Life Sciences, Vol. 41, pp. 1805-1814 Pergamon Journals Printed in the U.S.A.

SHOCK-INDUCED MODULAI[ON OF LYMPHOCYIE REACT[V[IY: SUPPRESSION, HABIIUAIION, AND RECOVERY

Donald I. Lysle*, Mark Lyte**, Harry Fowler ***, and Bruce S. Rabin**

Departments of *Psychiatry, **Pathology, and ~**Psychology, University of Pittsburgh, Pittsburgh, PA, USA

(Received in final form August ii, 1987)

Summary

The present study was designed to evaluate the suppressive effect of different frequencies of signaled-shock presentations on mitogenic reactivity of lymphocytes in Lewis rats, and to assess the recovery of that reactivity at varying times after the shocks. [he results showed that the magnitude of decreased reactivity in both the spleen and whole-blood lymphocytes, as determined by mitogenic reactivity to Concanavalin A (Con A), was directly related to the number of shock presentations within a daily session. However, the suppressed reactivity for the spleen cells diminished with repeated sessions of frequent shocks, in contrast to the whole-blood lymphocytes which did not show any habituation. Furthermore, the imposition of different periods of recovery following a single session of frequent shocks showed that the decreased reactivity for the whole-blood lymphocytes extended beyond the immediate period of the shock experience, and took 48 to 96 hours to recover completely. In contrast, the spleen lymphocytes showed complete recovery within 24 hours following the administration of shock. These results establish that the rate of habituation to and recovery from a shock-induced decrease in mitogen reactivity is more rapid For the spleen than whole-blood lymphocytes.

Recent research has indicated that exposure to aversive stimulation can appreciably alter immune function. For example, presentations of electric shock to rats have been shown to decrease the responsiveness of lymphocytes to stimulation by plant lectins (I), decrease natural killer cell activity (2), and increase susceptibility to tumor challenge (),4). Although all such reports agree that electric shock can suppress immune function, there have been few systematic evaluations of the parameters of the shock experience and the compartments of the immune system that are affected. Keller and colleagues (1,5) showed that intense shocks induce a greater reduction in the responsiveness of whole-blood and isolated lymphocytes than do shocks of a weaker intensity. However, their findings indicated that splenic lymphocytes were not affected even by the presentation of strong shocks (5). While these results demonstrate that the immune system is sensitive to variations in aversive stimulation, they also indicate that different compartments of the immune system are differentially affected by that stimulation.

Correspondence and requests for reprints should be addressed to Donald T. Lysle, Ph.D., Division of Clinical Immunopathology, Department of Pathology, Room 5725, One Children's Place, Pittsburgh, PA 15213-3417.

0024-3205/87 $3.00 + .00 Copyright (c) 1987 Pergamon Journals Ltd.

1806 Shock-lnduced Immunomodulation Vol. 41, No. 15, 1987

Studies investigating the relationship between aversive stimulation and immune function have also been restricted by their focus on the immune response immediately following presentation of the aversive event. To understand the relationship between aversive stimulation and disease susceptibility, it is important to consider the recovery of immune Function following the aversive experience. Several investigations with human subjects have examined the recovery of immune function following the immunosuppression induced by conjugal bereavement (e.g., 6). However, in the case of bereavement, the temporal characteristics of the stressful situation are not well defined, and therefore it is difficult to determine the onset of recovery and/or termination of bereavement. Only one study, which used rats as experimental subjects, has purportedly examined the recovery of lymphocyte reactivity following exposure to electric shock (7). In that investigation, different groups of rats were initially given electric-shock exposure for 14, 20, or 28 hours per week for a 6-month period, and then immune function was assessed one month after cessation of the shock experience. The results showed that the decrease in lymphocyte responsiveness to mitogenic stimulation was more pronounced with longer shock exposure. However, the findings provided no evidence of any recovery of immune function following exposure since there was no evaluation of the immediate effects of the shock. Thus, there is presently no systematic investigation of the recovery of immune function following aversive stimulation.

Given the lack of systematic research, the present study was designed to evaluate the effect of different frequencies of aversive stimulation on immune function, as indexed by mitogen reactivity, and to assess the recovery of that function at varying times following the aversive experience. The first manipulation examined the effect of the number of electric shock sessions, as well as the number of shocks administered within a session, lhe response of both whole-blood and splenic lymphocytes to Con A was assessed to provide a broader index of immunomodulation, lhe second manipulation evaluated the recovery of responsiveness of both blood and spleen Iymphoeytes at different times following a single session of extended shock presentations.

