대한류마티스학회지 Vol. 17, No. 3, September, 2010 DOI:10.4078/jkra.2010.17.3.238 원저 IL-15 에의한류마티스관절염환자활막섬유모세포에서의 SDF-1 유도 부산대학교의학전문대학원내과학교실 1, 가톨릭대학교류마티스연구센터 2 박영은 1 ㆍ김성일 1 ㆍ박성후 1 ㆍ백승훈 1 ㆍ오혜좌 2 ㆍ허양미 2 ㆍ조미라 2 =Abstract= IL-15 Induced an Increased SDF-1 Expression in the Synovial Fibroblasts of Patients with Rheumatoid Arthritis Young-Eun Park 1, Sung-Il Kim 1, Seong-Hu Park 1, Seung-Hoon Baek 1, Hye-Jwa Oh 2, Yang-Mi Heo 2, Mi-La Cho 2 Department of Internal Medicine, School of Medicine, Pusan National University, Busan 1, The Rheumatism Resarch Center, Catholic Research Institute of Medical Science, The Catholic University of Korea, Seoul 2, Korea Objective: Interleukin-15 (IL-15) recruits and activates synovial T cells, and IL-15 plays an important role in amplifying and perpetuating inflammation in the pathogenesis of rheumatoid arthritis (RA). Stromal cell-derived factor-1 (SDF-1) is a potent chemoattractant for memory T cells in the inflamed RA synovium. This study investigated the effect of IL-15 on SDF-1 production in RA fibroblast-like synoviocytes (FLS). Methods: The expressions of IL-15 and SDF-1 were determined from the synovium of patients with RA and osteoarthritis (OA) by performing immunohistochemistry. The expressions of SDF-1 was measured from the RA FLS that were cultured with IL-15 and IL-17 by real-time RT-PCR and ELISA. The SDF-1 expression was also measured, via ELISA, from the RA FLS stimulated by IL-15 together with the inhibitors of such intracellular signal molecules as phosphatidylinositol 3-kinase (PI 3-kinase, LY294002), STAT3 (AG490), MAP Kinase (PD98059), NF-κB (parthenolide) and activator protein 1 (AP-1, curcumin). Results: IL-15 and SDF-1 were mainly expressed in the RA synovium compared to that of the OA synovium. IL-15 increased the SDF-1 expressions and it, and had an additive effect with <접수일 :2010년 5월 7일, 수정일 :2010년 5월 31일, 심사통과일 :2010년 6월 2일> 통신저자 : 김성일부산시서구아미동1가부산대학교의학전문대학원내과학교실 Tel:051) 240-7580, Fax:051) 241-7580, E-mail:ksimd@pusan.ac.kr 238
