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1 Journal of Rheumatic Diseases Vol. 20, No. 2, April Original Article 류마티스관절염동물모델에서메토트렉세이트의 STAT3-TH17/STAT5-Treg Axis 조절을통한관절염의치료효과 박은미 1,2 ㆍ박미경 1,2 ㆍ이동건 1,2 ㆍ백승예 1,2 ㆍ우정원 2 ㆍ곽승기 1,2,3 ㆍ조미라 1 ㆍ박성환 1,2,3 가톨릭대학교의과학연구소류마티스연구센터 1, 서울성모병원면역질환융합연구사업단자가면역연구소 2, 가톨릭대학교의과대학류마티스내과학교실 3 Reciprocal Regulation of TH17 and Regulatory T Cells by Methotrexate and Its Therapeutic Effects in Collagen-induced Arthritis (CIA) Eun-Mi Park 1,2, Mi-Kyung Park 1,2, Dong-Gun Lee 1,2, Seung-Ye Baek 1,2, Jung-Won Woo 2, Seung-Ki Kwok 1,2,3, Mi-La Cho 1, Sung-Hwan Park 1,2,3 Rheumatism Research Center, Catholic Institutes of Medical Science 1, Immune Tolerance Research Center, Convergent Research Consortium for Immunologic Disease, Seoul St. Mary s Hospital 2, Division of Rheumatology, Department of Internal Medicine, The Catholic University of Korea College of Medicine 3, Seoul, Korea Objective. Methotrexate is the first-line drug in treatment of rheumatoid arthritis (RA) exhibiting higher efficacy and better tolerability than most other DMARDs. To have a better understanding of the anti-arthritic mechanism of methotrexate, we investigated the effect of methotrexate on suppressing the autoimmune inflammatory and destructive arthritis in collagen-induced arthritis (CIA) mice. Methods. The effects of methotrexate on joint inflammation were assessed by clinical scoring and histologic analysis. Levels of cytokines and autoreactive antibodies were analyzed by immunohistochemistry and ELISA. The population of TH17 and Foxp3 + regulatory T (Treg) cells and phosphorylation of their critical transcription activators, STAT3 and STAT5, were examined by fluorescence microscopy and flow cytometry, respectively. Results. Treatment with methotrexate significantly alleviated joint inflammation and cartilage destruction in CIA. Serum levels of total immunoglobulins G, G1, G2a specific to type II collagen were also reduced considerably in methotrexate-treated mice. The drug inhibited the expression of proinflammatory cytokines such as IL-1β, TNF-α, IL-6 and IL-17 in arthritic joints ex vivo as well as by splenocytes in vitro. Moreover, methotrexate treatment resulted in reciprocal modulation of TH17 cells and Foxp3 + regulatory T (Treg) cells in spleen tissues, in which TH17 cells were decreased and Treg cells in number were increased. Subsequent analysis of CD4 + T cells showed that phosphorylation of STAT3 was decreased whereas phosphorylation of STAT5 was increased in methotrexate-treated mice. Conclusion. Methotrexate treatment effectively suppressed autoimmune arthritis and restored homeostasis of the immune system by reciprocal regulation of TH17 and Treg cells in a mouse model of collagen-induced arthritis. Key Words. Methotrexate, Collagen-induced arthritis, Cytokines, TH17 cells, Regulatory T cells <Received:November 7, 2012, Revised (1st:November 19, 2012, 2nd:November 23, 2012), Accepted:November 24, 2012> Corresponding to:sung-hwan Park, Division of Rheumatology, Department of Internal Medicine, The Catholic University of Korea School of Medicine, Seoul St. Mary s Hospital, 505, Banpo-dong, Seocho-gu, Seoul , Korea. rapark@catholic.ac.kr pissn: X, eissn: Copyright c 2013 by The Korean College of Rheumatology This is a Free Access article, which permits unrestricted non-commerical use, distribution, and reproduction in any medium, provided the original work is properly cited. 94

