HEALTH PSYCHOLOGY, 1992,11(4), 223-232 Copyright © 1992, Lawrence Erlbaum Associates, Inc.
Stress, Reactivity, and Immune Function in Healthy Men Sandra G. Zakowski Uniformed Services University of the Health Sciences
Cathy G. McAllister University of Pittsburgh
Marlene Deal and Andrew Baum
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Uniformed Services University of the Health Sciences We examined the effects of acute psychological stress on lymphocyte proliferation and circulating levels of interleukin-1 and - 2 . Healthy men were exposed to two viewings of a gruesome surgery film and were asked to recall details of the film twice during a 30-min period. These subjects were compared to a nonstress control group. Lymphocyte proliferation to the mitogen concanavalin A (Con A; 5 ug/ml) was decreased during and after exposure to the stressor when compared to the control group. This decrease was more pronounced in subjects exhibiting greater blood pressure reactivity while viewing the film than in subjects showing smaller blood pressure responses. None of the other immunological measures was significantly affected by the stressor. Cortisol was not correlated with lymphocyte responsiveness. Possible explanations for these results and implications for further research are discussed. Key words: stress, reactivity, lymphocyte proliferation, interleukin
Despite rapid growth in research on the relation between stress and immune function, several fundamental questions remain partly or completely unanswered. Many believe, for example, that changes in immune function mediate the association between stress and infectious illness (e.g., Jemmott & Locke, 1984), but studies providing definitive evidence of clinical outcomes of stress-related immunosuppression have not been reported. Likewise, the causal role of stress in these changes or the mechanisms by which they occur remain to be demonstrated under controlled laboratory conditions. The clearest evidence of stress-induced immunomodulation that we have is drawn from studies of various stressors on animals (e.g., Riley, 1981) and from studies of naturalistic stressors in humans (e.g., Kiecolt-Glaser & Glaser, 1987; McKinnon, Weisse, Reynolds, Bowles, & Baum, 1989). We have no clear information on several aspects of how long these changes take to occur, how long they last, or whether they have any clinical significance. The present study was designed to establish a stable model of acute stress that could be used to examine temporal and mechanistic properties of stress-induced changes in immune function. We sought (a) to determine whether this procedure could induce stress and produce reliable changes in immune function and (b) to elaborate relations among bodily systems. The effects of exposure to a 30-min passive stressor on mood, blood pressure (BP), plasma cortisol levels, and immune measures were assessed. The possible mediating role of arousal in immunological outcomes—represented by BP changes—was also examined.
STRESS AND IMMUNE FUNCTION For the most part, research on stress and immune function in humans has examined the effects of naturalistic stressors such as bereaveRequests for reprints should be sent to Andrew Baum, Hebert School of Medicine, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814.
ment, examinations, or marital distress. Bereavement is associated with reduced lymphocyte responsiveness to mitogen challenge (e.g., Bartrop, Luckhurst, Lazarus, Kiloh, & Penny, 1977; Schleifer, Keller, Camerino, Thornton, & Stein, 1983). Similarly, divorce and separation, providing long-term care to Alzheimer's patients, and chronic stress associated with living near the Three Mile Island nuclear power plant after the accident there have been associated with alterations in immune function reflecting suppressed activity (e.g., Kiecolt-Glaser, Fisher et al., 1987; Kiecolt-Glaser, Glaser et al., 1987; McKinnon et al., 1989). Depressed mood appears to be correlated with decreased lymphocyte responsiveness to phytohemagglutinin (PHA) and allogeneic cells in men with or without recent experience of family death or serious family illness (e.g., M. W. Linn, B. S. Linn, & Jensen, 1984). Less extreme stressors are also associated with changes in immune function. Studies of medical students facing major examinations have indicated that examination stress is associated with suppression of lymphocyte proliferation to PHA mitogen challenge, depression of natural killer cell activity, and increases in antibody titers to Epstein-Barr virus, suggesting suppression of cellular control of the latent virus (Glaser et al., 1987; Kiecolt-Glaser & Glaser, 1987; Workman & La Via, 1987). These changes were also related to perceived distress. Another study found elevated numbers of T and B cells and impaired mitogen responsiveness in trainees in psychiatry before final examinations (Dorian et al., 1982), and stress and anxiety due to hospitalization have been associated with decreased lymphocyte responsiveness as well (B. S. Linn, M. W. Linn, & Jensen, 1981). These findings are consistent with a large literature on stress and immunity in animals that suggests that stress causes changes in a variety of immune markers. These studies indicate that stress affects the immune system in many ways, usually reducing functional capabilities of cells or systems. Given that one of the primary foci of health psychology is the clinical application of research findings in natural settings, why step back into the laboratory to examine the short-term effects of a mild experimental stressor? Because most of the studies we have noted
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were correlational, it can be argued that we have little or no experimental evidence of stress-caused immune system changes in humans. In addition, the development of stable, controlled models of human stress that can be used to study the properties of stress-induced immune changes has lagged behind the development of animal models of these phenomena. This may be due to the belief that most immunological changes require extended stressor exposure or take days or weeks to become evident (Glaser et al., 1987). There now is evidence, however, of more rapid change: Several studies have reported immunological changes as a result of a brief (i.e. 30-min or less) stressor or exercise period (Eskola et al., 1978; Hedfors, Holm, & Ohnell, 1976; Manuck, Cohen, Rabin, Muldoon, & Bachen, 1991; Naliboff et al., 1991). Weisse et al. (1990), for example, reported decreases in lymphocyte proliferation to the mitogen concanavalin A (Con A) 50 min and 2Vi hr after exposure to controllable shock and noise stressors. Other studies have detected decreases in immune function 1 hr or less after exposure to the stressor (Eskola et al., 1978; Hedfors et al., 1976; Manuck et al., 1991). Only one study (Naliboff et al., 1991), conducted on women of two age groups, found an increase in immune function in the younger women after exposure to an acute stressor. Research has suggested that stressors varying in the degree to which active coping is possible may elicit different patterns of cardiovascular response. Lacey (1959) found that responses to some challenging tasks, such as mental arithmetic, were similar to general arousal reactions, including increases in heart rate (HR). Other challenges, such as listening for tones, produced HR decreases at the same time that other indicators of sympathetic arousal increased. Lacey argued that tasks involving thought or rejection of outside stimuli produced HR acceleration, whereas those involving attention and sensory intake caused HR to decrease. Obrist (1976) expanded on this distinction and referred to active coping, in which it is possible to have meaningful effects on outcomes and which involves HR increases. Passive coping, associated with tasks on which one has little control over outcomes, was more like Lacey's depiction of environmental intake. More recently, research has indicated that some challenges, such as mirror tracing, appear to be vascular stimuli, producing increases in peripheral resistance and BP but not reliably increasing HR (Kasprowicz, Manuck, Malkoff, & Krantz, 1990). Other stressful tasks, such as mental arithmetic, seem to affect resistance and function of the heart and therefore produce increases in both BP and HR. By establishing a controlled setting in which stress-related immune system changes can be produced, we can begin to examine several important questions. The present study was designed to determine whether and how quickly a 30-min stressor produced changes in lymphocyte proliferation to two commonly used mitogens and in the amount of circulating interleukin-1 and - 2 (IL-1 and IL-2). This study also considered passive and active stressors.
