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Effect of the Nonsteroidal Anti-inflammatory Drug Indomethacin on Th1 and Th2 Immune Responses in Mice
Effect of the Nonsteroidal Anti-inflammatory Drug Indomethacin on Th1 and Th2 Immune Responses in Mice KOUYA YAMAKI,1 HIROYUKI UCHIDA,1 YOSHIKI HARADA,1 RIE YANAGISAWA,2 HIROHISA TAKANO,2 HIDEYUKI HAYASHI,3 YOKI MORI,4 SHIN YOSHINO1 1
Department of Pharmacology, Kobe Pharmaceutical University, 4-19-1 Motoyamakita-machi, Higashinada-ku, Kobe 658-8558, Japan 2
Pathophysiology Research Team, National Institute for Environmental Studies, Tsukuba, Ibaraki 305-8506, Japan
3
Department of Clinical Toxicology and Metabolism, Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Tobetsu, Hokkaido 061-02, Japan
4
Department of Immunology and Microbiology, Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Tobetsu, Hokkaido 061-02, Japan
Received 23 August 2002; revised 26 December 2002; accepted 29 December 2002
ABSTRACT: The present study was undertaken to study the effect of the nonsteroidal anti-inflammatory drug indomethacin on Th1 and Th2 immune responses. For this study, mice were immunized by s.c. injection of ovalbumin (OVA) emulsified with complete Freund’s adjuvant into the base of the tail (day 0). Varying doses of indomethacin were orally administrated daily from days 0 to 20. On day 21, anti-OVA IgG2a and interferon-g as an indicator of Th1 responses and anti-OVA IgG1 and interleukin-10 as that of Th2 responses were measured. The results showed that treatment with indomethacin was followed by decreases in OVA-specific IgG and proliferation of spleen cells to the antigen. Indomethacin inhibited both Th1 and Th2 responses, although the nonsteroidal antiinflammatory drug suppressed the former more effectively than the latter. Administration of indomethacin resulted in suppression of antigen (OVA)-induced arthritis that was associated with inhibition of anti-OVA IgG2a but not IgG1 production. These results suggest that nonsteroidal anti-inflammatory drugs may downregulate Th1 and, to a lesser extent, Th2 immune responses. ß 2003 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 92:1723–1729, 2003
Keywords: Th1/Th2 immune response; nonsteroidal anti-inflammatory drug; indomethacin; ovalbumin; antibody; arthritis; cytokine
INTRODUCTION Helper T cells are mainly divided to two subsets, types I (Th1) and II (Th2).1,2 Th1 cells produce interleukin-2 (IL-2) and interferon-g (IFN-g) involved in cellular immunity, whereas Th2 cells secrete ILs-4, -5, and -6 implicated in humoral Correspondence to: Shin Yoshino (Telephone/Fax: þ81-78441-7572; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 92, 1723–1729 (2003) ß 2003 Wiley-Liss, Inc. and the American Pharmacists Association
immune responses. These Th1 and Th2 cytokines have also been shown to play an important role in a number of diseases in humans. For example, IFN-g is produced in joints of patients with rheumatoid arthritis3 and neutralization of IFN-g attenuates the severity of the disease.4 On the other hand, an increased expression of IL-4 mRNA is observed in sputum of patients with asthma5 and neutralization of IL-4 blocks the development of acute airway hypersensitivity in a murine asthma model.6 Because IFN-g inhibits Th2 immune responses7 and ILs-48 and -10
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downregulate Th1 responses,9 it is anticipated that modulation of either Th1 or Th2 responses may influence such diseases. Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used to treat patients with rheumatoid arthritis and other inflammatory diseases. Treatment with NSAIDs results in marked inhibition of cyclooxygenase associated with suppression of prostanoid production.10 NSAIDs also modulate immune responses including proliferation11,12 and differentiation13 of lymphoid cells as well as production of antigen-specific antibodies14,15 in vivo. However, the effect of NSAIDs on Th1 and Th2 immune responses was not previously investigated in detail. In the present study, we show that treatment with the NSAID indomethacin inhibited both Th1 and, to a lesser extent, Th2 responses.
MATERIALS AND METHODS Animals Female DBA/1J mice, 7 to 9 weeks of age, were used in all experiments. The mice were maintained in a temperature-controlled environment with free access to standard rodent chow and water.
Table 1. Weights
Effect of Indomethacin on Gaining Body
Body Wt. (g) Dose (mg/kg) 0 0.1 0.5 2.5
Day 0
Day 21
22.0 0.563 22.9 0.161 23.3 0.609 22.0 0.787
23.5 0.699 24.9 0.440 24.7 0.541 22.8 0.939
Mice were orally given indomethacin dissolved in 0.5% carboxymethylcellulose at the time of immunization with OVA (day 0) and then daily up to day 20. Values are shown as mean SEM of five to six mice.
