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The role of stress hormones in exercise-induced suppression of alveolar macrophage antiviral function
Journal of Neuroimmunology 81 Ž1998. 193–200
The role of stress hormones in exercise-induced suppression of alveolar macrophage antiviral function M.L. Kohut, J.M. Davis ) , D.A. Jackson, L.H. Colbert, A. Strasner, D.A. Essig, R.R. Pate, A. Ghaffar, E.P. Mayer Department of Exercise Science, School of Public Health and the Department of Microbiologyr Immunology, School of Medicine, UniÕersity of South Carolina, Columbia, SC 29208, USA Received 25 April 1997; revised 2 September 1997; accepted 2 September 1997
Abstract We hypothesized that a previously observed exercise-induced suppression of alveolar macrophage antiviral resistance results from increases in corticosterone andror epinephrine. Mice ŽCD-1. were run to fatigue on a treadmill Žexercise., or placed in Plexiglas lanes above the treadmill Žcontrol.. The role of corticosterone was assessed by further dividing mice into groups receiving one of the following treatments; sham surgery, adrenalectomy, or adrenalectomy plus corticosterone replacement. Macrophage antiviral function was suppressed in the exercised mice compared to the control mice. However, macrophage antiviral function was not suppressed in the exercised mice that underwent adrenalectomy or adrenalectomy plus corticosterone replacement. We tested whether another adrenal factor Žepinephrine. may be involved by dividing mice into exercise and control groups treated with either saline or propranolol. Macrophage antiviral function was again suppressed in the saline-treated exercised mice compared to saline-treated control mice, but no differences were found between the exercised mice receiving propranolol, control mice receiving propranolol, or saline-treated control mice. Isoproterenol, when added to alveolar macrophages in culture, also suppressed antiviral resistance. These findings suggest that decreased macrophage antiviral function following exercise may be due to increased release of adrenal catecholamines. q 1998 Elsevier Science B.V. Keywords: Lung; Glucocorticoid; Catecholamines; Herpes simplex virus; Stress
1. Introduction There is growing evidence that stress can influence immune function and alter susceptibility to upper respiratory infection. In humans, a wide range of stressors such as negative life events, final exams and running a marathon have been shown to suppress specific immune responses andror increase susceptibility to upper respiratory infection ŽNeiman et al., 1990; Cohen et al., 1991; Glaser et al., 1992.. Various parameters of antiviral immunity and resistance to viral infection are also suppressed in animals following exposure to numerous stressors including forced exercise, restraint stress and social stress ŽRasmussen et al., 1957; Reyes and Lerner, 1976; Ilback et al., 1989.. Stress activates the hypothalamic–pituitary–adrenal axis ŽHPA. and the sympathetic nervous system. As a result of ) Corresponding author. Tel.: q1-803-7776881; fax: q1-803-7778422; e-mail: [email protected]
0165-5728r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 5 7 2 8 Ž 9 7 . 0 0 1 7 9 - 3
this activation, catecholamines are released from sympathetic nerve terminals and the adrenal medulla, while corticosteroids are released from the adrenal cortex. Numerous reviews of neuroendocrine–immune interactions report that catecholamines andror corticosteroids can alter immune function ŽDantzer and Kelley, 1989; Kusnecov and Rabin, 1994.. Furthermore, several studies have shown that the release of catecholamines andror corticosteroids during various stress paradigms can result in immunosuppression ŽCunnick et al., 1990; Bonneau et al., 1993; Brown and Zwilling, 1994.. Many studies focusing on stress and immunity have examined the response of circulating or splenic leukocytes, however very little has been published on stress and any antiviral defense system within the respiratory tract, one of the most common sites of viral infection. The lung has a vast array of host defense mechanisms to cope with the inhaled pathogens that can potentially result in upper respiratory infection. Cells of the innate immune system
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located within the respiratory tract, such as the alveolar macrophage, constitute the first line of defense against invading organisms until specific immune responses are initiated. The alveolar macrophage has the ability to ingest and degrade viruses, facilitating the clearance of the virus from initial sites of infection ŽMogensen, 1979.. A murine model of HSV-1 infection has been used in our lab to study the effects of prolonged exercise to fatigue on alveolar macrophage intrinsic resistance to viral infection ŽDavis et al., 1997.. Intrinsic resistance by the macrophage is manifested in the cells ability to take up viral particles and limit or stop their replication ŽShellam and Flexman, 1986; Morahan et al., 1995.. This type of resistance to viral infection may be completely independent of extrinsic resistance. Macrophage extrinsic resistance refers to the ability of the macrophage to inactivate extracellular viruses via cytokine release or macrophage mediated antibody dependent cellular cytotoxicity ŽMorahan et al., 1995.. The findings from our laboratory demonstrate that a single, prolonged bout of exercise to fatigue increases susceptibility to intranasal HSV-1 infection and suppresses alveolar macrophage intrinsic resistance to HSV-1 infection ŽDavis et al., 1997.. To elucidate the mechanisms of this exercise-induced suppression of macrophage antiviral resistance, we evaluated the roles of adrenal hormones and catecholamines. We hypothesized that blocking either the release of, or effect of, corticosterone andror catecholamines during exercise would eliminate the suppression of macrophage antiviral resistance. Adrenalectomized mice were used to determine if adrenal hormones were involved in the suppression of alveolar macrophage antiviral function. The beta-adrenergic receptor antagonist propranolol was used to inhibit catecholamine-receptor binding to establish the possible role of catecholamines.
