Innate and adaptive immune responses in a social conflict paradigm

CLINICAL

IMMUNOLOGY

AND

IMMUNOPATHOLOGY

57,

137-147 (1990)

Innate and Adaptive Immune Responses in a Social Conflict Paradigm’ MARK LYTE,*"

SCOTT G.NELSON,*

AND MICHAEL

L. THOMPSON~

*Department of Biological Sciences, Mankato State University, Mankato, Minnesota 56002, and TDepartment of Pharmacology, Tufts University School of Medicine, Boston, Massachusetts 021 I1 Social conflict stress was examined for its effects on in vitro and in vivo immunity in mice. Adaptive immunity, as measured by the generation of primary IgM antibody responses to the T-dependent antigen keyhole limpet hemocyanin, was suppressed following chronic (> 1 day), but not acute (< 1 day), stress periods while the IgM response to the T-independent antigen polyvinylpyrrolidone was not affected. In vitro proliferative responses of splenocytes to the T cell mitogen concanavalin A and the B cell mitogen lipopolysaccharide were unaffected. Acute (cl day) stress dramatically increased innate immunity as measured by a luminol-dependent chemiluminescence assay of phagocytic cell function. DBA/2J mice averaged a 26% increase in phagocytosis as compared to a 412% increase in C57BW6J. This differential effect of stress on immune responsiveness indicates that alterations in innate immunity in addition to adaptive immunity should also be considered when evaluating neuroendocrine and immune interactions in response to stress. 0 1990 Academic Press. Inc.

INTRODUCTION

Stress has been demonstrated in numerous rodent model systems to affect immune responsiveness (for review see Refs. (1, 2)). In attempting to substantiate a role for stress in the mediation of immunological responsiveness, numerous experimental systems employing stressors ranging from immobilization (3, 4) to centrifugation (5) to electric shock (6-8) have been used by investigators to induce “stressful” conditions in rodents. The results of such studies have in large part concluded that “stress” can affect immune competence, although depending on the specific experimental conditions, stress has been shown to increase, decrease, or not affect immunity. Innate immune responses such as natural killer cell activity (8) and phagocytosis (4) have been shown in rodents to be suppressed by acute exposures to electric shock and immobilization, respectively. Measures of adaptive immunity such as in vitro lymphocyte response to mitogens have shown that mice subjected to an auditory stress evidence a pattern of initial suppression followed by an enhancement of the proliferative response (9). Electric shock has also been demonstrated to suppress production of antigen-specific antibody formation (6) while immobilization has been shown to decrease delayed-type hypersensitivity response while increasing cell-mediated contact sensitivity (3). The neuroendocrinological mechanisms responsible for such stress-induced immunomodulation are still incompletely understood. i This study was supported by Grant MH45246 (M.L.) from the NIMH. ’ To whom correspondence should be addressed. 137 0090-1229190 $1.50 Copyright All rights

Q 1990 by Academic Press, Inc. of reproduction in any form reserved.

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The present study, unlike other studies utilizing artificial laboratory stressors, employs an experimental system which closely approximates stress as that encountered in nature in order to examine the effects of stress on a number of in vitro and in vivo measures of innate and adaptive immunity. Two strains of inbred mice were used in order to identify possible strain-specific differences in immune response to stress. These strains were chosen based on previously published reports which have documented differences in the neuroendocrinological responses to stress between the strains. Both the innate and adaptive components of the immune response were studied in the same set of experiments in order to provide a more complete examination of the possible effects of stress on immunity. MATERiALS