METHOD Subjects Twenty-nine men, ages 18 to 46 years (M = 31.3 years), participated in the study. Subjects were recruited through newspaper advertisements asking for volunteers for a study assessing the effects of task performance on physiological functioning. Eligibility for participation was determined by a standard telephone screening procedure.
All subjects were nonsmokers and had indicated that they were not using illicit drugs or taking any prescription or nonprescription drugs that could affect the physiological measures taken in this study (e.g., beta-blockers, steroids). In addition, volunteers who had chronic or current acute health problems (e.g., cancer, diabetes, high BP) or who were being treated for psychiatric problems (e.g., depression or anxiety) were excluded from the study. On the day before the scheduled session, subjects were asked if they had any illnesses or infections (e.g., cold or flu) and/or if they had been exposed to someone with any such illness. If the subject reported that he had recently been in contact with someone who was ill, the session was postponed to a later date. This was done in order to avoid the possible effects of already-occurring immune changes on the physiological measures collected in this study. All subjects provided informed consent and were paid $30 for participation. Procedures Participants were randomly assigned to one of two conditions: the experimental condition (n - 20), in which subjects were exposed to the stressor, or the control condition (« = 9), which served to control for the effects of the blood-drawing procedures and for possible circadian variations in the physiological measures (Tavadia, K. A. Fleming, Hume, & Simpson, 1975). Conducting all sessions at the same time of day provides some control for circadian effects but does not control for possible changes in the 2 hr between baseline and the final blood samples. Without a control condition, which could reflect any effects of time, results from the stress group would not be interpretable. All sessions were started between 7 and 8 a.m. and lasted approximately 3 hr. Subjects were asked not to eat any foods high in fat or cholesterol content the morning of the session. Blood-drawing procedures. Five 20-ml blood samples were drawn during the course of the study (see Figure 1). The first was taken at the time that the catheter was inserted. Because adrenal cortical response is not immediate but rather requires some time (5 to 15 min for modest stressors) to become apparent, this baseline sample was drawn to precede any effects of venipuncture that may have occurred. This also provided a reasonable estimate of baseline immune status. The second sample (stress sample) was drawn midway through the stressor (15 min after initiation of the stressor and 40 min after insertion of the catheter). The third sample was drawn 30 min after the stressor period ended (+30 min), 85 min after baseline. The fourth and fifth samples were taken 60 and 90 min (+60 and +90 min) after the stressor ended (i.e., 115 and 145 min after baseline). Experimental procedures. At the beginning of the session, subjects were given a brief description of the procedures and were asked to read and sign the consent form. After 10 min of rest, three baseline BP and HR measures were taken with an automated BP monitor programmed to inflate every 2 min. An intravenous catheter was then inserted into the subject's antecubital fossa, and the first 20-ml blood sample was taken. The catheter remained in the subject's vein for the entire 3-hr session; periodic infusions of 1 ml of Heparin (100 units/ml) prevented coagulation of blood in the line. A 20-min rest period followed insertion of the catheter, during which four more baseline BP and HR readings were taken. Cardiovascular measures were then recorded throughout the 30-min task.
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Rest 0 min
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baseline blood sample -cortisol -proliferation -IL-1 &2 -WBC
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145 min
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+ 90 min sample -cortisol -prolif. -IL-1&2 -WBC
V blood sample drawn
FIGURE 1 Timeline for blood draws, and measures taken at each point in time.
Subjects were told that they would be shown a videotape and were asked to pay close attention to the film because they would be asked later to recall aspects of it. Subjects were then shown a videotape depicting combat surgery in Vietnam. Immediately after the film, subjects completed a brief measure of how they felt and how aversive the film viewing had been. After this, subjects were asked to recall the details of the film, and their recollections were audiotaped. The stress questionnaire was then readministered, and the second blood sample (stress sample) was taken. Then, subjects were again asked to view the film and recall aspects of it. One subject refused to complete the second half of the stressor session. After the second viewing, subjects completed a questionnaire asking about their reactions to the film, and the recall task and stress questionnaires were readministered. After completion of the film and recall tasks, subjects were then told that they could relax and read magazines for the remaining 90 min of the session, during which the experimenter would take blood samples every 30 min (+30-, +60-, and +90-min samples). After the final blood sample was drawn, subjects were debriefed, paid, and thanked. Approximately 3 days after the session, subjects were called and asked whether they had come down with a cold or flu to further determine possible effects of infections on the physiological measures taken during the session. None of the subjects had become ill. The control group underwent identical procedures as the experimental group, with the exception of the stressor period. Instead of the surgery film and the recall tasks, subjects in the control condition were asked to watch a nonstressful film for the same amount of time. This film, showing landscape scenes from Africa with calming background music, has been used in other studies and has not been stressful. After the first half of the film, subjects completed the stress and film questionnaires, and the second blood sample was taken. Then the second half of the film was shown, and the two questionnaires were readministered. The procedures for the remainder of the session were the same as those used in the experimental condition.
