A DITHIOTHREOTOL-DEPENDENT
ANTIBACTERIAL FACTOR FROM
LYSATE AND LYSATE FRACTIONS OF
PERITONEAL SELLS FROM MICE INFECTED
WITH MYCOBACTERIUM BOVIS. BOG ' '

Dissertatien far the Degree of Ph. D.
MSCHIGAN STATE UNWERSIW
DONNA Y. MUIRHEAD
E9T4 ,

LIBRARY

Michigansma
U .A .

 

This is to certify that the
thesis entitled
A Dithiothreotol-Dependent Antibacterial Factor

from Lysate and Lysate Fractions of Peritoneal Gen?"
from Mice Infected with Mycobacterium bovis, BCG

 

presented by

Donna Y . Mu irhead

has been accepted towards fulfillment
of the requirements for

Ph.D. Microbiology

degree in

 

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ABSTRACT

A DITHIOTHREOTOL-DEPENDENT ANTIBACTERIAL FACTOR
FROM LYSATE AND LYSATE FRACTIONS OF PERITONEAL CELLS
FROM MICE INFECTED WITH MYCOBACTERIUM BOVIS, BCG

By

Donna Y. Muirhead

Antilisterial activity was detected in lysates of mouse peritoneal
mononuclear cells. Retention of the activity after collection was
dependent on a reducing agent, dithiothreotol (DTT), but could be
regenerated in stored lysates with DTT.

Lysates of peritoneal cells from mice immunized and prestimulated
with Mycobacterium bovis, BCG, had slightly more activity than lysates
from control mice. Peritoneal cells from BCG-immunized and prestimu-
lated mice had a greater reduction potential than controls as measured
by the ability to reduce nitro blue tetrazolium in yiggg.

Activity was found in the pellet fraction containing lysosomes
from homogenized peritoneal cells fractionated by differential centri-
fugation and correlated with acid phosphatase, a lysosomal enzyme marker.

Lysates of both glass adherent and nonadherent peritoneal cells
from Swiss Webster mice had antilisterial activity; the former had more.
Lysates of purified populations of peritoneal macrophages and spleen
lymphocytes from CBA/J mice possessed low levels of antilisterial activ-

ity. Lysates of normal peritoneal macrophages which had been incubated

Donna Y. Muirhead

with culture fluid of BCG-sensitive spleen lymphocytes possessed anti-
listerial activity at much higher titers in the presence of DTT. Both
lymphocyte culture supernatant fluid and lysates had slight activity.

Antilisterial activity in crude lysates was stable at 100 C for
10 minutes, but was partially lost after lyophilization or aeration.
The material was acid labile in the absence of DTT.

Inactivation of stationary phase listeria over time in the
presence of crude lysates was biphasic. A rapid decrease in the
number of colony forming units occurred initially within 1% hours and
was followed by a slower rate of inactivation. There was no detectable
effect on listeria in log phase.

TWO fractions with antilisterial activity were detected in
eluates from DEAE-cellulose chromatography of peritoneal cell lysates
of BCG-immunized and prestimulated mice. The first fraction (DEAE-I)
was found in eluates from cell lysates of control mice. The second
(DEAE-II) was detected only in lysates from BCG-immunized mice. The
fraction had optimal activity near neutral pH.

DEAE-II was less stable than the unfractionated lysate to heating
and differed in listerial inactivation kinetics. Combining the two
active DEAE fractions did not restore the original kinetic pattern.
Although DEAE fraction II does not completely account for the activity
found in unfractionated whole lysates, it does appear to be unique to
lysates of peritoneal cells from BCG-immunized and prestimulated mice
and may partially account for the increased antimicrobial properties

of activated macrophages in antibacterial cell-mediated immunity.

A DITHIOTHREOTOL-DEPENDENT ANTIBACTERIAL FACTOR
FROM LYSATE AND LYSATE FRACTIONS OF PERITONEAL CELLS
FROM MICE INFECTED WITH MYCOBACTERIUM BOVIS, BCG

by

Donna Y. Muirhead

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Microbiology and Public Health

1974

ACKNOWLEDGMENTS

There are a great number of people I must thank for their
assistance and support during my graduate school years. First I
wish to thank Dr. Virginia H. Mallmann for her help and guidance
in this project.

Much appreciation goes to my husband, Dennis, and children,
Kelly and Todd, for their enlightened attitudes and continuous
encouragement.

I also wish to thank Dr. Doris Beck for her moral support and
friendship through our years together as graduate students.

I thank Mrs. Litty Moore for her encouragement and patience in
preparing this thesis.

I am grateful to Drs. Ronald Patterson and Robert Brubaker and
members of my committee, Harold Miller, Norman McCullough and Donald
Twohy for their time, help, and many useful suggestions.

I am indebted to the Michigan Tuberculosis and Respiratory

Disease Association for their financial assistance.

11

TABLE OF CONTENTS

Page

LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . v
LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . vii
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . 1
LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . . . . . 3
I. Humoral antimicrobial systems . . . . . . . . . . . . . 3

A. "Normal" and nonspecific humoral factors . . . . . . 3

B. Specific induced antibodies . . . . . . . . . . . . 7

II. Cellular antibacterial systems . . . . . . . . . . . . 8
A. Normal cellular systems . . . . . . . . . . . . . . 8
l. Neutrophils . . . . . . . . . . . . . . . . . . . 8

2. Eosinophils . . . . . . . . . . . . . . . . . . . 10
3. Lymphocytes . . . . . . . . . . . . . . . . . . . 10
4. Monocytes and macrophages . . . . . . . . . . . . 10
B. Cell-mediated antibacterial immunity . . . . . . . . ll

1. Immunological aspects of cell mediated
imunity I O O O O O O O O O O O O O O O O 0 O 11

2. Cell-mediated immunity in microbial
diseases 0 O O O O O O O O O O O O O O O O O O 12

3. Cellular mechanisms . . . . . . . . . . . . . . . 14
a. Lymphocytes in cell-mediated immunity . . . . 14

b. Cell-cell interactions and biological
mediators . . . . . . . . . . . . . . . . . 19

iii

Page

1. Specificity and relationship with
delayed-type hypersensitivity . . . . . 19

2. Factors from lymphocytes affecting
macrophages . . . . . . . . . . . . . . 24

3. Factors from macrophages affecting
lymphocytes . . . . . . . . . . . . . . 31

4. Cytophilic antibody . . . . . . . . . . . . 32

c. Macrophages in antimicrobial cell-
mediated immunity . . . . . . . . . . . . . 33

WRENQS . O O O O O O O O O O O I O O O O O O O O O O O O C O 3 9
MANUSCRIPT l . . . . . . . . . . . . . . . . . . . . . . . . . . 59
An Antilisterial Factor from Mouse Peritoneal Cells
MANUSCRIPT 2 . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Dithiothreotol-Dependent Antilisterial Activity of
Lysates from Normal Macrophages Exposed in vitro
to Culture Fluids from Spleen Cells of BCG-
Immunized Mice
MAMJSCRIPT 3 0 O O O O O O O O O O O O O O O O O O O O O O O O O 108
Isolation and Characterization of an Antibacterial

Factor from Peritoneal Cell Lysates of Mice
Immunized with Viable Mycobacterium bovis, BCG

iv

TABLE

LIST OF TABLES

LITERATURE REVIEW
Antimicrobial factors in normal cells and serum

Distinguishing characteristics of T lymphocytes,
B lymphocytes, and macrophages . . . . . . . . .

Factors from lymphocytes or associated with
delayed hypersensitivity-ccllular immunity . . . . .

MANUSCRIPT 1

Acid phosphatase activities of high speed supernatant
fractions from lysates of 10 macrophages per ml

Antilisterial activities of high speed supernatant
fractions from peritoneal cell lysates from BCG-
immunized and control mice diluted to equivalent
acid phosphatase activity . . . . . . . .

MANUSCRIPT 2

Antilisterial activity with or without DTT of culture
fluids from mouse spleen lymphocytes, incubated
in vitro with or without BCG, from normal and
BCG-immunized mice . . . . . . . . . . . . . .

Antilisterial activity with or without DTT of lysates
of mouse spleen lymphocytes, incubated.ig vitro
with or without BCG, from normal and BCG-immunized
mice 0 O O C O O O O O I O O O O O l O O O C C C O

Antilisterial activities with or without DTT of lysates
of normal mouse peritoneal macrophages after incuba-
tion in vitro with culture fluids from spleen
lymphocytes, cultured with or without BCG, from
normal or BCG-immunized mice . . . . . . . . . . . .

Page

15

25

89

90

105

106

107

TABLE Page

MANUSCRIPT 3
1. Stability of antilisterial factor in crude lysates . . . . 133

2. Amount of antilisterial activity recovered in
fractions from peritoneal cell lysates . . . . . . . . . 134

3. Antilisterial activity of crude lysates and
DEAE-cellulose fraction II after heating . . . . . . . . 135

4. Antibacterial activities of unfractionated lysate
and DEAR-cellulose fraction II . . . . . . . . . . . . . 136

vi

LIST OF FIGURES

FIGURE Page
MANUSCRIPT l

1. Restoration by 10 mM dithiothreotol (DTT) of
antilisterial activity of mouse peritoneal
cell lysate lost by overnight storage at 2 C . . . . . . 78

2. Antilisterial activity of lysates of subpopulations
of mouse peritoneal cells . . . . . . . . . . . . . . . 80

3. Nitro blue tetrazolium reduction by mouse
peritoneal macrophages . . . . . . . . . . . . . . . . . 82

4. Acid phosphatase and antilisterial activities of
high speed supernatant fractions of mouse
peritoneal cells homogenized 2, 4, 6 and
8 minutes . . . . . . . . . . . . . . . . . . . . . . . 84

5. Acid phosphatase and antilisterial activities of
low speed pellet fractions of mouse peritoneal
cells homogenized 2, 4, 6 and 8 minutes . . . . . . . . 86

6. Acid phosphatase and antilisterial activities of
high speed pellet fractions of mouse peritoneal
cells homogenized 2, 4, 6 and 8 minutes . . . . . . . . 88

MANUSCRIPT 3

l. Fractionation diagram for purification of
antilisterial factor . . . . . . . . . . . . . . . . . . 122

2. DEAR-cellulose chromatography fractionation of
high speed supernatant fluid . . . . . . . . . . . . . . 124

3. Relative absorbance patterns of stained acrylamide
gels of unfractionated high speed supernatant
fluid and DEAE-cellulose fractions I and II
separated electrophoretically . . . . . . . . . . . . . 126

4. Sephadex fractionation of high speed supernatant
fluid 0 O O O I O O O O O O O O O O C O O O O O O O O O 128

vii

FIGURE Page
5. Antilisterial activity of DEAR-cellulose
fraction II at different pH . . . . . . . . . . . . . . 130
6. Rate of listerial inactivation by unfractionated

lysate and fractions I and II from DEAE-
cellulose chromatography . . . . . . . . . . . . . . . . 132

viii

INTRODUCTION1

Antibacterial cell-mediated immunity is a host response to
infection with a facultative, intracellular parasite. Animals
which have mounted a cellular response against agents such as
mycobacteria possess "activated" macrophages. These cells are
nonspecifically more active against not only the same microorganisms,
but also against many others. Whereas the macrophage is the cell
which directly provides this protection and by which the response
is observed, an immunologically specific sensitized lymphocyte popu-
lation provides the stimulus for activating these cells. The lympho-
cytes are sensitized during the initial contact and the specificity
of antibacterial cell-mediated immunity is the subsequent reaction
between the same antigen and sensitized lymphocytes. The lymphocytes
then elaborate "lymphokines" which activate macrophages. The greater
antibacterial efficiency of these cells is measured by increased
survival times of the host and decreased numbers of viable bacterial
units. The bactericidal or bacteristatic mechanisms of activated
macrophages are unknown.

The purpose of this study was to examine a phenomenon observed
in previous research in which antistaphylococcal activity was
detected in lysates of peritoneal cells from mice immunized and

1The material presented in this Introduction is discussed more

completely and referenced in the following Literature Review.

1

prestimulated with Mycgbggterium.bggi§, BCG, but not from control
mice. This observation held promise for a possible correlation with
antibacterial cellular immunity and study at a subcellular level.
This thesis is composed of four sections. The first is a
literature review describing antibacterial systems of cells and
tissues of normal and immune animals. The other three sections
consist of manuscripts prepared for publication. The first paper
describes the activity present in murine peritoneal cell lysates,
the second the effect of BCG-sensitive lymphocytes on activity of
normal peritoneal macrophages and the third the purification and

characterization of active fractions.

LITERATURE REVIEW

I. Humoral antimicrobial systems.

A. "Normal" or nonspecific humoral factors.

The large number of antimicrobial substances and systems that
have been identified in normal cells and sera are summarized in Table l.

The early reports, dating back to the late 1800's, are often
confusing and ill-defined. One of the earliest humoral substances
reported was beta-lysin which is bactericidal, primarily for gram-
positive microorganisms (166). Beta-lysin is a system composed of at
least two components, released during blood coagulation, and probably
is only activated in sites where tissue damage occurs and fibrin is
formed (200).

Lysozyme is an enzyme, muramidase, found in the granule fraction
of macrophages and polymorphonuclear leucocytes and in body secretions.
It is bactericidal to some organisms (31, 147). Its range of activity
can be increased by pretreatment of the organisms with specific
antibody and complement (200), by an acidic environment or by lipid
solvents (153).

Histone, protamines and other basic proteins and polypeptides
have antimicrobial properties, primarily against gram-positive organisms
(136). Histones are extracted from nuclei as complexes with nucleic
acids (136). Protamines may be obtained from a variety of sources

(135, 224). The mode of action appears similar to the cationic

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proteins of polymorphonuclear leukocytes which is discussed in a
later section (II.A.1).

Complement is perhaps the best studied humoral component, first
described by Nuttall in 1888 (155). With antibody, complement is
bactericidal to gram-negative organisms. Complement, with antibody
may be lethal to some viruses (81), protozoa (198), and spirochetes
(109) and may enhance phagocytosis (201) and immune aggregation (105).

The exact mechanism of the complement bactericidal reaction is
unknown. Muschel (145) postulated the bacterial cell membrane was
damaged. He theorized the complement activating antigen-antibody
reaction took place at the cell wall and the activated complement
components were transferred through the wall to the bacterial membrane.