25 mL of PBS containing 50 mg of OVA into left ankle joints on day 21. The right ankle joints were injected with 25 mL of PBS alone as a control. To evaluate the severity of arthritis, the thickness of the both ankles was measured using a dial gauge caliper calibrated with 0.01-mm graduations. The net increase in joint thickness attributable to the antigenic challenge was calculated by subtracting the increase in thickness of the right ankle from that of the left ankle. There was no net joint swelling after injection of OVA in nonimmunized mice. Measurement of Antibodies to OVA
Immunization with Ovalbumin (OVA) One hundred micrograms of OVA (Sigma-Aldrich Fine Chemicals, St. Louis, MO) was dissolved in 50 mL of phosphate-buffered saline (PBS) and emulsified with an equal volume of complete Freund’s adjuvant (CFA; Difco Laboratories, Detroit, MI.). One hundred microliters of the emulsion was injected s.c. into the base of the tail (day 0). Administration of Indomethacin Varying doses including 0.1, 0.5, and 2.5 mg/kg of indomethacin were dissolved in 500 mL of 0.5% carboxymethylcellulose and orally administrated daily from days 0 to 20. Five hundred microliters of 0.5% carboxymethylcellulose alone was given as a control. No effect of indomethacin on gaining body weights was observed (Table 1). Induction of Antigen-Induced Athritis (AIA) Mice immunized with OVA as described above were challenged by intraarticular injection of JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 8, AUGUST 2003
Sera were obtained on day 21 and heat inactivated at 568C for 30 min. Anti-OVA IgG, IgG1, and IgG2a were measured using an ELISA. In brief, 96-well flat-bottom microtiter plates were incubated with 100 mL/well of OVA (100 mg/mL) at 378C for 1 h and washed three times with PBS containing 0.05% Tween-20. The wells were then blocked by incubation with 100 mL of PBS containing 1% casein (Sigma) at 378C for 30 min. After washing, the plates were incubated with 100 mL of a 1:5000 (for IgG and IgG1 measurement) or 1:500 (for IgG2a measurement) dilution of each serum sample at 378C for 30 min. The plates were washed, and 100 m:/well of a 1:2000 dilution of alkaline phosphatase-labeled rat antimouse IgG (Sigma) or 1:1000 dilution of alkaline phosphatase-labeled antimouse IgG1 and IgG2a (PharMingen, San Diego, CA) was added and incubated at 378C for 1 h. After washing, 100 m:L of 3 mM p-nitrophenylphosphate (Bio-Rad, Richmond, CA) was added per well and the plates were incubated in the dark at room temperature for 15 min. The absorbance was then measured at 405 nm by IMMUNO-MINI NJ-2300 (Nalge
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Nunc International K.K., Tokyo, Japan). The results were expressed as absorbance units at OD405 SEM.
Table 2. Effects of Indomethacin on Production of Anti-OVA IgG and Proliferative Responses of Spleen Cells to the Antigen
Measurement of Cytokines
Dose (mg/kg)
Spleens were removed on day 21 and cell suspensions prepared. Erythrocytes in the cells were lysed with Tris-NH4Cl. A total 5 106 cells in 1 mL of RPMI1640 (Sigma) containing 1 mM L-glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, 5 105 M 2-mercaptoethanol, and 2% heat inactivated autologous mouse serum were cultured in 24-well tissue culture plates either in medium alone or with 50 mg/mL OVA. Forty-eight hours later supernatants were harvested and stored at 208C until assayed. Cytokine production was quantified by commercially available ELISA kit (Endogen, Inc., Woburn, MA) for IL-10 and IFN-g.
0 (control) 0.1 0.5 2.5
Proliferation Assay 5
A total 5 10 of spleen cells in 0.1 mL of RPMI1640 (Sigma) containing 1 mM L-glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, 5 105 M 2-mercaptoethanol, and 2% heat inactivated autologous mouse serum were cultured with 50 mg/mL of OVA in each well of 96-well tissue culture plates. Seventy-two hours later, each well was pulsed with 0.5 mCi of tritiated thymidine, and the cells were cultured for another 17 h. The cultures were harvested onto fiberglass filters using multiharvester and counted using standard liquid scintillation techniques. Results, expressed in cpm, are the average of triplicate cultures of cells pooled from five to six mice. Statistics To analyze data statistically, the Mann-Whitney U-test was used as one of nonparametric statistical methods because sample sizes in our experiments were small; therefore, the normality obtained was poor.
RESULTS Effect of Indomethacin on OVA-Specific Immune Responses Treatment with indomethacin was followed by a decrease in anti-OVA IgG antibodies in sera in a dose-related fashion (Table 2). To examine
Anti-OVA IgG (A405)
Proliferation (cpm)
0.7616 0.09067a 0.6272 0.02679 0.5577 0.05222 0.4590 0.04475c
55556 3228.8b 10231 1285.9 25467 1564.6 13341 440.19
Mice were orally given indomethacin dissolved in 0.5% carboxymethylcellulose at the time of immunization with OVA (day 0) and then daily up to day 20. On day 21, anti-OVA IgG in sera and proliferative response of spleen cells to the antigen were measured as described in Materials and Methods. The background counts of spleen cells cultured without OVA were between 3000 to 5000 cpm, and there was no effect of indomethacin on the spontaneous proliferation of the lymphoid cells. a Mean SEM of five to six mice. b Mean SEM of triplicated cultures of cells pooled from five to six mice. Data are representative of three experiments. c p < 0.05 versus control.
