Emotional stress accompanied by social interactions and conflict occupies an important place in modern human life. Social conflict, especially when chronic, can.
Neuroscience and Behavioral Physiology, Vol. 31, No. 1, 2001
Changes in Various Measures of Immune Status in Mice Subject to Chronic Social Conflict N. I. Gryazeva,* A. V. Shurlygina, L. V. Verbitskaya, E. V. Mel’nikova, N. N. Kudryavtseva, and V. A. Trufakin Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 85, No. 8, pp. 1035–1042, August, 1999. Original article submitted November 21, 1998. Revised version received March 25, 1999. Levels of the major regulatory subpopulations of lymphocytes in the thymus and spleen and blood lymphocyte dehydrogenase levels were measured in male mice with aggressive and submissive patterns of behavior formed over 10 or 20 aggressive confrontations leading to repeated experience of victory or defeat. The results showed that repeated experience of social confrontation non-specifically increased the proportion of segmented neutrophils and lactate dehydrogenase activity in both participants in aggressive encounters, and decreased the numbers of CD4+ and CD8+ T-lymphocytes in the spleen. Succinate dehydrogenase specifically decreased in the lymphocytes of aggressive mice and increased in the lymphocytes of submissive mice. The proportion of CD4+ T-lymphocytes in victims’ thymuses also decreased. Changes in metabolic measures and percentage ratios of lymphocyte contents were dynamic and depended on the duration of confrontational interactions. KEY WORDS: Psychosocial stress, aggressive behavior, submissive behavior, immunocompetent cells.
along with the development of derangement of immune status in the body [1, 15, 19, 20, 22, 25]. However, the experimental data on the interaction between social stress and the body’s immune status are fragmentary and largely involve integral measures of immune system function – the humoral immune response – and do not cover the interactions between the central and peripheral parts of the immune system. In addition, our previous data from studies of rats genetically predisposed to pathological depression [3] suggested that the enzyme spectrum of lymphoid cells can be regarded as a measure with diagnostic and prognostic significance in studies of higher nervous activity pathology associated with disruption of behavioral reactions. Thus, the aim of the present work was to study the effects of emotional status – formed during chronic confrontations in mice using a sensory contact model – on the subpopulation spectrum and activity of energy metabolism of thymus, spleen, and blood lymphocytes.
Emotional stress accompanied by social interactions and conflict occupies an important place in modern human life. Social conflict, especially when chronic, can result in dysfunction of many organs and systems, including the immune system, leading to weakening of the body’s resistance to unfavorable environmental factors and infections. A large amount of factual data have been accumulated from studies of the cellular immunity (analysis of lymphocyte subpopulation compositions, lymphocyte proliferative activity in response to mitogen stimulation, levels of natural killer cells) in patients with psychogenic and endogenous depression [5, 8]. Chronic emotional stress, which in mice is accompanied by the development of aggressive and submissive behavior, induces in humans and animals a state of fear and anxiety, *
Deseased
Institute of Clinical and Experimental Lymphology, Siberian Division, Academy of Medical Sciences, 2 Academician Timakov Prospekt, 630117 Novosibirsk, Russia; Institute of Cytology and Genetics, Siberian Division, Russian Academy of Sciences, 10 Academician Lavrent’ev Prospekt, 630090 Novosibirsk, Russia.
METHODS Experiments were performed on adult male C57Bl/6J mice aged 2.5–3 months and weighing 22–25 g. 75 0097-0549/01/3101-0075$25.00 ©2001 Plenum Publishing Corporation
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Fig. 1. Blood lymphocyte dehydrogenase activities in mice with different experience of social confrontation. The vertical axes show enzyme activity in arbitrary units. *Different from control, p < 0.05, +significant difference between victims with different durations of experience of social confrontation, p < 0.05.