2. Materials and methods 2.1. Mice Male CD-1 mice, 4 weeks of age, were purchased from Charles River Labs and acclimated to our facility for at least three days prior to any experimentation. Mice were purchased as pathogen free stock and periodic antibody screening of sentinel mice yielded negative results for common murine viral or bacterial pathogens. Mice were maintained on a 12:12 h light–dark cycle in a low stress environment Ž228C, 50% relative humidity, low noise. and given food ŽPurina Chow. and tap water ad libitum. 2.2. Adrenalectomy In the first series of experiments, mice were anesthetized with an intraperitoneal injection of avertin Ž2-2-2-
tribromoethanol, Polysciences, Warrington, PA.. A dorsolateral incision was made superior to each kidney, kidneys were exposed and the adrenal gland was removed. Sham adrenalectomized mice underwent the same procedure with the exception of adrenal gland removal. Mice were then returned to their cages and were allowed to recover for one full week prior to further experimentation. All adrenalectomized mice received saline drinking water Ž0.9% NaCl. supplemented with corticosterone Ž50 mgrl, Sigma, St. Louis. to provide basal level replacement of this hormone. 2.3. Treadmill acclimation and exercise protocol In the first series of experiments, mice were randomly assigned to one of the following five groups Ž n s 10 per group.: CON, control treatment, sham adrenalectomy; ADX CON, control treatment, adrenalectomy; EX, exercise treatment, sham adrenalectomy; ADX EX, exercise treatment, adrenalectomy; ADX EX q C, exercise treatment, adrenalectomyq corticosterone replacement during exercise equivalent to that level obtained in normal exercising mice. In the second series of experiments, mice were randomly assigned to one of the following groups Ž n s 10 per group.: CON, control treatment; CON q PRO, control treatment plus propranolol; EX, exercise treatment; EX q PRO, exercise treatment plus propranolol. Each series of experiments was repeated three times for a total n s 30 in each group. The exercise treatment consisted of one run to fatigue at gradually increasing speeds, 18–40 m P miny1 , 5% grade. Fatigue was defined as the point at which mice fail to maintain pace with the treadmill despite being given mild physical prodding continuously for 5 min ŽWoods et al., 1994.. Electric shock was never used in these experiments as mice readily respond to a gentle tap of the tail or hindquarters. With this protocol mice typically fatigued in 2.5–3 h. Mice in the control groups were contained in well ventilated Plexiglas lanes above the treadmill for an equivalent period of time, exposed to similar handling, noise and treadmill vibration. All mice were acclimated to treadmill running for three days prior to the actual experiment. During the treadmill acclimation period, mice were allowed to briefly explore the control lanes and the treadmill followed by a run at slow speeds Ž1–5 m P miny1 . for a 5 to 10 min period. Mice were acclimated twice per day at the beginning and the end of the 12 h dark period. 2.4. Drug preparation and administration In the first series of experiments, all adrenalectomized mice received a basal replacement of corticosterone, 50 mgrl, in the drinking water. In addition, mice in the ADX EX q C group received a 0.1 ml subcutaneous injection of corticosterone, 8 mgrkg dissolved in saline, administered at the onset of exercise and again 90 min into the exercise session. Mice in the remaining four groups received a 0.1
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ml subcutaneous injection of saline at the same time points. In the second series of experiments, a 10 mgrkg dose of DL-propranolol ŽSigma, St. Louis. was administered intraperitoneally 15 min prior to the start of the exercise or control sessions to mice in the CON q PRO and EX q PRO groups. Mice in the EX and CON groups received an intraperitoneal injection of saline. In a third experiment Ž".isoproterenol-bitartrate salt was added to the wells of microtiter plates containing pooled alveolar macrophages obtained from 20 control mice at the following concentrations: 1, 0.2 and 0.02 m mol P ly1 . 2.5. AlÕeolar macrophage collection and preparation All animals were sacrificed in a bell jar containing ether immediately after reaching fatigue during exercise or at an equivalent period of time in the control Plexiglas lanes. Sacrifice by overetherization occurred in less than 2 min. Lungs were removed and alveolar macrophages were obtained by gentle lavage of the lungs with 15–20 ml of physiological media. The culture media used in all experiments was RPMI 1640 ŽGibco, Grand Island, New York. containing 100 Urml penicillin, 100 m grml streptomycin and 20 mmolP ly1 glutamine. Lung lavage cells were washed once with RPMI, and any remaining red cells were lysed with Tris ammonium chloride, pH s 7.2. Cells from mice in each group Ž n s 10. were pooled due to the limited number of cells from each mouse and adjusted to a concentration of 4 = 10 6 cellsrml in RPMI media supplemented with 10% fetal bovine serum ŽCRPMI-Environmental Diagnostics, Burlington, North Carolina.. Viability was determined using trypan blue exclusion and the percentage of macrophages was calculated from a cytocentrifuge preparation followed by Diff Quick ŽBaxter Scientific, Chicago, IL. stain. Subsequently, 0.2 ml of the cell preparation was added to the wells of a 96 well microtiter plate and allowed to adhere for 3 h at 378C. After three hours each well was washed gently with prewarmed Ž378C. RPMI to remove nonadherent cells. 2.6. HSV-1 Õirus HSV-1 KOS strain virus was propagated in Vero cells. Stocks of virus were titrated on Vero cells by a plaque assay and contained 1.2 = 10 8 pfurml. Aliquots of virus were stored at y808C until use. 2.7. AlÕeolar macrophage antiÕiral resistance: Neutral red assay
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virus was allowed to absorb in a small volume Ž50 m l. for 90 min. Prewarmed CRPMI media was added to each well and the plates were incubated at 378C, 5% CO 2 for 48 h until a cytopathic effect was observed. The cytopathic effects were then quantified by a neutral red dye uptake assay ŽLazdins et al., 1990.. Briefly, cell monolayers were washed twice with RPMI and stained for 2 h with 0.006% neutral red diluted in RPMI. The stained monolayers were washed and the dye was extracted by lysing cells with 0.2 ml of Sorensens citrate buffer ŽpH 4.1. containing 50% ethanol. The optical density was read at 530 nm on a Dynatech MR5000 microplate reader. The cytopathic effect was evaluated by calculating a viability index which is expressed as the ratio of dye uptake by infected cells to dye uptake by uninfected cells. Healthy uninfected cells have a greater dye uptake than infected dying cells. 2.8. Plasma corticosterone and free fatty acid analysis At the time of sacrifice, blood was collected from the inferior vena cava in syringes containing 0.05 ml of 100 Urml heparin. After centrifuging, the plasma was collected and stored at y208C until analysis of corticosterone and free fatty acids. Corticosterone was analyzed using a rodent corticosterone RIA kit ŽDiagnostic Products Corp., Los Angeles.. Free fatty acids were determined using a standard spectrophotometric technique ŽNoma et al., 1973.. 2.9. Statistics The results from the experiments assessing macrophage intrinsic antiviral resistance were compared with a one way ANOVA Ž4–5 groups.. Due to the limited number of alveolar macrophages obtainable from one mouse, cells from the 10 mice per group were pooled and the experiment was replicated three times for a total n s 3. Plasma corticosterone and free fatty acids were measured in each mouse for a total n s 30 per group. Differences in corticosterone and free fatty acids between groups were analyzed with a one way ANOVA Ž4–5 groups.. In the final experiment, various amounts of isoproterenol were added to pooled cells in wells of microtiter plates. Four wells per concentration of isoproterenol were measured and differences between wells containing the various concentrations of isoproterenol were analyzed with a one way ANOVA. For all experiments, when significant differences were detected, the Student–Neuman–Keuls post hoc test was used to analyze group differences.
3. Results 3.1. Exercise treatment
After nonadherent cells were removed from the wells of the microtiter plate by washing, macrophages were infected at 7–10 pfurcell with the HSV-1 KOS virus. The
Run time to fatigue did not significantly differ across groups in any of the experiments. The experiment using
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adrenalectomized animals was performed three times and the average run time to fatigue across experiments was as follows: 162 " 31 min for EX, 154 " 28 min for ADX EX and 151 " 39 min for ADX EX q C Žmean " S.D., n s 30 per group.. Mice receiving propranolol also exhibited no difference in run time to fatigue compared to mice receiving saline, 152 " 26 and 158 " 28 min, respectively. 3.2. Corticosterone leÕels A single bout of exercise to fatigue significantly elevated plasma corticosterone levels ŽEX ) CON, p - 0.01, Fig. 1.. Replacement of corticosterone by subcutaneous injection in ADX EX q C resulted in plasma levels similar to that of EX. In addition, replacement of corticosterone in the drinking water of adrenalectomized mice, ŽADX CON and ADX EX. yielded basal levels of corticosterone. Although these levels appear to be on the low side of the normal resting physiological range, these samples were taken approximately 3 to 4 h after the active dark period and mice had been water deprived for the 2–3 h on the treadmill or in control lanes. Mice tend to consume water during the dark active period, and in other experiments, we found that replacement levels are higher during this active period Ž300–500 nmol P ly1 . and gradually decline during the day. There is some degree of variability among the replacement levels as they reflect the amount of water consumed by an animal in the last several hours before sacrifice. As expected, EX and ADX EX q C exhibited high plasma corticosterone whereas ADX CON and ADX EX had low levels due to adrenalectomy ŽFig. 1..
