International Immunopharmacology 1 2001. 12191226 www.elsevier.

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Tacrolimus suppressed the production of cytokines involved in atopic dermatitis by direct stimulation of human PBMC system. Comparison with steroids/
Shozo Sakuma a, ) , Yasuyuki Higashi a , Natuki Sato b, Tatsuya Sasakawa a , Takanori Sengoku a , Yoshitaka Ohkubo a , Tadahiro Amaya b, Toshio Goto a
a

Medicinal Biology Research Laboratories, Fujisawa Pharmaceutical Co., Ltd. 1-6, Kashima 2-chome, Yodogawa, Osaka 532-8514 Japan b Deelopment Diision, Fujisawa Pharmaceutical Co., Ltd. 1-6, Kashima 2-chome, Yodogawa, Osaka 532-8514 Japan Received 13 November 2000; received in revised form 5 February 2001; accepted 9 February 2001

Abstract Tacrolimus FK506. ointment showed remarkable efficacy against atopic dermatitis in animal models and clinical trials. The suppressive effect of tacrolimus on the production of the cytokines involved in atopic dermatitis IL-2, IL-3, IL-4, IL-5, IFN-g and GM-CSF. from human peripheral blood mononuclear cells PBMC. was investigated. We constructed a new cytokine production system in which T cells are activated by direct stimulation in vitro with anti-CD3rCD2 or anti-CD3rCD28 antibody combination. Tacrolimus inhibited the production of these cytokines by both stimulations. In a comparative study with steroids alclometasone dipropionate and betamethason valerate. in anti-CD3rCD2 system, tacrolimus and both steroids inhibited Th1 cytokines IL-2, IFN-g .,Th2 cytokines IL-4, IL-5. and IL-3,GM-CSF produced by both Th1 and Th2.. The suppressive effect of tacrolimus on cytokine production was stronger than that of alclometasone dipropionate and equal to or stronger than that of betamethason valerate. The effective dose of tacrolimus IC 50 , 0.020.11 ngrml. is almost the same as for Th1 and Th2 cytokines, and 1 ngrml of tacrolimus suppressed all cytokines completely. These results suggest that tacrolimus suppresses the allergic cytokines from T cells, and that tacrolimus ointment is effective against atopic dermatitis through the inhibition of cytokine production. q 2001 Elsevier Science B.V. All rights reserved.
Keywords: Tacrolimus; Atopic dermatitis; Th1 cytokine; Th2 cytokine; CD3rCD2; CD3rCD28

1. Introduction Atopic dermatitis is a common skin disease, but the pathophysiological mechanism has not been well understood. There are many reports stating that
Corresponding author. Tel.: q 81-6-6390-1145; fax: q 81-66304-5367. E-mail address: shozo sakuma@po.fujisawa.co.jp S. Sakuma..
)

cytokine-mediated response in the immune system to allergens is thought to be an important pathogenetic factor w14x. Th2 cytokines are mainly related to the immediate allergic reaction. For instance, IL-4 enhances IgE production from B cells w5,6x, and IgE on mast cell releases the mediators, such as histamine, by antigen re-stimulation. IL-5 activates eosinophils, which is one of the major effector cells in the late-phase allergic reactions w7,8x. On the other hand, Th1 cytokines are related to the delayed-type hyper-

1567-5769r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 1 5 6 7 - 5 7 6 9 0 1 . 0 0 0 5 9 - 5

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sensitivity DTH. reactions. Recently, some reports showed that DTH reaction is one of the causes of chronic atopic dermatitis w2,9x. IL-2 activates DTH T cells and IFN-g activates macrophages. IL-3 and GM-CSF are produced from both Th1 and Th2 cells, which proliferate and activate mast cells, basophils and eosinophils w10,11x. Tacrolimus is a potent immunosuppressant for preventing rejection in organ transplantation. The immunosuppressive effect is thought to depend on the suppression of cytokine production, mainly IL-2 w12x. Tacrolimus ointment is effective against atopic dermatitis in animal models w1315x, and clinical trials w16,17x. Many reports showed that tacrolimus inhibits cytokines from T cells in vitro w1820x. Now, we focus on cytokines involved in atopic dermatitis from human T cells. It is well known that T cell receptor signal and co-stimulatory signal are necessary for the activation of T cells. We selected anti-CD3 antibody for T cell receptor stimulation and anti-CD2 or anti-CD28 antibody for co-stimulating stimulation w2124x. To stimulate T cells directly without the effects of antigen-presenting cells, stimulating antibodies were fixed to microplate through anti-Fc antibody. Tacrolimus inhibited the production of many cytokines IL-2, IL-3, IL-4, IL-5, IFN-g and GM-CSF. from both anti-CD3rCD2 and anti-CD3rCD28 stimulations. We also tested the effects of tacrolimus compared with well-known steroids such as alclometasone dipropionate and betamethason valerate in antiCD3rCD2 system. The IC 50 values of tacrolimus for each cytokine inhibition are almost the same 0.020.11 ngrml. and the order in potency was tacrolimus, betamethason valerate and alclometasone dipropionate. According to these results, tacrolimus and steroids suppressed cytokine production in atopic dermatitis, and reflected the efficacy in clinical use.

