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Inhibitory influences of tranilast on multinucleated giant cell formation from monocytes by supernatant of concanavalin A-stimulated mononuclear cells
Journal of Dermatological Science 24 (2000) 166 – 170 www.elsevier.com/locate/jdermsci
Inhibitory influences of tranilast on multinucleated giant cell formation from monocytes by supernatant of concanavalin A-stimulated mononuclear cells Kana Mizuno, Hiroyuki Okamoto *, Takeshi Horio Department of Dermatology, Kansai Medical Uni6ersity, 10 -15 Fumizono, Moriguchi, Osaka 570 -8507, Japan Received 13 January 2000; received in revised form 7 February 2000; accepted 7 February 2000
Abstract Tranilast is an anti-allergic drug that inhibits the release of chemical mediators from mast cells. There have been cases-reports showing that tranilast is effective for the treatment of granulomatous diseases such as granuloma annulare and cutaneous sarcoidosis. Here we examined the in vitro effects of tranilast on the formation of multinucleated giant cells (MGCs) from human peripheral monocytes. Supernatant of concanavalin A (Con A)-stimulated mononuclear cells induced Langhans-type and foreign body-type MGCs and the addition of 10 or 100 mg/ml tranilast inhibited the formation of total MGCs and foreign body-type MGCs. Tranilast decreased the number of MGCs with 16 Bnuclei and increased that of MGCs with three to five nuclei. Fluorescence-activated cell sorting analysis showed that tranilast-treated monocytes had lower expressions of intercellular adhesion molecule-1 (ICAM1). These findings suggest that tranilast is effective for cutaneous lesions in some cases of granulomatous disorders partly through a direct effect on monocyte/macrophage-lineage cells. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Tranilast; Granulomas; Multinucleated giant cells; Monocytes; ICAM-1
1. Introduction Tranilast has been used for the treatment of atopic dermatitis because of its anti-allergic action [1]. It affects the membrane of mast cells, resulting in an inhibition of the liberation of chemical mediators such as histamine and prostaglandins * Corresponding author. Tel.: + 81-6-69921001, ext: 3301; fax: + 81-6-69963299. E-mail address: [email protected] (H. Okamoto).
[2]. Mast cells are key cells not only in allergic disorders but also fibrotic conditions. Therefore, tranilast has also been used for the treatment of patients with keloid and hypertrophic scars [3,4]. Recently there have been some reports showing the effectiveness of tranilast in treating cutaneous lesions in some cases of granulomatous disorders such as cheilitis granulomatosis, granuloma annulare, and sarcoidosis [5–7]. Although the decreased release of chemical mediators by tranilast may be associated with its efficacy even in such
0923-1811/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 9 2 3 - 1 8 1 1 ( 0 0 ) 0 0 0 9 4 - 3
K. Mizuno et al. / Journal of Dermatological Science 24 (2000) 166–170
diseases, the precise action mechanism is unknown. Histologically, multinucleated giant cell (MGC) is one of the characteristic features of granulomatous disorders. The cells are also produced in vitro, utilizing peripheral blood mononuclear cells and bone marrow cells by various stimuli, including cytokines and growth factors [8 – 11]. Supernatants from concanavalin A (Con A)-stimulated lymphocytes also have the capacity to produce MGCs [11]. Here we examined the effects of tranilast on the formation of MGCs produced from human monocytes by Con A-stimulated lymphocyte supernatants.
2. Materials and methods
2.1. Reagent Tranilast (N-(3,4-dimethoxycinnamoyl) anthranilic acid) was obtained from Kissei Pharmaceutical (Matsumoto, Japan). For in vitro examinations, tranilast was dissolved in 1% NaHCO3 solution, and all wells examined included the same dose of NaHCO3. Because the plasma concentration of tranilast at clinical dose is 22.2 mg/ml, 1, 10 or 100 mg/ml tranilast was used in our studies.