2 메토트렉세이트에의한류마티스관절염의치료효과 95 서론메토트렉세이트 (methotrexate) 는류마티스관절염의치료에있어서단독혹은다른약제와의복합치료제로서가장흔히사용되는항류마티스약물이다. 메토트렉세이트는 50년전항암제로처음사용되기시작하였으며. 1980년대들어류마티스관절염뿐아니라다른전신류마티스질환에흔히사용되는약제로엽산유사체이다. 메토트렉세이트는세포내로이동하여 polyglutamated되어세포내의효소인 aminoimidazole carboximide ribonucleotide (AICAR), transformylase (ATIC), thymidylate synthetase (TYMS), dihydrofolate reductase (DHFR) 등의작용을억제하며, 그결과아데노신 (adenosine) 의증가와피리미딘 (pyrimidine) 합성감소, transmethylation 반응억제를나타내는것으로알려져있다. 즉 DHFR의억제와엽산의존효소의억제를통한세포내퓨린 (purine) 수치, 피리미딘대사, DNA 합성에영향을미치는것으로알려져왔으나그기전은정확히알려지지않았다 (1). 동물모델및세포주를이용한연구에서메토트렉세이트는단핵구와대식세포에서염증을유발하는사이토카인인인터루킨-12 (IL-12), 종양괴사인자 (TNF)-알파, 인터루킨-6 (IL-6), 인터루킨-8 (IL-8) 의생성을억제하며항염증작용사이토카인인인터루킨-10 (IL-10) 의생성을촉진하는것으로알려져있다. 또한메토트렉세이트는류마티스관절염의관절파괴의중요한인자로알려진프로스타글란딘 (prostaglandin) 과루코트리엔 (leukotriene) 을억제조절하는것으로알려져있으나, 메토트렉세이트의류마티스관절염질병제어와관련된작용기전은충분히밝혀지지않았다 (2-4). 최근인터루킨-17 (IL-17) 을생산하는 TH17 세포가류마티스관절염의병인에주도적인역할을하며특히관절염증과골파괴를직접유도하는것이밝혀지면서이들의제어를위해서 TH17 세포의활성을유도하는 IL-6 사이토카인을억제하거나 forkhead box protein 3 (Foxp3) 를발현하는면역조절 T 세포 (regulatory T cells; Treg) 의증식과활성을유도하여병인 T 세포의제어및면역관용을유도하는것이류마티스관절염의치료에중요한것으로알려지고있다. 이러한 TH17 세포또는조절 T 세포의동시균형조절은핵심적인사이토카인과이에상응하는전사인자의상호조절을통해나타나는것으로밝혀졌다. TH17 세포의분화및활성에핵심전사인자로알려진 STAT3는직접적으로 IL-17의로커스 (locus) 를조절할수있고 TH17 분화와관련된많은전사인자들의발현에필수적인요소로써역할을수행하는것으로알려져있다 (5,6). TH17 세포내에서이러한 STAT3의활성을억제하면 TH17 세포는제어되고면역조절 T 세포의활성은동시에유도되는이상적인면역조절이이루어졌다 (7). 면역조절 T 세포에서는 Foxp3와 STAT5가주요전사인자로알려져있으며 TH17 세포에서 STAT3 활성을억제하면 동시에 STAT5 활성이유도되는기전이밝혀지면서 STAT3 와 STAT5와상호조절이매우중요한면역조절기전으로예측되고있다. 따라서면역질환제어를위해연구자들은이들이상적면역조절을유도하기위해 STAT3 제어및 STAT5 유도관련유도체들에대한연구가질병제어전략으로주목받고있다 (8-10). 메토트렉세이트가류마티스관절염대표적치료제로알려져있으나사이토카인 IL-17과병인 TH17 세포에미치는영향에대한연구는매우제한적인상황이다 (11). 저자들은류마티스관절염동물모델인콜라겐유도관절염모델 (type II collagen-induced arthritis; CIA) 에서메토트렉세이트처치에따른관절염치료효과와염증에관여하는사이토카인의변화와함께메토트렉세이트의 TH17 세포에미치는영향을조사하고, 조절 T세포에미치는영향을조사하여작용기전을조사하고자하였다. 대상및방법제2형콜라겐유발관절염동물모델확립과메토트렉세이트처리 분양받은 6 7주령의수컷 DBA/1J 마우스 (Jackson Laboratory, Bar Harbor, ME) 는 2주일동안실험실환경에적응시킨후실험에사용하였다. 동물사육실의조건은 conventional system으로 22.0±2 o C, 1일중 12시간은 Lux 로조명하고, 12시간은모든빛을차단하였다. DBA/1J 마우스에 Complete Freund's Adjuvant (CFA; DIFCO, Detroit, MI) 와 1:1 (v/v) 비율로섞은 Bovine Type II collagen (Chondrex Inc., Redmond, WA) 100 μg을꼬리부분에피하로주사하였다. 주사후일주일후 phosphate buffered saline (PBS) 를먹인대조군과메토트렉세이트 (Sigma Aldrich, St. Louis, MO) 를투여한실험군 (1 mg/kg, 7.5 mg/kg) 으로나눈후일주일에 3회각각 100 μl씩복강내주사하였다. 동물실험에사용된메토트렉세이트의농도는이전실험논문을토대로결정하였다 (12). 관절염의육안관찰및측정 관절염병변의육안적관찰은참고문헌에근거하여다음과같은스코어를사용하여실시하였다 (13). 0점 : 부종이나종창이없음, 1점 : 발또는발목관절에국한된경한부종과발적, 2점 : 발목관절에서족근골에걸친경한부종과발적, 3점 : 발목관절에서족근골에걸친중등도의부종과발적, 4점 : 발목에서다리전체에걸쳐부종과발적이있고관절경직이나타난경우이다. 따라서, 관절염병변의최고스코어는마우스한마리당 16이며, 꼬리에주사한시점으로부터 7주까지일주일에 3회씩실시하였고, 평가자료는실험과관계없는 3인이작성하였다. 관절의조직학적변화 각그룹의마우스를희생하여관절조직을채취하여 10%