Several measures were collected. Some were to determine subjects' background characteristics, some were to determine whether the film and memory tasks in the experimental condition were stressful, and others were to determine extraneous sources of stress. Last, immune measures were the primary dependent variables in this study. During the rest periods before and after the task, subjects were asked to complete questionnaires assessing life stress, health status, sociodemographic variables, and other background measures that could influence measures collected during the study. These questionnaires included a modification of the Life Change Inventory (Holmes & Rahe, 1967), the Perceived Stress Scale (PSS; Cohen, Kamarck, & Mermelstein, 1983), a questionnaire on perceived control and social support (R. Fleming, Baum, Gisriel, & Gatchel, 1982), the Symptom Check List-90-R (SCLr-90-R; Derogatis, 1977), the Beck Depression Inventory (BDI; Beck, Ward, Mendelson, Mock, & Erbaugh, 1961), the Cook-Medley Hostility Scale (Cook & Medley, 1954), a questionnaire asking about eating habits, and a checklist of foods consumed during the preceding 24 hr. Income, educational achievements, age, marital status, and socioeconomic status were also measured. Assessments of sleep and alcohol and caffeine consumption were made and were used to ensure that these factors did not affect the study results.
Stressor. The stressor in this study was a gruesome combat surgery film. It was used because it has produced reliable increases in hemodynamic and self-reported measures of stress in previous work in our laboratory. Many subjects react to the film by turning away or not watching closely. In order to discourage this, subjects were told that they would be asked to recall key aspects of the film
Stress measures. The stressfulness of the tasks was assessed in several ways. Self-reports of distress were gathered on two questionnaires. The first asked about feelings during the task. Subjects rated each item on a 5-point rating scale ranging from not at all (0) to extremely (4). This inventory was designed for this study and appeared to include five factors reflecting comfort (e.g., feeling
after they viewed it. The film, depicting scenes of amputation, debridement, and other surgical procedures, lasted 8 min, and the recall of details about the film lasted another 7 min. After a break for collection of measures, subjects saw the film again and were again asked to recall details of it. The second administration of the stressor was intended to extend the period of stress: Because acute immune system changes had not been heavily studied, we did not have a clear idea of "how much" exposure would be needed to cause changes if any occurred.
Measures
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ZAKOWSKI, MCALLISTER, DEAL, BAUM
relaxed, at ease, calm), somatic complaints (e.g., nausea, cold spells), lack of energy (e.g., feeling faint, low in energy), nervousness (e.g., feeling worried, tense, scared), and fear (e.g., feeling restless, shaky). These factors were reasonably internally consistent (as = .91, .85, .52, .37, and .43, respectively), and use of the factors reduced the number of comparisons to be made and minimized analysis of individual related measures. This questionnaire was administered four times during the session: before presentation of the films, after the first viewing of the film, and after each of the two recall periods. Subjects in the control groups were given this questionnaire before presentation of the films and after each of the nonstressful film segments. The second questionnaire consisted of five questions asking specifically about the subject's reaction to the film, including anger, inability to watch certain parts of the film, perceived stress, nausea, and nervousness. The subject was asked to rate each reaction on a 5-point scale ranging from not at all (0) to extremely (4). This survey was given twice during the session, after each viewing of the film. BP and HR measurements were taken before and during completion of the tasks using an automated BP monitor (Model SD700A, IBS Corporation) calibrated to a mercury column. An automatically inflating BP cuff was attached to the subject's nondominant arm and was inflated every 2 min during the first part of the session. These measures served as an additional manipulation check in order to ensure that the tasks were stressful to the subject and to measure the extent of cardiovascular reactivity of each subject to the stressor. BP and HR values were averaged within periods of the experimental session: Baseline values reflect the mean of baseline measures, and so on. Plasma cortisol levels were also measured at baseline, 30 min after completion of the tasks (+30 min), and at the end of the session (+90 min). Blood samples were frozen and later assayed using a Gammacoat competitive binding radioimmunoassay kit (Baxter Healthcare Corporation, Cambridge, MA).
pulsed with 20 \i\ of tritiated thymidine (50 nCi/ml; ICN Pharmaceuticals, Inc., Irvine, CA). After an additional incubation time of approximately 17 hr, the cultures were harvested onto glass-fiber filter paper using a cell harvester (Cambridge Technology, Inc., Watertown, MA). A Beckman LS5801 liquid scintillation counter, counting radioactive decays per minute (dpm), determined the incorporation of tritiated thymidine into the DNA present in the cultures. In order to determine the responsiveness to the mitogens alone, an average was taken from each of the triplicates, and the background counts from the cultures incubated in complete media without mitogens were subtracted. Levels of circulating IL-1 and IL-2 were measured at baseline and 90 min after completion of the tasks. This was done using ELISA (enzyme-linked immunosorbent assay) kits (Assay Research, Inc., College Park, MD). Purified antiserum against either IL-1 or IL-2 was diluted 1:10,000 in coating buffer (15.9 g/L Na2CO3; 29.3 g/L NaHCO3; pH = 9.6), and 100 (il/well were plated in an ElA plate and incubated for 2 hr. The wells were then washed with wash buffer (0.2% Twee 20 solution in phosphate-buffered saline [PBS]), and either 50 \il of each standard (100 ng/ml, 25 ng/ml, 6.25 ng/ml, 1.563 ng/ml, 0.39 ng/ml, and 0.098 ng/ml) or unknown samples were added to appropriate wells and incubated for 1 hr. To account for possible matrix effects, the standards were diluted in PBS containing 0.1% bovine serum albumin and 0.1% NaN3 or in a 50% serum solution for which endogenous IL-1 or IL-2 had been preabsorbed out. Following the 1-hr incubation, conjugated IL-1 or IL-2 (Assay Research, Inc.) was added to the wells and allowed to compete with IL-1 or IL-2 in the standards or samples for an additional hour. The wells were then washed and incubated for 45 min with conjugated alkaline-phosphatase (Assay Research, Inc.) followed by the addition of substrate. The resultant optical density was determined at 405 nm, and the data for the standard curve, as well as the potency estimates for the unknowns, were analyzed by computer-assisted four-parameter loglogit curve-fitting software (Microplate Manager, Bio-Rad).