박영은외 : IL-15 Induced SDF-1 in RA FLS IL-17 on the SDF-1 expressions in the cultured RA FLS. The IL-15 induced increase of the SDF-1 expression in the cultured RA FLS was blocked by the inhibitors of PI 3-kinase, NF-κB and AP-1. Conclusion: The SDF-1 expression was increased in the RA synovium and it was up-regulated by IL-15 in the RA FLS through the PI 3-kinase, NF-κB, and AP-1 pathways. These results imply that the IL-15 induced increase of the SDF-1 expressions may be involved in the immunopathogenesis of RA. Key Words: IL-15, Stromal cell-derived factor-1, IL-17, Fibroblast-like synoviocytes, Rheumatoid arthritis 서론류마티스관절염 (rheumatoid arthritis, RA) 은관절활막의지속적인염증으로연골및골을포함한관절조직의파괴가발생하는전신성만성염증성자가면역질환으로, 명확한병인은밝혀져있지않으나, 선천면역세포 ( 항원인지세포 ), 후천면역세포 (T 및 B 림프구 ) 및활막섬유모세포 (fibroblast-like synoviocyte, FLS) 등의활성과이들세포에서유리되는사이토카인및케모카인들을포함한염증매개물질들이병인에중요한역할을한다 (1). Stromal cell-derived factor 1 (SDF-1; CXCL12) 은 T 및 B 림프구, 단핵세포 / 대식세포들을염증성활막내로이동하도록촉진하는케모카인이다. SDF-1은 RA 환자의골수기질세포, FLS, 대식세포및혈관내막세포에서생성되며, RA 환자의혈액, 활액및활막에서발현이증가된다 (2-5). SDF-1은 CD4 + T 세포의이동및축적과단핵세포의활막내이동을촉진하고, 혈관생성유도및연골세포와파골세포를활성화시켜관절파괴를악화시킨다 (2,6). SDF-1 및 SDF-1 수용체인 CXCR4의억제제들은 collagen-induced arthritis (CIA) 에서관절염발생빈도및중증도를감소시킨다 (7,8). IL-15는 RA의병인에중요한염증성사이토카인으로, RA 환자의활액및활막에서발현이증가되고, 주로대식세포, FLS 및혈관내막세포에서발현된다 (9-11). IL-15는대식세포와 memory T 세포의상호접촉에의해 T 림프구를염증반응부위로이동시키고증식시킨다 (9). IL-15에의하여 T 림프구에서생성이증가된 tumor necrosis factor (TNF), interferon (INF)-γ 및 interleukin (IL)-17은 FLS에서 IL- 15, IL-8 및 IL-6의생성을자극하여염증을악화시킨다 (12,13). CIA에서 IL-15의기능차단시관절염발생및 TNF, IL-1β, IL-6 및 IL-17 생성이감소하고, RA 환자에게 IL-15에대한항체투여는질병활성을감소시킨다 (14-16). 이러한결과들은 IL-15가 RA에서염증반응을지속시키고확장시키는데중요한역할을함을암시하며, 치료전략의표적사이토카인으로서의가능성을시사한다. 저자들은 RA 환자의활막에서 IL-15가 SDF-1의발현에미치는영향에대하여조사하고자하였다. 대상및방법 1. 대상미국류마티스학회 (American College of Rheumatology) 의 RA의진단기준에부합되는환자중무릎관절치환술을받는 4명 ( 평균연령은 51±11세 ) 에서활막조직을채취하였고, 대조군은성별, 연령이일치하는골관절염 (osteoarthritis, OA) 환자 4명에서활막조직을채취하였다. 본연구는임상연구윤리위원회로부터승인받았으며, 모든환자로부터동의서를획득하였다. 2. 시약 Recombinant human IL-15, IL-17, IL-1β, anti-il-15 antibody (Ab) 및 anti-sdf-1 Ab는 R&D System (Minneapolis, MN) 으로부터구입하였다. 3. 면역조직화학염색파라포름알데하이드 (4%) 에고정된 RA 및 OA 활 239
대한류마티스학회지제 17 권제 3 호 2010 막을파라핀에포매한후절편기를이용하여 7μm 절편을만들어슬라이드에부착하였다. 면역조직화학염색은 ABC (Vector laborites, Burlingame, CA, USA) kit를사용하여염색하였으며, 슬라이드에부착된절편을자일렌과에탄올로탈파라핀과함수를시킨후 3% H 2O 2 로내인성과산화효소를차단시키고, 인산화완충액 (phosphate buffered saline, PBS) 으로수세한다. 비특이적인반응을차단할목적으로 anti-mouse serum을 30분반응시킨후일차항체인 recombinant human anti-il-15 Ab와 anti-sdf-1 Ab (R&D systems, 1:100) 를 4 o C에서다음날 (16시간) 까지반응시켰다. 일차항체반응후결합이안된항체를 PBS로수세하고바이오틴이결합된이차항체와과산화효소가결합된 streptavidin반응을시킨후 DAB으로발색시켰다. Mayer s 해마톡실린대조염색한후수세하고봉입하여광학현미경으로관찰하였다. 