3 96 박은미외 중성완충포르말린에서 1일간고정한후 Calci-Clear Rapid (National Diagnostics, Atlanta, Georgia) 으로 7시간동안탈회하였다. 일반적인방법에의하여수세후 Ethyl alcohol로탈수하고 xylene으로명화한후파라핀으로포매하였다. 포매후 7 μm 두께로절편한후 H-E (Hematoxylin-Eosin) 염색을시행하였다. 면역조직화학염색파라핀포매조직을 7 μm 두께로자른후 xylene으로파라핀을제거하고 Ethyl alcohol로함수시킨후, Digest-All 3 (Zymed Laboratories, South San Francisco, CA) 처리후 3% hydrogen peroxide를 15분간처리하여 endogenous peroxidase를제거하였다. 조직절편을 PBS로 15분간세척후면역조직화학염색은 Vector Elite ABC kit (Vector Laboratories, Inc., Burlingame, USA) 를사용하였다. 조직절편을 1.5% normal goat serum으로 30분간반응한다음 1차항체인 rabbit anti-il-17 (Santa Cruz Biotechnology, Inc, CA), goat anti-tnf-α (Santa Cruz Biotechnology), rabbit anti-il-6 (Abcam Inc., Cambridge, MA), rabbit anti-il-1β (Santa Cruz Biotechnology) 또는 isotype control 항체 (rabbit IgG, goat IgG) 를 4 o C에서 14 16시간동안반응시켰다. 반응이끝난후 biotin이결합한 2차항체를실온에서 40분반응시킨다음 ABC reagent를실온에서 1시간적용하였다. 3,3'-diaminobenzidine (Thermo Shandon Co., Pittsburgh) 를사용하여발색반응을보았고 hematoxylin으로대조염색을실시한후최종적으로봉입을시행하였다. 콜라겐특이적인 IgG 및 IgG 아형측정콜라겐을 dilution buffer (Chondrex) 에희석한후 96-well plate의각 well에 4 o C에서 12시간반응하였다. 1% bovine serum albumin (Sigma) 가함유된 phosphate buffered saline (PBS) 를 200 μl/well씩넣고 1시간상온에둔채 blocking 하였다. 3회 washing 한후 0.05% Tween이함유된 PBS로희석한혈청 (1:25,000) 을 100 μl/well씩넣고 4 o C에서 12시간반응하였다. 3회 washing 후 anti-mouse IgG1/IgG2a peroxidase conjugate (Bethyl Lab Co., Montgomery, TX, USA) 를 1:40,000으로희석한후 30분간상온에두었다. 3회 washing 후 TMB substrate reagent 100 μl를가한후 30분이지나서 1 M H 2SO 4 50 μl를첨가하였다. Microplate reader (Molecular Devices, US) 로파장 nm에서측정하였다. 공초점현미경 OCT compound (Sakura Finetech USA, Torrance, CA) 에포매시킨뒤액체질소로동결된비장조직을 7 μm 두께의연속적인조직절편을 Coated slide에부착하였다. 조직절편은공기중에서한시간건조시킨후 4% paraformaldehyde solution (Sigma) 에넣어실온에서차가운 acetone으로 15분간고정시킨뒤 PBS로 15분간씻어주었다. 불특정결합을억제하기 위해 10% normal goat serum을넣고상온에서 30분간반응시켰다. Anti-CD4 PerCP 또는 FITC 항체 (BD Pharmingen, San Diego, CA), anti-il-17 FITC 항체 (ebioscience, San Diego, CA) 와 anti-foxp3 PE (ebioscience), anti-cd25 APC, anti-stat3 phospho Y705 항체 (BD Pharmingen), anti-stat3 phospho S727 항체 (BD Pharmingen) 와 anti-stat phospho Y694 FITC 항체 (BD Pharmingen) 를넣고 4 o C에서 18시간동안반응시켰다. 반응이끝난후실온에서 1시간동안반응시킨후 PBS로 30분간씻어주었다. Cover-slip 위에수용성봉입제 (Dako cytomation, Denmark) 를한방울떨어뜨린뒤조직부위에살포시덮었다. 그리고공초점현미경 (Zeiss, LSM 510 Meta, Germany) 으로관찰하였다. 세포분리및자극마우스의비장을적출하고 PBS 용액으로세척후가위로자르고이를 Cell strainer (BD Falcon, Bedford, MA) 에통과시켰다. 4 o C에서 1,300 rpm으로 5분간원심분리후상층액을제거한비장세포들중에서, 적혈구는적혈구용액 (2.06% Tris와 0.83% NH4Cl을 1:9로섞음 ) 과실온에서 5 분간반응시킨후 Cell strainer (BD Falcon, Bedford, MA) 에통과시켰다. 마우스비장세포는 5% heat-treated 우태아혈청 (fetal bovine serum; FBS, Gibco BRL, Grand Island, NY) 및 1% penicillin/streptomycin이첨가된 RPMI (Gibco BRL) 에부유되어 cell/ml로 24 well culture plate (NUNC, Roskilde, Denmark) 에분주하고 anti-cd3 항체 1 μg/ml, anti-cd28 항체 2 μg/ml (BD Pharmingen), lipopolysaccharide (LPS, Sigma) 1 μg/ml와메토트렉세이트 0.5, 1 μg/ml로 3일간자극되었다. 사이토카인측정세포로부터배양한상층액을모아 IL-10, IL-6를 sandwich ELISA를이용하여농도를측정하였다. Sandwich ELISA용 96 well plate (NUNC) 에단클론성 anti-il-6 항체, anti-il-17 항체와 anti-tnf-α 항체 (R&D, Minneapolis, MN, USA) 를각각 4 μg/ml로 100 μl/well씩넣고 4 o C에서밤새반응시킨다음차단용액 (1% BSA/PBST) 을 200 μl/well씩넣고실온에서 2시간반응시켰다. Standard로는재조합 IL-6, IL-17와 TNF-α (R&D) 를이용하여각각 10,000 pg/ml pg/ml 농도를사용하였다. 표준시료와함께측정할세포배양상층액을 100 μl/well씩넣고실온에서 2시간반응시켰다. 반응용기를세척용액 (0.05% Tween 20/PBS) 으로세척후 biotinylated anti-il-6 항체 (R&D) 를 200 ng/ml로, anti-il-17 항체와 anti-tnf-α 항체 (R&D) 를 400 ng/ml로희석하여 100 μl/well씩넣어실온에서 2시간반응시킨후세척하였다. 마지막으로는 Extravidin-Alkaline phosphatase conjugate (Sigma) 를 1:2,000으로희석하여 100 μl/well씩넣고실온에서 2시간반응시키고세척후 phosphate diso-

