cells. Late in necrosis DNA cleavage can occur, but results in
indistinct fragmentation, in contrast to the nucleosomal cleavage pattern seen in apoptosis
(Bicknell and Cohen, 1995).

Early studies in apoptosis depended on the morphologic analysis of the dying
cells, therefore more sophisticated research such as the analysis of cell death in
heterogeneous tissue was difficult if not impossible. Later an electrophoretic method was
developed that could determine the degree of apoptosis in cell populations by the
presence of a DNA “ladder”, indicative of intemucleosomal DNA cleavage. This
methodology aided somewhat in studying apoptosis, but it only provided quantitative
data and not qualitative identiﬁcation of dying cells. This lab made a signiﬁcant
contribution in methodology for the identiﬁcation of apoptosis in whole cells (Telford et
al., 1991; Telford et al., 1994). Cells could be ﬁxed in ethanol and the DNA stained with
propidium iodide whereby cells containing hypodiploid DNA were determined to be
apoptotic. This method allowed for the analysis of heterogeneous tissues, since mixed

populations could be phenotyped for speciﬁc cell types and analyzed on an individual

43

cell basis. Prior to this, apoptosis in bone marrow B lymphocytes, the major topic of this
thesis, could not be adequately studied unless complex puriﬁcation protocols or cell lines

were utilized.

Apoptosis Activation (Caspases and Mitochondria)

In the 1990’s intensive research efforts were applied to the identiﬁcation of a
central biochemical pathways that lead to the commitment to and execution of apoptosis.
Many intracellular processes were implicated as central death effectors, but further
investigation showed apoptosis initiation could occur independent of the proposed
effectors. Calcium inﬂux, generation of reactive oxygen species and endonuclease
activation were just a few processes implicated as central to the activation of apoptosis
(reviewed in Schwartzman and Cidlowski, 1993). Each of these were later shown to be
common features of apoptosis under many circumstances but not necessary for death. In
recent years with the effort of many laboratories a model of the central pathway of
apoptosis has been generated (Figure 1.6). It integrates both mitochondrial changes and
the activation of a class of proteases called caspases. The model also deﬁnes two distinct
types of apoptosis, receptor-mediated death that is initiated in healthy cells (caspase
dependent) and death that occurs in damaged or neglected cells (mitochondria
dependent). A brief overview of receptor-mediated apoptosis verses apoptosis in
damaged cells will be presented below, followed by a detailed overview of the central
death pathway. Brieﬂy, a classic example of cells that are healthy but eliminated is
evident in the immune response. This type of autoregulatory death occurs via the

crosslinking of death receptors of the tumor necrosis factor receptor (TNFR) superfamily

44

(Nagata, 1997). This results in the activation of the caspase cascade and ultimately
apoptosis. Mitochondrial depolarization and the release of cytochrome c is a common
feature seen in this type of death, but it is not necessary for it to occur (Los et al., 1995).
On the other hand, cells that undergo apoptosis due to neglect or damage depend on
mitochondrial changes but caspase activation is not required for death. The activation of
caspases is common and responsible for the morphology associated with apoptosis, but
blocking caspase activity does not block cell death. Neglect by serum deprivation or
DNA damage are two situations whereby blockage of caspase activity does not inhibit
cell death, and the death that occurs is more similar to necrosis in morphology (McCarthy
et al., 1997; Mills et al., 1998).

Research on cell death in the nematode, Caenarhabditis elegans, has led to
fundamental discoveries of the central molecular mechanisms of apoptosis. Genetic
mutants with cell death defects were created and two proteins called CED-3 and CED-4
were found to be activators of apoptosis. CED-3 was later cloned, and shown to have
homology to the mammalian cysteine protease, interleukin-1B converting enzyme (ICE)
(Yuan et al., 1993). At the time, ICE was known to process IL-l B into an active form,
but it had not been implicated in apoptosis. Further studies showed, indeed, that ICE
overexpression could itself induce apoptosis (Miura et al., 1993). Since then several ICE-
like proteases have been identiﬁed and were renamed caspases. To date 13 caspases have
been identiﬁed. A unique characteristic of the caspase family is their speciﬁcity for
cleavage after aspartate residues in proteins (Howard et al., 1991). There are many
intracellular substrates for caspase cleavage that once cleaved can elicit a biological

effect. The cleavage and activation of these proteins leads to the distinct morphology

45

seen in apoptosis. For example, caspase 3 has been shown to cleave and degrade proteins
involved in the cytoskeleton, such as actin (Kayalar et al., 1996). In addition to
generating morphological changes, the caspases can also act to amplify the death
pathway by cleaving anti-apoptotic proteins such as Bel-2, rendering them pro-apoptotic
(Cheng et al., 1997). Therefore many of the cellular changes associated with apoptosis
are due to caspase activity.

Along with action on cellular substrates caspases can cleave and activate
themselves, thus rendering a cascade of activated caspases. The caspases exist in an
inactive procaspase form. Both cytochrome c from the mitochondria and the newly
identiﬁed CED-4 homologue, apoptotic protease activating factor (APAF-1), are
necessary for caspase activation in apoptosis due to neglect or cell damage (Zou et al.,
1997). Cell neglect, such as lack of growth factors or cell damage can result in the
depolarization of the mitochondrial membrane and the release of cytochrome c (Vander
Heiden et al., 1997). Cytochrome c is thought to bind APAF-l and activate binding to
procaspase 9 resulting in autocleavage of the procaspase and thus formation of an active
caspase 9 (Srinivasula et al., 1998). It is then believed that caspase 9 activates caspase 3,
ultimately resulting in a cascade of caspase activity (Hakem et al., 1998).

Caspase activation due to TNF R family mediated apoptosis (death in otherwise
healthy cells) initiation proceeds by a different mechanism. TNFR family members that
promote death have a homologous cytoplasmic region termed the death domain (DD)
(Nagata, 1997; Tartaglia et al., 1993). The receptors include FAS (CD95), TNFR-I and
the death receptors DR3, 4 and 5. The DD allows recruitment of adaptor molecules that

can directly bind caspase 8 (Boldin et al., 1996; Muzio et al., 1996). Receptor

46

crosslinking via ligand binding is then thought to promote the cleavage of procaspase 8
into its active form, followed by activation of the caspase cascade. Mitochondrial
membrane depolarization and the release of cytochrome c typically occurs in this death
pathway, but it is not essential. Therefore it appears that there are at least two distinct
mechanisms for initiating the caspase cascade, one that is dependent on the mitochondrial
release of cytochrome c for caspase activation and another that can directly activate

caspases via receptor ligation.

Apoptosis Inhibition (Bel-2 Family of Proteins)

Before the discovery of caspases the homologue for the C. elegans cell survival
protein CED-9 was discovered. As previously stated, a gene translocation in B cell
follicular lymphoma resulted in the discovery of Bel-2. It was later discovered that CED-
9 and Bel-2 shared sequence and functional homology (Hengartner and Horvitz, 1994).
Prior to this observation it was discovered that Bcl-2 could functionally replace CED-9 as
a cell survival factor in C. elegans (V aux et al., 1992). Bel-2 is part of a large family of
proteins. The proteins are deﬁned as having at least one of four Bel-2 homology domains
(BHl, 2, 3 or 4). Interestingly, several members of the family have anti-apoptotic
potential, whereas others can induce apoptosis. Family members that promote survival
(e. g. Bel-2 and Bcl-X long isoform; Bel-XL) contain each of the BH domains. Death
promoting members (e. g. Bax, Bad and Bel-X short isoform; Bcl-Xs) vary in the number
of BH domains. Rather than functioning independently, it is commonly accepted that the
ratio of pro.death versus anti-death factors determines the activity. It is thought that

heterodimerization, between pro-apoptotic and anti-apoptotic proteins, disrupts activity

47

by disturbing homodimerization. Therefore, the protein in excess will form functional
homodirners. It is also believed that the majority of Bel-2 family members are targeted to
membranes via a transmembrane tail (Nguyen et al., 1993). In fact early observations
suggested that Bel-2 elicited its effects by maintaining the mitochondrial membrane
potential.

The exact function of the Bel-2 family members has not been fully elucidated, but
in recent years progress has been made. In 1996 the structure of Bcl-XL was determined
using x-ray crystallography and nuclear magnetic resonance (Muchmore et al., 1996).
This led to speculation that Bel-XL could potentially control mitochondrial membrane
homeostasis via pore formation due to structure similarities to bacterial toxins that
function by forming membrane pores. Since then Bel-XL, as well as Bel-2 and pro-
apoptotic Bax, have been shown to form ion channels in synthetic lipid bilayers (Minn et
al., 1997). Functionally, Bel-2 has been shown to maintain mitochondrial polarization
and block the release of cytochrome c into the cytosol (Marchetti et al., 1996). Bax, on
the other hand, promotes mitochondrial membrane depolarization and cytochrome c
release from the inner mitochondrial membrane space (Jurgensmeier et al., 1998).
Observations that Bax-regulated release of cytochrome c could occur prior to membrane
depolarization had also been made. This suggested that perhaps Bax might function as a
channel for cytochrome c release, but this has not been proven and remains a subject of
debate. Although the mechanism of action of these proteins have not been completely
deﬁned, it has become widely accepted that the Bel-2 family members do play an

important role in regulating mitochondrial activity in apoptosis.

48

Recent studies on the regulation of Bel-2 family members, have provided
evidence that their function can be modulated by intracellular signaling cascades. It was
commonly believed that the major regulatory mechanism was via expression levels of the
anti and pro-apoptotic molecules. Studies on the pro-apoptotic member, Bad, have
indicated phosphorylation as another regulatory mechanism (Zha et al., 1996). Bad was
discovered in 1995 (Yang et al., 1995). It varies from other pro-apoptotic family member
in that it contains only the BH3 domain and does not have a membrane binding motif.
Bad has been shown to be phosphorylated on the BH3 domain. This results in
association with the cytosolic protein 14-3-3. Further investigation showed that in an IL-
3 dependent cell line, IL-3 addition resulted in activation of the protein kinase AKT (or
protein kinase B) (Datta et al., 1997). AKT activation led to the phosphorylation and
cytosolic sequestration of Bad by 14-3-3. Removal of IL—3 resulted in the
dephosphorylation of Bad and subsequent heterodirnerization with Bel-XL. This resulted
in inactivation of Bcl-XL function and allowed for Bax homodimerization and pro-
apoptotic function. Interestingly, the cytokine lL-4 has also been shown to activate AKT
and promote thymocyte survival (Cerezo et al., 1998). A proposed model of Bad
function is presented in Figure 1.7. These fairly recent ﬁndings stimulate the hypothesis
that perhaps cytokine induced survival may at least in part proceed by the inactivation of
Bad activity. Cytokine induced survival will be discussed further later in this chapter.

In addition to phosphoryl-regulation through Bad, direct phosphorylation of Bel-2
may be another regulatory mechanism. The Raf-l serine/threonine kinase has been
shown to associate with Bel-2 at the mitochondrial membrane (Wang et al., 1994). It can

phosphorylate Bel-2 leading to its inactivation. This presents an interesting paradox,

49

since the Raf-l activating MEK pathway promotes cell survival, rather than cell death.
The SAPK pathway has also been indicated in Bel-2 phosphorylation (Maundrell et al.,
1997). Considering the death promoting activity of the SAPK pathway, inactivation of
Bel-2 is a logical progression. It is clear that the regulation of Bel-2 family members via
cell signaling cascades has not been fully elucidated and will remain an active area of

research.

Bel-2 Family Members in B Cell Lymphopoiesis

The role of Bel-2 family members in the development of mammals has been
addressed by creating gene knockout mice. Both Bcl-2 -/- and Bel-XL -/- mice result in
lethality (Kamada et al., 1995; Motoyama et al., 1995; Veis et al., 1993). Bel-2 -/- mice
die around three weeks after birth as a result of renal failure. In contrast, Bel-XL
knockout mice die prior to birth at approximately embryonic day 13. Lethality appears to
be due to faulty development of the brain. The immune system in both models also
exhibits dysregulation. In Bel-XL -/- mice, lymphocyte development in the embryonic
liver is blocked. To determine the effects of Bel-XL on lymphopoiesis, Bel-XL deﬁcient
embryonic stem cells were used to generate chimeric mice (Ma et al., 1995). In this case,
survival of developing thymocytes and B lymphocytes was dramatically reduced. The
Bel-2 -/- mice initially generated mature B and T lymphocytes, but by three weeks of age
both B and T cells were signiﬁcantly reduced. These results suggested that both Bel-XL
and Bel-2 were independently involved in generating functional lymphocytes. Bel-XL
appears to play a signiﬁcant role in early generation of lymphocytes, whereas Bel-2

functions in survival after the initial generation of the lymphoid compartment.

50

The expression of Bel-2 in murine B lymphocytes was shown to correlate with
developmental populations that were shown to be resistant to apoptosis in vitra. High
levels of Bel-2 were shown to be present in pre-pro and mature B lymphocytes ﬁom the
BM; early-pro B cells showed lesser, yet signiﬁcant expression levels (Li et al., 1993;
Merino et al., 1994). These and other experiments have indicated that normal in vitra
apoptosis and dex-induced apoptosis is greatest in pre B cells (Garvy et al., 1993; Nunez
et al., 1990). In transgenic mice and B cell lines with Bel-2 expression induced
throughout the developmental stages, the pre B cells were resistant to apoptosis. This
indicated that Bel-2 was likely important to normal B cell development and that its varied
expression might be important in selection processes. Although some in vivo studies by
our lab had looked at B lymphocyte sensitivity in IgM' verses lgM+ B cells (as described
earlier), the more deﬁned B lymphocyte populations had not been studied (Garvy et al.,
1993). Therefore this thesis addresses the sensitivity the various speciﬁc B lymphocyte
developmental substages to Cs elevation in viva, to determine if cell populations that

express Bel-2 are more resistant to losses.

Cytokine-Withdrawal Induced Apoptosis

In multicellular organisms each cell depends on signals from its environment to
survive. The context in which a cell is maintained provides for the homeostasis and
identity of the cell. When a cell is removed from its environment, and functioning
properly, it undergoes apoptosis. It has been proposed that each cell contains the
necessary components to undergo apoptosis and that extracellular signals are critical for

maintaining survival signals that repress the death process (Raff, 1992). This theory was

51

developed by observations that removing cells from their environment or by blocking
gene transcription results in apoptosis. A classic model for this has been observed in
immune cells, by withdrawal of cytokines that are necessary for survival (Boise et al.,
1993; Rebollo et al., 1995; Sohur et al., 2000). This is a commonly observed form of
apoptosis termed factor-withdrawal cell death. The recent discovery of IL-4 induced
suppression of Bad activity via the P13 Kinase/AKT kinase pathway, indicated that
cytokine receptors may function as survival signals by a common mechanism. Recent
studies had also shown that both lL-3 and IL-7 could induce translocation of the pro-
apoptotic Bax protein from the cytosol to the mitochondrial membrane (Khaled et al.,
1999). It has been proposed that the removal of these cytokines in relevant cell types
resulted in an intracellular increase in pH that caused a conformational change in Bax,
allowing for its membrane binding and ultimately apoptosis. In addition to pro-apoptotic
actions following withdrawal, the presence of several cytokines (including IL-7) had also
been indicated in the upregulation of the anti-apoptotic Bc1-2 protein (Dancescu et al.,
1992; Karawajew et al., 2000). These observations suggested that cytokine signals can
result in direct activation of proteins involved in the control of apoptosis, yet the ability
of cytokines to protect cells from apoptosis induction from other sources had not been
studied in depth. This thesis will explore the potential of lL-7 and SCF to protect normal
BM B lymphocytes ﬁom factor-withdrawal induced apoptosis and Cs-induced apoptosis.
Another molecule that appeared to have signiﬁcant anti-apoptotic function via
cytokine signaling was STATS (signal transducers and activators of transcription)
(Nosaka et al., 1999; Rosa Santos et al., 2000). In studies using STATS deﬁcient B and T

cell lines, the readdition of the molecule resulted in the upregulation of c-myc, Bel-2 and

52

Bcl-x (Lord et al., 2000). STATS activation is promoted by signaling through the janus
kinase 3 (J ak3) phosphorylation pathway. The IL-2 common y receptor had been shown
to recruit and activate J ak3 upon ligand binding, suggesting that the cytokines that share
this domain (IL-2, IL-4, lL-7, IL-9 and IL-1 5) may send this common signal, promoting
cell survival. This was also suggested by the lymphoid deﬁciencies observed in Jak3
knockout mice, since these mice are deﬁcient in B cell lymphopoiesis. (Thomis et al.,
1997). It therefore appeared that certain cytokines, especially those sharing the common
y chain receptor, could potentially promote survival by inhibiting apoptotic factors and

promoting anti-apoptotic factors.