Fig. 2. Viability index of macrophages infected with HSV-1 virus. Results are meansqSE for pooled macrophages in wells of microtiter plates 48 h post infection. One value is obtained per group per experiment and means from each group are compared across experiments Ž ns 3.. Significant differences from control ŽCON. value indicated by ) P - 0.01.
3.3. Exercise, adrenal hormones, and macrophage antiÕiral resistance In Fig. 2, the results from the three experimental trials designed to assess the role of the adrenal hormone corticosterone are shown. Exercise decreases the ability of the alveolar macrophage to resist viral infection in vitro as demonstrated by the decrease in percent viability; EX CON, p - 0.01. The percent viability of the macrophages from ADX EX and ADX EX q C is also not different from CON or ADX CON. These results suggest that although products released from the adrenal gland may be involved, it is not corticosterone that acts to suppress macrophage antiviral resistance during exercise because no suppression was observed in the ADX EX q C group even though this group demonstrated corticosterone levels similar to EX. 3.4. Effect of catecholamines during exercise on macrophage antiÕiral resistance
Fig. 1. Plasma corticosterone levels obtained immediately post exercise or control treatment. Results are means"SE for 30 mice. Significant differences from control ŽCON. values indicated by ) P - 0.01; CONs control treatment, sham adrenalectomy; ADX CON s control treatment, adrenalectomy; EX sexercise treatment, sham adrenalectomy; ADX EX sexercise treatment, adrenalectomy; ADX EXqC sexercise treatment, adrenalectomyqcorticosterone replacement during exercise.
Epinephrine and norepinephrine increase during exercise ŽDavies and Rew, 1973; Kjaer, 1992. and the betaadrenergic receptor antagonist propranolol was used to block the effects of these catecholamines. In the second series of experiments, propranolol Ž10 mgrkg. was administered just prior to the exercise andror control treatments to determine whether catecholamines released during fatiguing exercise act to suppress macrophage antiviral resistance. Again, macrophage antiviral resistance reflected by % viability is decreased in EX compared to CON ŽFig. 3, EX - CON, p - 0.01. Administration of propranolol prior to exercise completely eliminated the suppressive effects
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Fig. 3. Viability index of macrophages infected with HSV-1 virus. Results are meansqSE for pooled macrophages in wells of microtiter plates 48 h post infection. One value is obtained per group per experiment and means from each group are compared across experiments Ž ns 3.. Significant differences from control ŽCON. value indicated by ) P - 0.01. CON s control treatmentqsaline, CONqPROs control treatmentq propranolol; EX s exercise treatmentqsaline; EXqPRO s exercise treatmentqpropranolol.
of exercise on macrophage antiviral resistance; CON, CON q PRO, EX q PRO ) EX, p - 0.01. In order to establish whether propranolol was effective in blocking peripheral beta-adrenergic receptors, free fatty acids were measured. It is well known that the exercise-induced increase in plasma free fatty acids is primarily mediated via beta-adrenergic stimulation of lipolysis within adipocytes. Fig. 4 demonstrates that propranolol administration suppressed the typical increase in plasma free fatty
Fig. 4. Plasma free fatty acid concentration measured immediately post exercise or control treatment. Results are meansqSE for the 30 mice in each group. Significant difference from control value indicated by ) P 0.01. Significant difference from control ŽCON. and exercise ŽEX. indicated by q P - 0.01.
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Fig. 5. In vitro effect of isoproterenol ŽISO. on macrophage antiviral resistance assessed by viability index. Pooled alveolar macrophages were incubated with ISO for 3 h and then infected with HSV-1 virus. Results are meansqSE of individual wells in microtiter plates 48 h post infection. Significant difference from 0.0 isoproterenol is indicated by an ) Ž P - 0.05..
acids observed during prolonged exercise ŽEX q PRO EX, p - 0.01.. These findings suggest that propranolol was effective in blocking peripheral beta-adrenergic receptors during exercise. 3.5. In Õitro beta-adrenergic agonist (isoproterenol) and macrophage antiÕiral resistance To verify that beta-adrenergic receptor stimulation can suppress macrophage antiviral resistance, we added the beta-adrenergic agonist isoproterenol directly to cultures of alveolar macrophages obtained from CON mice. As expected, all wells infected with HSV showed a cytopathic effect Ždecreased percent viability.. When isoproterenol was added to these cultures of infected macrophages, the percent viability declined in a dose-dependent manner ŽFig. 5.. Both 1 and 0.2 m mol P ly1 concentrations of isoproterenol significantly suppressed macrophage antiviral resistance Ž p - 0.05.. These results suggest that direct activation of beta-adrenergic receptors in culture suppresses macrophage intrinsic antiviral function as measured by neutral red dye uptake. The findings obtained in culture in vitro complement the ex vivo findings demonstrating that activation of beta-adrenergic receptors during exercise suppresses macrophage resistance to viral infection. 4. Discussion The results of these studies confirm our previous findings that exercise to fatigue increases alveolar macrophage susceptibility to viral infection with HSV-1 ŽDavis et al.,
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1997.. A decrease in oxygen dependent antimicrobial activity in alveolar macrophages has also been reported in horses following strenuous exercise ŽWong et al., 1990.. In contrast to the immunosuppression observed in alveolar macrophages, exhaustive exercise appears to enhance peritoneal macrophage phagocytosis and antitumor cytotoxicity ŽOrtega et al., 1993; Woods et al., 1994.. Macrophage responses to exercise can vary depending upon the location of the cell and the particular function being assessed. The increased susceptibility to upper respiratory infection following prolonged intense exercise may be due in part, to the suppression of alveolar macrophage function. However, the mechanisms mediating these changes in macrophage function following exercise have not been addressed. The findings from these experiments suggest that alveolar macrophage resistance to viral infection following prolonged exercise to fatigue may be mediated by catecholamine binding to beta-adrenergic receptors. Other investigators have also reported that catecholamines may mediate alterations in various immune responses during exercise. For example Murray et al., 1992, found that prior administration of propranolol blunted the exercise induced decrease in concanavalin A ŽConA. stimulated IL-2 receptor expression and w3 Hx thymidine incorporation in human peripheral blood mononuclear cells. Others report that changes in plasma levels of epinephrine are likely associated with alterations in circulating numbers of lymphocyte subsets ŽNeiman et al., 1992.. In our study, evidence from the adrenalectomy experiments and from the experiments using propranolol lends support to the conclusion that elevated catecholamines can mediate macrophage antiviral resistance during exercise. Adrenalectomized-exercised