Animals were kept in standard animal-house conditions with unrestricted food and water. The model of aggressive and submissive types of behavior was formed in adult male mice by initially keeping them individually for five days to release the effects of group interactions. The heaviest individuals for different litters were then placed in pairs in metal experimental cages of size 28 × 14 × 10 cm, divided into two equal sectors by a transparent separator with openings – the animals could see each other, hear each other, and perceive each other’s odors, but were unable to make physical contact (sensory contact conditions). Behavioral testing was started two days after adaptation of the animals to the new keeping conditions and sensory acquaintance with each other. During tests, which lasted 10 min and were performed daily, the separator was removed, leading to antagonistic interactions. Experience of victory or defeat in the first tests (over the first three days) in antagonistic interactions with the same partner was reinforced on subsequent days by repeating the conflicts with a partner displaying the opposite type of behavior. Thus, after tests, a vanquished male would be placed in an unfamiliar cage with fresh litter with another aggressive male on the other side of the separator. Aggressive males stayed in their own cages. The result of these manipulations was that groups of mice with the aggressive type of behavior were formed by sequential experience of victory in social confrontations (aggressors, victors), which demonstrated marked aggression in the daily tests, falling upon and attacking the unfamiliar partner. The protocol also formed a group of animals with the submissive type of behavior due to acquiring sequential experience of social defeats and demonstrating completely submissive poses in or
running away from aggressive interactions (victims, vanquished). Controls for both groups of animals, with the alternative types of social behavior, were provided by individuals kept alone for five days in individual cages. Thus, the experiment formed five groups of animals: controls, A10 and A20 aggressors (experience of victory in 10 and 20 confrontations), and V10 and V20 victims (experience of defeat in 10 and 20 confrontations) [9, 22]. Mice were sacrificed by decapitation the day after the last confrontation; blood was collected, thymuses and spleens were removed, blood smears were prepared, along with suspensions of thymus and spleen cells, and these were stained for lymphocyte enzyme activity and the levels of CD4+, CD8+, and CD25+ T-cells. Blood lymphocyte dehydrogenase activities (succinate dehydrogenase and lactate dehydrogenase) were measured cytochemically using a quantitative method based on p-nitrotetrazolium violet. Enzyme activity was assessed in terms of the mean number of granules of formazan formed per lymphocyte (arbitrary units). The number of granules per 40 cells was determined in each smear [14]. The blood leukocyte formula was determined in smears stained by the Romanovskii method [12]. The levels of lymphocyte CD4+, CD8+, and CD25+ subpopulations were estimated in smears of thymus and spleen smears using an immunocytochemical method based on the appropriate monoclonal antibodies (Boehringer Mannheim), with a peroxidase-labeled extravidin-biotin kit (Sigma) as the second layer. Peroxidase was detected using diaminobenzidine HCl (Sigma). Experimental data were analyzed statistically using the Wilcoxon–Mann–Whitney non-parametric test (the U criterion).
Changes in Various Measures of Immune Status in Mice Subject to Chronic Social Conflict
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Fig. 2. Relative lymphocyte and segmented neutrophil counts in mice with different experience of social confrontation. **Difference compared with controls, p < 0.01. For further explanation see caption to Fig. 1.
Fig. 3. Numbers of CD4+ and CD8+ T-lymphocytes and their ratios in mice with different experience of social confrontation. oDifference between groups with different social status, p < 0.05. 1) Controls; 2) group A10; 3) group A20; 4) group V10; 5) group V20. For further explanation see caption to Fig. 1.
RESULTS The data obtained here on the effects of acquisition of social status on blood lymphocyte dehydrogenase activities are presented in Fig. 1. Blood lymphocyte succinate dehydrogenase and lactate dehydrogenase activities depended on the type of behavior acquired by the animal and the period of time over which it was acquired. Succinate dehydrogenase activity in A10 mice decreased significantly, but returned to control levels in A20 mice. In submissive mice, changes were opposite in nature: the
V10 group showed no change in succinate dehydrogenase while the V20 group showed a sharp increase. The dynamics of lactate dehydrogenase levels were rather different: in aggressors, activity increased in the A10 group and persisted in the A20 group. In submissive mice of the V10 group, enzyme activity was also increased, but returned to control in the V20 group. Acquisition of psychoemotional status also changed the percentage composition of blood lymphocytes and segmented neutrophils (Fig. 2). These measures changed with opposite patterns in animals with different types of
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Fig. 4. Levels of interleukin-2 receptors in the thymus and spleen in mice with different experience of social conflict. For further explanation see caption to Fig. 1.