whether proliferative responses of lymphoid cells to OVA are modulated by indomethacin, spleen cells from mice treated with the NSAID were cultured with OVA in vitro. As shown in Table 2, the splenic cell proliferation against OVA was markedly suppressed in mice given all doses of the drug used. Approximately 75% inhibition (versus control) was observed in mice treated with 2.5 mg/ kg of indomethacin. Effect of Indomethacin on Th1 and Th2 Immune Responses Administration of indomethacin resulted in dosedependent decreases in both OVA-specific IgG2a and IgG1, although the decrease in IgG2a appeared to be greater than that in IgG1 (Figure 1). The highest dose (2.5 mg/kg) of indomethacin showed 73% inhibition of IgG2a production, whereas 33% inhibition of IgG1 production was seen in mice given the same dose of the NSAID. The Th1 cytokine IFN-g and the Th2 cytokine IL10 secreted by spleen cells were also measured to examine the effect of indomethacin on Th1 and Th2 immune responses, respectively. As shown in Figure 2, indomethacin markedly suppressed IFN-g production (up to 76% inhibition), whereas this NSAID failed to modulate IL-10 secretion. Effect of Indomethacin on AIA To examine whether indomethacin can modulate AIA induced by immunization of OVA (day 0) and JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 8, AUGUST 2003
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Figure 1. Effect of indomethacin on anti-OVA IgG1 and IgG2a antibody production. Mice were immunized with OVA on day 0. Five hundred microliters of 0.5% carboxymethylcellulose containing the indicated doses of indomethacin were orally administered daily from days 0 to 20. Five hundred microliters of 0.5% carboxymethylcellulose alone was given as a control. On day 21, the animals were sacrificed and anti-OVA IgG1 and IgG2a antibodies were measured as described in Materials and Methods. Bars show the mean SEM of five to six mice. Data are representative of three experiments. *p < 0.05, **p < 0.01 versus control, MannWhitney analysis.
Figure 3. Effect of indomethacin on AIA. Mice were immunized with OVA on day 0. Five hundred microliters of 0.5% carboxymethylcellulose containing indicated doses of indomethacin were orally administered daily from days 0 to 20. Five hundred microliters of 0.5% carboxymethylcellulose alone was given as a control. On day 21, AIA was induced by injection of OVA into ankle joints. The severity of arthritis was determined by measuring joint swelling. Closed square, 0.5% carboxymethylcellulose alone (control); open square, 0.5% carboxymethylcellulose containing indomethacin (2.5 mg/kg). Vertical bars show SEM of five to six mice. Data are representative of three experiments. *p < 0.05, **p < 0.01 versus control, Mann-Whitney analysis.
then challenged intraarticularly with the same antigen on day 21, mice were orally given indomethacin over a period of 21 days commencing on day 0. As shown in Figure 3, treatment with indomethacin was followed by significant suppression of OVA-induced joint swelling when it was measured at 3, 5, and 24 h after challenge injection. The suppressive effect of indomethacin on the joint inflammation was associated with significant decreases in proliferative responses to OVA and production of anti-OVA IgG2a but not IgG and IgG1 antibodies (Table 3).
DISCUSSION
Figure 2. Effect of indomethacin on IFN-g and IL-10 production by spleen cells. Mice were immunized with OVA on day 0. Five hundred microliters of 0.5% carboxymethylcellulose containing the indicated doses of indomethacin were orally administered daily from days 0 to 20. Five hundred microliters of 0.5% carboxymethylcellulose alone was given as a control. On day 21, the animals were sacrificed and spleen cells were incubated with or without OVA for 48 h. IFN-g and IL-10 were measured as described in Materials and Methods. Bars show the mean SEM of five to six mice. Data are representative of three experiments. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 8, AUGUST 2003
The present study suggests that NSAIDs may have inhibitory effects on Th1 and, to a lesser extent, Th2 immune responses because administration of indomethacin to mice was followed by a marked decrease in Th1 responses including anti-OVA IgG2a and IFN-g production and by a moderate decrease in Th2 responses such as antiOVA IgG1 and IL-10 secretion. Previous in vivo studies showed that indomethacin suppressed splenic T cell proliferation11 and antigen-specific IgG production.14 In contrast, this NSAID was also previously shown to have the opposite effects in vitro. For instance, in vitro
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Table 3. Effects of Indomethacin on Th1 and Th2 Responses in Mice with AIA Anti-OVA (A405) Dose (mg/mL) 0 (control) 2.5
Anti-OVA IgG (A405) 1.10 0.049 0.92 0.073
a
Proliferation (cpm) b
68203 3832 35826 882.3
IgG2a
IgG1 a
0.93 0.063 0.50 0.070c
0.98 0.031a 0.95 0.035
Mice were orally given indomethacin dissolved in 0.5% carboxymethylcellulose at the time of immunization with OVA (day 0) and then daily up to day 20. On day 21, OVA was injected into ankle joint. On day 22, anti-OVA IgG, proliferative responses, and anti-OVA IgG2a and IgG1 were measured as described in MaterialS and Methods. The background counts of spleen cells cultured without OVA were between 3000 to 5000 cpm, and there was no effect of indomethacin on the spontaneous proliferation of the lymphoid cells. a Mean SEM of five to six mice. b Mean SEM of triplicated cultures of cells pooled from five to six mice. Data are representative of three experiments. c p < 0.01 versus control.