The resistance of gram-positive microorganisms to complement has
been explained by a thicker cell wall and a lower phospholipid content.
Protoplasts of gram-positive Bacillus subtilus are affected by anti- .
body and complement (146). The action of complement on the cell
membrane may be an intermediate rather than the final reaction leading
to bacteriolysis. Escherichia coli B can be made nonviable through
the complement-antibody reactions but the cells do not lyse unless
lysozyme is added (3).

Properdin, a protein present in normal serum, is bactericidal in
the presence of complement against gram-negative organisms, and also
neutralizes some viruses (169). The properdin system is now generally
regarded as normal or natural antibody that is of low specificity.

Properdin antibactericidal activity is not as effective when excessive

amounts are present and is inhibited by specific antibody to Shigella
dysenteriae (220).

leukins (73), plakin (70), hematin and mesohematin (87), and
lactenin have been reported but not well-characterized. A comprehen-
sive review written by Skarnes and Watson (200) describe these factors.

Other antimicrobial agents such as fatty acids, high CO2 tension
and lactic acid accumulation in inflammatory sites can also contribute
to host resistance by acting singly or in concert with another anti-

bacterial factors (47). Ionic environment and pH can also affect the

outcome of an infection (51).

B. Specific induced antibodies.

Humoral immunity is important in most, if not all, types of
infections. Sera of artificially immunized or convalescent animals
contain antibodies, immunoglobulins which may confer protection when
exposed to the same or similar cross-reacting microorganisms.

Immunoglobulins, or antibodies, provide protection through several
mechanisms: 1) the neutralization of toxins secreted or associated
with microorganisms, 2) neutralization of viruses, 3) with complement,
bactericidal and 1ytic activity of gram-negative bacteria, and 4)
opsonization of microorganisms for enhanced phagocytosis and removal
from the host's system.

Potentially pathogenic agents such as Diplococcus pneumoniae and
Haemophilus influenzae are generally disposed of by the humoral response.
Antibodies are formed against facultative or obligate intracellular par-

asites, but apparently are ineffective in resolving these infections (122).

II. Cellular antibacterial systems

A. Normal cellular systems

1. Neutrophils

White cells (leucocytes) phagocytose, inactivate, kill and degrade
many types of microorganisms. Polymorphonuclear leucocytes (PMN's)
are present in large numbers in the tissues and circulation and are of
prime importance in preventing infection. Phagocytosis by PMN's begins
by ingestion of the microorganism, through the invagination of the cell
membrane and formation of a vacuole (phagosome). Adjacent granules
(primary lysosomes) fuse with the phagosome (now a secondary lysosome)
and release their contents. This is generally followed by the death
and degradation (digestion) of the ingested organism (94).

There are two or more separate populations of primary lysosomes
in PMN's, each containing a somewhat different content of enzymes or
factors. The populations differ as to size, density, enzyme content,
and time and mode of origin (225).

Increased metabolic activity is associated with phagocytosis.
Oxygen and glucose consumption and lactic acid production are increased
as is increased glucose l-C oxidation, a measure of hexose monophos-
phate shunt activity (192). The increased lactic acid production lowers
the intravacuolar pH (127) which may contribute to antibacterial action
on acid sensitive microorganisms such as the pneumococci.

The increased respiration is associated with H 0 formation (10).

2 2

The H202 formed by leucocytes may be bactericidal independently or

as part of the myeloperoxidase system which is very effective against

many bacteria, fungi and viruses (14, 93, 114, 209). The system

requires myeloperoxidase enzyme, H202, and iodide. Other halide
cofactors such as bromide or chloride may substitute for iodide.
Optimal activity occurs at pH 4.5 to 5.0 and the mode of action was
originally postulated to be a halogenation of the infectious agent
(93). More recently, it has been suggested that aldehydes are produced
by amino acid decarboxylation and deamination. The myeloperoxidase
system may catalyze formation of H001 from C1- and H202 in an acid
medium. The HOCl may then react with amino acids producing chloramines
which spontaneously decompose to NH3, C02, Cl' and the bactericidal
aldehyde (193, 241).

A number of cationic proteins with antbmicrobial activities have
been extracted from PMN granules. A crude preparation, termed phago-
cytin by Hirsch (77, 78) has an acid pH optimum and purified material
is heat stable (100 C, 90 min.). The cationic proteins can be
separated electrophoretically into a number of fractions with different
antimicrobial specificities, including both gram-positive and gram-
negative organisms (240). Unfractionated preparations also inactivate
actinomyces (39). The effect of the material is dependent on the
number of bacteria (77). There is an initial, rapid decrease in the
numbers of viable bacteria followed by no additional killing. The
proteins reportedly are bound by strong electrostatic forces, causing
bacterial clumping, inhibition of oxygen consumption and damage to
the cell membrane (239).

An iron-binding protein, lactoferrin, in leucocyte granules is

bacteriostatic (128). Lysozyme, also present in leucocyte granules

may lyse certain bacteria. Its antibacterial properties may be

lO

enhanced by pretreatment of bacteria with specific antibody and
complement (139), although its major contribution may be the degrada-

tion of microorganisms after they are killed by other mechanisms.

2. Eosinophils

Eosinophils may phagocytose and kill bacteria by mechanisms
similar to those of neutrophils. These systems operate more slowly
and less efficiently than do those of neutrophils and probably do not
offer a major contribution to antiinfectious systems (26, 37).
Morton et al. (143) presented evidence for an antibacterial system

at the cell membrane which does not require phagocytosis.

3. Lymphocytes

If lymphocytes have phagocytic properties is a point of debate.
Reports that lymphocytes phagocytose microorganisms (176, 242) are
countered with those claiming they do not (22). Strauss et al. (210)
reported spleen cells (95% lymphocytes) have a marked hexose mono-
phosphate shunt activity when incubated with phagocytosable particles
and suggested bactericidal activity may also be occurring at the cell
surface. They did not observe phagocytosis, but by light microscopy,
bacteria were seen in close association with the spleen cells. They
proposed that lymphocytic antimicrobial systems are similar to PMN's,

with differences being chiefly quantitative.

4. Monocytes and macrophages
It has been amply demonstrated that cells of the monocyte-macrophage

series phagocytose, kill and degrade many infectious agents (28, 211).

11

The systems and mechanisms will be discussed later and compared with
the antibacterial mechanisms of "activated" macrophages of antimicrobial

cell-mediated immunity (AM-CMI).

B. Cell-mediated antibacterial immunity
1. Immunological aspects of cell-mediate immunity

Antimicrobial cellular immunity refers to the increased capacity
of macrophages to kill or inhibit microorganisms after a host has
been infected. The macrophages from immune animals are better able
to inactivate and/or destroy microorganisms than macrophages from
normal animals.

The criteria of the response categorize it as an immunologic
reaction because 1) the host requires a finite period of time to
respond, 2) immune animals slowly lose their resistance but respond
more quickly to the second contact with the same organism, 3) resistance
may be increased by additional injections with the same organism (120),
and 4) the response requires a particular lymphocyte population (124).

The formal separation of cell-mediated immunity from humoral
immunity is based on the capability of the latter system to passively
transfer immunity with serum from hmmune animals. Cellular immunity
may be passively transferred to normal recipients with lymphocytes
from immune animals (113).

Antibody, or humoral immunity which can be passively transferred
with serum, is more effective against acute bacterial infections
whereas cell-mediated immunity is associated with chronic bacterial

infections (121). Classical circulating antibody has no apparent

12

effect on the resolution of infections with most facultative or
obligate intracellular parasites (172). In addition, antimicrobial
cell-mediated immunity appears to be more of a local or centralized
response rather than the systemic response observed in humoral or
antibody-mediated immunity. For example, the activated macrophages
in cellular immunity achieve the greatest activation at local tuber-
culous lesions (41, 222).

Although the cellular response requires a specific recall with
the original infecting microorganism, once elicited the enhanced
resistance is effective against other microorganisms. Immunization
with Mycobggtegigm tuberculosis produces cell-mediated immunity which
protects not only against M. tuberculosis but also against listeria,
brucellae and staphylococci (35, 48, 113, 119, 123) and phylogenetically
diverse organisms. Resistance to Toxoplasma_gondii produces protection

against listeria, salmonellae, mengovirus and besnoitia (187).

2. Cell-mediated immunity in microbial diseases

Cellular immunity is of primary importance in many bacterial (33,
83, 91, 124, 227), fungal (81), viral (141), rickettsial (221),
protozoan (137, 208), metazoan (45) and mycoplasma (46) diseases. A
prime requirement for the elicitation of cell-mediated immunity appears
to be infection with viable, facultative intracellular parasites.
Mycobacterium may be an exception in that killed cells induce some
resistance although less effectively than a comparable number of
viable cells (35, 237). The organisms must persist for some time in

the tissues for acquired resistance to develop (34).

13

Although viable organisms are required to elicit the response,
the anamnestic response can be recalled with killed organisms or
specific antigenic fractions (71).

The prototype of cell-mediated immunity is anti-tuberculous
immunity, but studies have been made with many types of facultative
intracellular bacteria. Studies can be complicated by the activity
of multiple systems. The resolution of salmonella infections (15, 33,
119) can include cell-mediated immunity and the concomitant humoral
response that arises (32). Some investigators have shown a bactericidal
effect of serum from animals infected with whole cell vaccines of §,
typhimurium in giyg and in 31539 (92). Others report humoral and
cellular systems are important in the immune elimination of salmonella
(215, 216). These latter reports show that serum from mice immunized
with attenuated.§, typhimurium, heat-killed bacterial suspensions or
ribosomal preparations from S. Ayphimurium passively transferred
protection to normal mice (215). The same antigenic preparations were
capable of eliciting a cell-mediated immunity (216). Although this
appears to be in direct contradiction to original observations for
the requirement of viable intracellular parasites, there are an in-
creasing number of reports which indicate that antibacterial cell-
mediated immunity can be elicited with antigenic preparations from
microorganisms (104), especially ribosomal fractions (36, 236, 238).

Development of AB-CMI in yiyg is dependent on the size of the
inoculum and the infecting organism. Listeria monocytogenes, which
multiples more rapidly than M. tuberculosis, produces an immunity in

mice four days after injection (118). Mice inoculated with large

14

numbers (107 viable units) of BCG develop a high level of resistance at
12-15 days (121). Inoculation of mice with small numbers (104 viable
units) of BCG induces delayed-type hypersensitivity (DTH), but little
protective response. The immunity persists only as long as the
original antigenic stimulus persists, suggesting that specific activa-

tion of macrophages requires frequent or continuous stimulation (120).

3. Cellular mechanisms
a. Lymphocytes in cell-mediated immunity

Acquired resistance to infection with intracellular parasites
depends on the interaction of at least two cell types: specifically
committed lymphocytes and cells of the monocyte-macrophage series
(122, 131).

There are two major types of lymphocytes involved in the body's
immunological system, each with distinctive features and different
functions. Bone-marrow derived lymphocytes (B-lymphocytes or B-cells)
are thymus-independent cells and represent the cellular branch of the
immune response responsible for antibody production and secretion.
Thymus-dependent lymphocytes (T-lymphocytes or T-cells) arise from
stem cells in the bone marrow and mature under the influence of the
thymus, circulate through the body and respond to specific antigenic
stimuli but do not secrete antibody. T-cells play a role in rejection
of tumors and allografts (164), delayed-hypersensitivity reactions
(117, 323), activating macrophages to resist infection (16, 79, 106)
and cooperation with B-cells in some antibody responses (13). Table II

lists characteristic properties of T-cells, B-cells and macrophages.

15

Tablella Distinguishing characteristics of T lymphocytes, B lymphocytes

and macrophages. (Taken from reference 230).

 

 

Membrane markers T lympho- B lympho- Macro-
cytes cytes phages

lgG - + -

Receptor for C 3 (erythrocyte—antibody- - + +

complement (EAC) rosettes)
Receptor for lg or Ab-Ag complexes (Fc) - + +

Thymus-specific antigens (9, mouse thymocyte
leukaemia antigen, etc.) + - -

Receptors for sheep red blood cells
(erythrocyte (E) rosettes) + - _

In vitro stimulation of DNA synthesis
by mitogensa

Phytohaemagglutinin (PHA) + -b _
Concanavalin A (Con A) + - -
Lipopolysaccharide (bacterial endotoxin)c — + _
Anti-1g - + -
Specific binding to antigen-coated beads - + _
Mixed lymphocyte culture reactivity 1+ - —
Graft-versus-host reaction inducing capacity + - -
Adherence to surfaces (glass, plastic) -d _e +
Phagocytic — - +

 

8These data derive mainly from experiments in mice, and their extrapolation
to an is questionable.

Some B lymphocytes may be recruited to divide secondarily by factors ela-
borated by activated T lymphocytes. B cells may also be stimulated when the
mitogen is attached to a solid support.

In mice.

Except for blast cells.

Except for mature plasma cells or when immune complexes are attached to
B cells.

16

This is an active area of research. Recent reviews (90, 212) are
available describing these cells and their interactions in the immune
response.

Although antibacterial cell-mediated immunity is expressed by the
macrophages, immunologically committed lymphocytes are the mediators
responsible for the specificity of AM-CMI. Cellular immunity can be
passively transferred by viable lymphocytes from immune donors (118,
142) but not with lysed cell preparations (58, 125). Animals at the
peak of a cellular immune response, either during a primary infection
to BCG or after a secondary challenge with BCG, have activated macro-
phages which are much more resistant to L. monocytogenes, B. abortus
and.§. typhimurium. When lymphoid cells from BCG-immunized donors are
transferred to normal recipients, they confer no measurable protection
to L. monocytogenes unless BCG is also injected (18, 113).

Immune lymphocytes have no direct effect against bacteria (121),
however, recent reports in anti-tumor cell-mediated immunity indicate
there is direct cytotoxic activity by immune lymphocytes against target
cells operating without phagocytosis at the membrane level (164). A
minor role for direct lymphocytic antibacterial activity may remain as
a possible mechanism in AM-CMI.

Rats infected with.L. monocytogenes produce a population of newly
formed lymphocytes (100). In the afferent limb of the response, un-
committed lymphocytes transform, divide and differentiate to committed
cells upon contact with the infecting organism, a similar sequence
found in humoral mechanisms (125). This population of newly formed

lymphocytes arises primarily from the spleen or regional lymph nodes

17

in animals injected intravenously or subcutaneously, respectively,
with listeria or mycobacteria. Approximately 48 hours after injection,
large numbers of pyroninophilic blast cells arise in the paracortical
regions of the responding lymph nodes, these areas containing thymus-
derived lymphocytes (63, 218). The blast cells continue to divide for
10-12 days, migrate to the lymph node medulla and then pass into the
efferent lymph, developing into pyroninophilic small lymphocytes (124).