AND METHODS

Animals Six- to 7-week-old male inbred mice of the C57BW6J and DBA/2J strains were purchased from Jackson Laboratories, Bar Harbor, Maine. Six- to g-month-old male CF-1 retired breeders were obtained from Charles River Laboratories, Wilmington, Massachusetts. Upon receipt from the breeder facilities, mice were immediately placed under reversed day-night light cycle (dark: 6 AM-~ PM; light: 6 PM-~ AM). Animals were housed in Nalgene units complete with barrier filter tops at a density of five animals per cage (group housed) except for retired breeders which were housed individually. All animals were maintained in the animal facility for a minimum of 4 weeks before being employed in an experimental protocol. Food and water were available ad libitum. Two animal rooms were solely dedicated to the project in which one room served as the holding facility and the other (with a connecting door in between) served as the procedure room. Access to the animal rooms was severely restricted to only those individuals who were performing experiments that day. Cage and bedding changes were performed at least 1 day in advance of any planned experiment. Animals were solely handled by the tail with the use of long handled forceps. At no time were animals in contact with human skin. Individuals performing experiments were always gowned, gloved, and wore particle masks. Preliminary results strongly indicated that these precautions were essential for ensuring experimental reproducibility. Social Conflict Procedure Social conflict is defined as the agonistic encounters and aggressive interactions which occur between a territorial resident animal and an intruder animal (IO). As a part of the normal behavior repertoire of the mouse, these encounters, which do not usually result in significant harm to either participant, serve a variety of functions such as a means by which to disperse reproductively active males within the species (11, 12). In the laboratory the social conflict paradigm was carried out essentially as described by Teskey et al. (13) in which a group-housed male mouse, the intruder, was placed into the cage of a singly housed older male, the resident. Once placed into the cage of a resident, the intruder mouse displays a behavioral response consisting of an initial attempt to retaliate. After being en-

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gaged by the resident, the intruder then exhibits a number of defensive postures such as an upright position. Following repeated attacks by the resident, the intruder will assume a submissive or “defeat” posture which is characterized by an upright body posture, limp forepaws, upwardly angled head, and retracted ears (10-14). At this point the resident will disengage from the intruder. The intruder will not orient itself toward the movement of the resident and may remain in a defeat posture for as long as a minute. A single defeat session consisted of first introducing the intruder into a resident’s cage. Immediately following the assumption of a defeat posture the intruder was removed from the resident’s cage and placed into a second resident’s cage. The assumption of a defeat posture was usually more rapid in the second resident’s cage than the first. The intruder was then removed from the second resident’s cage and placed back into the first resident’s cage where a defeat posture for the third time was assumed. A single defeat session thus consisted of three individual defeats and usually took 2-4 min to perform. The time between individual defeat sessions on days in which multiple defeats were experienced was between 0.5 and 2 hr. Approximately 15-25 bites were received by the intruder on the flank and back regions until the appearance of the first defeat posture with approximately O-15 bites received on succeeding defeats for a cumulative total of 15-55 bites per defeat session. For all experiments two control groups consisting of a home cage control group and a handled control group were used. The handled control group consisted of individual mice which were removed from the group-housed setting and placed in a sequence of empty cages for a time approximately equal to that required for an intruder to achieve defeat when placed in a resident’s cage. Animals on chronic (>I day) stress protocols which were observed to develop abscessed wounds following social conflict, which was a very rare occurrence, were eliminated from the studies. All social conflict procedures were videotaped under dim red light using a black and white video camera capable of 0.5 lux sensitivity (Model WVBL200, Panasonic) coupled to a video cassette recorder equipped with a built-in time and date generator (Model AG-1050, Panasonic). All tapes were reviewed by an independent investigator following each experiment to assure consistency of interpretation of behavioral responses, i.e., assumption of the defeat posture. Media and Chemicals

Roswell Park Memorial Institute 1640 medium (RPMI) supplemented with 1% L-glutamine was purchased from MediaTech (Washington, DC). All RPM1 containing medium was supplemented with 25 mM Hepes and 0.005% gentamicin sulfate. Virus and mycoplasma negative fetal bovine serum (FBS) was purchased from GIBCO Laboratories (Long Island, NY). Concanavalin A (Con A) and lipopolysaccharide W (LPS, Escherichia coli 055:B5) were purchased from Difco Laboratories (Detroit, MI). Polyvinylpyrrolidone (PVP) of 360,000 molecular weight, luminol, and zymosan were obtained from Sigma Chemical (St. Louis, MO). Keyhole limpet hemocyanin (KLH) was obtained from Calbiochem (San Diego, CA). Phenol red-free Hanks buffered salt solution (HBSS) was prepared from powdered media obtained from GIBCO. NaCl (0.85% NaCl in distilled wa-

