Handbook of Human Stress and Immunity

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PREFACE

There is increasing evidence that the central nervous system (CNS) can influence the immune response, the body’s defense against infectious and malignant diseases. This book focuses on human studies, as well as relevant animal models for human infectious and autoimmune illness. We chose to highlight these particular animal models because they demonstrate the direct impact of various stressors on the immune system and the subsequent modulation of infectious and autoimmune diseases. In addition, these animal studies help to demonstrate the range of physiological changes associated with stressors. We believe that the chapters in this book will provide the reader with a current summary of the field of psychoneuroimmunology as it pertains to stress and illness outcomes.

Psychological distress can lead to adverse immunological changes providing one physiological pathway through which major and minor life changes might result in an increased incidence of infectious and malignant disease. However, most individuals who experience major life changes do not become ill, or they only experience short illness episodes. Actual organically based episodes of illness are a function of differential exposure to pathogens and/or carcinogens, genetics, as well as the prior health of the individual, particularly regarding immune function. Thus, in individuals with equal exposure to an infectious agent (e.g., a virus), the probability of clinical illness and the intensity and duration of the illness are, in part, the product of the prior status of the individual’s immune system. Following this line of reasoning, individuals who are presumably most likely to show health changes in response to stressors are those whose immune function is already compromised to some extent, particularly the elderly, since aging is associated with impaired immune function.

The adrenal cortex produces glucocorticoids in response to stressors through the hypothalamic–pituitary axis. Individuals with an adrenocortical insufficiency such as Addison’s Disease have physiological difficulty handling stress. In addition, an overabundance of glucocorticoids during stress also can have major physiological consequences. Research on the relationships among the CNS, immune system and endocrine systems provides evidence of physiological pathways, including the glucocorticoids, and other ‘stress hormones" such as the catecholamines, prolactin, and growth hormone, through which distress may modulate immune function.

There also are other pathways through which the CNS may influence immune function, such as direct innervation of both primary and secondary lymphoid organs. Our knowledge about the interactions among the CNS, the immune system, and the endocrine system in regard to mechanisms of action is still limited. However, there is an active and growing literature describing such relationships, and their health-related consequences. These and other issues are covered in the chapter in this book by well-known experts in their respective areas.

Ronald Glaser

Janice K. Kiecolt-Glaser

1

Stress-Induced Modulation of Immune Function in Mice

Jan A. Moynihan, Gary J. Brenner, Robert Cocke, Jonathan D. Karp, Stephen M. Breneman, Joel M. Dopp, Robert Ader, Nicholas Cohen, Lee J. Grota and Suzanne Y. Felten

Publisher Summary

Stress, which is the response of an organism to stimulation or change, is characterized by activation of the autonomic nervous system and the hypothalamo-pituitary-adrenal (HPA) axis. The resulting neurochemical changes have been demonstrated to affect immune function directly and indirectly. Direct effects are possible because lymphocytes bear receptors on their surfaces for many neurohormones and transmitters, and lymphocytes are exposed to neurochemicals in lymphoid organs and in peripheral blood. More indirect mechanisms for neural-immune interactions might involve changes in lymphocyte trafficking resulting from changes in sympathetic vascular tone. The elucidation of pathways of communication between the central nervous system and the immune system has advanced, to a large extent, from studies utilizing stressor administration in animal models. Application of stressor in animals results in diverse changes in specific humoral and cell-mediated immune parameters, and in non-specific natural killer (NK) cell number and/or activity.

I INTRODUCTION

Stress, which is broadly defined as the response of an organism to stimulation or change (Selye, 1950), is characterized by activation of both the autonomic nervous system and the hypothalamo-pituitary-adrenal (HPA) axis. The resulting neurochemical changes have been demonstrated to affect immune function both directly and indirectly. Direct effects are possible because lymphocytes bear receptors on their surfaces for many neurohormones and transmitters, and lymphocytes are exposed to neurochemicals in lymphoid organs and in peripheral blood (Carr & Blalock, 1991; Roszman & Carlson, 1991; Feiten & Feiten, 1991). More indirect mechanisms for neural-immune interactions may involve changes in lymphocyte trafficking resulting from changes in sympathetic vascular tone (Ottaay, 1991).

