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The interactive effect of exercise and immunosuppressant cyclosporin A on
immune function in mice
Miau-Hwa Koa; Chen-Kang Changb; Ching-Lin Wuc; Yu-Chi Houd; Wei Honge; Shih-Hua Fangf a Department of Anatomy, China Medical University, Taichung b Sport Science Research Center,
National Taiwan Sport University, Taichung c Graduate Institute of Sports and Health Management,
National Chung Hsing University, Taichung d School of Pharmacy, China Medical University,
Taichung e Department of Exercise and Health Science, National Taiwan Sport University, Taichung f
Institute of Athletics, National Taiwan Sport University, Taichung, Taiwan First published on: 18 June 2010
To cite this Article Ko, Miau-Hwa , Chang, Chen-Kang , Wu, Ching-Lin , Hou, Yu-Chi , Hong, Wei and Fang, Shih-Hua(2010) 'The interactive effect of exercise and immunosuppressant cyclosporin A on immune function in mice', Journal of Sports Sciences,, First published on: 18 June 2010 (iFirst)
To link to this Article: DOI: 10.1080/02640414.2010.481306
URL: http://dx.doi.org/10.1080/02640414.2010.481306
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The interactive effect of exercise and immunosuppressant cyclosporin A
on immune function in mice
MIAU-HWA KO
1, CHEN-KANG CHANG
2, CHING-LIN WU
3, YU-CHI HOU
4,
WEI HONG
5, & SHIH-HUA FANG
61
Department of Anatomy, China Medical University, Taichung,2Sport Science Research Center, National Taiwan Sport University, Taichung,3Graduate Institute of Sports and Health Management, National Chung Hsing University, Taichung, 4
School of Pharmacy, China Medical University, Taichung,5Department of Exercise and Health Science, National Taiwan Sport University, Taichung, and6Institute of Athletics, National Taiwan Sport University, Taichung, Taiwan
(Accepted 25 March 2010)
Abstract
Cyclosporin A (CsA) is an effective immunosuppressive agent and exerts its actions by interfering with the activation of T cells. There is growing evidence that regular exercise improves immune function. However, the effects of exercise on immune functions in patients taking CsA are unclear. Here, we examine the interactive effects of CsA administration and regular exercise on immune function in mice. Forty-eight BALB/c mice were randomly assigned to one of six groups with eight mice per group: 0-Ex (no CsA/no exercise), 0þ Ex (no CsAþ exercise), 10-Ex (10 mg kg71 day71 CsA/no exercise), 10þ Ex (10 mg kg71 day71 CsAþ exercise), 20-Ex (20 mg kg71 day71CsA/no exercise), and 20þ Ex (20 mg kg71 day71 CsAþ exercise). The three exercise groups were trained for 8 weeks, three times a week, at approximately 75% maximum oxygen uptake ( _V O2max). Nitric oxide and interferon-gamma secretions by mitogen-activated macrophages and spleen cells, respectively, were higher in exercise groups than in non-exercise groups receiving the same doses of CsA. The results of this study indicate that regular exercise may enhance Type I helper T cell functions in mice receiving 10 or 20 mg kg71 day71
CsA. Our results demonstrate that moderate regular exercise modulates the immune function of CsA-treated mice. However, whether this exercise-induced immunomodulatory effect is beneficial or detrimental to CsA-treated patients needs to be clarified.
Keywords: Cyclosporine, exercise, immune response, Type I helper T/Type II helper T cytokines
Introduction
Cyclosporin A (CsA), a lipophilic cyclic peptide isolated from the fungus Hypocladium inflatum gams (Borel, Feurer, Gubler, & Stahelin, 1976), is a potent immunosuppressant that is extensively prescribed for the treatment of autoimmune diseases and allograft transplantation (Faulds, Goa, & Benfield, 1993; Opelz & Dohler, 2001). It is well known that CsA binds to cyclophilin to form an active complex that inhibits the enzyme calcineurin phosphatase (Liu et al., 1991). Without being dephosphorylated by calcineurin, nuclear factor of activated T cells family members are unable to translocate into the nucleus to activate cytokine genes in T cells and subsequently suppress immune responses (Matsuda & Koyasu, 2000; Rao, Luo, & Hogan, 1997).