Immunologic measures. Mitogen-stimulated lymphocyte proliferation was the primary measure of immune function considered in this study. We chose this measure due to its frequent use in previous studies of stress and immune function. Because we sought to test a model of acute stress and immunity in the laboratory, a widely used measure seemed the most appropriate choice. Blood samples were drawn into tubes prepared with ethylenediamene tetra-acetic acid at five times during the session. Quantitative and functional assays were performed in vitro in order to assess immune changes in response to the stressor. Immune function was measured by assessing lymphocyte proliferation in response to mitogen challenge. Lymphocytes were separated under sterile conditions by Ficoll-Hypaque sedimentation and adjusted to a final concentration of 2 x 10 mononuclear cells/ml of RPMI1640 medium with Hepes (Mediatech, Washington, DC) supplemented with 2 mM glutamine, 100 units/ml penicillin, 100 ng/ml streptomycin, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 5 x 10 M 2mercaptoethanol, and 5% heat inactivated fetal calf serum. Con A (Sigma Medical Company, St. Louis, MO) was used at concentrations of 5 and 10 fig/ml, and PHA (Sigma Medical Company) was used at 10 and 50 jig/ml. Background proliferation was measured by incubating cells in complete media only. Each assay was performed in triplicate. One 10th of a milliliter of PHA or Con A was added to 2 x 105 lymphocytes (0.1 ml complete medium) in 96-well plates. After a 53-hr incubation time in a humidified incubator with 5% CO2 at 37 "C, the cell cultures were
Two quantitative assays were also performed. White blood counts (WBCs) were done yielding the total number of white blood cells per cubic millimeter. Further, cell differential counts determined numbers of lymphocytes, monocytes, and granulocytes. RESULTS Data analyses were first directed toward establishing that the experimental and control groups were comparable and that no major differences between the groups were influential during the session. Self-reported mood, BP, HR, and cortisol levels before, during, and after the stressor period were used to determine whether the stressor was effective in inducing stress, and analyses comparing experimental and control subjects on these variables were conducted. Next, analyses considered the primary hypotheses being tested in this study—comparing the two groups' lymphocyte proliferation values over time to determine whether exposure to the stressor affected these values. After these analyses were completed, internal, post hoc analyses were conducted to determine whether BP reactivity during the stressor was related to changes in lymphocyte proliferation. Additional analyses explored the possibility that the different parts of the stressor (film vs. recall) affected proliferation values differently. Last, the possibility that differences in cortisol reactivity or in self-reported distress were associated with these outcomes was examined.
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STRESS, REACTIVITY, AND IMMUNE FUNCTION
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Group Comparability There were no differences between groups for any demographic or psychosocial variables—including occurrence of stressful life events and scores on the SCL-90-R, the PSS, and the BDI. Similarly, baseline BP, HR, self-reported distress, and lymphocyte responsiveness to the mitogens were comparable across the two groups. Subjects in the control group reported more symptoms of alienation (Ms = .34 and .14), F(l, 27) = 6.19, p < .05. However, this was not significantly correlated with any immune measures. Reported intensity of alienation by both groups was low and in the normal range, and differences in social support, which should be related, were not significant. The two groups were not significantly different on their hostility scores on the Cook-Medley Hostility Scale, and hostility scores were not significantly correlated with the immune measures or with BP reactivity during the stressor. There were no differences in consumption of caffeine during the 12 hr before the experimental session. Consumption was low, averaging less than 1 cup of coffee. Alcohol consumption and sleep the night before were also comparable. Manipulation Checks Several measures of stress responding were used to assess the effectiveness of the stress manipulation. Self-report data indicated that subjects shown the surgery film and asked to recall parts of it thought that these procedures were stressful (see Table 1). Because initial levels of reported distress could affect subsequent reports and because variance in these measures was homogeneous and the distribution apparently normal, repeated-measures analysis of covariance (ANCOVA) using baseline values as covariates were done. The two groups reported comparable affect before presentation of the films, but, during and after the stressor, subjects in the experimental group reported significantly more discomfort, F(\, 24) = 18.24,/> < .001, tension, F(l, 24) = 17.03,/? < .001, fear, F ( l , 23) = 3.99,/? < .05, and somatic distress, F(l, 24) = 6.81,/? < .01, than did control subjects. The groups also differed on how they rated the film; subjects in the experimental group rated it as significantly more
stressful than subjects in the control group rated the film they saw, F(l, 24) = 28.89,p < .001 (see Table 1). BP changes during the stressor also indicated that the session was stressful (see Figure 2). For the same reasons as noted earlier, repeated-measures ANCOVAs were performed on the cardiovascular measures covarying for baseline BP and HR responses. Systolic BP (SBP) was significantly higher in experimental subjects than in control subjects, F(l, 25) = 8.37, p < .01, but this was qualified by a Group x Time interaction, F(3, 78) = 5.67, p < .001. Tukey post hoc analyses indicated that, although SBP for the experimental and control subjects was comparable before the stressor and during the first 8-min film, experimental subjects exhibited significantly higher SBP during the second film viewing and both recall periods (all ps < .05). For diastolic BP (DBP), there was a significant time effect, F(3, 78) = 7.23, p < .001, and a Group x Time interaction, F(3, 78) = 4.56,p < .005, suggesting that experimental subjects showed DBP increases larger than those of the control subjects during the two recall periods. However, mean comparisons did not yield significant differences between the groups. No significant effects were found for HR. For cortisol, repeated-measures ANCOVA yielded a significant Group x Time interaction, with the experimental group (M = 138.5 ng/ml, SD = 35.7 ng/ml) showing higher values than the control group (M= 116.7 ng/ml, SD = 20.1 ng/ml) 30 min after the stressor, F(l, 22) = 5.06, p < .04. A significant time effect was also found, suggesting that cortisol decreased for both groups during the entire study, F(l, 22) = 5.05, p < .04. 135 I
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Stress, Reactivity, and Immune Function in Healthy Men Sandra G. Zakowski Uniformed Services University of the Health Sciences
Cathy G. McAllister University of Pittsburgh
Marlene Deal and Andrew Baum
This document is copyrighted by the American Psychological Association or one of its allied publishers. This article is intended solely for the personal use of the individual user and is not to be disseminated broadly.