The elucidation of pathways of communication between the central nervous system and the immune system has advanced, to a large extent, from studies utilizing stressor administration in animal models. Application of a stressor in animals results in diverse changes in both specific humoral and cell-mediated immune parameters, and in nonspecific natural killer (NK) cell number and/or activity (reviewed in Kiecolt-Glaser & Glaser, 1991; Shavit, 1991; Moynihan & Cohen, 1992; Ader & Cohen, 1993). Many of the studies that have addressed stress-induced changes in disease outcome and/or immune function also illustrate the complexities of the interactions between the central nervous and immune systems. It is certainly apparent that: (1) not all stressors produce the same pattern of neurochemical responses; (2) not all strains, species, or sex of animals respond to a given stressor in the same behavioral, neurochemical or immunological manner; and (3) not all immune effector functions are equally affected by stressor application. Thus, in response to stressors, immune effector functions are suppressed, enhanced, or unchanged. As outlined in Table 1, several variables have been demonstrated to be important determinants of the immunological consequences of stressor administration. These variables reflect the complexity of both the neuroendocrine responses to a stressor, and the interaction of these responses with the cascade of molecular and cellular immune responses to an antigen. This neuroendocrine complexity can be highlighted by pointing out that, although in some studies, stressor-associated immunomodulation correlates with altered levels of glucocorticoids (Blecha, Kelley, & Satterlee, 1982; Keller, Weiss, Schleifer, Miller, & Stein, 1983; Okimura, Ogawa, Yamauchi, & Sasaki, 1986; Coe, Rosenberg, Fisher, & Levine, 1987) or opioids (Shavit, Lewis, Terman, Gale, & Liebeskind, 1984; Shavit, 1991), altered levels of these glucocorticoids and/or opioids have not been found in all studies that report effects of stressors on immune responses (Cunnick, Lysle, Armfield, & Rabin, 1988; Ben-Eliyahu, Yirmiya, Shavit, & Liebeskind, 1990; Rabin, Cunnick, & Lysle, 1990; Moynihan & Cohen, 1992; Ader & Cohen, 1993). Further, at least 17 neuroendocrine peptides have immunomodulatory potential (Khansari, Murgo, & Faith, 1990), and many of these hormones or peptides are altered by stressors. Finally, hypothalamic hormones such as corticotropin-releasing hormone (CRH) may exert immunomodulatory effects via both the pituitary-adrenal axis (Munck & Guyre, 1991) and the sympathetic and parasympathetic nervous system release of norepinephrine and other catecholamines (Sundar, Cierpial, Kilts, Ritchie, & Weiss, 1990; Irwin, Hauger, Jones, Provencio, & Britton, 1990; Irwin, Vale, & Rivier, 1990). Thus, multiple neuroendocrine pathways are undoubtedly involved in stress-induced alteration of immune function.

TABLE 1

Interacting Variables in Stress-Induced Immune Modulation

Although it is critical to study stress-induced neurochemical changes from the perspective of the immunologist it is also necessary to understand what events in the immune cascade that are triggered by antigen or pathogen are changed as a consequence of the stress response. Experimental mechanistic approaches from this perspective are based on the awareness that the immune system has levels of complexity that rival those of the nervous system. This chapter will discuss our recent findings using stressors to alter antigen-driven events that lead to cellular and humoral immune responses; illustrate some of the interactions between the variables listed in Table 1; and highlight some of the possible immunological mechanisms involved in stress-induced changes in immune function.

II THE EFFECTS OF HANDLING ON IMMUNE FUNCTION AND ON TUMOR METASTASES

Early stress studies often used a simple, yet effective, method of psychosocial stimulation, that is, picking up and holding an animal for a short period. Many of these studies examined the effects of handling neonatal rats or mice (Solomon, Levine, & Kraft, 1968; Friedman, Glasgow, & Ader, 1970; LaBarba, 1970; Michaut, Dechambre, Doumerc, Lesourd, Devillechabrolle, & Moulias, 1981; Lown & Dutka, 1987). However, many studies that have investigated handling of neonatal or adult rodents have not examined immune function per se, but, for example have looked at changes in survival time following tumor or viral challenge (Newton, Bly, & McCrary, 1962; Ader, 1965; Friedman et al., 1970). Our approach has been to study the effects of repeated daily handling of adult mice on a number of specific and nonspecific immune effector mechanisms or an antibody response, as well as a tumor challenge (Moynihan, Brenner, Koota, Breneman, Cohen, & Ader, 1990; Brenner, Cohen, Ader, & Moynihan, 1990).

