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ORIGINAL ARTICLES |
From the School of Psychology, University of Western Sydney (A.J.S); School of Psychiatry, University of New South Wales (U.V-C., B.B.); Brain and Mind Institute, Sydney University (I.B.H.); and Inflammation Research Unit, School of Medical Sciences, University of New South Wales (A.R.L.).
Address correspondence and reprint requests to A.J. Smith, PhD, School of Psychology, University of Western Sydney, Locked Bag 1797 Penrith South DC, NSW, 1797 Australia. E-mail: aj.smith{at}uws.edu.au
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ABSTRACT |
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METHODS: Subjects (N = 166) were immunized (baseline) with the novel antigen, keyhole limpet hemocyanin (KLH), and DTH skin test responses against KLH were assessed 3 weeks later (follow-up). The CMI Multitest (Merieux, France), which evaluates DTH responses to a panel of seven antigens, was also administered at follow-up. Emotional distress was assessed at baseline and follow-up by the Profile of Mood States.
RESULTS: Distress levels at baseline were associated with a reduced likelihood of developing DTH responses against KLH at follow-up (r = –0.22, p = .01). There was no relationship between distress at follow-up and cutaneous DTH in response to KLH (r = 0.09, p = .24) or in the Multitest (r = –0.03, p = .70). In addition, higher levels of alcohol consumption at baseline (r = –0.19, p = .02) and at follow-up (r = –0.20, p = .01) were associated with a decreased likelihood of developing cutaneous DTH against KLH.
CONCLUSIONS: Everyday levels of distress predicted the capacity of the cellular arm of the immune system to exhibit recall responses to an antigen, when the experimental paradigm allowed the assessment of distress at the time of antigen sensitization. Moderate alcohol consumption independently affected cutaneous DTH.
Key Words: Keyhole limpet hemocyanin, • alcohol, • psychoneuroimmunology, • distress, • delayed-type hypersensitivity.
Abbreviations: DTH = delayed-type hypersensitivity;; KLH = keyhole limpet hemocyanin;; POMS = profile of mood states.
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INTRODUCTION |
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One such measure is delayed-type hypersensitivity (DTH), a systemic, cellular immune response that can be conveniently assessed via skin tests that provide a "window" on those systemic processes (4). Cellular immunity is of particular interest because there is evidence that stress results in a shift of cytokine patterns from those associated with a cellular immune response to those associated with a humoral response (5). In addition, alterations in DTH may be of clinical significance (6). To date, the assessment of cutaneous DTH in psychoneuroimmunology research has relied largely on the CMI Multitest (Merieux, France). This test comprises a convenient plastic applicator with 7 ubiquitous antigens (tetanus, diphtheria, streptococcus, tuberculin, candida, trichophyton, and proteus) and a glycerine control preloaded in eight tined heads (7). A decline in immune function, associated with severe (melancholic) depression (8,9), wintering in the Antarctic (10), and perceived distress (11), has been demonstrated using this apparatus.
Despite the ease of use and the standardization of the Multitest, a considerable limitation in interpreting DTH results is the lack of control over prior exposure to the antigens (12). Consequently, variations in responsiveness between individuals and/or those from different geographical locations observed in psychoneuroimmunology research may reflect differences in prior immunization or natural exposure rather than the effects of psychosocial factors (7,9,13). Interpretation of brain/immune relationships is therefore difficult. A better technique of assessing the effects of psychosocial factors on DTH would be to immunize subjects with a novel antigen and to assess responses to this antigen. This would permit assessment of psychological and social functioning both at the time of sensitization with the antigen and the later induction of a response. The method adopted in this study permitted an examination of the relationship between distress at both these time points and DTH responses. The potential differential effects resulting from the uncontrolled nature of exposure to the Multitest antigens was assessed by comparing the relationship between distress at follow-up and DTH responses in the CMI Multitest and in response to a novel antigen.
Keyhole limpet hemocyanin (KLH) is ideal for this purpose (6). KLH is a purified protein derived from the hemolymph of a marine mollusc, the keyhole limpet (Megathura crenulata). It has been extensively used with human subjects in both clinical and experimental immunology (14). DTH skin test responses against KLH can be reliably elicited in human subjects (6,15), and an appropriate immunization protocol has been developed (6). In addition, cutaneous DTH against KLH may be indicative of overall cellular immune competence since responsiveness to KLH has been shown to correlate with responsiveness to a battery of prognostic antigens (16,17).
We have previously reported that cutaneous DTH responses against KLH at follow-up were related to levels of distress at baseline (6). Distressed participants (medical students) were less likely than nondistressed individuals to develop cutaneous DTH against KLH. Variance in levels of distress was promoted by immunizing half of the subjects at the time of an important examination, and half at the time of low university-related distress. In the current study, a deliberate decision was taken to immunize participants early in the university session, when university-related stressors were likely to be minimal. This provided an opportunity to evaluate the effect of the vicissitudes of everyday life on cellular immune competence in this group of healthy, young students.
