In vivo indomethacin reverse exercise-induced immunosuppression in rats

Int. J. Immunopharmac., Vol. 18, No. S/9, pp. 491497, 1996 Copyright 0 1996 International Society for Immunopharmacology Published by ElsevierScienceLtd. Printed in Great Britain

Pergamon

0192-0561/96$15.00+ .OO PII: S0192-0561(%)00024-0 IN VW0

INDOMETHACIN REVERSE EXERCISE-INDUCED IMMUNOSUPPRESSION IN RATS*

PIERRE ASSELIN, CORINNE BENQUET, KRZYZSTOF KRZYSTYNIAK, PAULINE BROUSSEAU, ROLAND SAVARD and MICHEL FOURNIERT Dtpartement des Sciences Biologiques et TOXEN, UniversitC du Qutbec &Montrkal, Mont&al, Qubbec, Canada (Received 14 November 1995 and infinalform 1 April 1996)

Abstract-The effect of oral indomethacin on the immunosuppressive effect of exercise was examined in exercised untrained female Wistar rats immunized with sheep red blood cell (SRBC) antigens. Intensity of the 1 h exercise was controlled by &50 kPa air pressure, generated by a compressor located at the bottom of a water tank, during continuous swimming of the rats, previously immunized with SRBC. After 48-72 h, depending on the ip (intraperitoneal) or iv (intravenous) route of SRBC immunization, the exercise suppressed humoral PFC response and augmented phagocytosis of peritoneum macrophages. These effects occurred only when exercise was performed at 48 h after antigen injection. Animals receiving indomethacin, however, did not show any exercise-related suppression of the PFC response. The data suggest a relationship between exercise-induced immunosuppression and possible increased in vivo prostaglandin synthesis during the intense exercise. Overall, exercise-related suppression of humoral PFC response was dependent on the intensity of the exercise, was time specific, and was reversible by pharmacological blockade of the cyclooxygenase pathway of prostaglandin synthesis. Copyright 0 1996 International Society for Immunopharmacology. Keywords: exercise, immunosuppression,

prostaglandins, indomethacin, humoral response, phagocytosis.

Possible negative consequences of intense exercise are questioned in relation to the popularity of physical activities. Several athletes were reported to miss international competitions because of benign infections (Fitzgerald, 1988). It is postulated that exhaustive exercise can produce different effects, such as oxidative

DNA damage in humans (Hartmann et al., 1995) and temporary immune depression (Mahan and Young, 1989; Garagiola et al., 1995). Several points remain unclear how physical exercise could affect the immune system (Fitzgerald, 1988; Oshida et al., 1988, Benquet et al., 1994; Bouix et al., 1995). First, it is not clear how the intensity of the exercise and the fitness level could affect the immune response (Fitzgerald, 1988; Mahan and Young, 1989; Bouix et al., 1995). For example, moderate joggers claimed to be in better health which made them less susceptible to bacterial infections, thus pointing to the possibility of activating/stimulating the host immune system by moderate exercise (Goodin and Shephard, 1985; Fitzgerald, 1988). Second, intense exercise appeared to induce rather short and transient immunosuppression (Fitzgerald, 1988; Fry et al., 1992;

Benquet et al., 1994). The nature and duration of this transient suppression was unclear. Third, it is not clear how intense exercise could alter proportions of the lymphoid subsets (Keast et al., 1988; Lewicki et ol., 1988; Smith et al., 1993). Fourth, the target cells are poorly defined. For example, exercise reduced NK cell activity (Pedersen et al., 1988) but activated macrophage phagocytosis (Fehr et al., 1989). Finally, it is not clear what the relationship is between exerciserelated changes in the levels of corticosteroids, endorphins and prostaglandins and subsequent changes in humora and cellular responses (Hashimoto et al., 1983; Lijnen et al., 1983; Khansary et al., 1990; Bouix et al., 1995). A variety of hormones including neuropeptides, and chemicals released during exercise such as cortisol, catecholamines and prostaglandins can possibly redistribute the immune cells and modulate/suppress lymphocyte functions (Hashimoto et al., 1983; Lijnen et al., 1983; Khansary et al., 1990; Nieman and Nehlsen-Cannarella, 1994). Some clinical situations associated with depressed cellular immunity have been linked with overproduction of PGE, or

t Author to whom correspondence should be addressed at: DCpartement des Sciences Biologiques, Universitt du Quebec ii Montrtal, C.P. 8888, Succ. A., Montreal, Qukbec, Canada H3C 3P8. Phone: (514) 987 4027; Fax: (514) 9874647. 491