Group-housed adult BALB/c. ByJ female mice were held gently without restraint for 2 min/day for 2 weeks prior to intraperitoneal (ip) injection of the T cell-dependent protein antigen keyhole limpet hemocyanin (KLH). Mice were bled at various times following immunization to measure specific anti-KLH antibody responses. Mice that were handled for two weeks prior to immunization had a significant and reproducible decrease in lgG anti-KLH antibody responses compared to unhandled control mice. The results of one such experiment are shown in Figure 1. Handling was also associated with a significant decrease in antigen-induced in vitro spleen cell production of the cytokine interleukin (IL)-2, produced by a subset of mature T helper cells designated T helper 1 (TH1) cells (Street & Mosmann, 1991) (Figure 2). Additionally, the proliferative response of spleen cells from nonimmunized mice to the T cell mitogen concanavalin A (ConA) was diminished in cell cultures from handled mice. The proliferative response to the B cell mitogen lipopolysaccharide (LPS) was unaffected. Thus, handling mice for 2 weeks resulted in both depressed T cell function and diminished lgG antibody production to a T cell-dependent antigen.

FIGURE 1 The IgG anti-KLH antibody response in handled versus control BALB/c female mice. Mice in both groups (n = 12/group) were immunized following two weeks of daily handling (2 min/day) with 100 μg of KLH. Mice were bled to measure serum antibody titers on Days 5, 10, and 15 following immunization (Moynihan et al., 1990). Data are expressed as mean ± SEM.

FIGURE 2 In vitro KLH-induced IL-2 production in spleen cell cultures from handled versus control BALB/c female mice. Mice (n = 6/group/time point) were handled as described earlier, immunized following handling with 100 μg of KLH, and sacrificed 6, 9, 12, and 15 days following immunization. Spleen cells were cultured in 96-well plates at 2 × 10⁵ cells/well with 80 μg/ml of KLH. Supernatants were removed 24 h later and assayed for IL-2 using the IL-2-dependent cell line CTLL (Mosmann, Cherwinski, Bond, Giedlin, & Coffman, 1986). Data are expressed as mean ± SEM.

We have also examined the effects of 2 weeks of daily handling on the response to intravenous injection of a tumor cell line, resulting in lung metastases. The line 1 alveolar carcinoma (Yuhas, Toya, & Wagner, 1975; Yuhas, 1977) was injected into syngeneic BALB/c female mice following 2 weeks of handling. Mice were sacrificed 8 to 21 days later (the variability was between, not within, experiments) and the numbers of lung tumors were enumerated. Figure 3 illustrates that numbers of lung metastases were reproducibly increased in handled mice, and that this increase was statistically significant regardless of the overall numbers of metastases (i.e., an average of 149 metastases in Experiment 3 versus 5.4 in Experiment 2).

FIGURE 3 Pulmonary metastases in handled or unhandled control BALB/c female mice. Mice (n = 7–12/group/experiment) were injected intravenously following 2 weeks of handling with 10⁵ line 1 cells. Mice were sacrificed 8 to 21 days later (the variability was between, not within, experiments) and pulmonary metastases were enumerated (Brenner et al., 1990). Four replications are shown (a–d). Data are expressed as mean±SEM.

The line 1 tumor is reported to be sensitive to lysis by NK cells, and is not particularly immunogenic, presumably due to poor expression of class I major histocompatibility antigens (Cerosaletti, Bleiden, Harwell, Welsh, Frelinger, & Lord, 1990). In a number of studies in which we examined both in vivo and in vitro NK cell cytotoxicity against either the YAC-1 lymphoma line or line 1 cells, we did not observe any decrease in NK cell function in handled mice. Thus, although immune function (as evidenced by decreased T cell function and antigen-specific lgG production) is altered in handled mice, these immunological changes may not necessarily be involved in the increased metastases following tumor challenge.