treatment with indomethacin reverses the suppression of spontaneous IgG and IgM production and PHA-induced T cell proliferation seen in patients with rheumatoid arthritis16 and psoriasis,17 respectively. Our present studies demonstrated that administration of indomethacin was followed by inhibition of both anti-OVA IgG production and the antigen-specific T cell proliferation, supporting the in vivo results reported previously. The discrepancy of the effects of indomethacin between in vivo and in vitro immune responses may be in part explained by the difference in the timing of treatment with the NSAID. In most of previous in vivo experiments, indomethacin was administrated at the time of immunization with antigen so that the NSAID excerted its effect on the development of immune reactions. On the other hand, in in vitro studies indomethacin was tested for its effect on T cells isolated after immune responses had been established. Thus, indomethacin may act differently on mature and immature T cells. There are only a few studies demonstrating the effect of indomethacin on Th1 and Th2 responses. For instance, treatment with indomethacin of BALB/c mice infected with Leishmania major resulted in augmentation of Th1 responses.18 In vitro experiments showed that prostaglandin E2 (PGE2), which production was downregulated by indomethacin inhibited IL-2 and IFN-g secretion but failed to affect ILs-4 and -5 secretion.19–21 In addition, IFN-g production by Staphylococcus aureus Cowan I-sensitized spleen cells was increased following indomethacin pretreatment, whereas addition of PGE2 to the lymphoid cells suppressed IFN-g production.22 These results indicate that indomethacin may promote Th1 responses and that the promoting effect of the
NSAID on the immune responses may be explained by its inhibitory effect on PGE2 production. In contrast, administration of indomethacin suppresses the severity of myelin basic proteininduced experimental allergic encephalomyelitis in rats in which Th1 responses are critically involved,23 suggesting that indomethacin inhibits Th1 cell-mediated immune responses. Moreover, indomethacin enhances antidinitrophenyl IgE production in mice24 that is strictly depending on the Th2 cytokine IL-4. Our present study showed that indomethacin inhibited both Th1 and Th2 responses to OVA, although the inhibitory effect of the drug on Th1 seemed to be greater than that on Th2. Taken together, indomethacin appears to either inhibit or enhance Th1 responses, depending on the experimental systems used. Indomethacin might enhance Th1 responses to bacteria such as Leishmania major and Staphylococcus aureus, while the cyclooxygenase inhibitor might augment the immune responses to soluble proteins including myelin basic protein as well as OVA. Indomethacin also seems to either downregulate or upregulate Th2 responses. Th2 responses to haptens including dinitrophenyl appear to be upregulated by indomethacin while the native soluble antigen OVA-specific Th2 responses might be downregulated by this NSAID, although the suppression of Th2 responses by indomethacin appeared to be relatively resistant compared to that of Th1 reactions by the drug. Further studies are required before concluding the effect of indomethacin on Th1 and Th2 responses. Treatment with indomethacin was followed by suppression of AIA known as one of models of rheumatoid arthritis because there are histological similarities between AIA and the human joint inflammatory disease.25 In addition, the suppresJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 8, AUGUST 2003
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sion of AIA by indomethacin was associated with the downregulation of anti-OVA IgG2a but not IgG1antibody production, suggesting that AIA induction may be Th1- but not Th2-cell dependent. To our knowledge, this is the first demonstration that Th1 responses played a pivotal role in AIA. A role for Th1 responses including specific IgG2a production in joint inflammation was also shown in collagen-induced arthritis in mice,26 which was another experimental model of rheumatoid arthritis. In addition, the ratio of IFN-g to IL-4 produced by T cells is significantly higher in patients with rheumatoid arthritis than in healthy subjects. These results indicate that Th1 immune responses may be critically involved in rheumatoid arthritis, and that the downregulation of Th1 responses by drugs such as indomethacin may lead to the improvement of the autoimmune disease. In summary, indomethacin inhibited Th1 and, to a lesser extent Th2 immune responses. Treatment with this drug was followed by downregulation of AIA which was associated with suppression of Th1 responses including anti-OVA IgG2a antibody production. The anti-inflammatory effect of NSAIDs on rheumatoid arthritis may be at least in part due to the suppression of Th1 immune responses involved in the disease.
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eosinophils, is required in a murine model of acute airway hypersensitivity. J Exp Med 183:109–117. Dickensheets HL, Venkataraman C, Schindler U, Donnelly RP. 1999. Interferons inhibit activation of STAT6 by interleukin 4 in human monocytes by inducing SOCS-1 gene expression. Proc Natl Acad Sci USA 96:10800–10805. van Roon JA, van Roy JL, Duits A, Lafeber FP, Bijilsma JW. 1995. Proinflammatory cytokine production and cartilage damage due to rheumatoid synovial T helper-1 activation is inhibited by interleukin-4. Ann Rheum Dis 54:836–840. Yin Z, Braun J, Neure L, Wu P, Liu L, Eggens U, Sieper J. 1997. Crucial role of interleukin-10/ interleukin-12 balance in the regulation of the type 2 T helper cytokine response in reactive arthritis. Arthritis Rheum 40:1788–1797. Turini ME, DuBois RN. 2002. Cyclooxygenase-2: A therapeutic target. Annu Rev Med 53:35–57. Seng GF, Benensohn J, Bayer BM. 1990. Changes in T and B lymphocyte proliferative responses in adjuvant-arthritic rats: antagonism by indomethacin. Eur J Pharmacol 178:267–273. Barasoain I, Rojo JM, Portoles A. 1980. ‘‘In vivo’’ effects of acetylsalicylic acid and two other derived compounds on primary immune response and lymphoblastic transformation. Immunopharmacology 2:293–300. Shibuya T, Izuchi K, Koga Y, Nomoto K, Shirakawa K. 1986. Prostaglandin modulation of the postnatal development of T and B lymphocytes in the