Glucocorticoid induced Apoptosis

Although Gc-induced apoptosis has been studied for decades, the exact
mechanism by which these steroids induce death in cells of the immune system has not
been elucidated. This may be because it affects several cell survival and death processes
within the cell. The biological effect of Go on immune cells was studied long before
apoptosis became an active area of interest. It was observed that Go downregulated
metabolism in lymphoid cells. Cells treated with Gc showed decreased glucose uptake,
decreased intracellular ATP levels and down regulation in protein and nucleotide
synthesis was observed (Montague and Cidlowski, 1995). Although a general decrease in
transcription and translation was observed, Gc was also known to enhance the
transcription of many genes (reviewed in (Bumstein and Cidlowski, 1989). Experiments
that blocked Gc—induced transcription by coaddition of actinomycin D or protein

synthesis with cycloheximide, eliminated Gc-induced death in thymocytes (McConkey et

53

al., 1989). This led to the hypothesis that Gc-induced apoptosis caused, at least in part,
by inducing what were termed “death genes”. Many efforts were put into identifying and
deﬁning genes that were upregulated or downregulated in Gc-induced apoptosis. Further
investigation was not able to indicate any one of these genes as sufﬁcient or central to
cell death.

More recent efforts have been applied to determine how Gc may induce apoptosis
by affecting speciﬁc signaling pathways. Using mouse thymocytes, studies have
indicated that ceramide release, by sphingomyelinase (Smase) activity, may be one way
that Go effects death (Cifone et al., 1999). One group used several indirect methods to
inhibit Smase activity, thus inhibiting ceramide production and apoptosis. In addition,
they showed that blocking phosphatidylinositol phospholipase C (PI-PLC) activity
blocked the activation of Smase and ceramide production and apoptosis. These events
were shown to be upstream of GcR mediated transcription. This data contrasts other
observations indicating that Gc-induced apoptosis is dependent on GcR mediated
transcription (McConkey et al., 1989). One transcriptionally regulated pathway that has
been suggested in the mediation of Go induced apoptotic effects is the NFKB pathway. It
has been shown that downregulation of NFKB activity and one of its gene targets, c-myc,
by GcR mediated upregulation of IKB may be another mechanism by which Gc induces
apoptosis. Overexpression of NFKB or c-myc in dex treated thymocytes resulted in a
decrease in Gc-mediated apoptosis (Wang et al., 1999). Clearly, more research is needed
to fully elucidate the mechanism of Gc-induced apoptosis, but it is likely that the

mechanism will involve multiple intracellular processes.

54

Summary

Over the past two decades the study of apoptosis has shown that cell death is
critical in the homeostasis of multicellular organisms. Intensive research efforts were
applied to discovering the mechanism of this process. In the currently proposed models,
both caspases and mitochondrial dysregulation are the effectors of cell death.
Mitochondrial release of cytochrome c can induce the activation of caspases due to death
initiated by damage or neglect. Caspase activation is not required for death in this case,
but normally occurs and provides the apoptotic phenotype. Death via death receptor
ligation can directly initiate the caspase cascade and death, but mitochondrial release of
cytochrome c enhances this process. There is also a family of intracellular proteins (the
Bel-2 family) that have either pro-apoptotic or anti-apoptotic function. It is believed that
they function as regulators of the mitochondrial membrane. Less is known about how
cell death is initiated. Factor withdrawal induced death is one form of apoptosis
commonly observed. Certain cytokines (such as IL-4) can promote survival by inhibiting
the expression and activity of pro-apoptotic proteins. Their withdrawal then allows the
activation of pro-apoptotic proteins and results in the downregulation of anti-apoptotic
proteins. Glucocorticoid induced apoptosis induces death as seen in neglected or
damaged cells. Developing B and T lymphocytes are two cell types that undergo massive
apoptosis due to Go exposure. It is thought that this may play a role in regulating
negative selection of these cell types during lymphopoiesis. Cmrent evidence suggests
that Gc-induced apoptosis is a complex process that likely occurs via mediating
transcription of genes directly involved in the death process, by downregulating survival

pathways and by inducing other signaling pathways to induce death. Here Cs-induced

55

apoptosis in B lymphocytes will be thoroughly studied in vitra. Additionally, the effect
of other factors, such as cytokine signaling by IL-7 and SCF or the effect of stromal cells
as a whole, will be investigated for the ability to modify Cs action on the developing B
cells. This is a whole new approach to studying Cs-induced death in normal B cells that
explores how survival-inducing and death-inducing pathways affect B lymphocyte

responses when they are co-initiated.

56

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61

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62

 

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()3

CHAPTER 2: THE IN VI V0 EFFECT OF CORTICOSTERONE ON

DEVELOPING B LYMPHOCYTES: FROM COMMITTED PROGENIT OR TO

MATURITY

64

ABSTRACT

Chronic increases in circulating glucocorticoids (Gc), resulting from activation of
the stress axis, can cause decreased levels of circulating B lymphocytes and ultimately
compromise host defense. Reduced B cell development in the bone marrow (BM) has
been indicated as a mechanism contributing to Gc-induced lymphopenia, yet the affect of
Go on each B cell developmental substage was not known. Utilizing multipararneter ﬂow
cytometry, the effect of the natural Gc, corticosterone (Cs), on B lymphocyte
development, from progenitor cells to maturity, was investigated. The results showed
that a modest elevation in plasma Cs concentrations, analogous to concentrations seen
during physiological stress, caused a signiﬁcant reduction in the BM B cell population as
early as 12 hours; by 24 and 36 hours nearly 50% of the developing B lymphocytes were
lost. Thirty-six hours after increased exposure to Cs, 80% of the surviving cells were
IgMIgD+ mature B cells. The populations exhibiting the greatest losses were those that
had been reported to be undergoing active lg gene rearrangement; the early-pro through
the pre B cell stages of development. The earliest committed progenitors, the pre-pro B
cells, with Ig genes in the germline conﬁguration, were somewhat resistant to losses,
decreasing by only 23% aﬁer 24 hours and 34% after 36 hours. Immature B cells did not
show signiﬁcant losses after 24 hours, but were nearly eliminated by 36 hours. The
remaining pro and pre B cells, normally cycling populations, decreased in cells in the
S/GZ/M phases of the cell cycle by 74% and 85%, respectively. Therefore, Cs appeared
to negatively affect developing BM B lymphocytes by severely depleting all but the

earliest progenitors of developing B cells and by preventing their expansion by inhibiting

65

cell cycling. Additionally, analysis of major hematopoietic lineages of the bone marrow
indicated that whereas Cs had a negative effect on BM B lymphocytes, it appeared to
promote myelopoiesis by expanding the granulocyte compartment. Therefore modest
increases in Cs, comparable to concentrations seen during the induction of the
neuroendocrine stress axis, can result in the selective reduction of developing B
lymphocytes in as little as 12 hours of exposure. Interestingly, after 36 hours the other
developing blood cell lineages in the BM were either not affected or actually expanded in
response to increased Cs. Thus, it appears that during stress the energy-expensive
process of B cell development is downregulated, but perhaps compensated for by an

upregulation in the production of cells that provide the ﬁrst line of immune defense.

66

INTRODUCTION

There are important physiological and pharmacological reasons for interest in the
effect of glucocorticoids (Gc) on the immune system. The natural steroid hormones, like
corticosterone (Cs) or cortisol, are produced and released from the adrenal gland at basal
levels and can become elevated in response to stress (Selye, 1947). Zinc deﬁciency,
trauma, burns and neuroendocrine diseases are examples of chronic stress that can cause
enhanced production of Ge (DePasquale-Jardieu and F raker, 1980; Raber, 1998).
Conversely, synthetic Gc are commonly used at pharmacological concentrations as anti-
inﬂammatory drugs for diseases such as arthritis, asthma and autoimmune diseases.
Natural or pharmacological increases in Go concentrations can cause a decline in the
number of peripheral B and T cells. This lab has shown that a major cause of this
decrease could be the downregulation of B and T cell development in the primary
immune tissue (Garvy et al., 1993). Using Cs implants, Garvy et a1, produced
concentrations of circulating Cs in mice analogous to those seen during stress, potentially
resulting in a compromised host defense. This resulted in a dramatic decrease in thymus
weight and a decrease in developing B lymphocytes in the bone marrow. It was also
noted that early B lymphocytes, not expressing IgM, were very sensitive to Cs induced
losses. However little was known about how Cs affected the various subpopulations of B
lymphocytes as they matured from progenitor to maturity. Therefore it was important to
determine whether the losses were speciﬁc to certain developmental stages or if Cs

negatively affected all developing B cells to the same extent.

67

Such studies have been limited due to the complexity associated with the
identiﬁcation of subsets of cells within the B cell lineage, especially since they reside in a
heterogeneous tissue, the bone marrow (BM). All cells of the hematopoietic lineage
originate and most mature in the BM of adult mammals. One exception are the T
lymphocytes, which are generated in the BM, then migrate to the thymus to mature. B
lymphocytes develop entirely in the marrow in adults and comprise approximately 30%
of murine BM cells. Over the last decade ﬂuorochrome conjugated antibodies against B
cell surface proteins has been utilized for the ﬂow cytometric identiﬁcation of distinct
stages in B lymphocyte development. The B lymphocyte lineage is identiﬁed by an
antibody that recognizes CD45RA (8220) (Cofﬁnan and Weissman, 1981). Using the
phenotypic scheme developed by Hardy et al. (Hardy et al., 1991) 8220+ B cells can be
further subdivided, from the earliest progenitor to maturity, as follows: pre-pro
(S7+HSA“BP1’), early-pro (S7+HSA+BP1'), late-pro (S7+HSA+BP1+), pre (S7'IgM'),
immature (IgM+IgD') and mature (IgM+IgD+). The status of Ig gene rearrangement has
been determined for each of these stages of development (Faust et al., 1993; Li et al.,
1993). Pre-pro B cells have Ig genes in a germline conﬁguration; recombination of the
heavy chain begins in early-pro and is completed in late-pro B cells. Pre B cells contain a
completed heavy chain and undergo light chain gene rearrangements. At the immature
stage of development lgM rearrangement is complete and it is expressed on the surface of
the cell. This well developed phenotypic system provides the tools needed to better
determine the effect of Ge on cells of the B lineage.

During the Ig rearrangement process it is estimated that nearly 80% of developing

B cells are lost due to faulty or anti-self recombination events (Osmond, 1986). A

68

downregulation of the anti-apoptotic protein, Bcl-2, has been observed in cells
undergoing Ig recombination, suggesting that induction of apoptosis may be a major
mechanism of precursor B cell deletion of unwanted cells in the bone marrow (Li et al.,
1993; Merino etal., 1994; Nunez et al., 1990). Only the pre-pro and mature B cells
express substantial amounts of Bel-2, therefore, it was of interest to determine if
increased endogenous production of Cs might adversely affect those stages in B cell
development that do not express Bcl-2. This would suggest that apoptosis might play a
role in the Gc mediated downregulation of B cell lymphopoiesis seen both in vivo and in
vitro (Garvy et al., 1993; Garvy et al., 1993; Merino et al., 1994).

Subdermal implantation of a Cs containing pellet can cause increased
concentrations of circulating Cs being analogous to concentrations produced during
physiological stress. Previous studies from this lab focussed on the effect of Cs on B
lymphocyte after days or weeks of exposure. Beyond two days maximum depletion of B
lymphocytes was apparent and remained virtually unchanged for two weeks. Thus the
studies here were performed at 12, 24 and 36 hours after Cs implantation, to determine
the initial effects of increased concentrations of circulating Cs on developing B
lymphocytes and the thymus. A comprehensive investigation of the onset of Cs-induced
suppression of B cell lymphopoiesis has not been investigated. These experiments
demonstrate that the stages during B lymphocyte development where 1g gene
rearrangement is occurring, undergo dramatic cell losses aﬁer just a few hours of
exposure to steroid. Indeed losses began as early as 12 hours in early-pro and pre B cell
populations and maximum effects were seen in these and late-pro and immature

populations by 36 hours. In contrast, the populations that had been shown to contain

69

signiﬁcant levels of Bel-2 (pre-pro and mature B cells) were more resistant to losses due
to chronic exposure to Cs. Therefore in the time period of 12 to 36 hours the negative
effect of Cs on B cell lymphopoiesis in mice was dramatic.

Previous in vitro studies have shown that Gc causes lymphoid cells to undergo
cell cycle arrest in 60/61 (Andreau et al., 1998; King and Cidlowski, 1998). Pro and pre
B cells normally undergo expansion, whereas the immature and mature B cells are
quiescent in the bone marrow. Although a decrease in cycling lgM' precursors after
prolonged Cs exposure was previously noted, the initial affect on the individual pro and
pre B cells was not investigated. These experiments show a rapid and similar decrease in
cycling of surviving pro and pre B cells. Therefore in addition to inducing B cell losses,
Cs also rapidly reduced cell proliferation.

Interestingly the BM cellularity did not change subsequent to exposure to Cs, yet
there was a signiﬁcant decrease in B lymphocytes. It seemed probable that other cell
lineages were expanding during chronic exposure to Cs. Using a recently deﬁned
phenotypic labeling system, the cellular composition of key populations of cells of the
marrow was determined (de Bruijn et al., 1994). Differential expression of the cell
surface proteins ERMP12 (CD31) and ERMP20 (CD59), allows for the delineation of
granulocytes, monocytes, hematopoietic progenitors, erythroid progenitors and
lymphocytic cells. Interestingly, mice with Cs implants had a signiﬁcant increase in the
percent of granulocytic cells concomitant with a decrease in lymphocytic cells whereas
the other lineages appeared unaffected. These observations suggested that Cs, while
downregulating B cell development, might promote the generation of granulocytic cells.

Therefore a reduction in B cell development can occur within 12 hours of modest

70

physiological increases in Cs. After 24-36 hours B cell development is nearly completely
eliminated except for the earliest progenitors, yet the development of the other blood cell
lineages is not downregulated and in some cases their development is upregulated. Thus
it appears that very early after the induction of the stress axis the development of blood

cells shiﬁs ﬁom lymphopoiesis towards myelopoiesis.

71

MATERIALS AND METHODS

Mice and Cs Implantation
Young adult male Balbc/J mice were purchased from Jackson Laboratories (Bar Harbor,
ME), and were used from 8 to 12 weeks of age. Mice were housed in the laboratory
animal facility in the Biochemistry department and all protocols were approved by the
University Animal Use Committee at Michigan State University, East Lansing, MI. The
facility was maintained at 25°C with 12-hour light and dark cycles.

Methoxyﬂurane inhalation was used to anesthetize the mice and tablets containing
20 mg corticosterone (Sigma, St. Louis, MO) and 20 mg cholesterol were implanted
subcutaneously. Sham controls received tablets containing 40 mg of cholesterol. Post-
surgery the mice were housed in the animal facility on sterile bedding and fed

commercial rodent chow (Purina, St. Louis, MO) and acidiﬁed water.