III HANDLING RESULTS IN HABITUATION OF THE STRESS RESPONSE

We have speculated that handling results in a classic stress response in mice, resulting in activation of the HPA axis and the sympathetic nervous system (SNS). We began these experiments with the bias that the handled mice were the stressed group, and that the unhandled controls were nonstressed. However, we have observed that following the administration of an ip injection (a stressor in itself), previously handled mice have attenuated plasma corticosterone and catecholamine responses compared to previously unhandled mice (Figures 4a and 4b). Indeed, it has been reported in the literature that habituation to handling is associated with higher gammabutyric acid (GABA) receptor binding in certain brain areas associated with an increase in number of GABA receptors (Biggio, Corda, Concas, Demontis, Rossetti, & Gessa, 1981), lower levels of cerebellar cGMP and cAMP (Corda, Biggio, & Gessa, 1980), reduced opioid-induced analgesia (Fanselow & Sigmundi, 1986), and reduced glucocorticoid responses compared to unhandled animals (Ader, Friedman, Grota, & Schaefer, 1968; Ader & Grota, 1969; File, 1982; Moynihan et al., 1990). Indeed, ablation of the peripheral SNS using the drug 6-hydroxydopamine (6-OHDA) prior to, but not following, injection of line 1 tumor cells resulted in increased numbers of pulmonary metastases (Brenner, Feiten, Feiten, & Moynihan, 1992; see Figure 5). Thus, the increase in pulmonary metastases observed in either handled or sympathectomized mice may be a function of the attenuation, rather than the elicitation, of a stress or catecholamine response. Although increased metastases are associated with neuroendocrine changes, there is no evidence that increased metastases in either handled or sympathectomized mice are associated with altered immune effector mechanisms (Brenner et al., 1990, 1992). Thus, it may be that not all stress-induced changes in tumor or disease progression are associated with altered immunity.

FIGURE 4 Plasma epinephrine and corticosterone responses in handled versus control BALB/c female mice. Mice (n = 6/group/time point) were injected with saline intraperitoneally and sacrificed at the indicated times to measure plasma epinephrine and corticosterone. The mice sacrificed at time zero did not receive an injection. Data are expressed as the mean±SEM.

FIGURE 5 Pulmonary metastases in chemically sympathectomized versus control BALB/c female mice. Mice (n = 18–20/group) were injected intraperitoneally with either 125 mg/kg of the neurotoxin 6-hydroxydopamine (6-OHDA) or vehicle at the times indicated. Mice were injected intravenously with 10⁵ line 1 tumor cells on Day 0, sacrificed 14 days later, and pulmonary metastases were enumerated (Brenner et al., 1992). Data are expressed as mean±SEM.

IV THE EFFECTS OF STRESS ON VIRAL INFECTION AND IMMUNITY

Although the increased number of lung metastases associated with handling does not appear to be immunologically mediated, there are clear instances in which stress-induced changes in disease pathogenesis have been correlated directly with immune function. For example, recent studies have demonstrated that the viral-specific immune responses of C57Bl/6 female mice following primary infection in the footpad with herpes simplex virus-type 1 (HSV-1) can be altered by either restraint stress (Bonneau, Sheridan, Feng, & Glaser, 1991a,b) or mild electric footshock (Kusnecov, Grota, Schmidt, Bonneau, Sheridan, Glaser, & Moynihan, 1992). Using a footshock model, we (Kusnecov et al., 1992) have observed that stressor administration results in depression of spleen and popliteal lymph node cell number in infected mice; in vitro cytotoxic T cell responses to virally-infected cells; IL-2 production following stimulation with virally-infected cells in vitro; and humoral immune responses. In addition, infectious virus recovered from the footpads of the shocked mice differs dramatically from both the apparatus control and the home cage mice (44.5± 1.9, 9.1± 3.5, and 1.0± 0 plaque-forming units × 10⁵, respectively, for the shock, apparatus control, and home cage mice). Thus, impaired antiviral responses are associated with impaired clearance of virus from the site of infection.