mice do not exhibit the decrease in macrophage antiviral resistance that is observed in the exercised mice receiving sham surgery, suggesting that the adrenal glands produce some factor during exercise that suppresses macrophage function. However, this factor does not appear to be corticosterone, as adrenalectomized-exercised mice receiving corticosterone replacement have plasma corticosterone levels similar to that in the adrenal intact mice during exercise. If the level of corticosterone produced during exercise suppressed macrophage function, a decline in macrophage antiviral resistance should have been observed in adrenalectomized mice receiving high doses of corticosterone during exercise. Instead these results are consistent with the hypothesis that catecholamines mediate the exercise-induced decline in macrophage antiviral resistance. The adrenal medulla is a major source of epinephrine during fatiguing exercise ŽDavies and Rew, 1973; Kjaer, 1992.. Removal of the adrenal gland attenuated the suppression of macrophage function. Even more convincing are the experiments which show that prior administration of the beta-adrenergic receptor antagonist completely eliminated the exercise-induced suppression. The dose of propranolol administered was clearly
sufficient to elicit a physiological response Žinhibition of free fatty acid release via blockade of peripheral betaadrenergic receptors on adipocytes. and, therefore, could conceivably block the effect of catecholamines on betaadrenergic receptors known to be present on macrophages ŽAbrass et al., 1985; Liggett, 1989.. The results from the first series of experiments suggest that the adrenal is the source of an agent Žlikely epinephrine. that may suppress macrophage antiviral function. Although these experiments were not designed to determine the source of catecholamines that may alter macrophage antiviral function, it is possible that the release of catecholamines from sympathetic nerve endings also played a role in modulating macrophage function, considering the numerous studies reporting interactions between the sympathetic nervous system and the immune system ŽMadden et al., 1995.. It is possible that short periods of stress may alter immune function primarily through elevated catecholamines while chronic, long term stress modulates immune response through prolonged elevations in corticosteroids. The results from our experiments and others ŽBoranic et al., 1990. suggest that several hours of stress may alter certain parameters of immune function via elevated catecholamines. Yet it appears that both catecholamines and corticosteroids may act to alter different parameters of immunity in other stress paradigms ŽDobbs et al., 1993.. For example, in one model of shock-induced immunosuppression, adrenalectomy attenuated the peripheral blood lymphocyte suppression whereas propranolol and nadolol attenuated the splenic lymphocyte suppression ŽCunnick et al., 1990.. Other studies employing long term stress, 16 to 18 h of restraint stress daily for one to two weeks, demonstrate that the stress-induced release of corticosteroids acts to increase macrophage susceptibility to Mycobacterium aÕium growth and suppresses the generation of HSV-specific cytotoxic lymphocytes ŽBonneau et al., 1993; Brown and Zwilling, 1994.. Future studies comparing long term and short term stress and differing immune parameters may help to clarify the particular roles of catecholamines and corticosteroids in stress-induced immunomodulation. However, these types of studies can be difficult to execute as animals may adapt to chronic stress and fail to secrete high levels of corticosteroids. The mechanisms responsible for the decrease in macrophage antiviral function following catecholamine binding to adrenergic receptors are unknown. Several investigators have shown that macrophage function can be altered by epinephrine, norepinephrine, or isoproterenol and that these beta-adrenergic agonists appear to exert their effects through elevations in intracellular cAMP ŽKoff and Dunegan, 1986; Chelmicka-Schorr et al., 1992.. One study showed that norepinephrine and epinephrine elevated cAMP in peritoneal macrophages and subsequently blocked the capacity of these macrophages to be activated to a lytic state capable of killing HSV infected cells. Exogenous dibutyryl cAMP had the same effect ŽKoff and Dunegan,
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1986.. Similarly, macrophages treated with isoproterenol prior to LPS treatment resulted in suppressed tumor necrosis factor a ŽTNFa . production and the same response occurred when dibutyryl cAMP was used. In addition, the beta-adrenergic antagonist pindobind blocked the effect of isoproterenol on macrophages ŽChelmicka-Schorr et al., 1992.. These findings suggest that the effect of isoproterenol on macrophages is mediated through beta-adrenergic receptors and that beta-adrenergic agonists exert their effects by elevating intracellular cAMP. Although the evidence from these studies supports the concept that elevated intracellular cAMP can suppress some macrophage functions, it is not clear how intrinsic antiviral resistance may be altered by elevated cAMP. Given that the intrinsic antiviral activity of murine macrophages to HSV-1 appears to be strongly influenced by increases in interferon-beta ŽPyo et al., 1991., it is possible that elevations in cAMP alter the expression of interferon-beta or other important antiviral cytokines, including TNFa which has been shown to be suppressed after exposure to isoproterenol ŽChelmicka-Schorr et al., 1992.. However, our recent findings suggest that interferon-beta mRNA levels are actually elevated in infected macrophages from exercised mice, apparently in response to a greater degree of viral infection Žunpublished observations.. It is therefore possible that catecholamines act in some other manner leading to greater viral replication, and elevated interferon levels simply reflect this greater degree of viral infection. The data from these studies suggest that catecholamines may act to suppress macrophage intrinsic antiviral resistance during exercise and provides one of the few in vivo indications of mechanisms operating during prolonged, fatiguing exercise that may decrease resistance to viral infection within the lung. This decrease in innate immunity may contribute to increased susceptibility to viral respiratory infections following prolonged, strenuous exercise. Acknowledgements This work was supported in part by a Grant-in-Aid of Research from Sigma Xi, The Scientific Research Society and the American College of Sports Medicine FoundationrCybex research grant. References Abrass, C.K., O’Connor, S.W., Scarpace, P.J., Abrass, I.B., 1985. Characterization of the beta-adrenergic receptor of the rat peritoneal macrophage. J. Immunol. 135, 1338–1341. Bonneau, R.H., Sheridan, J.F., Feng, N., Glaser, R., 1993. Stress-induced modulation of the primary cellular immune response to Herpes simplex virus infection is mediated by both adrenal-dependent and independent mechanisms. J. Neuroimmunol. 42, 167–176. Boranic, M., Pericic, D., Poljak-Blazi, M., Maney, H., Sverko, W., Gabrilovac, J., Radacic, M., Pivac, N., Miljenovic, G., 1990. Immune response of stressed rats treated with drugs affecting serotoninergic and adrenergic transmission. Biomed. Pharmacother. 44, 381–387.