Tissue Harvest and Processing

At 12, 24 or 36 hours aﬁer tablet implantation, mice were bled under anesthesia
and sacriﬁced by cervical dislocation. The plasma was separated from the blood and Cs
concentrations were determined as previously described (DePasquale-Jardieu and Fraker,
1980). Thymuses were removed to determine the amount of thymic atrophy by weight.
Bone marrow was ﬂushed from femurs with approximately 1 ml harvest buffer (Hanks’
balanced salts, 1 mM HEPES pH 7.2 and 4% FBS) using a syringe and 22 gauge needle.
The marrow was processed into a single cell suspension and red blood cells were

removed by lysis. The cells were then washed in harvest buffer, resuspended in 0.5 ml

label buffer (Hanks’ balanced salts, 1 mM HEPES pH 7.2, 0.1% sodimn azide and 2%
FBS) and placed on ice. Cell counts were performed and trypan blue exclusion was used
to determine bone marrow cell number and viability. Following cell counts, two million
cells were aliquoted into 5 ml polystyrene tubes (Becton Dickinson, Franklin Lakes, NJ)

for phenotypic analysis.

lmmunophenotyping and DNA Staining

Three separate phenotypic protocols were used to determine the various stages in
B lymphocyte development. All antibodies were used at a dilution predetermined to
provide optimum labeling. To identify pro, pre and lgM+ cells the following antibodies
were used: phycoerythrin (PE) conjugated rat anti-mouse CD45RA (8220), ﬂuorescein
(F ITC) conjugated rat anti-mouse CD43 (S7) and biotinylated (biotin) goat anti-mouse
lgM F (ab’); (lgM) were added simultaneously to cell aliquots. Antibodies against 8220
and S7 were purchased from Pharmingen (San Diego, CA) and anti-lgM was purchased
from Jackson Immunoresearch Laboratories (West Grove, PA). Cells were incubated for
thirty minutes, then washed two times with label buffer. The ﬂuorochrome red 670
(R670) conjugated to streptavidin (Av) (Gibco, Grand Island, NY) was added to cells for
conjugation to biotin-anti-IgM. Cells were incubated for 20 minutes, washed and ﬁxed
with 1 ml of 1.25% paraforrnaldehyde for 40 minutes at room temperature. For DNA
staining cells were washed with label buffer two times and resuspended in 0.5 ml 1 ug/ml
4’, 6-diamidino-2-phenylindole (DAPI) (Sigma, St. Louis, MO) and incubated for at least
one hour at room temperature. For the identiﬁcation of immature and mature B cells PE

conjugated anti-B220, biotin conjugated anti-lgM and FITC conjugated rat anti-mouse

73

IgD (Pharmingen, San Diego, CA) were added simultaneously. Samples were incubated
for 25 minutes, washed and incubation with Av-R670 for 20 minutes. Following
phenotyping cells were ﬁxed with paraforrnaldehyde and stained with DAPI, as described

above.

To identify the pro B cell subsets (pre-pro, early-pro and late-pro) four-color
phenotypic analysis was used. The antibodies (all purchased from Pharmingen) were as
follows: PE conjugated anti-8220, FITC conjugated anti-S7, biotin rat-anti-mouse CD24
(HSA) and puriﬁed rat anti-mouse LY-51 (BP-l, 6C3 clone). Cells were ﬁrst incubated
with anti-BP-l for 30 minutes and washed. An arninomethylcoumarin (AMCA)
conjugated goat anti-rat IgG (Jackson lrnmunoresearch Laboratories, West Grove, PA)
was added for identiﬁcation of the BP-l primary antibody. Cells were washed two times
and 10 ug of Rat lg was added for 10 minutes to block any unbound anti-IgG antibody.
The antibodies against 3220, S7 and HSA were then added at their appropriate dilutions
and the cells were incubated for 25 minutes and washed. Av-R67O was then added for
conjugation to HSA-biotin. Following labeling and washing, the cells were ﬁxed with

0.5 ml of 1.25% paraformaldehyde and stored at 5°C until ﬂow cytometric analysis.

To determine the composition of the major cell lineages within the BM, additional
antibodies were used to determine the percent of granulocytes, monocytes, lymphocytes
(as a whole), hematopoietic progenitors and erythroid progenitors. For this purpose
biotinylated rat anti-mouse CD31 (ERMP12) and F ITC conjugated rat anti-mouse CD59
(ERMP20), (Bachem, King of Prussia, PA) were utilized. Differential surface expression

of CD31 and CD59 on the various cell lineages allowed for their identiﬁcation as

74

follows: granulocytes(ERMP12'ERMP20MW‘3, monocytes
(ERMplzmd‘mERMPmb’igh‘), lymphocytes (ERMPlzmwmmERMPZO'), hematopoietic
progenitors (ERMP12W‘ERMP20WW) and erythroid progenitors (ERMPIZ‘ERMPZO'
). To label BM cells, ERMP12 and ERMP20 were added simultaneously to 2 X 106 cells
on ice at a predetermined dilution. Samples were incubated for 30 minutes then washed
with label buffer. Following phenotyping, cells were ﬁxed by the addition of 0.5 ml

1.25% paraforrnaldehyde and stored at 5°C until F ACS analysis

Flow Cytometry and Data Analysis

Samples were analyzed on a Becton Dickinson FACS Vantage ﬂow cytometer.

F ITC, PE and R670 ﬂuorochromes were excited at 488 nm, and emission was detected at
530, 575 and 670, respectively. DAPI and AMCA were excited at 365 nm and emission
was detected at 470 nm and 450 nm, respectively. To reduce the spectral overlap of
ﬂuorochromes voltage compensation was performed by subtracting non-speciﬁc
ﬂuorescence ﬁ'om each wavelength analyzed.

For the 3-color phenotypic and DNA analysis, debris and cellular aggregates were
excluded from analysis by gating using size and DNA content. Cell size was determined
by forward and side light scatter and DNA content was determined by a DAPI pulse
processed width versus area signal; cells with hypodiploid DNA were considered
apoptotic, as previously described (Telford et al., 1991). A region was drawn around
8220+ cells to identify the B lymphocytes of the marrow and depending on the antibody

combination used, the 3220+ subpopulations were determined as follows: pro B

75

(S7+IgM'), pre B (S7'IgM’), immature (IgM+IgD‘) and mature (IgM+IgD+) B cells.
lsotype-matched samples were used as negative controls.

To determine the subpopulations of pro B cells, the 4-color phenotypic samples
were analyzed by ﬂow cytometry. Cells were gated based on size to exclude debris and
aggregates. To deﬁne pro B cells a region was drawn around 82204'87+ cells and then
analyzed as follows: pre-pro (HSA'BPI'), early-pro (HSA+BP1') and late-pro
(HSA+BP1+). The percent of each population was determined and isotype-matched
control samples were used to determine negative ﬂuorescence.

Most samples were run in duplicate and averaged. Data was processed and
analyzed using PC-LYSIS (Becton Dickinson) and WinList (Verity Software House, Inc.,

Topsham, ME) software. Microsoft Excel was used for statistical analysis and graphing.

Statistics

The Student’s t-test was used to determine signiﬁcant differences (p < 0.05)

between experimental and sham mice where mean data :1: the standard deviations were

reported.

76

RESULTS

Plasma Cs Concentrations, Thymic Weight and BM Cellularity

Under conditions of chronic stress in mice the plasma Cs increases 2 to lO-fold
above concentrations seen in non-stressed animals. Therefore the concentration of
plasma Cs was determined for both sham-control mice and mice receiving a Cs-implant
(Figure 2.1). Mice containing the Cs tablet showed increased concentrations of plasma
Cs at 12 hours of approximately 95 ung. At 24 and 36 hours serum Cs plateaued
between 60 and 70 ug/dl. These concentrations of Cs are consistent with those seen
under conditions of stress (Osati-Ashtiani et al., 1998). The slightly higher concentration
of Cs, apparent in sham mice at 12 hours, is likely due to some contribution of steroid
due to surgical stress.

Thymic atrophy is a common observation under conditions of chronic increases in
circulating Cs (Garvy et al., 1993). The thymuses of mice that underwent a sham surgery
with a cholesterol tablet implant or mice that had a Cs-containing tablet implant were
weighed to determine the affects on thymic integrity. Much of the thymus is composed
of developing T cells and this lab has shown that a decrease in weight is often correlated
with a decrease in thymic cellularity (personal communication, King and Fraker). Figure
2.2 shows the thymic weights for each mouse. As early as 12 hours after surgery, the
thymus of mice containing the Cs tablet implant had decreased by 25%. After 24 and 36
hours thymic weight had decreased 54% and 69%, respectively. This is of interest since
BM, in contrast, did not decrease in total cell numbers. Table 2.1 shows the total

cellularity of mouse femurs 12 and 36 hours after sham or Cs implantation. No

77

signiﬁcant change in BM cellularity was seen and the absolute cell numbers did not
change in the BM of mice exposed to Cs. Since the BM is composed of many different
cell lineages, this suggested that the reduction in a population such as B lymphocytes

might have been compensated for by an expansion in another cell lineages.

Signiﬁcant Changes in B-Lineage Cells of the Bone Marrow Induced by Cs
Exposure

Although distinct stages in B lymphocyte development have been deﬁned based
on surface expression of proteins, Ig gene rearrangement status and the expression of
proteins relevant to B cell lymphopoiesis, the in viva effect of Cs on the various stages
from committed progenitor to maturity had not been assessed. Figure 2.3 and Figure 2.4
show the dramatic changes in the composition of cells of the B lineage subsequent to
only a few hours of exposure to Cs. Figure 2.3 shows actual ﬂow cytometric proﬁles of
the various B lymphocyte developmental populations 24 hours following implantation of
a sham or Cs tablet. Chronic Cs exposure resulted in a decrease of almost 60% in pre B
cells and a decrease of about 55% in pro B cells. Further analysis of the pro B cell
substages showed that pro B cell response to Cs varied between the populations. Pre-pro
B cells were relatively resistant to losses, decreasing by only 17%. Early-pro B cells, in
contrast, showed extensive losses, decreasing 70% and late-pro B cells decreased 33%.
Analysis of the immature and mature B cell populations indicated that, while immature
cells were not substantially different from the negative control, mature cells actually
increased to 164% of the proportion noted in sham mice. These proﬁles are from

representative mice and indicate that the pre B cells (where lg light chain rearrangement

78

is initiated) and early-pro B cells (where heavy chain rearrangement is initiated) undergo
dramatic losses in less than 24 hours of exposure to somewhat modest increases in Cs
that were analogous to those produced by the stress axis.

The effect of chronic exposure to Cs on the composition of the B lymphocyte
developmental substages over the course of 36 hours is shown in Figure 2.4. The earliest
committed B cell progenitors, the pre-pro B cells, were not signiﬁcantly affected by
enhanced exposure to Cs in the ﬁrst 12 hours. Relative to the other precursor populations
the decrease in these cells after 24 or 36 hours of 23% and 34%, respectively, were
modest. Early-pro B cells, in contrast, were dramatically decreased by 60% after only 12
hours of heightened Cs exposure. These cells were nearly eliminated, at 24 and 36 hours,
decreasing by approximately 80%. The next developmental stage, the late-pro B cells,
did not show signiﬁcant decreases until 24 hours post tablet implant. At 24 a decrease of
62% was observed and they continued to decrease by 72% through 36 hours. The pre B
cells, like the early-pro B cells, were signiﬁcantly decreased after only 12 hours of Cs
elevation. They continued to decrease by 70% and 94% after 24 and 36 hours,
respectively. Immature B cells were somewhat resistant to losses during early exposure,
not showing signiﬁcant decreases until 36 hours post-implantation. At 36 hours, these
cells were nearly eliminated, they decreased ﬁom approximately 7% of the BM
population to less than 1% of the BM. Interestingly, mature B cells were not adversely
affected by Cs and as early as 12 hours after heightened exposure to Cs these cells
actually increased in the BM. They continued to increase through 36 hours, where
experimental mice displayed nearly 3-fold more mature B cells in their BM compared to

sham controls. Mature B cells in sham mice made up around 4% of the BM, whereas

79

they made up 12% of the marrow in Cs-treated mice. Clearly, Cs severely affected the
development of B lymphocytes in the EM. Early-pro, late-pro, pre and immature B
lymphocytes were so dramatically decreased that together they made up less than 3% of
the BM as compared to 24% of the BM in sham mice. In contrast the pre-pro B cells
were somewhat resistant to losses and mature B cells actually increased dramatically in
the BM. Since the total cellularity of the bone marrow of sham and experimental mice
did not change these increases represent actual increases in the overall cell numbers for
mature B cells in the bone marrow. Therefore Cs exposure can begin to negatively affect

B cell lymphopoiesis as early as 12 hours and maximum losses were noted by 36 hours.

Cs Induced Changes in Cell Cycle Status of Pro and Pre B Lymphocytes

The reduction in the B cell compartment of the marrow was largely due to losses
in numbers of developing cells, but a decrease in proliferation of the remaining cells
might have also played a role. To investigate this, the cycling status of pro and pre B
cells (lgM+ B cells have very few cells in a cycling state) after Cs exposure was
determined. For this purpose the BM was phenotyped with DAPI to determine the DNA
content. A dramatic decrease in the percent of pro and pre B cells in the S/G2/M phases
of the cycle was observed 24 hours after Cs implantation. Figure 2.5 shows both the
change in the cell cycle distribution of these cells as compared to sham mice as well as
the overall percentage of cells in the S/Gz/M phases for both treatment groups. The
average percent of pro B cells in the S/Gz/M phases of the cell cycle in sham controls was
25.5 :1: 2.8% whereas mice exposed to increased Cs had only 6.2 i 0.2% cells in the

cycling phases (a 75% decrease). The remaining pre B cells displayed a dramatic 84%

80

decrease in cells in the S/G2/M phases of the cell cycle 24 hours after Cs exposure.
Therefore chronic Cs exposure, analogous to that produced by the stress axis, resulted in
dramatic decreases in cell cycling in the remaining pro and pre B cells in the BM. This
suggested that at 24 hours the remaining cells were not proliferating and could not
reconstitute the B cell compartment. Taken together this data shows that chronically
elevated Cs can not only cause dramatic decreases in pro and pre B cells, but also

substantially reduced the proliferative capacity of the surviving cells during exposure.

The Effect of Cs on Major Hematopoietic Cell Lineages of the BM

Considering that approximately half of the B lymphocytes in the BM are
eliminated due to Cs exposure, an analysis of the composition of the key hematopoietic
cell lineages of the BM was performed to determine why the overall BM cellularity was
unchanged. Using the ERMP12 and ERMP20 labeling system granulocytes, monocytes,
lymphocytes, hematopoietic progenitors and erythroid progenitors could be differentiated
(de Bruijn et al., 1994). The composition of the BM of sham mice and mice containing a
Cs tablet insert alter 36 hours is shown in Figure 2.6. The lymphocyte compartment that
represented 32% of the cells of the marrow of sham mice had been reduced by 40%, to
19% of the BM, in mice with heightened exposure to Cs. Interestingly, the granulocyte
population increased by 31%, comprising 42% of the marrow in sham mice and 55% of
the marrow in mice with a Cs tablet implant. These results strongly suggested that the
overall BM cellularity did not change due to Cs, because the granulocyte population
expanded almost proportionately to the decrease in lymphocytes. Thus Cs can have two

different effects on hematopoiesis by negatively affecting the generation of B

81

lymphocytes while positively affecting the generation of granulocytes, thereby potentially
skewing the immune system towards myeloid defense as lymphoid mediated immune

responses are decreased.

82

DISCUSSION

The experiments herein showed that increased concentrations of circulating Cs
caused substantial losses of developing B lymphocytes and reduced proliferation among
surviving cells. The early-pro through pre B cell stages of development, where lg gene
rearrangement has been shown to occur, were the most sensitive to Cs-induced losses.
These populations are likely intrinsically susceptible to apoptosis, due to the natural
elimination of up to 80% of developing B cells that occurs during selection for correct lg
gene rearrangement and against faulty rearrangements. These are also the developmental
populations where the expression of the anti-apoptotic protein, Bel-2, had been shown to
be downregulated (Merino et al., 1994). This suggests that Cs might, at least in part,
elicit its negative effect on developing B cells by inducing apoptosis in cells that are
sensitive to cell death, due to low levels of Bcl-2. In fact Chapters three and four of this
thesis will show in vitro studies where Cs directly induced apoptosis in bone marrow B
lymphocytes with pre B cells exhibiting the greatest susceptibility to cell death.

The loss of B lymphocyte cellularity also appears to be due to a reduction in cell
cycling. These data showed that the remaining pro and pre B cells, normally cycling
populations, displayed dramatically reduced cycling after 24 hours of Cs exposure.
Considering that newly generated cells would likely be eliminated by apoptosis due to
heightened Cs, proliferation of these cells would be a futile process. The data presented
later in this thesis will also demonstrate that, in vitro, Cs caused reduced cell cycling in
the non-apoptotic pro and pre B cells. It is not known whether the decrease in

proliferating cells in vivo was due to a selective elimination of cycling cells and/or as a

83

result of a direct reduction in cycling by Cs. The latter scenario would be congruent with
the literature that reports a direct link between Cs and cell cycle downregulation (King
and Cidlowski, 1998).