V THE CENTRAL NERVOUS SYSTEM RESPONSE OF A RECIPIENT MOUSE TO THE ODORS EMITTED BY A STRESSED CONSPECIFIC ALTERS CELL-MEDIATED AND HUMORAL IMMUNE FUNCTION DIFFERENTLY

Thus far, this chapter has discussed the effects of physical stressors on immune function. In addition to these studies, we have recently focused on a paradigm that does not involve physical pain or trauma, and is of clear ethologic relevance to the rodent. The paradigm involves pheromonal stimulation of conspecifics. The olfactory system of a rodent is highly developed, and is critical for communication among these animals (Harrington & Rosario, 1992). Other investigators have determined that odors (such as botanical odors) can be immunoregulatory (Shibata, Fujiwara, Iwamoto, Matsuoka, & Yokoyama, 1990). Further, there is some evidence that the odors of predators or of stressed conspecific animals can alter the number of antibody forming cell to sheep red blood cells (SRBC, Zalcman, Kerr, & Anisman, 1991). We have tested the hypothesis that odors or pheromones produced by footshock-stressed mice result in altered immune function in conspecific odor recipients. We hypothesize that this ethologically relevant model of stress mimics components of a natural predator-prey situation.

Initially, we examined the responses of BALB/c male mice to odors produced by conspecifics that were subjected to brief electric footshock and had their tails clipped to release blood odors (Cocke, Moynihan, Cohen, Grota, & Ader, 1993); the olfactory stimulation of recipient mice is, presumably, a complex set of total body odors. A number of immune parameters were measured following 24 hr of odor exposure. ConA-induced IL-2 production by spleen cells from stress odor-exposed mice was suppressed (Fig. 6a) compared with IL-2 production by splenocytes from either apparatus control or home cage control mice. In addition, NK cell cytotoxicity to the YAC-1 lymphoma line in spleen cell cultures from mice that were exposed to odors from stressed mice was suppressed (Figure 6b). In contrast to the observed suppression in cell-mediated responses, stress odor-exposed BALB/c mice had enhanced humoral immune responses to KLH (Figure 7). Thus, stressor administration does not necessarily result in blanket immunosuppression and it does not affect all immune measures in a given strain of animals in the same direction. We hypothesize that this differential alteration of cell-mediated versus cellular immunity is likely due to differential activation of the two TH cell subsets, TH1 and TH2 cells.

FIGURE 6 (a) IL-2 production by spleen cells from stress versus control odor-exposed BALB/c male mice. Mean (±SEM) absorbance for IL-2 produced by Con A-stimulated (0.3–5 μg/ml) spleen cells obtained from control odor-exposed (n = 12) and footshock stress odor-exposed (n = 12) mice following a 24 h odor exposure (Cocke et al., 1993). IL-2 was assayed using the CTLL bioassay. (b) NK cell cytotoxicity of spleen cells from odor-exposed mice. Varying numbers of spleen cells from either stress odor-exposed (n = 12) or control odor-exposed (n = 12) mice were incubated with YAC-1 lymphoma cells in a standard 6-h ⁵¹Cr-release assay. Data are expressed as mean±SEM.

FIGURE 7 IgM and IgG anti-KLH titers in stress versus control odor-exposed BALB/c male mice. Mice (n = 8/group) were immunized with 100 μg of KLH 24 h prior to odor exposure as described earlier. Animals were bled 6 days following immunization to assay for anti-KLH antibody levels. Data are expressed as mean±SEM.