Methods

Animals. Male rats of the Lewis strain, 65 days old and 250-300 grams in weight, were purchased from Charles-River Laboratories. Upon arrival, the subjects were individually caged in a colony room where a reversed day-night (12-hour) cycle was maintained through artificial illumination. They received free access to both food (Wayne Lab Blox) and water throughout the experiment, and a two-week acclimation period prior to the experimental manipulations.

Shock Apparatus. Four identical rodent chambers (Coulbourn Instruments Model EI0-I0), measuring 25 x 30 x 37 cm, served as the shock apparatuses. The chambers had clear Plexiglas side wails, sheet-metal top and end walls, and a grid floor consisting of bars 0.24 cm in diameter, spaced 0.87 cm apart. An 8.5-cm, 3.5-ohm speaker (Quam model 3A05), externally mounted on the wall of each chamber, was connected to an audio generator (BRS/LVE Model AU-902) to provide an auditory signal: an 78-dB (re .0002 dyne/cm 2) clicking sound that pulsed at the rate of 8 clicks/s. [he grid floor of each chamber was connected through timer circuitry to the output of a shock generator and scrambler (8RS/LVE Models 903 and SC 902) to provide a 5.0-second, 1.6-mA, footshock. [he chambers were individually housed in identical sound-attenuating cubicles, 50 x 60 x 88 cm, that were located in a room adjacent to the programming equipment. A tOO-W, 120-V bulb, recessed behind a frosted plate in the ceiling of each cubicle, was operated at 85 V, ac, to provide diffuse illumination of the chamber. An ambient sound level of 72 dB was provided by operating the cubicle's ventilating fan at 57 V, ac.

Vol. 41, No. 15, 1987 Shock-lnduced In~unomodulatlon 1807

Shock Manipulation. lhe experimental subjects were assigned to nine groups (n : 4) of a 3 x 3 factorial design that involved 4, 8, or 16 signaled shocks per session for I, ), or 5 daily sessions. Each daily session began one hour into the dark phase of the day-night cycle. For all groups, the shocks were delivered on a variable basis, every 2-6 minutes, with the time between shocks averaging 4 minutes. Each shock was always preceded by the 15-second clicker signal. Control subjects remained undisturbed in their home cages.

Immediately following the last shock experience, each experimental subject was rapidly sacrificed by cervical dislocation with a clamp, lhe ~mimal was placed on its back and sprayed with alcohol, and then a mid-abdominal incision was made to expose the abdominal aorta. Blood was collected into heparinized syringes through 21 gauge needles, lhe spleens were removed and placed in polypropylene tubes containing 7 ml of RPMI-1640 tissue culture medium that was supplemented with 10 mM hopes, 2 mM glutamine, 50 ug gentamicin/ml, lhe control subjects were removed for their home cages and sacrificed in the same manner and at the same time as the experimental subjects. A group of control subjects (n = 4) was sacrificed with each of the two sets of experimental groups that received 4 and 8 shocks per session. A separate control group was sacrificed with each of the three experimental groups that received 16 shocks per session. Ihat procedure enabled a group of control subjects to be sacrificed and assayed under the same conditions (e.g., time and culture) as the experimental subjects.

Mitogen Assay. A mitogenic stimulation assay was performed with both the whole-blood and spleen lymphocytes, using the non-specific, I-cell mitogen, Con A. A single suspension of spleen cells was prepared by gently pressing the spleens between the ends of frosted microscope slides. The leukocytes were counted in a Coulter counter (Model ZBI) and their concentration was adjusted to 5 x 106 cells/ml with supplemented RPMI-1640 without further washing. Con A (Difco) was prepared in supplemented RPMI-1640 at a concentration of 5.0 ug/ml, and 100 ul was added in triplicate to the wells of a 96-well, flat-bottom, m icrotiter plate (Costar #3596). Preliminary investigations showed that a Con A concentration of 5.0 ug/ml provided optimal lymphocyte stimulation. To background culture wells, 100 ul of supplemented RPMI-1640 was added. Both Con A stimulated and background wells were supplemented with 10 ul of fetal calf serum, lhen, 100 ul of the adjusted spleen-cell suspension was added to each well and the plates were incubated at 37 C in a humidified incubator with 5 percent CO 2. [he cultures were pulsed with I uCi ~H-thymidine in 50 ul of RPMI-1640 during the last 5 hours of a 48 hour incubation, the Con A stimulated and background cultures were harvested onto glass filter paper and the incorporation of 3H-thymidine was determined with a liquid scintillation counter (Packard Model 1500).