Uniformed Services University of the Health Sciences We examined the effects of acute psychological stress on lymphocyte proliferation and circulating levels of interleukin-1 and - 2 . Healthy men were exposed to two viewings of a gruesome surgery film and were asked to recall details of the film twice during a 30-min period. These subjects were compared to a nonstress control group. Lymphocyte proliferation to the mitogen concanavalin A (Con A; 5 ug/ml) was decreased during and after exposure to the stressor when compared to the control group. This decrease was more pronounced in subjects exhibiting greater blood pressure reactivity while viewing the film than in subjects showing smaller blood pressure responses. None of the other immunological measures was significantly affected by the stressor. Cortisol was not correlated with lymphocyte responsiveness. Possible explanations for these results and implications for further research are discussed. Key words: stress, reactivity, lymphocyte proliferation, interleukin
Despite rapid growth in research on the relation between stress and immune function, several fundamental questions remain partly or completely unanswered. Many believe, for example, that changes in immune function mediate the association between stress and infectious illness (e.g., Jemmott & Locke, 1984), but studies providing definitive evidence of clinical outcomes of stress-related immunosuppression have not been reported. Likewise, the causal role of stress in these changes or the mechanisms by which they occur remain to be demonstrated under controlled laboratory conditions. The clearest evidence of stress-induced immunomodulation that we have is drawn from studies of various stressors on animals (e.g., Riley, 1981) and from studies of naturalistic stressors in humans (e.g., Kiecolt-Glaser & Glaser, 1987; McKinnon, Weisse, Reynolds, Bowles, & Baum, 1989). We have no clear information on several aspects of how long these changes take to occur, how long they last, or whether they have any clinical significance. The present study was designed to establish a stable model of acute stress that could be used to examine temporal and mechanistic properties of stress-induced changes in immune function. We sought (a) to determine whether this procedure could induce stress and produce reliable changes in immune function and (b) to elaborate relations among bodily systems. The effects of exposure to a 30-min passive stressor on mood, blood pressure (BP), plasma cortisol levels, and immune measures were assessed. The possible mediating role of arousal in immunological outcomes—represented by BP changes—was also examined.
STRESS AND IMMUNE FUNCTION For the most part, research on stress and immune function in humans has examined the effects of naturalistic stressors such as bereaveRequests for reprints should be sent to Andrew Baum, Hebert School of Medicine, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814.
ment, examinations, or marital distress. Bereavement is associated with reduced lymphocyte responsiveness to mitogen challenge (e.g., Bartrop, Luckhurst, Lazarus, Kiloh, & Penny, 1977; Schleifer, Keller, Camerino, Thornton, & Stein, 1983). Similarly, divorce and separation, providing long-term care to Alzheimer's patients, and chronic stress associated with living near the Three Mile Island nuclear power plant after the accident there have been associated with alterations in immune function reflecting suppressed activity (e.g., Kiecolt-Glaser, Fisher et al., 1987; Kiecolt-Glaser, Glaser et al., 1987; McKinnon et al., 1989). Depressed mood appears to be correlated with decreased lymphocyte responsiveness to phytohemagglutinin (PHA) and allogeneic cells in men with or without recent experience of family death or serious family illness (e.g., M. W. Linn, B. S. Linn, & Jensen, 1984). Less extreme stressors are also associated with changes in immune function. Studies of medical students facing major examinations have indicated that examination stress is associated with suppression of lymphocyte proliferation to PHA mitogen challenge, depression of natural killer cell activity, and increases in antibody titers to Epstein-Barr virus, suggesting suppression of cellular control of the latent virus (Glaser et al., 1987; Kiecolt-Glaser & Glaser, 1987; Workman & La Via, 1987). These changes were also related to perceived distress. Another study found elevated numbers of T and B cells and impaired mitogen responsiveness in trainees in psychiatry before final examinations (Dorian et al., 1982), and stress and anxiety due to hospitalization have been associated with decreased lymphocyte responsiveness as well (B. S. Linn, M. W. Linn, & Jensen, 1981). These findings are consistent with a large literature on stress and immunity in animals that suggests that stress causes changes in a variety of immune markers. These studies indicate that stress affects the immune system in many ways, usually reducing functional capabilities of cells or systems. Given that one of the primary foci of health psychology is the clinical application of research findings in natural settings, why step back into the laboratory to examine the short-term effects of a mild experimental stressor? Because most of the studies we have noted
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were correlational, it can be argued that we have little or no experimental evidence of stress-caused immune system changes in humans. In addition, the development of stable, controlled models of human stress that can be used to study the properties of stress-induced immune changes has lagged behind the development of animal models of these phenomena. This may be due to the belief that most immunological changes require extended stressor exposure or take days or weeks to become evident (Glaser et al., 1987). There now is evidence, however, of more rapid change: Several studies have reported immunological changes as a result of a brief (i.e. 30-min or less) stressor or exercise period (Eskola et al., 1978; Hedfors, Holm, & Ohnell, 1976; Manuck, Cohen, Rabin, Muldoon, & Bachen, 1991; Naliboff et al., 1991). Weisse et al. (1990), for example, reported decreases in lymphocyte proliferation to the mitogen concanavalin A (Con A) 50 min and 2Vi hr after exposure to controllable shock and noise stressors. Other studies have detected decreases in immune function 1 hr or less after exposure to the stressor (Eskola et al., 1978; Hedfors et al., 1976; Manuck et al., 1991). Only one study (Naliboff et al., 1991), conducted on women of two age groups, found an increase in immune function in the younger women after exposure to an acute stressor. Research has suggested that stressors varying in the degree to which active coping is possible may elicit different patterns of cardiovascular response. Lacey (1959) found that responses to some challenging tasks, such as mental arithmetic, were similar to general arousal reactions, including increases in heart rate (HR). Other challenges, such as listening for tones, produced HR decreases at the same time that other indicators of sympathetic arousal increased. Lacey argued that tasks involving thought or rejection of outside stimuli produced HR acceleration, whereas those involving attention and sensory intake caused HR to decrease. Obrist (1976) expanded on this distinction and referred to active coping, in which it is possible to have meaningful effects on outcomes and which involves HR increases. Passive coping, associated with tasks on which one has little control over outcomes, was more like Lacey's depiction of environmental intake. More recently, research has indicated that some challenges, such as mirror tracing, appear to be vascular stimuli, producing increases in peripheral resistance and BP but not reliably increasing HR (Kasprowicz, Manuck, Malkoff, & Krantz, 1990). Other stressful tasks, such as mental arithmetic, seem to affect resistance and function of the heart and therefore produce increases in both BP and HR. By establishing a controlled setting in which stress-related immune system changes can be produced, we can begin to examine several important questions. The present study was designed to determine whether and how quickly a 30-min stressor produced changes in lymphocyte proliferation to two commonly used mitogens and in the amount of circulating interleukin-1 and - 2 (IL-1 and IL-2). This study also considered passive and active stressors.