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Shellam, G.R., Flexman, J.P., 1986. Genetically determined resistance to murine cytomegalovirus and Herpes simplex virus in newborn mice. J. Virol. 58, 152. Wong, C.W., THompson, H.L., Thong, Y.H., Thornton, J.R., 1990. Effect of strenuous exercise stress on chemiluminescence response of equine alveolar macrophages. Equine Vet. J. 22, 33–35. Woods, J.A., Davis, J.M., Mayer, E.P., Ghaffar, A., Pate, R.R., 1994. Effects of exercise on macrophage activation for antitumor cytotoxicity. J. Appl. Physiol. 76, 2177–2185.
The role of stress hormones in exercise-induced suppression of alveolar macrophage antiviral function M.L. Kohut, J.M. Davis ) , D.A. Jackson, L.H. Colbert, A. Strasner, D.A. Essig, R.R. Pate, A. Ghaffar, E.P. Mayer Department of Exercise Science, School of Public Health and the Department of Microbiologyr Immunology, School of Medicine, UniÕersity of South Carolina, Columbia, SC 29208, USA Received 25 April 1997; revised 2 September 1997; accepted 2 September 1997
Abstract We hypothesized that a previously observed exercise-induced suppression of alveolar macrophage antiviral resistance results from increases in corticosterone andror epinephrine. Mice ŽCD-1. were run to fatigue on a treadmill Žexercise., or placed in Plexiglas lanes above the treadmill Žcontrol.. The role of corticosterone was assessed by further dividing mice into groups receiving one of the following treatments; sham surgery, adrenalectomy, or adrenalectomy plus corticosterone replacement. Macrophage antiviral function was suppressed in the exercised mice compared to the control mice. However, macrophage antiviral function was not suppressed in the exercised mice that underwent adrenalectomy or adrenalectomy plus corticosterone replacement. We tested whether another adrenal factor Žepinephrine. may be involved by dividing mice into exercise and control groups treated with either saline or propranolol. Macrophage antiviral function was again suppressed in the saline-treated exercised mice compared to saline-treated control mice, but no differences were found between the exercised mice receiving propranolol, control mice receiving propranolol, or saline-treated control mice. Isoproterenol, when added to alveolar macrophages in culture, also suppressed antiviral resistance. These findings suggest that decreased macrophage antiviral function following exercise may be due to increased release of adrenal catecholamines. q 1998 Elsevier Science B.V. Keywords: Lung; Glucocorticoid; Catecholamines; Herpes simplex virus; Stress
1. Introduction There is growing evidence that stress can influence immune function and alter susceptibility to upper respiratory infection. In humans, a wide range of stressors such as negative life events, final exams and running a marathon have been shown to suppress specific immune responses andror increase susceptibility to upper respiratory infection ŽNeiman et al., 1990; Cohen et al., 1991; Glaser et al., 1992.. Various parameters of antiviral immunity and resistance to viral infection are also suppressed in animals following exposure to numerous stressors including forced exercise, restraint stress and social stress ŽRasmussen et al., 1957; Reyes and Lerner, 1976; Ilback et al., 1989.. Stress activates the hypothalamic–pituitary–adrenal axis ŽHPA. and the sympathetic nervous system. As a result of ) Corresponding author. Tel.: q1-803-7776881; fax: q1-803-7778422; e-mail: [email protected]
0165-5728r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 5 7 2 8 Ž 9 7 . 0 0 1 7 9 - 3
this activation, catecholamines are released from sympathetic nerve terminals and the adrenal medulla, while corticosteroids are released from the adrenal cortex. Numerous reviews of neuroendocrine–immune interactions report that catecholamines andror corticosteroids can alter immune function ŽDantzer and Kelley, 1989; Kusnecov and Rabin, 1994.. Furthermore, several studies have shown that the release of catecholamines andror corticosteroids during various stress paradigms can result in immunosuppression ŽCunnick et al., 1990; Bonneau et al., 1993; Brown and Zwilling, 1994.. Many studies focusing on stress and immunity have examined the response of circulating or splenic leukocytes, however very little has been published on stress and any antiviral defense system within the respiratory tract, one of the most common sites of viral infection. The lung has a vast array of host defense mechanisms to cope with the inhaled pathogens that can potentially result in upper respiratory infection. Cells of the innate immune system
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located within the respiratory tract, such as the alveolar macrophage, constitute the first line of defense against invading organisms until specific immune responses are initiated. The alveolar macrophage has the ability to ingest and degrade viruses, facilitating the clearance of the virus from initial sites of infection ŽMogensen, 1979.. A murine model of HSV-1 infection has been used in our lab to study the effects of prolonged exercise to fatigue on alveolar macrophage intrinsic resistance to viral infection ŽDavis et al., 1997.. Intrinsic resistance by the macrophage is manifested in the cells ability to take up viral particles and limit or stop their replication ŽShellam and Flexman, 1986; Morahan et al., 1995.. This type of resistance to viral infection may be completely independent of extrinsic resistance. Macrophage extrinsic resistance refers to the ability of the macrophage to inactivate extracellular viruses via cytokine release or macrophage mediated antibody dependent cellular cytotoxicity ŽMorahan et al., 1995.. The findings from our laboratory demonstrate that a single, prolonged bout of exercise to fatigue increases susceptibility to intranasal HSV-1 infection and suppresses alveolar macrophage intrinsic resistance to HSV-1 infection ŽDavis et al., 1997.. To elucidate the mechanisms of this exercise-induced suppression of macrophage antiviral resistance, we evaluated the roles of adrenal hormones and catecholamines. We hypothesized that blocking either the release of, or effect of, corticosterone andror catecholamines during exercise would eliminate the suppression of macrophage antiviral resistance. Adrenalectomized mice were used to determine if adrenal hormones were involved in the suppression of alveolar macrophage antiviral function. The beta-adrenergic receptor antagonist propranolol was used to inhibit catecholamine-receptor binding to establish the possible role of catecholamines.