In summary, the aims of the current study were: 1) to examine the effects of everyday levels of distress on cutaneous DTH against KLH; and 2) to compare the effects of distress on DTH against a specific antigen and in the CMI Multitest.
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METHODS |
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Approval for this research was granted by the Committee on Experimental Procedures Involving Human Subjects of the University of New South Wales. Informed, written consent was obtained from all subjects before the commencement of procedures. The exclusion criteria adopted were: 1) a history of seafood allergy; 2) pregnancy; 3) a significant illness or operation in the past month; 4) a history of major immune related illness (eg, diabetes, cancer); 5) use of immunosuppressive medication; and 6) symptoms of a likely infective illness at the commencement of the study.
Measure of Distress
Current distress was assessed using the profile of mood states (POMS) questionnaire. Six subscales are provided: depression-dejection (referred to as depression), tension-anxiety (tension), fatigue-inertia (fatigue), anger-hostility, confusion/bewilderment, and vigor-activity. Interest in this research focused on the depression, anxiety, and fatigue subscales because these states represent the most prevalent psychological disturbances reported in the community (18). A total POMS score was also calculated by summing across the six subscales (vigor is subtracted) (19). Subjects were asked to rate how well 65 adjectives (eg, tense, blue, exhausted) described them over the past week, including the current day on a 5-point rating scale from 0 (not at all) to 4 (extremely). The scale has been shown to be reliable, and its construct validity has been demonstrated (19).
Behavioral Assessment
Behavioral factors that affect immune function (20,21) were monitored. The number of cigarettes smoked daily, average weekly alcohol consumption, amount of aerobic exercise over the past week, hours of sleep loss, and any weight loss over the previous month were reported. Female students recorded the stage of their menstrual cycle. The exclusion criteria ensured that participants with either acute or chronic illnesses, or taking immunosuppressive medication were not included in the study.
Immune Measures
Subjects were immunized with 0.1 mg of KLH (1.25 mg/mL) adsorbed to 0.9 mg of the adjuvant alum (45 mg/mL) administered into the deltoid muscle (6). The DTH skin test against KLH involved the intradermal administration of 0.001 mg of KLH in 0.01 mL of saline solution to the volar aspect of the arm. The response was read 48 hours after administration by measuring induration in millimeters for two diameters at right angles. The CMI Multitest was also administered, and the development of DTH skin test responses was measured at 48 hours in the same manner as for the DTH responses against KLH. A positive cutaneous DTH response against KLH was defined as an induration of greater than 2 mm (17). The criterion for nonresponse (anergy) on the CMI Multitest was no response of at least 2 mm (7). The number of responses in the Multitest was also examined.
Procedure
Subjects were seen on two occasions: at baseline and 3 weeks later for follow-up. At baseline, subjects were immunized with KLH/alum, and the distress and behavioral measures were administered. At follow-up, DTH against KLH and in the CMI Multitest were assessed, and the distress and behavioral measures were re-administered. Participants were trained to read their own DTH responses. They were instructed to ignore any erythema and to identify the area of induration by carefully approaching the immunization site with a ballpoint pen. Typically, the pen stops at the edge of the induration (16). Readings were taken using a ruler, and the participants were phoned to obtain their results. All subjects administered a DTH skin test provided this information.
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RESULTS |
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Distress and DTH
There was considerable variability in the total scores obtained by subjects on the POMS, both at immunization (M = 31.1, SD = 27.3) and follow-up (M = 29.5, SD = 28.7). A paired sample t test indicated that there was no significant difference between scores on the POMS at the two time points (t (163) = 0.63, p = .53), and the scores were moderately correlated (r = 0.63, p = .00).
Participants who were more distressed at the time of baseline immunization were less likely to develop DTH skin test responses against KLH than were less distressed subjects, as indicated by the total POMS score (r = –0.22, p = .01). The total score at follow-up did not predict these DTH responses (r = 0.09, p = .24).
To better understand the factors contributing to the relationship between distress and the development of cutaneous DTH against KLH, the correlations between the three subscales of interest and the measure of cellular immune competence were examined. The participants scored below college norms and were more homogeneous on all three subscales (19) (Table 1). Fatigue (r = –0.19, p = .01) and tension (r = –0.17, p = .03) were both predictors of DTH against KLH, while depression was not predictive (r = –0.14, p=.07) (Table 2). However, neither tension nor fatigue predicted the immune measure independently. When tension was controlled, a partial correlation indicated that fatigue no longer predicted DTH responses (r = 0.11, p = .15); when fatigue was controlled, tension did not predict DTH (r = 0.07, p = .36). There was a tendency for participants who were more fatigued at the time of immunization to have had less sleep in the month before immunization (r = –0.16, p = .05).