METHOD Subjects Twenty-nine men, ages 18 to 46 years (M = 31.3 years), participated in the study. Subjects were recruited through newspaper advertisements asking for volunteers for a study assessing the effects of task performance on physiological functioning. Eligibility for participation was determined by a standard telephone screening procedure.
All subjects were nonsmokers and had indicated that they were not using illicit drugs or taking any prescription or nonprescription drugs that could affect the physiological measures taken in this study (e.g., beta-blockers, steroids). In addition, volunteers who had chronic or current acute health problems (e.g., cancer, diabetes, high BP) or who were being treated for psychiatric problems (e.g., depression or anxiety) were excluded from the study. On the day before the scheduled session, subjects were asked if they had any illnesses or infections (e.g., cold or flu) and/or if they had been exposed to someone with any such illness. If the subject reported that he had recently been in contact with someone who was ill, the session was postponed to a later date. This was done in order to avoid the possible effects of already-occurring immune changes on the physiological measures collected in this study. All subjects provided informed consent and were paid $30 for participation. Procedures Participants were randomly assigned to one of two conditions: the experimental condition (n - 20), in which subjects were exposed to the stressor, or the control condition (« = 9), which served to control for the effects of the blood-drawing procedures and for possible circadian variations in the physiological measures (Tavadia, K. A. Fleming, Hume, & Simpson, 1975). Conducting all sessions at the same time of day provides some control for circadian effects but does not control for possible changes in the 2 hr between baseline and the final blood samples. Without a control condition, which could reflect any effects of time, results from the stress group would not be interpretable. All sessions were started between 7 and 8 a.m. and lasted approximately 3 hr. Subjects were asked not to eat any foods high in fat or cholesterol content the morning of the session. Blood-drawing procedures. Five 20-ml blood samples were drawn during the course of the study (see Figure 1). The first was taken at the time that the catheter was inserted. Because adrenal cortical response is not immediate but rather requires some time (5 to 15 min for modest stressors) to become apparent, this baseline sample was drawn to precede any effects of venipuncture that may have occurred. This also provided a reasonable estimate of baseline immune status. The second sample (stress sample) was drawn midway through the stressor (15 min after initiation of the stressor and 40 min after insertion of the catheter). The third sample was drawn 30 min after the stressor period ended (+30 min), 85 min after baseline. The fourth and fifth samples were taken 60 and 90 min (+60 and +90 min) after the stressor ended (i.e., 115 and 145 min after baseline). Experimental procedures. At the beginning of the session, subjects were given a brief description of the procedures and were asked to read and sign the consent form. After 10 min of rest, three baseline BP and HR measures were taken with an automated BP monitor programmed to inflate every 2 min. An intravenous catheter was then inserted into the subject's antecubital fossa, and the first 20-ml blood sample was taken. The catheter remained in the subject's vein for the entire 3-hr session; periodic infusions of 1 ml of Heparin (100 units/ml) prevented coagulation of blood in the line. A 20-min rest period followed insertion of the catheter, during which four more baseline BP and HR readings were taken. Cardiovascular measures were then recorded throughout the 30-min task.
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Rest 0 min
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baseline blood sample -cortisol -proliferation -IL-1 &2 -WBC
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FIGURE 1 Timeline for blood draws, and measures taken at each point in time.
Subjects were told that they would be shown a videotape and were asked to pay close attention to the film because they would be asked later to recall aspects of it. Subjects were then shown a videotape depicting combat surgery in Vietnam. Immediately after the film, subjects completed a brief measure of how they felt and how aversive the film viewing had been. After this, subjects were asked to recall the details of the film, and their recollections were audiotaped. The stress questionnaire was then readministered, and the second blood sample (stress sample) was taken. Then, subjects were again asked to view the film and recall aspects of it. One subject refused to complete the second half of the stressor session. After the second viewing, subjects completed a questionnaire asking about their reactions to the film, and the recall task and stress questionnaires were readministered. After completion of the film and recall tasks, subjects were then told that they could relax and read magazines for the remaining 90 min of the session, during which the experimenter would take blood samples every 30 min (+30-, +60-, and +90-min samples). After the final blood sample was drawn, subjects were debriefed, paid, and thanked. Approximately 3 days after the session, subjects were called and asked whether they had come down with a cold or flu to further determine possible effects of infections on the physiological measures taken during the session. None of the subjects had become ill. The control group underwent identical procedures as the experimental group, with the exception of the stressor period. Instead of the surgery film and the recall tasks, subjects in the control condition were asked to watch a nonstressful film for the same amount of time. This film, showing landscape scenes from Africa with calming background music, has been used in other studies and has not been stressful. After the first half of the film, subjects completed the stress and film questionnaires, and the second blood sample was taken. Then the second half of the film was shown, and the two questionnaires were readministered. The procedures for the remainder of the session were the same as those used in the experimental condition.