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ter) and phosphate-buffered saline (PBS) were prepared in the laboratory. Opsonized zymosan particles were prepared by incubating zymosan in mouse serum at a concentration of 20 mg zymosamml of serum at 37°C for 30 min. Opsonized zymosan was then spun down at 3000g for 8 min, the supernatant was discarded, and the pellet was resuspended in HBSS. The opsonized zymosan was washed twice more and resuspended to a final volume of 4 ml of HBSS and either used immediately or stored at -20°C until use. A stock 2 X low3 M luminol solution was prepared in dimethyl s&oxide. Immunization

Protocols

All immunizations were given as 0.2-ml intraperitoneal injections at times indicated in figure legends. KLH was dissolved in 0.85% NaCl at a concentration of 20 p,g KLH/ml and sterilized by passage through a sterile 0.45p,rn filter, PVP was dissolved in 0.85% NaCl at a concentration of 50 p,g PVP/ml and sterilized by passage through a sterile 0.45pm filter. Enzyme-Linked

Zmmunosorbent

Assays

(ELISA)

ELISA for the detection of KLH-specific and PVP-specific IgM were performed using a biotin-streptavidin detection system modified from Smith and Hayes (15). In brief, flat-bottomed 96-well ELISA microtiter plates (Corning, Corning, NY) were coated overnight with 0.1 mg of either KLH or PVP/ml of PBS. Plates were extensively washed with PBS containing 5% (w/v) of Tween 20 and unoccupied sites were blocked with 5% Carnation nonfat dry milk. Doubling dilutions of mouse serum were added into duplicate wells and incubated at room temperature for 3 hr. Plates were extensively washed and biotinylated goat antimouse IgM (p-chain specific, 0.5 mg/ml, Southern Biotechnology, Birmingham, AL) was used at a 15000 dilution. Plates were incubated for 2.5 hr at room temperature, washed, and peroxidase-labeled streptavidin at a 1: 1000 dilution (0.5 mg/ml, Southern Biotechnology) was added. Following a 1S-hr incubation, plates were washed and a commercially prepared one-step color development reagent. ABTS peroxidase substrate (Kirkegaard and Perry Laboratories, Gaithersburg, MD), was added and the resulting color reaction was read using a Bio-Tek Model 312 kinetic reader (Bio-Tek Instruments, Winooski, VT). Results are presented as optical density (OD) at 405nm wavelength. Tissue Preparation

Groups were sacrificed by cervical dislocation in the following order immediately after completion of the last defeat session: home cage control, handled control, and defeat. The amount of elapsed time from completion of the last defeat session to removal of tissue from the last member of the defeat group was usually 10-25 min. Spleens were removed using aseptic technique and placed into polystyrene tubes containing 10 ml of cold RPMI. Results represent data derived from individual mice. A single cell suspension of spleen cells was prepared by gently pressing the spleen between the frosted ends of two microscope slides, The cell suspension was allowed to settle in a 15ml conical tube and any large particulate matter was removed from the bottom of the tube. All tubes were then spun down

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at 8OOg for 8 min and the supernatant was discarded. Cells were resuspended with 5 ml of cold RPM1 + 10% FBS and then counted in a Coulter counter (Model ZBi). Since chemiluminescence assays were performed using both red blood cell (rbc) lysed and unlysed specimens, both red and white blood cell counts were made. For lymphocyte mitogen studies the leukocytes were adjusted to a concentration of 5 x 106/ml of RPM1 + 10% FBS. For chemiluminescence experiments, the splenocytes in RPM1 + 10% FBS containing medium were centrifuged as described above and resuspended at 1 x 10’ leukocytes/ml of HBSS. Additionally, for chemiluminescence studies, an aliquot of splenocytes was subjected to hypotonic lysis by the addition of 0.9 ml of DDW to a packed cell pellet; 0.1 ml of 10X HBSS was then added and the tube was respun, the supernatant was discarded, and the cells were recounted and then resuspended at a concentration of 1 x IO’ leukocytes/ml in HBSS. Cytospin preparations of between 1 x lo4 and 1 x lo5 spleen cells were prepared using a Shandon Cytospin II cytocentrifuge for those experiments in which chemiluminescence was determined. Duplicate slides were stained for nonspecific esterase (16) and Wright’s stain. Two hundred cells were enumerated for esterase positive and negative cells and Wright’s staining of leukocytes. Serum was obtained by cardiac puncture from mice anesthetized by the use of Metafane (methoxyflurane, Pittman-Moore, Washington-Crossing, NJ). Mitogenic