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increased sensitivity of lymphocytes to this feedback inhibitor (Ceuppens and Goodwin, 1982). Indomethacin, a cyclooxygenase inhibitor used for pharmacological blockade of prostaglandin synthesis (Vane, 1971; Webb and Osheroff, 1976), decreased in vitro mitogen-stimulated immunoglobulin (Ig) synthesis (Ceuppens and Goodwin, 1982). Conversely, addition of PGEz to the lymphocyte cultures reversed the indomethacin-related reduction of the Ig synthesis (Ceuppens and Goodwin, 1982). In a previous study, we demonstrated modulation of the exercise-related immunosuppression by dietary factors, such as by polyunsaturated fatty acids (PUFA)(Benquet et al., 1994). In this study, we examblockade of ined the effect of in uivo indomethacin prostaglandin synthesis on exercise-induced immunosuppression in rats.

EXPERIMENTAL

PROCEDURES

Animals Young adult female Wistar rats weighing 200-250 g were obtained from the Charles River (St-Constant, Quebec, Canada). Upon arrival, the rats were quarantined for 1 week and then either allowed to remain sedentary or were used in the exercise program. The rats were housed by groups of 2-3 animals and given water and food ad libitum. All procedures used in this study were in agreement with the policy of the Canadian Council on Animal Care. Exercise protocol Exercise consisted of 1 h of continuous swimming, as previously reported (Benquet et al., 1994) with modifications. Briefly, the rats were randomly divided into two groups. At day 0 of the experiment, rats from one group were immunized with the SRBC antigen, while the other group served as non-immunized controls. Two sedentary control groups were randomly selected from the immunized and non-immunized animals. Except for the sedentary controls, all animals performed single 1 h swimming exercise, at controlled intensity, at different time intervals, i.e. either immediately after the SRBC immunization (day 0) or at l5 days after the immunization. A propylene tank containing warm water (36.5Cf0.5”C) was used for the swimming exercise. The intensity of the swimming exercise was controlled by increasing the air pressure generated by a central compressor located at the bottom of the water tank. At no air pressure, the lowintensity exercise was performed in waveless conditions, whereas at 4 pounds (17 kPa) continuous

e/ al.

air pressure (medium intensity) and 12pounds (50 kPa) continuous air pressure (high intensity), wave swimming conditions were created. Each experimental group consisted of six animals. Immediate effects on immunocompetent cells were monitored within hours, and delayed effects were determined daily, up to 5 days following the swimming exercise. All animals were killed at 4 or 5days after the SRBC immunization (day 0), depending on the intravenous (iv) or intraperitoneal (ip) introduction of the SRBC antigen. All assays were performed individually on each rat. Cells Peritoneal exudate cells (PEC) were collected as previously described (Benquet et al.. 1994). Briefly, peritoneal cavities of the rats were washed with heparinized RPM1 1640 medium (Gibco, Grand Island, NY). The cells were centrifuged at 250x g at 4°C for 10 min, resuspended in RPM1 1640 and counted. Spleens were aseptically removed, washed twice with phosphate-buffered saline (PBS) and splenocyte suspensions were obtained by teasing the spleens into the Hanks balanced salt solution (HBSS, Flow Laboratories, Toronto, Ontario). The viable nucleated spleen cells were counted using the trypan blue excluding method or by ethidium bromide staining and cytofluorometric assay (Brunet et al., 1993). The anti-SRBC

humoral response

Exercised and sedentary animals were injected ip with 15 x IO9 or iv with 5 x lo9 SRBC (Cunningham and Szenberg, 1968). The number of IgM plaque forming cells (PFC) was determined 4d after iv immunization or 5 d after ip immunization of animals with SRBC (Jerne et al., 1974) (Diagram 1). Single-cell iv

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Diagram I, Protocol of intravenous (iv) or intraperitoneal (ip) immunization of Wistar rats with sheep red blood cells (SRBC), subsequently exercised in a single bout of intense swimming, O-5 days thereafter. Animals were sacrificed immediately after exercise, spleens removed, and humoral IgM response to SRBC was assayed by plaque-forming cells (PFC)-Circles mark time intervals for the exercise-related suppression of PFC.