Several measures were collected. Some were to determine subjects' background characteristics, some were to determine whether the film and memory tasks in the experimental condition were stressful, and others were to determine extraneous sources of stress. Last, immune measures were the primary dependent variables in this study. During the rest periods before and after the task, subjects were asked to complete questionnaires assessing life stress, health status, sociodemographic variables, and other background measures that could influence measures collected during the study. These questionnaires included a modification of the Life Change Inventory (Holmes & Rahe, 1967), the Perceived Stress Scale (PSS; Cohen, Kamarck, & Mermelstein, 1983), a questionnaire on perceived control and social support (R. Fleming, Baum, Gisriel, & Gatchel, 1982), the Symptom Check List-90-R (SCLr-90-R; Derogatis, 1977), the Beck Depression Inventory (BDI; Beck, Ward, Mendelson, Mock, & Erbaugh, 1961), the Cook-Medley Hostility Scale (Cook & Medley, 1954), a questionnaire asking about eating habits, and a checklist of foods consumed during the preceding 24 hr. Income, educational achievements, age, marital status, and socioeconomic status were also measured. Assessments of sleep and alcohol and caffeine consumption were made and were used to ensure that these factors did not affect the study results.
Stressor. The stressor in this study was a gruesome combat surgery film. It was used because it has produced reliable increases in hemodynamic and self-reported measures of stress in previous work in our laboratory. Many subjects react to the film by turning away or not watching closely. In order to discourage this, subjects were told that they would be asked to recall key aspects of the film
Stress measures. The stressfulness of the tasks was assessed in several ways. Self-reports of distress were gathered on two questionnaires. The first asked about feelings during the task. Subjects rated each item on a 5-point rating scale ranging from not at all (0) to extremely (4). This inventory was designed for this study and appeared to include five factors reflecting comfort (e.g., feeling
after they viewed it. The film, depicting scenes of amputation, debridement, and other surgical procedures, lasted 8 min, and the recall of details about the film lasted another 7 min. After a break for collection of measures, subjects saw the film again and were again asked to recall details of it. The second administration of the stressor was intended to extend the period of stress: Because acute immune system changes had not been heavily studied, we did not have a clear idea of "how much" exposure would be needed to cause changes if any occurred.
Measures
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ZAKOWSKI, MCALLISTER, DEAL, BAUM
relaxed, at ease, calm), somatic complaints (e.g., nausea, cold spells), lack of energy (e.g., feeling faint, low in energy), nervousness (e.g., feeling worried, tense, scared), and fear (e.g., feeling restless, shaky). These factors were reasonably internally consistent (as = .91, .85, .52, .37, and .43, respectively), and use of the factors reduced the number of comparisons to be made and minimized analysis of individual related measures. This questionnaire was administered four times during the session: before presentation of the films, after the first viewing of the film, and after each of the two recall periods. Subjects in the control groups were given this questionnaire before presentation of the films and after each of the nonstressful film segments. The second questionnaire consisted of five questions asking specifically about the subject's reaction to the film, including anger, inability to watch certain parts of the film, perceived stress, nausea, and nervousness. The subject was asked to rate each reaction on a 5-point scale ranging from not at all (0) to extremely (4). This survey was given twice during the session, after each viewing of the film. BP and HR measurements were taken before and during completion of the tasks using an automated BP monitor (Model SD700A, IBS Corporation) calibrated to a mercury column. An automatically inflating BP cuff was attached to the subject's nondominant arm and was inflated every 2 min during the first part of the session. These measures served as an additional manipulation check in order to ensure that the tasks were stressful to the subject and to measure the extent of cardiovascular reactivity of each subject to the stressor. BP and HR values were averaged within periods of the experimental session: Baseline values reflect the mean of baseline measures, and so on. Plasma cortisol levels were also measured at baseline, 30 min after completion of the tasks (+30 min), and at the end of the session (+90 min). Blood samples were frozen and later assayed using a Gammacoat competitive binding radioimmunoassay kit (Baxter Healthcare Corporation, Cambridge, MA).
pulsed with 20 \i\ of tritiated thymidine (50 nCi/ml; ICN Pharmaceuticals, Inc., Irvine, CA). After an additional incubation time of approximately 17 hr, the cultures were harvested onto glass-fiber filter paper using a cell harvester (Cambridge Technology, Inc., Watertown, MA). A Beckman LS5801 liquid scintillation counter, counting radioactive decays per minute (dpm), determined the incorporation of tritiated thymidine into the DNA present in the cultures. In order to determine the responsiveness to the mitogens alone, an average was taken from each of the triplicates, and the background counts from the cultures incubated in complete media without mitogens were subtracted. Levels of circulating IL-1 and IL-2 were measured at baseline and 90 min after completion of the tasks. This was done using ELISA (enzyme-linked immunosorbent assay) kits (Assay Research, Inc., College Park, MD). Purified antiserum against either IL-1 or IL-2 was diluted 1:10,000 in coating buffer (15.9 g/L Na2CO3; 29.3 g/L NaHCO3; pH = 9.6), and 100 (il/well were plated in an ElA plate and incubated for 2 hr. The wells were then washed with wash buffer (0.2% Twee 20 solution in phosphate-buffered saline [PBS]), and either 50 \il of each standard (100 ng/ml, 25 ng/ml, 6.25 ng/ml, 1.563 ng/ml, 0.39 ng/ml, and 0.098 ng/ml) or unknown samples were added to appropriate wells and incubated for 1 hr. To account for possible matrix effects, the standards were diluted in PBS containing 0.1% bovine serum albumin and 0.1% NaN3 or in a 50% serum solution for which endogenous IL-1 or IL-2 had been preabsorbed out. Following the 1-hr incubation, conjugated IL-1 or IL-2 (Assay Research, Inc.) was added to the wells and allowed to compete with IL-1 or IL-2 in the standards or samples for an additional hour. The wells were then washed and incubated for 45 min with conjugated alkaline-phosphatase (Assay Research, Inc.) followed by the addition of substrate. The resultant optical density was determined at 405 nm, and the data for the standard curve, as well as the potency estimates for the unknowns, were analyzed by computer-assisted four-parameter loglogit curve-fitting software (Microplate Manager, Bio-Rad).