음의대조조직은 (negative control tissue) 은 isotype control antibody (Mouse IgG1:R&D Systems, Minneapolis, MN) 를사용하였다. 4. 활막섬유모세포 (fibroblast-like synoviocyte, FLS) 의분리및자극 FLS는 RA 및 OA 환자의활막에서효소를이용하여분리하였으며, 조직은 2 3 mm 조각으로잘게자른후 Dulbecco s modified Eagle s medium (DMEM) 에 type I collagenase (Worthington, Freehold, NJ) 와함께 37 o C, 5% CO 2 에서 4시간동안반응시켰다. 분리된세포를 500 g에서원심분리하여 10% fetal calf serum (FCS), 2 mm L-glutamine, penicillin (100 units/ml), streptomycin (100 μg/ml) 를가한 DMEM 에재부유시켜 75 cm 2 플라크스에분주하였다. 하루가지난후배양기에부착되어있는세포만을 10% FBS를가한 DMEM에넣어 5% CO 2 와 37 o C의조건에서배양하고배양액은매 3일마다교환하였다. 플라스크바닥의 90 95% 가 FLS로충만되면신선한배양액으로 1:3 희석하여계대배양하였다. 실험에는 4 8 계대의 FLS를사용하였다. 배양된세포가 FLS임을확인하기위해항 CD14 PE 항체 (PharMingen, SanDiego, CA), 항 CD3 FITC 항체또는항 Thy-1 (CD90) 항체 (PharMingen) 를이용하여유세포분석을시행하였다. 세포를카운트하여 5 10 4 cells/well을 1 ml의 serum free DMEM/insulin-transferrin-selenium A (Life Technologies, Gaithersburg, MD) 배양액에섞어 24-well plate에분주하여준다. 다양한농도의 IL-15 및 IL-17을각각혹은함께자극하거나, 신호전달억제물질들 (LY294002, AG490, PD98059, parthenolide, 그리고 curcumin) 을처리후 IL-15 (10 ng/ml) 로자극하였다. 5. SDF-1의단백질발현측정 (ELISA) FLS 자극후 48시간째에세포배양상층액을모아 SDF-1 정량을위한 ELISA를시행하였다. Sandwich ELISA용 96 well plate (Nunc, Roskilde, Denmark) 에 human anti-sdf-1 Ab (4 ug/ml; R&D Systems) 에넣고 4 o C, 밤새반응시킨후, 차단용액 (1% bovine serum albumin (BSA)/0.05% Tween200) 이함유된 phosphate buffered saline (PBS) 를첨가하여실온에서 2 시간반응시켰다. Biotinylated SDF-1 polyclonal antibody (400 ng/ml; R&D Systems), streptavidin-alkaline phosphate (1:2,000, Sigma) 와 avidin-peroxidase (50 μl, 1:2,000), 501 tetramethylbenzidine substrate solution (Kirkegaard & Perry, Guildford, UK) 을차례대로각 well에첨가하여반응시켰다. 6. SDF-1 mrna 측정 (real-time RT-PCR) FLS를다양한농도의 IL-15로자극후 16시간째에세포의 mrna를 RNAzol B (Biotecx, Houston, TX) 를이용하여추출하였다. 총 2 μg의추출된 mrna를 Superscript reverse transcription system (Takara, Shiga, Japan), 42 o C에서역전사시켰다. SYBR Green I (Roche Diagnostic, Mannheim, Germany) 를이용하여 real-time PCR을시행하였다. Real-time PCR 전용 capillary tube 를사용하여 LightCycler FastStart DNA mastermix (SYBR Green I) 2 μl, 각각의 primer 0.5 μm, MgCl 2 4 mm, 그리고 template DNA 2 μl을포함하여총 20 μl가되도록하였다. SDF-1과 GAPDH 시발체의염기서열은다음과같았다 ; SDF-1 (5 -ATG-AAC-GCC- AAG-GTC-GTG-GTC-3 (sense), 5 -TGG-CTG-TTG-TGC- TTA-CTT-GTT-T-3 (antisenss)), GAPDH (5 -CGA-TGC- TGG-GCG-TGAGTA-C-3 (sense), 5 -CGT-TCA-GCT-CAG- GGA-TGACC-3 (antisense)). SDF-1과 GAPDH에서, 변성을위해 94 o C에서 30초, annealing은 56 o C에서 1분, 240