Activated TH1 cells produce the cytokines IL-2 and interferon (IFN)-γ (Mosmann, Cherwinski, Bond, Giedlin, & Coffman, 1986; Mosmann & Coffman, 1987; Street & Mosmann, 1991; Janeway & Goldstein, 1991; Kapsenberg, Wierenga, Bos, & Jansen, 1991). These cytokines are required for generating cell-mediated immune responses, including NK cell cytotoxicity. TH2 cell-derived cytokines IL-4, IL-5, IL-6, and IL-10 are important in supporting growth of B cells and immunoglobulin isotype switching to IgG1, IgA, and IgE (Mosmann & Street, 1991). The TH1- and TH2 cell-derived cytokines are reciprocally controlled; high levels of IFN-γ, for example, suppress production of IL-4; IL-10 can suppress IFN-γ production. Although IL-4 appears to act directly on T cells to activate TH2 cells, IL-10 appears to suppress TH1 cell activity by inhibiting production of the macrophage-derived cytokine IL-12, which promotes TH1 cell development (Hseih, Macatonia, Tripp, Wolf, O’Garra, & Murphy, 1993). Thus, the suppression of cell-mediated immune function and enhancement of humoral immunity in BALB/c stress odor-exposed mice is consistent with the hypothesis that the odor stimulus results in activation of TH2 cells and down-regulation of TH1 cells.

VI DIFFERENT STRESSORS CAN AFFECT IMMUNE FUNCTION DIFFERENTLY

There is no evidence to indicate that different stressors either do or do not evoke the same series of neurochemical changes in the experimental subject. Logically, then, it is not surprising that different stressors affect immune parameters differently in the same strain of mouse. We have examined the effects of handling (or handling–habituation) on immune functions in BALB/c female mice, and have examined the effects of stress odor exposure on immunity in BALB/c male mice. In the handling paradigm, both IL-2 production and IgG anti-KLH antibody were similarly affected (suppressed in the habituated mice, or enhanced in the previously unmanipulated animals). However, odor exposure resulted in suppressed IL-2 production and enhanced anti-KLH antibody. These observations may reflect differences in gender or the different neurochemical responses to the different stimuli. Such studies may be helpful for elucidating the role of a given neurochemical in mediating stress effects on immune function.

VII DIFFERENT STRAINS OF MICE RESPOND DIFFERENTLY TO THE SAME STRESSOR

The number of animals housed in a cage affects behavioral responses, as well as physiological and immunological processes (Fauman, 1987; Jessop & Bayer, 1989; Bohus & Koolhaus, 1991). It has been demonstrated that there is a strain-specific effect on immunological changes associated with differential housing; individual housing of C3H/HeJ, but not C57Bl/6, male mice resulted in enhanced primary antibody responses to SRBC (Rabin, Lyte, Epstein, & Caggiula, 1987; Rabin & Salvin, 1987). Using the protein antigen KLH, however, we have demonstrated that although group and individually housed C57Bl/6 male mice did not differ in their primary antibody response to KLH, singly housed mice had significantly increased secondary IgM and IgG anti-KLH antibody titers (Figure 8). Interestingly, individually housed BALB/c male mice had higher secondary IgM titers than group-housed BALB/c mice, however there was no difference in the antigen-specific IgG response between the groups (Figure 8). Thus, housing conditions did not affect the level of antibody produced during the primary response in two strains of mice, but did affect the secondary responses, with differences observed between the strains (Karp, Moynihan, and Ader, 1993). The altered secondary response might be a function of the number of memory cells that are generated following priming, or may reflect the elaboration of cytokines following secondary challenge.

FIGURE 8 Secondary IgG anti-KLH antibody titers of individually housed (n = 9/strain) and group housed (n = 12/strain) male C57Bl/6 and BALB/c mice. Mice were housed for 2 weeks prior to immunization with 100 μg of KLH. On Day 23, mice were again immunized with 100 μg of KLH, and were bled 6, 9, and 14 days later to measure IgG anti-KLH antibody. Data are expressed as mean±SEM.

VIII STRESS EFFECTS ON ANTIBODY RESPONSES MAY DEPEND ON THE CONCENTRATION OF ANTIGEN USED TO PRIME MICE

We have hypothesized that the use of optimal concentrations of antigen, which by definition elicit robust immune responses, to study stress-associated changes in humoral immunity might mask somewhat subtle behavioral and neuroendocrine influences. Further, it can be argued that immunization with such suprathreshold antigen concentrations is not reflective of antigen exposure in the real world. We have investigated the effects of footshock stress administered at the time of priming with low doses of KLH on the secondary antibody response to a low dose of KLH (Moynihan, Schachtman, Grota, Cohen, & Ader, 1990).