The newly formed large lymphocytes have a propensity to be drawn
into inflamed tissues and accumulate in an induced exudate in the
peritoneal cavity (100). Rats injected with vinblastine, an antimitotic
agent, at the peak of their response against L, monocytggenes do not
possess the population of specifically sensitized effector cells in
their thoracic ducts nor are they found in inflammatory exudates in-
duced in the peritoneal cavity (133). The drug must be injected within
5 days of the primary infection to interfere with the host response.
After this time, the protective lymphocytes are vinblastine resistant
and are presumably transformed to immunologically committed small
lymphocytes. The numbers of large lymphocytes in the thoracic duct
lymph of untreated animals have decreased to "normal" values at this
time (133). The cells which transfer resistance have a short circulat-
ing life span. After intravenous transfer into uninfected recipients,
protective donor lymphocytes do not recirculate to the thoracic duct
in sufficient numbers to confer protective immunity to a second set of
normal recipients (132).

Protective cells with blast cell morphology are seen to accumulate

in nonspecifically induced inflammatory foci. Similar protective cells

18

are absent or present in small numbers in unstimulated peritoneal
cavities (101). It has been suggested, that these cells respond to
an inflammatory stimulus and migrate to the area where they provide
the stimuli to attract monocytes and macrophages into the area and
affect enhanced macrophage microbicidal activity. The large lympho-
cytes may transform into small lymphocytes which provide immunological
memory. Upon future contact with the antigen, the cells are again
stimmlated to divide and transform into the large effector cells
which would preferentially localize in infected tissues (133).
Little is known about the life history of the progenitors of
effector T-cells or requirements of cooperation among cell types in
the induction of AM-CMI. On the basis of delayed hypersensitivity
and graft-versus-host reactions in mice, it has been proposed that
induction can require two different classes of T-cells (7, 170).
The two types may belong to different cell types or may represent
different stages of maturation within a single cell line. The first
type of T-cell postulated (T1) may be normally present in the thymus
and spleen and decrease in number in secondary lymphoid tissue 2-6
weeks after adult thymectomy, i.e., it might be in an early stage of
post-thymic maturation in these tissues. The second type postulated
(T2) may be scarce in the thymus and may not decrease in number in
secondary lymphoid tissue after adult thymectomy, i.e., it might be
in a late stage of post-thymic maturation. It is suggested that T1
cells are the progenitors of effector cells and that T cells act as

2

amplifiers of the response. Investigations of cellular requirements

19

for cell-mediated immune reactions in other systems suggest cooperation
between T1 and T2 cell types is not essential (17, 206).

By definition, cell-mediated immunity may be passively transferred
to normal recipients with lymphocytes, not with serum (58). One argu-
ment for the involvement of AMrCMI in a particular infection is the
abrogation of the protective response by antilymphocyte or antithymocyte
sera. Mice treated with antithymocyte serum develop little or no
protective response to ectromelia virus (16) or vaccinia virus (79).
There is also clinical evidence of a participatory role of T-cell
dependence in cell-mediated resistance. Patients with immunological
deficiency diseases lacking a thymus or thymus function have progressive
and often fatal diseases with vaccinia (59). Similarly, passive trans-
fer of infectious immunity may be eliminated by treating immune spleen
cells lg giggg with anti-9 serum and complement (106). This is not
conclusive evidence for cell-mediated immunity, however. There are
thymus-dependent antibody responses (13) and abrogation of an immune

response after treatment with antithymocyte serum may not be indicative

of a cell-mediated immunity.

b. Cell-cell interactions and biological mediators
1. Specificity and relationship with delayed-type hyper-
sensitivity
The effect of lymphocyte mediators on macrophages must be discussed
with delayed-type hypersensitivity although any relationship between
delayed-type hypersensitivity and antimicrobial cell-mediated immunity

has not been resolved. There are two opposed viewpoints:

20

l) delayed-type hypersensitivity is related and may provide the
specificity for AM-CMI and 2) there is no relationship.

Delayed-type hypersensitivity.igngiyg is defined in terms of
morphological observations and time sequence of events. The Arthus
and anaphylactic-type skin reactions in immediate hypersensitivity
develop early and are mediated by various classes of immunoglobulins.
Histologic examination of the skin shows the predominant cell types to
be granulocytes.

In delayed-hypersensitivity reactions, such as a tuberculin skin
test, the reaction does not reach a maximum until 24-48 hours, and
is characterized by a mononuclear cell infiltration including both
lymphocytes and monocytes and macrophages. When normal macrophages
with antigen are injected into the skin of a sensitized animal, a
delayed-type reaction develops sooner and more intensely than when
antigen only is injected. This suggests a major factor in the delayed
reaction is the time required for accumulation of macrophages (76).
Skin tests are used to measure DTH.in.giyg in many species, including
man. In the mouse, a variation of the skin test, the foot pad test, is
used.

At the time an animal begins to mount a cell-mediated immunity,
it also generally develops a delayed-type hypersensitivity to the same
microorganism (195) and like AM-CMI, delayed-type hypersensitivity is
cell-mediated. It may be passively transferred to normal recipients
with lymphoid cells but not with immune serum. Antilymphocyte serum
(217) or antithymocyte serum (213) inhibits DTH reactions and thoracic

duct lymphocytes can transfer sensitivity (27).

21

Like AMPCMI, there appears to be a reaction with antigen and
specific effector cells followed by recruitment of noncommitted
passive cell types. In transfer studies with donor cells labeled
with 3H-thymidine, the majority of cells which accumulate at the
reaction site are those of the recipient animal (72, 148). Using
isotopically labeled leucocytes and cell transfer techniques, McClusky
(129) demonstrated that the majority of cells infiltrating the site of
a dermal cellular hypersensitivity reaction were not specifically
sensitized. Labeled lymphocytes from sensitized donors were always
present in small numbers in the infiltrates (72, 148). Inhibition of
macrophage migration (MIF test), an 13.1iggg test for DTH which cor-
relates with the_ig.yiyg skin test, can be obtained when 97.5% of the
peritoneal exudate cells were from the unsensitized donor and only
2.5% were lymphocytes from a sensitized animal (44).

There appears to be two separate populations of cells involved
in DTH, a thymus-dependent antigen sensitive lymphocyte population and
a thymus-independent bone marrow-derived population (117, 232). Al-
though the latter is generally believed to be monocytes, there is
some evidence it may be a T-cell population. Youdin et al. (233)
eliminated passive transfer of DTH by treatment with anti-0 serum and
complement.

Passive transfer of AM-CMI with lymphocytes also transfers the
corresponding DTH (118). Delayed-type hypersensitivity can be
transferred in man with transfer factor from cell-free extracts from
lysed leucocytes (107). With the appearance of delayed sensitivity,

there is often a coincident recovery from a systemic infection such

22

as in the case of patients with generalized vaccinia, moniliasis and
leprosy (108).

While DTH generally accompanies AM-CMI (195) it is also possible
to have one response and not the other (223). Animals may be desensi-
tized to tuberculin hypersensitivity (180, 229) or made hypersensitive
without causing an increase in resistance (171). Delayed hypersensitivity
can be elicited to Staphylococcus aureus without eliciting the increased
resistance of AM-CMI (115). Goihman-Yahr et a1. (62) reported that
guinea pigs with delayed-type hypersensitivities,reactions of infectious
allergy, graft rejection or allergic dermatitis did not possess enhanced
listericidal activity and only macrophages from animals sensitized to
BCG had a significant capacity to inactivate L. monocytogenes.

Certain fractions from salmonellae (215) and the tubercle bacillus
(40, 238) give a measurable degree of resistance without producing
cutaneous hypersensitivity. Youman's group (150, 238) reported mice
immunized with ribosomal fractions of mycobacteria developed a specific
immunity to mycobacteria without the development of delayed-type hyper-
sensitivity. They argued against a role for tuberculin hypersensitivity
in cell-mediated immunity on this basis. Mice and guinea pigs injected
with ribosomal fractions and tested for DTH by the footpad test (238)
and the 13 giggg test for MIF (150) had only slightly greater reactions
over controls. The same authors reported a specific immunity to tuber-
culosis was induced with these fractions (35, 36). In contrast, reports
by other authors noted that ribosomal preparations of mycobacteria are

active in provoking DTH reactions (11, 12, 156).

23

Although skin sensitivity may be suppressed by daily injections
of tuberculin, acquired resistance persists and may even increase (229).
Mackaness argues that the absence of skin sensitivity implies nothing
in respect to immunologic reactivity at the cellular level and the
process of desensitization might only cause the temporary withdrawal
of the sensitized cells involved in DTH from the circulation (121).
Results of skin tests in humans may not be definitive. Lympho-
cytes from patients with diseases associated with depressed or absent
delayed hypersensitivity, such as sarcoidosis, Hodgkin's disease and
chronic mucocutaneous candidiasis, were tested for hypersensitivity
by theligqxiggg methods of antigen-induced MIF production and 3H-
thymidine incorporation (24, 181, 182, 184). Of the 37 patients
tested, 16 exhibited cutaneous hypersensitivity to one or more antigens
and had positive MIF and proliferation assays. Six patients had no
cutaneous hypersensitivity reactions and had negative in yiggg tests,
indicating the lack of sensitive lymphocytes. Four of the remaining
15 patients had consistently negative skin reactions and repeatedly
positive MIF and proliferation assays, indicating that despite the
lack of skin reactivity, sensitive lymphocytes were present (24, 182).
It was suggested that the lack of an 13.3319 response was a result of
some dysfunction other than a lack of sensitive lymphocytes, possibly
an abnormal macrophage response. The other 11 patients had consistently
negative skin and MIF tests, but did show normal antigen-induced thymi-
dine incorporation, showing a dissociation between MIF production and
cell proliferation. Obviously DTH is not a simple response and seems

comparable to biochemical lesions, i.e., any single enzyme deletion or

24

alteration in a biosynthetic pathway may result in the loss of the end

product. Skin test results should therefore be interpreted with caution.

2. Factors from lymphocytes affecting macrophages

Delayed-type hypersensitivity may provide the basis for the speci-
ficity for AM-CMI and the ensuing nonspecific aspects due to reactions
of macrophages, effector cells, after stimulation by lymphokines (120).
A number of factors or lymphokines with biological activity have been
detected from sensitive lymphocytes of animals with DTH (49). A list
of these mediators with their characteristic properties are listed in
Table III. It is not known whether the same cell can produce all these
factors or whether different cell populations are involved. Flanagan
et al. (54) report an MIF-like material in supernatant fluids from
monkey kidney cells infected with mumps or Newcastle disease virus and
suggest these mediators may be produced by a number of cell types.

Lymphocytes or lymphokines may affect macrophages in several ways.

In vivo events of DTH may be postulated to occur in the following

 

manner. After stimulation, small numbers of specifically committed
lymphocytes undergo blaazformation and produce and secrete factors
which cause an inflammatory response and effect the accumulation first,
of a granulocytic cell population followed by a monocytic infiltrate
(chemotactic factor(s)). Once the monocyte-macrophage population has
entered the area, they may be prevented from leaving by migration-
inhibition factor (MIF). The macrophages may also be stimulatedlto
divide (mitogenic factor(s)) and, in AM-CMI, stimulated to enhanced

antimicrobial capabilities (macrophage resistance factor(s)).

255

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29

In diseases such as tuberculosis in which the infectious agents are
exceptionally resistant to inactivation and degradation, a granuloma
may be formed in which large differentiated macrophages prevent dis-
semination by walling off the organisms.

A soluble factor(s) elaborated by antigen-stimulated lymphocytes
enhances certain.ig.!i££9 macrophage functions. Increased macrophage
adherence, membrane ruffled border activity, phagocytosis and hexose
monophosphate oxidation appears after 3 days of incubation in MIF rich
fractions (83). Lymphocytes from M. tuberculosis-immunized mice,
when cultured ig.yi££9*with the same organism, produce supernatants
which inhibit the growth of M. tuberculosis in normal macrophages
(95, 158). Godal et a1. (61) found that supernatant from rabbit mixed
leucocyte cultures suppressed the multiplication of mycobacteria in
normal peripheral blood monocytes after 10 to 17 days. Krahenbuhl and
Remington (103) reported that resistance to listeria was conferred to
normal guinea pig macrophages by supernatant fluids from spleen cells
of toxoplasma-infected animals incubated with toxoplasma antigen. They
also reported MIF activity in these supernatant fluids. The supernatant
fluids had no direct effect on bacteria and did not inhibit the extra-
cellular multiplication (55, 95). Pearsall et al. (161) reported some
direct activity against certain yeasts by lymphokine preparations.

Supernatant fluids from splenic lymphocytes of mice immunized
with L. monocytogenes cultured‘igiyiggg with listeria cause normal
macrophages to inhibit the intracellular multiplication of virulent

mycobacterium (96). However, if listeria is added to lymphocytes from

30

nonimmunized mice or to lymphocytes from mice immunized with H37Ra
.M. tuberculosis the supernatant fluids are still able to inhibit the
intracellular growth of virulent tubercle bacilli to some degree.
These in giggg studies mimic.in.xigg studies (122) in which lymphocytes
react specifically with the antigen and confer on macrophages the
ability to nonspecifically inactivate microorganisms.

Partially purified peritoneal lymphocyte preparations from immunized

mice mediated a suppression of intracellular growth of Histqplasma

 

capsulatgm in normal mouse macrophages in xiggg (81). An outbred
strain of mice (Swiss-Webster) was used, however, and the presence of
mediators produced from a mixed lymphocyte reaction cannot be eliminated.

Lymphocytes from peritoneal exudates of guinea pigs with delayed
hypersensitivity to bovine gamma globulin (BGG) cultured with and
without BCG confer no enhanced listericidal activity to normal peri-
toneal macrophages (196). The supernatants do have migration inhibitory
activity, however. It is interesting in these experiments in which
viable facultative parasites were not used, DTH was elicited but not
AM-CMI. These data reflect lg 3119 situations in which AM-CMI may
involve a response over and above DTH.