박영은외 : IL-15 Induced SDF-1 in RA FLS 신장을위해 72 o C에서 1분의과정을 30회반복하였다. 형광커브는 LightCycler software v. 3.0에서분석하였다. 7. MTT assay 96 well plate에 1 10 3 /ml의농도로세포를배양하여, ITSA가포함된 serum free DMEM 배양액으로 starvation한다. LY294002, AG490, PD98059, parthenolide 혹은 curcumin을처리후 IL-15는 10 ng/ml의농도로처리하였으며, 48시간동안 37 o C, 5% 배양기에서배양하였다. 여기에 MTT 용액 (Thiazolyl blue, 2.5 mg/ml, indeiodinized H2O) 10 μl를혼합하여 4시간동안반응시킨후 5분간원심분리하고, 0.04 M HCl 100 μl를첨가하여 5분동안실온에방치하였다가 ELISA 판독기로 595 nm로측정하였다. 8. 통계적유의성의검증결과는평균과표준오차로표현하였고통계적유의성은 SPSS 통계프로그램 (version 10.0) 을사용하여 Mann-Whitney U test나 Wilcoxon s signed rank test를실시하였고 p값이 0.05 이하일때통계적으로유의하다고분석하였다. 결과 1. RA 활막조직에서 IL-15 및 SDF-1의발현증가 RA 환자의활막조직에서 IL-15과 SDF- 1의발현이대조군 ( 골관절염환자활막조직 ) 에비하여증가되어있음을면역조직화학염색법을통해서관찰하였다. SDF-1과 IL-15은모두류마티스관절염환자활막에서과발현되어있었다 ( 그림 1). 2. RA (FLS) 에서 IL-15에의한 SDF-1 발현증가 RA 환자 FLS에서 IL-15에의한 SDF-1 발현이조절되는지알아보고자, FLS 1 10 5 cell/ml 로배양된세포에 IL-15을 0.1, 1, 10, 50 ng/ml의농도로처리하여 12시간의세포와 48시간세포배양액에서각각 SDF-1의 mrna와단백질발현양을 real- time PCR과 ELISA 방법으로평가하였다. 그결과 IL-15 농도의존적으로 SDF-1의 mrna ( 그림 2A) 와단백질발현양 ( 그림 2B) 이 FLS에서증가되는것을관찰하였다 (*p<0.05 compared to nil, p<0.01 compared to nil). IL-15과 IL-17을함께자극하였을때 SDF-1의단백질발현에부가적인효과 (additive effect) 가관찰되었다 (*p<0.01 compared to nil, p<0.05 compared to Fig. 1. Immunohistochemical staining for stromal-cell derived factor-1 (SDF-1) and interleukin-15 (IL-15) on the synovium of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). IL-15 and SDF-1 were significantly more expressed in the RA synovium compared to that of the OA synovium (original magnification, 400). 241
대한류마티스학회지제 17 권제 3 호 2010 Fig. 2. The expressions of SDF-1 from the RA synovial fibroblasts stimulated with various concentrations of IL-15 (0, 0.1, 1, 10 and 50 ng/ml) were determined by real-time RT-PCR (A) and ELISA (B). IL-15 increased the SDF-1 expression in a dose-dependent manner. *p<0.05 compared to nil, p<0.01 compared to nil. inhibitor, AG490; JAK2-STAT3 inhibitor, PD98059; MAPK inhibitor, parthenolide; NF-κB inhibitor, curcumin; AP-1 inhibitor) 를처리하였다. 이때 PI3-kinase 억제제 (LY294002), NF-κB 억제제 (parthenolide) 및 AP-1 억제제 (curcumin) 는 IL-15에의한활막섬유모세포의 SDF-1 단백질발현을유의하게억제하였으나, STAT3 억제제 (AG490) 및 MAPK 억제제 (PD98059) 는 SDF-1 단백발현에영향이없었다 (*p<0.05 compared to IL-15 10 ng/ml, p<0.01 compared to IL-15 10 ng/ml) ( 그림 4A). 