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ORIGINAL ARTICLES |
Production After Acute Psychological Stress, Exercise, and Infused Isoproterenol: Differential Effects and Pathways
From the Departments of Psychiatry (M.U.G., P.J.M., M.R.I.) and Medicine (M.G.Z.), University of California, San Diego, La Jolla, CA; Department of Medical Psychology (M.U.G.), University of Essen, Essen, Germany; and Veterans Affairs Medical Center (M.R.I.), La Jolla, CA.
Address reprint requests to: Marion Goebel, Department of Medical Psychology, Medical Faculty, University of Essen, Hufelandstr. 55, D-45122 Essen, Germany. Email: marion.goebel{at}uni-essen.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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(TNF-). METHODS: Thirty-two healthy volunteers performed speech and exercise tasks and underwent a 30-minute infusion of isoproterenol.
RESULTS: As expected, acute activation of the sympathetic nervous system led to leukocytosis, including increases in lymphocyte, monocyte, and granulocyte populations (p values < .05). Lipopolysaccharide-induced IL-6 production was increased after both the speaking and exercise tasks (p values < .001), whereas TNF- production was elevated only after exercise (p < .05). In contrast, infusion of isoproterenol inhibited TNF- production (p < .001) and caused no change in IL-6 production.
CONCLUSIONS: In response to the challenges, IL-6 and TNF- production showed different profiles. Purely ß-agonist stimulation led to downregulation of TNF- production, providing evidence of the antiinflammatory effect of in vivo ß-receptor activation. The enhanced production of both cytokines after exercise, and of IL-6 after the speech task, can be best explained by a simultaneous upregulation of proinflammatory and inflammation-responding mediators. These effects may have an important role in controlling the immune response to acute psychological and physical stress.
Key Words: sympathetic nervous system activation • psychological stress • exercise • isoproterenol • cytokine production
Abbreviations: cAMP = cyclic adenosine monophosphate; IL = interleukin; LPS = lipopolysaccharide; SNS = sympathetic nervous system; TNF- = tumor necrosis factor-; VO2max =maximum oxygen consumption.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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and IL-6, have a pivotal role in coordinating the body’s response to inflammation (1, 2). TNF- effectively induces a local inflammatory response and helps to control infections. On the other hand, systemic release of TNF- can lead to sepsis, shock, and death. IL-6 causes lymphocyte activation and antibody production. IL-6 and TNF-, along with IL-1, synergistically control infection by regulating the production of acute-phase proteins and by raising body temperature (3). Because macrophages are the major source of TNF- and IL-6, stimulation with the endotoxin LPS provokes the release of these cytokines (4). Although IL-6 is commonly described as a proinflammatory cytokine, it is more accurate to call it an inflammation-responding cytokine because it increases inhibitory mediators, such as the IL-1 receptor, and stimulates the hypothalamic-pituitary-adrenal axis (2, 5). Many studies have demonstrated the interaction of the SNS, through psychological or physical stress and infusions with adrenergic agonists, and cytokine responses (6–11). Although the pathways of these interactions are not fully clarified, the link between the SNS and proinflammatory cytokines is of clinical interest in inflammatory diseases (7–15).
Among studies of the effects of psychological or physical stressors and proinflammatory cytokine secretion, there is a noticeable heterogeneity in methods and materials. Animal studies investigating psychological stress models generally show increased in vitro production after stimulation with endotoxin and/or increased plasma levels after exposure to restraint stress, an open field, or electric foot shocks (16–19). Human studies in this area are rare; however, it has been shown that after public speech tasks or examinations, TNF- production is elevated, especially in volunteers with higher stress perception (20, 21). Exposure to influenza A elevates IL-6 plasma levels, and probands who report being more stressed are more likely to develop greater symptoms of illness (22).
Strenuous acute physical activity also induces activation of the SNS, resulting in a ß2-receptor–mediated leukocytosis (23, 24), and is considered a model of inflammation-like processes (25). In general, studies demonstrate marked increases in stimulated and circulating plasma levels of IL-6 (26, 27). Studies of stimulated and circulating plasma levels of TNF- after exercise have produced inconsistent results (28–31); this inconsistency is likely related to variations in duration and intensity of the exercise tasks.
Many studies have investigated the effects of ß-agonists in vitro and have shown that epinephrine blunts TNF- production (32–36), whereas results for IL-6 are contradictory (37, 38). Only a few studies have investigated the in vivo effects of ß-agonist infusion (39–41); these studies have also indicated that adrenergic stimulation impairs TNF- production. The in vivo effects on IL-6 production in humans are unknown.