The negative effect of Cs on the BM, appeared to be speciﬁc for the B
lymphocyte lineage. Results here suggest that increased Cs exposure might actually have
a positive effect on the generation of other cell lineages, speciﬁcally granulocytes. This
generates an intriguing hypothesis on how the immune system might respond to stress.
Heightened Cs might selectively reduce the highly error-prone B cell development
process while promoting granulopoiesis to maintain and enhance a ﬁrst line of defense to
try to compensate for a decreased second line of defense. Clearly, this area of research
needs to be studied further to determine the effect of Cs on granulopoiesis for longer time
periods and to determine the mechanism whereby Cs enhances the granulocyte
population. This might simply be due to a non-responsiveness of granulocytes to Cs,
resulting in expansion since loss of B lymphocytes would allow for more physical space
in the BM. Expansion may also result if Cs has a proliferative and/or cell survival effect
on BM granulocytes. Relatively recently studies have shown that Gc, in contrast to its
negative effect on lymphocytes, promotes the survival of neutrophils (Liles et al., 1995).
Therefore further investigation of Ge effects on the myeloid lineage is clearly warranted.

The experimental system used here has proven to be a reliable and reproducible
model for inducing physiological-like increases in serum Cs. Surprisingly little was
known of the effects that the induction of the stress axis could have on developing cells
of the immune system. Most studies had focussed on the effects of Gc on peripheral,

mature immune cells. Here it was shown that the development of B cells is reduced, thus

84

likely contributing to the lymphopenia often seen during chronic stress. Considering the
early onset of BM B cell downregulation and the modest increases in Cs, it appears that
lymphocyte development is one of the initial immune functions to be negatively affected
by the steroid. It therefore seems likely that somehow protecting these cells from
heightened Cs exposure might provide immune resilience to stress.

With the advent of B lymphopoiesis supporting long term bone marrow cultures
and more advanced technology for studying cells of the marrow, it has become clear that
stromal cells can play a major role in both lymphopoiesis and myelopoiesis by the
production of critical cytokines (Whitlock and Witte, 1982). It is also clear that the
addition of Gc to long term cultures, where stromal cells are present, selectively promotes
the development of the myelopoietic lineage. Considering the results presented herein, it
might be that Cs modiﬁes the production of key molecules produced by stromal cells to
support a more myelopoietic-like environment. It may also be that the exogenous
addition of lymphopoiesis supporting factors could override Cs negative effects on B cell
development.

Interleukin-7 is one cytokine produced by stromal cells that can act as a critical
proliferative, survival and differentiation signal in early stages of B cell development
(Namen et al., 1988). It might be that this cytokine could potentially act as an
immunotheraputic agent to protect B cell development against increases in Ge. Therefore
studies investigating the effect of IL-7 and other factors, critical to lymphopoiesis, on Cs-
induced downregulation of B cells could have important implications in the treatment of
stress-related immunodeﬁciencies. Later chapters in this thesis investigate the potential

of stromal cells and/or exogenous lL-7 to modulate the negative effects of Cs on B

85

lymphocytes and also investigate the direct effect of Cs on the production of two key
cytokines involved in blood cell development, IL-7 and SCF.

Further understanding of the effect of Go on immune cell development could
provide important advances in health studies, since both physiological stress and
pharmacological administration of Go is widespread. In vivo studies to determine the
effect of Cs on the development of the myeloid lineage is warranted, as are studies
investigating possible immunotheraputic agents on B lymphocyte development.
Additionally, in vitro experiments could provide insight into the mechanism whereby Gc
downregulates B cell development and how various factors might modulate that
mechanism. Clearly the work presented here has provided some key insight into the
effect of Go on blood cell development and likely could provide a foundation for further

investigations.

86

Figure 2.1 The plasma Cs levels for mice receiving a 40 mg cholesterol implant (sham)
(solid bars) or a 20 mg Cs plus 20 mg cholesterol implant (dotted bars) at 12, 24 and 36
hours are shown. The data are expressed as ug/dl and standard deviations are shown
(n=4). Signiﬁcant differences between control and experimental mice were established
using the Student’s t-test (p < 0.05) and are indicated (*).

87

 

 

 

 

 

 

 

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88

Figure 2.2 The individual thymic weights for sham control mice (X) and mice receiving
a Cs implants (O) for 12, 24 and 36 hours are shown. Six mice were analyzed per group
where some individual data points are hard to distinguish due to overlap. The mean of
each group is indicated and an asterisk indicates signiﬁcant differences between sham
control and experimental mice as determined by the Student’s t-test (p < 0.05).

89

 

 

 

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90

Table 2.1 Bone Marrow cellularity of Cs or sham implanted mice

 

 

 

 

 

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group.

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Figure 2.3 F low cytometric data for pre-pro through mature B lymphocytes where
representative results from a sham control mouse and a Cs exposed mouse 24 hours post-
implantation are shown. Panel (A) shows the 8220+ gated cells and gives the percent of
pro (B220+S7'IgM’), pre (B220+S7'IgM') and lgM“ (B220+S7‘IgM+) cells in the BM of
sham controls and Cs-treated mice. Regions were drawn around each population and the
cell type and percent are shown. Panel (B) shows the B220+ gated cells and the
percentage of immature (B220+IgM+IgD') and mature (B220+IgM+IgD‘) B cells in the
BM. Panel (C) shows BZZO+S7+ cells and the percent of pre-pro (B220+S7+HSA'BP'1'),
early-pro (3220*S7+HSA+BP-l') and late-pro (3220*s7+HSA*BP-1*) B cells.
Percentages were based on total nucleated BM cells. Data is representative of at least six
mice per treatment group.

92

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lymphocytes

93

Figure 2.4 The percentage of pre-pro, early-pro, late-pro, pre, immature and mature B
cells in the BM are shown for sham (solid lines) and Cs-implanted mice (dashed line) at
12, 24 and 36 hours after implantation. Six mice were averaged per treatment group and

the mean i standard deviations are shown.

94

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95

Figure 2.5 The cell cycle distribution for pro and pre B cells of sham and Cs-treated
mice at 24 hours was determined. The main panel shows the change in the percentage of
cells found in the S/GZ/M phases of the cell cycle for pro and pre B cells. The actual
percentages of cells in S/szM are shown in the panel insert. The data are for six mice
from both sham and Cs-implanted mice where the mean i stande deviations are shown
and signiﬁcance is indicated with an asterisk.

96

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Figure 2.5 Cs-induced cell cycle changes in pro and pre B cells

97

Figure 2.6 The cellular composition of the BM from sham and Cs-treated mice after 36
hours is shown. Percentages were determined by ﬂow cytometric analysis of the
phenotypic distribution of granulocyte, lymphocyte, hematopoietic progenitors, monocyte
and erythroid progenitors. These results are based on the expression of the ERMP12 and
ERMP20 cell surface antigens. The means for sham control mice (n=5) and Cs-
implanted mice (n=3) are shown.

98

l Sham Implant
l 36 Hours/Bone Marrow Cellularity

Monocyte

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99

CHAPTER 3: STROMAL CELL PROTECTION AGAINST

CORTICOSTERONE-INDUCED APOPTOSIS IN BONE MARROW B

LYMPHOCYTES

100

ABSTRACT

Glucocorticoids (Gc) can induce apoptosis and cell cycle arrest in developing
bone marrow (BM) B cells both in vitro and in viva. Stromal cells are critical to B
lymphocyte development in the BM and in long-term BM cultures. They can provide
support to the developing B cells through direct cell-cell interactions and by the
production of lymphopoietic cytokines. Here it is shown that soluble factors produced by
and direct interactions with stromal cells can protect BM B lymphocytes from short-term
exposure to a natural Gc, corticosterone (Cs). Indeed, stromal cells could reduce the
ability of the steroid to initiate apoptosis by up to 30-50% among pro, pre and IgM+ B
cells. However, neither soluble factors nor direct interactions with the stromal cells
restored cell cycling in developing B cells exposed to Cs. However, the exogenous
addition of the lymphopoiesis promoting cytokine, interleukin-7 (IL-7), augmented the
stromal cell protection against Cs-induced apoptosis in pro and pre B cells and also
restored cycling to normal levels among pro B cells. In addition the potential of Ge to
directly modulate the expression of genes important in hematopoiesis was investigated.
Stromal cell expression of IL-7 and stem cell factor (SCF), a cytokine that supports early
blood cell progenitors from many lineages, was therefore determined. After one day, Cs
caused SCF mRN A to increase 2-3 fold, but it did not have an affect on IL-7 expression.
Therefore the direct affect of Cs on BM B lymphocytes can be inﬂuenced by other cells
normally found in the BM microenvironment, namely stromal cells. Additionally Cs
appeared to also directly effect stromal cells by causing the upregulation of a general

hematopoiesis supporting cytokine, SCF. Taken together these experiments demonstrate

101

the complex nature of studying BM B cells, since cells and factors that would normally
be present in vivo can modulate the responses of developing cells presented with a death
inducing molecule such as Cs. Thus, some of the negative effects of Gc on B cell
development during physiological stress could potentially be alleviated by factors

produced by stromal cells and/or exogenous IL—7.

102

INTRODUCTION

The successful generation of mature B lymphocytes is a complex multi-stage
process consisting of differentiation and proliferation. It is accepted that during B cell
development apoptosis plays an important role by eliminating the high population of
precursor cells with faulty immunoglobulin (lg) gene rearrangements. Detection of
apoptosis in vivo has been very difﬁcult due to the rapid phagocytosis of dying cells and
the difﬁculties associated with studying subsets of cells in heterogeneous tissues like the
bone marrow. Short-term in vitro culture systems and ﬂow cytometry have allowed for
the phenotypic analysis of the various stages in B cell development to include DNA
analysis for cell cycle status and apoptosis (Garvy et al., 1993; Hardy et al., 1991; Telford
et al., 1994). Our lab and others have shown that, in vitro, precursor B cells normally
undergo low but signiﬁcant levels of apoptosis in short-term cultures (less than 24 hours)
(Garvy et al., 1993; Merino et al., 1994). Additionally, glucocorticoids (Gc), such as
dexamethasone or corticosterone (Cs), have been shown to cause a dramatic increase in
apoptosis among these cells. Therefore in vitro models have proven valuable in studies
that otherwise could not be performed in vivo.

A major concern when studying B lymphocyte apoptosis and cell cycle in vitro is
that in vivo B lymphocytes normally develop in close association with ﬁbroblast-like
cells, termed stromal cells, that support their development (Osmond, 1990). In the mid-
1980’s Whittlock and Whitte developed a long-term B lymphopoiesis supporting culture
system (Whitlock and Witte, 1982). Stromal cells were key to the successful long-term

maintenance of lymphopoiesis in this culture system. Later a variety of stromal cell lines

103

were generated that could help support B cell lymphopoiesis for long periods of time
(Collins and Dorshkind, 1987; Dorshkind et al., 1986). Without stromal cells,
lymphocytes do not survive beyond a few days (Borghesi et al., 1997). Interestingly,
with the modiﬁcation of culture conditions B lymphopoietic long-term bone marrow
cultures and stromal cell lines could be converted from the promotion of B cell
lymphopoiesis to the production of granulocytes (Dorshkind et al., 1986). Myelopoiesis
supporting cultures were ﬁrst described by Dexter (Dexter et al., 1977) and vary from
lymphopoietic cultures in that exogenous Go is added to the media. This suggested that
the presence of Ge aided in changing the environment from the support of lymphocytes to
the support of granulocytes in long-term cultures. Therefore Cs, in a period of weeks,
could dramatically modify the culture environment. However, few studies have
investigated the more immediate effect of stromal cells on lymphoid cells treated with an
apoptosis inducing factor such as Cs. One study by Borghesi, et al showed that a stromal
cell line, BMSZ, could protect precursor B lymphocytes (IgM') from dexamethasone
induced apoptosis, but effects on the speciﬁc pro, pre and lgM+ populations was not
investigated.

In addition to directly inducing apoptosis in developing B cells, it may be that Cs
directly modulates stromal cell gene transcription. Many effects of Ge on their target
cells are mediated by transcription modulation of certain genes via Gc receptor binding to
glucocorticoid response elements. Under B lymphopoiesis supporting conditions,
stromal cells produce certain cytokines, such as interleukin 7 (IL-7) and stem cell factor
(SCF) that promote B cell development. Exogenous IL-7 can promote the proliferation

and survival of pro and pre B cells without stromal cell support (N amen et al., 1988;

104

Namen et al., 1988). Elimination of IL-7 in culture resulted in the loss of B cells.
Moreover it was determined that IL-7 deﬁcient transgenic mice do not produce B cells
beyond the pro-B cell stage (von Freeden-Jeffry et al., 1995). SCF, in combination with
IL-7, promoted commitment to the B lineage and enhanced proliferation of the pro B cell
population (McNiece et al., 1991). Whereas IL-7 was shown to be speciﬁc and critical
for lymphocyte development, SCF has been shown to promote the development of
progenitors of several cell lineages (Ashman, 1999). Therefore it might be that IL-7
plays an important role in Whitlock-type cultures whereas SCF supports both B
lymphopoietic and myelopoietic Dexter-type cultures. Since the presence of CC is the
main difference between these culture systems it is possible that the steroid can modulate
these cytokines that are involved blood cell development.

The stromal cell lines 810 and $17 and stromal-like cells sorted from long term
bone marrow cultures were used to determine the effect of stromal cells on Cs-induced
apoptosis and cell cycle status in pro, pre and IgM+ B cells. Additionally, the effect of
810 and 817 stromal cell derived soluble factors, alone, on Cs-induced apoptosis was
determined by culturing the BM B cells suspended over stroma with a membrane insert.
These soluble factors modestly protected each B lymphocyte population ﬁom Cs-induced
apoptosis while direct interactions with the stromal cells afforded slightly more
protection. Although stromal cells demonstrated some protection against Cs-induced
apoptosis they did not protect against the Cs-induced cell cycle arrest. The exogenous
addition of IL-7 augmented stromal cell protection against Cs-induced apoptosis in pro
and pre B cells and restored cycling among pro B cells. Additionally, the direct effect of

Cs on the expression of SCF and IL-7 mRNA was determined. In the time frame studied

105

here, Cs caused a signiﬁcant increase in SCF mRNA levels; the cytokine that promotes
both lymphoid and myeloid development. Interestingly, the cytokine that is speciﬁc for
lymphoid development, IL-7, did not increase. Therefore Cs might promote myelopoietic

cultures by upregulating cytokines involved in this process.

106

MATERIALS AND METHODS

Harvesting of Bone Marrow from Mice

Young adult male Balb c/J mice were purchased from Jackson Laboratories (Bar
Harbor, ME) and housed in the animal facility in the Biochemistry department at
Michigan State University, East Lansing, MI; protocols were approved by the All-
University Committee on Animal Use and Care.

Mice were used from 6 to 14 weeks of age and marrow was ﬂushed from femurs
with approximately 1 ml harvest buffer per bone (Hanks’ balanced salts, 1 mM HEPES
pH 7.2 and 4% F BS) using a 22 gauge needle. The marrow was processed into a single

cell suspension and red blood cells were removed by lysis.

Cell Culture/Cell Lines

Bone marrow derived murine stromal cell lines 310 and S l 7 were generous gifts
from the laboratory of Dr. Kenneth Dorshkind. These lines were grown at 37°C with
7.5% C02 and maintained in RPMI 1640 containing 5 x 10'5 M 2-mercaptoethanol, 1 mM
HEPES pH 7.2, 1000 units/ml penicillin, 0.1 mg/ml streptomycin, 2 mM glutamine and
5% fetal bovine serum (PBS). PBS was purchased from HyClone (Logan, UT) and was
tested for optimal performance; the same lot was used throughout. $10, $17 or sorted
stromal cells were plated at 1x105 cells/ml in 24-well (lml/well) or 12-well (2ml/well)
Corning Costar (Corning, NY) tissue culture plates and were incubated overnight to
insure complete adherence. To obtain stromal cells sorted from long-term bone marrow

cultures, cultures were initiated and maintained as previously described (Whitlock and

107

Witte, 1982) and VCAM-1+ (Southern Biotechnology Associates, Inc., Birmingham, AL)
cells were sorted via ﬂuorescence activated cell sorting (F ACS). FACS sorted cells were
maintained under the conditions used for the stromal cell lines; the sorted VCAM-1+ cells
had typical stromal cell morphology and were MAC—1'(Pharmingen, San Diego, CA).
Bone marrow was plated at 1-2 x 106 cells/well either in 24-well plates in media
only, in 24 well plates directly onto conﬂuent stroma or onto 0.4 uM transwell inserts
suspended over stroma in 12-well plates. Corticosterone (Cs), purchased from Sigma (St.
Louis, MO), and/or recombinant murine interleukin 7 (IL-7), purchased ﬁom R&D
Systems (Minneapolis, MN), were added to cultures at 0.1 11M Cs and at 0.1 ng/ml IL-7.