Immunologic measures. Mitogen-stimulated lymphocyte proliferation was the primary measure of immune function considered in this study. We chose this measure due to its frequent use in previous studies of stress and immune function. Because we sought to test a model of acute stress and immunity in the laboratory, a widely used measure seemed the most appropriate choice. Blood samples were drawn into tubes prepared with ethylenediamene tetra-acetic acid at five times during the session. Quantitative and functional assays were performed in vitro in order to assess immune changes in response to the stressor. Immune function was measured by assessing lymphocyte proliferation in response to mitogen challenge. Lymphocytes were separated under sterile conditions by Ficoll-Hypaque sedimentation and adjusted to a final concentration of 2 x 10 mononuclear cells/ml of RPMI1640 medium with Hepes (Mediatech, Washington, DC) supplemented with 2 mM glutamine, 100 units/ml penicillin, 100 ng/ml streptomycin, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 5 x 10 M 2mercaptoethanol, and 5% heat inactivated fetal calf serum. Con A (Sigma Medical Company, St. Louis, MO) was used at concentrations of 5 and 10 fig/ml, and PHA (Sigma Medical Company) was used at 10 and 50 jig/ml. Background proliferation was measured by incubating cells in complete media only. Each assay was performed in triplicate. One 10th of a milliliter of PHA or Con A was added to 2 x 105 lymphocytes (0.1 ml complete medium) in 96-well plates. After a 53-hr incubation time in a humidified incubator with 5% CO2 at 37 "C, the cell cultures were
Two quantitative assays were also performed. White blood counts (WBCs) were done yielding the total number of white blood cells per cubic millimeter. Further, cell differential counts determined numbers of lymphocytes, monocytes, and granulocytes. RESULTS Data analyses were first directed toward establishing that the experimental and control groups were comparable and that no major differences between the groups were influential during the session. Self-reported mood, BP, HR, and cortisol levels before, during, and after the stressor period were used to determine whether the stressor was effective in inducing stress, and analyses comparing experimental and control subjects on these variables were conducted. Next, analyses considered the primary hypotheses being tested in this study—comparing the two groups' lymphocyte proliferation values over time to determine whether exposure to the stressor affected these values. After these analyses were completed, internal, post hoc analyses were conducted to determine whether BP reactivity during the stressor was related to changes in lymphocyte proliferation. Additional analyses explored the possibility that the different parts of the stressor (film vs. recall) affected proliferation values differently. Last, the possibility that differences in cortisol reactivity or in self-reported distress were associated with these outcomes was examined.
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Group Comparability There were no differences between groups for any demographic or psychosocial variables—including occurrence of stressful life events and scores on the SCL-90-R, the PSS, and the BDI. Similarly, baseline BP, HR, self-reported distress, and lymphocyte responsiveness to the mitogens were comparable across the two groups. Subjects in the control group reported more symptoms of alienation (Ms = .34 and .14), F(l, 27) = 6.19, p < .05. However, this was not significantly correlated with any immune measures. Reported intensity of alienation by both groups was low and in the normal range, and differences in social support, which should be related, were not significant. The two groups were not significantly different on their hostility scores on the Cook-Medley Hostility Scale, and hostility scores were not significantly correlated with the immune measures or with BP reactivity during the stressor. There were no differences in consumption of caffeine during the 12 hr before the experimental session. Consumption was low, averaging less than 1 cup of coffee. Alcohol consumption and sleep the night before were also comparable. Manipulation Checks Several measures of stress responding were used to assess the effectiveness of the stress manipulation. Self-report data indicated that subjects shown the surgery film and asked to recall parts of it thought that these procedures were stressful (see Table 1). Because initial levels of reported distress could affect subsequent reports and because variance in these measures was homogeneous and the distribution apparently normal, repeated-measures analysis of covariance (ANCOVA) using baseline values as covariates were done. The two groups reported comparable affect before presentation of the films, but, during and after the stressor, subjects in the experimental group reported significantly more discomfort, F(\, 24) = 18.24,/> < .001, tension, F(l, 24) = 17.03,/? < .001, fear, F ( l , 23) = 3.99,/? < .05, and somatic distress, F(l, 24) = 6.81,/? < .01, than did control subjects. The groups also differed on how they rated the film; subjects in the experimental group rated it as significantly more
stressful than subjects in the control group rated the film they saw, F(l, 24) = 28.89,p < .001 (see Table 1). BP changes during the stressor also indicated that the session was stressful (see Figure 2). For the same reasons as noted earlier, repeated-measures ANCOVAs were performed on the cardiovascular measures covarying for baseline BP and HR responses. Systolic BP (SBP) was significantly higher in experimental subjects than in control subjects, F(l, 25) = 8.37, p < .01, but this was qualified by a Group x Time interaction, F(3, 78) = 5.67, p < .001. Tukey post hoc analyses indicated that, although SBP for the experimental and control subjects was comparable before the stressor and during the first 8-min film, experimental subjects exhibited significantly higher SBP during the second film viewing and both recall periods (all ps < .05). For diastolic BP (DBP), there was a significant time effect, F(3, 78) = 7.23, p < .001, and a Group x Time interaction, F(3, 78) = 4.56,p < .005, suggesting that experimental subjects showed DBP increases larger than those of the control subjects during the two recall periods. However, mean comparisons did not yield significant differences between the groups. No significant effects were found for HR. For cortisol, repeated-measures ANCOVA yielded a significant Group x Time interaction, with the experimental group (M = 138.5 ng/ml, SD = 35.7 ng/ml) showing higher values than the control group (M= 116.7 ng/ml, SD = 20.1 ng/ml) 30 min after the stressor, F(l, 22) = 5.06, p < .04. A significant time effect was also found, suggesting that cortisol decreased for both groups during the entire study, F(l, 22) = 5.05, p < .04. 135 I
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