valerate and alclometasone dipropionate were purchased from Sigma St. Louis, MO. and U.S.P.C., respectively. These compounds were dissolved in ethanol at 1 or 0.5 mgrml alclometasone dipropionate. and further diluted in complete culture medium. Anti-mouse IgG Fc antibody AQ-127. was obtained from Chemicon. Stimulating mouse mAb to human CD3 OKT3. was obtained from Kyowa Hakko Kogyo, CD2 Leu-5b. and CD28 Leu-28. were obtained from Japan Becton Dickinson. The ELISA kits for quantitative analysis of human cytokines were obtained from Cayman IL-2., R & D IL-1 b , IL-3, IL-5, IL-6, IL-8, TNF-a , IFN-g and GM-CSF. and BioSource IL-4., respectively. 2.2. Cell isolation Human peripheral blood mononuclear cells PBMC. were isolated from venous whole blood of normal volunteers by FicollrHypaque centrifugation. Adherent cell-depleted T cells were isolated through a nylon wool column Waco 142-05791.. PBMC and purified T cells were washed and re-suspended in a culture medium made of RPMI 1640 medium supplement with 2.5% heat-inactivated human AB-type serum, 2 = 10y5 M 2ME, 50 IUrml penicillin and 50 m grml streptomycin. 2.3. Cell culture Cell cultures for cytokine production were set up in a flat-bottomed 24-well plate. Anti-mouse IgG Fc antibody was diluted at 25 m grml in D-PBS and added to each well at 0.4 mlrwell. The plates were incubated for 1 h at 378C, after that the non-reactive antibody was removed and washed in the plate with D-PBS. Anti-CD3 antibody and co-stimulate antibody anti-CD2 or anti-CD28. 300 ngrml in 0.5% BSAPBS. were added in equal volume in mixture 0.2 mlrwell for each antibody., incubated for 1 h at 378C and washed with D-PBS. Immediately, a culture medium or drug twofold concentration of final dose. was added at 1 mlrwell and cell suspension 3 = 10 6 cellsrml. was added at 1 mlrwell. After a 24-h culture at 378C in 5% CO 2 incubator, the culture supernatant was collected and separate small quantities were kept at y208C until the measure-

2. Materials and methods 2.1. Reagents Tacrolimus hydrate was prepared by Fujisawa Pharmaceutical Osaka, Japan.. Betamethasone

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ment of cytokine. In this experiment, only the antimouse IgG Fc antibody treatment was a negative control and anti-CD3rco-stimulate antibody treatment without drug was a positive control.

2.5. Calculation of the %inhibition and IC50 alue The %inhibition is calculated by the next formula %Inhibition s The amount of positive control

2.4. Measurement of cytokine by ELISA The measurement of cytokine was performed according to the procedure of the accompanying document of each ELISA kit. The amount of cytokine is determined from a standard curve and multiplied by the dilution folds.

yThe amount with drug . r The amount of positive control yThe amount of negative control. = 100 The amount of positive control is the amount without drug stimulation only.. The amount of nega-

Fig. 1. Comparison of PBMC and purified T cell in cytokine production by anti-CD3rCD2 stimulation. PBMC or purified T cell is cultured with anti-CD3rCD2 antibody q. or without stimulation y.. The amount of each cytokine after a 24-h culture was measured by ELISA.

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S. Sakuma et al. r International Immunopharmacology 1 (2001) 12191226 Table 2 IC 50 values of tacrolimus in cytokine production from human PBMC IC 50 values are shown in nanogram per milliliter. Anti-CD3rCD2 stimulation IL-2 IL-3 IL-4 IL-5 IFN-g GM-CSF 0.02 0.02 0.02 0.07 0.11 0.07 Anti-CD3rCD28 stimulation 0.05 0.05 0.15 0.38 0.12 0.97

tive control is the amount without stimulation. The IC 50 value is calculated by Probit analysis. 2.6. Statistical analysis The statistical analyses were calculated by Dunnetts multiple comparison test against the positive control a. or the same concentration of tacrolimus ) .. P values less than 0.05 were considered statistically significant.