BM cells were dislodged from culture by the addition of 0.02% EDTA.

lmmunophenotyping/DNA staining

Antibodies to B cell surface antigens were added to samples at a predetermined
dilution to phenotypically label different stages involved in B cell lymphopoiesis.
phycoerythrin (PE) conjugated anti-CD45R (B220), ﬂuorescein (F lTC) conjugated anti-
CD43 (S7) and biotinylated anti-lgM F(ab’)2 (lgM) were added to samples
simultaneously and incubated for 25 minutes. Antibodies against S7 and B220 were
purchased from Pharmingen (San Diego, CA) and anti-lgM was purchased ﬁom Jackson
Immunoresearch Laboratories (West Grove, PA). Following primary staining,
streptavidin-red670 (R670), purchased from Gibco (Grand Island, NY), was added at a
predetermined dilution for conjugation to biotinylated anti-lgM. Samples were incubated
with R670 for 20 minutes and then washed two times with label buffer. Following

phenotyping, cells were suspended in 50% FBS and ﬁxed with the slow addition 1.2 ml

108

of ice cold 70% ethanol. To determine DNA content for cell cycle and apoptosis,
samples were stained with 0.5 to 1.0 ml of 4’, 6-diamidino-2-phenylindole (DAPI)

(Sigma, St. Louis, M0) for at least one hour prior to ﬂow cytometric analysis.

Flow Cytometry

Samples were analyzed on a Becton Dickinson FACS Vantage ﬂow cytometer.

F ITC, PE and R670 ﬂuorochromes were excited at 488 nm, and emission was detected at
530, 575 and 670, respectively. DAPI was excited at 365 nm and emission was detected
at 470 nm.

Debris and cellular aggregates were excluded from analysis by gating based on
size and DNA content. Cell size was determined by forward and side light scatter. DNA
content was determined by a DAPI ﬂuorescence and cells with hypodiploid DNA were
considered apoptotic, as previously described (Telford et al., 1994). 8220' B lymphocyte
subsets were deﬁned as follows: pro B cells were S7+IgM', pre B cells were S7'IgM' and
IgM B cell were 87'. Flow cytometric data was processed using either PC-LYSIS

(Becton Dickinson) or WinList (Verity Software House, Inc.) software

Ribonuclease protection assay

RNA was isolated ﬁ'om 810, 817 or sorted stromal cells using Pharrningen’s
Total RNA Isolation Kit and protocol (San Diego, CA). In general 5 X 106 to 1 X 107
cells were used per isolation yielding approximately 80-150 ug total RNA. RNA was

resuspended in 0.6 ml RNase-free water and stored at -80°C until analysis.

109

The IL-7 and SCF probe templates were purchased from Pharmingen. Probe
synthesis was performed as described in Pharmingen’s Riboquant Multi-Probe RNase
Protection Assay (RPA) System. Brieﬂy, the probe template was transcribed with 32F
labeled uridine triphosphate (New England Biolabs). The radiolabeled probe was then
added to 50 ug stromal cell RNA and hybridized overnight. For positive control samples
10 pg of RNA from IL-7 overexpressing cell lines Psi 5-20 and N59 and the SCF
overexpressing BHK-MKI cell line were used. Control lines were very generous gifts

from the laboratory of Dr. Richard Schwartz. Negative control samples contained 2 ug

of tRN A. RNA samples and radiolabeled probe were hybridized overnight at 56°C.
Following hybridization, ribonuclease treatment was performed as described by
Pharmingen. Samples were electrophoresed on a 5% denaturing polyacrylamide gel and
visualized by phosphorimaging. To obtain a standard curve for fragment mobility, the
probe was run at 2000 counts per minute per lane of the gel. The housekeeping genes
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and L32 were used to normalize
variances in the amount of sample applied to the gels. Phosphorimaging was used for

quantitation and Excel was used to analyze the data.

Data Analysis and Statistics
Data analysis was performed using Microsoft Excel software and mean i standard
errors are reported. Statistical signiﬁcance was determined using the Student’s t-test (p <

0.05).

110

RESULTS

Pro, Pre and lgM+ B Lymphocytes Undergo Spontaneous and Cs-Induced Apoptosis
To determine the effect of stromal cells on spontaneous verses Cs-induced
apoptosis it was important to assess the degree of apoptosis in each cell type. To choose
an appropriate culture time frame where signiﬁcant levels of apoptosis were induced but

where necrosis or cell losses were minimal, the degree of spontaneous and Cs-induced
apoptosis at 15, 24, and 48 hours was determined. Figure 3.1 shows a time course of
apoptotic pro, pre and lgM+ B lymphocytes incubated in media alone (Panel A) or with
0.1 M Cs (Panel B). The data show that over time, the percent of spontaneous apoptosis
increased in each developmental population from 8%, 14% and 9% in pro, pre and lgM
cells at 15 hours to 29%, 36% and 23% in the pro, pre and lgM+ populations at 48 hours,
respectively. Freshly isolated marrow contained fewer than 2% apoptotic B lymphocytes
(data not shown). The addition of Cs to culture caused a signiﬁcant increase in apoptosis.
At 15 hours Cs caused a modest induction of apoptosis in pro, pre and IgM+ of 24%, 44%
and 36%. Greater levels of death were apparent at 24 hours; 32%, 64% and 48% in the
pro, pre and IgM+ cells, respectively. Aﬁer 48 hours nearly all of the pre and IgM+ cells
were apoptotic. Therefore a 15 hour culture period resulted in a signiﬁcant induction of
spontaneous and Cs-induced apoptosis, yet these levels were more modest compared to
later time points, thus making the modulation of B cell death by stromal cells more

apparent.

lll

Protective Effect of Stromal Cell Lines on Spontaneous and Cs-Induced Apoptosis
in B Lymphocytes

Pro, pre and IgM+ B lymphocytes underwent low levels of spontaneous apoptosis
at 15 hours and the addition of Cs caused increased apoptosis, as shown above.
Therefore, whether stromal cells and/or the soluble factors they produce could modulate
this apoptosis in the B cell populations was determined. BM cells were cultured as a
single cell suspension either in media alone, directly on conﬂuent stromal cells or
suspended above the stromal cells using a 0.4 11M permeable membrane insert. Samples
were incubated for 15-16 hours after which the composition of cells of the B lineage and
degree of apoptosis were determined by ﬂow cytometry. Figure 3.2 (A) shows the effect
of either soluble factors from and/or direct interactions of BM B lymphocytes with the
817 stromal cell line on spontaneous apoptosis. Direct incubation with $17 resulted in a
signiﬁcant decrease in spontaneous apoptosis of 40-50% in each pro, pre and lgM+ B
cells. However soluble factors, alone, did not reduce spontaneous apoptosis in any stage
of development. The 810 stromal cell line also had similar effects on the BM B cells
(data not shown). Therefore, direct interaction with the stromal cell lines, but not soluble
support alone, could reduce spontaneous apoptosis in pro, pre and IgM B cells.

Figure 3.2 (B) shows the effect of soluble factors from and/or direct interactions
with S17 on Cs-induced apoptosis in pro, pre and lgM+ B cells. Direct interaction with
817 resulted in the greatest protection against Cs-induced apoptosis in pro B cells (44%
reduction). lgM+ cells were also signiﬁcantly protected with a 39% decrease and pre B
cells showed a more modest 29% decrease. Soluble factors produced by S17 also

protected B lymphocytes from Cs-induced apoptosis. A 31%, 20% and 28% reduction in

112

apoptosis by soluble factors alone was observed in pro, pre and IgM+ cells, respectively.
Although this trend suggested lesser protection by soluble factors, the reduction was not
statistically different from those obtained from direct stromal contact. S 10 stromal cells
had similar protective effects on the B lymphocytes (data will be presented below). The
data suggests that soluble factors produced by these stromal cell lines provided
signiﬁcant protection to each stage of B lymphocyte development from Cs-induced cell
death in short-term cultures. Interestingly, soluble factors from the stromal cell lines did
not appear to protect B lymphocytes from spontaneous apoptosis, suggesting that either
one or more soluble molecules produced by stromal cells could speciﬁcally protect

against Cs-induced apoptosis without affecting basal levels of apoptosis.

Stromal Cell Effect on Cs-Induced Cell Cycle Arrest in Pro and Pre B Lymphocytes

Data here and in Chapter two showed that along with causing apoptosis in B
lymphocytes, Cs also caused a reduction in cycling pro and pre B cells. The extent to
which these two Gc-induced phenomenon are interrelated is not fully understood. Here
experiments were performed to determine the effect of S17 on Cs-induced cell cycle
arrest. BM B lymphocytes were incubated with or without S17 as previously described
and in media alone. Table 3.1 shows the percent of pro, pre or lgM+ cells in the S/Gle
phases of the cell cycle. Samples incubated without Cs showed that neither soluble nor
direct interaction with S17 changed the proportion of cycling cells in control cultures.
Addition of 0.1 M Cs, on the other hand, resulted in a signiﬁcant decrease in the percent
of cycling pro and pre B cells similar to the decreases seen in in viva studies. Cells in

S/Gz/M decreased by 54% in pro B and 71% in pre B cells when cultured with Cs.

113

Neither soluble factors nor direct interaction with S17 were able to restore the cycling
population in the pro or pre B cells. S10 similarly did not effect the cell cycle status and
these results will be shown below. Therefore, although stromal cells could protect B
lymphocytes from Cs-induced apoptosis they were not able to protect the developing pro

and pre cells from the Cs-induced cycling decrease.

The Effect of Exogenous IL-7 on BM B Lymphocytes Cultured with Stromal Cells

Chapter 4 in this thesis shows the effect of exogenous addition of IL-7 alone on B
lymphocytes exposed to Cs. The cytokine protected pro B cells from Cs-induced
apoptosis and cell cycle arrest and also modestly protected pre B cells ﬁom the Cs-
induced apoptosis, but not the cell cycle arrest. It was therefore important to determine if
the exogenous addition of IL—7 to cells incubated with either S10 or S l 7 could reproduce
the effect on B lymphocytes seen with IL-7 alone. Figure 3.3 shows actual ﬂow
cytometric data for apoptosis and S/Gz/M in pro, pre and IgM cells cultured with no
treatment, 0.1 uM Cs, Cs and $10 or Cs, S10 and IL—7 for 16 hours. As compared to
background levels, Cs clearly induced substantial induction of apoptosis in pro, pre and
IgM cells. Additionally signiﬁcant reductions in pro and pre B cell cycling were
apparent. Direct incubation with S10 resulted in a 50%, 51% and 55% decrease in Cs-
induced apoptosis in pro, pre and lgM+ cells, respectively. The exogenous addition of
IL-7 further reduced the percent of apoptotic pro B cells by an additional 62%, bringing
the percent apoptosis to below background levels. IL-7 caused a more modest decrease
in apoptosis in pre B cells of 43% as compared to pre B cells cultured with S10 alone.

The cytokine did not cause a further reduction in apoptosis in lgM cells. These data are

114

consistent with the independent effect of IL-7 on these cell types. IL-7 addition to B
lymphocytes cultured with S 17 resulted in similar responses to those seen with S10 (data
not shown).

As seen with the S l 7 data above, S 10 alone did not affect the decrease in pro and
pre B cells in S/G2/M induced by treatment with Cs. The addition of IL-7 to cells
incubated with S 1 0, on the other hand, resulted in a restoration in pro B cell cycling to
that seen in background cultures. Again the effect of exogenous IL-7 addition to these
cultures reproduced the effect of IL-7 alone on pro B cell cycling. Therefore
recombinant IL-7 has effects on Cs-induced cycle arrest that S10 alone could not provide
and S10 alone appears to protect lgM cells from Cs-induced apoptosis whereas IL-7 did

not augment this response.

The Effect of Cs on Stromal Cell Expression of SCF and IL-7

The major mechanism whereby Cs elicits its effects in cells is by transcription
regulation via the Ge receptor. Although the protective effect of 810 and 817 on B
lymphocytes could be attributable to soluble factors other than IL-7, it was important to
determine whether this cytokine was expressed in each stromal cell type and whether that
expression was modiﬁed by Cs-treatment. Additionally the cytokine SCF could be a
target for regulation by Cs, since it generally promotes hematopoietic progenitors. In
contrast to IL-7, SCF likely could play a role in long term myelopoietic cultures, where
Cs is part of the culture medium. Figure 3.4 shows the relative RNA expression of IL-7
and SCF in 817, S10, sorted stroma and from positive control cell lines. Clearly message

for both cytokines was present in each stromal cell type, with sorted stromal cells

115

expressing higher levels of IL-7. Relative to IL-7, SCF was expressed at dramatically

higher levels.

Figure 3.5 (A) shows the change in SCF mRNA levels in 810, S17 and sorted
stromal cells treated with 0.1 M or 10 1.1M Cs for 20-24 hours. Cs treatment (10 1.1M)
resulted in a 2-3 fold elevation in SCF mRNA expression in all stromal cell types tested.
Although the increase caused by 0.1 uM Cs was less overall it was not statistically
different ﬁ'om the increases induced by 10 11M treatment. These data show that Cs

caused an upregulation of SCF as early as one day after treatment.

IL-7 mRNA was detectable in SI 7, S10 and sorted stroma. $17 and 810 showed
very low but similar expression levels. Sorted stroma, in contrast, had approximately 5-
10 fold higher production of IL-7 mRNA (as shown in Figure 3.4). Figure 3.5 (B) shows
that there was no signiﬁcant change in IL-7 mRN A expression with Cs treatment.
Therefore, Cs does not appear to regulate IL-7 expression after 20-24 hours exposure to
Cs, suggesting that the modest protection provided by stromal cells was not due to

upregulated IL—7 expression.

116

DISCUSSION

The experiments presented here have shown that in short-term primary bone
marrow cultures signiﬁcant but low levels of spontaneous apoptosis occurs in pro, pre
and IgM+ B cells. The addition of Cs at concentrations analogous to physiological rather
than pharmacological concentrations resulted in substantial increases in apoptosis, with
pre B cells being the most susceptible to death. Additionally the steroid dramatically
reduced cycling pro and pre B cell numbers. Soluble factors produced by, or direct
interactions with, the $10 or S 17 stromal cell lines caused a modest reduction in
apoptosis in each B cell population, but in contrast the stromal cells did not affect the Cs-
induced reduction in cell cycling. This suggests that factors produced by stromal cells
could potentially protect developing B cells from Cs-induced apoptosis over a short
period of time.

At least one soluble factor produced by the stroma appeared to be responsible for
the decrease in Cs-induced apoptosis. IL—7 would be surmised to be one likely candidate
for this protective function, since it is critical and speciﬁc for lymphopoiesis rather than
myelopoiesis in the bone marrow (Namen et al., 1988; von Freeden-Jeffry et al., 1995).
The S 1 7 cell line had been reported to not produce biologically active levels of this
cytokine but here these cells protected the B lymphocytes to the same extent as $10,
which has been reported to produce biologically active IL-7 (Henderson et al., 1990).
However, 8 1 7 was also reported to not produce IL—7 mRN A by RT-PCR but the
experiments herein show that S17 in fact displayed low levels of IL-7 gene expression

being comparable to that seen in S 10 (Faust et al., 1993). Therefore these 817 stromal

117

 

cells did express IL-7 mRN A in contrast to previous publications. The difference could
potentially arise from a reversion of the cell line to an IL-7 producing stromal cell. Both
810 and S17 were derived from multiple passages of long term bone marrow cultures to
obtain a self-renewing clonal cell type, therefore there is no enforced selection of cells to
maintain characteristics such as the lack of expression of certain cytokines.

