대한임상병리학회지 : 제 19 권제 2 호 1999 Korean J Clin Pathol 1999; 19: 234-8 진단면역학 전신성홍반성낭창환자에서혈청내 sfas 및 sfas Ligand 측정 신정원 김현숙 송정식 * 이수곤 * 연세대학교의과대학임상병리과학교실, 내과학교실 * Determinations of Soluble Fas and Soluble Fas Ligand in Patients with Systemic Lupus Erythematosus Jeong Won Shin, M.D., Hyon-Suk Kim, M.D., Jeongsik Song, M.D.*, and Soo Kon Lee, M.D.* Departments of Clinical Pathology and Internal Medicine*, Yonsei University College of Medicine, Seoul, Korea Background : The Fas/Fas ligand (FasL) system plays an important role in apoptosis by involvement in various immunologic functions, especially the removal of autoreactive and activated T- cells. sfas is a variant of the Fas receptor molecule, which lacks the transmembrane domain by alternative splicing of Fas mrna and has an inhibitory effect in apoptosis by inhibition of the Fas/FasL pathway. sfasl is a coverted form of FasL by metalloproteinase and is increased in various malignant and autoimmune diseases. In this study, we investigated the expression of sfas and sfasl in systemic lupus erythematosus (SLE) and evaluated their usefulness as markers of disease activity. Methods : The concentration of sfas and sfasl in sera from 43 patients with SLE, 17 with rheumatoid arthritis (RA) and 15 normal healthy persons were measured using sfas (S) ELISA Kit and sfas Ligand ELISA Kit (MBL Co., LTD., Nagoya, Japan), respectively. Twenty of 43 SLE sera were paired samples of 10 patients obtained on admission and discharge. Results : The concentration of sfas in SLE (3.12±2.28 ng/ml) was significantly higher than in RA (2.23±0.37 ng/ml) and in the normal control (2.12±0.33 ng/ml). In particular, the concentration of sfas in sera on admission (4.35±3.68 ng/ml) was significantly higher than in the sera on discharge (2.89±0.66 ng/ml), but, the concentration of sfasl among the 3 groups was not statistically different. Conclusions : These results suggest that apoptosis is involved in the pathogenesis of SLE and sfas might be a useful marker as a predictor of disease activity. Further study on the correlation between sfas and other disease activity markers, such as CRP, CH50, CD4 cell count and autoantibody titer is needed. Also, the evalution of sfas as a predictor of disease progression on follow-up studies of these patients is needed. (Korean J Clin Pathol 1999; 19: 234-8) Key words : Apoptosis, sfas, sfas ligand, Systemic lupus erythematosus (SLE) 서 론 Fas/APO-1/CD95 및 Fas ligand (FasL) 는여러가지면역 접수 : 1998 년 10 월 12 일접수번호 : KJCP1228 수정본접수 : 1998 년 11 월 9 일교신저자 : 김현숙우 135-720 서울시강남구도곡동 146-92 영동세브란스병원임상병리과전화 : 02-3497-3531, Fax : 02-3462-9483 기능과조절에관여하여 apoptosis에중요한역할을하며, 특히자가반응성 T 세포및활성화된 T 세포의제거에관여한다 [1-3]. Soluble Fas (sfas) 는 Fas 유전자의 alternative splicing에의해 transmembrane domain을포함하는 21개의아미노산이제거된형태로서, Fas-FasL 결합의억제인자로작용함으로써 apoptosis를억제하는것으로알려져있다 [4-6]. 