Lymphokines can be generated from antigen-induced sensitive
lymphocytes or from lymphocytes incubated with mitogens, such as
Conconavalin A (Con-A) and phytohemagglutinin (PHA). Both PHA and
Con-A are plant mitogens that induce an increase in DNA synthesis
by lymphocytes (96). Both substances stimulate lymphocytes to produce

MIF and lymphotoxin (38, 66, 167, 194). Macrophages incubated with

31

antigen-induced lymphokine preparations inactivate 95% of the listeria
to which they are exposed. However, little or no inactivation of
listeria occurs in macrophages treated with FHA-induced lymphokines
(189). Macrophages incubated with MIF-rich fractions of supernatants
from Con-A stimulated guinea pig lymphocytes have 4-6 times fewer
listeria than controls (55). The authors reported a bacteristatic
rather than bactericidal medhanism. Klun and Youmans (96) found both
mitogens cause the uptake of 3H-thymidine by lymphocytes, but only
Con-A stimulated lymphocytes to secrete a product which inhibited the
intracellular growth of virulent tubercle bacilli within normal macro-
phages. Perlmann et al. (165) noted PHA, but not Con-A, was capable
of inducing normal human lymphocytes to become cytotoxic to chicken
erythrocytes. Stobo et al. (207) reported Con-A stimulated spleen
cells from thymectomized mice but PHA did not. There were approxi-
mately 10% 0-positive cells present in spleens from thymectomized
animals and if this same cell population was again incubated with
anti-9 serum, all responsiveness to Con-A was abolished. Thus, it
appears that Con-A may stimulate a separate population of T-cells than
PHA, and it is the former population that is responsible for inhibition

of facultative intracellular parasites.

3. Factors from macrophages affecting lymphocytes

Digested sheep erythrocytes recovered after various periods of
time from guinea pig peritoneal macrophages incubated in yiggg no
longer induce antibody formation and preferentially induce delayed-

type hypersensitivity responses_in vivo (163).

32

A glass-adherent helper cell population is required for activation
of lymphocytes in some cell-mediated responses. Removal of macrophages
and monocytes from peripheral blood samples by glass bead columns
prevents lymphocyte transformation by PPD, streptokinase-streptodornase
and streptolysin O (9, 75). A glass-adherent cell population is required
in mixed lymphocyte reactions, but the requirement for these cells can
be replaced by supernatants of normal, unstimulated glass-adherent
cells (9). Whether the requirement in these responses is for macrophage
antigen processing, antigen presentation or production of nutrients is

unknown.

4. Cytophilic antibody

Boyden and Sorkin (21) reported immunized animals possess an
immunoglobulin, cytophilic antibody, most often described as IgG
(distinguished from IgE) which may play a role in cell-mediated immunity
(21) and delayed-type hypersensitivity (152). Cytophilic antibodies to
PPD may be present in animals immunized with BCG (152, 203) and Amos
et a1. (5) reported cytophilic antibodies to tuberculoprotein prevented
the migration of peritoneal exudate cells in the presence of the
antigen.

No observations have been reported of morphological or biochemical
changes occurring in macrophages as a result of binding of cytophilic
antibody. It is argued that the nonspecificity of antimicrobial cell-
mediated immunity would not be accomplished through a cytophilic anti-
body specific for a particular antigen (214), however it is possible

for changes in macrophage functions to occur as a result of a reaction

33

at the macrophage membrane. In any case, the relationship, if any,
of macrophage-cytophilic antibody and antimicrobial cell-mediated

immunity remains to be clarified.

c. Macrophages in antimicrobial cell-mediated immunity

Macrophages ingest and degrade a variety of soluble (29, 50) and
particulate (28, 211) materials and have been called the scavengers
of the body. Whereas PMN's play a major role in pyogenic infections,
monocytes and macrophages are more important in the control of intra-
cellular parasites (144). Mononuclear phagocytes are distinguished by
their nuclei, their phagocytic capacity, the nature and content of
their lysosomes (which differ from polymorphonuclear leucocytes),
their adherence to glass and other surfaces, and the presence on their
plasma membrane of receptors for certain immunoglobulins or immune
complexes (IgGl, IgG3, and IgM) (6) and complement (C'3) (84) (Table
III). In addition, macrophages possess considerably more synthetic
potential than PMN's and can be stimulated to form large amounts of
lysosomal and other enzymes.

Macrophages can be "activated” both specifically and nonspecifical-
ly. Nonspecifically activated macrophages can be obtained from peri-
toneal exudates of animals stimulated with intraperitoneal injections
of glycogen, casein hydrolysate, etc. These agents produce a general
inflammatory response in the peritoneal cavity. Two to three days
after prestimulation, a large population of macrophages arises with
altered morphological and biochemical characteristics such as changes

in cell size, motility, ”stickiness", pinocytotic rate, phagocytic

34

capacity, and the number, size and content of lysosomes (31, 134).
Nonspecifically activated macrophages do not possess the heightened
microbicidal capacities that specific, immunologically activated
macrophages possess, although they may appear similar morphologically
(191).

Using time-lapse phase-contrast cinematography, peritoneal macro-
phages from normal or tuberculin sensitive guinea pigs in the absence
of antigen (PPD) have numerous pseudopodia and migrate freely. Macro-
phages from normal or sensitized animals in the presence of sensitive
lymph node or thymus cells have fewer pseudopodia and are much less
motile. Nonsensitive macrophages aggregate and become associated
with lymphocytes in the presence of PPD and sensitive lymphocytes.

The migration of sensitive lymphocytes is not affected in the presence
of PPD unless they are aggregated with macrophages (191).

The macrophages of the body are the cells in which antimicrobial
immunity is expressed. These cells develop a greatly enhanced
microbicidal capacity not only for the original infecting organism,
but for a wide variety of microorganisms (121). This is reported in
iglgiyg and-£3.3iggg studies usually with peritoneal macrophages but
also with fixed phagocytes of the spleen, liver and lung (118).

Surprisingly, mice injected with anti-macrophage sera (AMS) during
infection with L. monocytogenes do not die sooner nor does the mortal-
ity rate increase. When treated with antithymocyte sera mice infected
with listeria have decreased survival time and increased mortality (174).

Howard et a1. (81) reported freshly harvested mononuclear phago-

cytes from immune Swiss albino mice inhibited the intracellular growth

35

of Histoplasma capsulggum, whereas cells maintained in culture for

48 hours did not. Hirt and Bonventre (80) noted freshly harvested
peritoneal macrophages obtained from BCG-immunized mice phagocytosed
staphylococci poorly but to a greater extent than control cells. The
phagocytic and bactericidal activities of peritoneal macrophages
cultured for 2 days was much greater than freshly harvested macrophages.
After 2 days in culture, killing of staphylococcus by macrophages from
control or BCG-immunized Swiss albino mice was the same.

MCGhee and Freeman (130) observed that within 6 hours after
infection of immune macrophages-ig.yi££g, 50% of surviving intracellular
brucellae become sensitive to osmotic conditions and required media
containing 0.2 M sucrose to survive. Osmotically sensitive brucella
were not produced in normal macrophages.

It is suggested that the increased microbicidal properties of
macrophages in AM-CMI is due to the increased lysosomal enzyme activity
of activated macrophages (8, 102), assuming the killing and degradation
of microorganisms takes place within these organelles (30). The
activities of some lysosomal enzymes increase in animals infected with
BCG (31), 57, 188) or Cornyebacterium ovis (74), although Franson and
White (57) reported a decrease in activities of phospholipases A and

1
A2 in BCG-infected rabbits. Lung macrophages possess higher activities
of lysozyme than peritoneal macrophages but the latter are more ef-
ficient in killing bacteria (160). Results from experiments performed
by Brown et a1. (23) suggested that increased lysosomal activity in

chemically activated macrophages is associated with vigorous bacterial

36

multiplication rather than bacterial inhibition. They postulated that
lysosomal enzymes are not detrimental but actually may be beneficial
to the survival of facultative intracellular parasites by providing
growth stimulating low-molecular weight nutrients as products of
hydrolysis.

Myeloperoxidase activity, a primary antibacterial system in PMN's
has been found in mouse peritoneal macrophages (159), however the
levels reported are barely above the lower limit of detectability.
Furthermore, phagocytosing peritoneal macrophages have less activity
than resting cells, which is the reverse of what is expected based
upon results for PMN's. Simmons and Karnovsky (197) reported almost
undetectable levels in peritoneal macrophages even though the cells
were very competent in their killing ability.

Phagocytosis of listeria by human blood monocyte derived macro-
phages is inhibited by glycolytic poisons such as NaF but is unaffected
by cyanide or 2,4-dinitrophenol (DNP), inhibitors of respiration (25).
Macrophages from mice infected with listeria or BCG have enhanced
phagocytic properties and glucose metabolism (175). Cohn (28) found
macrophages from immune hosts did not differ from normal cells in their
ability to degrade labeled bacteria. He noted the presence of immune
serum had an inhibitory action on the rate of degradation. Iodoactate,
arsenite and cyanide, inhibitors of glycolysis and respiration, had no
influence on the degradation of labeled bacteria, suggesting the
process is not dependent on energy requiring mechanisms (25, 28).

Cline (25) reported maximal killing of listeria by normal human

macrophages required oxygen but was unaffected by cyanide or DNP.

37

Miller (140) also found killing by normal mouse peritoneal
macrophages was inhibited by anaerobiosis but unaffected by cyanide,
antimycin A or amytal. Intracellular inactivation of bacteria was
unaffected by uncouplers of oxidative phosphorylation (DNP, oligomycin
or arsenite), by glycolytic (NaF and iodoacetate), Krebs cycle (sodium
malate), or phosphogluconate (quinacrin and phenylbutazone) pathway
inhibitors. Compounds with redox potentials of +0.22 volts or less
were inhibitory to killing of Pseudomonas aeruginosa. Material of
+0.36 volts and higher were not. He suggested the electron transport
chain is intimately involved in the mechanism of bactericidal activity
of normal mouse macrophages.

In metabolic studies with Con-A on polymorphonuclear leucocytes
and normal alveolar macrophages there is a rapid enhancement in the
rate of cell respiration, even in the presence of potassium cyanide
(185). There is a KCN-insensitive increase in the rate of l-IAC-
glucose oxidation to 14CO2 indicating a specific activation of the
hexose monophosphate shunt. Immune macrophages do not undergo sub-
stantial changes in their oxidative metabolism as compared to normal
macrophages even though fluorescent studies reveal a binding of Con-A.

It has been reported that macrophage lysates (186) and lysosomal
extracts of Kuppfer cells (98) and peritoneal macrophages (157)
possess no direct antibacterial activity. Outteridge et al. (157)
noted lysates of pulmonary alveolar macrophages from 5 out of 15 sheep

had some inhibitory, but no killing activity against listeria in

lysosomal fractions.

38

Ramseier and Suter (173) found antimycobacterial activity in
peritoneal mononuclear whole cell lysates. Activity in BCG-immunized
guinea pigs was four times that of control animals and was apparently
specific. There was no inhibition of Brucella abortus, Salmonella
typhimurium,‘§£éphylococcus aureus, or Escherichia coli. The material
was heat labile and associated with the nucleus (174).

Some antimycobacterial activity by macrophage fractions is re-
ported due to toxic free fatty acids produced as a result of hydrolsis
of lipoproteins or phospholipids by lipases (88, 97, 98) with
slightly higher levels in lysates from BCG-immune mice than in controls.

Gershon and Olitzki (60) reported a factor from mouse monocyte
lysates, monocytin, which was bactericidal for Salmonella typhi,

.§. paratyphi, g, ggli,.§. dysenteriae and_§. aureus. It was effective
in protecting mice against.§.._yphi if injected 4 hours before the
bacterial inoculum. The material was a basic protein, labile to acid
treatment and trypsin and stable to heating at 100 C for 1 hour.

The exact nature and mode of microbial inactivation or killing

in normal macrophages or those involved in AM-CMI are unknown.

10.

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233. Youdim, S., 0. Stutman, and R. A. Good. 1973. Thymus dependency
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235.

236.

237.

238.

239.

240.

241.

242.

58

Youmans, A. S. and G. P. Youmans. 1962. Effect of mycosuppressin
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Youmans, A. S. and G. P; Youmans. 1965. Immunogenic activity of
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J. Bacteriol. 82:1291-1298.

Youmans, G. P. and A. S. Youmans. 1969. Immunizing capacity of
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culous infection. J. Bacteriol. ‘21:107-113.

Youmans, G. P. and A. S. Youmans. 1969. Allergenicity of myco-
bacterial ribosomal and ribonucleic acid preparations in mice
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Zeya, H. I. and J. K. Spitznagel. 1966. Cationic proteins of
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‘2l:755-762.

Zeya, H. I. and J. K. Spitznagel. 1969. Arginine-rich proteins
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ficity and biochemical heterogeneity. J. EXp. Med. 127:927-941.

Zgliczynski, J. M., T. Stelmaszynska, J. Domanski, and W. Ostrowski.
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and lymphocytes. J. Exp. Med. 124:535-542.

AN ANTILISTERIAL FACTOR FROM MOUSE PERITONEAL CELLS

Donna Y. Muirhead and Virginia H. Mallmann

Department of Microbiology
and Public Health

Michigan State University

East Lansing, Michigan 48824

Running head: ANTILISTERIAL FACTOR

Michigan Agricultural Experiment Station Journal Article No. 6462.

Supported in part by the Michigan Tuberculosis and Respiratory Disease
Association and a Cooperative Agreement with Animal and Plant Health
Inspection Service, United States Department of Agriculture.

Please address request for reprints to Dr. Virginia H. Mallmann,

Department of Microbiology and Public Health, Michigan State University,
East Lansing, Michigan 48824.

ABSTRACT

Lysates of mouse peritoneal cells contained antilisterial
activity which was detectable in the presence of a reducing agent,
dithiothreotol. Lysates from mice immunized and prestimulated with
viable BCG had greater activity than lysates from mice immunized and
prestimmlated with heat-killed-BCG or control mice. Activity was
associated with both glass adherent and nonadherent cells, but to a
greater extent with the former. Peritoneal cells of mice immunized
and prestimulated with heat-killed or viable BCG were able to reduce
nitro blue tetrazolium (NBT) to a much greater extent than cells of
control mice. After mechanical disruption of cells and differential
centrifugation, antilisterial activity was found in the pellet fraction

containing lysosomes.