spleens of newborn mice. Dev Comp Immunol 10:419–428. Jaramillo A, Bhattacherjee P, Sonnenfeld G, Paterson CA. 1992. Modulation of immune responses by cyclo-oxygenase inhibitors during intraocular inflammation. Curr Eye Res 11:571–579. Franch A, Castellote C, Castell M. 1994. Effect of acetylsalicylic acid and dexamethasone on antibody production in adjuvant arthritis. Rheumatol Int 14: 27–31. Panush RS, Katz P, Longley S. 1983. In vitro immunoglobulin production by mononuclear cells in rheumatoid arthritis. Clin Immunol Immunopathol 28:252–264. Zarrabeitia MT, Farinas MC, Rodriguez-Valverde V, Riancho JA, Llaca HF. 1989. T and B cell function in psoriasis and psoriatic arthropathy. Allergol Immunopathol 17:155–159. De Freitas LA, Mbow LM, Estay M, Bleyenberg JA, Titus RG. 1999. Indomethacin treatment slows disease progression and enhanced a Th1 response in susceptible BALB/c mice infected with Leishmania major. Parasite Immunol 21:273–277. Betz M, Fox BS. 1991. Prostaglandin E2 inhibits production of Th1 lymphocytes but Th2 lymphocytes. J Immunol 146:108–113. Snijdewint FG, Kalinski P, Wierenga EA, Bos JD, Kapsenberg ML. 1993. Prostaglandin E2 differ-
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entially modulates cytokine secretion profiles of human T helper lymphocyte. J Immunol 150: 5321–5329. 21. Gold KN, Weyand CM, Goronzy JJ. 1994. Modulation of helper T cell function by prostaglandins. Arthritis Rheum 37:925–933. 22. Kuroda E, Sugiura T, Zeki K, Yoshida Y, Yamashita U. 2000. Sensitivity difference to the suppressive effect of prostaglandin E2 among mouse strains: A possible mechanism to polarize Th2 type response in BALB/c mice. J Immunol 164:2386– 2395. 23. Reder AT, Thapar M, Sapugay AM, Jensen MA. 1995. Eicosanoids modify experimental allergic encephalomyelitis. Am J Ther 2:711–720.
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24. Oh-hashi K, Watanabe S, Kobayashi T, Okuyama H. 1997. Reevaluation of the effect of high alphalinolenate and high linoleate diet on antigeninduced antibody and anaphylactic responses in mice. Biol Pharm Bull 20:217–223. 25. Cooke TD, Jasin HE. 1972. The pathogenesis of chronic inflammation in experimental antigeninduced arthritis. I. The role of antigen on the local immune response. Arthritis Rheum 15:327–337. 26. McIntyre KW, Shuster DJ, Gillooly KM, Warrier RR, Connaughton SE, Hall LB, Arp LH, Gately MK, Magram J. 1996. Reduced incidence and severity of collagen-induced arthritis in interleukin-12-deficient mice. Eur J Immunol 26:2933– 2938.
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Department of Pharmacology, Kobe Pharmaceutical University, 4-19-1 Motoyamakita-machi, Higashinada-ku, Kobe 658-8558, Japan 2
Pathophysiology Research Team, National Institute for Environmental Studies, Tsukuba, Ibaraki 305-8506, Japan
3
Department of Clinical Toxicology and Metabolism, Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Tobetsu, Hokkaido 061-02, Japan
4
Department of Immunology and Microbiology, Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Tobetsu, Hokkaido 061-02, Japan
Received 23 August 2002; revised 26 December 2002; accepted 29 December 2002
ABSTRACT: The present study was undertaken to study the effect of the nonsteroidal anti-inflammatory drug indomethacin on Th1 and Th2 immune responses. For this study, mice were immunized by s.c. injection of ovalbumin (OVA) emulsified with complete Freund’s adjuvant into the base of the tail (day 0). Varying doses of indomethacin were orally administrated daily from days 0 to 20. On day 21, anti-OVA IgG2a and interferon-g as an indicator of Th1 responses and anti-OVA IgG1 and interleukin-10 as that of Th2 responses were measured. The results showed that treatment with indomethacin was followed by decreases in OVA-specific IgG and proliferation of spleen cells to the antigen. Indomethacin inhibited both Th1 and Th2 responses, although the nonsteroidal antiinflammatory drug suppressed the former more effectively than the latter. Administration of indomethacin resulted in suppression of antigen (OVA)-induced arthritis that was associated with inhibition of anti-OVA IgG2a but not IgG1 production. These results suggest that nonsteroidal anti-inflammatory drugs may downregulate Th1 and, to a lesser extent, Th2 immune responses. ß 2003 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 92:1723–1729, 2003
Keywords: Th1/Th2 immune response; nonsteroidal anti-inflammatory drug; indomethacin; ovalbumin; antibody; arthritis; cytokine
INTRODUCTION Helper T cells are mainly divided to two subsets, types I (Th1) and II (Th2).1,2 Th1 cells produce interleukin-2 (IL-2) and interferon-g (IFN-g) involved in cellular immunity, whereas Th2 cells secrete ILs-4, -5, and -6 implicated in humoral Correspondence to: Shin Yoshino (Telephone/Fax: þ81-78441-7572; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 92, 1723–1729 (2003) ß 2003 Wiley-Liss, Inc. and the American Pharmacists Association
immune responses. These Th1 and Th2 cytokines have also been shown to play an important role in a number of diseases in humans. For example, IFN-g is produced in joints of patients with rheumatoid arthritis3 and neutralization of IFN-g attenuates the severity of the disease.4 On the other hand, an increased expression of IL-4 mRNA is observed in sputum of patients with asthma5 and neutralization of IL-4 blocks the development of acute airway hypersensitivity in a murine asthma model.6 Because IFN-g inhibits Th2 immune responses7 and ILs-48 and -10
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downregulate Th1 responses,9 it is anticipated that modulation of either Th1 or Th2 responses may influence such diseases. Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used to treat patients with rheumatoid arthritis and other inflammatory diseases. Treatment with NSAIDs results in marked inhibition of cyclooxygenase associated with suppression of prostanoid production.10 NSAIDs also modulate immune responses including proliferation11,12 and differentiation13 of lymphoid cells as well as production of antigen-specific antibodies14,15 in vivo. However, the effect of NSAIDs on Th1 and Th2 immune responses was not previously investigated in detail. In the present study, we show that treatment with the NSAID indomethacin inhibited both Th1 and, to a lesser extent, Th2 responses.