It has been suggested that exercise affects various components of the immune system (Hoffman-Goetz & Pedersen, 1994; Nieman, 1998). For example, the
secretion of peripheral interferon-gamma was signifi-cantly lowered in patients with cardiovascular disease following 6 months of regular aerobic exercise (Castaneda et al., 2004). In addition, Goldhammer et al. (2005) observed that the concentrations of several pro-inflammatory cytokines such as interleu-kin-6 and interferon-gamma were reduced, whereas that of interleukin-10, an anti-inflammatory cytokine, was increased in coronary heart disease patients who underwent long-term aerobic training. It has been reported that regular exercise plays an important role in the rehabilitation of patients with autoimmune rheumatic disease (Minor, Hewett, Webel, Anderson, & Kay, 1989; Nordemar, Ekblom, Zachrisson, & Lundqvist, 1981). On the other hand, more vigorous exercise has been shown to suppress immune responses (Nehlsen-Cannarella, 1998). Therefore, it is important to determine whether moderate regular exercise is clinically beneficial for patients taking immunosuppressive agents, such as CsA.
Correspondence: S.-H. Fang, Institute of Athletics, National Taiwan Sport University, No. 16, Sec. 1, Shuan-Shih Road, Taichung 40404, Taiwan. E-mail: shfang@ntcpe.edu.tw
ISSN 0264-0414 print/ISSN 1466-447X online Ó 2010 Taylor & Francis DOI: 10.1080/02640414.2010.481306
Studies of organ transplant recipients receiving immunosuppressants have shown significant reduc-tions in exercise capacity and maximum oxygen consumption (Jensen, Yanowitz, & Crapo, 1991; Kavanagh et al., 1988). The reduction in exercise capacity may be caused by toxic effects of CsA on skeletal muscle cells. Research conducted by Mercier and colleagues (Mercier, Hokanson, & Brooks, 1995) and Biring and colleagues (Biring, Fournier, Ross, & Lewis, 1998) demonstrated that prolonged administration of CsA without regular exercise can considerably reduce the exercise capacity of rats. In addition, De Luca et al. (2005) reported that regular exercise produced beneficial effects on dystrophic Mdx mice that were taking CsA. Cyclosporin A has been widely used for the treatment of chronic inflammation and autoimmune diseases. Therefore, the determination of potential beneficial or detri-mental effects of moderate regular exercise on CsA-treated patients is an important clinical matter. In this study, to determine whether moderate regular exercise attenuates immune responses of CsA-administrated mice, different immunological para-meters were measured in CsA-treated and control mice with or without moderate regular exercise.
Methods Animals
Female BALB/c mice were purchased from the National Laboratory Animal Center (Taipei, Tai-wan) and maintained in the Animal Center of China Medical University. The animal room was main-tained at a 12-h light/12-h dark cycle with constant temperature (22+ 18C) and humidity (55 + 5%). The mice used were 8 weeks old and were housed in groups. The body weight of mice was recorded every 2 weeks (Figure 1). All procedures were performed according to the Principles of Laboratory Animal Care (NIH publication #86-23, revised 1985).
Drugs
Cyclosporin A (Neoral1, 100 mg ml71) was pro-vided by Novartis (Taiwan) Co. Ltd. Lipopolysac-charide, concanavalin A, and phosphate-buffered saline (PBS) were purchased from Sigma Chemical (St. Louis, MO, USA). Different doses of CsA were administered orally in one dose using gavage for 8 weeks. Recombinant interferon-gamma was purchased from PeproTech (Margravine, London, UK). The spleen cells and peritoneal excluded macrophages were maintained in Roswell Park Memorial Institute 1640 medium supplemented with 10% fetal calf serum, 1% penicillin, 1% streptomycin, and 200 mM L-glutamine (Gibco BRL, Grand Island, NY, USA).