억제제에의한세포독성효과가없음은 MTT assay로확인하였다 ( 그림 4B). Fig. 3. The productions of SDF-1 from the RA FLS stimulated with IL-15 and IL-17 were determined by ELISA. IL-15 and IL-17 up-regulated SDF-1 production. IL-17 had an additive effect with IL-15 on the SDF-1 production. *p<0.01 compared to nil, p<0.05 compared to IL-15 1 ng/ ml, p<0.01 compared to IL-15 10 ng/ml. IL-15 1 ng/ml, p<0.01 compared to IL-15 10 ng/ ml) ( 그림 3). 3. RA FLS에서 IL-15에의한 SDF-1 발현증가관련세포내신호전달 RA 환자 FLS에서 IL-15에의한 SDF-1 발현에관여하는신호전달경로를조사하기위하여세포에 IL-15와신호전달물질의억제제 (LY294002; PI3Kinase 고찰저자들은 IL-15와 SDF-1 발현이 OA에비하여 RA 활막의 lining layer에서주로발현되고, IL-15는 RA FLS에서 SDF-1의 mrna 및단백발현을농도의존적으로증가시키며, IL-15와 IL-17은 SDF-1 발현에부가적인효과가있음을확인하였다. 또한, IL-15에의한 FLS의 SDF-1 발현증가는 PI3-kinase, NF-κB 및 AP-1 경로에의함을확인하였다. SDF-1은 CXC 케모카인으로, 단독수용체인 CXCR4 와결합하여세포의이동및정체에중요한역할을한다 (17). SDF-1과 CXCR4의결합은인간면역결핍바이러스 (HIV), 암, 제1형당뇨병및 RA 등의다양한질병병인에관여한다 (18-20). RA에서 SDF-1은 T 림프구의활막내이동및혈관주위의침윤을 242
박영은외 : IL-15 Induced SDF-1 in RA FLS Fig. 4. (A) The IL-15 induced increase of SDF-1 was blocked by the inhibitors of PI3-kinase, NF-κB and AP-1. RA FLS that were, pre-treated with the inhibitors of PI3-kinase (LY294002), STAT3 (AG490), MAPK (PD98059), NF-κB (parthenolide) and AP-1 (curcumin), were cultured with IL-15; the production of SDF-1 was determined by ELISA. Up-regulation of SDF-1 by IL-15 was blocked by the inhibitors of PI3-kinase, NF-κB and AP-1. (B) MTT assay was done to confirm that the inhibitors did not have cell toxicity. This assay showed that there was no difference in cell viability after all the stimulations and/or inhibitions. *p<0.05 compared to IL-15 10 ng/ml, p<0.01 compared to IL-15 10 ng/ml. 촉진하고, T 림프구의활성에의한자가사멸 (apoptosis) 을억제하여활막내 T 림프구를축적시킨다 (6). 또한활막내 B 림프구를축적하여 ectopic germinal center 형성을촉진하고 FLS 활성, 신생혈관생성자극, 연골세포와파골세포의 matrix metalloprotease-3 (MMP-3) 와 MMP-9의분비를자극하여연골기질파괴및골흡수증가를야기하여 RA의병인에서중요한작용을한다 (2). RA에서 SDF-1의발현을조절하는기전에대한연구는적은데, 항 CD40 항체, 저산소증 (hypoxia), TGF-1β 및 IL-17은 RA FLS에서 SDF-1 발현을증가시키고, IL-1β와 TNF-α는 RA FLS에서의 SDF-1 발현에영향이없지만피부와구강내섬유모세포의 SDF-1 발현을감소시킨다 (6,21,22). IL-15는 RA에서염증지속에중요한역할을한다. IL-15는활막내로 T 림프구이동및활성을촉진시키고, 활성화된 T 림프구는대식세포와접촉하여 TNF-α의분비를유도함으로써염증반응을증폭, 지속시킨다 (9). 