Given the existing literature, we addressed how and to what extent acute activation of the SNS, through psychological stress, physical stress, or administration of a pharmacological agent, modulates the secretion of IL-6 and TNF- in humans. In contrast to prior studies, which generally gathered data on isolated measures, we compared the effects of three different methods of SNS activation within the same subject. We reasoned that this would decrease errors in comparing effects across different types of tasks and provide more reliable insight into the mechanisms of action. We used a public speaking task (a commonly used component of mental stress tests), a bicycle ergometer task, and infusion of the ß-agonist isoproterenol. For cytokine assessment, we measured in vitro LPS-induced TNF- and IL-6 production. This whole-blood culture system represents the capacity of macrophages to derive cytokines in response to LPS. We also assessed leukocyte and lymphocyte subpopulations to demonstrate changes in distribution (all three experiments) and the plasma level of catecholamine to demonstrate SNS activation (speaking and exercise tasks).
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METHODS |
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Procedure
All subjects were studied at the University of California, San Diego, Medical Center General Clinical Research Center between 8:15 AM and 2:00 PM. Subjects refrained from caffeinated beverages and smoking for 12 hours before study. On arrival at the laboratory, subjects were seated, a 19-gauge catheter was inserted into a forearm vein, and then they rested for 30 minutes. Starting at 9:00 AM, volunteers performed, in a fixed order, a 15-minute speaking task and a 15- to 18-minute bicycle ergometer exercise task. They then underwent a 30-minute isoproterenol infusion. There was a 120-minute interval between completion of each task and the next baseline period. The speaking task consisted of two back-to-back speeches (with the subject sitting) in which the subject prepared and then presented a speech on a hypothetical situation. Subjects were told that the speech would be evaluated and rated by experts. The two situations were to defend oneself from being falsely accused of shoplifting and a confrontation with an unscrupulous car dealer. If subjects stopped talking before the time was up, they were reminded to continue (eg, to reiterate and summarize their points) (42).
For the exercise task, subjects were informed that the exercise would last approximately 15 to 18 minutes and would begin with a series of 3-minute stages marked by increasing resistance and thus greater effort on their part (43). They were told that the peak level of effort would be challenging and that once that peak had been established the workload would actually be slightly reduced for the remainder of the exercise period. They were informed of warning signs of excess exertion (eg, faintness, shortness of breath, dizziness, and muscle cramps) and that although such complications were not expected, they should inform the investigator immediately if any occurred. Subjects were instructed to begin pedaling and to achieve and maintain a pedaling rate of 70 rpm (rate was indicated on the ergometer display panel in their view). VO2max was estimated using heart rate, and the workload was adjusted so that the exercise was completed at a level comparable to 75% of the estimated VO2max for each subject. After the test, wheel resistance was removed, and subjects continued to pedal with no resistance for 5 minutes (cool-down period).
For infusion of the ß-agonist, subjects were supine, and isoproterenol was infused in doses of 20 and 40 ng/kg per min for 15 minutes each (44). Blood was drawn before and immediately after each task. Subjects were monitored by electrocardiography throughout infusion. The half-life of isoproterenol in the body is 2 to 3 minutes.
Enzyme-Linked Immunosorbent Assay
Whole blood was collected in tubes with sodium heparin and incubated with LPS (100 ng/ml for 3 hours in a 37°C water bath). Samples were centrifuged at 1400 rpm for 15 minutes at 4°C. Supernatants (800 ml) of the six testing time points (before and after each experiment) were collected and stored at -70°C. Cytokines were analyzed by using commercially available ELISA kits (R&D Systems, Minneapolis, MN). To reduce within-subject variability, all samples for each subject were assayed together. The intraassay coefficient of variation for the TNF- assay was 4.4%, and that for the IL-6 assay was 3.0%. The interassay coefficient of variation for the TNF- assay was 8.7%, and that for the IL-6 assay was 2.5%.