2.2. Isolation and culture of monocytes Peripheral blood mononuclear cells (PBMC) were obtained from six healthy adult volunteers after obtaining informed consent. Mononuclear cells were isolated from heparinized blood by density gradient centrifugation with Lymphoprep™ (Nycomed, Oslo, Norway), and washed twice in phosphate-buffered saline (PBS). The cells were resuspended in RPMI 1640 medium (Nikken Bio Med, Kyoto, Japan) supplemented with 2 mmol/l glutamine (Gibco BRL, Rockville, MD) and 10% fetal calf serum (FCS; Gibco BRL), and then plated onto 48-well culture plates (Becton Dickinson Labware, Lincoln Park, NJ) at a concentration of 2×106 cells per well. After incubation for 1.5 h at 37°C, the nonadherent cells were removed by repeated vigorous wash-
167
ings. The resulting cells consisted of at least 90% monocytes, judged by nonspecific esterase staining and phenotype analysis. The cells were more than 95% viable, as determined by trypan blue (Wako Pure Chemical Industries Ltd., Osaka, Japan) dye exclusion.
2.3. Generation of MGCs The formation of MGCs was induced by supernatants of Con A-stimulated mononuclear cells (conditioned medium). PBMC were cultured for 72 h in RPMI 1640 supplemented with 10% FCS, 2 mmol/l glutamine and 16 mg/ml Con A (Sigma, St. Louis, MO) at a density of 2×106 cells/ml. The cell-free supernatant was stored at −20°C until added to monocyte cultures. Generation of MGCs was evaluated by staining with Giemsa (Merck, Darmstadt, Germany). MGCs were defined as cells with more than three nuclei per cell. Langhans-type MGC show a circular peripheral arrangement of nuclei and foreign body-type MGC have the nuclei scattered in an irregular fashion throughout the cell. The fusion rate of monocytes was expressed as fusion index. Fusion index (%) is determined as (the number of nuclei within MGC)/(the total number of nuclei counted)× 100. All nuclei were counted through the plates. Data are expressed as mean9 standard deviation (S.D.) from triplicate assays and Student’s t-tests were used for statistical analysis.
2.4. Flow cytometry The expression of adhesion molecules was assessed using the following monoclonal antibodies: mouse anti-CD11a (LFA-1) and anti-CD54 (ICAM-1) antibodies (Serotec Ltd., Oxford, UK). FITC-conjugated rabbit anti-mouse IgG antibody (Caltag, Burlingame, CA) was used as a secondstep antibody for indirect staining. Monocytes were placed in microcentrifuge tubes and fixed with 1% paraformaldehyde (Sigma). Cells were stained with 1:50 dilution of the monoclonal antibodies described above by incubation on ice for 60 min. The cells were washed and then incubated with a 1:50 dilution of FITC-conjugated rabbit anti-mouse IgG antibody. They were then washed
168
K. Mizuno et al. / Journal of Dermatological Science 24 (2000) 166–170
and analyzed by cytofluorography on a FACScan (Becton Dickinson Immunocytometry System).
3. Results
3.1. Effects of tranilast on generation of MGCs
Fig. 1. Fusion index of peripheral blood monocytes cultured in conditioned medium with or without 1–100 mg/ml tranilast. LGC, Langhans-type multinucleated giant cells; FGC, foreign body-type multinucleated giant cells.
MGC formation was maximal when 50% of the conditioned medium was used. Based on this result, all of the following experiments were conducted with that dose of conditioned medium. The fusion index of total, foreign body-type and Langhans-type MGCs by conditioned medium was 90.09 0.9, 65.694.8 and 24.59 5.1%, respectively (Fig. 1). One mg/ml tranilast did not influence the formation of MGCs. Ten and 100 mg/ml tranilast significantly attenuated the formation of total MGCs and foreign-body type MGCs; 85.99 2.8 and 55.59 3.6% in 10 mg/ml and 65.19 6.3 and 28.79 8.0% in 100 mg/ml, respectively. The fusion index of Langhans-type MGCs was slightly but not increased significantly by tranilast treatment in a dose-dependent manner; 27.196.7 in 1 mg/ml, 30.4 95.8 in 10 mg/ml and 36.5 9 6.3% in 100 mg/ml. The cellular viability was not influenced by the stimulation of 1–100 mg/ml tranilast.