메토트렉세이트에의한류마티스관절염의치료효과 97 dium salt hexahydrate (PNPP, Fluka)/Diethanolamine 용액 (DEA, 97 Ml, NaN3 0.

통계적유의성의검증실험결과는평균 ± 표준오차로나타냈으며, 통계적유의성은 Student s t-test를실시하였고 p값이 0.05 이하일때통 계적으로유의하다고분석하였다.

콜라겐 1차주입후 7일째일주일에 3번메토트렉세이트를복강투여한치료군과 PBS만투여한대조군의관절염중증도를평가하였다. 콜라겐유발관절염모델은 1차 Figure 1.

Arthritis was induced by immunization with CII in Freund s complete adjuvant on day 0.

(A) Severity of arthritis was determined as described in Materials and Methods.

4 메토트렉세이트에의한류마티스관절염의치료효과 97 dium salt hexahydrate (PNPP, Fluka)/Diethanolamine 용액 (DEA, 97 Ml, NaN3 0.2 g, MgCl2H2O 0.1 g, 1차증류수 800 ml) 을 1 mg/ml 농도로녹여 100 μl/well씩넣어 30분후 0.2 M NaOH로반응을멈추고 405 nm 파장에서흡광도를측정하였다. 통계적유의성의검증실험결과는평균 ± 표준오차로나타냈으며, 통계적유의성은 Student s t-test를실시하였고 p값이 0.05 이하일때통 계적으로유의하다고분석하였다. 결과콜라겐유발관절염동물모델에서메토트렉세이트의치료효과메토트렉세이트의관절염치료효과를평가하기위해제2 형콜라겐을이용하여콜라겐유발관절염마우스를확립하였다. 콜라겐 1차주입후 7일째일주일에 3번메토트렉세이트를복강투여한치료군과 PBS만투여한대조군의관절염중증도를평가하였다. 콜라겐유발관절염모델은 1차 Figure 1. Suppression of arthritis development in MTX-treated CIA mice. Arthritis was induced by immunization with CII in Freund s complete adjuvant on day 0. On day 7, mice also received PBS or MTX (1 mg/kg or 7.5 mg/kg) three times per week for 7 weeks. (A) Severity of arthritis was determined as described in Materials and Methods. (B) Representative histological analysis of knee joints and paws was assessed by H & E, toluidine blue, and safranin o staining. Original magnification: 40 and 200 for H&E staining and 200 for toluidine blue and safranin o staining. Values of histological scores of inflammation and cartilage damage were shown in the right panel. (C) Anti-collagen antibody levels in CIA mice. The levels of IgG anti-collagen antibodies were measured by ELISA. Values are expressed as the optical density (O.D.) *p<0.01, p<0.001 compared to the vehicle control mice.