Animals and drug administration
The 48 mice were randomly divided into six groups with eight mice in each group: 0-Ex (no CsA/ no exercise), 0þ Ex (no CsAþ exercise), 10-Ex (10 mg kg71 day71 CsA/no exercise), 10þ Ex (10 mg kg71 day71 CsAþ exercise), 20-Ex (20 mg kg71 day71 CsA/no exercise), and 20þ Ex (20 mg kg71 day71 CsAþ exercise). The three exercise groups underwent a progressive tread-mill run at increasing intensity and duration. Mice in the exercise groups performed progressive treadmill running that began at 10 m min71, 0% grade, for 10 min three times a week in weeks 1 and 2, and was systematically increased up to 18 m min71 at 0% grade for 45 min, three times a week for 8 weeks. The maximum oxygen consumption ( _V O2max) of the mice was estimated based on the equation developed by Fernando et al. (1993): V O_ 2max (ml min71)¼ 0.1276 weight (g)þ 0.040 6 running speed (m min71) 7 0.974. The final training intensity of the exercise groups was thus estimated at about 75%
_
V O2max(Fernando, Bonen, & Hoffman-Goetz, 1993; Jones et al., 2005). Mice were sacrificed 1 day after the last bout of exercise to avoid any influence of acute phase reactions.
Cell culture
Animals were sacrificed by cervical spine dislocation. The spleen was removed and crushed into a single cell suspension, and red blood cells were lysed with Tris-buffered ammonium chloride before being washed three times with HBSS. The numbers of cells were determined with a hemocytometer, and viabilities were assessed by trypan blue dye exclu-sion. Cells were seeded at a density of 26 106
Figure 1. The weight of mice throughout the study. The 0þ Ex, 10þ Ex, and 20 þ Ex groups exercised at approximately 75%
_
V O2maxthree times a week for 8 weeks. The weight of mice was monitored every 2 weeks. The data are expressed as mean-s+ standard deviations. 0-Ex (no CsA/no exercise), 0þ Ex (no CsAþ exercise), 10-Ex (10 mg kg71
day71CsA/no exercise), 10þ Ex (10 mg kg71
day71 CsA
þ exercise), 20-Ex (20 mg kg71
day71 CsA/no exercise), and 20
þ Ex (20 mg kg71 day71CsAþ exercise).
2 M.-H. Ko et al.
cells ml71and incubated at 378C in humidified 5% CO2/95% air. All culture materials were disposable and free of endotoxin.
Cell viability assay
Cell viability was assayed by measuring cellular 3-(4, 5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bro-mide (MTT) reduction activity. Mitochondrial dehy-drogenases in viable cells reduce soluble MTT to insoluble purple formazan crystals. Four hours before the cells were harvested, MTT (final concentration 0.1 mg ml71) was added to each well. After remov-ing the culture medium, the cells were rinsed twice with PBS. Then acid isopropanol was added to dissolve the formazan crystals. The absorbance values at 570 nm were measured by a microplate reader (model 3550; BIO RAD, Richmond, CA, USA). The number of viable cells is proportional to the optical density values and was calculated by plotting against a standard curve.
Determination of nitric oxide
Peritoneal excluded macrophages were obtained from mice as described previously (Fang et al., 2003). Briefly, cells were harvested by lavage with 10 ml of cold HBSS per mouse 3 days after intraperitoneal injection of 3 ml 3% thioglycollate solution (Difco, Detroit, MI, USA). The cells were seeded in 96-well plates at a density of 26 106 cells ml71 and incubated at 378C in humidified 5% CO2/95% air to allow macrophage adherence. Two hours later, the non-adherent cells were removed by washing with warmed PBS and the remaining cells (90% macrophages, judged by non-specific esterase stain) were incubated in Roswell Park Memorial Institute 1640 medium in the presence or absence of lipopolysaccharide (2 mg ml71) plus interferon-gamma (10 U ml71). Nitric oxide was determined by measuring the accumulation of nitrite, a stable end product, in the culture supernatant using the Griess reaction (Green et al., 1982). Equal volumes of culture supernatant were mixed with Griess reagent and left for 10 min at room temperature. The optical density was measured with a microplate reader (model 3550; BIO-RAD, Richmond CA, USA) at 540 nm and the nitrite concentration was calcu-lated using sodium nitrite as a standard.