A standard whole-blood assay was used. lhe blood was diluted 1:10 with supplemented RPMI-1640, and leukocyte counts were determined using a Coulter counter, then, 100 ul of the diluted blood was added in triplicate to the wells of a 96-well, flat-bottom, microtiter plate (Costar #3596) containing 100 ul of the prepared Con A (5.0 ug/ml) or wells with just supplemented RPMI-1640. the incubation and harvest conditions were the same as those used for the spleen-cell assay, except the total incubation time was extended to 96 hours. Wright stained peripheral blood ~nears were made for those animals that received 16 shocks for 5 sessions and their control animals. A two hundred count cell differential was performed on those specimens.

Statistical Ireatment for Shock Manipulation. A one-way analysis of variance was used to assess differences among control groups. The leukocyte counts for both the blood- and spleen-cell suspensions were analyzed. [he means of the triplicate scintillation counts for the background and Con A stimulated wells were analyzed for both the blood and spleen. The percentage of lymphocytes determined from the cell differentials were compared by a t

1808 Shock-induced Immunomodulation Vol. 41, No. 15, 1987

test. To afford comparisons among the experimental groups that were unaffected

by variations in assay conditions, the leukocyte counts and the means of the triplicate background and Con A stimulated scintillation counts for each experimental subject were transformed into percentage scores based on the means for its respective control group, lhe percentage scores for the Coulter counts and scintillation counts were then separately analyzed in the form of a 3 x 3 factorial design consisting of 4, 8, or 16 signaled-shock presentations per session, and I, 3, or 5 sessions. Significance level was set at a probability of less than or equal to 0.05.

Recovery Manipulation. Other experimental subjects were assigned to three groups (n = 4) that received one session of exposure to 16 signaled shocks, followed by a recovery period in their home cages of either 24, 48, or 96 hours. The shocks were presented in the same manner as described above. The sessions were scheduled such that all groups were sacrificed at the same time, immediately after their respective recovery periods. A control group of subjects (n : 4) remained undisturbed in their cages, prior to being sacrificed at the same time as the experimental groups. A mitogen assay was performed with both the whole-blood and spleen lymphocytes, as described above.

Statistical Ireatment for Recover 7 Manipulation. For the experimental groups, the leukocyte counts and the mean of the triplicate background and Con A stimulated scintillation counts for both the blood and spleen were transformed into percentage scores based on the means of the control group. That afforded a camparison of spleen and blood effects. Thus, the percentage scores were analyzed in the form of a 3 x 2 factorial design consisting of 24, 48, or 96 hours of recovery, and whole-blood or spleen lymphocytes in assay. Significance level was set at a probability of less than or equal to 0.05.

Results

Shock Manipulation. Analyses of the blood and spleen leukocyte counts for the home-cage control groups showed no significant differences among the groups; the leukocyte counts for the blood and spleen were 7.3 x I06/mi and 45.9 x I06/mi, respectively. The scintillation counts for the home-cage controls showed excellent mitogenic stimulation of both the blood and spleen lymphocytes; the mean counts per minute for the blood and spleen were 94,445 and 304,789, respectively, lhe home-cage control subjects showed relatively low scintillation counts per minute for the unstimulated background cultures; the means for the blood and spleen were 109 and 9416, respectively. Analyses showed no significant differences among the home-cage control groups on either the background scintillation counts or the Con A stimulated scintillation coun ts .

Analyses of the percentage scores of the leukocyte counts for the experimental groups showed no significant main effects or interactions for either the blood- or spleen-cell counts; the mean percentage for the blood and spleen was 106 and 90, respectively. Thus, the different shock experiences did not differentially alter the percentage of leukocytes present in the whole blood or the spleen. The analyses of the percentage scores of background scintillation counts did not show any significant main effects or interactions for the blood or spleen scores.

Figure I displays the mean percentage scores of the experimental groups for the scintillation counts from the Con A stimulated wells in the whole blood assay. Analysis of those scores showed a significant effect of the number of shocks presented within a session, ~(2,27) = 15.51, p < .001. Orthogonal contrasts indicated that there was no difference between subjects receiving 8 or 16 shocks per session, ~ < I, but the level of stimulation for those animals was significantly suppressed by comparison with animals

Vol. 41, No. 15, 1987 Shock-induced Immunomodulation 1809

receiving 4 shocks per session, L (1,27) = 30.21, p < .001. There was no significant effect of the number of shock sessions, nor a significant interaction between the number of shocks per session and the number of sessions, Fs < I. Accordingly, the present resuits indicate that mitogenic reactivity--by the whoIe-bIood lymphocytes decreases with the number of shocks per session, but is unaffected by the number of shock sessions.