98 박은미외 면역후약 3주부터관절염이발병하기시작하였으며메토트렉세이트 1 mg/kg

5 mg/kg 치료군에서는치료기간동안지수가지속적으로낮은상태를유지하였다 (Figure 1A).

5 mg/kg 치료군의관절조직에서조직학적차이를조사하기위해헤마톡실린-에오신법으로염색하였으며,

관절염유도군의관절은파괴가심하고, 염증부위에면역세포들이침윤, 판누스 (pannus) 형성이나타난반면,

1B). 게다가메토트렉세이트를주입한동물모델의경우에혈청중콜라겐에특이적인 Total IgG 항체뿐만아니라아형인 IgG1과

메토트렉세이트주입에의한콜라겐유발관절염동물모델의염증억제효과염증성사이토카인인 IL-1β, TNF-α, IL-6와

따라서 대조군과치료군의콜라겐유발관절염마우스의관절조직에서대표적인염증성사이토카인인 IL-1β, TNF-α, IL-6와

관절염유도군의관절은침윤된세포내많은양의 IL-1β, TNF-α, IL-6와

메토트렉세이트주입에의한콜라겐유발관절염모델V내염증 TH17 세포및면역조절 T

5 98 박은미외 면역후약 3주부터관절염이발병하기시작하였으며메토트렉세이트 1 mg/kg 치료군에서중증도는비교적낮은상태를유지하였다이에비해메토트렉세이트 7.5 mg/kg 치료군에서는치료기간동안지수가지속적으로낮은상태를유지하였다 (Figure 1A). 치료적효과가가장크게나타난메토트렉세이트 7.5 mg/kg 치료군의관절조직에서조직학적차이를조사하기위해헤마톡실린-에오신법으로염색하였으며, 연골파괴정도를확인하기위하여톨루이딘블루와사프라닌 O 염색을시행하였다. 관절염유도군의관절은파괴가심하고, 염증부위에면역세포들이침윤, 판누스 (pannus) 형성이나타난반면, 메토트렉세이트처리군은관절염유도군에비해관절파괴, 염증정도가적고면역세포들의침윤, 연골파괴및뼈침식이현저히적게관찰되었다 (Figure 1B). 게다가메토트렉세이트를주입한동물모델의경우에혈청중콜라겐에특이적인 Total IgG 항체뿐만아니라아형인 IgG1과 IgG2a의함량이대조군과비교시처치군에서유의적으로감소하였다 (Figure 1C). 메토트렉세이트주입에의한콜라겐유발관절염동물모델의염증억제효과염증성사이토카인인 IL-1β, TNF-α, IL-6와 IL-17는자가면역관절염의병인과관련된중요인자들이다. 따라서 대조군과치료군의콜라겐유발관절염마우스의관절조직에서대표적인염증성사이토카인인 IL-1β, TNF-α, IL-6와 IL-17에특이적인항체를이용하여면역조직화학염색을시행하였다. 관절염유도군의관절은침윤된세포내많은양의 IL-1β, TNF-α, IL-6와 IL-17이분포되어있었으나치료군의관절내에는이러한염증성사이토카인의발현이관찰되지않았다 (Figure 2). 메토트렉세이트주입에의한콜라겐유발관절염모델V내염증 TH17 세포및면역조절 T 세포의조절효과최근염증성자가면역질환의치료패러다임은병인세포를억제하는동시에조절세포의분포및활성을증가시켜면역체계의이상을정상화하는것에초점이맞춰지고있다. 메토트렉세이트에의한치료효과규명을위해처리군의비장내존재하는 TH17 세포와조절 T 세포의분포를조사하기위해면역형광염색을시행하고공초점현미경으로관찰, 분석하였다. 관절염발병군의비장조직상에는많은양의 CD4+IL-17+ 세포의분포가관찰된반면메토트렉세이트치료군에서는현저하게감소되어있었다 (Figure 3). 메토트렉세이트에의한 STAT3/STAT5 의존적인 TH17 세포및면역조절 T 세포의변화 TH17 세포와조절 T 세포의분화는각각 STAT3와 STAT5 Figure 2. Immunohistologies of joints tissues from MTX treated CIA mice. Tissue sections from mice joints of each group were stained with anti-tnf-α, anti-il-1β, anti-il-6, anti-il-17 antibodies or isotype antibodies, respectively. Cells stained with each antibody are shown in brown. Original magnification: 400.

regulatory T cells in MTX-treated CIA mice.