Cytokine assay
Spleen cells, 56 106 ml71, were incubated with and without 5 mg ml71 concanavalin A in 24-well plates for 48 h. The culture supernatants were collected and stored at 7808C before analysis by
enzyme-linked immunosorbent assay (PharMingen, San Diego, CA, USA), as described previously (Fang, Hwang, Chen, & Chiang, 2000). Briefly, 96-well plates were coated with monoclonal antibody with specificity for interferon-gamma or interleukin-4 and incubated overnight at interleukin-48C, washed with 0.05% Tween 20 in PBS, and blocked by Roswell Park Memorial Institute 1640 medium supplement with 10% fetal calf serum for 1 h at room tempera-ture. Serially diluted culture supernatants and standards prepared from recombinant mouse interferon-gamma or interleukin-4 separately (Phar-Mingen, San Diego, CA, USA) were added for 2 h at room temperature. The wells of the plates were washed, and biotin-conjugated rat anti-mouse inter-feron-gamma or interleukin-4 was added for another 1 h at room temperature. After proper washing, avidin-horseradish peroxidase was added and incu-bated for 1 h at room temperature. After aspirating and washing, substrate (tetramethylbenzidine and hydrogen peroxide) was added for 30 min at room temperature in the dark. The optical density was measured with a microplate reader (model 3550; BIO-RAD, Richmond, CA, USA) at 450 nm. The detection sensitivity of interferon-gamma and interleukin-4 was 31.3 and 7.8 pg ml71, respectively.
Statistical analysis
All data are expressed as means+ standard devia-tions. Statistical analysis was performed using two-way analysis of variance followed by Dunnett’s post-hoc test. Significant differences are reported between an exercise group and a non-exercise group administered the same dose of CsA, and between no exercise (0-Ex, 10-Ex, and 20-Ex) groups.
Results
Effect of exercise and CsA on the weight of mice To investigate the possible interactions between exercise and CsA administration on immune activities in vivo, we used an optimal dose of CsA, 10 mg kg71 day71, as well as 20 mg kg71 day71, as described in our previous study (Fang, Hou, & Chao, 2005). To monitor growth of mice, we recorded their weight every 2 weeks. Mice in the three exercise groups (0þ Ex, 10þ Ex, and 20þ Ex) performed exercise at approximately 75% V O_ 2max three times a week for 8 weeks. Although the differences were not statistically significant, we consistently observed that mice in the exercise groups exhibited a lower weight gain than those in the non-exercise groups (Figure 1).
Effect of exercise and CsA administration on the function of peritoneal macrophages
Eight weeks after the initiation of the experiment, the function of peritoneal macrophages was determined by monitoring the amount of nitric oxide released from peritoneal macrophages under lipopolysacchar-ide/interferon-gamma stimulation. The results showed that nitric oxide secretion by mitogen-stimulated macrophages was higher in both CsA-administered groups with exercise (10þ Ex and 20þ Ex) than in CsA-administered groups without exercise (10-Ex and 20-Ex) (Figure 2).
Effect of exercise and CsA on the function of spleen cells Spleen is one of the major secondary immune organs and CsA is known to inhibit T cell activation. Therefore, we investigated whether the effects of CsA on the functions of spleen cells were altered by exercise. The mitogenic activity of T cells was monitored by stimulating spleen cells with 5 mg ml71 concanavalin A for 3 days and cell proliferation was measured by the 3-(4, 5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bro-mide method. As shown in Figure 3, in the three non-exercise groups (0-Ex, 10-Ex, 20-Ex), mitogen-stimulated cell proliferation was significantly sup-pressed by administration of CsA (P5 0.05; Figure 3, open bars). However, when animals were sub-jected to regular exercise (0þ Ex, 10 þ Ex, 20 þ Ex), the inhibition of mitogen-stimulated cell prolifera-tion was not observed in CsA-treated mice (P¼ 0.07;
Figure 3, hatched bars). Results of cytokine secretion by concanavalin A-stimulated spleen cells of non-exercise groups revealed that the secretion of Type I helper T cytokine (interferon-gamma; Figure 4a, open bars) but not Type II helper T cytokine (interleukin-4; Figure 4b, open bars) was decreased in a dose-dependent manner by prolonged adminis-tration of CsA. We found that regular exercise resulted in a significant increase in interferon-gamma secretion (Figure 4a, hatched bars). However, no significant difference for interleukin-4 secretion (Figure 4b, hatched bars) was detected among exercise groups.