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Behavioral/Demographic Measures and DTH
Relationships are reported with cutaneous DTH against KLH for both baseline and follow-up behavioral and demographic measures, because cutaneous responses to KLH are potentially affected by health-related factors at either the sensitization or the induction phase of the immune response. Relationships between the CMI Multitest and follow-up demographic and behavioral measures are reported because only behaviors at this phase of the response can be assessed. The timing of sensitization is unknown.
The mean number of standard drinks consumed per week was 3.1 (SD = 5.5) at baseline and 2.9 (SD = 5.7) at follow-up. DTH skin test responses against KLH were correlated with alcohol consumption at immunization (r = –0.19, p = .02) and at follow-up (r = –0.20, p = .01). These alcohol measures were themselves positively correlated (r = 0.80, p = .00). Partial correlations with alcohol consumption at baseline and DTH against KLH, controlling for alcohol consumption at follow-up, revealed no relationship (r = –0.01, p = .82). Alcohol consumption at follow-up predicted DTH against KLH independently of consumption at baseline (r = 0.17, p = .03). Thus it seems that alcohol consumption only at the time of induction of a DTH response is related to the immune measure. Similarly, alcohol consumption at follow-up was correlated with CMI Multitest categories (r = –0.16, p = .04) and number of responses (r = –0.20, p = .01).
Gender was not associated with the measure of anti-KLH immunity. However, as expected from previous research (7), men were less likely than women to be anergic on the CMI Multitest [
2 (1, 158) = 8.72; p = .00] and developed greater numbers of responses (r = –0.36, p = .00). There were no differences between Asians and Caucasians in the likelihood of developing a DTH response in against KLH [2 (1, 161) = 1.81; p = .19] or in the likelihood of responding the CMI Multitest [2 (1, 154) = 0.17; p = .81]. However, Asians developed more responses to the Multitest than did Caucasians (r = 0.18, p = .03).
None of the remaining behavioral or demographic factors were related to the development of cutaneous DTH, either against KLH or in the Multitest. No correlation exceeded 0.11 (p = .18) and no 2 exceeded 1.81 (p = .18).
Logistic Regression
Logistic regression was used to explore the capacity of distress at baseline and alcohol consumption at follow-up to predict the likelihood of developing DTH responses against KLH (22).
Data were available for 165 participants. The model was significantly different from the constant only model [2 (2, 166) = 18.82; p = .0001] and correctly predicted the DTH skin test responder status in 67% of cases (77% of responders and 56% of nonresponders). The Wald statistic was significant for both the POMS (z = 10.05, p = .00) and alcohol consumption (z = 7.46, p = .01) (Table 3).
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Relationships Between Immune Measures
Subjects who were anergic on the CMI Multitest were less likely to develop cutaneous DTH responses against KLH [2 (1, 158) = 7.18; p = .007]. Of the 23 subjects who were anergic on the CMI Multitest, 17 failed to develop DTH skin test responses against KLH. Similarly, KLH-induced DTH skin test responses correlated positively with the number of responses on the Multitest (r = 0.35, p = .000).
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DISCUSSION |
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This study, by virtue of its correlational nature, does not say anything about the direction of causality of the relationship between distress and immune function. In particular, the correlation between fatigue and DTH responsiveness against KLH (Table 2) may be the result of an underlying, sub-symptomatic infection (26). Alternatively, the relationship between distress and cellular immune function may reflect alterations in health behaviors, although no such behavior was identified in this study. Baseline fatigue was marginally associated with baseline sleep disturbance, but this sleep disturbance was not related to immune function. In the case of alcohol consumption, partial correlations indicated that baseline distress levels predicted DTH against KLH independently of follow-up alcohol consumption, indicating that this behavior was not responsible for the correlation between distress at baseline and anti-KLH cellular immunity.
Neither the DTH responses against KLH or in the Multitest were related to distress measured at the time of follow-up, although the levels of distress were comparable at the two time points. In the case of the anti-KLH DTH responses, this is consistent with our past research and suggests that immunological processes at the time of immunization are more sensitive to the effects of psychological factors (6). A number of mechanisms that might mediate the relationship between distress at immunization and immune function have been identified. Research with animal models, immunized with a T-dependent antigen at the time of stressor exposure, suggests that T-cell functions are involved in these stressor-dependent effects. Consistent with this conclusion, the antibody response to a T-dependent antigen, but not to a T-independent antigen, was reduced by stressor exposure (27). Macrophage function has been implicated in these effects, since both phagocytosis (28) and expression of class II molecules (29) are reduced in these models. A role has also been suggested for IL-2 production by monocytes (30,31). The development of antibodies to KLH in immunized rats has been associated with alterations in draining lymph nodes of T–cell subsets (32) and a reduction in TH1 helper T-cells (33), possibly as a result of increased macrophage production of nitric oxide (34). Alterations in T-cell trafficking and activation may also be responsible for modulation of immune responses.