Stimulation

One hundred microliters of splenocyte suspensions (5 x lo5 cells) in RPM1 + 10% FBS was added to wells of 96-well flat-bottomed microtiter plates (Nunc, Naperville, IL) containing 0.1 pg Con A, 0.5 pg Con A, or 10 p,g LPS per culture well in 100 ~1 of RPMI. Cells were harvested at 48 hr following a 5-hr pulse with 1 l&i of [3H]thymidine (6.7 G/r&Q. The average incorporation of [3H]thymidine was determined as disintegrations per minute (dpm) using a Packard 1600CA scintillation counter. Chemiluminescence

Assay

Luminol-dependent chemiluminescence resulting from the phagocytosis of opsonized zymosan particles was performed essentially as described by Easmon et al. (17). In brief, 0.3 ml of spleen cells containing 3 x lo6 leukocytes was added to a 37°C preheated polypropylene tube containing 0.6 ml of 5 x 10e5 M freshly prepared luminol in HBSS. All chemiluminescence measurements utilized an LKB 125 1 luminometer capable of 37°C continuous processing of 25 samples controlled by the LKB Phagocytosis program running on a Epson Equity I+ computer. Following a lo-min preincubation period to allow for temperature equilibration, 0.2 ml of 37°C preheated opsonized zymosan particles was added. Chemiluminescence results are presented in millivolts. Statistical

Analysis

One-way analysis of variance (ANOVA) and post-hoc analysis were performed using the SYSTAT statistical package (Systat, Evanston, IL).

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RESULTS Generation of Anti-KLH

and Anti-PVP

Primary Responses

KLH. The immunization of DBA/2J mice with the T-dependent antigen KLH (Fig. 1) resulted in significant production of antigen-specific IgM. Mice subjected to one defeat session per day for 3 days (DEF 1 x 3) or one defeat session per day for 5 days (DEF 1 x 5) experienced significant suppression of anti-KLH formation. It should be noted that in the acute stress group, DEF 1 x 1, there was no significant alteration in the primary antibody response to KLH. Similar results were obtained with C57BL/6J mice. Further increases in the number of social conflict sessions per day as well as total days stressed, for both strains, resulted in an even greater magnitude of suppression of the primary antibody-forming response (data not shown). PVP. The immunization of DBA/2J and C57BL/6J mice with the T-independent antigen PVP resulted in significant production of antigen-specific IgM. However, in contrast with the results obtained with KLH immunization (Fig. l), social conflict stress was not observed in either strain to have any significant effect on the primary antibody response (data not shown). Mitogenesis Assays

As shown in Fig. 2 the mitogen-induced proliferative responses of splenocytes from defeated DBA/2J mice did not differ significantly from control. These results represent 4 representative experiments performed for the DBA/2J strain. Two concentrations of Con A representing a suboptimal (1 &ml) and an optimal (5 l&ml) proliferative response were used. Due to the relatively flat dose-response curve for LPS, only one concentration of LPS was used. Similar results were 0.800

-

?

0.600

-.

z V

0.400

--

0.200

--

d 6

0.000 1

-I-

** 1 I1IL

-+CON

:

HC

DEF 1x1

DEF 1 x3

DEF 1x5

GROUPS 1. KLH-specific IgM responses in home cage control (CON), handled control (HC), and defeated (DEF) DBA/2J mice. Mice were- defeated prior to KLH immunization (Day 1) for one session on Day 1 (DEF 1 x l), one session per day on Days - 1 through 1 (DEF 1 x 3), or one session per day on Days - 3 through 1 (DEF 1 x 5). HC group was handled each day for Days - 3 through 1 prior to immunization and all mice were sacrificed on Day 7 postimmunixation and serum was assayed for KLH-specific IgM as described under Materials and Methods. Each bar represents mean OD 2 SEM for five mice. Asterisks denote P G 0.05. Data are representative of four experiments. FIG.

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300 -250 -. 200 -. 150-w loo-. 50..