were stained by anti-cd4 (green), anti-cd25

CD4 + IL-17 + T cells were analyzed using laser

Spleens from mice in each group were stained

white, lower), anti-foxp3 (red), phospho-stat3

6 메토트렉세이트에의한류마티스관절염의치료효과 99 Figure 3. Reciprocal regulation of TH17 and Foxp3+ regulatory T cells in MTX-treated CIA mice. Spleen from vehicle and MTXtreated CIA mice were stained by anti-cd4 (green), anti-cd25 (blue), and anti-il-17 (red, upper), anti- Foxp3 (red, lower) antibodies. Populations of CD4 + CD25 + Foxp3 + T cells and CD4 + IL-17 + T cells were analyzed using laser confocal microscopy. Original magnification: 400. The graphs represent the number of positive cells. *p<0.05 compared to the vehicle control mice. Figure 4. STATs-dependent regulation of TH17 and Foxp3+ regulatory T cells in MTX-treated CIA mice. Spleens from mice in each group were stained with antibodies against anti-cd4 (green, upper; white, lower), anti-foxp3 (red), phospho-stat3 (red) or phospho-stat5 (blue). Original magnification: 400. The graphs represent the number of positive cells. Data are expressed as the mean±sd. *p<0.05 compared to the vehicle control mice.

7 100 박은미외 에의존적으로조절된다. 메토트렉세이트처리에의한 TH17 세포의증가및조절 T 세포의감소가이러한주요전사인자들의발현과연관성이있는지보고자 CD4+ T 세포내 STAT3 또는 STAT5의인산화부위를면역형광염색을통해조사하였다. TH17 세포및조절 T 세포의분포와동일한양상으로인산화된 STAT3 (Y705와 S727) 는관절염발병군에서증가하고, 반면인산화된 STAT5 (Y694) 는메토트렉세이트치료군에서증가되어있었다 (Figure 4). 메토트렉세이트에의한염증성사이토카인의생성억제효과 In vivo 상의메토트렉세이트의항염증효과를시험관내에서확인하고자정상마우스의비장세포를 LPS 또는 CD3/CD28 자극하였을때메토트렉세이트에의한염증성사이토카인 TNF-α, IL-6와 IL-17의생성억제효과를조사하였다. TNF-α, IL-6와 IL-17는모두 LPS 또는 CD3/ CD28 자극에의해증가되었으며추가적인메토트렉세이트의처리는증가된 TNF-α, IL-6와 IL-17의생산을억제 하였다 (Figure 5). 고찰본연구결과메토트렉세이트투여가대표적인류마티스관절염동물모델인콜라겐유발관절염동물모델에서대조군과비교하여관절염지수및관절염발병률을낮추고조직학적으로연골파괴, 염증정도를개선시키는등의치료효과가있음을보여주었다. 메토트렉세이트는콜라겐유발관절염마우스의관절조직에서류마티스관절염의병인과밀접한관련이있는염증성사이토카인인 IL-1β, TNF-α, IL-6와 IL-17의발현을억제하였다. 메토트렉세이트투여는콜라겐유발관절염마우스의비장내염증 TH17 세포를감소시키고조절 T 세포를증가시켰으며이는각각염증 TH17 세포와조절 T 세포의대표적인전사인자인 STAT3와 STAT5 의존적임을증명하였다. 또한시험관내에서메토트렉세이트투여는 LPS혹은 anti-cd3/ CD28로자극된정상마우스비장의염증성사이토카인의 Figure 5. Suppressive effect of MTX on production of inflammatory cytokines. Splenocytes of DBA/1J mice were cultured with LPS (100 ng/ml) (A) or anti-cd3 (1 μg/ml)/cd28 (2 μg/ml) (B) in the presence or absence of MTX (0.5 μg/ml) for 24 hours in vitro. TNF-α, IL-6 and IL-17 in the cell supernatant was determined by ELISA. Data are expressed as the mean±sd. *p<0.01, p <0.001 compared to the LPS or CD3/CD28-treated cells.