6O

INTRODUCTION

Phagocytes of the body provide a primary defense mechanism
against infection. Antibacterial systems and mechanisms of action
have been identified from polymorphonuclear leucocytes, including
lysosomal cationic proteins (1) and a myeloperoxidase system (2).
Macrophages phagocytize and kill many types of bacteria (3) and
activated macrophages of cell mediated immunity have an increased
antibacterial capacity (4, 5). With the exception of lysozyme, the
mechanism(s) by which macrophages inhibit or kill microorganisms is
unknown. This paper reports an oxygen sensitive factor from mouse
peritoneal mononuclear cells which is bactericidal (or bacteriostatic)

for Listeria monocytogenes.

 

61

MATERIALS AND METHODS

Microorganisms. Listeria monocytogenes was maintained on Brain
Heart Infusion Agar (Difco Laboratories, Detroit, Mich.). Cultures
used for measuring antilisterial activity were grown in Brain Heart
Infusion Broth (Difco) at 37 C overnight with shaking. Attenuated
Mycobacteriqm bovis (BCG) was grown in Dubos Broth base without
enrichment or Tween 80 (Difco) supplemented with 0.5% dextrose at
37 C for two weeks.

.Mige, immunization and prestimulation. Female Swiss albino mice
(20-22g) (Carworth Farms, Portage, Mich.) were divided into four
groups. The mice in the first group were inoculated ip with 0.2 ml
of viable BCG suspension homogenized by mortar and pestle at a concen-
tration equivalent to a #7 McFarland tube («.4 x 108 cells/mouse).
Three to four weeks later, the mice were prestimulated by inoculating
ip 0.2 m1 of homogenized viable BCG at a concentration equivalent to
a #2 McFarland tube («al.Z x 108 cells/mouse). The mice in the
second group were immunized and prestimulated with heat-killed BCG
by the same method. The mice in the third group were not immunized
but were prestimulated with 1.5 ml of 0.1% glycogen (Matheson, Coleman
and Bell, Cincinnati, 0.). The mice in the fourth group were not
immunized or prestimulated.

,lelgcollection. Peritoneal cell exudates were collected three
days after prestimulation of Groups 1, 2 and 3 by injecting 3-4 ml of
Hank's balanced salt solution (HBSS) (Microbiological Associates,

Bethesda, Md.) with 10 units/m1 ammonium heparin (Scientific Products,

62

63

Evanston, 111.). The abdomens were massaged and the exudates withdrawn
with a syringe and a 22 gauge stainless steel needle with additional
holes drilled in the shaft. Cells of mice in each group were pooled
in siliconized centrifuge tubes on ice. The cell suspensions were
centrifuged at 200 x g for 10 min in a refrigerated centrifuge (IEC,
Needham, Mass.) and resuspended in HBSS. Measured aliquots of the
cell suspension were added to 0.1 m1 of a solution of 2.1% citric
acid-0.1% crystal violet and counted in a Neubauer hemocytometer
(Clay Adams, New York, NY). Peritoneal cells from control mice were
collected and treated in the same manner. All exudates contained
50-90% macrophages, 10-40% lymphocytes and less than 2% granulocytes.
Cell lysis and fractionation. Unfractionated lysates were ob-
tained from cells washed 2X and resuspended in citrate-phosphate

buffer (6), pH 7.0 (PCB) and lysed with 20 HD unitsl/ml of staphylo-

50
coccal delta hemolysin2 by incubating for 20 min at ambient tempera-
ture. Cell lysis in all experiments was greater than 98%.

Cells from which lysates were to be fractionated were washed 2X
in cold 0.25 M sucrose in 0.05 M Tris-acetate buffer at pH 7.4 and
resuspended in 16 ml of the same solution. Four 4-m1 aliquots were
ground with a teflon-coated tissue homogenizer (TRI-R Instrument Inc.,
Rockville Center, NY) on ice at 600 rpm for 2, 4, 6, or 8 min.

After homogenation, two 0.5 ml buffer rinses of the tissue grinder
were added to the lysed cell suspension for a final volume of 5.0 m1.
From 70 to 95% of the cells were disrupted within 2 to 8 min time

periods. The homogenized lysates were fractionated by differential

centrifugation by the method of Cohn and Wiener (7).

64

Antilisterial assay. The assay system used to measure anti-
listerial activity was a modification of that by Hirsch (8). All
lysates and fractions were dialyzed against 1:10 PCB before testing.
Serial 3-fold dilutions of the material to be tested were made in PCB
containing 0.01% bovine serum albumin (Difco). After preliminary
experiments indicated that the factor was oxygen-labile, the reducing
agent, dithiothreotol (DTT) (Calbiochem, San Diego, Calif.) was included
in the buffer system at a concentration of 10 mM. The final volume of
each dilution tube was 1.0 ml. The control was a tube containing 1.0 ml
of the diluent only. An 18 hour broth culture of Listeria monocytogenes
was centrifuged, resuspended in 0.85% saline and diluted to ca. 2 x 105
cfufiml. One-tenth milliliter was added to each dilution and the control
tube. Each tube was flushed with argon and incubated at 37 C for 2 hrs.
The suspensions were diluted and aliquots of each dilution added to
20 m1 of warm (50 C), melted Brain Heart Infusion agar. After mixing,
the contents were poured into petri dishes, allowed to solidify and
incubated overnight at 37 C. The number of colonies were counted and
expressed as the per cent kill calculated from the control.

Separation of glas -adhgr§nt and nongdherent cells. Peritoneal
cells from mice immunized and prestimulated with viable BCG were
suspended in 5 m1 Eagles MEM (Microbiological Associates) with 20%
fetal calf serum, supplemented with 1% each vitamins, amino acids,
and glutamic acid, 100 unitsfiml of penicillin and 100 ug/ml streptomycin.
The cell suspension was placed in plastic petri dishes and incubated
for 2 hrs at 37 C in 95% air and 5% C02. After incubation, the non-

adherent cells were removed. The cells were gently washed 2X with

65

HBSS and removed from the plastic dishes with a plastic policeman and
salt solution of 0.8% NaCl, 0.02% KHZPO , 0.02% KCl, 0.115% NaZHPO4
and 0.02% NaEDTA. Both groups of cells were centrifuged, washed 2X
and resuspended in HBSS. Cell counts were made (65% macrophages)

and the cells lysed with delta hemolysin. The nonadherent cells were

centrifuged, washed 2X with HBSS, counted (99% lymphocytes) and lysed

with delta hemolysin. The lysates were assayed for antilisterial

activity.
Enzyme and protein assays. Acid phosphatase activity was measured

 

using p-nitrophenyl phosphate (Sigma Chemical Co.) as substrate ac-
cording to the method of Dipietro and Zengerle (9). Protein was
measured by the method of Lowry et a1. (10).

Nitro blue tetrazolium reduction by peritoneal cells. Peritoneal
cells were collected from unstimulated or prestimulated mice immediately
after sacrifice by cervical dislocation. The abdominal skin was re-
flected, and cells were withdrawn after each of two 5 m1 injections
of cold HBSS. The cells from each mouse were treated separately and
bloody suspensions were discarded. The cells were centrifuged at
250 x g for 10 min at 4 C and resuspended in 0.5 to 1.0 m1 of phosphate
buffered saline (without magnesium or calcium) with 2 mg/ml dextrose
and 5 units/m1 heparin. The cells were counted and adjusted to 4 x 107
macrophages/m1. One-tenth milliliter of nitro blue tetrazolium (NBT)
(Sigma, catalog #N 51293)3 and 0.05 ml of 0.81 micron latex particles
(Difco) were added to a siliconized test tube and prewarmed in a 37 C

water bath for 2 min. One-tenth milliliter of the cell preparation

was added, mixed, covered and incubated for 15 min at 37 C. One-half

66

milliliter of 0.5 N HCl was then added and the cells were centrifuged

at 250 x g for 10 min at 4 C. The supernatant was removed by aspiration.
One milliliter of pyridine (Reagent Grade, Mallinckrodt) was added to

the cell pellet, mixed and extracted in a 95 C water bath, in a hood,

for 10 min. The extracted cells were centrifuged at 500 x g for 10 min

and the supernatant was measured spectrophotometrically at 515 nm.

RESULTS

Antilisterial activity of peritoneal cell lysates. The antilis-

terial activity of lysates was lost if stored overnight either at 2 C
or -56 C. The addition of DTT restored the activity (figure 1). The
initial concentration of lysate before dilution was 108 peritoneal
cells/ml and had a protein concentratration of 6.7 mg/ml after dialysis.
There was some inhibition of activity in the higher lysate concentra-
tions and all graphs plotting activity versus lysate dilution were
U-shaped when the higher concentrations were tested.

,Agtivity from glass-adherent_gpggnonadperent cells. More antilis-
terial activity was associated with lysates from glass-adherent cells
than from nonadherent cells (figure 2). Cell concentrations for
adherent and nonadherent cells after separation were 3.1 x 107 cells/ml
(65% macrophages) and 7.6 x 107 cells/m1 (99% lymphocytes), respectively.

Nitro blue tetrazolipm reduction by peritoneal cell . Optical
density readings for extracted, reduced NBT are plotted for individual
mice in figure 3. Very little reduction of NBT was found in control,
unstimulated mice or mice prestimulated with glycogen. Mice injected
and prestimulated with heat-killed BCG had 0.D. readings 3-9 times
greater than control values. Mice injected and prestimulated with

viable BCG had even higher values of 8-14 times greater than controls.

 

Comparison of antilisterial activity and acid phosphatase in

lysate fractions. From hemolysin disrupted cells, 90% of the total
acid phosphatase activity was in the high speed supernatant fluid after

differential centrifugation. This was interpreted to mean that delta

67

68

hemolysin disrupted lysosomes. For this experiment then, cells were
disrupted mechanically to obtain lysates for fractionation. Antilis-
terial activity in the supernatant fluid left after high speed centri-
fugation correlated with acid phosphatase activity at all dilutions
tested (figure 4). In the low speed (nuclear) and high speed lysosomal)
pellet fractions of homogenized cell suspensions, antilisterial activity
did not parallel enzyme activity until the fractions had been diluted
(figures 5 and 6). This effect corresponded to the inhibition observed
in the higher concentrations of unfractionated lysate (figure 1).

Apid phosphatase and antilisterial activity of immunized:gnd control

mice. Total acid phosphatase activity per 106 macrophages/m1 was greater

 

from.mice prestimulated with glycogen than from unstimulated control
Inice (table 1). Enzyme activity was highest from mice which were im-
xnunized and prestimulated with viable BCG. An intermediate level of
activity was found for mice receiving heat-killed BCG. When high speed
supernatant fractions from homogenized cell suspensions of each of the
four groups of mice were adjusted to equivalent acid phosphatase enzyme
activities (1 x 10"4 uM substrate hydrolyzed/hr) comparable numbers of

listeria were inactivated (table 2).

DISCUSSION

Macrophages from animals exhibiting cell-mediated immunity are
known to possess greater antibacterial capacities than macrOphages
from normal animals. Activated macrophages from animals which have
mounted a specific cellular inmune response against such facultative
parasites as mycobacteria, listeria and salmonella characteristically
exhibit a nonspecific heightened capacity to inactivate and degrade a
wide range of apparently unrelated organisms (4, 5, ll, 12). The
nature of the biochemical agent(s) responsible for this activity is,
at present, unknown.

Intracellular degradation of radio-actively labeled bacteria takes
place in the lysosomes of macrophages (3). Heat-killed bacteria are
degraded more readily than viable bacteria and organisms such as
Bacillus subtilus and Micrococcus lysodiekticus are degraded more
rapidly than Staphylococcus aureus.

Greater numbers of lysosomes and higher activities of lysosomal
enzymes were found in macrophages of animals with cellular immunity
than in normal animals (7, 13). It was suggested that this, in part,
may be responsible for the heightened antibacterial response in cell-
mediated inmunity. Contrary evidence suggested that lysosomal enzymes
may be beneficial to facultative intracellular parasites by providing
growth-stimulating low molecular weight compounds, as products of the
l'Wclrolytic enzymes (14). In addition, alveolar macrophages have higher
1Ilfsosomal enzyme activities than peritoneal macrophages (7, 15), yet

the latter are more efficient in killing bacteria (16).

69

70

The removal of bacteria from a system by a phagocyte is probably
dependent on a three step process: 1) phagocytosis and fusion of
lysosomes with phagosomes, 2) inactivation or killing, and 3) degrada-
tion by lysosomal enzymes (3, 18). Little is known of the biochemical
events responsible for step two. We are reporting an antibacterial
material which may contribute to elucidation of this step.

The activated macrophage in cell-mediated immunity is associated
with an increased metabolic activity and a higher rate of phagocytosis
(12). ‘Miller (17) found killing required an intact respiratory electron
transport chain in normal unstimulated mouse peritoneal macrophages,
although an uncoupler of oxidative phosphorylation, 2, 4-dinitrophenol,
had little or no effect on bactericidal activity (17). Cyanide, anti-
mycin A, or amytal, inhibitors of respiration, interfered with bacteri-
cidal activity to some extent. Cohn (18) found no interference in bac-
terial degradation by cyanide. Inhibitors of glycolysis, the citric
acid cycle, and the phosphogluconate oxidative pathway did not interfere
significantly. The latter pathway is important for the generation of
H202 in the bactericidal myeloperoxidase system in PMNs. Although
Paul et a1. (2) reported low levels of activity in macrophages from
various sources, Miller (17) and Cline (19) found no evidence for a
significant myeloperoxidase system in macrophages. Anaerobiosis has no
apparent effect on phagocytosis by mononuclear cells and, although some
antibacterial activity is expressed, maximal activity requires the
presence of oxygen in.human peripheral (19) and mouse peritoneal mono-

nuclear phagocytes (l7).

71

Cell lysates from all groups of mice had antilisterial activity
when tested in the presence of DTT. Higher activities were found in
lysates from prestimulated than nonstimulated control mice and the
highest level of activity was in lysates from mice injected and pre-
stimulated with viable BCG.

The antilisterial factor is probably bactericidal rather than
bacteriostatic in nature. Continued incubation of petri plates caused
no increase in the number of colonies of listeria.

Antilisterial activity was associated with lysates from both
glass-adherent (65% macrophages) cells and with nonadherent cells (99%
lymphocytes). More than 99% of the cells in the nonadherent cell popu-
lation were morphologically lymphocytes but it is possible this activity
may have been derived from the small percentage of contaminating mono-
cytes and macrophages.