NONE

CON A Amo;m

CON A

LPS 100

(/q5,mi)

2. Splenocyte proliferative responses to the nonspecific T cell mitogen Con A and the nonspecific B cell mitogen LPS in defeated DBA/2J mice as compared to control mice. Concentration of mitogens used, as well as values for background unstimulated cultures (NONE), are as shown. Defeated mice were defeated for I to 3 sessions per day for 5 to 7 days which had been shown to affect antibody production to the T-dependent antigen KLH, but not the T-independent antigen, PVP. Four representative experiments are shown. Each point represents data from an individual experiment employing five mice per group. [3H]Thymidine incorporation values for unstimulated cultures averaged I-7000 dpm and for stimulated cultures averaged 50-300,000 dpm. Data from each experiment are depicted by a different symbol. FIG.

obtained for the C57BL/6J strain (data not shown). While individual experiments did at times show significant differences between groups, this effect was not reproducible from experiment to experiment (Fig. 2). Analysis of 19 experiments involving 365 mice of the C57BL/6J and DBA/2J strains revealed that no reproducible significant changes in mitogen-stimulated proliferation could be detected. It should be noted that every effort was made to control inherent variability in the assay system. For example, the same lots of Con A and LPS, as well as fetal bovine serum, were used throughout all experiments. Phagocytosis Phagocytic ability of splenic leukocytes was evaluated by luminometry (see Materials and Methods). As shown in Figs. 3 and 4, splenocytes rapidly phagocytize opsonized zymosan particles with a maximal rate of phagocytosis occurring within 10 min. As little as one defeat session resulted in significant increases in phagocytosis when compared to control groups. Figures 4A and 4B, which each represent data from five experiments, show that significant increases of up to 520% of control and handled control values were observed in splenocytes from defeated animals. The greatest increase in the phagocytic capability of splenocytes from defeated animals compared to’ controls was observed in C57BL16J (Fig. 4B) which showed a 412% average increase as compared to a 269% average increase in DBA/2J (Fig. 4A). Since Figs. 3 and 4 present data that were obtained over a 25- to 40-min time course, only the rate of phagocytosis, and not the time at which the phagocytosis occurred, differed between defeated and control

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THOMPSON C57BL,‘6J

DBA/2J zo-

A

A-A o-o ===CON HC O-0 OEF I *:

z+c a--a = CON o-o O-0==HC OEF 1x1

0

5

10

15

20

25

30

35

40

0

5

10

TIME (minutes)

15 20 25 TIME (minutes)

30

35

40

FIG. 3. (A) Phagocytosis of opsonized zymosan particles by splenocytes from home cage control (CON, A), handled control (HC, O), and defeated I x 1 (DEF 1 X 1, q ) DBA/ZJ mice. Each point represents mean -t SEM for five mice. Asterisks denote P s 0.05. (B) Phagocytosis of opsonized zymosan particles by splenocytes from home cage control (CON, A), handled control (HC, 0), and defeated 1 x 1 (DEF 1 x 1, 0) C57BU6J mice. Each point represents mean 5 SEM for five mice. Asterisks denote P s 0.05.

groups. This strain-specific difference in immune responsiveness to stress is discussed in the Discussion section in terms of possible neuroendocrine mechanisms. Microscopic examination of duplicate slides stained for nonspecific esterase and Wright’s stain as described under Materials and Methods revealed no significant differences between defeated and control groups in the percentages of esterase positive and negative cells and the percentage classes of Wright-stained cells (data not shown). Although stress-induced trafficking and redistribution of cells must still be considered, the ability to effect large changes in phagocytic cell function in such a short time frame (~4 min of stress) suggests that the assay system was reporting results from cells that were already present in the organ site 400

A

DBA/2J o-o 0-0

d

E300 $0 I

C57BL,‘6J = HC = DEF I-5 x 1

* * * * I 1 i I ~--o-o--o~-o 1 I I I

a TIME

(minutes)

* 1 I

*

400 -300 -. 200 -loo--

o-o

T *

HC O-0 ==OEF,--5X1

i TIME

1

I

(minutes)

4. (A) Phagocytosis of opsonized zymosan particles by splenocytes from handled control (HC. 0) and defeated one to five sessions per day for 1 day (DEF l-5 X 1, 0) DBA/2J mice as compared to nonhandled home cage control mice (100% response). Each point represents data from five experiments employing five mice per group per experiment. Asterisks denote P s 0.05. (B) Phagocytosis of opsonized zymosan particles by splenocytes from handled control (WC, 0) and defeated one to five sessions per day for 1 day @EF l-5 x 1, 0) C57BL/6J mice as compared to nonhandled home cage control mice (100% response). Each point represents data from five experiments employing five mice per group per experiment. Asterisks denote P S 0.05. FIG.