3.2. Effects of tranilast on the number of the nuclei in MGCs
Fig. 2. Comparison of the number of nuclei in MGCs generated from peripheral blood monocytes cultured in conditioned medium with or without 1–100 mg/ml tranilast. LGC, Langhans-type multinucleated giant cells; FGC, foreign body-type multinucleated giant cells.
Tranilast influenced the number of nuclei in MGCs (Fig. 2). The ratios of MGCs with six to 15 nuclei among total-MGC were not influenced significantly by 1–100 mg/ml tranilast. However, the ratio of MGCs with five or less nuclei was increased by 10 or 100 mg/ml of tranilast; 32.2 9 9.4 in 10 mg/ml, 44.3918.8% in 100 mg/ml. Such doses of tranilast decreased the rates of MGCs with 16B nuclei; 8.59 9.1 in 10 mg/ml and 4.29 7.2% in 100 mg/ml. While 1 mg/ml tranilast increased the rate of Langhans-type with 16B nuclei of Langhans-type MGCs, 100 mg/ml tranilast prevented the formation of Langhans-type MGCs with 16 B nuclei. The rates of foreign body-type MGCs with six to 15 nuclei for all foreign body-type MGCs were not influenced significantly by tranilast. The rates of foreign bodytype MGCs with five or less nuclei were increased greatly by 10 or 100 mg/ml of tranilast; 16.39 10.2 in 10 mg/ml, 35.2919.4% in 100 mg/ml. However, the rates of foreign body-type MGC with 16B
K. Mizuno et al. / Journal of Dermatological Science 24 (2000) 166–170
Fig. 3. A representative FACS analysis of monocytes cultured in conditioned medium with or without 100 mg/ml tranilast for 1 day. This is a representative experiment out of the three independent assays. (A) CD11a (LFA-1), (B) CD54 (ICAM-1).
nuclei were decreased by such doses of tranilast; 13.29 12.8 at 10 mg/ml and 9.5 916.5% at 100 mg/ml.
3.3. Expression of ICAM-1 and LFA-1 on monocytes To examine the effect of tranilast on adhesion molecules of monocytes, cells were cultured in conditioned medium with or without 100 mg/ml tranilast for 1 day. FACS analysis showed that a very low level of ICAM-1 and LFA-1 expression was detected in the untreated monocytes (Fig. 3). Monocytes cultured with conditioned medium showed high expression of ICAM-1 and LFA-1. The expression of ICAM-1 decreased on monocytes treated with tranilast, while LFA-1 expression was not changed.
4. Discussion Tranilast is a widely used anti-allergy drug in Japan and has been adapted for the treatment of atopic dermatitis patients [1]. This drug is also
169
used to treat keloid and hypertrophic scars [3,4], because it has been shown to inhibit collagen synthesis of fibroblasts from the lesions [12]. Recently, there have been case reports showing the efficacy of tranilast in granulomatous disorders. Chiba et al. [5] reported a case of cheilitis granulomatosis successfully treated with tranilast. They showed that mast cells, particularly those with ongoing degranulation, increased in number in the lesions and suggested that tranilast was effective for granulomatous inflammation partly through inhibition of mediators released from mast cells. Yamada et al. [6,7] reported that tranilast resolved cutaneous lesions in three cases of granuloma annulare and two cases of sarcoidosis. Because mast cells are not critical in such disorders, the precise action mechanisms of tranilast in these granulomas remain unknown. The key cells in granulomatous lesions are monocyte macrophage-lineage cells such as activated macrophages, epithelioid cells, and MGCs. There have been reports showing that MGCs are produced in vitro from monocytes stimulated by cytokines such as interleukin(IL)-4 [8], IL-13 plus macrophage colony-stimulating factor (M-CSF) [9] and interferon(IFN)-g [12]. Supernatant of Con A stimulated-mononuclear cells also induced MGCs from monocytes [11] but which cytokine in the supernatant controls MGC formation has not been defined. There are several reasons we utilized