또한 soluble FasL (sfasl) 는세포표면의 FasL가 metalloproteinase에의해혈청으로유리된형태로서, 여러악성종양과자가면역질환에서 234
전신성홍반성낭창환자에서의혈청내 sfas 및 sfas ligand 측정 235 증가하여조직손상을매개하는것으로보고되어있다 [7-9]. 본연구에서는자가면역질환중특히 apoptosis가발병기전에중요한역할을하는것으로알려져있는전신성홍반성낭창 (systemic lupus erythematosus, SLE) 환자혈청에서 sfas 및 sfasl의발현양상을살펴보고, 질병활성도예측에있어서의유용성을평가하고자하였다. 대상및방법 1. 대상 American College of Rheumatology (ACR) 의개정기준 [10] 에따라 SLE 로진단된환자의혈청 43 검체와역시 ACR revised criteria[11] 를만족하는류마티스관절염 (rheumatoid arthritis, RA) 환자혈청 17 검체및정상건강인 15명의혈청을대상으로하였다. SLE 환자검체중 20 검체는 10명의환자의입원시와퇴원시에얻은쌍검체였다. Fas ligand 항체가 coating되어있는 microplate well에희석액으로 2배희석한환자혈청 100 L를넣고실온에서 60분간반응시킨후, peroxidase가 conjugation 되어있는단클론성항 Fas ligand 항체 100 L를넣고역시실온에서 60분간반응시켰으며, 여기에기질액 100 L를첨가하여실온에서 30분간반응시킨후반응정지액 100 L를넣고 450 nm에서흡광도를측정하였다. 검사시 7가지농도 (5, 2.5, 1.25, 0.625, 0.3125, 0.15625, 0 ng/ml) 의표준물질을함께시행하여표준곡선를구하였으며, 이를이용하여환자혈청내의 sfasl 농도를계산하였다. 3. 통계분석 SLE와 RA 및정상대조군간의 sfas와 sfasl 값비교를위해 t-test 를이용하였으며, P < 0.05를통계학적유의수준으로하였다. 결과 2. 방법 1. 각군간의 sfas 값비교 1) sfas 측정 sfas (S) ELISA Kit (MBL Co., LTD., Nagoya, Japan) 를이용하여제조사의지시된방법에따라시행하였다. 검사방법을간단히요약하면, 다클론성항 Fas 항체가 coating되어있는 microplate well에검체희석액으로 5배희석한환자혈청 150 L를넣고실온에서 60분간반응시킨후, peroxidase가 conjugation 되어있는단클론성항 Fas 항체 100 L를넣고역시실온에서 60분간반응시켰으며, 여기에기질액 100 L를첨가하여실온에서 30분간반응시킨후반응정지액 100 L를넣고 450 nm에서흡광도를측정하였다. 검사시 8가지농도 (2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, 0 ng/ml) 의표준물질을함께시행하여표준곡선을구하였으며, 이를이용하여환자혈청내의 sfas 농도를계산하였다. 2) sfasl 측정 sfas Ligand ELISA Kit (MBL Co., LTD., Nagoya, Japan) 을이용하여다음과같이시행하였다. 즉, 단클론성항 sfas값 (Mean±SD) 은 SLE 환자군에서 3.12±2.28 ng/ml 로 RA 환자군의 2.23±0.37 ng/ml나정상대조군의 2.12±0.33 ng/ml보다유의하게증가되어있었으며, 특히 10명의환자에서얻은입원시와퇴원시의쌍검체를비교해본결과입원시혈청의 sfas가 4.35±3.68 ng/ml로퇴원시의 2.89±0.66 ng/ml 보다 sfas (ng/ml) 10 8 6 4 2 0 SLE Fig. 1 The distributions of serum sfas in SLE, RA and normal controls. RA NC Table 1. Serum levels of sfas and sfasl in systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and normal controls (NC) on admission SLE on discharge RA NC sfas 4.35 ± 3.68* ng/ml 2.89 ± 0.66 ng/ml 2.23 ± 0.37 ng/ml 2.12 ± 0.33 ng/ml sfasl 0.42 ± 0.01 ng/ml 0.43 ± 0.00 ng/ml 0.39 ± 0.05 ng/ml 0.46 ± 0.00 ng/ml * mean ± SD.