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Exercise-induced Immunosuppression suspensions were prepared by coincubation with SRBC in the Cunningham chambers, and the number of cells secreting anti-SRBC IgM antibodies determined in duplicate (Jerne et al., 1974).

RESULTS Exercise-induced a suppression of PFC response

The effects of 1 h swimming exercise on primary IgM antibody response to the T-dependent SRBC Blockade of cyclooxygenase pathway by indomethacin antigen were determined by the PFC assay and compared to non-exercised controls. The animals were Indomethacin (ICN, Costa Mesa, CA) was disimmunized ip with the SRBC antigen and the exercise solved in a mixture of 5% ethanol-water (v/v). Animals were gavaged twice on the exercise day, at 1O:OO was performed &5 d thereafter. As shown in Fig. 1, a significant reduction in the PFC response was a.m. and 3:00 at indomethacin doses of OSmg and observed only when exercise was performed 48 h after 0.25 mg, respectively. Control animals were also ip immunization and 3 d prior to the PFC assay. Simigavaged twice with water only. The 1 h swimming lar results were obtained when the latter response was exercise at 17 kPa air pressure was performed between expressed as a number of PFC/spleen (results not the two indomethacin administrations, i.e. 2 h before shown). Furthermore, when the animals were immuthe second gavage. Four groups of rats were examnized iv with the SRBC antigen, the suppression of ined: (1) water-gavaged, sedentary controls; (2) waterPFC response was also noted when the exercise was gavaged, exercised controls; (3) indomethacinperformed 48 h after immunization and 2 d prior to gavaged, sedentary control; and (4) indomethacinthe PFC assay (Fig. 2, Diagram 1). Generally then, gavaged, exercised rats. All animals were immunized suppression of PFC response was inducible only when iv with the SRBC antigen 2d before the exercise exercise was performed at 48 h post-immunization. (Benquet et al., 1994). Rats were killed 2d after the Cytofluorometric analysis of the microsphere exercise, and the PFC assay was performed, as uptake in the sedentary controls during a 30 min incudescribed above. Additional negative control of nonbation of cells at 37°C showed a marked phagocytic immunized, exercised animals was examined for the activity (240% of cells engulfed three or more micnumber of spontaneous PFC. rospheres/cell) over the negative control of phagocytosis at 4°C (14.0% of engulfing cells)(p
Cells were centrifuged at 25Og at 4°C for lOmin, resuspended in RPM1 1640 and counted. Cell suspensions ( lo6 cells/ml RPM1 1640, GIBCO) were incubated at 4°C (negative control) and at 37°C for 30 min with fluorescent microspheres (ratio 100: 1 microspheres/cell; Cat. NO. 17687, Polyscience, Warrington, PA), previously determined to he optimal for phagocytosis assay (Krzystyniak et al., 1989). The percentage of cells with engulfed particles was determined by flow cytometry, using FACScan (BectonDickinson Immunocytometry Systems, Mountain View, CA) (Steward et al., 1987). Individual particulate burdens within each cell were expressed as the total frequency of phagocytic cells and as the frequency of phagocytic cells with three or more internalized particles.

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Statistical analysis

Data are expressed as arithmetical means + S.D. Two-way analysis of variance (ANOVA) was used to establish significant differences between control and experimental groups. Significance was concluded at p I 0.05. For the significantly different groups, a oneway ANOVA was carried out between the sedentary and exercised groups. For the statistical analysis of percentages, the x2 test was used (Lutz, 1987).