the supernatant to produce MGCs in this study. First, it can induce a high rate of MGCs. Another reason is that the supernatant could induce two types of MGCs, Langhans-type and foreign bodytype cells. These characteristics can facilitate quantitative and qualitative analysis of the effect of tranilast on MGC formation. This study showed that tranilast inhibited the formation of total MGCs and foreign body-type MGCs. The mechanism by which tranilast inhibited the formation of MGCs is unknown. Not only the above cytokines but also adhesion molecules are considered to play a crucial role in the process of MGC formation. For example, MGC formation was blocked by anti-ICAM-1 and anti-LFA-1 monoclonal antibodies which reduced the aggregation of monocytes [13]. Intestinal macrophages from uninflamed surgical specimens, with poor
170
K. Mizuno et al. / Journal of Dermatological Science 24 (2000) 166–170
expression of adhesion molecules such as ICAM-1 and LFA-1, had less capacity to form MGCs, while the cells from Crohn’s disease highly expressed both surface markers and formed MGCs spontaneously [14]. Also, the change in ICAM-1 expression and cellular distribution has been reported to be involved in the mechanisms of IFNg-induced MGC formation [12]. Thus, ICAM-1 is considered to be one of the surface molecules that play a role in MGC generation. The current studies showed that tranilast decreased ICAM-1 expression on monocytes. The effects of tranilast on cytoplasmic membranes have been reported in mast cells and are considered the most important event in the action mechanisms of the chemical in allergic disorders [2]. Tranilast was reported to inhibit the release of transforming growth factor (TGF)-b, IL-1b and prostaglandin E2 from human monocytes–macrophages [15]. Expression of HLA-DR and -DQ antigens on macrophages was significantly suppressed by treatment with tranilast [16,17]. Taken together with these findings, the present studies suggested that tranilast directly affects the monocyte macrophage-lineage cells, resulting in resolution of the granulomatous lesions.
References
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[1] Gondo A, Saeki N, Yamamoto E, Hirabayashi T, Tokuda Y. Efficacy of tranilast administration and specific hyposensitization therapy of atopic dermatitis. Arerugi 1985;34:310 – 9 (in Japanese). [2] Komatsu H, Kojima M, Tsutsumi N, Hamano S, Kusama H, Ujiie A, Ikeda S, Nakazawa M. Study of the mechanism of inhibitory action of tranilast on chemical mediator release. Jpn J Pharmacol 1988;46:43–51. [3] Suzawa H, Kikuchi S, Arai N, Koda A. The mechanism involved in the inhibitory action of tranilast on collagen biosynthesis of keloid fibroblasts. Jpn J Pharmacol 1992;60:91 – 6. [4] Shigeki S, Murakami T, Yata N, Ikuta Y. Treatment of keloid and hypertrophic scars by iontophoretic transdermal delivery of tranilast. Scand J Plast Reconstr Surg Hand Surg 1997;31:151–8. [5] Chiba M, Kobayashi M, Shiohara T, Nagashima M. Successful treatment of cheilitis granulomatosis with po-
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[14]
[15]
[16]
[17]
tent inhibitors of mediator release — possible involvement of mast cells in the pathogenesis. Nippon Hifuka Gakkai Zasshi 1989;99:883 – 9 (in Japanese). Yamada H, Ide A, Sugiura M, Kurihara S, Tajima S. Treatment of granuloma annulare with tranilast. J Dermatol 1995;22:354 – 6. Yamada H, Ide A, Sugiura M, Tajima S. Treatment of cutaneous sarcoidosis with tranilast. J Dermatol 1995;22:149 – 52. McInnes A, Rennick DM. Interleukin 4 induces cultured monocytes/macrophages to form giant multinucleated cells. J Exp Med 1988;167:598 – 611. Ikeda T, Ikeda K, Sasaki K, Kawakami K, Hatake K, Kaji Y, Norimatsu H, Harada M, Takahara J. IL-13 as well as IL-4 induces monocytes/macrophages and a monoblastic cell line (UG3) to