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before the initiation of the stress procedure. Lysis of rbc by hypotonic lysis (see Materials and Methods) yielded results approximately the same as obtained with unlysed samples (data not shown). DISCUSSION

The results presented in this study demonstrate that the stress of social conflict can result in a significant differential modulation of the immune system. A significant suppression of anti-KLH formation was observed in two strains of inbred mice subjected to chronic (>l day), but not acute (
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concluded that the lack of reproducibility inherent in the use of the in vitro mitogenesis assay makes it a less than desirable assay with which to measure the effects of stress on immune functioning. Stress, per se, has been amply documented in rodents as to its immunosuppressive effects (for review see Refs. (1, 2)). The vast majority of reports employing rodents have used artificial laboratory stressors such as immobilization (3, 4), electric shock (6-g), and centrifugation (5). The present study, as well as a recent report by Fleshner et al. (20), has also found suppressive effects of social conflict stress on certain measures of adaptive immunity such as antigen-specific antibody formation. However, the data presented herein have demonstrated that social stress can also significantly enhance innate immunity. Figures 3 and 4 demonstrate that the stress of social contlict resulted in a highly signiIicant and consistently reproducible enhancement of phagocytic function in cells from the spleen. As shown in Fig. 4. splenocytes from defeated C57BU6J (Fig. 4B) mice displayed greater phagocytic activity than did splenocytes from defeated DBAQJ (Fig. 4A) mice. This result may be due to strain-specific differences in the neuroendocrine response to stress (21). The release of p-endorphin has been demonstrated to occur as a consequence of defeat (21, 22). The possible role for the regulation of immune responsiveness by @endorphin, as well as other opioid peptides, has recently been reviewed (23). For example, Deitch ef al. (24) have shown that p-endorphin can alter resting and zymoSan-stimulated oxygen consumption by neutrophils . The two- to four-fold enhancement of phagocytosis in the acute stress groups is important since a valid criticism of the social conflict paradigm is that the animal may be wounded during the induction of stress and that a subsequent infection may confound any immunological measurements. While this criticism may be relevant to experiments involving chronic exposure to social conflict, it seems an unlikely possibility to explain effects observed 2-4 min after the initiation of the encounter. Further, wounded animals were eliminated from the experiment. When the evolutionary implications of stress are considered, it may be argued that it is in the animal’s best interest not to respond to acute stress with a suppression of immune responsiveness. Such a suppression would undoubtedly render the animal more susceptible to the bacterial challenge that often occurs when an animal is wounded or may exacerbate an existing infection. Accordingly, such a line of reasoning would predict that stress should result in an initial enhancement of immune responsiveness, particularly those cells that constitute the first line of defense against infection. Repeated or prolonged challenge to the immune system would be more likely to be followed by a suppression of the immune system. It may therefore be argued that the results presented in this paper may support such a hypothesis. ACKNOWLEDGMENTS The authors gratefully acknowledge the skilled technical assistance of Sharon M. Ernst, Carol Paronis, Beniam Baissa, and Danie Zawadzka.