Discussion
To our knowledge, this is the first in vivo study to investigate the effects of moderate regular exercise on CsA-induced immunosuppression. The evidence provided in this study suggests that moderate regular exercise stimulates immune function in CsA-treated mice. In addition, exercise also stimulated the secretion of a Type I helper T-type cytokine, interferon-gamma, in a CsA dose-dependent man-ner. The results of this study clearly show that moderate regular exercise can attenuate CsA-in-duced immunosuppression and other side-effects. However, whether these changes are beneficial or detrimental to CsA-treated patients needs further investigation.
Effects of regular exercise on immune functions of mice Previous reports indicated that CsA exerts its im-munosuppressive effects by reducing the concentrations of inducible nitric oxide synthase,
Figure 2. Effects of different combinations of CsA and exercise on nitric oxide production from macrophages. Murine peritoneal macrophages were added with lipopolysaccharide (2 mg ml71) plus interferon-gamma (10 U ml71) and supernatants were collected 48 h after the initiation of the cultures to determine the amount of nitric oxide by the Griess method. The data are expressed as means+ standard deviations. Significant difference between an exercise group and a non-exercise group administered the same dose of CsA: *P5 0.05; **P 5 0.01. Significant difference between 0-Ex and 10-Ex (or 20-Ex): #P5 0.05. NO¼ nitric oxide. Open bars¼ exercise groups; hatched bars¼ non-exercise groups.
Figure 3. The cell proliferation of concanavalin A-stimulated spleen cells. 56 106spleen cells were incubated with or without 5 mg ml71
concanavalin A in 24-well plates for 72 h. The culture supernatants were collected and analysed using a sandwich enzyme-linked immunosorbent assay as described in the ‘Materials and methods’. The data are expressed as means+ standard deviations. Significant difference between 0-Ex and 10-Ex (or 20-Ex):#P5 0.05. Open bars¼ exercise groups; hatched bars ¼ non-exercise groups.
4 M.-H. Ko et al.
nitric oxide, and cyclooxygenase-2 produced by lipopolysaccharide-activated RAW264.7 cells (Attur et al., 2000). In line with these findings, we also observed that when mice are not subjected to moderate regular exercise (0-Ex, 10-Ex and 20-Ex), prolonged CsA treatment inhibits immune functions. First, nitric oxide production by lipopolysaccharide/ interferon-gamma activated macrophages in mice without regular exercise (Figure 2, open bars) was inhibited by administration of 10 or 20 mg kg71 day71of CsA. Second, the mitogen-stimulated T cell proliferation by spleen cells in non-exercise groups (Figure 3; pen bars) was suppressed by administration of 10 or 20 mg kg71 day71 of CsA. Furthermore, our results revealed that nitric oxide production and mitogen-stimulated T cell proliferation were not significantly different in the two groups of mice without CsA treatment (0-Ex and 0þ Ex) (Figures 2 and 3). Previous studies revealed that nitric oxide production is affected by the intensity of exercise
(Goto et al., 2003; Green, Maiorana, O’Driscoll, & Taylor, 2004). The intensity of exercise performed in this study may be insufficient to induce significant changes in the basal level of nitric oxide production during the study period.
When mice were subjected to moderate regular exercise during the course of CsA treatment, we observed that the CsA-induced immunosuppressive effects were diminished or even reversed. As shown in Figure 2, in the three exercise groups (hatched bars), nitric oxide production was higher in CsA-treated mice (10þ Ex and 20 þ Ex) than in control mice (0þ Ex). Furthermore, results indicated that CsA had a suppressive effect on T-cell proliferation in non-exercising animals (Figure 3, open bars; P5 0.05). However, this suppressive effect was diminished when animals were subjected to regular exercise (Figure 3, hatched bars; P¼ 0.07). These results demonstrate that the CsA-induced immuno-suppressive effect in mice is partially diminished or even reversed by moderate regular exercise. Although the underlying mechanisms are still un-clear, this observation suggests that the suppressed immunity in CsA-treated patients can be modulated by moderate regular exercise. Further studies are needed to determine whether this exercise-induced immunomodulatory effect is beneficial or detrimen-tal to the health of CsA-treated patients.