behavior. Thus, while the proportion of lymphocytes in group A10 decreased significantly, with a further decrease in the A20 group, the numbers of neutrophils in the A10 group, contrarily, increased, staying elevated in the A20 group. In submissive mice of the V10 group, lymphocyte levels also decreased, but returned to the baseline level in the V20 group, while the number of neutrophils increased sharply in V10 and then, like the relative lymphocyte count, returned to baseline in the V20 group. Changes in the numbers of CD4+ and CD8+ cells also depended on the animal’s social status and showed different patterns in the thymus and spleen (Fig. 3). In the thymus of animals with the neurophysiological status of aggressors, the number of CD4+ and CD8+ subpopulations remained unchanged in both the A10 and A20 groups. In submissive mice, thymus cell subpopulations were unchanged in the V10 group, though there was some tendency to increase, especially in CD8+ thymocytes; in the V20 group, the number of CD4+ cells decreased significantly. The numbers of both subpopulations in victims showed a significant relationship with the number of defeats. In the spleens of A10 mice, the number of CD4+ lymphocytes decreased significantly while the content of CD8+ cells remained at the control level. In group A20, there were significant decreases in both CD4+ and CD8+ splenocytes. In submissive mice, the number of spleen CD4+ cells decreased sharply in the V10 group and returned to baseline in the V20 group. The level of CD8+ cells decreased in the V10 and V20 groups. The CD4+/CD8+ ratio remained at the control level in all groups in both the thymus and the spleen, though
the value of this ratio in the aggressor thymus was significantly dependent on the number of victories: the more victories, the greater the ratio. In addition, the CD4+/CD8+ ratio was greater in the A20 group than the V20 group. The levels of cells expressing interleukin-2 receptors (CD25+ cells) (Fig. 4) remained at the control levels in the spleens and thymuses of aggressor and victim mice, though the number of CD25+ cells was significantly greater in the V10 group than in the A10 group. In the spleen, the percentage content of CD25+ lymphocytes was greater than control in submissive mice regardless of the number of defeats; the level was also increased in the A10 group, returning to baseline in the A20 group (Fig. 4).
DISCUSSION Analysis of these data shows that repeated experience of social victory or defeat in daily intermale confrontations affects the population composition and metabolism of immunocompetent blood cells. Many of the changes appear to be non-specific in nature, as they were found in animals with both alternative social types of behavior. The same comment applies to the decrease in the percentage composition of lymphocytes and the increase in the composition of segmented neutrophils in the blood of aggressors and victims as compared with controls, as well as to the increase in lactate dehydrogenase activity in blood lymphocytes. Both aggressors and victims showed decreases in CD4+ and CD8+ T-lymphocytes and increases in the numbers of cells expressing
Changes in Various Measures of Immune Status in Mice Subject to Chronic Social Conflict interleukin-2 receptors (CD25+) in the spleen. This suggests that in mice taking part in confrontations, the formation of each type of social behavior represents a state of chronic stress. It has, for example, been shown [26] that chronic stress induced by immobilization of rats for 3 h/day for 11 days also decreased the relative lymphocyte count but increased the neutrophil count, these changes being accompanied by decreases in CD4+ and CD8+ cells, though the ratio of these subpopulations, as in our experiments, showed no significant change. It is likely that the decrease in the blood relative lymphocyte count and the CD4+ and CD8+ lymphocyte counts in the spleen in both aggressors and victims are due to the redistribution of these cells in the bone marrow [4, 7] and restriction of their migration from the central immune organs to the periphery, because of chronic stress and stable increases in glucocorticoid levels [6, 21]. The increase in the content of immature and/or activated CD25+ cells in the spleens of experimental animals accords with data obtained by Teshima et at. [27], also showing a shift in the composition of thymus, blood, and spleen cell populations in mice, with predominance of immature forms, in conditions of immobilization stress. Increases in lactate dehydrogenase activity in lymphocytes may reflect a state of tension in the immune system, as switching of lymphocytes to the anaerobic pathway of energy metabolism would appear to be compensatory, aimed at producing additional energy equivalents [2, 11]. However, the dynamic pattern of changes in these measures from test to test and the extents of the changes in aggressors and victims were often different. Thus, while aggressors showed a typical gradual increase in lactate dehydrogenase activity, as well as in the relative content of segmented neutrophils and lymphocytes from test to test, victims subjected to 20 confrontational interactions showed increases in a number of measures as compared with controls. This can evidently be interpreted as adaptation of the system to functioning in conditions of prolonged unavoidable stress [4, 6]. One of us has previously shown that 20 days of experience of sequential defeat induces a near-psychopathological depression state in victim mice [9]. The behavior of these mice shows the following changes: 1) An increase in the time spent demonstrating the submissive pose of lying on the back, which typifies the state of the animal’s being in emotional tension. 2) Fixed forms of inadequate behavior appear, showing that borderline states develop. 3) “Freezing” poses are observed, which are similar to immobile states whose appearance is associated with the development of catatonic states. 4) Most animals show “depression” poses, when they keep their noses in the corner of the cage or litter and are completely immobile. Additional testing of