differentiate into multinucleated giant cells in the presence of M-CSF. Biochem Biophys Res Commun 1998;253:265 – 72. Enelow RI, Sullivan GW, Carper HT, Mandell GL. Induction of multinucleated giant cell formation from in vitro culture of human monocytes with interleukin-3 and interferon-g: comparison with other stimulating factors. Am J Respir Cell Mol Biol 1992;6:57 – 62. Mo¨st J, Spo¨tl L, Mayr G, Gasser A, Sarti A, Dierich MP. Formation of multinucleated giant cells in vitro is dependent on the stage of monocyte to macrophage maturation. Blood 1997;89:662 – 71. Fais S, Burgio VL, Silvestri M, Capobianchi MR, Pacchiarotti A, Pallone F. Multinucleated giant cells generation induced by interferon-g. Changes in the expression and distribution of the intercellular adhesion molecule-1 during macrophages fusion and multinucleated giant cell formation. Lab Invest 1994;71:737 – 44. Kazazi F, Chang J, Lopez A, Vadas M, Cunningham AL. Interleukin 4 and human immunodeficiency virus stimulate LFA-1-ICAM-1-mediated aggregation of monocytes and subsequent giant cell formation. J Gen Virol 1994;75:2795 – 802. Fais S, Pallone F. Inability of normal human intestinal macrophages to form multinucleated giant cells in response to cytokines. Gut 1995;37:798 – 801. Suzawa H, Kikuchi S, Ichikawa K, Koda A. Inhibitory action of tranilast, an anti-allergic drug, on the release of cytokines and PGE2 from human monocytes – macrophages. Jpn J Pharmacol 1992;60:85 – 90. Kawano Y, Noma T. Cell action mechanism of tranilast — effect on the expression of HLA-class II antigen. Int J Immunopharmacol 1993;15:487 – 500. Matsumura T, Kugiyama K, Sugiyama S, Ota Y, Doi H, Ogata N, Oka H, Yasue H. Suppression of atherosclerotic development in Watanabe heritable hyperlipidemic rabbits treated with an oral antiallergic drug, tranilast. Circulation 1999;99:919 – 24.
.
Inhibitory influences of tranilast on multinucleated giant cell formation from monocytes by supernatant of concanavalin A-stimulated mononuclear cells Kana Mizuno, Hiroyuki Okamoto *, Takeshi Horio Department of Dermatology, Kansai Medical Uni6ersity, 10 -15 Fumizono, Moriguchi, Osaka 570 -8507, Japan Received 13 January 2000; received in revised form 7 February 2000; accepted 7 February 2000
Abstract Tranilast is an anti-allergic drug that inhibits the release of chemical mediators from mast cells. There have been cases-reports showing that tranilast is effective for the treatment of granulomatous diseases such as granuloma annulare and cutaneous sarcoidosis. Here we examined the in vitro effects of tranilast on the formation of multinucleated giant cells (MGCs) from human peripheral monocytes. Supernatant of concanavalin A (Con A)-stimulated mononuclear cells induced Langhans-type and foreign body-type MGCs and the addition of 10 or 100 mg/ml tranilast inhibited the formation of total MGCs and foreign body-type MGCs. Tranilast decreased the number of MGCs with 16 Bnuclei and increased that of MGCs with three to five nuclei. Fluorescence-activated cell sorting analysis showed that tranilast-treated monocytes had lower expressions of intercellular adhesion molecule-1 (ICAM1). These findings suggest that tranilast is effective for cutaneous lesions in some cases of granulomatous disorders partly through a direct effect on monocyte/macrophage-lineage cells. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Tranilast; Granulomas; Multinucleated giant cells; Monocytes; ICAM-1
1. Introduction Tranilast has been used for the treatment of atopic dermatitis because of its anti-allergic action [1]. It affects the membrane of mast cells, resulting in an inhibition of the liberation of chemical mediators such as histamine and prostaglandins * Corresponding author. Tel.: + 81-6-69921001, ext: 3301; fax: + 81-6-69963299. E-mail address: [email protected] (H. Okamoto).