8 메토트렉세이트에의한류마티스관절염의치료효과 101 합성을억제하였다. 류마티스관절염의병인에 T세포, 그중에서도 CD4+ T세포가중요한역할을담당하고있다는것은주지의사실이다. CD4+ T 세포는 1986년 Mosmann 등이 CD4+ T세포가상반된기능을가지는 TH1/TH2 세포로분화한다는사실을밝힌이후 (14), TH1/TH2 개념이지속되어서다양한자가면역질환의병인을 TH1/TH2 개념에서해석하고자하는많은시도가 2005년 TH17 세포가규명될때까지지속되었다 (15,16). TH17 세포는염증성사이토카인인 IL-17 (IL-17A) 의합성을특징으로하며다양한사이토카인 (IL-6, TGF-β, IL-23, IL-21, IL-1β) 이 TH17 세포의분화및증폭에관련됨이알려져있다 (17,18). IL-17과 TH17 세포는류마티스관절염, 기관지천식, 염증성장질환, 건선등의다양한자가면역질환의발병에중요한역할을한다 (18-20). 특히류마티스관절염의경우 TH17 세포와 TH17 세포에서분비하는 IL-17이질환의발병에결정적인역할을하는것으로밝혀지면서현재 IL-17 억제제가류마티스관절염환자들을대상으로임상실험중이다. TH17 세포와대조적으로면역조절 T 세포는그분화에 Foxp3라는전사인자의발현을특징으로하며면역관용및조절에결정적인역할을하는 CD4+ T 세포의아형이다 (21,22). 그러므로, TH17 세포를감소시키고조절 T 세포를증가시키는물질이류마티스관절염의치료에새로운후보물질이될수있을것이다. 메토트렉세이트는엽산유사체로서다양한류마티스관절염동물모델에서치료효과가입증되었고류마티스관절염환자들을대상으로한 randomized controlled trial에서치료효과및골파괴억제효과가증명되어현재가장많이사용되고있는항류마티스약제이다 (23). 최근병인에근거한표적치료제인항 TNF-α로대표되는생물학적제제가기존의항류마티스약제보다치료효과가우수함이입증되면서류마티스관절염치료의패러다임에큰변화를가지고왔다. 그러나, 생물학적제제들도메토트렉세이트와병용시치료효과및골파괴억제효과가증가함이알려지면서거의모든생물학적제제들도메토트렉세이트와의병용요법이추천되는만큼메토트렉세이트는류마티스관절염치료의근간이되는약제라할수있다. 그러나, 메토트렉세이트의정확한작용기전은아직밝혀지지않았다. 메토트렉세이트가류마티스관절염의핵심병인세포인 TH17 세포에관한연구는거의보고된바없다. Li 등이류마티스관절염환자의말초혈액단핵구를대상으로시험관내에서메토트렉세이트처리시 IL-17의합성이억제됨을보고하였고 (11), 이는메토트렉세이트를투여한류마티스관절염동물모델의비장에서 TH17 세포의감소및비장세포에서 IL-17의합성억제를관찰한본연구결과와일맥상통하는결과라할수있을것이다. 메토트렉세이트가조절 T 세포에미치는영향에관한연구도거의이루어진바없다. Oh 등은정상인의말초혈액 에서 CD4+CD25+ 조절 T 세포를분리하여시험관내에서메토트렉세이트를처리시조절 T 세포의기능에영향이없음을보고하였다 (24). 한편 Kinoshita 등은동물실험에서엽산이결핍된식이섭취시 Foxp3를발현하는조절세포가유의하게감소함을보고하여엽산이조절 T 세포의유지에중요하고주장하였다 (25). 이러한결과들은메토트렉세이트가류마티스관절염동물모델의비장에서조절세포의수를증가시키는본연구결과와상반된다고할수있을것이다. 이러한차이점에관하여아직명확한원인을알수없으며향후추가적인연구가필요할것이다. 본연구에서류마티스관절염동물모델에서 TH17 세포를감소시키고조절 T 세포를증가시킴을보여주었고이는 STAT3/STAT5의발현변화와관련있음을증명하였다. 그러나이러한메토트렉세이트의 TH17/Treg 조절효과가메토트렉세이트가직접적으로 STAT3, STAT5의발현에영향을주는것인지아니면메토트렉세이트의항염증효과에의하여 TH17 세포의분화에영향을주는염증성사이토카인의발현을억제함에의한 2차적인현상인지에관하여는추가연구가필요하다고하겠다. 결론본연구를통하여메토트렉세이트에의한류마티스관절염치료가핵심병인세포인 TH17 세포및조절 T 세포의조절과관련되어있음을보여주었다. 이러한연구는자가면역관절염의면역체계의이해와그치료법을연구하는데중요한초석이될것으로기대된다. 감사의글본연구는보건복지가족부선도형연구중심병원사업의지원에의하여이루어진것입니다 ( 과제고유번호 A092258). 참고문헌 1. Firestein GS, Kelley WN. Kelley's textbook of rheumatology. In: Cannella AC, O'dell JR, eds. Methotrexate, leflunomide, sulfasalazine, hydroxychloroquine, and combination therapies. 8th ed. p. 883, Philadelphia, PA, Saunders Elsevier, Link AA, Kino T, Worth JA, McGuire JL, Crane ML, Chrousos GP, et al. Ligand-activation of the adenosine A2a receptors inhibits IL-12 production by human monocytes. J Immunol 2000;164: Haskó G, Szabó C, Németh ZH, Kvetan V, Pastores SM, Vizi ES. Adenosine receptor agonists differentially regulate IL-10, TNF-alpha, and nitric oxide production in RAW macrophages and in endotoxemic mice. J Immunol 1996;157: Mello SB, Barros DM, Silva AS, Laurindo IM, Novaes GS. Methotrexate as a preferential cyclooxygenase 2 inhibitor in whole blood of patients with rheumatoid arthritis. Rheumatology (Oxford) 2000;39: Chen Z, Laurence A, Kanno Y, Pacher-Zavisin M, Zhu