3. Result 3.1. Construction of cytokine production system We constructed a new cytokine production system in which T cells are stimulated directly with antiCD3rCD2 antibodies. In this system, anti-CD3rCD2 antibodies were fixed to a 24-well flat-bottomed plate through the anti-mouse IgG Fc antibody. We can detect not only IL-2, IL-3, IL-4, IL-5, IFN-g but also IL-1 b , IL-6, IL-8, TNF-a from PBMC. We cannot detect IL-1 b , IL-6, IL-8 from purified T cells through a nylon wool column and TNF-a was partially suppressed from purified T cells Fig. 1.. This means that T cells were stimulated directly in this system and produced cytokines such as Th1 IL-2, IFN-g . Th2 IL-4, IL-5. and IL-3. The other cytokines, such as IL-1 b , IL-6, IL-8, were produced from non-T cells. TNF-a was produced from both T cells and non-T cells Fig. 1.. Thus, we can estimate the effect of the immunosuppressant on cytokine production from the T cell in this PBMC system. 3.2. Comparison with anti-CD3 r CD2 and anti-CD3 r CD28 stimulation CD2 and CD28 are well known co-stimulatory molecules to T cell activation. We compared the cytokine production profiles with anti-CD3rCD2 and anti-CD3rCD28. Table 1 showed the amount of each cytokine produced by both stimulations. Only IL-2 production by anti-CD3rCD28 stimulation is much greater about 200 times. than that by antiCD3rCD2 stimulation. The amounts of IFN-g produced are almost the same between both stimulations but those by anti-CD3rCD28 stimulation are slightly higher in IL-3, IL-4, IL-5 and GM-CSF. 3.3. Tacrolimus inhibits the production of both Th1 and Th2 cytokines by anti-CD3 r CD2 or anti-CD2 r CD28 stimulation Tacrolimus inhibited these six cytokine IL-2, IL-3, IL-4, IL-5, IFN-g , and GM-CSF. productions that are thought to be closely related to atopic dermatitis by both stimulations. The IC 50 values are shown in Table 2. Although the IC 50 values of tacrolimus for each cytokine in both stimulations are slightly different from those of the cytokines, each production was suppressed by tacrolimus under the 1 ngrml concentrations. Even in the IL-2 production by anti-CD3rCD28, which is a very high amount Table 1., tacrolimus also inhibited as low as the 0.05 ngrml concentration of the IC 50 value.

Table 1 Amount of cytokines produced by anti-CD3rCD2 or antiCD3rCD28 stimulation Amount of cytokines are shown in picogram per milliliter. Anti-CD3rCD2 stimulation IL-2 IL-3 IL-4 IL-5 IFN-g GM-CSF 3786 6. 635 6. 822 6. 819 3. 11403730 6. 161325 6. Anti-CD3rCD28 stimulation 1062415201 3. 28533 3. 28119 3. 1384 3. 10175494 3. 3041378 3.

. shows the number of examples.

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3.4. Tacrolimus suppressed cytokine production more effectiely than steroids In the next experiment, we compared the efficacy of tacrolimus on cytokine production with steroids such as betamethasone valerate and alclometasone dipropionate that are used in the clinical treatment by topical use. For this experiment, we used the antiCD3rCD2 stimulation system. Tacrolimus and steroids inhibited every cytokine examined in a dose-dependent manner Fig. 2.. One nanogram per milliliter of tacrolimus inhibited all cytokines completely. The IC 50 values of each drug are shown in Table 3. Th1 cytokine IL-2, IFN-g ., Th2 cytokine IL-4, IL-5. and IL-3 and GM-CSF produced by both Th1 and Th2. production were inhibited by

Table 3 Comparison of the IC 50 values of tacrolimus and steroids in cytokine production from human PBMC, stimulated by antiCD3rCD2 IC 50 values are shown in nanogram per milliliter. IC 50 values were calculated from Fig. 2 by Probit analysis. Tacrolimus IL-2 IL-3 IL-4 IL-5 IFN-g GM-CSF 0.02 0.02 0.02 0.07 0.11 0.07 Betamethasone valerate 0.27 0.17 0.16 0.08 0.72 0.10 Alclometasone dipropionate 5.54 1.76 2.09 0.89 3.26 0.76

tacrolimus and steroids. Tacrolimus is the most potent of the three drugs investigated.

Fig. 2. Comparison of tacrolimus and steroids in cytokine production from human PBMC stimulated with anti-CD3rCD2. PBMC are stimulated by anti-CD3rCD2 with different concentrations of drugs. The amount of each cytokine after a 24-h culture was measured by ELISA. The %inhibition was calculated by the formula described in Section 2. The mean SE. was calculated from six volunteers in the case of IL-5: n s 3.. a, aa: Significant differences from the positive control group at P - 0.05 and P - 0.01 Dunnetts multiple comparison test., respectively. ) , ) ) : Significant differences from the same concentration of the tacrolimus-treated group at P - 0.05 and P - 0.01 Dunnetts multiple comparison test., respectively.