또한 IL-15는 TNF와는독립적으로 auto-reactive CD4 + CD28 - T cell에서자연살상세포 (natural killer cell, NK cell) 수용체인 NKG2Ddml 발현을유도하여 RA의 self-perpetuating inflammation에관여하므로, IL-15가항 TNF 제제와는무관하게 memory T 세포의확장과유지를지속시킨다 (23). RA 활막에서 IL-15 발현이항 TNF 제제에의하여감소하지않으므로항 TNF제제에반응이불충분한환자에서 IL-15의기능차단은 RA의새로운치료법이될수있음을시사하였다 (24). 염증성활막내의 CD4 + T 세포축적은 RA의중요한병인중하나이다. Toshihiro 등은활막 CD4 + memory T 세포에서발현된 CXCR4와활막세포에서발현된 SDF-1의상호작용이 CD4 + T 세포의이동에중심적인역할을하며, IL-15가 T 림프구의 CXCR4 의발현을증가시킴을보고하였다 (6). 저자들은 IL- 15가 RA FLS에서 SDF-1의 mrna 및단백발현을농도의존적으로증가시키며, IL-15와 IL-17은 SDF-1 발현에부가적인효과가있음을확인하였다. 이러한결과들은 IL-15가 T 림프구의 CXCR4 및 FLS의 SDF-1를증가시켜활막내 T 림프구의축적에관여함을암시한다. 활막의활성화된 T 림프구와대식세포및단핵세포와의상호작용에의하여증가된 TNF, IFN-γ 및 IL-17은 FLS를자극하여 IL- 15를포함하는염증성사이토카인및매개물질의생성을증가시킨다. 활막에서증가된 IL-17과 IL-15는 FLS에서 SDF-1의생성을증가시켜 T 림프구의활막내이동및활성화를더욱촉진하고, 혈관생성, 연골파괴및골흡수를야기하여관절파괴가지속적으로 243
대한류마티스학회지제 17 권제 3 호 2010 진행됨을암시한다. Kim 등은 RA FLS에서 IL-17이 SDF-1의발현을증가시키고, 이를매개하는세포내신호전달과정으로 NF-κB, PI 3-kinase 그리고 AP-1과연관됨을보고하였다 (22). IL-15 receptor를통한 IL-15의세포내신호전달과정으로는 NF-kB, JAK1, JAK3, STAT3, STAT5 그리고 MAPK 등여러신호전달물질을매개하는것으로알려져있고, 본연구에서는 RA FLS 에서 IL-15가 NF-κB, PI 3-kinase 및 AP-1과연관하여 SDF-1의발현을증가시킴을확인하였다 (25,26). 결 본연구는 SDF-1과 IL-15가 RA 활막에서발현이증가하고, IL-15가 RA FLS에서 SDF-1의발현을증가시키며, IL-15에의한 SDF-1의증가는 PI-3 kinase, NF-κB 및 AP-1의신호전달과정을통함을확인하였다. 이러한결과들은 IL-15에의한 FLS의 SDF-1 증가가 RA의염증지속과관절파괴에주요한역할을하며, IL-15 기능차단을이용한치료법개발에새로운근거가될것으로생각된다. 론 참고문헌 1) Müller-Ladner U, Pap T, Gay RE, Neidhart M, Gay S. Mechanisms of disease: the molecular and cellular basis of joint destruction in rheumatoid arthritis. Nat Clin Pract Rheumatol 2005;1:102-10 2) Mittal GA, Joshi VR, Deshpande A. Stromal cellderived factor-1 alpha in rheumatoid arthritis. Rheumatology (Oxford) 2003;42:915-6. 3) Kanbe K, Takagishi K, Chen Q. Stimulation of matrix metalloprotease 3 release from human chondrocytes by the interaction of stromal cell-derived factor 1 and CXC chemokine receptor 4. Arthritis Rheum 2002; 46:130-7. 4) Grassi F, Cristino S, Toneguzzi S, Piacentini A, Facchini A, Lisignoli G. CXCL12 chemokine upregulates bone resorption and MMP-9 release by human osteoclasts: CXCL12 levels are increased in synovial and bone tissue of rheumatoid arthritis patients. J Cell Physiol 2004;199:244-51. 