The independent protective effect of exogenous IL—7 on B lymphocytes is
thoroughly presented in Chapter 4 of this dissertation. The cytokine clearly protected pro
and pre B cells from Cs-induced apoptosis and eliminated the cell cycle arrest in pro B
cells. Those experiments suggested that 1L-7 could potentially have immunotheraputic
value in lymphocyte development during stress. Here the exogenous co-addition of this
cytokine to stromal cell supported cultures, reproduced the protective effects seen by the
cytokine alone in addition to stromal cell protection. Therefore the immunotheraputic
potential of IL-7 was further supported by these experiments.

Regardless of the potential effects that low levels of lL-7 produced by stromal
cells might have, it seems likely that other soluble factors produced by the stromal cells
have a protective effect on B cells exposed to Cs. Evidence for this comes from the
protection ﬁom Cs-induced apoptosis observed in IgM+ cells. IL-7 alone or in
combination with stromal cells did not protect this more mature cell population and
previous studies have indicated that lgM+ cells in the marrow are mostly unresponsive to
IL-7 (Sudo et al., 1993). One stromal cell derived cytokine that could potentially elicit
this effect is thymic stromal lymphopoietin (TSLP). Recent studies on the positive
effect of TSLP on developing B lymphocytes indicated that this cytokine could

potentially support B cells from Cs—induced apoptosis, because it supports B cell

118

 

development throughout I gM+ stages (Levin et al., 1999). Further studies to identify
active factors that can protect all stages of B lymphocyte development from Cs-induced
apoptosis would therefore be of value.

The studies on the expression of SCF message levels have shown that Cs can
directly affect bone marrow stromal cells and potentially can modulate the hematopoietic
environment by transcriptionally regulating cytokines important to blood cell
development. Cs treatment for one day signiﬁcantly upregulated SCF gene expression.
Since SCF is a cytokine that positively affects a variety of progenitor blood cell types and
that Cs promotes myelopoiesis, it may be that the upregulation of SCF acts to promote
the initiation of a myelopoietic-like environment. IL-7 on the other hand, which does not
promote myelopoiesis, was not upregulated in response to Cs. Since long-term exposure
to Cs causes the stromal cell environment to promote myelopoiesis it is logical that the
steroid would cause the upregulation of a general cytokine involved in blood cell
development, but would not upregulate a cytokine speciﬁc for lymphopoiesis which the
environment does not support. It could be that after extended culture with Cs, stromal
cells might actually downregulate IL—7 expression. Advances in technology have created
the potential to screen the expression of a variety of cytokine genes simultaneously and
Cs treatment of stromal cells could be a valuable area of investigation.

It is clear that the study of B lymphocyte responses to physiological factors is a
very complex area of study with several variables to take into account when studying
cells in vitra. Nevertheless, the complete understanding of B cell development and how

that development can be modiﬁed remains a very important area in health and disease.

119

 

Therefore it is important to study these cells and attempt to understand the potential roles

of factors that would normally be found in the BM on them.

120

Figure 3.1 The percent of apoptotic cells in pro, pre and lgM+ B lymphocytes from BM
are shown. Apoptosis was determined after culturing for 15, 24 or 48 hours, by DNA
staining and analysis for hypodiploid DNA. Panel A shows apoptosis among cells
cultured in media only and Panel B shows the percent apoptosis in the B cell populations
cultured with 0.1 uM Cs. Data are the average of duplicate samples i the standard
deviations and is representative of several experiments. Error bars not seen are smaller
than the data symbols.

121

 

 

 

,-s from 811
by DNA
cells
populations
ldard

'6 smaller

50% "
40%

30% ‘

Apoptosis

20% *'

10% *

0% .L - . _.__

10096.
80%

60% l

Apoptosis

40%
20%

0% 7‘

Figure 3.1 Spontaneous and Cs-induced apoptosis over time in pro, pre and

IgM+ B cells in vitro.

 

 

Cs Treatment

Time (Hours)

122

Figure 3.2 Apoptosis was measured among pro, pre and IgM+ B cells with and without
Cs to determine potential protective effects of stromal cells (direct contact) or factors
produced by stromal cells (membrane insert). BM was processed into a single cell
suspension and plated in media alone (solid bars), on a 0.4 uM transwell insert suspended
over S l 7 stromal cells (dots) or directly onto a conﬂuent layer of S 1 7 stromal cells
(stripes). Following 15-16 hours in culture the BM cells were harvested and phenotyped
for pro, pre and lgM+ B cells, ﬁxed and stained with DAPI, a DNA dye, for analysis of
apoptosis. Panel (A) shows the change in apoptosis in cells cultured alone or with or
without direct stromal cell contact in media and (B) shows the change in apoptosis among
cells exposed to 0.1 M Cs alone or when in direct or indirect contact with stromal cells.
All data were normalized to the percent of apoptosis among cells cultured without S17
and are the averages of at least three experiments. The actual percent apoptosis for pro,
pre and IgM cells in control cultures without Cs or stromal cells were 7%, 14% and
12%, respectively. Control samples treated with Cs were 21%, 51% and 41% apoptotic
in pro, pre and IgM+ cells, respectively. Standard error bars are shown and signiﬁcant
differences from control samples was determined using the Student’s t-test (p < 0.05) and
indicated by an asterisk.

123

wt

1 A No Treatment
i 140%

“ 120% ‘
1 100% *
1 ' 80% - s *
l 60%
l 40% a
j 20%
l 0% ,
Pro Pre IgM+

l I No s17 [:1 Soluble s17 IDirect S17

Apoptosrs

 

1 B Cs Treatment

 

‘ 120%?

1 100% . s *

é a 80% i * * * *

l 1.

‘ a

1 40% j

1 20% 1

l ‘ ‘

i 0% . , 7, ,
Pro Pre IgM+

I No 817 El Soluble Sl7 IDirect 817
l

l. _ hm, , _

Figure 3.2 Stromal cell modulation of spontaneous and Cs-induced apoptosis

124

Table 3.] Bone marrow B cells in S/Gz/M phases of the cell cycle

 

 

 

 

 

 

 

 

 

 

 

 

Cell Type . N o Stroma Soluble Factors‘l Direct Contact”
1"" 16.6 i- 8.5%* 153 i 6.8%“ 13.1 i- 4.6% c
N” C’ P“ 12.6 i 1.2% 10.6 i 1.9% 11.9 i 1.8%
lgM 4.8 3: 2.0% 4.8 i 0.8% 4.2 i 1.7%
Pro 7.7 i 0.5% 8.0 i 1.2% 6.25 i 2.1%
0.1 M
Pre 3.6 :1: 2.0% 3.8 i 1.5% 3.4 i 1.0%
Cs
IgM+ 3.6 i 1.4% 3.6 i 03% 3.3 i 1.1%

 

* Data are presented as the averages of three experiments i standard deviations
a B lymphocytes cultured suspended over S 1 7 stromal cells by a 0.4 11M membrane

insert

b B lymphocytes cultured directly on S17 stromal cells
c There were no statistical differences between cells incubated in media alone, with

soluble factors from the S17 stromal cell line or with direct contact with the stromal cells

125

 

 

Figure 3.3 Flow cytometric DNA histograms show the Cs-induced change in apoptosis
and cell cycle status for pro, pre and IgM B cells and modulation of these Cs-induced
changes by stromal cells or a combination of stromal cells and IL-7. BM was processed
into a single cell suspension and incubated for 16 hours in culture media only (A), with
0.1 11M Cs (B), directly on a conﬂuent S10 stromal cell layer and with 0.1 uM Cs (C) or
with S l 0, Cs and 0.1 ng/ml IL-7 (D). Following culture BM cells were harvested and
phenotyped for pro, pre and IgM+ cell types, the samples were ﬁxed with ethanol and the
DNA stained with DAPI. Cells were analyzed by ﬂow cytometry as follows: pro B
(B220+S7+IgM’), pre B (B220+S7'lgM') and IgM cells (B220+S7'IgM+). Debris and cell
aggregates were excluded from analysis by gating based on cell size and each population
was analyzed for apoptosis (A0) and cell cycling (S/Gz/M) as indicated. These data are
representative of several experiments.

126

Relative Cell Number

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DNA Content

Figure 3.3 Flow cytometry of 810 and/or lL-7 effects on apoptosis and
cycling of pro B cells

127

 

\

Figure 3.4 The relative RNA expression between stromal cell types is shown as the
actual phosphoirnage obtained from the ribonuclease protection assay. RNA was
extracted and the ribonuclease protection assay were performed as described by
Pharmingen using 50 1.1g stromal cell RNA and 10 pg positive control cell line RNA.

The samples are shown as follows: Lane (A) unprotected probe, (B) S17 stromal cells,
(C) 810 stromal cells, (D) sorted stromal cells, (E) IL-7 positive control cell line N59, (F)
SCF positive control cell line BHK-MKI. The approximate electrophoretic mobility for
each is indicated on the left hand side of the ﬁgure.

A B C D E F
IL-7 Probe—p g

IL-7 —>

SCF Probe—-p w

SCF—p
L32 Probe/v

GAPDH Probe—p

L32 —D

GAPDH

 

Figure 3.4 Relative expression of IL-7 and SCF message

129

 

Figure 3.5 The relative change in messenger RNA expression levels of SCF (A) and IL-
7 (B) for stromal cells treated with 0.1 11M or 10 11M Cs for 20-24 hours. Stromal cells
tested were the S10 and S17 cell lines and stromal-like cells sorted from long term bone
marrow cultures. Cells were sorted based on large size and VCAM-1 surface expression.
These cells had ﬁbroblast like morphology typical of stromal cells and did not express
MAC-l on their surface. The stromal cells were grown just to conﬂuence and spent
media was replaced with fresh media for approximately one day before Cs addition.
Following culture, whole cell RNA was extracted from approximately 5 X 106 - 1 X 107
stromal cells using Pharmingen’s Total Cell RNA Isolation Kit. RNA analysis was
performed using the ribonuclease protection assay and quantiﬁed using phosphorimaging.
The results shown are the average changes relative to the corresponding non-treated cells
for at least two experiments and standard deviation bars are shown. Asterisks represent
signiﬁcant differences as determined by the Student’s t-test (p < 0.05) from untreated
samples.

130

’ A SCF gene expression
1 440% \
300% T
250% :
20096 <
15096 i
100% 1
50% 1'
0% 1

 
  
  
  
 

RNA Expresssion

 

 

l INoCs EJ'0.1|.1M'Cs I110uMCs

 

1L7 gene expression

25096 1
20096 ;
15096 ~

100% *1

 

RNA Expresssion

50% 2

096 ‘

INo Cs 110.1 11M Cs I110 11M Cs

Figure 3.5 Cs effect on stromal cell expression of SCF and IL-7 RNA

131

 

 

 

CHAPTER 4: INTERLEUKIN-7 PROTECTS EARLY STAGES IN B

LYMPHOYCTE DEVELOPMENT FROM CORTICOSTERONE-INDUCED

APOPTOSIS AND CELL CYCLE ARREST

132

ABSTRACT

Independent and combined effects of the cytokines interleukin-7 (IL-7) and stem
cell factor (SCF) on spontaneous verses glucocorticoid (Gc) induced apoptosis in
developing B lymphocytes was investigated. These cytokines could potentially protect
developing B cells from the negative effects of cultming and Ge exposure, since they act
as positive factors during the early stages of normal B cell lymphopoiesis. Murine bone
marrow was cultured as a single cell suspension, without stromal cell support, and the
various B lymphocyte developmental stages, levels of apoptosis and cell cycle status
were determined by ﬂow cytometry. Exogenous addition of IL-7 reduced spontaneous
apoptosis in the pro B cell population by nearly half, but had little effect on the survival
of the more mature pre and IgM+ B cells. This cytokine also dramatically protected the
pro B cell population from apoptosis induced by the natural murine Gc, corticosterone
(Cs), decreasing levels of apoptosis by 60%. IL-7 also afforded modest protection to pre
B cells from Cs-induced apoptosis but only minimal protection was provided to lgM
bearing cells. Cs exposure resulted in a profound decrease in cycling among pro and pre
B cells but once again IL-7 protected pro B cells by eliminating this cell cycle arrest
while providing little cell cycle protection to pre B cells. SCF, which can augment
lymphopoietic development, similarly caused a 40% decrease in background apoptosis in
pro B cells when added alone to short-term cultures. However, in contrast to IL-7, it did
not appear to have a signiﬁcant affect on apoptosis induced by Cs. When SCF was used
in combination with IL-7, it did not augment the protection against Cs-induced apoptosis

in pro B cells. Therefore although both cytokines could protect pro B lymphocytes ﬁ'om

133

 

Spontaneous apoptosis, only IL-7 protected early B lymphocytes from Cs-induced
apoptosis. Additionally, I L-7 also provided substantial protection of pro B cells from Cs-
induced cell cycle arrest. Clearly this cytokine has the potential to dramatically protect
early stages in B cell development from agents that alter proliferation or induce
apoptosis. It reinforces the importance of IL-7 to the early phases of lymphopoiesis and

indicates it has immunotheraputic potential.

134

 

 

INTRODUCTION

Chronic increases in circulating levels of glucocorticoids (Gc) are evident during
physiological stresses such as in burn victims, trauma patients, the aged and the
malnourished and can cause impaired immune function and lymphopenia (Schleirner et
al., 1989). In vitra, dexamethasone (a synthetic Gc) has been shown to induce apoptosis
and decrease cycling in developing lymphocytes in the marrow, with precursor cells
(IgM') showing greater sensitivity to these negative effects (Borghesi et al., 1997; Garvy
et al., 1993; Merino et al., 1994). Our lab has also demonstrated, in viva, that chronically
elevating corticosterone (Cs), resulted in a selective reduction in early B lymphocytes
that had not begun to express surface irmnunoglobulin (Garvy et al., 1993). Therefore Cs
might elicit its lymphopenic effect in viva, at least in part, by inducing apoptosis in the
early stages of B cell development. Recent data also showed that Cs could dramatically
reduce cycling among both pro and pre B cells, suggesting this as another mechanism
whereby the steroid might downregulate B cell development (Laakko and Fraker,
unpublished observation; King and Cidlowski, 1998).

Blocking Gc-induced cell death and cell cycle reduction in B lymphocytes could
potentially help protect against the lymphopenia that can occur under conditions of stress.
Cytokines that are critical to B lymphocyte development might be of beneﬁt in protecting
these cells from Gc induced apoptosis. IL-7 and SCF are cytokines that could potentially
protect early stages in B lymphocyte development from apoptosis. IL-7 was originally
identiﬁed as a factor produced by stromal cells in long term bone marrow cultures that,

independently, promoted proliferation of precursor B cells in vitra (N amen et al., 1988).

135

When added to B lymphocyte cultures, with no other apparent cytokine or stromal cell
support, I L-7 promoted the survival of pro B cells that would otherwise rapidly undergo
apoptosis in culture (Gibson et al., 1996; Lee et al., 1989). In addition, IL-7 caused
dramatic increases in cycling in early B cell progenitors, not expressing surface lgM.
Recent evidence suggested that IL-7 might promote survival via phosphatidyl inositol 3
kinase (PI3K) activation of the AKT pathway and/or by upregulation of the anti-apoptotic
Bel-2 protein (Lavagna-Sevenier et al., 1998; Lu et al., 1999). Another stromal cell
derived cytokine, SCF, also can promote B cell lymphopoiesis. In combination with IL-7
it promoted the commitment of progenitor cells to the B lineage and it increased IL-7
induced proliferation among pro B cells (McNiece etal., 1991). In addition to
amplifying lymphopoiesis, SCF had also been shown to positively affect many
progenitors from various lineages when in combination with growth factors speciﬁc for
those lineages (Ashman, 1999). PI3K mediated activation of the AKT cell survival
pathway has also been indicated in SCF signaling via its receptor c-kit (Blurne-Jensen et
al., 1998). Although both IL-7 and SCF have shown positive effects on precursor B cell
development and survival, what effects these cytokines might have on apoptosis among
early B cells has not been determined. Moreover the effect of these cytokines on
developing B lymphocytes exposed to a potent death-inducing factor, such as Cs, are not
known.