MATERIALS AND METHODS Animals Female DBA/1J mice, 7 to 9 weeks of age, were used in all experiments. The mice were maintained in a temperature-controlled environment with free access to standard rodent chow and water.
Table 1. Weights
Effect of Indomethacin on Gaining Body
Body Wt. (g) Dose (mg/kg) 0 0.1 0.5 2.5
Day 0
Day 21
22.0 0.563 22.9 0.161 23.3 0.609 22.0 0.787
23.5 0.699 24.9 0.440 24.7 0.541 22.8 0.939
Mice were orally given indomethacin dissolved in 0.5% carboxymethylcellulose at the time of immunization with OVA (day 0) and then daily up to day 20. Values are shown as mean SEM of five to six mice.
25 mL of PBS containing 50 mg of OVA into left ankle joints on day 21. The right ankle joints were injected with 25 mL of PBS alone as a control. To evaluate the severity of arthritis, the thickness of the both ankles was measured using a dial gauge caliper calibrated with 0.01-mm graduations. The net increase in joint thickness attributable to the antigenic challenge was calculated by subtracting the increase in thickness of the right ankle from that of the left ankle. There was no net joint swelling after injection of OVA in nonimmunized mice. Measurement of Antibodies to OVA
Immunization with Ovalbumin (OVA) One hundred micrograms of OVA (Sigma-Aldrich Fine Chemicals, St. Louis, MO) was dissolved in 50 mL of phosphate-buffered saline (PBS) and emulsified with an equal volume of complete Freund’s adjuvant (CFA; Difco Laboratories, Detroit, MI.). One hundred microliters of the emulsion was injected s.c. into the base of the tail (day 0). Administration of Indomethacin Varying doses including 0.1, 0.5, and 2.5 mg/kg of indomethacin were dissolved in 500 mL of 0.5% carboxymethylcellulose and orally administrated daily from days 0 to 20. Five hundred microliters of 0.5% carboxymethylcellulose alone was given as a control. No effect of indomethacin on gaining body weights was observed (Table 1). Induction of Antigen-Induced Athritis (AIA) Mice immunized with OVA as described above were challenged by intraarticular injection of JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 8, AUGUST 2003
Sera were obtained on day 21 and heat inactivated at 568C for 30 min. Anti-OVA IgG, IgG1, and IgG2a were measured using an ELISA. In brief, 96-well flat-bottom microtiter plates were incubated with 100 mL/well of OVA (100 mg/mL) at 378C for 1 h and washed three times with PBS containing 0.05% Tween-20. The wells were then blocked by incubation with 100 mL of PBS containing 1% casein (Sigma) at 378C for 30 min. After washing, the plates were incubated with 100 mL of a 1:5000 (for IgG and IgG1 measurement) or 1:500 (for IgG2a measurement) dilution of each serum sample at 378C for 30 min. The plates were washed, and 100 m:/well of a 1:2000 dilution of alkaline phosphatase-labeled rat antimouse IgG (Sigma) or 1:1000 dilution of alkaline phosphatase-labeled antimouse IgG1 and IgG2a (PharMingen, San Diego, CA) was added and incubated at 378C for 1 h. After washing, 100 m:L of 3 mM p-nitrophenylphosphate (Bio-Rad, Richmond, CA) was added per well and the plates were incubated in the dark at room temperature for 15 min. The absorbance was then measured at 405 nm by IMMUNO-MINI NJ-2300 (Nalge
INDOMETHACIN EFFECT ON Th1 and Th2 IMMUNE REPSONSES IN MICE
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Nunc International K.K., Tokyo, Japan). The results were expressed as absorbance units at OD405 SEM.
Table 2. Effects of Indomethacin on Production of Anti-OVA IgG and Proliferative Responses of Spleen Cells to the Antigen
Measurement of Cytokines
Dose (mg/kg)
Spleens were removed on day 21 and cell suspensions prepared. Erythrocytes in the cells were lysed with Tris-NH4Cl. A total 5 106 cells in 1 mL of RPMI1640 (Sigma) containing 1 mM L-glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, 5 105 M 2-mercaptoethanol, and 2% heat inactivated autologous mouse serum were cultured in 24-well tissue culture plates either in medium alone or with 50 mg/mL OVA. Forty-eight hours later supernatants were harvested and stored at 208C until assayed. Cytokine production was quantified by commercially available ELISA kit (Endogen, Inc., Woburn, MA) for IL-10 and IFN-g.
0 (control) 0.1 0.5 2.5
Proliferation Assay 5
A total 5 10 of spleen cells in 0.1 mL of RPMI1640 (Sigma) containing 1 mM L-glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, 5 105 M 2-mercaptoethanol, and 2% heat inactivated autologous mouse serum were cultured with 50 mg/mL of OVA in each well of 96-well tissue culture plates. Seventy-two hours later, each well was pulsed with 0.5 mCi of tritiated thymidine, and the cells were cultured for another 17 h. The cultures were harvested onto fiberglass filters using multiharvester and counted using standard liquid scintillation techniques. Results, expressed in cpm, are the average of triplicate cultures of cells pooled from five to six mice. Statistics To analyze data statistically, the Mann-Whitney U-test was used as one of nonparametric statistical methods because sample sizes in our experiments were small; therefore, the normality obtained was poor.