2. Materials and methods 2.1. Mice Male CD-1 mice, 4 weeks of age, were purchased from Charles River Labs and acclimated to our facility for at least three days prior to any experimentation. Mice were purchased as pathogen free stock and periodic antibody screening of sentinel mice yielded negative results for common murine viral or bacterial pathogens. Mice were maintained on a 12:12 h light–dark cycle in a low stress environment Ž228C, 50% relative humidity, low noise. and given food ŽPurina Chow. and tap water ad libitum. 2.2. Adrenalectomy In the first series of experiments, mice were anesthetized with an intraperitoneal injection of avertin Ž2-2-2-
tribromoethanol, Polysciences, Warrington, PA.. A dorsolateral incision was made superior to each kidney, kidneys were exposed and the adrenal gland was removed. Sham adrenalectomized mice underwent the same procedure with the exception of adrenal gland removal. Mice were then returned to their cages and were allowed to recover for one full week prior to further experimentation. All adrenalectomized mice received saline drinking water Ž0.9% NaCl. supplemented with corticosterone Ž50 mgrl, Sigma, St. Louis. to provide basal level replacement of this hormone. 2.3. Treadmill acclimation and exercise protocol In the first series of experiments, mice were randomly assigned to one of the following five groups Ž n s 10 per group.: CON, control treatment, sham adrenalectomy; ADX CON, control treatment, adrenalectomy; EX, exercise treatment, sham adrenalectomy; ADX EX, exercise treatment, adrenalectomy; ADX EX q C, exercise treatment, adrenalectomyq corticosterone replacement during exercise equivalent to that level obtained in normal exercising mice. In the second series of experiments, mice were randomly assigned to one of the following groups Ž n s 10 per group.: CON, control treatment; CON q PRO, control treatment plus propranolol; EX, exercise treatment; EX q PRO, exercise treatment plus propranolol. Each series of experiments was repeated three times for a total n s 30 in each group. The exercise treatment consisted of one run to fatigue at gradually increasing speeds, 18–40 m P miny1 , 5% grade. Fatigue was defined as the point at which mice fail to maintain pace with the treadmill despite being given mild physical prodding continuously for 5 min ŽWoods et al., 1994.. Electric shock was never used in these experiments as mice readily respond to a gentle tap of the tail or hindquarters. With this protocol mice typically fatigued in 2.5–3 h. Mice in the control groups were contained in well ventilated Plexiglas lanes above the treadmill for an equivalent period of time, exposed to similar handling, noise and treadmill vibration. All mice were acclimated to treadmill running for three days prior to the actual experiment. During the treadmill acclimation period, mice were allowed to briefly explore the control lanes and the treadmill followed by a run at slow speeds Ž1–5 m P miny1 . for a 5 to 10 min period. Mice were acclimated twice per day at the beginning and the end of the 12 h dark period. 2.4. Drug preparation and administration In the first series of experiments, all adrenalectomized mice received a basal replacement of corticosterone, 50 mgrl, in the drinking water. In addition, mice in the ADX EX q C group received a 0.1 ml subcutaneous injection of corticosterone, 8 mgrkg dissolved in saline, administered at the onset of exercise and again 90 min into the exercise session. Mice in the remaining four groups received a 0.1
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ml subcutaneous injection of saline at the same time points. In the second series of experiments, a 10 mgrkg dose of DL-propranolol ŽSigma, St. Louis. was administered intraperitoneally 15 min prior to the start of the exercise or control sessions to mice in the CON q PRO and EX q PRO groups. Mice in the EX and CON groups received an intraperitoneal injection of saline. In a third experiment Ž".isoproterenol-bitartrate salt was added to the wells of microtiter plates containing pooled alveolar macrophages obtained from 20 control mice at the following concentrations: 1, 0.2 and 0.02 m mol P ly1 . 2.5. AlÕeolar macrophage collection and preparation All animals were sacrificed in a bell jar containing ether immediately after reaching fatigue during exercise or at an equivalent period of time in the control Plexiglas lanes. Sacrifice by overetherization occurred in less than 2 min. Lungs were removed and alveolar macrophages were obtained by gentle lavage of the lungs with 15–20 ml of physiological media. The culture media used in all experiments was RPMI 1640 ŽGibco, Grand Island, New York. containing 100 Urml penicillin, 100 m grml streptomycin and 20 mmolP ly1 glutamine. Lung lavage cells were washed once with RPMI, and any remaining red cells were lysed with Tris ammonium chloride, pH s 7.2. Cells from mice in each group Ž n s 10. were pooled due to the limited number of cells from each mouse and adjusted to a concentration of 4 = 10 6 cellsrml in RPMI media supplemented with 10% fetal bovine serum ŽCRPMI-Environmental Diagnostics, Burlington, North Carolina.. Viability was determined using trypan blue exclusion and the percentage of macrophages was calculated from a cytocentrifuge preparation followed by Diff Quick ŽBaxter Scientific, Chicago, IL. stain. Subsequently, 0.2 ml of the cell preparation was added to the wells of a 96 well microtiter plate and allowed to adhere for 3 h at 378C. After three hours each well was washed gently with prewarmed Ž378C. RPMI to remove nonadherent cells. 2.6. HSV-1 Õirus HSV-1 KOS strain virus was propagated in Vero cells. Stocks of virus were titrated on Vero cells by a plaque assay and contained 1.2 = 10 8 pfurml. Aliquots of virus were stored at y808C until use. 2.7. AlÕeolar macrophage antiÕiral resistance: Neutral red assay
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virus was allowed to absorb in a small volume Ž50 m l. for 90 min. Prewarmed CRPMI media was added to each well and the plates were incubated at 378C, 5% CO 2 for 48 h until a cytopathic effect was observed. The cytopathic effects were then quantified by a neutral red dye uptake assay ŽLazdins et al., 1990.. Briefly, cell monolayers were washed twice with RPMI and stained for 2 h with 0.006% neutral red diluted in RPMI. The stained monolayers were washed and the dye was extracted by lysing cells with 0.2 ml of Sorensens citrate buffer ŽpH 4.1. containing 50% ethanol. The optical density was read at 530 nm on a Dynatech MR5000 microplate reader. The cytopathic effect was evaluated by calculating a viability index which is expressed as the ratio of dye uptake by infected cells to dye uptake by uninfected cells. Healthy uninfected cells have a greater dye uptake than infected dying cells. 2.8. Plasma corticosterone and free fatty acid analysis At the time of sacrifice, blood was collected from the inferior vena cava in syringes containing 0.05 ml of 100 Urml heparin. After centrifuging, the plasma was collected and stored at y208C until analysis of corticosterone and free fatty acids. Corticosterone was analyzed using a rodent corticosterone RIA kit ŽDiagnostic Products Corp., Los Angeles.. Free fatty acids were determined using a standard spectrophotometric technique ŽNoma et al., 1973.. 2.9. Statistics The results from the experiments assessing macrophage intrinsic antiviral resistance were compared with a one way ANOVA Ž4–5 groups.. Due to the limited number of alveolar macrophages obtainable from one mouse, cells from the 10 mice per group were pooled and the experiment was replicated three times for a total n s 3. Plasma corticosterone and free fatty acids were measured in each mouse for a total n s 30 per group. Differences in corticosterone and free fatty acids between groups were analyzed with a one way ANOVA Ž4–5 groups.. In the final experiment, various amounts of isoproterenol were added to pooled cells in wells of microtiter plates. Four wells per concentration of isoproterenol were measured and differences between wells containing the various concentrations of isoproterenol were analyzed with a one way ANOVA. For all experiments, when significant differences were detected, the Student–Neuman–Keuls post hoc test was used to analyze group differences.