The increase in reduction potential in macrophages from mice re-
ceiving BCG was striking. Macrophages from mice in these groups reduced
NBT to a much greater extent than controls. NBT reduction has been
shown to occur in the electron transport system via an interaction
with ubiquinone at E; = +1.00 volt (20). Miller (17) found various
dyes and electron acceptors with redox potentials of less than E; = 0.22
volt interfered with bactericidal activity of normal macrophages and
suggested the electron transport system was involved in macrophage
antibacterial mechanisms. In our system, DTT, a reducing agent or
electron donor with a redox potential of E; - -33 volt, activates an

antilisterial factor. The almost minimal levels of reduction potential

in control mice may mean the factor may be present, but all or most of

72

it perhaps in the inactive, oxidized form. The increased metabolism

of the macrophages in mice with cell-mediated immunity may be genera-
ting the elevated reduction potentials required to bring the factor to
the active reduced form. In previous experiments (21), only peritoneal
cell lysates from BCG-immunized mice had antibacterial activity when
assayed without DTT immediately after cell collection and lysis.

The requirements for a reducing compound, dithiothreotol, for
antibacterial activity to be retained after release from the cell may
also account for the lack of its detection in other studies. Enzymes
have been isolated which have this same requirement. Some of these,
such as arylamidases (22) and cathepsins (23), are present in lysosomes
of rat liver and kidney.

All fractions from homogenized peritoneal cell lysates obtained
by differential centrifugation had antilisterial activity. The relative
activity found in the pellet obtained by centrifugation at low speed
steadily decreased as homogenation time was increased and more cells
were broken. Antilisterial activity paralleled phosphatase activity in
the high speed supernatant fractions at each dilution tested. Enzymes
such as phosphoprotein phosphatase can interfere with the assay for
acid phosphatase (24) and the factor(s) may correlate with the cyto-
plasmic enzyme. However, antilisterial activity was found in the pellet
fraction obtained by high speed centrifugation which may suggest a
lysosomal origin for the factor(s). Noncorrelation of antilisterial and
enzyme activities in the pellet fraction at the higher concentrations
suggests the presence of an inhibitor, perhaps a membrane or membrane
component.

Studies to isolate and characterize this factor(s) are in progress.

REFERENCES

1.

Zeya, H. I., Spitznagel, J. K. Arginine-rich proteins of poly-
morphonuclear leukocyte lysosomes. J. Exp. Med. 121:927-941, 1968.

Paul, B. B., Strauss, R. R., Jacobs, A. A., Sbarra, A. J. Function
of H202, myeloperoxidase, and hexose monophosphate shunt enzymes
in phagocytizing cells from different species. Infect. Immunity
.l:338-344, 1969.

Cohn, Z. A. The fate of bacteria within phagocytic cells. I. The
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Blanden, R. V., Lefford, M. J., Mackaness, G. B. The host response
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Mackaness, G. B. The immunological basis of acquired cellular
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Gomori, G. Preparation of buffers for use in enzyme studies. 1p
S. P. Colowick and N. 0. Kaplan (ed.) Methods in Enzymology, Vol.
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Cohn, Z. A., Wiener, E. Particulate hydrolases of macrophages.

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Hirsch, J. G. Bactericidal action of histone. J. Exp. Med. ‘128:
925-944, 1958.

Dipietro, D. L., Zengerle, F. S. Separation and properties of three

acid phosphatases from human placenta. J. Biol. Chem. 242:3391-

3396, 1967.
73

74

10. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., Randall, R. J.

Protein measurement with the folin phenol reagent. J. Biol. Chem.
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11. Mackaness, G. B. Resistance to intracellular infection. J. Infect.
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12. Ratzan, K. R., Musher, D. M., Keusch, G. T., Weinstein, L. Cor-
relation of increased metabolic activity, resistance to infection,
enhanced phagocytosis, and inhibition of bacterial growth by macro-
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499-504, 1971.

13. Saito, K., Suter, E. Lysosomal acid hydrolases in mice infected
with BCG. J. Exp. Med. 121:727-738, 1965.

14. Brown, D. A., Draper, P., Hart, P. D. Mycobacteria and lysosomes:
A paradox. Nature (London)‘ggl:658-660, 1969.

15. Myrvik, Q. N., Leake, E. S., Fariss, B. Lysozyme content of alveolar
and peritoneal macrophages from the rabbit. J. Immunol. ,§p:133-
136, 1961.

16. Pavillard, E.R.J. In vitro phagocytic and bactericidal activity
of alveolar and peritoneal macrophages of normal rats. Aust. J.
Exp. Biol. Med. Sci. _l;265-274, 1963.

17. Miller, T. E. Metabolic event involved in the bactericidal activity
of normal mouse macrophages. Infect. Immunity §;390-397, 1971.

18. Cohn, Z. A. The fate of bacteria within phagocytic cells. II. The
modification of intracellular degradation. J. Exp. Med. 117:43-

53, 1953.

19.

20.

21.

22.

23.

24.

75

Cline, M. J. Bactericidal activity of human macrophages: Analysis
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Slater, T. F., Sawyer, B., Strauli, U. Studies on succinate-
tetrazolium reductase systems. II. Points of coupling of four
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1963.

Muirhead, D. Y. Studies on a possible cellular response in mice
immunized with Staphylococgpp aureus, Smith strain diffuse.
Master's Thesis. Michigan State University, 1971.

Mahadevon, S., Tappel, A. L. Arylamidases of rat liver and kidney.
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Huisman, W., Bonma, J.M.W., Gruber, M. Involvement of thiol
enzymes in the lysosomal breakdown of native and denatured proteins.
Biochim. Biophys. Acta 221:98, 1973.

Revel, H. R. Enzymes of phosphate metabolism. Phosphoprotein
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Enzymology, Vol. VI. Academic Press Inc., New York, 1963, p. 214.

FOOTNOTES

1HD50 unit/m1 is the concentration which lyses 50% of the cells.

2Generously donated by Dr. Frank A. Kapral, Ohio State University.

3Prepared as suggested by manufacturer. A request for the exact

concentration of NBT was refused by Sigma Chemical Co.

76

77

Figure 1. Restoration by 10 mM dithiothreotol (DTT) of antilisterial
activity of mouse peritoneal cell lysate lost by overnight storage

at 2 C (lysate dilutions with DTT 0 , lysate dilutions without DTT I ).
Undiluted lysate equivalent to 1 x 107 cells/m1 (6.5 x 106 macrophages

and 3.5 x 106 lymphocytes per m1).

 

78

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79

Figure 2. Antilisterial activity of lysates of subpopulations of

mouse peritoneal cells, glass adherent cells 0 , and nonadherent cells A .

— __.__.__,______ _ ——.4

100

70

40

30

PER CENT LISTEIII KILLED

20

10

80

105 10‘ 107

Figure 2.

LYSATE CONCENTRITIHTIOI EQUIVALENT

T0 NUMBER OF CELLS PER ML

81

Figure 3. Nitro blue tetrazolium reduction by mouse peritoneal
macrophages. Optical density values of extracts of reduced NBT from

4 x 106 macrophages.

82

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83

Figure 4. Acid phosphatase and antilisterial activities of high speed

supernatant fractions of mouse peritoneal cells homogenized 2, 4, 6,

and 8 minutes. Antilisterial activities of fractions diluted 1:10,() ;

1:30,<>; 1:90,A; and 1:270, D .
C .

Undiluted acid phosphatase activities,

84

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85

Figure . Acid phosphatase and antilisterial activities of low speed

pellet fractions of mouse peritoneal cells homogenized 2, 4, 6, and

8 minutes. Antilisterial activities of fractions diluted 1:10, 0 ;

1:30, 0; 1:90, A ; and 1:270, u . Undiluted acid phosphatase

activities, 0 .

86

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87

Figure 6. Acid phosphatase and antilisterial activities of high speed
pellet fraction of mouse peritoneal cells homogenized 2, 4, 6, and
8 minutes. Antilisterial activities of fractions diluted 1:10,(3 ;

1:30, 0 ; 1:90, A ; and 1:270, D . Acid phosphatase activities of

undiluted fractions, 0 .

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88

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DITHIOTHREOTOL-DEPENDENT ANTILISTERIAL ACTIVITY OF LYSATES FROM
NORMAL MACROPHAGES EXPOSED IN VITRO T0 CULTURE FLUIDS

FROM SPLEEN CELLS OF BCG-IMMUNIZED MICE

Donna Y. Muirhead and Virginia H. Mallmann
Department of Microbiology and Public Health
Michigan State University

East Lansing, Michigan 48824

Michigan Agricultural Station Journal Article No. 6689

ABSTRACT

The effect of culture fluids from mouse lymphocytes on the anti-
listerial activity of normal macrophages.ipqyippp was determined.
Titers of lysates of macrophages incubated with or without culture
fluids from lymphocytes from control mice had titers of 10-270 with or
without a reducing agent, dithiothreotol (DTT). Lysates of macrophages
after incubation with culture fluids of spleen lymphocytes from BCG-
immunized mice had antilisterial titers with and without DTT of
197,000 and 810 respectively. This may indicate a DTT-dependent
antilisterial system inherent in activated macrophates of cell-mediated
immunity. Lysates of incubated spleen cells and their culture fluids

also had slight antilisterial activity.

92

INTRODUCTION

Culture supernatant fluids from lymphocytes of immunized animals
incubated ip'yippp with specific antigen reportedly affects the activity
of macrophages ip'yippp. Nathan et a1. (7) reported increased macro-
phage adherence, phagocytosis and hexose monophoSphate oxidation in
macrophages from normal guinea pigs incubated with supernatants of
lymphocytes from o-chlorobenzoyl bovine ‘Y-globulin immunized animals
after the lymphocytes were incubated with the specific antigen.
Patterson and Youmans (8) found a significant decrease in the number
of virulent Mycobacterium tuberculosis H37Rv in normal macrophages
when splenic lymphocytes from mice immunized with avirulent M.
tuberculosis H37Ra were added. They also observed intracellular
inhibition when culture fluids from immune spleen cells incubated 1p
,yippp with specific antigen were added to normal macrophages. Howard
et a1. (3) reported similar observations when lymphocytes from
.fliptoplasma capsulatum immunized mice were added to normal macrophages.

The increased macrophage antimicrobial activity is not specific
for the immunizing organism. Culture supernatant fluids of lymphocytes
from listeria-immunized mice incubated.;p,yippp with listeria confer
the ability to inhibit virulent mycobacteria on normal macrophages (5),
and culture filtrates of BCG-immune lymphocytes incubated with BCG

activate normal macrophages to greater killing of listeria (10).

93

94

We detected antilisterial activity in mouse peritoneal cell

lysates which depends on the presence of a reducing agent, dithio-
threotol (DTT) (6). This is a report of the detection of this activity
in lysates of normal macrophages and an increased activity in lysates

of macrophages incubated with culture fluids from BCG-immune lymphocytes.

MATERIALS AND METHODS

 

CBA/J female mice approximately nine weeks old were obtained from
Jackson Laboratories, Bar Harbor, Maine. They were housed 5 per cage

with water and food pellets, pd libitum.

Bacterialjpreparations

Mycobgcterium bovis, BCG was grown at 37 C for 2 weeks in Dubos
Broth base without enrichment or Tween 80 (Difco Laboratories, Detroit,
Mich.) with 0.5% dextrose, centrifuged and homogenized with mortar
and pestle. Cells were suspended in tissue culture media, the concen-
tration estimated by centrifugation in a Hopkin's tube, and adjusted
to approximately 1 x 108 organisms/ml.

Listeria monocytogenes was maintained on Brain Heart Infusion agar
(Difco). Cultures used for measuring antilisterial activity were
grown for 18 hrs in Brain Heart Infusion broth (Difco at 37 C with

shaking.

Immunizations
Mice were inoculated once intraperitoneally with approximately
2 mg (wet weight) of BCG in physiological saline in a total volume of

0.2 ml.

95

96

Lypphocyte cultures

Spleens from control mice and mice immunized 4 weeks previously
with BCG were removed aseptically and placed in cold Hank's basal salt
solution (HBSS) (Microbiological Associates, Bethesda, Md). Both groups
were treated in the same manner. The spleens were punctured at various
sites, injected with cold HBSS and the cells expressed with sterile
forceps.

The cells were dispersed by forcing them first through a 20-gauge
and then through a 27-gauge syringe needle. The cells were centrifuged
at 5 C for 10 min at 250 x,g and resuspended in HBSS with 10% fetal
calf serum and supplemented with 1% each glutamic acid, sodium pyruvate,
glutamine, vitamin, essential and nonessential amino acid pools, and
100 ug/ml streptomycin and 100 units/ml penicillin.

Cell numbers were determined by hemocytometer. Differential cell
counts were made in a solution of 2.1% citric acid-0.1% crystal violet.
Viability counts were made by the trypan blue exclusion technique.

Spleen cell suspensions were adjusted to l x 107 viable lympho-
cytes per ml and 15 ml were placed in 100 x 15 mm plastic tissue
culture dishes. BCG was added to one-third of the spleen cell cultures
for a final concentration of 106 organisms per ml. The cell cultures
‘were incubated for 2 hrs at 37 C in an atmosphere of 95% air and 5%
(X32. Nonadherent cells in the culture medium were transferred to new
tissue culture dishes, incubated 3 days and centrifuged. The culture
Ifluids were decanted and set aside. The lymphocytes were washed 1x and

r'esuspended to l x 107 cells/ml in 0.1 M phosphate buffer, pH. 7. Cell

8llapensions after incubation were 93-99% lymphocytes.

97

Macrophgge collection,gpd culture

A modification of the method of Klun and Youmans (4) was used.
Peritoneal cells of normal, unstimulated mice were collected as described
previously (6). The cells were pooled, centrifuged 10 min at 5 C at
250 x,g and resuspended in the above medium with 10% horse serum added.
Differential cell counts and viability were determined as above and
cell numbers adjusted to 1 x 107 macrophages/m1. Fifteen ml were
placed in plastic tissue culture dishes and incubated at 37 C in 95%
air and 5% C02. After two days, the medium and unattached cells were
removed, fresh medium was added and the macrophages incubated for an
additional 24 hrs.

After a total of 3 days incubation, the medium was removed and
replaced with 10 ml fresh medium and 5 ml of lymphocyte culture fluid.
The cell cultures were incubated 24 hrs. The media was decanted and
the adherent cells rinsed quickly and gently with a 1:1 trypsin-EDTA
at room temperature. The cells were covered with cold trypsin-EDTA
and removed with a rubber policeman. The macrophages were washed,

counted and resuspended to 2 x 106 macrophages/m1 in 0.1 M phosphate

buffer, pH 7.0. The cell suspensions were 99% macrophages.