236 신정원 김현숙 송정식외 1 인 유의하게높은값을보였다. 각군에서의 sfas 분포는 Fig. 1과같다. RA와정상대조군간에는통계학적으로유의한차이가없었다 (Table 1). 2. 각군간의sFasL 값비교 sfasl 값 (Mean±SD) 은 SLE 환자군에서 0.42±0.01 ng/ ml, RA에서 0.39±0.05 ng/ml, 그리고정상대조군에서는 0.46±0.00 ng/ml로각군간에유의한차이가없었으며, SLE 환자중입원시와퇴원시검체간에도통계학적으로유의한차이가없었다. 고찰 Apoptosis란염색질의응축현상 (chromatin condensation) 과 DNA strand의분절및 apoptotic body 형성등을특징으로하는능동적인세포사멸현상을말하며, 1972년 Kerr에의해처음기술된후여러가지질환의발병기전및경과에중요한역할을하는것으로알려져왔다 [12]. Fas/APO-1/CD95는 tumor necrosis factor (TNF)/nerve growth factor receptor family에속하는 45 kda의 I형막단백으로서흉선이나간, 난소, 폐등의다양한조직에서발현되며 [7, 13-14], 항 Fas 항체나 FasL와결합함으로써 apoptosis를유발한다 [15-17]. Fas 분자의 soluble form인 sfas는 Fas mrna의 alternative splicing에의해 Fas cdna의 700 base pair (bp) 에서 762 bp까지 63개의염기서열이제거된형태로서제거된염기서열중에 Fas 유전자의 transmembrane domain이포함되기때문에 secreted, soluble form으로발현되며, Fas-FasL 결합시억제인자로작용한다 [4, 6, 18]. 따라서 sfas는특히 SLE 등의자가면역질환에서증가하여자가반응성 T 세포의 apoptosis 를억제함으로써이질환의발병기전에중요한역할을하며 [4, 6], SLE의질병활성지수 (disease activity index)[19] 와도유의한상관성이있는것으로알려져있다 [20-22]. 그러나, Goel 등 [23] 은 SLE와 RA 등자가면역질환환자들에서의 sfas 값은정상대조군과통계적으로유의한차이가없었으며, 항 dsdna 항체나 CH50, erythrocyte sedimentation rate (ESR) 및 rheumatoid factor (RF) 등질병활성도를반영하는다른검사종목과도상관성이없었다고보고하였으며, Knipping 등 [24] 의연구에서도 SLE와 juvenile rheumatoid arthritis (JRA) 환자혈청의 sfas 값이정상대조군과통계학적으로차이가없었다. 본연구에서는 SLE 환자혈청에서의 sfas 값이 RA나정상대조군에서보다유의하게증가되어있었고, 특히 SLE 환자의입원시와퇴원시쌍검체비교에서입원시의값이퇴원시보다유의하게증가되어있어 sfas가질병활성도예측에도움을줄수있을것으로생각되었다. FasL는 TNF family에속하는 40 kda의 II형막단백이며 [13, 15, 25], sfasl는 metalloproteinase-like enzyme에의해 FasL 가전환된 26 kda의단백으로 natural killer cell lymphoma나 large granular lymphocytic leukemia 등에서증가되어 apoptosis에의한조직손상을매개하는것으로알려져있다 [8, 26, 27]. Nozawa 등 [22] 은 sfas가증가되어있는 SLE와 RA 환자에서역시 sfasl도증가되어있었다고보고하였으며, 이에따라 sfasl도 sfas와마찬가지로 Fas-FasL 매개성 apoptosis를 downregulation하는억제인자로간주하였다. 그러나, 본연구에서의 sfasl 값은 SLE와 RA, 그리고정상대조군간에통계학적으로유의한차이가없어 sfas에비해자가면역질환의질병활성도예측인자로서의유용성은떨어지는것으로생각되었다. 한편 sfasl 값이 SLE나 RA, 그리고정상대조군모두에서표준물질중가장낮은농도인 0.15625 ng/ml 보다낮았기때문에측정이다소부정확했을가능성을배제할수없었고, 따라서표준곡선의범위조정등을포함한보다많은연구가필요할것으로생각되었다. 결론적으로, SLE 환자에서 sfas는 RA나정상대조군에비하여유의하게증가되어있어이질환의발병기전에 apoptosis가관여함을시사하였으며, SLE의질병활성도를예측하고치료효과를판정하는데유용하게이용될수있을것으로생각되었다. 앞으로 sfas와함께 SLE의질병활성도를반영할수있는 C-반응성단백 (C-reactive protein, CRP) 과 CH50 등보체활성도및 CD4 양성세포수, 그리고다른자가항체와의상관성등에관한연구가더필요할것으로생각되었고, 이런환자들의추적관찰을통해 sfas의예후판정인자로서의유용성도함께검토되어야할것으로사료되었다. 요약배경 : Fas 및 FasL는여러가지면역기능과조절에관여하여 apoptosis에중요한역할을하며, 특히자가반응성 T 세포및활성화된 T 세포의제거에관여한다. sfas는 Fas 유전자의 alternative splicing에의해 transmembrane domain을포함하는 21개의아미노산이제거된형태로서, Fas-FasL 결합시억제인자로작용하여 apoptosis를억제하며, sfasl는세포표면의 FasL 가 metalloproteinase에의해유리된형태로서여러악성질환및자가면역질환에서증가되는것으로보고되어있다. 본연구에서는 SLE 환자혈청에서 sfas 및 sfasl의발현양상을살펴보고, 질병활성도예측에있어서의유용성을평가하고자하였다. 방법 : SLE 환자혈청 43 검체, RA 17 검체및정상인 15명의혈청을대상으로하였다. SLE 환자중 20 검체는 10명의환자의입원시와퇴원시에얻은쌍검체였다. sfas와 sfasl 측정은각각 sfas (S) ELISA Kit와 sfas Ligand ELISA Kit (MBL Co., LTD., Nagoya, Japan) 를이용하였다. 각군간의 sfas와 sfasl 값비교를위해서는 t-test를이용하였다.