Mild footshock was delivered for 90 min daily for 7 days before and 7 days after priming of male C3H/HeJ mice with 1 μg of KLH ip. No primary antibody response was detected in either the footshock or apparatus control group. After approximately 3 weeks, mice were given a secondary immunization with 1 μg of KLH and were bled 5 and 15 days later. Both IgM and IgG anti-KLH antibody responses were suppressed in the mice that were exposed to footshock (Figure 9). However, the same footshock protocol did not affect the anti-KLH response elicited following a secondary challenge with 5 μg (rather than 1 μg) of KLH (data not shown). Thus, stress-induced modulation of antibody responses to a nonreplicating protein antigen may depend on the amount of antigen used to challenge; physiological rather than suprathreshold concentrations of antigen may be more useful for defining pathways of neuroendocrine-immune system interactions.

FIGURE 9 Secondary IgM and IgG anti-KLH antibody titers in footshocked versus control mice. C3H/HeJ male mice were subjected to 90 min of footshock (n = 7) of exposure to the apparatus (n = 5) for 7 days before and 7 days after primary immunization with 1 μg of KLH. On Day 23 post priming, mice were immunized with an additional 1 μg of KLH and bled for detection of anti-KLH antibody 5 and 15 days later. The data (mean±SEM) for the 1:25 dilution of sera are expressed collapsed across assay days.

IX STRESS AND AUTOIMMUNE DISEASE

Given that stressors can affect the immune system in a normal animal, it is logical to hypothesize that the neuroendocrine system might play a role in a dysregulated immune state, such as in autoimmune disease. There is evidence that the SNS, which is activated during a stress response, can modulate experimentally induced autoimmune diseases in rodents. Levine, Dardick, Roizen, Helms, and Basbaum (1986) demonstrated that blockade of β2 adrenergic receptors prior to induction of adjuvant-induced arthritis (AIA) is associated with delayed onset and decreased severity of disease in rats. In contrast, Chelmicka-Schorr, Kwasniewski, and Wollmann (1992) observed that neonatally sympathectomized rats had more severe experimental allergic encephalomyelitis (EAE) than control rats.

To date, few studies have examined the effects of stressors on autoimmune disease in animal models. Levine and Saltzman (1987) have demonstrated that physical restraint following initial onset of EAE significantly reduces relapses of paralysis compared to control rats. Rabin, Cohen, Ganguli, Lysle, and Cunnick (1989) showed that footshock stress prior to induction of AIA decreases disease severity, but increases disease severity when administered at the initial onset of symptoms. Thus, the effects of stress on an autoimmune disease process may be dichotomous.

We have studied the effects of stress on the naturally occurring systemic lupus erythematosus-like disease of the MRL-lpr/lpr mouse. These mice develop an aggressive autoimmune disease at approximately 8 to 12 weeks of age, and there is approximately 50% mortality at 20 weeks (reviewed by Theofilopoulos & Dixon, 1985). The disease is characterized by a wide spectrum of autoantibodies, altered cytokine production, and a massive proliferation of an aberrant T cell population (characterized as CD3+, CD4–, CD8–, and B220+) in secondary lymphoid organs (Shirai, Abe, Yagita, Okumura, Morse, & Davidson, 1990). We subjected MRL-lpr/lpr mice to 60–10 sec signaled 0.6-mA footshocks over a 90-min period for 5 days/week beginning at 19 weeks of age, when there is already severe disease in most of the mice. After 7 weeks of shock, 1 of 16 (6.25%) of the footshocked mice had died, compared to 6 of 16 (37.5%) of the apparatus control mice (animals that were placed in the shock boxes but were not shocked) and 8 of 12 (66.7%) of the home cage control mice (Figure 10). Thus, it appears that stress can prolong survival of autoimmune animals, perhaps by an ameliorating effect on autoimmune disease in the MRL-lpr/lpr model. However, there have been populations of MRL-lpr/lpr mice that did not respond in the same way to footshock. We hypothesize that there is a discrete window in disease development in which footshock must be administered to affect disease, since there is heterogeneity in the age of onset of disease in the MRL-lpr/lpr mouse, it is possible to miss the age at which footshock is immunomodulatory or therapeutic.