Prepgration of cell lysates and lymphocyte culture fluids for assay

Macrophage and lymphocyte suspensions were lysed with staphylococcal
delta-hemolysin as described previously (6). When microscopic examina-
tion determined greater than 95% lysis had occurred, aliquots were

removed and assayed immediately for antilisterial activity.

98

Supernatant fluids from cultured lymphocytes were dialyzed in
distilled water which had been degassed and flushed with argon. The
dialyzed culture fluids were assayed immediately.

Aliquots of cell lysates and dialyzed lymphocyte culture fluids

were stored at 4 C and reassayed after 3 weeks.

Antiligterigl assay

Antilisterial activity was measured using a modification of the
assay described in a previous paper (6). Serial 3-fold dilutions of
the material to be tested were made in two diluents, 0.1 M phosphate
buffer pH 7 containing 0.01% bovine serum albumin with and without DTT
at a concentration of 10 mM. The controls were tubes containing 1.0 m1
of the diluent. An 18-hour broth culture of.L. monocytogenes was
centrifuged, resuspended in 0.85% saline and diluted to ca. 2 x 106
colony forming units/m1. One-tenth ml was added to each dilution and
control tubes. The tubes were incubated for 2 hrs at 37 C. The
suspensions were diluted and aliquots of each dilution added to 15 ml
of warm, melted Brain Heart Infusion agar. After mixing, the contents
were poured into petri dishes, allowed to solidify and incubated over
night at 37 C. The number of colonies were counted and the titer taken

as the highest dilution which inactivated 50% of listeria when compared

to the buffer control.

RESULTS

Anpilisterial activity of lymphocyte culture fluids

In the absence of DTT, no detectable antilisterial activity was
found in fresh lymphocyte culture fluids except in those from BCG
stimulated lymphocytes of BCG-immunized mice (Table 1). Without DTT,
no antilisterial activity in culture fluids was detected after 3 weeks
storage. Slight activity was detected in the presence of DTT culture
fluids from control lymphocytes which had been incubated with BCG, and

from BCG-immune lymphocytes incubated with or without BCG.

Antilisterial activity of lymphocyte lysates
There was antilisterial activity in lysates from lymphocytes from
all groups, with and without DTT (Table 2). Titers of stored lysates

were similar to initial titers.

Antilisterial activigy of macrophage lysates

Endpoints were not reached in several of the initial titrations
(Table 3). When reassayed, the greatest activity was found in lysates
of macrophages incubated with culture fluids from BCG-immune lymphocytes
incubated with BCG. In the presence of DTT, lysates in this group had
titers of 197,430. Without DTT, the titer was 810. Macrophage
lysates from cells incubated with culture fluids of BCG-immune lympho-
cytes, unstimulated lp yippp, had similar titers. Endpoints reached

with macrophage lysates in other groups were not greater than 270.

99

DISCUSSION

The lymphocyte culture fluids and lysates of spleen cells inacti-
vated listeria to some extent. Whether the activity is due to a
material produced and secreted by lymphocytes or whether this base-
line activity is a result of the small number of macrophages present
could not be determined.

Low levels of antilisterial activity were detected in lysates of
macrophages with or without culture fluids from unsensitized lymphocytes.
This was interpreted to represent the normal antibacterial factors of
macrophages, although an effect by pinocytosed antibiotics cannot be
eliminated. Titers with or without DTT did not differ markedly.

In the presence of DTT, lysates of macrophages incubated with
culture fluids of BCG-stimulated lymphocytes from BCG-immunized mice
had greater antilisterial activity than the above controls. Unpublished
data indicates this is a qualitative difference, not quantitative.
Culture fluid from BCG-immune spleen cells cultured 1p yippp without
BCG were also able to stimulate macrophages and high titers were obtained
with lysates in this group when DTT was included in the assay system.
Spleen cells had been collected from mice one month after injection
with BCG and, therefore, the cells may still have been near the peak
of their immune response. Blanden et a1. (1) found significant re-
sistance to L. monocytogenes in mice infected with BCG four weeks

earlier.

100

101

The nature of the stimulus affecting macrophages is unknown. The
macrophages may be pinocytosing and concentrating an active lymphocyte
product or receiving a signal to produce and/or activate a macrophage
antilisterial product(s). The latter is more probable, assuming
complete uptake of an active factor(s) from culture fluid and the action
additive to normal macrophage antibacterial systems. There was not
sufficient total activity in the volume of culture fluid added to
macrophages to account for the increased activity, even using least
favorable data. It is possible that a DTT-dependent antibacterial
factor produced by lymphocytes is taken up and is acting synergistically
with macrophage systems. Lysates of macrophages incubated with culture
fluids of BCG-sensitized lymphocytes, incubated 1p.yippp‘with or with-
out BCG, had titers of 197,430 and 65,610, respectively. In this same
test group, titers were 270 with or without DTT when BCG was added to
culture fluid at the time of transfer to macrophages. This suggests
the removal of an active factor(s) by the BCG.

We have reported a DTT-requiring antilisterial factor(s) from mouse
peritoneal cell lysates. Slightly greater activities were detected in
cell lysates from BCG-immunized and prestimulated mice (6). The depend-
ency of the antilisterial factor on a reducing agent for activity may
explain why it has not been detected by previous investigators. The
elevated antilisterial titer, with DTT, of lysates of macrophages
exposed to culture fluid of BCG-immune lymphocytes suggests the DTT-
dependent factor(s) may be important in antibacterial cell-mediated

immunity.

102

Other authors have reported an inhibitory influence of culture
supernatant fluids from immune, stimulated lymphocytes on intracellular
parasites in normal, infected macrophages (2, 3, 4, 5, 8, 9, 10).
Pearsall et a1. (9) reported a direct effect on yeast cells by lymphokine-
containing lymphocyte supernatants. Klun and Youmans (4) and Fowles et
a1. (2) found no direct effect on extracellular mycobacteria or listeria
by lymphocyte culture fluids but suggested these results may not be
definitive. Fowles et a1. (2) found the addition of sensitive lympho-
cytes had a greater effect on macrophage bacteriostasis than did
supernatant fluids and suggested the mediators produced by lymphocytes
may be labile. In their experiments, the effect of lymphocytes on
macrophages was detected by determining the bacterial inactivation in
intact macrophages. Our system offers the advantage of measuring
bacterial inactivation in a cell free system and examining the process

at a subcellular level.

REFERENCES

Blanden, R. V., M. J. Lefford, and G. B. Mackaness. 1969. The
host response to Calmette-Guerin bacillus infection in mice.
J. Exp. Med. 1;_:1079-1101.

Fowles, R. E., J. M. Fajardo, J. L. Leibowitch, and J. R. David.
1973. The enhancement of bacteriostasis by products of activated
lymphocytes. J. Exp. Med. ‘l§_:952-964.

Howard, D. H., V. Otto, and R. K. Gupta. 1971. Lymphocyte-mediated
cellular immunity in histoplasmosis. Infect. Immun. .4:605-610.

Klun, C. L., and G. P. Youmans. 1973. The effect of lymphocyte
supernatant fluids on the intracellular growth of virulent tubercle
bacilli. J. Reticuloendothel. Soc. .13:263-274.

Klun, C. L., and G. P. Youmans. 1973. The induction by Listeria
monogytogenes and plant mitogens of lymphocyte supernatant fluids
which inhibit the growth of Mycobacterium tuberculosis within
macrophages 1p.y;pgp. J. Reticuloendothel. Soc. 13:275-285.

Muirhead, D. Y., and V. H. Mallmann. 1973. Antilisterial factor
from mouse peritoneal cells. Submitted to J. Infect. Dis.

Nathan, C. F., M. L. Karnovsky, and J. R. David. 1971. Alterations

of macrophage functions by mediators from lymphocytes. J. Exp.

Med. 13 :1356-1376.

103

104

8. Patterson, R. J., and G. P. Youmans. 1970. Demonstration in
tissue culture of lymphocyte-mediated immunity to tuberculosis.
Infect. Immun. Ilz600-603.

9. Pearsall, N. N., J. S. Sundsmo, and R. S. Weiser. Lymphokine
toxicity for yeast cells. J. Immunol. ll_;1444-l446.

10. Salmon, B. J., J. I. Kreisberg, and G. M. Middlebrook. 1973.
Listericidal activities of macrophages from Bacillus Calmette-

Guerin immunized guinea pigs by specific and nonspecific mitogens.

Fed. Proc. ._2:1039 (Abs.).

105

TABLE 1
ANTILISTERIAL ACTIVITY WITH OR WITHOUT DTT OF CULTURE FLUIDS

FROM MOUSE SPLEEN LYMPHOCYTES, INCUBATED l_ VITRO WITH OR

 

WITHOUT BCG, FROM NORMAL AND BCG-IMMUNIZED MICE

 

 

 

 

 

Added to Antilisterial Antilisterial
Source lymphocytes Included in titerd when titer
of for incubation assayed assayed of supernatants
lymphocytesa ip vitro solutions immediately after storage
0 < 10 < 10
0
DTTC < 10 < 10
Unimmunized
b 0 <110 <I10
BCG
DTT < 10 270
0 < 10 < 10
0
DTT > 270 810
BCG-immunized
0 > 270 < 10
BCG
DTT 90 270

 

.Lymphocyte concentration in in vitro culture - l x 107 cells/m1
b . . . 6

BCG concentrat1on 1n,lp vxtrq culture - 1 x 10 cells/ml

cDTT concentration in assay solutions = 10 mM

dTiter - highest dilution which inactivated 50% of listeria

106

TABLE 2
ANTILISTERIAL ACTIVITY WITH OR WITHOUT DTT OF LYSATES

OF MOUSE SPLEEN LYMPHOCYTES, INCUBATED l_.VITRO WITH

 

0R WITHOUT BCG, FROM NORMAL AND BCG-IMMUNIZED MICE

 

 

 

 

 

 

Added to . Antilisterial Antilisterial
Source lymphocytes Included in titerd when titer
of for incubation assayed assayed of supernatants
lymphocytesa 1p vitro solutions immediately after storage
0 30 30
0
DTT 90 270
Unimmunized
b 0 30 3O
BCG
DTT 90 90
0 > 270 270
0
DTT 30 10
BCG-immunized
0 > 270 90
BCG
(DTT 30 10
7

aLymphocyte concentration in 13 vitro culture n l x 10 cells/m1

 

bBCG concentration in ip vitro culture I l x 106 cells/ml

cDTT concentration in assay solutions = 10 mM

dTiter - highest dilution which inactivated 50% of listeria

107

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/08

ISOLATION AND CHARACTERIZATION OF AN ANTIBACTERIAL FACTOR
FROM PERITONEAL CELL LYSATES OF MICE IMMUNIZED
WITH VIABLE MYCOBACTERIUM BOVIS, BCG

Donna Y. Muirhead and Virginia H. Mallmann

Department of Microbiology and Public Health
Michigan State University
East Lansing, Michigan 48824

ABSTRACT

Crude homogenates of peritoneal cells from BCG-infected mice
contained antilisterial activity when a reducing compound, dithio-
threotol (DTT), was included in the assay system. The material was
oxygen sensitive, heat stable and active against Listeria monocytogenes,

Staphylococcus aureus and Mycobacterium fortuitum but not Pseudomonas

 

aeruginosa. Inactivation of.L. monocytogenes in stationary phase was
biphasic and continuous over time. There was no detectable inactiva-
tion of log phase L. monocytogenes.

Lysates from peritoneal cells of control mice had antilisterial
activity in only one DEAE-cellulose fraction (DEAE-I), lysates from
BCG-infected mice had an additional fraction with antilisterial
activity (DEAE-II). The additional fraction had the same antibacterial
spectrum as crude lysate. DEAE-fraction II was most active at a pH
near neutrality. Inactivation rate kinetics were not the same as those
of crude lysates, i.e., a short period of listerial inactivation was
followed by no further decrease in viable units over time instead of
steady continued inactivation. No additive effects were observed when
fractions I and II were combined.

It is concluded that the active fraction unique for lysates of
BCG-immunized mice only partially accounts for the antimicrobial
properties in macrophage lysates of BCG-immunized and prestimulated

mice.

109

INTRODUCTION

Lysates of mouse mononuclear cells were capable of inactivating
Listeria monocytogenes (5). Only peritoneal cell lysates from mice
immunized and prestimulated intraperitoneally with BCG had antibac-
terial activity when assayed immediately after cell collection and
homogenation (4). When a reducing agent, dithiothreotol (DTT), was
included in the assay system, lysates from all groups of mice immunized
or control, had some antilisterial activity (5). Peritoneal macrophages
from mice immunized and prestimulated with BCG had high reduction
potentials when assayed in yiggg, but cells from control mice or mice
prestimulated with glycogen had little or none. This suggested that
if the antilisterial factor(s) is present in normal cells, it may only
be present in an inactive, oxidized form.

We have recently reported DTT-requiring antilisterial activity in
lysates of normal macrophages which had been incubated_ig.yi££2 with
supernatant fluids from lymphocytes of BCG-immunized mice (6). DTT-
dependent activity was present at much higher titers in these lysates
than controls.

This is a report of partial separation attempts and characteriza-

tion of the active factor(s).

llO

MATERIALS AND METHODS

Animals. Female Swiss-Webster albino mice (Carworth Farms,
Portage, Michigan) were housed 5 per cage and fed mouse pellets and
water‘gd libitum. Immunization with viable BCG, peritoneal cell
collection and homogenation procedures were as described previously (5).
A11 fractionation and testing, unless indicated otherwise, was with
cell lysates from mice immunized and prestimulated with viable BCG.

Microorganisms. Listeria monocytogenes, Staphylococcus aureus,
and Pseudomonas aerugenosa were maintained on Brain Heart Infusion
agar (Difco). Mycobacterium fortuitum and Mycobacterium bovis, BCG
were maintained on Dubos oleic agar (Difco).