Regular exercise specifically enhances the Type I helper T-dependent response
As shown in Figure 4a, in the non-exercise groups (0-Ex, 10-Ex, and 20-Ex) prolonged administration of CsA suppressed the secretion of a Type I helper T cytokine, interferon-gamma, in a CsA dose-dependent manner. The interferon-gamma secretion was not significantly different between the two groups that were not administered CsA (0-Ex and 0þ Ex). Previous studies have shown that the secretion of interferon-gamma can be modulated by strenuous exercise or long-term moderate regular exercise (Baum, Muller-Steinhardt, Liesen, & Kirchner, 1997; Castaneda et al., 2004). The results presented here indicate that exercise undertaken by the mice in this study may be insufficient to significantly affect the basal interferon-gamma secretion.
However, when mice were treated with CsA, regular exercise strongly enhanced interferon-gamma secre-tion (10þ Ex and 20 þ Ex). A similar effect was not observed when the concentrations of a Type II helper T cytokine, interleukin-4, were measured (Figure 4b). This indicates that regular exercise specifically en-hances the Type I helper T-dependent but not the Type II helper T-dependent response when given with CsA. A large body of evidence suggests that interferon-gamma acts as a key pro-inflammatory cytokine
Figure 4. The concentrations of interferon-gamma (a) and interleukin-4 (b) cytokines secreted by concanavalin A-stimulated spleen cells. 56 106spleen cells were incubated with or without 5 mg ml71
concanavalin A in 24-well plates for 48 h. The culture supernatants were collected and analysed using a sandwich enzyme-linked immunosorbent assay as described in the ‘Materials and methods’. The data are expressed as means+ standard deviations. Significant difference between an exercise group and a non-exercise group administered the same dose of CsA: **P5 0.01. Significant difference between 0-Ex and 10-Ex (or 20-Ex): #P5 0.05; ##
P5 0.01. IFN-g¼ interferon-gamma; IL-4¼ interleukin-4; Open bars¼ exercise groups; hatched bars¼ non-exercise groups.
(Billiau & Matthys, 2009). Thus, an elevated secretion of interferon-gamma may elicit detrimental effects in patients with autoimmune diseases. However, recent studies have revealed that interferon-gamma may play a paradoxical protective role in autoimmune diseases (Kelchtermans, Billiau, & Matthys, 2008; Kim, Chi, Bouziane, Gaur, & Moudgil, 2008). Therefore, the exact clinical and pathological features of the exercise-enhanced Type I helper T-dependent response still need to be clarified.
Whether moderate regular exercise indirectly decreases CsA absorption or increases CsA clearance due to the induction of metabolizing enzymes, increased P-glycoprotein activity, changed protein binding, or induced extra-hepatic metabolism which results in a reduction in CsA bioavailability requires further study. Based on the results of this study, the interactive effects of CsA administration and regular exercise on immune function in a mouse model with Type II helper T- dominant autoimmune disease is currently being investigated.
Conclusion
In conclusion, moderate regular exercise in mice significantly decreased CsA-induced immunosup-pression and resulted in higher macrophage and Type I helper T-type activities than the administra-tion of CsA alone. The results of this study provide new insights into the in vivo interactions between moderate regular exercise and CsA administration on immune activities in a mouse model. However, direct transfer of the findings from a mouse model to human patients is not recommended at the present stage. Further clinical studies are required to determine whether moderate regular exercise exerts beneficial effects in CsA-treated patients.
Acknowledgements
This study was supported by NSC 95-2320-B-028-001-MY2 granted by National Science Council, R.O.C. and 97DG0008 granted by National Taiwan Sport University. The authors thank Pei-Yu Shih for her expert technical assistance.
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