Leukocyte Subsets
Whole blood was preserved with ethylenediaminetetraacetic acid and maintained at room temperature (23°C). Flow cytometry (FACSCalibur, Becton-Dickinson, San Jose, CA) using CellQuest software was used to quantify lymphocyte, monocyte, and granulocyte populations. Blood was processed within 3 hours of collection, and whole blood was stained with monoclonal antibodies conjugated to various fluorochromes (44). The lysing reagent was FACS brand lysing solution (Becton-Dickinson), which results in simultaneous lysis of red blood cells and partial fixation of leukocytes. Positive four-color staining was used with monoclonal antibodies conjugated to either fluorescein isothiocyanate, phycoerythrin, peridinin chlorophyll protein, or allophycocyanin (Becton-Dickinson and PharMingen, San Jose, CA). Fluorescence compensation was performed using CaliBRITE beads (Becton-Dickinson) and FACSComp software. Optimal amounts of antibodies were used, and 8000 to 15,000 events were analyzed per tube. Isotypic controls were used for each assay to determine nonspecific staining. Phenotypes were expressed as the percentages of total cells analyzed by flow cytometry.
Catecholamines
Blood samples for catecholamines were obtained before and immediately after the speech and exercise tasks. We did not assess catecholamines for the isoproterenol infusion experiment because of the redundancy of infused and internal catecholamine levels. Blood was collected on ice and separated in a refrigerated centrifuge, and plasma was stored at -80°C until assay. Epinephrine and norepinephrine were determined by radioenzymatic assay (45). The intra- and interassay coefficients of variation for the assay are 6.5% and 11%, respectively.
Data Analysis
Data were analyzed using repeated-measures analysis of variance. For multiple comparisons, Bonferroni adjustments were performed.
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RESULTS |
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LPS-stimulated IL-6 production was enhanced on the speaking (F = 27.59, p < .001) and exercise tasks (F = 32.74, p < .001), whereas TNF- production was moderately but significantly increased on exercise only (F = 5.84, p < .05) (Figure 1). Overall, no significant gender differences were observed; women, however, had consistently lower levels of cytokines.
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production was lower at baseline for the isoproterenol infusion than for the other two tasks (F = 15, p < .001). In response to infused isoproterenol, LPS-stimulated TNF- production was significantly decreased (F = 7.40, p = .01). In contrast, IL-6 production showed no variation (Figure 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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was differentially affected depending on the type of stimulus, with psychological stress and exercise leading to an increase of IL-6 in vitro production. TNF- production increased only on exercise but was blunted by isoproterenol infusion.
Our study replicated results of several studies showing an exercise-induced increase in IL-6 production (28, 29); however, our observation of a moderate but significant increase in TNF- production contrasts with findings of other studies (30, 31). The physiological mechanism of IL-6 and TNF- elevation in plasma and of in vitro production of these cytokines in response to exercise has not been fully revealed. Several similarities exist between high-intensity exercise and immune reactions in inflammatory processes, such as mobilization and activation of leukocytes, induction of acute-phase response, increases in proinflammatory cytokine production, cellular infiltration, and tissue damage (46). Despite these events, other symptoms, such as vasodilatation, organ dysfunction, and leukocyte aggregation, do not occur (47). Therefore, exercise is considered to be a model of limited inflammatory response. Exercise also affects the number of circulating leukocytes, predominantly natural killer cells, which corresponds to their ß2-receptor expression (48). These changes are thought to be a short-term enhancement of immune function (49). In the intact host, TNF- and IL-6 work synergistically to stimulate antiinflammatory processes, such as antiinflammatory cytokines and cytokine receptors (50, 51). IL-6 also has antiinflammatory action (eg, it induces acute-phase proteins and stimulates the hypothalamic-pituitary-adrenal axis) and therefore controls the inflammatory response and contributes to the maintenance of homeostasis (5, 52). Some studies have also suggested that high-intensity exercise and IL-6 secretion are related to local muscle damage and that damaged muscle fibers trigger local IL-6 production (53–55). Given our data of an increase in IL-6 in response to psychological stress, it seems that enhanced IL-6 production is not solely a result of a mechanical event. Brenner et al. (46) also reported an association between changes in plasma IL-6 levels and cardiovascular and humoral changes on exercise but noted that markers such as creatine kinase and muscle soreness ranking were not associated with cytokine levels. Given the elevated plasma norepinephrine and epinephrine levels and the mobilization of leukocytes (48), we suggest an association between catecholamine levels in vivo and IL-6 production in vitro. In contrast to IL-6, TNF- was less responsive to the neuroendocrine changes on the speech task, which could be due to the smaller changes in hormone levels and leukocyte numbers (56).
Even though physical activity and psychological stress affect neuroendocrine and immunological parameters differently, both are potent activators of the central nervous system and alter the immune response (48). Both stressors increase intracellular production of cAMP through stimulation of ß-receptors on immune-competent cells (57, 58). The beneficial effects of regular and moderate exercise are well documented (59). Thus, immunological performance is continuously trained and might therefore protect effectively against infections (60). This may be applicable to limited psychological stressors, but chronic stress is thought to suppress immune function (61).