9 102 박은미외 BM, Tato C, et al. Selective regulatory function of Socs3 in the formation of IL-17-secreting T cells. Proc Natl Acad Sci U S A 2006;103: Durant L, Watford WT, Ramos HL, Laurence A, Vahedi G, Wei L, et al. Diverse targets of the transcription factor STAT3 contribute to T cell pathogenicity and homeostasis. Immunity 2010;32: Yang XP, Ghoreschi K, Steward-Tharp SM, Rodriguez- Canales J, Zhu J, Grainger JR, et al. Opposing regulation of the locus encoding IL-17 through direct, reciprocal actions of STAT3 and STAT5. Nat Immunol 2011;12: Zhou L, Lopes JE, Chong MM, Ivanov II, Min R, Victora GD, et al. TGF-beta-induced Foxp3 inhibits T(H)17 cell differentiation by antagonizing RORgammat function. Nature 2008;453: Elias KM, Laurence A, Davidson TS, Stephens G, Kanno Y, Shevach EM, et al. Retinoic acid inhibits Th17 polarization and enhances FoxP3 expression through a Stat-3/ Stat-5 independent signaling pathway. Blood 2008;111: Mucida D, Park Y, Kim G, Turovskaya O, Scott I, Kronenberg M, et al. Reciprocal TH17 and regulatory T cell differentiation mediated by retinoic acid. Science 2007;317: Li Y, Jiang L, Zhang S, Yin L, Ma L, He D, et al. Methotrexate attenuates the Th17/IL-17 levels in peripheral blood mononuclear cells from healthy individuals and RA patients. Rheumatol Int 2012;32: Fiehn C, Wunder A, Krienke S, Max R, Ho AD, Moehler T. Lack of evidence for inhibition of angiogenesis as a central mechanism of the antiarthritic effect of methotrexate. Rheumatol Int 2005;25: Barnett ML, Kremer JM, St Clair EW, Clegg DO, Furst D, Weisman M, et al. Treatment of rheumatoid arthritis with oral type II collagen. Results of a multicenter, double-blind, placebo-controlled trial. Arthritis Rheum 1998; 41: Mosmann TR, Cherwinski H, Bond MW, Giedlin MA, Coffman RL. Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. J Immunol 1986;136: Harrington LE, Hatton RD, Mangan PR, Turner H, Murphy TL, Murphy KM, et al. Interleukin 17-producing CD4+ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages. Nat Immunol 2005;6: Park H, Li Z, Yang XO, Chang SH, Nurieva R, Wang YH, et al. A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17. Nat Immunol 2005;6: Dong C. Differentiation and function of pro-inflammatory Th17 cells. Microbes Infect 2009;11: Tesmer LA, Lundy SK, Sarkar S, Fox DA. Th17 cells in human disease. Immunol Rev 2008;223: Dong C. IL-23/IL-17 biology and therapeutic considerations. J Immunotoxicol 2008;5: Iwakura Y, Nakae S, Saijo S, Ishigame H. The roles of IL-17A in inflammatory immune responses and host defense against pathogens. Immunol Rev 2008;226: Sakaguchi S. Naturally arising CD4+ regulatory t cells for immunologic self-tolerance and negative control of immune responses. Annu Rev Immunol 2004;22: Sakaguchi S, Wing K, Onishi Y, Prieto-Martin P, Yamaguchi T. Regulatory T cells: how do they suppress immune responses? Int Immunol 2009;21: Kremer JM. Toward a better understanding of methotrexate. Arthritis Rheum 2004;50: Oh JS, Kim YG, Lee SG, So MW, Choi SW, Lee CK, et al. The effect of various disease-modifying anti-rheumatic drugs on the suppressive function of CD4+CD25+ regulatory T cells. Rheumatol Int 2013;33: Kinoshita M, Kayama H, Kusu T, Yamaguchi T, Kunisawa J, Kiyono H, et al. Dietary folic acid promotes survival of Foxp3+ regulatory T cells in the colon. J Immunol 2012;189:

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α α α α α α α α 太陰調胃湯加減方 dbdb 마우스 肝에 대한 아디포사이토카인 및 발현에 미치는 영향 SREBPs 와 섞어서 하고 마커 또한 하여 전기 영동을 하였다 전기영동을 한 후에 에 를 쪼여서 각 를 확인하였다 이 를 프로그램을 이용해 수치화하 여 분석하였다 肝 조직 동결 절편 분리한 조직은 로 시간 동안 고정 시킨 후 에 세척한 후 물기를