The potential of IL-7 or SCF to suppress spontaneous or Cs-induced apoptosis in
a short-term (16 hour) culture system was determined here. Murine bone marrow was
used as the source for developing B lymphocytes and the degree of apoptosis and cell

cycle status was determined using ﬂow cytometry. An established phenotypic scheme

136

 

using ﬂuorochrome conjugated monoclonal antibodies to cell surface proteins, developed
by Hardy et a1 (Hardy et al., 1991), was used to identify three stages in B lymphocyte
development. The stages are listed in order of least to most mature as follows: pro B
cells, pre B cells and IgM+ cells consisting of immature and mature B cells. The degree
of apoptosis and cell cycle status was also determined for each population by measuring
the DNA content. The results show that IL—7 and SCF individually provided substantial

protection against background apoptosis among pro B cells prior to the induction of cell

 

proliferation, but there was no appreciable protection provided by either cytokine to pre
or IgM+ cells undergoing spontaneous death. Interestingly, IL-7 and SCF did not have
the same effect on Cs-induced apoptosis. IL-7 protected pro B cells, and to a lesser
extent pre B cells, from Cs-induced apoptosis. In the pro B cell population IL—7 also
inhibited the cell cycle arrest induced by Cs, but this was not the case for the pre B cell
population. SCF did not appear to have a signiﬁcant affect on Cs-induced apoptosis
either alone or in combination with IL-7. Therefore, both of these cytokines appear to
promote survival of cells early in B lymphocyte development but only IL-7 can protect
early B cells from Cs-induced apoptosis. This is the ﬁrst direct evidence that a cytokine

may be able to help protect early stages in B cell lymphopoiesis from Cs exposure.

137

MATERIALS AND METHODS

Bone Marrow Preparation and Cell Culture

Young adult male Balbc/J mice were purchased from Jackson Laboratories (Bar
Harbor, ME) and housed in a temperature and light controlled facility. All protocols used
herein were approved by the All-University Committee on Animal Use and Care at
Michigan State University.

Bone marrow was ﬂushed from femurs with approximately 1 ml of harvest buffer
(Hanks’ balanced salts, 1 mM HEPES pH 7.2 and 4% F BS) per bone using a 22 gauge
needle. The marrow was processed into a single cell suspension and red blood cells
were removed by lysis. Bone marrow cells (1-2 x 106 cells/ml) were cultured at 37°C in
7.5% C02 using RPMI 1640 containing 5 x 10'5 M 2-mercaptoethanol, 1 mM HEPES pH
7.2, 1000 units/ml penicillin, 0.1 mg/ml streptomycin, 2 mM glutamine and 5% fetal
bovine serum (F BS). FBS was purchased from HyClone (Logan, UT) and the same lot of
serum was used throughout.

Corticosterone was purchased from Sigma (St. Louis, MO) and both recombinant
murine IL-7 (rrnIL-7) and recombinant murine SCF (rmSCF) were purchased from R&D
Systems (Minneapolis, MN). Cs was used at 0.1 11M, unless otherwise stated. Both SCF
and IL-7 were titered for maximum activity and used at 100 ng/ml and 0.1 ng/ml,

respectively, unless otherwise stated. The standard culture time was 15 to 16 hours.

lmmunophenotyping/DNA staining

Antibodies to B cell surface antigens were added to samples at optimum

concentrations and samples were maintained at approximately 5°C throughout

138

 

phenotyping. Cells were incubated for 25 minutes, simultaneously, with phycoerythrin
(PE) conjugated anti-CD45R (8220), ﬂuorescein (F ITC) conjugated anti-CD43 (S7) and
biotinylated anti-lgM F (ab’)2 (IgM) in label buffer (HBSS containing 2% FBS, lmM
HEPES and 0.1% sodium azide). Antibodies against S7 and B220 were purchased from
Pharmingen (San Diego, CA) and anti-lgM was purchased ﬁ'om Jackson
Immunoresearch Laboratories (West Grove, PA). Following primary staining,
Streptavidin-Red670 (R67 0), purchased from Gibco (Grand Island, NY), was added for
conjugation to biotinylated anti-lgM for 20 minutes and then washed two times with label
buffer.

Following phenotyping, cell pellets were resuspended in 50% FBS and ﬁxed by
slow addition of 1.2 ml of ice cold 70% ethanol with gentle mixing. Cells were leﬁ in the
ﬁxative for at least one hour and for as long as three days. At least one hour before
FACS analysis, ﬁxed samples were incubated at room temperature in 0.5-1.0 ml of DAPI

staining solution (1 [lg/m1 DAPI and 0.01 mM EDTA in phosphate buffered saline) for

DNA analysis of apoptosis and cell cycle.

Flow Cytometry

Samples were analyzed on a Becton Dickinson FACS Vantage ﬂow cytometer.
F ITC, PE and R670 ﬂuorochromes were excited at 488 nm, and emission was detected at
530, 575 and 670 nm, respectively. DAPI was excited at 365 nm and emission was
detected at 470 nm.

Debris and cellular aggregates were excluded ﬁom analysis by gating based on

size and DNA content and ﬂuorochrome conjugated isotype antibodies were used as

139

 

negative controls. Cell size was determined by forward and sideward light scatter and
DNA content was determined by DAPI width versus area. According to the phenotypic
scheme developed by Hardy (Hardy et al., 1991), B lymphocyte subsets were deﬁned as
follows: pro B cells were B220+S7+IgM', pre B cells were 8220+S7'lgM' and immature
and mature cells were deﬁned as B220+IgM. Each population was gated and analyzed
for apoptosis (hypodiploid DNA) and for cell cycle status. PClysis (Becton Dickinson)
or WinList (Verity Software House, Inc., Topsham, ME) soﬁware were used to process

ﬂow cytometric data.

Statistics

Microsoft Excel software was used to analyze data and perform statistics. Data
are presented as the mean i the standard deviation and signiﬁcance was established using

the Student’s t-test (p < 0.05).

140

 

RESULTS

Analysis of the Effect of IL-7 and Cs on Short-Term Marrow Cultures

Cytokine signals are often required for the survival of cells of the immune system.
Early stages in B cell lymphopoiesis depend on IL-7 for proliferation, differentiation and
long-term survival (Lee et al., 1989; Mertsching etal., 1996; Sudo et al., 1989;
Valenzona et al., 1998). Here a short-term culture system where stromal cells and their
effects were negligible was used to directly analyze the effect of IL-7 on developing B
cells. For this type of culture system it was also important to use a rather short time
period where necrosis and cell losses due to culturing and Cs addition were minimal.
This was to ensure accurate comparisons between treatment groups and to determine IL-7
effects independent of its proliferative potential. Thus the majority of studies were
performed in 16 hours.

Table 4.1 shows the recovery and the viability of nucleated cells from bone
marrow after 16 hours in culture with media alone, 0.1 ng/ml lL-7, 0.1 uM Cs or IL-7 in
combination with Cs. Microscopic cell counts were performed to determine recovery and
viability by trypan blue exclusion; apoptotic cells exclude trypan blue and were
considered viable in cell counts. The viability at the initiation of cultures was
approximately 90% (data not shown). At the end of a typical 16 hour period there was
excellent viability in control cultures (93%) with some expansion of cells (126%
recovery). Cells incubated with Cs alone had only a slight reduction in viability,
decreasing from 90% viable at culture initiation to 82% post-culture. Therefore under

these culture conditions there was a modest expansion in B cells and the viability did not

141

 

 

differ by more than 10% of the viability of freshly plated cells. The effect of IL-7, alone,
on recovery and viability was analogous to untreated cultures, however it provided good
protection to Cs-treated cells keeping proliferation and survival to levels analogous to

media alone.

Protective Effects of IL-7 on Spontaneous and Cs-Induced Apoptosis Among Pro B
Cells

To determine the speciﬁc effect of IL-7 on the survival of cells of the B lineage,
the marrow was phenotyped and DNA analysis of the pro, pre and lgM+ cells was
assessed by ﬂow cytometry. Figure 4.1 shows that the addition of IL-7 resulted in a 40%
decrease in spontaneous apoptosis among pro B cells (Panel A). However, this
protection from apoptosis was not evident in either the pre B cell or lgM+ cell
populations. Panel B of Figure 4.1 shows IL-7 also provided dramatic protection from
Cs-induced apoptosis causing a 60% decrease in apoptosis among pro B lymphocytes.
The IL-7 reduction in Cs-induced apoptosis in pre and IgM B lymphocytes was much
less than that seen in pro B cells; there was a 30% reduction in pre B cell apoptosis and
only a 15% decrease in lgM)r cell apoptosis. Therefore, IL-7 protected pro B
lymphocytes from both spontaneous apoptosis and Cs-induced apoptosis dining a 16 hour
culture period. Interestingly, although IL-7 had been shown to act as a growth factor for
pre B cells (Mertsching et al., 1996; Sudo et al., 1993), it did not appear to inhibit the
background apoptosis that occurred when these cells were incubated in culture media
alone, but did modestly protect the pre B cells from Cs—induced apoptosis. Therefore the

mechanism of IL-7 protection varies between protection from spontaneous apoptosis

142

 

 

verses protection against Cs-induced apoptosis, since the cytokine reduced Cs-induced

apoptosis without affecting spontaneous apoptosis.

The Ability of IL-7 to Maintain Cell Cycling of B cells in the Presence of Cs

While IL-7 can enhance proliferation in early B lymphocytes, Cs can induce cell
cycle arrest in Go/Gl in some cell types, especially early B cells (King and Cidlowski,
1998). Since the above data showed that IL-7 dramatically protected pro B cells, and to a
lesser extent pre B cells, from Cs-induced apoptosis it was important to determine if it
could protect against changes in cell cycle induced by Cs. Data in Figure 4.2 shows the
change in cycling status of pro and pre B cells when IL—7 was added alone, Cs was added
alone or Cs and IL-7 were added in combination to short-term BM B lymphocyte
cultures. Data was normalized to control levels of pro and pre B cells in the S/G2/M
phases of the cell cycle cultured in media alone. During 16 hours of culture, the addition
of IL-7 did not enhance the number of pro or pre B cells in the S/Gz/M phases of the cell
cycle. A signiﬁcant increase in IL-7 induced cycling in pro and pre B cells was not
observed until 24 hours after culture; by 48 hours there was nearly a 4-fold increase in
pro B cells in S/Gz/M and a 2.7-fold increase in pre B cells in S/G2/M in cells treated
with IL-7 (data not shown). The latter showed that the IL-7 used was biologically active
on precursor B cells. Addition of Cs to cultures resulted in a substantial decrease in pro
and pre B cells in the S/Gz/M phases of the cell cycle. Cells in a cycling phase were
decreased by nearly 50% in pro B cells and by 57% in pre B cells. Interestingly, when
IL-7 and Cs were added to cultures simultaneously, IL-7 was able to completely override

the cell cycle arrest induced by Cs in pro B cells, maintaining the level of cycling to that

143

”A.

 

 

 

seen in media or IL-7 only treated cultures. By contrast, IL-7 had no effect on the Cs-
induced decrease in cycling observed in pre B cells. These results show that IL-7 totally
protected pro B cells from the Cs-induced cell cycle arrest. In contrast, IL-7 did not
inhibit the cell cycle arrest induced by Cs in pre B cells, suggesting a variable effect by

the cytokine on changes in cycling in pro and pre B cells caused by Cs.

DNA Histograms of IL-7 Protected Pro B cells

The above data indicate that IL-7 elicited its greatest effect on the earliest
committed B cell progenitors, the pro B cell, both by protecting the cells ﬁ'om
spontaneous and Cs-induced apoptosis and by eliminating the Cs-induced cell cycle
arrest. Figure 4.3 shows the DNA distribution for representative pro B cells
(B220+S7+lgM‘) as determined by ﬂow cytometry. The percent of apoptotic cells
(hypodiploid DNA content) and cells in the S/Gz/M phases of the cell cycle (hyperdiploid
DNA content) are indicated for each treatment group. Following 16 hours in culture in
media alone, pro B lymphocytes underwent low levels of spontaneous apoptosis (less
than 10% of pro B cells) and maintained a signiﬁcant percentage of cells in the S/G2/M
phases of the cell cycle (18.5% of pro B cells). The addition of 1 ng/ml IL-7 resulted in a
reduction in background apoptosis of approximately 40%, but it did not dramatically
affect the percent of pro B cells in S/G2/M phases. Therefore, IL-7 induced survival does
not appear to be due to any enhancement of proliferation at 16 hours.

As discussed, the addition of Cs at 0.1 11M to short-term BM cultures resulted in
an increase in apoptosis in pro B cells from 10% in background cultures to 25%

apoptosis. Cs also dramatically reduced the percentage of pro B cells in the S/G2/M

144

 

phases of the cell cycle by more than 50%. Whether Cs resulted in selective induction of
apoptosis in cycling cells or induced an arrest of cycling cells in Go/Gl was not
determined here, but previous studies have shown that Cs induces cell cycle arrest prior
to induction of apoptosis. However as before, the addition of IL-7 to Cs-treated cultures
resulted in a dramatic decrease in the percent of apoptotic pro B cells (Figure 4.3 D). In
fact, the protection from Cs-induced apoptosis in this cell type appeared to be complete,
since IL-7 reduced levels of apoptosis to less than levels seen in background cultures
(Figure 4.3 A). Interestingly, the percent of cycling pro B cells was unchanged by IL-7 at
the 16 hour time point but as before IL—7 offset the reduction in cycling created by Cs in
pro B cells (Figure 4.3 D). Therefore, IL-7 appeared to inhibit both the cell cycle arrest

and apoptosis induction by Cs in the pro B cell population after 16 hours in culture.

Continued Protection of Pro B cells by IL-7 at Higher Concentrations of Cs

The culture conditions presented in the above experiments were used to ascertain
the potential of IL-7 to protect against the effects of Cs on developing B lymphocytes at
concentrations analogous to that observed during physiological stress. In the next set of
experiments higher concentrations of Cs, some that were analogous to pharmacological
levels, were used to determine whether IL-7 continued to protect B cells against apoptosis
while maintaining cell cycle status. Phenotypic distribution, degree of apoptosis, and cell
cycle status were determined following 15 hours exposure to 0.1 11M, 1 11M or 10 11M Cs
(Figure 4.4). At 1 11M Cs, apoptosis appeared to plateau and levels at 10 11M Cs were
32%, 73% and 46% apoptosis for pro, pre and lgM+ B cells, respectively. IL-7 addition

to cultures containing 0.1 11M or 1 uM Cs resulted in similar protection against apoptosis

145

 

among the B cell subsets. Apoptosis among pro B cells was decreased by approximately
60% with the addition of IL-7 at either 0.1 11M or 1 11M Cs. Pre and lgM+ B cells treated
with 0.1 1.1M or 1 11M Cs exhibited IL-7 induced decreases in apoptosis of 35% and 19%,
respectively. The degree of protection afforded by IL-7 to cells treated with 10 11M Cs
for pro and pre B-lymphocytes was not as extensive as for lower concentrations of
steroid. IL-7 caused a 43% reduction in pro B cell apoptosis and a 27% decrease in pre B
cell apoptosis of cell treated with 10 11M Cs. However 10 11M is a pharmacological level
of this steroid. Consistent with the previous cell cycle data presented, IL-7 overrode the
decrease in pro B cell cycling caused by Cs at each concentration. However pre B
cycling, as before was substantially reduced by Cs and remained unprotected by the
addition of IL—7 (data not shown). These results show that IL-7, even at high
concentrations of Cs, maintained its ability to substantially protect pro B lymphocytes

from apoptosis and decreases in cell cycling.