FIGURE 10 Survival of autoimmune mice subjected to footshock. MRL-lpr/lpr female mice were subjected to 90 min of footshock 5 days/week beginning at 19 weeks of age; n = 16 for the footshock and apparatus control groups, and n = 12 for the home cage control groups. Shock was continued until the mice were 26 weeks of age.

As noted earlier, there is evidence that the SNS, which includes postganglionic nerve fibers in the parenchyma of the spleen, may play a role in experimentally induced autoimmune diseases. We have examined splenic noradrenergic innervation in the MRL-lpr/lpr mouse at 6, 12, 18, and 24 weeks of age using high performance liquid chromatography (HPLC) and catecholamine (CA) histofluorescence. We have observed a loss of detectable CA-containing nerve fibers in the MRL-lpr/lpr spleen over time, with no fibers detectable by 24 weeks of age (Breneman, Moynihan, Grota, Feiten, & Feiten, 1993). As shown in Table 2, there was a significant loss of splenic norepinephrine (NE) as early as 12 weeks of age compared to age-matched congenic MRL–+/+ mice (which lack the lymphoproliferative gene, and which develop an autoimmune disease late in life). No difference in catecholamine levels in nonlymphoid organs was observed between the substrains.

TABLE 2

Splenic Norepinephrine Content in MRL‐lpr/lpr and M R L ‐ + / + Mice (Mean±SEM)

Note. MRL‐lpr/lpr and MRL‐+/+ spleens (n=8–17/group) were prepared for HPLC aspreviously described (Brenner et al., 1992); substrains are significantly different at all ages (p<0.05).

It is tempting to hypothesize that loss of splenic catecholamines is directly correlated with disease onset in the MRL-lpr/lpr mouse, and that stress ameliorates the autoimmune disease by causing increased release of immunomodulatory catechols (by the SNS and the adrenals) which percolate through the spleen and lymph nodes. Studies in progress using the MRL-lpr/lpr model of human systemic lupus erythematosus address this hypothesis.

X CONCLUSIONS

The studies discussed in this chapter have demonstrated that a stressor can either suppress or enhance different immune responses in the same animal; that different stressors might have different effects on the same immune parameter; and that there are a number of variables that interact to determine the immunological outcome of stressor administration. Using the stress odor model, for example, we have observed that cell-mediated immunity is suppressed in BALB/c mice, whereas humoral immune function is enhanced. We have hypothesized that such an alteration of immunity may be the result of stress-induced activation of TH2 cells, with resulting down-regulation of TH1 cells.

Recently, Shearer et al., 1992, 1993; reviewed by Ezzell, 1993) have observed that there is a shift from a predominance of TH1 to TH2 cells in humans that develop acquired immunodeficiency syndrome (AIDS). They hypothesize that individuals with strong cell-mediated immunity (due to a predominance of TH1 cells) can keep the human immunodeficiency virus (HIV) in check, and that infected individuals may only become anti-HIV antibody positive, and later develop AIDS, following a shift to a predominance of TH2 cells. These investigators suggest that cofactors might be important in regulating the balance of TH1 and TH2 cells. We hypothesize that one such cofactor might be stress. Support for this hypothesis comes from recent publications by Daynes and Araneo (1989) and Daynes, Araneo, Dowell, Huang, & Dudley (1990) These investigators have demonstrated that corticosteroids (which are elevated in a classic stress response) can enhance IL-4 production, and suppress IL-2 production in vitro.

Stressful life experiences and the accompanying neurochemical changes have clear, but complex, effects on immunity. Because stressors are so prevalent in human life, their role in pathogenesis deserves further study. Future work from our laboratory will investigate stress-induced changes in TH1 versus TH2 cell activation following exposure to both protein antigens and infectious disease.

ACKNOWLEDGMENTS

The authors thank Susan Schmidt and Tracey Smith for their expert technical assistance. The studies reported from our laboratory were supported by the Whitehall Foundation and MH45681 from the National Institute of Mental Health. R.C., J.D.K., and J.M.D. were supported by T32MH18802; J.M.D. and G.J.B. were supported by T32A107285; G.J.B. was supported by the Howard Hughes Foundation and MH10144. R.A. was supported by MH06318.

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