110 BLOOD

100

9O u J ¢ / )

i 80 70 60

o o 5 0 u .

o 40

2O

10

O

I I i l

1 3 5 SHOCK SESSIONS

o 4 SHOCKS/SESSION

• e SHOCKS/SESSION

• 1@ SHOCKS/SESSION

FIC. I

Percent of control response in the whole-blood assay as a function of the number of shocks per session and the number of sessions.

The above conclusions on the effect of the number of shocks per session can be challenged because that veriabIe was confounded with the length of the shock session. To clarify the issue, we conducted a separate experiment involving four groups of subjects (n = 4). lwo groups were given 4 or 16 shocks, as described for the previous study. However, a third group received 4 shocks over the course of a session identical in length to that of the 16-shock subjects. The fourth group received 4 shock presentations, as described for the previous study, but remained in the shock chambers for the same period of time as the 16-shock subjects, lhe results showed no significant differences among the 4-shock groups in mitogenic reactivity of the whole-blood Iymphocytes to Con A, Fs < I. However, the 16-shock group showed significantIy reduced mitogenic reactivity by comparison with the 4-shock groups, ~(1,12) = 5.96, p < .05. Hence, the critical variable is the number of shocks and not mereIy the session length or the density of shock presentations within a session.

]he results of the leukocyte differential showed a 10% decrease in the lymphocyte percentage, t(6) = 5.49, p < .05. That finding is consistent with the work of other investigators (I). However, it seems unlikely that a greater than 90 percent reduction in mitogenic reactivity would be due to such a slight decrement in total lymphocytes, unless there was a selective loss of a subpopulation of immunocompetent cells. On that matter, our preliminary

1810 Shock-induced Immunomodulation Vol. 41, No. 15, 1987

results, using monoclonal antibodies, have not shown any differences in the number of T-helper and T-suppressor cells for shocked and nonshocked subjects.

Figure 2 displays the mean percentage scores of the experimental groups for the scintillation counts from the Con A stimulated wells in the spleen-cell assay. Analysis of those scores also showed a significant effect of the number of shocks per session, ~(Z,27) = 23.17, p < .001. Again, orthogonal contrasts indicated that there was no difference between subjects receiving 8 or 16 shocks per session, F(1,27) = 1.56, p < .05, but the level of mitogenic reactivity for those a~mals was significantly suppressed relative to animals receiving 4 shocks per session, ~(1,27) = 45.08, p < .001. In addition, the analysis showed a significant attenuating effect of the number of shock sessions, ~ (2 ,27) = 19.79, p < .001. 0rthogonal contrasts revealed a significant decrement across sessions, linear F(1,27) = 38.01, p < .001, and a nonsignificant quadratic trend, ~(1,27)--= 1.56, p > .05, indicating that the suppressive effect induced by the electric shocks decreased progressively as the number of shock sessions increased. The F-test results also showed a significant interaction between the number of shocks pec session and the number of sessions, ~(4,27) = 9.56, p < .001. As indicated in Figure 2 and confirmed by an orthogonal contrast, the attenuation of suppression across sessions occurred for subjects receiving 8 or 16 shocks per session, as those receiving 4 shocks per session failed to show any suppression, ~(1,27) = 25.61, p < .05. Furthermore, the attenuation was more rapid for subjects receiving 8 than 16 shocks per session, as indicated by a significant quadratic interaction effect, F(1,27) = 9.61, p < .05.

110

100

9O w

i 80 ,.,- 70

60

4O

3O

20

10

SPLEEN

T

t O _ _ ~ j I J

1 3 5

0 4 SHOCKS/SESSION

• 8 SHOCKS~SESSION

• 16 SHOCKS/SESSION

SHOCK SESSIONS

FIG. 2

Percent of control response in the spleen assay as a function of the number of shocks per session and the number of sessions.

Thus, the results of the spleen assay also indicate that the decrease in mitogenic responsiveness is a function of the number of shocks within a session. However, that conclusion can be challenged as well by the fact that

Vol. 41, No. 15, 1987 Shock-induced Immunomodulation 1811

session length was confounded with the number of shocks per session. Therefore, in the separate experiment described above, the spleen cells were also subjected to a mitogen stimulation assay. The results showed no significant differences among the 4-shock groups in mitogenic reactivity of the spleen lymphocytes to Con A, ~s < 1. However, the 16-shock group showed significantly reduced mitogenic reactivity by comparison with the 4-shock groups, ~(1,12) = 18.11, p < .05. Hence, the variable determining the amount of decreased mitogenic responsiveness in the initial shock session is the number of shocks and not the session length or the density of shock presentations.