RESULTS Effect of Indomethacin on OVA-Specific Immune Responses Treatment with indomethacin was followed by a decrease in anti-OVA IgG antibodies in sera in a dose-related fashion (Table 2). To examine
Anti-OVA IgG (A405)
Proliferation (cpm)
0.7616 0.09067a 0.6272 0.02679 0.5577 0.05222 0.4590 0.04475c
55556 3228.8b 10231 1285.9 25467 1564.6 13341 440.19
Mice were orally given indomethacin dissolved in 0.5% carboxymethylcellulose at the time of immunization with OVA (day 0) and then daily up to day 20. On day 21, anti-OVA IgG in sera and proliferative response of spleen cells to the antigen were measured as described in Materials and Methods. The background counts of spleen cells cultured without OVA were between 3000 to 5000 cpm, and there was no effect of indomethacin on the spontaneous proliferation of the lymphoid cells. a Mean SEM of five to six mice. b Mean SEM of triplicated cultures of cells pooled from five to six mice. Data are representative of three experiments. c p < 0.05 versus control.
whether proliferative responses of lymphoid cells to OVA are modulated by indomethacin, spleen cells from mice treated with the NSAID were cultured with OVA in vitro. As shown in Table 2, the splenic cell proliferation against OVA was markedly suppressed in mice given all doses of the drug used. Approximately 75% inhibition (versus control) was observed in mice treated with 2.5 mg/ kg of indomethacin. Effect of Indomethacin on Th1 and Th2 Immune Responses Administration of indomethacin resulted in dosedependent decreases in both OVA-specific IgG2a and IgG1, although the decrease in IgG2a appeared to be greater than that in IgG1 (Figure 1). The highest dose (2.5 mg/kg) of indomethacin showed 73% inhibition of IgG2a production, whereas 33% inhibition of IgG1 production was seen in mice given the same dose of the NSAID. The Th1 cytokine IFN-g and the Th2 cytokine IL10 secreted by spleen cells were also measured to examine the effect of indomethacin on Th1 and Th2 immune responses, respectively. As shown in Figure 2, indomethacin markedly suppressed IFN-g production (up to 76% inhibition), whereas this NSAID failed to modulate IL-10 secretion. Effect of Indomethacin on AIA To examine whether indomethacin can modulate AIA induced by immunization of OVA (day 0) and JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 8, AUGUST 2003
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Figure 1. Effect of indomethacin on anti-OVA IgG1 and IgG2a antibody production. Mice were immunized with OVA on day 0. Five hundred microliters of 0.5% carboxymethylcellulose containing the indicated doses of indomethacin were orally administered daily from days 0 to 20. Five hundred microliters of 0.5% carboxymethylcellulose alone was given as a control. On day 21, the animals were sacrificed and anti-OVA IgG1 and IgG2a antibodies were measured as described in Materials and Methods. Bars show the mean SEM of five to six mice. Data are representative of three experiments. *p < 0.05, **p < 0.01 versus control, MannWhitney analysis.
Figure 3. Effect of indomethacin on AIA. Mice were immunized with OVA on day 0. Five hundred microliters of 0.5% carboxymethylcellulose containing indicated doses of indomethacin were orally administered daily from days 0 to 20. Five hundred microliters of 0.5% carboxymethylcellulose alone was given as a control. On day 21, AIA was induced by injection of OVA into ankle joints. The severity of arthritis was determined by measuring joint swelling. Closed square, 0.5% carboxymethylcellulose alone (control); open square, 0.5% carboxymethylcellulose containing indomethacin (2.5 mg/kg). Vertical bars show SEM of five to six mice. Data are representative of three experiments. *p < 0.05, **p < 0.01 versus control, Mann-Whitney analysis.
then challenged intraarticularly with the same antigen on day 21, mice were orally given indomethacin over a period of 21 days commencing on day 0. As shown in Figure 3, treatment with indomethacin was followed by significant suppression of OVA-induced joint swelling when it was measured at 3, 5, and 24 h after challenge injection. The suppressive effect of indomethacin on the joint inflammation was associated with significant decreases in proliferative responses to OVA and production of anti-OVA IgG2a but not IgG and IgG1 antibodies (Table 3).