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DAYS Fig. 1. Uptake of 23 fluorescent microspheres by phagocyting cells from peripheral blood (-•-) and humoral PFC response of spleen lymphocytes (-¤-) from ip SRBC-immunized Wistar rats, after a single bout of intense swimming exercise at 17 kPa air pressure for 1h, O-5 days after the immunization. Sedentary controls of phagocytosis are represented by upper broken line. Lower broken line represents sedentary controls f S.D. (crossed space) of the PFC assay. Data are mean from five animals + S.D.; ‘~50.05,

**p20.01.

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80

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air pressure

CkPa)

determined by the PFC assay and compared to sedentary controls. The animals were immunized iv with the SRBC antigen 2d prior to the exercise and the PFC assay was performed 2 d after the exercise (Fig. 2A). A marked reduction in the PFC response was observed for all groups exercising at different O-50 kPa air pressures (Fig. 2A). When compared to the immunized sedentary controls, the PFC response was reduced by 48%, 62% and 83% in the respective exercise groups depending on the increasing air pressure, thus forcing the animals to do more intense exercise (*p
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Effect of in vivo indomethacin administration on exercise-induced immunosuppression

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Fig. 2. (A) Effect of different swimming intensifies, at continuous f&50 kPa air pressure during 1h exercise, on the PFC response, determined at 4 days after the iv immunization of Wistar rats with the SRBC antigen. Sedentary controls are represented by solid line k SD. (crossed space). Data are mean from six animals k S.D.; *po.o1.

Indomethacin, when administered in vivo to the exercised rats, affected the exercise-induced immunosuppression of primary IgM antibody response to the T-dependent SRBC antigen (Fig. 2B). The animals were immunized iv with the SRBC antigen, the 1 h exercise at 17 kPa air pressure was performed 2 d later and the animals were killed 4d after the immunization. In other words, the time interval between iv immunization with the SRBC antigen and the exercise was 2 d, and the time interval between the exercise and the PFC assay was also 2d. As expected, the PFC response in exercised animals gavaged with water only, was significantly reduced (Fig. 2B). However, a normal PFC response was noted both for indomethacin-exposed non-exercised animals and for the indomethacin-exposed exercised group (Fig. 2B).

DISCUSSION Except for the animals exercised 48 h after the SRBC the phagocytic activity of macimmunization, rophages from all experimental groups was comparable to the sedentary controls (p>O.5). At 48 h after the ip SRBC immunization, twice as many (78%) cells engulfed three or more microspheres/macrophage (Fig. 1). This increase in phagocytic: activity was highly significant over the sedentary controls (p > 0.0 1). Overall, both macrophage phagocytosis and PFC response were affected by exercise 48 h after SRBC immunization. Correlation between the intensity of exercise and subsequent suppression qf the PFC response The effect of different intensifies of exercise on primary IgM antibody response to SRBC antigen was

Our study showed alterations of functions of immune cells by intense exercise, including indomethacin-reversible suppression of humoral PFC response. Indomethacin is a noncompetitive and irreversible pharmacological inhibitor of the cyclooxygenase pathway of prostaglandin synthesis (Vane, 1971; Webb and Osheroff, 1976). One study confirmed the immunosuppressive effects of exercise on PFC (Misefari et al., 1991). In addition, this study also demonstrated that indomethacin in cyclooxygenase inhibition could reverse the inhibitory effect of exercise on PFC. This suggests that exercise could cause suppression of PFC via increased PG synthesis. Increased PG synthesis following exercise has been demonstrated in man (Pedersen et al., 1988) rats