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review. Ann. Rech. Vet. 11,445-4?8,

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2. Ader, R., “Psychoneuroimmunology,” Academic Press, New York, 1981. 3. Belcha, F., Barry, R. A., and Kelley, K. W., Stress-induced alterations in delayed-type hypersensitivity to SRBC and contact sensitivity to DNFB in mice. Proc. Sot. Exp. Bio/. Med. 16, 239-246, 1982. 4. Steplewski, Z., and Vogel, W. H., Total leukocytes, T cell subpopulation and natural killer (NK) cell activity in rats exposed to restraint stress. Life Sci. 38, 2419-2427, 1986. 5. Gisler, R. H., Bussard, A. E., Mazie, J. C., and Hess, R., Hormonal regulation of the immune response. I. Induction of an immune response in vitro with lymphoid cells from mice exposed to acute systemic stress. Cell. Immunol. 2, 634-645, 1971. 6. Zalcman, S., Minkiewicz-Janda, A., Richter, M., and Anisman, H., Critical periods associated with stressor effects on antibody titers and on the plaque-forming cell response to sheep red blood cells. Brain Behav. Zmmun. 2, 2.54-266, 1988. 7. Lysle, D. T., Lyte, M., Fowler, H., and Rabin, B. S., Shock-induced modulation of lymphocyte reactivity: Suppression, habituation, and recovery. Life Sci. 41, 1805-1814, 1987. 8. Shavit, Y., Lewis, J. M., Terman, G. W., Gale, R. P., and Liebeskind, J. C., Opioid peptides mediate the suppressive effects of stress on natural killer cell cytotoxicity. Science 223, 188-190, 1984. 9. Monjan, A. A., and Collector, M. I., Stress-induced modulation of the immune response. Science 196, 307-308, 1977. IO. Miczek, K. A., and O’Donnell, J. M., Intruder-evoked aggression in isolated and nonisolated mice: Effects of psychomotor stimulants and I-dopa. Psychopharmacology 57, 47-55, 1978. 11. Brain, P. F., Differentiating types of attack and defense in rodents. In “Multidisciplinary Approaches to Aggression Research” (P. F. Brain and D. Benton, Eds.), pp. 53-78, Elsevier, Amsterdam, 1981. 12. Crowcroft, P., “Mice All Over,” Foulis, London, 1966. 13. Teskey, G. C., Kavaliers, M., and Hirst, M., Social conflict activates opioid analgesic and ingestive behaviors in male mice. Life Sci. 35, 303-315, 1984. 14. Miczek, K. A.. Thompson, M. L., and Shuster, L.. Opioid-like analgesia in defeated mice. Science 215, 1520-1522, 1982. 15. Smith, S. M., and Hayes, C. E., Contrasting impairments in IgM and IgG responses of vitamin A-deficient mice. Proc. Natl. Acad. Sci. USA 84, 5878-5882, 1987. 16. Li, C. Y., Lam, K. W., and Yam, L. T., Esterases in human leukocytes. J. Histochem. Cytochem. 21, l-12, 1973. 17. Easmon, C. S. F., Cole, P. J., Williams, A. J., and Hastings, M., The measurement of opsonic and phagocytic function by luminol-dependent chemiluminescence. Zmmunology 41, 67-74, 1980. 18. Beden, S. N., and Brain, P. F., Studies of the effect of social stress on measures of disease resistance in laboratory mice. Aggressive Behav. 8, 126129, 1982. 19. Maier, S. F., and Laudenslager, M. L., Inescapable shock, shock controllability, and mitogen stimulated lymphocyte proliferation. Brain Behav. Zmmun. 2, 87-91, 1988. 20. Fleshner, M., Laudenslager, M. L., Simons, L., and Maier, S. F., Reduced serum antibodies associated with social defeat in rats. Physiol. Behav. 45, 1183-l 187, 1989. 21. Siegfried, B., Frischknecht, H.-R., and Waser, P. G., Defeat, learned submissiveness and analgesia in mice: Effect of genotype. Behav. Neural Biol. 42, 91-97, 1984. 22. Miczek, K. A., and Thompson, M. L., Analgesia resulting from defeat in a social confrontation: The role of endogenous opioids in brain. In “Modulation of Sensorimotor Activity during Alterations in Behavioral States” (R. Bandler, Ed.), pp. 431-456, A. R. Liss, New York, 1984. 23. Fischer, E. G., Opioid peptides modulate immune functions: A review. Zmmunopharmacol. Zmmunotoxicol. 10, 26.5-326, 1988. 24. Deitch, E. A., Xu, D., and Bridges, R. M., Opioids modulate human neutrophil and lymphocyte function: Thermal injury alters plasma B-endorphin levels. Surgery 104, 41-18, 1988. Received December 18, 1989; accepted with revision May 24, 1990