DISCUSSION
Figure 2. Effect of indomethacin on IFN-g and IL-10 production by spleen cells. Mice were immunized with OVA on day 0. Five hundred microliters of 0.5% carboxymethylcellulose containing the indicated doses of indomethacin were orally administered daily from days 0 to 20. Five hundred microliters of 0.5% carboxymethylcellulose alone was given as a control. On day 21, the animals were sacrificed and spleen cells were incubated with or without OVA for 48 h. IFN-g and IL-10 were measured as described in Materials and Methods. Bars show the mean SEM of five to six mice. Data are representative of three experiments. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 8, AUGUST 2003
The present study suggests that NSAIDs may have inhibitory effects on Th1 and, to a lesser extent, Th2 immune responses because administration of indomethacin to mice was followed by a marked decrease in Th1 responses including anti-OVA IgG2a and IFN-g production and by a moderate decrease in Th2 responses such as antiOVA IgG1 and IL-10 secretion. Previous in vivo studies showed that indomethacin suppressed splenic T cell proliferation11 and antigen-specific IgG production.14 In contrast, this NSAID was also previously shown to have the opposite effects in vitro. For instance, in vitro
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Table 3. Effects of Indomethacin on Th1 and Th2 Responses in Mice with AIA Anti-OVA (A405) Dose (mg/mL) 0 (control) 2.5
Anti-OVA IgG (A405) 1.10 0.049 0.92 0.073
a
Proliferation (cpm) b
68203 3832 35826 882.3
IgG2a
IgG1 a
0.93 0.063 0.50 0.070c
0.98 0.031a 0.95 0.035
Mice were orally given indomethacin dissolved in 0.5% carboxymethylcellulose at the time of immunization with OVA (day 0) and then daily up to day 20. On day 21, OVA was injected into ankle joint. On day 22, anti-OVA IgG, proliferative responses, and anti-OVA IgG2a and IgG1 were measured as described in MaterialS and Methods. The background counts of spleen cells cultured without OVA were between 3000 to 5000 cpm, and there was no effect of indomethacin on the spontaneous proliferation of the lymphoid cells. a Mean SEM of five to six mice. b Mean SEM of triplicated cultures of cells pooled from five to six mice. Data are representative of three experiments. c p < 0.01 versus control.
treatment with indomethacin reverses the suppression of spontaneous IgG and IgM production and PHA-induced T cell proliferation seen in patients with rheumatoid arthritis16 and psoriasis,17 respectively. Our present studies demonstrated that administration of indomethacin was followed by inhibition of both anti-OVA IgG production and the antigen-specific T cell proliferation, supporting the in vivo results reported previously. The discrepancy of the effects of indomethacin between in vivo and in vitro immune responses may be in part explained by the difference in the timing of treatment with the NSAID. In most of previous in vivo experiments, indomethacin was administrated at the time of immunization with antigen so that the NSAID excerted its effect on the development of immune reactions. On the other hand, in in vitro studies indomethacin was tested for its effect on T cells isolated after immune responses had been established. Thus, indomethacin may act differently on mature and immature T cells. There are only a few studies demonstrating the effect of indomethacin on Th1 and Th2 responses. For instance, treatment with indomethacin of BALB/c mice infected with Leishmania major resulted in augmentation of Th1 responses.18 In vitro experiments showed that prostaglandin E2 (PGE2), which production was downregulated by indomethacin inhibited IL-2 and IFN-g secretion but failed to affect ILs-4 and -5 secretion.19–21 In addition, IFN-g production by Staphylococcus aureus Cowan I-sensitized spleen cells was increased following indomethacin pretreatment, whereas addition of PGE2 to the lymphoid cells suppressed IFN-g production.22 These results indicate that indomethacin may promote Th1 responses and that the promoting effect of the
NSAID on the immune responses may be explained by its inhibitory effect on PGE2 production. In contrast, administration of indomethacin suppresses the severity of myelin basic proteininduced experimental allergic encephalomyelitis in rats in which Th1 responses are critically involved,23 suggesting that indomethacin inhibits Th1 cell-mediated immune responses. Moreover, indomethacin enhances antidinitrophenyl IgE production in mice24 that is strictly depending on the Th2 cytokine IL-4. Our present study showed that indomethacin inhibited both Th1 and Th2 responses to OVA, although the inhibitory effect of the drug on Th1 seemed to be greater than that on Th2. Taken together, indomethacin appears to either inhibit or enhance Th1 responses, depending on the experimental systems used. Indomethacin might enhance Th1 responses to bacteria such as Leishmania major and Staphylococcus aureus, while the cyclooxygenase inhibitor might augment the immune responses to soluble proteins including myelin basic protein as well as OVA. Indomethacin also seems to either downregulate or upregulate Th2 responses. Th2 responses to haptens including dinitrophenyl appear to be upregulated by indomethacin while the native soluble antigen OVA-specific Th2 responses might be downregulated by this NSAID, although the suppression of Th2 responses by indomethacin appeared to be relatively resistant compared to that of Th1 reactions by the drug. Further studies are required before concluding the effect of indomethacin on Th1 and Th2 responses. Treatment with indomethacin was followed by suppression of AIA known as one of models of rheumatoid arthritis because there are histological similarities between AIA and the human joint inflammatory disease.25 In addition, the suppresJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 8, AUGUST 2003
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sion of AIA by indomethacin was associated with the downregulation of anti-OVA IgG2a but not IgG1antibody production, suggesting that AIA induction may be Th1- but not Th2-cell dependent. To our knowledge, this is the first demonstration that Th1 responses played a pivotal role in AIA. A role for Th1 responses including specific IgG2a production in joint inflammation was also shown in collagen-induced arthritis in mice,26 which was another experimental model of rheumatoid arthritis. In addition, the ratio of IFN-g to IL-4 produced by T cells is significantly higher in patients with rheumatoid arthritis than in healthy subjects. These results indicate that Th1 immune responses may be critically involved in rheumatoid arthritis, and that the downregulation of Th1 responses by drugs such as indomethacin may lead to the improvement of the autoimmune disease. In summary, indomethacin inhibited Th1 and, to a lesser extent Th2 immune responses. Treatment with this drug was followed by downregulation of AIA which was associated with suppression of Th1 responses including anti-OVA IgG2a antibody production. The anti-inflammatory effect of NSAIDs on rheumatoid arthritis may be at least in part due to the suppression of Th1 immune responses involved in the disease.
7.
8.
9.
10. 11.
12.
13.
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