[2]. Mast cells are key cells not only in allergic disorders but also fibrotic conditions. Therefore, tranilast has also been used for the treatment of patients with keloid and hypertrophic scars [3,4]. Recently there have been some reports showing the effectiveness of tranilast in treating cutaneous lesions in some cases of granulomatous disorders such as cheilitis granulomatosis, granuloma annulare, and sarcoidosis [5–7]. Although the decreased release of chemical mediators by tranilast may be associated with its efficacy even in such
0923-1811/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 9 2 3 - 1 8 1 1 ( 0 0 ) 0 0 0 9 4 - 3
K. Mizuno et al. / Journal of Dermatological Science 24 (2000) 166–170
diseases, the precise action mechanism is unknown. Histologically, multinucleated giant cell (MGC) is one of the characteristic features of granulomatous disorders. The cells are also produced in vitro, utilizing peripheral blood mononuclear cells and bone marrow cells by various stimuli, including cytokines and growth factors [8 – 11]. Supernatants from concanavalin A (Con A)-stimulated lymphocytes also have the capacity to produce MGCs [11]. Here we examined the effects of tranilast on the formation of MGCs produced from human monocytes by Con A-stimulated lymphocyte supernatants.
2. Materials and methods
2.1. Reagent Tranilast (N-(3,4-dimethoxycinnamoyl) anthranilic acid) was obtained from Kissei Pharmaceutical (Matsumoto, Japan). For in vitro examinations, tranilast was dissolved in 1% NaHCO3 solution, and all wells examined included the same dose of NaHCO3. Because the plasma concentration of tranilast at clinical dose is 22.2 mg/ml, 1, 10 or 100 mg/ml tranilast was used in our studies.
2.2. Isolation and culture of monocytes Peripheral blood mononuclear cells (PBMC) were obtained from six healthy adult volunteers after obtaining informed consent. Mononuclear cells were isolated from heparinized blood by density gradient centrifugation with Lymphoprep™ (Nycomed, Oslo, Norway), and washed twice in phosphate-buffered saline (PBS). The cells were resuspended in RPMI 1640 medium (Nikken Bio Med, Kyoto, Japan) supplemented with 2 mmol/l glutamine (Gibco BRL, Rockville, MD) and 10% fetal calf serum (FCS; Gibco BRL), and then plated onto 48-well culture plates (Becton Dickinson Labware, Lincoln Park, NJ) at a concentration of 2×106 cells per well. After incubation for 1.5 h at 37°C, the nonadherent cells were removed by repeated vigorous wash-
167
ings. The resulting cells consisted of at least 90% monocytes, judged by nonspecific esterase staining and phenotype analysis. The cells were more than 95% viable, as determined by trypan blue (Wako Pure Chemical Industries Ltd., Osaka, Japan) dye exclusion.
2.3. Generation of MGCs The formation of MGCs was induced by supernatants of Con A-stimulated mononuclear cells (conditioned medium). PBMC were cultured for 72 h in RPMI 1640 supplemented with 10% FCS, 2 mmol/l glutamine and 16 mg/ml Con A (Sigma, St. Louis, MO) at a density of 2×106 cells/ml. The cell-free supernatant was stored at −20°C until added to monocyte cultures. Generation of MGCs was evaluated by staining with Giemsa (Merck, Darmstadt, Germany). MGCs were defined as cells with more than three nuclei per cell. Langhans-type MGC show a circular peripheral arrangement of nuclei and foreign body-type MGC have the nuclei scattered in an irregular fashion throughout the cell. The fusion rate of monocytes was expressed as fusion index. Fusion index (%) is determined as (the number of nuclei within MGC)/(the total number of nuclei counted)× 100. All nuclei were counted through the plates. Data are expressed as mean9 standard deviation (S.D.) from triplicate assays and Student’s t-tests were used for statistical analysis.