The Effect of SCF on Background and Cs-Induced Apoptosis in BM B Lymphocytes

Since SCF also has beneﬁcial effects on early B cell development, its effects on
background and Cs-induced apoptosis in B lymphocytes were also investigated. Figure
4.5 shows the effect of SCF on background and Cs-induced apoptosis in pro, pre and
IgM+ cells. SCF was added to cultures at predetermined concentration of 100 ng/ml and
Cs was used at a concentration of 0.1 M. The addition of SCF to cultures, resulted in a
decrease in spontaneous pro B cell apoptosis of 40% but pre and IgM bearing cells were
not signiﬁcantly protected. In contrast to the dramatic effect of SCF on background

apoptosis in pro B cells, only a small protective effect was seen on Cs-induced apoptosis

146

 

in these cells. In fact, the decrease of 17% in pro B cell apoptosis might be attributed to
SCF protection from basal apoptosis as described above. Additionally, pretreatment of
cells with SCF for 20 hours did not result in decreased apoptosis beyond that presented
here (data not shown). Therefore, although SCF appeared to protect pro B cells from
basal apoptosis in culture it did not necessarily protect these cells ﬁ'om Cs-induced
apoptosis, in contrast to the effect of IL-7. This suggests that SCF might promote in vitra
survival, similar to IL-7, but it does not protect these cells from the negative effect of Cs.
Since SCF acts synergistically with IL-7 to promote lymphopoiesis, it might
synergize with IL-7 to protect pro B cells from Cs-induced apoptosis. To determine this,
SCF together with IL-7 was added to cultures and their effects on apoptosis in pro B cells
analyzed. Table 4.2 shows the percent of apoptosis in pro B cells cultured alone or with
SCF, IL-7 and Cs in various combinations. IL-7 was also added at more dilute
concentrations to obtain a lower level of protection from Cs-induced apoptosis, since at
0.1 ng/ml it reduced apoptosis to background levels in pro B cells therefore any effect by
SCF may not have been apparent. The data indicated that SCF did not appear to
synergize with IL-7 to protect pro B cells from Cs-induced apoptosis. Additionally the
combination of SCF and IL-7 did not result in more protection from background
apoptosis. Pretreatment with SCF for 20 hours also did not provide enhancement of the
protective effect by IL—7 (data not shown). Clearly, although both SCF and IL-7 promote
the survival of pro B cells in culture these cytokines do not synergize in the protection of

be.

these cells ﬁ'om Cs-induced apoptosis.

147

 

DISCUSSION

The survival of hematopoietic cells often depends on signals sent from cytokines.
The limiting amounts of cytokines present in short-term marrow cultures can result in
enhanced cell death. Here we have shown that both SCF and IL-7 appear to act as
survival factors speciﬁcally for the pro B cell stage during B cell development by
reducing the degree of background or spontaneous apoptosis in these cells aﬁer
approximately 16 hours in culture. This reduction in spontaneous apoptosis preceded the
enhancement of proliferation by either cytokine, which did not occur until 24 hours. This
suggests separate mechanisms for cell cycle control and cell survival.

The promotion of pro B cell survival in culture did not necessarily mean that
either IL-7 or SCF could protect these cells from the induction of apoptosis by Cs. Here
it was shown that IL-7 dramatically reduced Cs-induced cell death in pro B lymphocytes
and it also inhibited the cell cycle arrest caused by the steroid. Therefore IL-7 appears to
block Cs signaling of apoptosis and/or to promote cell survival signals that overrides Cs
death promoting and cycle blocking activity. Even though IL-7 prevented the cycle block
it did not cause increased proliferation, above background levels, until after 24 hours in
cultm'e, suggesting that protection from apoptosis occurred earlier than the enhancement
of cell cycling.

Gc-induced apoptosis has been studied for many years, yet its exact mechanism of
eliciting death has not been established. It is becoming clear that Gc signaling through
the GcR can affect many other intracellular signaling pathways. For example, Gc has

been shown to negatively affect the nuclear factor kappa B (N FKB) and AP-l

148

transcription factors, both of which can promote survival and proliferation of
lymphocytes (Auphan et al., 1995; Heck etal., 1994). Additionally, some evidence exists
that Gc may promote apoptosis by the activation of phosphatidyl inositol dependent
phospholipase C and subsequent activation of sphingomyelinase and cerarrride release
(Cifone et al., 1999). It is apparent that much more investigation will be needed to
determine the exact mechanism of Gc-induced cell death and it is likely that different cell
types may use different pathways to induce death.

The recently described AKT pathway, which promotes survival by causing the
cytosolic sequestration of the pro-apoptotic protein Bad, may be a viable mechanism for
the survival induced by both lL-7 and SCF. The receptors for both IL—7 (IL-7R) and SCF
(c-kit) have been previously reported to have phosphatidyl inositol 3-kinase (PI3K)
activity upon ligand binding (Blume-Jensen et al., 1998). AKT has been shown to be
phosphorylated by the PI3K pathway, thus rendering it active to phosphorylate the
cytosolic protein 14-3-3 that sequesters Bad (Datta et al., 1997).

Reports on the expression of the IL-7R on B lymphocytes have varied, but it now
seems clear that the high afﬁnity form of the receptor is present on IgM' cells (pro and
pre B cells) (Sudo et al., 1993). If receptor activity was the same in pro and pre B cells,
the differences in IL-7 mediated protection from apoptosis and cell cycle arrest must be
attributed to variations in intracellular conditions and signaling. Clearly, the use of
phenotypic and apoptotic analysis provided great insight into the response of primary
developing B lymphocytes from the BM to speciﬁc cytokines. The use of primary cells

for these studies was very important in determining responses such as survival, since

149

 

normal cell survival, cell proliferation and cell differentiation are often compromised in
cell lines.

Interestingly, IL-7 elicited a modest, yet signiﬁcant protective effect against Cs-
induced apoptosis in pre B lymphocytes even though it did not promote background
survival in this cell type. This suggests that IL-7 may be utilizing different mechanisms
to promote background survival (perhaps the AKT pathway) than that used in promoting
protection from Cs-induced apoptosis. Since Bel-2 upregulation by IL-7 has been
previously reported, it could be that protection from Cs is mediated by increased
expression of this anti-apoptotic factor (Hemandez-Caselles et al., 1995; Karawajew et
al., 2000). Also of interest, although apoptosis was decreased, IL-7 did not inhibit the
Cs-induced cycle decrease in pre B cells. Therefore it appears from the pre B cell results,
that the protective effect of IL-7 on Cs-induced apoptosis can be separate from its ability
to inhibit cell cycle arrest.

Considering the similar ability of IL—7 and SCF to provide protection of pro B
cells from spontaneous cell death, it would be reasonable to assume similar effects on Cs-
induced apoptosis. This was not the case. SCF provided no appreciable protection ﬁom
Cs treatment, above that seen in background cultures, in any cell type. Also when used in
combination with lL-7 it did not appear to augment the protective effects of IL—7. Poor
activity of the recombinant SCF could be one explanation for the lack of apparent effects,
but this is unlikely since titrations were performed to determine the maximum effect of
SCF and SCF elicited protective effects on background apoptosis. Had there been no
activity or low activity, it would be likely that SCF-induced protection from spontaneous

apoptosis would not have been apparent.

150

 

By using methodology that allowed for the direct analysis of apoptosis in primary
developing B lymphocytes it was determined that IL-7, in fact, could protect early B
lymphocytes from Cs—induced apoptosis. Considering the devastating affect that Cs
elevation can have on the immune system, IL-7 may warrant further investigation as an
immunotheraputic agent. The mechanisms by which Cs elicits its effect or whereby IL-7
can inhibit those effects does not appear to be a simple process, the differential effects on
apoptosis and cell cycle status between cell types suggests a complex interplay between
these two signaling systems. It does appear that IL-7 mediated protection from
background apoptosis and Cs-induced apoptosis utilizes at least two different pathways.
Additionally, protection from apoptosis clearly occurs prior to induction of proliferation
and the two processes appear to be regulated separately. SCF, while similar to IL-7 in
protection from background apoptosis, does not appear to protect B lymphocytes from
Cs-induced apoptosis. This suggests that either these cytokines initiate different modes
of survival or that a different mechanism exists whereby IL-7 can also promote protection
from Cs-induced death. A combinatorial approach between the analysis of biological
responses and cell signaling analysis may aid in the elucidation of some of the

interactions between Cs-mediated and IL—7-mediated responses.

151

 

Table 4.1 Recovery and viability of bone marrow cultured for 15 to 16 hours

 

 

 

 

 

 

 

 

Media only 0.1 M Cs 0.1 rig/m1 IL-7 Cs and IL-7
Recovery‘ 126 i 9%c 106 i 8% 127 :1: 8% 123 i 18%
Viability*" 93 :1: 3% 82 :1: 3% 90 i 3% 86 :1: 3%

 

" Recovery was determined by dividing the viable and apoptotic cells obtained following
culture by the number of viable cells added to the original culture
b Viability was determined by dividing the viable and apoptotic cells obtained following
culture by the total of viable, apoptotic and trypan blue+ cells (dead)
° Cells were cultured for 15 to 16 hours, the data are averaged from ﬁve separate
experiments and standard deviations are shown
* Cell viability was approximately 90% at the initiation of cell cultures

152

 

 

Figure 4.1 The effect of IL-7 on spontaneous levels of apoptosis verses Cs-induced
apoptosis in pro, pre and IgM+ B lymphocytes. BM was cultured for 16 hours either with
0.1 ng/ml lL-7 only, 0.1 1.1M Cs only or with a combination of IL-7 and Cs. Phenotypic
and DNA analysis were performed using ﬂow cytometry. Panel (A) shows the change in
spontaneous apoptosis elicited by the addition of IL-7. Data was normalized to the
percent of apoptosis in cells cultured in media alone, with actual percent apoptosis of 7%,
16% and 11% for the pro, pre and IgM+. Panel (B) shows the change in Cs-induced
apoptosis caused by IL-7. Data was normalized to the percent apoptosis in cells
incubated with Cs alone, with actual percentages of 23%, 53% and 39% for pro, pre and
lgM+ cells, respectively. lL-7 induced changes in apoptosis were determined for four
separate experiments and were averaged with standard error bars shown. Signiﬁcant
differences were determined by the Student’s t-test (p < 0.05) and indicated with an
asterisk.

153

No Cs

 

 

 

 

120%
100%
,2 80%
.8
g. 60%
9-
< 40%
20%
0% ,
ProB PreB
120% . Cs Treated
100%
U)
‘5 80%
8
Q.
a 60%
<
40%
20%
0% i
ProB PreB

INo IL7 El 111ng IL7

Figure 4.1 IL-7 modulation of spontaneous and Cs-induced apoptosis

154

Figure 4.2 Data show the effect of added IL-7 on the S/Gz/M phases of the pro and pre
B lymphocyte cell cycle of cells cultured in media alone or treated with Cs. BM was
cultured for 16 hours in media alone (solid bars), with 0.1 ng/ml IL-7 alone (sparse dots),
with 0.1 11M Cs alone (stripes) or with a combination of both IL-7 and Cs (dense dots).
Data was normalized to 100% percent for pro or pre B cells in the S/GZ/M phase of the
cell cycle when cultured in media alone; actual percentages were 17% for pro B cells and
15% for pre B cells (controls). The percentage S/G2/M was determined via ﬂow
cytometric analysis of the DNA content and pro and pre B cells were identiﬁed by
phenotyping. The data shown are the average of two separate experiments and stande
error bars are shown. Signiﬁcant differences were determined by the Student’s t-test (p <
0.05) and indicated with an asterisk.

155

 

 

 

 

 

 

 

160% I IControl r
a 1 E] IL-7
1 120% J ICs and IL-7
: E 100% ~ 1
N l
1 Q 80% « t
. m , l
i 60% — 1
are .
40% i
DEL % ‘ ’
it. 3 20% 1
1c ,
and ' 0% 1
rd ~7 4 , # ——e _, J
‘p <

Figure 4.2 IL—7 and/or Cs effect on cell cycle distribution among pro and pre B cells

156

 

Figure 4.3 A representative DNA ﬂow cytometric proﬁle of pro B lymphocytes cultured
for 16 hours was determined by DAPI staining of phenotyped BM B cells. The
hypodiploid, or apoptotic, populations are indicated by “A0”. Panel (A) shows the DNA
content of pro B cells incubated in media alone. Panel (B) shows the percent A0 and
S/Gz/M for cells incubated with 0.1 ng/ml lL-7 alone and panel (C) shows pro B cells
incubated with 0.1 uM Cs. Panel (D) is the DNA proﬁle of pro B cells incubated with
both IL-7 and Cs. These data are representative of several experiments.

157

 

Relative Cell Number

 

 

 

I llilll 1111111

 

 

 

 

  
   

1 D

1 06/6.

1

1 ji A0 S/szM
' 7.3% 17.7%

 

FL

 

 

1
1
1

DAPI ﬂuorescent (DNA Content)

Figure 4.3 Flow cytometry of lL-7 and/or Cs effect on pro B cell apoptosis
and cell cycle

158

 

 

 

Figure 4.4 Data show that IL7 protected B lymphocytes from Cs-induced apoptosis even
at high concentrations of the steroid. The percent apOptosis, as determined ﬂow
cytometrically by DAPI stained DNA, is shown for (A) pro B (B) pre B and (C) lgM+ B
cells. BM was cultured for 16 hours either with no Cs, 0.1 11M, 1 11M or 10 11M Cs (solid
line) and the percent apoptosis was plotted. The percent apoptosis for each population
incubated with the above concentrations of Cs and with 0.1 ng/ml IL7 (broken line) is
also shown. Standard error bars are plotted and the data shown are representative of at
least two separate experiments.

159

ttptosis even
ow

'0 13211.3
.\1 C s 150111
)pU1311011

1 11116115
nveofm

% Apoptosis
N
6

Pro B Cell

 

 

10
01“ *_,1____
N0 CS 0.1 pM Cs 1 ”M Cs 10 “M Cs
80 Pre B Cell

 

% Apoptosis
&
G

 

No Cs 0.1 ”M Cs 1.0 ”M Cs 10 11M Cs

IgM+ B Cell

    

% Apoptosis

No Cs 0.1 “M Cs 1.0 “M Cs 10 “M Cs
° N0 IL7 "a" lug/m1 IL7

Figure 4.4 IL-7 effect on apoptosis induced by varying concentrations
of Cs among B cells

160

 

 

Figure 4.5 The effect of SCF on spontaneous and Cs-induced apoptosis in bone marrow
B cells. BM was cultured for 15 to 16 hours, cells were phenotyped for B cell subsets
and DNA was stained with DAPI to determine the amount of apoptosis by ﬂow
cytometry. SCF was used at 0.1 rig/ml and Cs was used at 0.1 uM. Panel (A) shows the
effect of SCF on spontaneous apoptosis in media alone. Data were normalized to the
amount of apoptosis in cells cultured without SCF addition with actual percentages of
12%, 16% and 16% for pro, pre and lgM+ cells, respectively. Panel (B) shows the effect
of SCF on Cs-induced apoptosis. Data were normalized to the level of apoptosis in cells
cultured with Cs only with actual control percentages of 22%, 49% and 40%,
respectively. Standard error bars are shown for the average of three separate experiments
where signiﬁcant differences were determined by the Student’s t-test (p < 0.05) and
indicated by an asterisk.

161

tone marrow
:11 subsets
0w

\) shows the
:ed 10 1116
:ntagCS 01
.vs the 9113“
tests in 65115

t'expt’ﬁmm
051 and

125% ‘

10096 1
75% j *
50% j
25% 3
0% 1
Pro Pre I

Apoptosis

 

13M+

1 1
1 I No treatment B SCF only ‘

B j 1
1 125% 4 I
1 m 100%

§ 75% 1
~ 8
1 2- 50% 1
1 25%
i 0% 1
1 Pro Pre IgM+

3 CS C ”CS, 3995‘???

Figure 4.5 SCF effect on spontaneous and Cs—induced apoptosis

162

 

 

Table 4.2 Effects of IL—7 and SCF on apoptosis in pro B cells

 

 

 

 

 

 

 

No Treatment _- SCF‘ ’ CSF— CS and SCF

N" "’7 8.7 :1: 0.5% ° 43 :t 1.2%* 23.4 :1: 4.1% 16.3 i 1.9%"
"-1 “g’m' "’7 3.6 :1: 2.2% 3.6 :1: 0.9% 7.3 :1: 2.8% 7.1 i 1.1%
0.01 ng/ml IL-7 N/D N/D 14.1 :1: 0.7% 13.8 i- 23%
0.001 118/ml 1L'7 M) MD 18.1 :1: 2.8% 19.2 i 1.0%

 

 

 

 

 

' SCF was used at 100 ng/ml

b Cs was used at 0.1 MM

° Data are the averages of duplicate experiment :1: the standard deviations and represent at
least two experiments

. Signiﬁcantly different from control sample (p < 0.05)
" Signiﬁcantly different from Cs sample (p < 0.1)

 

 

163

 

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