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these animals also demonstrated decreases in investigative activity and increases in the duration of passive swimming in the Porsolt test [24], characteristics which also show high levels of depression. Thus, the increase in a number of parameters to normal levels in the V20 group seen in the present experiments may result not from normalization of immune status but an adaptive response in which the system functions at a new level, as the ratio of counts and enzyme activities in blood lymphocytes remained unchanged. In addition, the thymuses of submissive mice showed changes in the cellular subpopulation composition, with increases in CD25+ thymocytes (pre-T-cells) in the V10 group as compared with the A10 group and decreases in the CD4+ population in the V20 group compared with the A20 group and controls. This may be evidence that T-lymphocyte maturation is deranged in mice with different social status. Taking data obtained by Devoino et al. [19] into consideration, the decrease in the level of humoral immune responses in V20 mice suggests that acquisition of the social status of a “victim” is accompanied by disturbance (perhaps slowing) of central T-lymphocyte differentiation processes. In relation to various other measures, we also observed specific effects typical for the formation only of the aggressive or submissive types of behavior. Aggressors (group A10) showed a decrease and victims (group V20) an increase compared with controls in blood lymphocyte succinate dehydrogenase activity. Prolonged experience of defeat led to decreases in the CD4+ subpopulation in the spleen in the V20 group and had no significant effect on this measure in aggressors; the CD4/CD8 ratio in aggressors tended to increase from test day 10 to test day 20 and its value was significantly higher than in victims. This agrees with data [23] showing immunostimulation in “victor” mice. Thus, the changes described here appear to be induced not by chronic stress, which was common to both participants in conflicts, but by the formation of the different psychoemotional status: positive in aggressive males and negative in submissive males. The near-psychopathological depressive state produced deeper alterations in immune status, affecting its central component. This is likely to have occurred in response to stable changes in the neurohormonal balance, with a strengthening of immunosuppressor (glucocorticoids, serotonin) and weakening of immunostimulator (dopamine) influences [21]. Our data on the dynamics of the enzyme spectrum of immunocompetent cells may be evidence not only of changes in the animals’ immune status in conditions of repeated experience of social confrontation, but also of changes in energy metabolism in brain tissues, as a significant correlation between dehydrogenase activities in blood lymphocytes and brain tissues has been reported
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[12, 18], along with a correlation between brain succinate dehydrogenase activity and the type of behavior demonstrated by animals [17]. Additionally, succinate dehydrogenase, which catalyzes one of the final stages in the tricarboxylic acid cycle, is known to be associated with the GABA shunt, resulting in the formation of the main inhibitory mediator of the brain, GABA. The decreases in succinate dehydrogenase activity in aggressive animals and increases in submissive animals seen in our experiments sit well with general concepts of the effects of GABA-positive and GABA-negative agents on aggressive and submissive behavior in animals [16]. There are grounds for suggesting that blood lymphocyte succinate dehydrogenase activity may be a measure, admittedly indirect but nonetheless available and quite informative, for assessing metabolism in the CNS of animals in groups at risk for developing psychopathology. Thus, our results provide evidence of the effects of social status formed in mice using the sensory contact model on the state of the body’s immunological functions. Changes in measures of immune status were characteristic in animals with opposite types of behavioral responses and depended on the numbers of victories and defeats. The most significant changes, close to immunopathological in extent, were seen in submissive animals. However, positive psychoemotional status in aggressors was also formed on a background of chronic stress and was accompanied by a pattern of immunological dysbalance, which could lead to negative changes in the protective functions of the body.
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