3. Results 3.1. Exercise treatment
After nonadherent cells were removed from the wells of the microtiter plate by washing, macrophages were infected at 7–10 pfurcell with the HSV-1 KOS virus. The
Run time to fatigue did not significantly differ across groups in any of the experiments. The experiment using
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adrenalectomized animals was performed three times and the average run time to fatigue across experiments was as follows: 162 " 31 min for EX, 154 " 28 min for ADX EX and 151 " 39 min for ADX EX q C Žmean " S.D., n s 30 per group.. Mice receiving propranolol also exhibited no difference in run time to fatigue compared to mice receiving saline, 152 " 26 and 158 " 28 min, respectively. 3.2. Corticosterone leÕels A single bout of exercise to fatigue significantly elevated plasma corticosterone levels ŽEX ) CON, p - 0.01, Fig. 1.. Replacement of corticosterone by subcutaneous injection in ADX EX q C resulted in plasma levels similar to that of EX. In addition, replacement of corticosterone in the drinking water of adrenalectomized mice, ŽADX CON and ADX EX. yielded basal levels of corticosterone. Although these levels appear to be on the low side of the normal resting physiological range, these samples were taken approximately 3 to 4 h after the active dark period and mice had been water deprived for the 2–3 h on the treadmill or in control lanes. Mice tend to consume water during the dark active period, and in other experiments, we found that replacement levels are higher during this active period Ž300–500 nmol P ly1 . and gradually decline during the day. There is some degree of variability among the replacement levels as they reflect the amount of water consumed by an animal in the last several hours before sacrifice. As expected, EX and ADX EX q C exhibited high plasma corticosterone whereas ADX CON and ADX EX had low levels due to adrenalectomy ŽFig. 1..
Fig. 2. Viability index of macrophages infected with HSV-1 virus. Results are meansqSE for pooled macrophages in wells of microtiter plates 48 h post infection. One value is obtained per group per experiment and means from each group are compared across experiments Ž ns 3.. Significant differences from control ŽCON. value indicated by ) P - 0.01.
3.3. Exercise, adrenal hormones, and macrophage antiÕiral resistance In Fig. 2, the results from the three experimental trials designed to assess the role of the adrenal hormone corticosterone are shown. Exercise decreases the ability of the alveolar macrophage to resist viral infection in vitro as demonstrated by the decrease in percent viability; EX CON, p - 0.01. The percent viability of the macrophages from ADX EX and ADX EX q C is also not different from CON or ADX CON. These results suggest that although products released from the adrenal gland may be involved, it is not corticosterone that acts to suppress macrophage antiviral resistance during exercise because no suppression was observed in the ADX EX q C group even though this group demonstrated corticosterone levels similar to EX. 3.4. Effect of catecholamines during exercise on macrophage antiÕiral resistance
Fig. 1. Plasma corticosterone levels obtained immediately post exercise or control treatment. Results are means"SE for 30 mice. Significant differences from control ŽCON. values indicated by ) P - 0.01; CONs control treatment, sham adrenalectomy; ADX CON s control treatment, adrenalectomy; EX sexercise treatment, sham adrenalectomy; ADX EX sexercise treatment, adrenalectomy; ADX EXqC sexercise treatment, adrenalectomyqcorticosterone replacement during exercise.
Epinephrine and norepinephrine increase during exercise ŽDavies and Rew, 1973; Kjaer, 1992. and the betaadrenergic receptor antagonist propranolol was used to block the effects of these catecholamines. In the second series of experiments, propranolol Ž10 mgrkg. was administered just prior to the exercise andror control treatments to determine whether catecholamines released during fatiguing exercise act to suppress macrophage antiviral resistance. Again, macrophage antiviral resistance reflected by % viability is decreased in EX compared to CON ŽFig. 3, EX - CON, p - 0.01. Administration of propranolol prior to exercise completely eliminated the suppressive effects
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Fig. 3. Viability index of macrophages infected with HSV-1 virus. Results are meansqSE for pooled macrophages in wells of microtiter plates 48 h post infection. One value is obtained per group per experiment and means from each group are compared across experiments Ž ns 3.. Significant differences from control ŽCON. value indicated by ) P - 0.01. CON s control treatmentqsaline, CONqPROs control treatmentq propranolol; EX s exercise treatmentqsaline; EXqPRO s exercise treatmentqpropranolol.
of exercise on macrophage antiviral resistance; CON, CON q PRO, EX q PRO ) EX, p - 0.01. In order to establish whether propranolol was effective in blocking peripheral beta-adrenergic receptors, free fatty acids were measured. It is well known that the exercise-induced increase in plasma free fatty acids is primarily mediated via beta-adrenergic stimulation of lipolysis within adipocytes. Fig. 4 demonstrates that propranolol administration suppressed the typical increase in plasma free fatty
Fig. 4. Plasma free fatty acid concentration measured immediately post exercise or control treatment. Results are meansqSE for the 30 mice in each group. Significant difference from control value indicated by ) P 0.01. Significant difference from control ŽCON. and exercise ŽEX. indicated by q P - 0.01.
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Fig. 5. In vitro effect of isoproterenol ŽISO. on macrophage antiviral resistance assessed by viability index. Pooled alveolar macrophages were incubated with ISO for 3 h and then infected with HSV-1 virus. Results are meansqSE of individual wells in microtiter plates 48 h post infection. Significant difference from 0.0 isoproterenol is indicated by an ) Ž P - 0.05..
acids observed during prolonged exercise ŽEX q PRO EX, p - 0.01.. These findings suggest that propranolol was effective in blocking peripheral beta-adrenergic receptors during exercise. 3.5. In Õitro beta-adrenergic agonist (isoproterenol) and macrophage antiÕiral resistance To verify that beta-adrenergic receptor stimulation can suppress macrophage antiviral resistance, we added the beta-adrenergic agonist isoproterenol directly to cultures of alveolar macrophages obtained from CON mice. As expected, all wells infected with HSV showed a cytopathic effect Ždecreased percent viability.. When isoproterenol was added to these cultures of infected macrophages, the percent viability declined in a dose-dependent manner ŽFig. 5.. Both 1 and 0.2 m mol P ly1 concentrations of isoproterenol significantly suppressed macrophage antiviral resistance Ž p - 0.05.. These results suggest that direct activation of beta-adrenergic receptors in culture suppresses macrophage intrinsic antiviral function as measured by neutral red dye uptake. The findings obtained in culture in vitro complement the ex vivo findings demonstrating that activation of beta-adrenergic receptors during exercise suppresses macrophage resistance to viral infection. 4. Discussion The results of these studies confirm our previous findings that exercise to fatigue increases alveolar macrophage susceptibility to viral infection with HSV-1 ŽDavis et al.,