Recovery Manipulation. Analysis of the percentage scores for the leukocyte counts showed no significant main effects or interactions. Thus, the different recovery periods did not result in changes in the percentages of leukocytes in the whole blood or the spleen. The scintillation counts for the control group showed excellent mitogenic reactivity for both the blood and spleen lymphocytes; the mean counts per minute for the blood and spleen were 96,718 and 407,)60, respectively. Also, the control group showed relatively low scintillation counts per minute for the unstimulated background cultures; the means for the blood and spleen were 71 and 12,585, respectively. Analysis of the percentage scores of background scintillation counts did not show any significant main effects or interactions.

110

100

90

80

7O

8 80

40

30

20

10

0 I I I

24 48 96 HOURS RECOVERY

o BLOOD

FIG. }

Percent recovery of control response in the blood and spleen assays as a function of hours of recovery after a single shock session.

Figure 3 displays the percent of control responding shown by the experimental groups on both the spleen and whole-blood measures after 24, 48, and 96 hours of recovery following a single 16-shock session. The scores are based on the scintillation counts from the Con A stimulated wells. As indicated, there was complete recovery of the spleen lymphocytes within 24

1812 Shock-induced Immunomodulation Vol. 41, No. 15, 1987

hours of the shock session, whereas the blood lymphocytes did not recover fuliy until sometime between 48 and 96 hours. These different rates of recovery produced a significant overall difference between the spleen and blood, ~(1,18) = 15.55, p < .001, as well as a significant effect of hours of recovery, F(2,18) = 4.82, p < .05, and a significant interaction of the two factors, F_~2,18) = 10.99, p < .001. Attesting to the changes observed in the blood cells, orthogonal contrasts showed that there was a significant rise in recovery between 48 and 96 hours after the shock session, F(1,18) = 9.38, p < .05, and that the differences between the spleen and b-lood occurred onIy during the first 48 hours of recovery, ~(1,18) = 21.94, p < .005; residuai Fs < I. These results suggest that the decreased mitogenic reactivity induced~y electric shock is much ionger iasting for the whole-blood lymphocytes than for the spleen lymphocytes, but that both effects dissipate completely given a sufficient recovery period.

Discussion

the present results establish that the magnitude of decreased mitogenic reactivity induced by exposure to electric shock is positively related to the number of shock presentations within a daily session. That effect was not due to a greater density of shocks, but rather a greater frequency, &~ all groups experienced the same intershock intervals. Furthermore, the effect was not influenced by the length of the session. A separate experiment equating the length of the session but varying the number of shock presentations showed the same e f f e c t .

Desp i t e comparably pronounced suppression o f mi togenic responsiveness i n both the blood and spleen lymphocytes f o l l o w i n g a s i n g l e sess ion w i t h many shocks, the e f f e c t for the spleen c e l l s d iminished w i th repeated sessions, in evidence o f an hab i tua t i on e f f e c t , in c o n t r a s t , the s u p p r e s s i v e e f f e c t fo r the w h o l e - b l o o d l ymphocy tes d id not show any h a b i t u a t i o n over repeated sessions; i f anyth ing, i t tended to increase.

/he more p e r v a s i v e e f f e c t f o r the who le -b l ood l ymphocy tes was a lso apparent i n the r e s u l t s o f the recovery m a n i p u l a t i o n . ]hose r e s u l t s showed t h a t the supp ress i on o f m i t ogen i c r e a c t i v i t y induced by a s ing le session o f many shocks extended we l l beyond the immediate per iod o f the shock e x p e r i e n c e for the blood c e l l s , but not the spleen c e l l s . Whereas the spleen lymphocytes recovered f u l l y w i t h i n 24 hours o f the shock exper ience, the blood lymphocytes were s t i l l a p p r e c i a b l y suppressed 48 hours f o l l o w i n g the shock sess i on . Nonetheless, l i k e the spleen lymphocytes, the blood lymphocytes also r e t u r n e d to normal r espons i veness w i t h a recove ry period sometime between 48 and 96 hours. These r e s u l t s are important in showing tha t d i f f e r e n t compartments o f the immune system, a l t h o u g h d i f f e r e n t i a l l y s u s c e p t i b l e to a v e r s i v e s t imu la t i on , are capable o f recover ing f u l l y from tha t s t i m u l a t i o n . Thus, the present r e s u l t s are cons is ten t w i th the e a r l i e r noted f i nd ings on the recovery from conjugal bereavement (6 ) .