Exercise-induced Immunosuppression et al., 1983) and dogs (Zambraski and Dunn, 1980). It was suggested that physical stress caused lymphocytes to become more sensitive to prostaglandin E2 (PGEJ (Goodwin et al., 1981). Indomethacin, when added in vitro to the lymphocyte cultures, was shown to block the exercise-induced inhibition of PHA response in human peripheral blood cells (Smith et al., 1993), but failed to reverse the suppressive effect of exercise in other studies (Fry et al., 1992). Alternatively, it was suggested that postexercise suppression of mitogenic response to PHA was due to the release of serum factor(s) capable of inducing prostaglandin synthesis by circulating monocytes/ macrophages (Smith et al., 1993). Exogenous PGE2 strongly inhibited in uitro lymphocyte response to SRBC (Misefari et al., 1991). Nevertheless, indomethacin was shown to decrease in vitro mitogenstimulated immunoglobulin (Ig) synthesis, which could be reversed by subsequent addition of lo-* to 10m9M PGE2 to the lymphocyte cultures (Ceuppens and Goodwin, 1982). Levels of several potent immunomodulating hormones and cytokines such as epinephrin and cortisol were reported to be related to the exercise (Nieman and Nehlsen-Cannarella, 1994). However, the blood concentration of epinephrin dropped immediately postexercise, whereas the cortisol level could remain elevated for 2 or more hours (Nieman and Nehlsen-Cannarella, 1994). A recent study by Bouix et al. (1995) demonstrated that naloxone opiate blockade restored to normal an exercise-induced decreased T4/T8 ratio in rats (Bouix et al., 1995). Immunosuppression of PFC response appeared to be related to the intensity of exercise and to the 48 h lag period between in vivo SRBC immunization and the subsequent exercise. Low IL-2 production by splenic cells at O-48 h could possibly play at least a partial role in the suppression of PFC response (Asselin et al., in preparation). As a matter of fact, transient effects of intense exercise and subsequent recovery were reported by several authors (Fitzgerald, 1988; Lewicki et al., 1988; Fry et al., 1992; Benquet et al., 1994). The time period required for the lymphocyte activation leading to an increased sensitivity to PGE, could also be important (Steward et al., 1987; Sunder-PlaBmann et al., 199 1). Immediate and delayed effects of intense exercise were reported. For example, a reduced response of human lymphocytes to Con A mitogen, experienced (Hashimoto

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immediately after exercise, returned to normal levels within 2 h of recovery time (Fry et al., 1992). The NK

cell activity did not return to pre-exercise levels for 20h (Fitzgerald, 1988). Animal studies showed a reduced lymphocyte response to mitogens immediately after the exercise (Hoffman-Goetz et al., 1986; Mahan and Young, 1989; Ferry et al., 1991), which was, however, not observed in our studies (unpublished data). The decrease in glutamine content in skeletal muscles may also he involved in immunosuppression due to exercise (Parry-Billing et al., 1990; Sharp and Koutedakis, 1992; Newsholme, 1994). A high level of psychosocial stress due to the competition itself, the expectations of trainers, sponsors and the public, travel (changing time, zone, away from home, etc.) are other factors that could influence some aspects of the immune system (Kelly, 1980; O’Leary, 1990). Nevertheless, the exercise-induced immunomodulation appeared to he influenced by several factors including the intensity of the exercise. Depending on the intensity of training, beneficial or negative health effects could he expected (Goodin and Shephard, 1985; Fitzgerald, 1988; Mahan and Young, 1989; Smith and Weidemann, 1990; Mackinnon, 1994). The involvement of exercise-related increase in PGE, synthesis and the subsequent suppression of the immune response, postulated by several authors (Pedersen et al., 1988; Mahan and Young, 1989; Benquet et al., 1994) does not exclude the possible role of other hormones in exercise. A large number of hormones, as well as lymphokines and monokines in the bidirectional communication between the immune system and the neuroendocrine system should not be underestimated (Evans et al., 1986; Field et al., 1991; Nieman and Nehlsen-Cannarella, 1994). In conclusion, exercise-related prostaglandin overproduction could be a primary factor, however, but not necessarily an exclusive factor in exercise-induced immunomodulation. Moreover, exercise-related suppression of humoral PFC response was dependent on the intensity of the exercise and was time specific. authors thank Dr Jacques Bernier, Dr Felix 0. Omara, Mr Denis Flipo (TOXEN) and Mr Frederic Gagnon (Bio-Recherches, Senneville, Quebec) for helpful assistance and discussions. This work was supported by the Natural Sciences and Engineering Research Council of Canada, FODAR and TOXEN, Universitt du Quebec a Montreal. Acknowledgements-The

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