It is not possible to demonstrate the influence of distress at the time of sensitization with the Multitest because there is no control over timing of exposure to the Multitest antigens. No association between the Multitest and distress at follow-up were evident in the present study. However, relationships between emotional disturbance at the time of administration of the Multitest and the subsequent development of cutaneous DTH have been demonstrated (8–11). These associations are difficult to interpret in light of the influence of prior exposure to the seven antigens. This influence is illustrated by a comparison of results for different geographical locations. Rates of anergy on the CMI Multitest (no responses of at least 2 mm) in this study (12%) were considerably higher than the 1% anergy observed in the United States (7), but consistent with the levels of anergy observed in the healthy Australian population (1 to 3% in men and 5.6 to 11% in women) (35). The sample in the present study included both Asian and Caucasian subjects, whereas subjects in the other Australian study were principally Caucasian in background. However, the somewhat higher rates of anergy in the present sample are unlikely to be because of the inclusion of more Asian subjects, since the Asians tended to have more DTH responses to the Multitest antigens (r = 0.18, p = .03) than did the Caucasian subjects. Subjects in this sample were younger (mean age = 20.4 years) than in the Australian comparison group (mean age = 44.7 years). Differences in rates of response may reflect lower rates of exposure to the antigens in the younger sample, compared with the older group. Whatever the explanation for the variable rates of anergy in these samples, the differences in response rates to the Multitest antigens appear to be due to varying rates of exposure between individuals. This conclusion emphasizes the value of using a novel antigen in the assessment of immune function, and it is supported by the failure to detect racial differences for the cutaneous DTH responses to intradermal administration of KLH.
Care needs to be taken when assessing cutaneous DTH responses against a specific antigen to avoid problems associated with the procedure. These problems include batch-to-batch variation in the immunogenicity of the antigen, consistent administration of the same amount of the antigen, and accurate intradermal delivery (36,37). The Pierce (Rockford, IL) preparation of KLH used in this research is characterized by consistent immunogenicity for different lots of the antigen. However, as a further precaution against batch-to-batch variability, the same lot of KLH was used throughout this research. Considerable care was taken in the administration of the exact amount of the immunogen. The DTH skin tests for KLH were administered by medical staff with extensive experience in delivering the antigen intradermally. Correct administration into the dermal layer of the skin is characterized by a raised area (blip) containing the antigen immediately after delivery.
Those individuals who consumed more alcohol at follow-up were less likely to develop cutaneous DTH against KLH, and they displayed reduced DTH skin test responses in the Multitest. In vitro, physiological doses of alcohol can suppress cellular and humoral immune function (38–41). In vivo, a history of alcoholism has been associated with immune suppression (42–44) and with greater rates of bacterial infections (45). However, it is not clear whether these effects are due to the alcohol itself or to behaviors (such as inadequate nutrition and lifestyle factors) associated with alcoholism (45). In addition, the effects of everyday levels of alcohol consumption may be quite different from the effects of alcohol abuse (46).
Subjects who were anergic on the Multitest were more likely to be anergic on the DTH skin test against KLH. This relationship suggests that at least some of the participants who did not develop a cutaneous DTH response against KLH may have failed to do so because of a general impairment of cellular immune function, rather than because of mechanisms specifically associated with developing a response to KLH. This relationship between the two measures lends support to the validity of the KLH model because failure to respond to a battery of commonly encountered antigens (eg, the Multitest) may be expected to predict the capacity to respond to a single antigen (16). A further factor that may account for this similarity of responses was the level of alcohol consumption at follow-up, because it was correlated with DTH skin test responses to KLH and with the number of responses to the Multitest. In each case, alcohol consumption was associated with a reduction in cellular immune response.
In conclusion, the importance of this research is that DTH responses against KLH represent the capacity of the immune system to develop an orchestrated cellular response in vivo. We have argued elsewhere (6) that these responses are likely to be of clinical significance. No attempt was made to elevate levels of distress experienced by participants in this research. On the contrary, students participated in the research early in the semester when university-related stressors were likely to be at a minimum. Nevertheless, considerable variation in POMS scores was evident, and distress predicted the likelihood of developing a DTH response against KLH. Knowledge of rates of alcohol consumption at follow-up increased the ability to predict DTH responses against KLH. While the reported relationships here are modest, the results do show that the vicissitudes of life and the psychological states that ensue affect the development of an immune response. It is likely that the relationship between these variables may be greater in those individuals who are already immunocompromised (such as the elderly) or in those suffering more profound and enduring levels of distress. These likely associations need to be further explored.
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ACKNOWLEDGMENTS |
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Received for publication August 14, 2003.
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