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4. Discussion The pathogenesis of atopic dermatitis is not well known, but the T cell-mediated immune response is thought to have a key role. Especially, the cytokines produced from Th1 and Th2 are very important w14x. Th2 cytokines IL-4, IL-5. are necessary for the immediate allergy reaction through IgE production and eosinophils activation w58x. Th1 cytokines IL-2, IFN-g . are necessary for the late chronic inflammation in atopic dermatitis through macrophages and T cells activation w2,9x. IL-3 and GM-CSF are important for the maturation and activation of mast cells and eosinophils w10,11x. So, it is important to study the effect of tacrolimus on the production of cytokines relevant to pathogenesis in atopic dermatitis. It is well known that T cell receptor signal and co-stimulatory signals, such as CD2 and CD28, are necessary for the activation of T cells. In order to examine the suppressive effect of tacrolimus on the production of cytokines from human T cells, we selected an anti-CD3rCD28 or anti-CD3rCD2 costimulation system. Both anti-CD3rCD28 and antiCD3rCD2 stimulations produced Th1 and Th2 cytokines and tacrolimus suppressed Th1 and Th2 cytokines produced by both stimulations. In comparison with anti-CD3rCD28 stimulation and antiCD3rCD2 stimulation, the former produced more IL-2 than the latter, but the other cytokines IL-3, IL-4, IL-5, IFN-g and GM-CSF. were produced in almost the same amount by both stimulations. We thought the anti-CD3rCD28 stimulation converts the immune response to Th1 type, and anti-CD3rCD2 stimulation keeps the balance of Th1 and Th2-type immune responses. In the case of atopic dermatitis, Th2 response is thought to be more important in the early phase of pathogenesis, and Th1 response is more important in the chronic phase w2,4x. Thus, we compared the suppressive effect of tacrolimus and two steroids in anti-CD3rCD2 cytokine production system. Tacrolimus and steroids suppressed all cytokine productions, and the efficacy of tacrolimus was equal to or slightly stronger than that of betamethason valerate and much stronger than that of alclometasone dipropionate. The effective concentration IC 50 value. of tacrolimus is between 0.02 and 0.11 ngrml, and 1 ngrml of tacrolimus suppressed

all relevant cytokine production completely. The concentration is significantly lower than the toxic levels identified in a previous report that the IC 50 value of tacrolimus on the growth and maturation of myeloid progenitor cells in vitro, depending upon CSF, is 1400 nM w12x. Many authors have reported the suppressive mechanism of cytokine production by tacrolimus w2527x. Tacrolimus binds to the FKBP-12 which is tacrolimus-binding protein in the cytoplasm, thereafter, the complex inhibits the activity of calcineurin that possesses the Ca2q and calmodulin-dependent dephosphatase activity. Inhibition of the action of calcineurin results in a complete block of the translocation of the cytosolic component of the nuclear factor of activated T cells NF-AT. into nuclear. Blocking of the signal of NF-AT results in a failure to activate the genes that are responsible for the production of Th1 and Th2 cytokines like IL-2, IL-3, IL-4, IL-5, IFN-g , GM-CSF. These cytokines are necessary for the activation of T cells and inflammatory cells, as well as B cells. On the other hand, steroid ointments are used for the treatment of atopic dermatitis w28,29x. Steroid binds to the cytoplasmic glucocorticoid receptor, and these complexes bind to the glucocorticoid responsive element GRE. in the nuclear, which modulates the production of proteins such as lipocortin and some enzymes w30,31x. Moreover, steroids suppressed the activation of nuclear factors, AP-1 and NF-k B, which modulate a variety of inflammatory and immune responses w3235x. Although the immunosuppressive mechanism is different between steroids and tacrolimus, the latter showed greater effect in inhibiting cytokine production. In the clinical trial for atopic dermatitis, tacrolimus ointment is effective w16,17x as well as steroid ointments. These results suggest that Th1 and Th2 cytokines are major players in the pathogenic process of atopic dermatitis, and the inhibition of the production of these cytokines leads to excellent clinical benefit in the treatment of this recalcitrant skin disease. We focused on atopic dermatitis in this report, but we can expect that tacrolimus is also effective on other T cell mediated autoimmunerallergic inflammatory diseases such as rheumatoid arthritis, psoriasis and asthma through the similar suppressive effect of cytokine production. Since steroids have many actions besides anti-inflamma-

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tion and immune-suppression, they have many side effects in the whole body together with hormone-like actions, and on local skin skin atrophy and telangiectasia. w29,3638x. In contrast, as tacrolimus works selectively on T cells, it would be associated with less side effects, especially in topical use. These data suggest that topical use of non-steroidal tacrolimus exerts the innovative effect on atopic dermatitis without the side effects of steroids w39x.

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