2.4. Flow cytometry The expression of adhesion molecules was assessed using the following monoclonal antibodies: mouse anti-CD11a (LFA-1) and anti-CD54 (ICAM-1) antibodies (Serotec Ltd., Oxford, UK). FITC-conjugated rabbit anti-mouse IgG antibody (Caltag, Burlingame, CA) was used as a secondstep antibody for indirect staining. Monocytes were placed in microcentrifuge tubes and fixed with 1% paraformaldehyde (Sigma). Cells were stained with 1:50 dilution of the monoclonal antibodies described above by incubation on ice for 60 min. The cells were washed and then incubated with a 1:50 dilution of FITC-conjugated rabbit anti-mouse IgG antibody. They were then washed
168
K. Mizuno et al. / Journal of Dermatological Science 24 (2000) 166–170
and analyzed by cytofluorography on a FACScan (Becton Dickinson Immunocytometry System).
3. Results
3.1. Effects of tranilast on generation of MGCs
Fig. 1. Fusion index of peripheral blood monocytes cultured in conditioned medium with or without 1–100 mg/ml tranilast. LGC, Langhans-type multinucleated giant cells; FGC, foreign body-type multinucleated giant cells.
MGC formation was maximal when 50% of the conditioned medium was used. Based on this result, all of the following experiments were conducted with that dose of conditioned medium. The fusion index of total, foreign body-type and Langhans-type MGCs by conditioned medium was 90.09 0.9, 65.694.8 and 24.59 5.1%, respectively (Fig. 1). One mg/ml tranilast did not influence the formation of MGCs. Ten and 100 mg/ml tranilast significantly attenuated the formation of total MGCs and foreign-body type MGCs; 85.99 2.8 and 55.59 3.6% in 10 mg/ml and 65.19 6.3 and 28.79 8.0% in 100 mg/ml, respectively. The fusion index of Langhans-type MGCs was slightly but not increased significantly by tranilast treatment in a dose-dependent manner; 27.196.7 in 1 mg/ml, 30.4 95.8 in 10 mg/ml and 36.5 9 6.3% in 100 mg/ml. The cellular viability was not influenced by the stimulation of 1–100 mg/ml tranilast.
3.2. Effects of tranilast on the number of the nuclei in MGCs
Fig. 2. Comparison of the number of nuclei in MGCs generated from peripheral blood monocytes cultured in conditioned medium with or without 1–100 mg/ml tranilast. LGC, Langhans-type multinucleated giant cells; FGC, foreign body-type multinucleated giant cells.
Tranilast influenced the number of nuclei in MGCs (Fig. 2). The ratios of MGCs with six to 15 nuclei among total-MGC were not influenced significantly by 1–100 mg/ml tranilast. However, the ratio of MGCs with five or less nuclei was increased by 10 or 100 mg/ml of tranilast; 32.2 9 9.4 in 10 mg/ml, 44.3918.8% in 100 mg/ml. Such doses of tranilast decreased the rates of MGCs with 16B nuclei; 8.59 9.1 in 10 mg/ml and 4.29 7.2% in 100 mg/ml. While 1 mg/ml tranilast increased the rate of Langhans-type with 16B nuclei of Langhans-type MGCs, 100 mg/ml tranilast prevented the formation of Langhans-type MGCs with 16 B nuclei. The rates of foreign body-type MGCs with six to 15 nuclei for all foreign body-type MGCs were not influenced significantly by tranilast. The rates of foreign bodytype MGCs with five or less nuclei were increased greatly by 10 or 100 mg/ml of tranilast; 16.39 10.2 in 10 mg/ml, 35.2919.4% in 100 mg/ml. However, the rates of foreign body-type MGC with 16B
K. Mizuno et al. / Journal of Dermatological Science 24 (2000) 166–170
Fig. 3. A representative FACS analysis of monocytes cultured in conditioned medium with or without 100 mg/ml tranilast for 1 day. This is a representative experiment out of the three independent assays. (A) CD11a (LFA-1), (B) CD54 (ICAM-1).
nuclei were decreased by such doses of tranilast; 13.29 12.8 at 10 mg/ml and 9.5 916.5% at 100 mg/ml.