Con A i s a non -spec i f i c l - c e l l mitogen that s t imu la tes both l - h e l p e r and [ -suppressor c e l l popu la t ions . I t i s c o n t r o v e r s i a l whether t h a t s t i m u l a t i o n p r e d o m i n a n t l y a f f e c t s / - h e l p e r or l - s u p p r e s s o r c e l l s . [he s t i m u l a t i o n o f l - s u p p r e s s o r c e l l s by Con A cou ld r e s u l t i n a supp ress i on o f m i t o g e n i c r e s p o n s i v e n e s s . In t h a t even t , the supp ress ion o f m i t o g e n i c r e a c t i v i t y induced by e l e c t r i c shock would r e f l e c t an enhancement o f immune f u n c t i o n . However, we be l ieve tha t the present f i nd ings i nd i ca te a suppression of immune func t ion when they are taken in con junc t ion w i th other f i n d i n g s showing t h a t e l e c t r i c shock produces a supp ress ion o f tumor r e j e c t i o n (3,4) and na tu ra l k i l l e r c e l l a c t i v i t y (2 ) .

[he mechanism under ly ing the suppression o f mi togenic r e a c t i v i t y induced by e l e c t r i c shock was not the focus o f the p resen t r esea rch . However, some i n v e s t i g a t o r s (8) have reported tha t lymphocytes possess receptors for opiate molecules, and others have ind ica ted a r e l a t i o n s h i p between immunosuppression and endogenous op iate a c t i v i t y . For example, Shavi t and col leagues (2) found

Vol. 41, No. 15, 1987 Shock-induced Immunomodulation 1813

that shock conditions producing opioid analgesia also produced immunosuppression, whereas shock conditions that produced nonopioid analgesia did not produce immunosuppression. In that Iight, it is important to note that the parameters of the signaled shock experiences used in the present research are comparable to those reported as producing opioid analgesia in the rat (Lysle & Fowler, submitted for publication).

Other research has indicated a possible relation between immunomodulation and the animal's lack of control of an aversive event. For example, Laudenslager and coileagues (9) have suggested that shock-induced suppression of lymphocyte responsiveness is related to the organism's perception of the shocks as uncontrollable, lhe present resuIts challenge the sufficiency of that relation because the decrease in mitogenic responsiveness observed in the present study occurred after a relativeIy brief experience with the shock. It is well documented that Iearning about the uncontroilability of an aversive event requires relatively extensive experience with the event, lypically, for the rat, the deveIopment of that form of learning involves 60 to 80 presentations of electric shock (10). lhe present research indicates not only that decceased mitogenic responsiveness occurs with relatively few shock experiences, but also that the suppressive effect can diminish as the number of inescapable shock sessions is increased.

A currently popular view is that peripheral blood lymphocytes traffic between the blood and spleen. In that respect, the development of an habituation or tolerance effect for the spleen lymphocytes, but not the blood lymphocytes, was unexpected, lhe reason for a restriction of the habituation process to the splenic lymphocytes is not readily apparent, given that the phenomenon of habituation is common to both physiological and behavioral response systems (11). Furthermore, the relationship between the differential habituation and the differential rate of recovery for the blood and spleen lymphocytes is not known. One possible basis for the difference between the blood and spleen is simply quantitative, lhe spleen cell population is very dense relative to that of the whole blood, and any intercellular interactions could occur at a faster rate in the spleen. Ihus, the same habituation process might weIl occur in the whole blood, given an extended number of shock sessions. In like fashion, the rate of recovery of particular cell populations may depend on the density of the cells. A second possible explanation for the habituation effect is that continued shock experiences could result in a change in the spleen tissue. Ihat change might restrict the migration of particuiar cell populations or the flow of immunosuppressive humoral factors. However, the present data do not provide a basis for evaluating these different possibilities.

Acknowledgements

Donald I. Lysle is the recipient of a Clinical Research Center Postdoctoral Fellowship (MH#30915) from Western Psychiatric Institute and Clinic, Pittsburgh, PA USA.

The authors thank Ellen Hamill and Barbara Kucinski for their technical assistance, and Laura Dillman for her help in preparing the manuscript.

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1814 Shock-induced Immunomodulation Vol. 41, No. 15, 1987

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