Antihgcterialggssay system. The assay system was as described
previously (5) except phosphate buffer (PB), pH 7 replaced phosphate-
citrate buffer, pH 7. The assay system used to measure antilisterial
activity was a modification of that by Hirsch (1). All lysates and
fractions were dialyzed against 1:10 PB before testing. Serial 3-fold
dilutions of the materials to be tested were made in PB containing
0.01% bovine serum albumin (Difco). After preliminary experiments
indicated that the factor was oxygen-labile, the reducing agent,
dithiothreotol (DTT) (Calbiochem, San Diego, Calif.), was included in
the buffer system at a concentration of 10 mM. The final volume of
each dilution tube was 1.0 ml. The control was a tube containing
1.0 ml of the diluent. An 18 hour broth culture of ListerigLEpnocy-
togenes was centrifuged, resuspended in 0.85% saline and diluted to

111

112

ca. 2 x 106 CFU/ml. One-tenth milliliter was added to each dilution
and the control tube. Each tube was flushed with argon and incubated
at 37 C for 2 hrs. The suspensions were diluted and aliquots of each
dilution added to 20 ml of warm (50 C), melted Brain Heart Infusion
Agar. After mixing, the contents were poured into petri dishes,
allowed to solidify and incubated overnight at 37 C. The number of
colonies were counted and expressed as the percent kill calculated
from the control.

Ammonium sulfate precipitation. A saturated solution of ammonium
sulfate was adjusted to pH 7 with ammonium hydroxide. Homogenized cell
fractions were centrifuged at 20,000 x‘g for 20 minutes and dialyzed
against phosphate buffer, pH 7. Fractionation procedures were per-

formed at 0-4 C. Appropriate volumes of saturated (NH SOZ were

4)2
added with constant stirring to obtain the desired percent saturated
solutions. After stirring on ice for 1 hour the mixture was centri-
fuged at 20,000 x.g for 20 min. The precipitate obtained between

50-80% was used in further fractionation. This fraction was redissolved
and dialyzed against PB.

DEAR-cellulose chromatography. Diethylaminoethyl (DEAE)-cellulose
(Reeve Angel, Clifton, NJ)was equilibrated with 0.05 M tris (hydroxy-
methyl)-amino methane (Tris)-hydrochloric acid buffer, pH 7.8 that
contained 5 mM dithiothreotol (DTT) (Calbiochem, San Diego, CA). A
column 1.7 by 27 cm was prepared. The sample was applied and eluted
by pumping at a flow rate of 1 ml/min with 0.05 M Tris-H01, pH 7.8

with 5 mM DTT followed by a linear gradient of 0.05 M Tris HCl-SmM DTT,

pH 7.8 and 0.05 M Tris HCl-SmM DTT and 0.5 M NaCl, pH 7.8. The eluate

113

(3 m1/tube) was monitored at 280 nm using an Isco UV monitor. Individual
fractions with activity were pooled, precipitated in 80% (NH4)SO4 and
reconstituted and dialyzed against PB with lmM DTT.

Gel filtration chromatography. Sephadex G-200 (Pharmacia) was
swollen and equilibrated in 0.05 M PB pH 7. A column 2.8 x 39 cm was
prepared and 1 ml of whole cell lysate applied and eluted at a constant
head pressure of 10 cm. Fractions (3.0 ml) were collected and measured
for absorbance at 280 nm, sterilized by millipore filtrations and
tested for antilisterial activity.

Polyacrylamide gel electrophoresis. A modification of the method
of Maizel (3) was used to monitor purification of the lysates. Samples
and electrode buffers were prepared with 1 mM DTT. The gel was 7.5%
in acrylamide; no stacking gel was used. Migration was to the anode
in Tris-glycine buffer, pH 8.3 at a constant current of 3 ma per gel.
Bromphenol blue was used as the tracking dye. The gels were stained
with 0.25% coomassie blue (Schwarz/Mann, Orangeburg, NY) in 7.5% acetic
acid and destained in 7.5% acetic acid. Gels were scanned at 580 nm
with a modified Gilford spectrophotometer.

Heat stability studies. Dilutions of supernatant fluid from lysates
after high speed centrifugation and DEAE fraction II were assayed for
activity. Appropriate dilutions were prepared in phosphate-citrate
buffer, pH 7 with 10 mM DTT. Aliquots were heated at 37 C for 30 min,
45 C for 30 min, 56 C for 30 min and 100 C for 10 min. The fractions
were cooled on ice and tested for antilisterial activity.

Inactiyation rate kinetics. Cultures of L. monocytogenes were

inoculated into Brain Heart Infusion (BHI) broth (Difco) and grown to

114

stationary phase (18 hr culture) and mid-log phase (2.5 hrs or an 0.D.
of 0.22 at 620 nm). The cultures were centrifuged and resuspended in
saline. Aliquots of the resuspended cultures were added to solutions
to be tested in 0.07 M phosphate-citrate buffer at pH 7 with 0.1% bovine
serum albumin and 10 mM DTT and also to controls without lysate. The
suspensions were incubated at 37 C. Aliquots were removed at appro-
priate times and serially diluted. Aliquots of the dilutions were
added to 20 ml of warm (50 C), melted BHI agar, mixed and poured into
petri dishes. After solidifying, the plates were incubated overnight
at 37 C. The number of colonies were counted and the difference be-
tween control and test values was used to calculate percent inactivation.

Relative activity‘against heterologous organisms. Eighteen hour
cultures of.L. monocytogenes, S, aureus, and.§. aerugenosa grown in
BHI broth and a 48 hr culture of M. fortuitum grown in Dubos broth
with Tween 80 (Difco) were used. Bacterial cultures were centrifuged
and resuspended in saline to ca 106 CFU/ml. Fractions were assayed
as above with dilutions which inactivated 60% of listeria.

pH optima. After determining the optimal dilution for activity
at pH 7 in 0.1 M phosphate buffer with 10 mM DTT, concentrated material
purified by DEAR-cellulose chromatography was diluted in 0.1 M acetate
buffer (pH 4 & 5), 0.1 M phosphate buffer (pH 6, 7, 8), and 0.05 M
glycine-NaOH buffer (pH 9 & 10) and tested for activity. Each buffer
system contained 10 mM DTT.

Protein determination. Protein was measured by the method of

Lowry et a1. (2).

RESULTS

Stability_9f active factor in crude lysates. Antilisterial activity
was measured in lysate treated or stored under different conditions
(Table 1). Some activity was lost in storage without DTT. There was
substantial loss of activity in 1yophilized or aerated samples. All
activity was lost in samples stored in buffer at pH 5 without DTT. In
all succeeding experiments, lysates and fractions were stored at 4 C
or -56 C with 10 mM DTT.

Fractionation. A schematic diagram of the fractionation procedure
is shown in Figure l. Peritoneal cells from groups of 50 BCG-immunized
and BCG-prestimulated mice were washed and resuspended in 30 m1 of
0.25 M sucrose in 0.05 M Tris-acetate buffer, pH 7.4. The homogenized
cell preparation was clarified by centrifugation, brought to 50%
saturation with ammonium sulfate and stirred on ice for 1 hr. The
precipitate left after centrifugation was discarded and the remaining
supernatant fluid brought to 80% saturation with ammonium sulfate. The
resulting precipitate was redissolved, dialyzed and applied to a DEAE-
cellulose column. The elution pattern is shown in Figure 2.

Activity was detected in samples under peaks I and II. A similar
elution pattern was observed with lysates from control mice, but anti-
listerial activity was detected only in the samples comparable to
peak I. In several experiments with lysates from BCG-immunized mice,

the second peak did not register but assays indicated the presence of

115

116

antilisterial activity in the region of peak II. Fractions under peak
II were pooled and used in further testing.

The absorbance scan patterns of stained acrylamide gels from crude
high speed supernatant fluid and DEAE-cellulose fractions I and II are
given in Figure 3. DEAE-cellulose fraction I appears to be reduced to
a two component system. Fraction II contained very little protein and
attempts to resolve the sample by acrylamide gel chromatography were
generally unsuccessful.

Table 2 lists protein concentration, activity and recovery values
at each purification step. After DEAE-cellulose chromatography, approxi-
mately 0.2% of the original total protein was recovered in fraction II.

Figure 4 represents the elution pattern of whole, unfractionated
lysate on Sephadex G-200. Antilisterial activity was detected only in
the shoulder preceding the second major peak.

Heat stability. Material in crude lysate is heat stable (Table 3).
There was less than a 10% loss in activity of lysates placed in a boiling
water bath for 10 min. DEAE fraction II was less stable. There was a
40% loss in activity in samples heated at 100 C for 10 min.

Bacterial inactivation. The relative antibacterial activities of
lysate and DEAE fraction II are listed in Table 4. There appeared to be
some reduction in the numbers of g, aureus and'M, fortuitum in the pre-
sence of both crude and purified material. L, monocytogenes was most
sensitive. There was little or no decrease of P, aeruginosa with
either fraction.

Inactivation of listeria in stationary phase by crude material was

biphasic and continuous over time but there was no measurable effect on

117

listeria in log phase (Figure 6). Inactivation kinetics of stationary
phase listeria by DEAE fraction II did not correspond with those of the
crude material, in that listeria were inactivated for a short period
of time followed by no further decrease in colony forming units instead
of a continuous decrease. Combining DEAR-cellulose fractions I and II
did not reestablish the original kinetic pattern. There was no clump-
ing or decrease in numbers of listeria in preparations examined micro-
scopically.

pH optima. A biphasic curve was observed with DEAE-cellulose
fraction II in inactivation studies of L, monocytogenes at different
pH values (Figure 6). There was a slight depression of activity at
pH 7 with greatest activity at pH 6 and 8.

Except for Table 1, all experiments were run 2-3 times.

DISCUSSION

Antilisterial activity in murine peritoneal cyll lysates required
the presence of a reducing agent, DTT, for activity (5). As detected
in this study, the activity in crude lysates is oxygen-sensitive and
acid-labile in the absence of DTT and is heat stable. By sephadex
chromatography, activity was found in fractions eluting before a pro-
tein marker of 67,000 molecular weight. Listeria in stationary phase
were inactivated, but not listeria in log-phase. There was a continuous
biphasic reduction of listerial colony forming units over time.

A fraction with antilisterial activity was found in DEAE-cellulose
eluates of cell lysates from BCG-immunized and prestimulated mice. No
detectable activity was found at this position from lysates of control,
unimmunized mice. Because of the very low concentration of protein in
the pooled sample, it was not possible to make any conclusions on the
homogeneity of the sample by acrylamide gel disc electrophoresis.

Like the crude material, the fraction was heat stable. It was
most active at pH 6 and 8. Whether the fraction is affected directly
or whether the bacteria are more stable at neutral pH is not known.

Although the numbers of §, aureus and M, fortuitum were
reduced slightly by both crude lysate and DEAE fraction II at the con-
centration inactivating 60% of L, monocytogenes, these values were

not statistically different from controls. The concentrations optimal

118

119

for the inactivation of listeria may not be the most effective for
other bacteria. Additional studies will be required to determine if
Inicroorganisms other than listeria are affected.

Like the crude material, DEAE fraction II was effective against
listeria, but the kinetics of inactivation were different. Whereas
the crude material continued to inactivate more listeria, fraction II
(flatained through DEAE-cellulose chromatography reduced the number of
ilisteria colony forming units for only a brief time after which there
seas no further inactivation.

A second fraction with antilisterial activity, DEAR-cellulose
:Eraction I, was found in DEAE-chromatography eluates of both control
43nd BCG-immunized mice. This was found to contain at least two com-
]ponents by acrylamide gel disc electrophoresis. Combining fraction I
and fraction II produced no change in the kinetic pattern and the
effect on listeria was less than additive.

Fraction II obtained by DEAE-cellulose chromatography does not
«:ompletely account for the activity detected in crude lysate. It does
appear to be unique for peritoneal cell lysates of BCG-immunized and

BCG-prestimulated mice.

LITERATURE CITED

Hirsch, J. G. 1958. Bactericidal action of histone. J. Exp. Med.
1198:925-944.

Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall.

1951. Protein measurement with the folin phenol reagent. J.
Biol. Chem. ‘123:265-275.

Maizel, J. V. 1969. Acrylamide gel electrophoresis of protein and
nucleic acids. 1N Fundamental Techniques in Virology, pp 334-362,
Academic Press, NY.

Muirhead, D. Y. 1971. Studies on a possible cellular response
in mice immunized with Staphylococcus aureus, Smith strain diffuse.
Master's thesis. Michigan State University.

Muirhead, D. Y. and V. H. Mallmann. 1973. An antilisterial factor
from mouse peritoneal cells. Submitted to J. Infect. Dis.

Muirhead, D. Y. and V. H. Mallmann. 1973. Dithiothreotol-dependent
antilisterial activity of lysates from normal macrophages exposed
12 31539 to culture fluids from spleen cells of BCG-immunized mice.

To be submitted to Infect. Immun.

120

 

121

FIG. 1. Fractionation diagram for purification of antilisterial

factor.

 

122
FIG. 1.

Mouse peritoneal cells

Homegenation in 0.25 M sucrose-0.05 M Tris acetate buffer,

pH 7.4 and centrifuged at 20,000 x‘g 20 min

 

 

Pg; superhatant fluid
(discard) 50% (NH4)2804, centrifuge

 

 

 

 

r 1

ppt supernatant fluid

(discard) ' 80% (NH4)2804, centrifuge
i
F i l
supernatant ppt
fluid .
Redissolve and dialyze against
(discard) 0.05 M Tris-CCl, pH 7.8

[ DEAE-cellulose chromatography

 

-___._. -.~_._ |

4th peak
in pregradient buffer (DEAR-II)
I concentrate w/ 80% (NH4)2804
reconstitute and dialyze

 

antilisterial fraction

123

FIG. 2. DEAF-cellulose chromatography fractionation of high spemi

supernatant fluid in 0.05 M Tris-HCI, pH 7.8 with 5 mM DTT.

124

 

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125

FIG. 3. Relative absorbance patterns of stained acrylamide gels
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126

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FIG. 4. Sephadex fractionation of high speed supernatant fluid.

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FIG. 5. Antilisterial activity of DEAE-cellulose fraction II at

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FIG. 6. Rate of listerial inactivation by unfractionate lysate,
and fractions I and II from DEAF-cellulose chromatography. Inactivation
of log-phase listeria by unfractionated lysate, Cl; inactivation of
stationary-phase listeria by unfractionated lysate, I ; inactivation
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TABLE 1. Stability of antilisterial factor in crude lysates.

 

 

Percent activity remaining»

 

Treatment with DTT1 without DTT2
24 hrs at 4 C

in PCB, pH 7 100 75
24 hrs at -56 C '

in PCB, pH 7 100 89
Lyophilized

in PCB, pH 7 40 38
Aerated, 15 min

in PCB, pH 7 67 67
24 hrs at 4 C

in PCB, pH 53 94 0

 

 

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2Lysate treated without DTT; 10 mM DTT added to lysate for assay

3Dialyzed against phosphate citrate buffer (PCB), pH 7 for assay

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