Few studies have examined the effects of in vivo pharmacological ß-receptor stimulation on cytokine production in humans. Our finding of inhibition of LPS-induced TNF- production after infusion of isoproterenol is consistent with the findings of van der Poll et al. (41). Using a dosage of 30 ng/kg per min and a 24-hour infusion protocol, this group showed that TNF- production was blunted within the first 4 hours and that this blunted production persisted until the end of the infusion. The effect was consistent for whole-blood stimulation with 1, 10, and 100 ng/ml LPS. Because we used 100 ng/ml LPS for 3 hours, our infusion protocol of 20 ng/kg per min and then 40 ng/kg per min reflects almost exactly this decrease. This in vivo finding is in accordance with the findings of in vitro studies, which indicate that ß-receptor stimulation leads to an increase of intracellular cAMP, which subsequently inhibits cytokine production (34–36). We also noted that the baseline TNF- level before isoproterenol infusion was significantly lower than the baseline levels for the other tasks. One limitation of this study is that we did not counterbalance the order of challenges or use separate testing days to avoid possible carryover effects. Given the stability of the baseline IL-6 levels across the three experiments, it is reasonable to assume that there were no carryover effects on IL-6. Despite the lower TNF- baseline level before infusion, we are confident that the significant decrease in TNF- after infusion was caused by isoproterenol, although we cannot say for certain given the consistency in prior studies (34–36, 41).
Hence, this finding demonstrates the antiinflammatory potential of ß-agonist stimulation. Studies of the effect of ß-receptor stimulation on IL-6 production have reported both inhibitory (33) and enhancing effects (35). Some reasons have to be considered for the unchanged IL-6 production. First, it is possible that an additional mechanism, such as -receptors, is involved in IL-6 production (32). Second, a recent in vitro study suggests a biphasic effect for the ß-agonist terbutaline (62). Higher concentrations resulted in a rise of IL-6 production in renal macrophages, whereas lower concentrations blunted IL-6 production. Additional studies are needed to evaluate the general effects of in vivo infusion on proinflammatory cytokines with different - and ß-adrenergic agents in various concentrations.
It is possible that the highly complex interaction between the SNS and the cytokine network may become unbalanced. The mechanisms that alter proinflammatory cytokine production are of clinical relevance in diseases such as sepsis, atherosclerosis, and chronic inflammatory diseases. For some inflammatory diseases, a shift toward the proinflammatory Th1 cytokines has been reported (63). There is also evidence of an accumulation of Th1 cytokines in atherosclerotic plaques and evidence that a balance of pro- and antiinflammatory cytokines is critical for the course of atherosclerosis (64). Moreover, activation of the SNS, as in congestive heart failure, is associated with high IL-6 levels in plasma (65), which is thought being a valuable prognostic factor. The disturbed host might not be able to counterbalance the early response to stressors, thus enhancing the inflammatory mediators and increasing the vulnerability. Studies might further investigate this cytokine-SNS interaction in modulating cytokine synthesis by blocking receptor function or by behavioral interventions to improve treatment strategies. Moreover, although we did not find significant gender effects, we observed a tendency for women to have consistently lower cytokine levels. It would be intriguing to investigate possible gender-related effects on this issue.
In summary, we found that acute SNS activation by psychological stress and exercise showed different in vitro cytokine production patterns than activation caused by a pure ß-agonist infusion. These findings further connect the results of existing studies, which focused on SNS activation and proinflammatory cytokines. The differential effects could be in part explained by activation of adrenoceptor mechanisms. TNF- response to LPS is reduced after ß-receptor stimulation, which demonstrates the antiinflammatory characteristics of ß-agonist infusion. Because IL-6 production was unaffected by the infusion, we suggest that an additional mechanism is involved. IL-6 increased after the psychological and physical challenges. Given that IL-6 is rather an inflammation-responsive cytokine, a potent antiinflammatory and immunosuppressive mediator, these findings might refer to its role in maintaining homeostasis during stress. On the other hand, the enhanced TNF- production observed after the exercise task is likely to be related to an inflammatory response to exercise. The dynamics of cytokine responses with the SNS activation methods we used give partial insight into the ways in which hosts respond to challenges. Future studies might investigate how and to what extent the cytokine-SNS interaction leads to potential treatment strategies in inflammatory diseases.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Received for publication June 21, 1999.
Revision received January 14, 2000.
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