5) Pablos JL, Santiago B, Galindo M, Torres C, Brehmer MT, Blanco FJ, et al. Synoviocyte-derived CXCL12 is displayed on endothelium and induces angiogenesis in rheumatoid arthritis. J Immunol 2003;170:2147-52. 6) Nanki T, Hayashida K, El-Gabalawy HS, Suson S, Shi K, Girschick HJ, et al. Stromal cell-derived factor-1-cxc chemokine receptor 4 interactions play a central role in CD4+ T cell accumulation in rheumatoid arthritis synovium. J Immunol 2000;165: 6590-8. 7) De Klerck B, Geboes L, Hatse S, Kelchtermans H, Meyvis Y, Vermeire K, et al. Pro-inflammatory properties of stromal cellderived factor-1 (CXCL12) in collagen-induced arthritis. Arthritis Res Ther 2005; 7:R1208-20. 8) Tamamura H, Fujisawa M, Hiramatsu K, Mizumoto M, Nakashima H, Yamamoto N, et al. Identification of a CXCR4 antagonist, a T140 analog, as an anti-rheumatoid arthritis agent. FEBS Lett 2004;569: 99-104. 9) McInnes IB, al-mughales J, Field M, Leung BP, Huang FP, Dixon R, et al. The role of interleukin-15 in T-cell migration and activation in rheumatoid arthritis. Nat Med 1996;2:175-82. 10) Thurkow EW, van der Heijden IM, Breedveld FC, Smeets TJ, Daha MR, Kluin PM, et al. Increased expression of IL-15 in the synovium of patients with rheumatoid arthritis compared with patients with Yersinia-induced arthritis and osteoarthritis. J Pathol 1997;181:444-50. 11) Oppenheimer-Marks N, Brezinschek RI, Mohamadzadeh M, Vita R, Lipsky PE. Interleukin 15 is produced by endothelial cells and increases the transendothelial migration of T cells in vitro and in the SCID mouse-human rheumatoid arthritis model in vivo. J Clin Invest 1998;101:1261-72. 12) Miranda-Carús ME, Balsa A, Benito-Miguel M, Pérez de Ayala C, Martín-Mola E. IL-15 and the initiation of cell contact-dependent synovial fibroblast-t lymphocyte cross-talk in rheumatoid arthritis: effect of methotrexate. J Immunol 2004;173:1463-76. 13) Cho ML, Yoon CH, Hwang SY, Park MK, Min SY, Lee SH, et al. Effector function of type II collagenstimulated T cells from rheumatoid arthritis patients: cross-talk between T cells and synovial fibroblasts. Arthritis Rheum 2004;50:776-84. 14) Ruchatz H, Leung BP, Wei XQ, McInnes IB, Liew FY. Soluble IL-15 receptor alpha-chain administration prevents murine collagen-induced arthritis: a role for IL-15 in development of antigen-induced immuno- 244
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