3.3. Expression of ICAM-1 and LFA-1 on monocytes To examine the effect of tranilast on adhesion molecules of monocytes, cells were cultured in conditioned medium with or without 100 mg/ml tranilast for 1 day. FACS analysis showed that a very low level of ICAM-1 and LFA-1 expression was detected in the untreated monocytes (Fig. 3). Monocytes cultured with conditioned medium showed high expression of ICAM-1 and LFA-1. The expression of ICAM-1 decreased on monocytes treated with tranilast, while LFA-1 expression was not changed.
4. Discussion Tranilast is a widely used anti-allergy drug in Japan and has been adapted for the treatment of atopic dermatitis patients [1]. This drug is also
169
used to treat keloid and hypertrophic scars [3,4], because it has been shown to inhibit collagen synthesis of fibroblasts from the lesions [12]. Recently, there have been case reports showing the efficacy of tranilast in granulomatous disorders. Chiba et al. [5] reported a case of cheilitis granulomatosis successfully treated with tranilast. They showed that mast cells, particularly those with ongoing degranulation, increased in number in the lesions and suggested that tranilast was effective for granulomatous inflammation partly through inhibition of mediators released from mast cells. Yamada et al. [6,7] reported that tranilast resolved cutaneous lesions in three cases of granuloma annulare and two cases of sarcoidosis. Because mast cells are not critical in such disorders, the precise action mechanisms of tranilast in these granulomas remain unknown. The key cells in granulomatous lesions are monocyte macrophage-lineage cells such as activated macrophages, epithelioid cells, and MGCs. There have been reports showing that MGCs are produced in vitro from monocytes stimulated by cytokines such as interleukin(IL)-4 [8], IL-13 plus macrophage colony-stimulating factor (M-CSF) [9] and interferon(IFN)-g [12]. Supernatant of Con A stimulated-mononuclear cells also induced MGCs from monocytes [11] but which cytokine in the supernatant controls MGC formation has not been defined. There are several reasons we utilized the supernatant to produce MGCs in this study. First, it can induce a high rate of MGCs. Another reason is that the supernatant could induce two types of MGCs, Langhans-type and foreign bodytype cells. These characteristics can facilitate quantitative and qualitative analysis of the effect of tranilast on MGC formation. This study showed that tranilast inhibited the formation of total MGCs and foreign body-type MGCs. The mechanism by which tranilast inhibited the formation of MGCs is unknown. Not only the above cytokines but also adhesion molecules are considered to play a crucial role in the process of MGC formation. For example, MGC formation was blocked by anti-ICAM-1 and anti-LFA-1 monoclonal antibodies which reduced the aggregation of monocytes [13]. Intestinal macrophages from uninflamed surgical specimens, with poor
170
K. Mizuno et al. / Journal of Dermatological Science 24 (2000) 166–170
expression of adhesion molecules such as ICAM-1 and LFA-1, had less capacity to form MGCs, while the cells from Crohn’s disease highly expressed both surface markers and formed MGCs spontaneously [14]. Also, the change in ICAM-1 expression and cellular distribution has been reported to be involved in the mechanisms of IFNg-induced MGC formation [12]. Thus, ICAM-1 is considered to be one of the surface molecules that play a role in MGC generation. The current studies showed that tranilast decreased ICAM-1 expression on monocytes. The effects of tranilast on cytoplasmic membranes have been reported in mast cells and are considered the most important event in the action mechanisms of the chemical in allergic disorders [2]. Tranilast was reported to inhibit the release of transforming growth factor (TGF)-b, IL-1b and prostaglandin E2 from human monocytes–macrophages [15]. Expression of HLA-DR and -DQ antigens on macrophages was significantly suppressed by treatment with tranilast [16,17]. Taken together with these findings, the present studies suggested that tranilast directly affects the monocyte macrophage-lineage cells, resulting in resolution of the granulomatous lesions.
References
[6]
[7]
[8]
[9]
[10]
[11]
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