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1997.. A decrease in oxygen dependent antimicrobial activity in alveolar macrophages has also been reported in horses following strenuous exercise ŽWong et al., 1990.. In contrast to the immunosuppression observed in alveolar macrophages, exhaustive exercise appears to enhance peritoneal macrophage phagocytosis and antitumor cytotoxicity ŽOrtega et al., 1993; Woods et al., 1994.. Macrophage responses to exercise can vary depending upon the location of the cell and the particular function being assessed. The increased susceptibility to upper respiratory infection following prolonged intense exercise may be due in part, to the suppression of alveolar macrophage function. However, the mechanisms mediating these changes in macrophage function following exercise have not been addressed. The findings from these experiments suggest that alveolar macrophage resistance to viral infection following prolonged exercise to fatigue may be mediated by catecholamine binding to beta-adrenergic receptors. Other investigators have also reported that catecholamines may mediate alterations in various immune responses during exercise. For example Murray et al., 1992, found that prior administration of propranolol blunted the exercise induced decrease in concanavalin A ŽConA. stimulated IL-2 receptor expression and w3 Hx thymidine incorporation in human peripheral blood mononuclear cells. Others report that changes in plasma levels of epinephrine are likely associated with alterations in circulating numbers of lymphocyte subsets ŽNeiman et al., 1992.. In our study, evidence from the adrenalectomy experiments and from the experiments using propranolol lends support to the conclusion that elevated catecholamines can mediate macrophage antiviral resistance during exercise. Adrenalectomized-exercised mice do not exhibit the decrease in macrophage antiviral resistance that is observed in the exercised mice receiving sham surgery, suggesting that the adrenal glands produce some factor during exercise that suppresses macrophage function. However, this factor does not appear to be corticosterone, as adrenalectomized-exercised mice receiving corticosterone replacement have plasma corticosterone levels similar to that in the adrenal intact mice during exercise. If the level of corticosterone produced during exercise suppressed macrophage function, a decline in macrophage antiviral resistance should have been observed in adrenalectomized mice receiving high doses of corticosterone during exercise. Instead these results are consistent with the hypothesis that catecholamines mediate the exercise-induced decline in macrophage antiviral resistance. The adrenal medulla is a major source of epinephrine during fatiguing exercise ŽDavies and Rew, 1973; Kjaer, 1992.. Removal of the adrenal gland attenuated the suppression of macrophage function. Even more convincing are the experiments which show that prior administration of the beta-adrenergic receptor antagonist completely eliminated the exercise-induced suppression. The dose of propranolol administered was clearly
sufficient to elicit a physiological response Žinhibition of free fatty acid release via blockade of peripheral betaadrenergic receptors on adipocytes. and, therefore, could conceivably block the effect of catecholamines on betaadrenergic receptors known to be present on macrophages ŽAbrass et al., 1985; Liggett, 1989.. The results from the first series of experiments suggest that the adrenal is the source of an agent Žlikely epinephrine. that may suppress macrophage antiviral function. Although these experiments were not designed to determine the source of catecholamines that may alter macrophage antiviral function, it is possible that the release of catecholamines from sympathetic nerve endings also played a role in modulating macrophage function, considering the numerous studies reporting interactions between the sympathetic nervous system and the immune system ŽMadden et al., 1995.. It is possible that short periods of stress may alter immune function primarily through elevated catecholamines while chronic, long term stress modulates immune response through prolonged elevations in corticosteroids. The results from our experiments and others ŽBoranic et al., 1990. suggest that several hours of stress may alter certain parameters of immune function via elevated catecholamines. Yet it appears that both catecholamines and corticosteroids may act to alter different parameters of immunity in other stress paradigms ŽDobbs et al., 1993.. For example, in one model of shock-induced immunosuppression, adrenalectomy attenuated the peripheral blood lymphocyte suppression whereas propranolol and nadolol attenuated the splenic lymphocyte suppression ŽCunnick et al., 1990.. Other studies employing long term stress, 16 to 18 h of restraint stress daily for one to two weeks, demonstrate that the stress-induced release of corticosteroids acts to increase macrophage susceptibility to Mycobacterium aÕium growth and suppresses the generation of HSV-specific cytotoxic lymphocytes ŽBonneau et al., 1993; Brown and Zwilling, 1994.. Future studies comparing long term and short term stress and differing immune parameters may help to clarify the particular roles of catecholamines and corticosteroids in stress-induced immunomodulation. However, these types of studies can be difficult to execute as animals may adapt to chronic stress and fail to secrete high levels of corticosteroids. The mechanisms responsible for the decrease in macrophage antiviral function following catecholamine binding to adrenergic receptors are unknown. Several investigators have shown that macrophage function can be altered by epinephrine, norepinephrine, or isoproterenol and that these beta-adrenergic agonists appear to exert their effects through elevations in intracellular cAMP ŽKoff and Dunegan, 1986; Chelmicka-Schorr et al., 1992.. One study showed that norepinephrine and epinephrine elevated cAMP in peritoneal macrophages and subsequently blocked the capacity of these macrophages to be activated to a lytic state capable of killing HSV infected cells. Exogenous dibutyryl cAMP had the same effect ŽKoff and Dunegan,
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1986.. Similarly, macrophages treated with isoproterenol prior to LPS treatment resulted in suppressed tumor necrosis factor a ŽTNFa . production and the same response occurred when dibutyryl cAMP was used. In addition, the beta-adrenergic antagonist pindobind blocked the effect of isoproterenol on macrophages ŽChelmicka-Schorr et al., 1992.. These findings suggest that the effect of isoproterenol on macrophages is mediated through beta-adrenergic receptors and that beta-adrenergic agonists exert their effects by elevating intracellular cAMP. Although the evidence from these studies supports the concept that elevated intracellular cAMP can suppress some macrophage functions, it is not clear how intrinsic antiviral resistance may be altered by elevated cAMP. Given that the intrinsic antiviral activity of murine macrophages to HSV-1 appears to be strongly influenced by increases in interferon-beta ŽPyo et al., 1991., it is possible that elevations in cAMP alter the expression of interferon-beta or other important antiviral cytokines, including TNFa which has been shown to be suppressed after exposure to isoproterenol ŽChelmicka-Schorr et al., 1992.. However, our recent findings suggest that interferon-beta mRNA levels are actually elevated in infected macrophages from exercised mice, apparently in response to a greater degree of viral infection Žunpublished observations.. It is therefore possible that catecholamines act in some other manner leading to greater viral replication, and elevated interferon levels simply reflect this greater degree of viral infection. The data from these studies suggest that catecholamines may act to suppress macrophage intrinsic antiviral resistance during exercise and provides one of the few in vivo indications of mechanisms operating during prolonged, fatiguing exercise that may decrease resistance to viral infection within the lung. This decrease in innate immunity may contribute to increased susceptibility to viral respiratory infections following prolonged, strenuous exercise. Acknowledgements This work was supported in part by a Grant-in-Aid of Research from Sigma Xi, The Scientific Research Society and the American College of Sports Medicine FoundationrCybex research grant. References Abrass, C.K., O’Connor, S.W., Scarpace, P.J., Abrass, I.B., 1985. Characterization of the beta-adrenergic receptor of the rat peritoneal macrophage. J. Immunol. 135, 1338–1341. Bonneau, R.H., Sheridan, J.F., Feng, N., Glaser, R., 1993. Stress-induced modulation of the primary cellular immune response to Herpes simplex virus infection is mediated by both adrenal-dependent and independent mechanisms. J. Neuroimmunol. 42, 167–176. Boranic, M., Pericic, D., Poljak-Blazi, M., Maney, H., Sverko, W., Gabrilovac, J., Radacic, M., Pivac, N., Miljenovic, G., 1990. Immune response of stressed rats treated with drugs affecting serotoninergic and adrenergic transmission. Biomed. Pharmacother. 44, 381–387.
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