전신성홍반성낭창환자에서의혈청내 sfas 및 sfas ligand 측정 237 결과 : sfas는 SLE 환자군에서 3.12±2.28 ng/ml로 RA 환자군의 2.23±0.37 ng/ml이나정상대조군의 2.12±0.33 ng/ml 보다유의하게증가되어있었으며, 특히입원시혈청의 sfas는 4.35±3.68 ng/ml로퇴원시의 2.89±0.66 ng/ml보다유의하게높은값을보였다. sfasl의경우 SLE에서 0.42±0.01 ng/ml, RA에서 0.39±0.05 ng/ml, 정상대조군에서 0.46±0.00 ng/ml 로각군간에유의한차이가없었다. 결론 : sfas는 SLE 환자에서유의하게증가되어있어이질환의발병기전에 apoptosis가관여함을시사하였다. 또한입원시 sfas 값이퇴원시보다증가되어있어 sfas가 SLE의질병활성도를예측하는데유용하게이용될수있을것으로생각되었다. 앞으로 sfas와함께 SLE의질병활성도를반영할수있는 CRP 와 CH50 및 CD4 양성세포수, 그리고다른자가항체와의상관성등에관한연구가더필요할것으로생각되었으며, 이런환자들의추적관찰을통해 sfas의예후판정인자로서의유용성도검토되어야할것으로사료되었다. 참고문헌 1. Suzuki N, Ichino M, Mihara S, Kaneko S, Sakane T. Inhibiton of Fas/Fas ligand-mediated apoptotic cell death of lymphocytes in vitro by circulating anti-fas ligand autoantibodies in patients with systemic lupus erythematosus. Arthritis Rheum 1998; 41: 344-53. 2. Ettinger R, Panka DJ, Wang JK, Stanger BZ, Ju ST, Marshak RA. Fas ligand-mediated cytotoxicity is directly responsible for apoptosis of normal CD4+ T cells responding to a bacterial superantigen. J Immunol 1995; 154: 4302-8. 3. Hanabuchi S, Koyanagi M, Kawasaki A, Shinohara N, Matsuzawa A, Nishimura Y, et al. Fas and its ligand in a general mechanism of T- cell-mediated cytotoxicity. Proc Natl Acad Sci USA 1994; 91: 4930-4. 4. Cheng J, Zhou T, Liu C, Shapiro JP, Brauer MJ, Kiefer MC, et al. Protection from Fas-mediated apoptosis by a soluble form of the Fas molecule. Science 1994; 263: 1759-62. 5. Knipping E, Krammer PH, Onel KB, Lehman TJ, Mysler E, Elkon KB. Levels of soluble Fas/APO-1/CD95 in systemic lupus erythematosus and juvenile rheumatoid arthritis. Arthritis Rheum 1995; 38: 1735-7. 6. Jodo S, Kobayashi S, Kayagaki N, Ogura N, Feng Y, Amasaki Y, et al. Serum levels of soluble Fas/APO-1 (CD95) and its molecular structure in patients with systemic lupus erythematosus (SLE) and other autoimmune diseases. Clin Exp Immunol 1997; 107: 89-95. 7. Tanaka M, Suda T, Haze K, Nakamura N, Sato K, Kimura F, et al. Fas ligand in human serum. Nat Med 1996; 2: 317-22. 8. Tanaka M, Takashi S, Takahashi T, Nagata S. Expression of the functional soluble form of human Fas ligand in activated lymphocytes. EMBO J 1995; 14: 1129-35. 9. Hashimoto H, Tanaka M, Suda T, Tomita T, Hayashida K, Takeuchi E, et al. Soluble Fas ligand in the joints of patients with rheumatoid arthritis and osteoarthritis. Arthritis Rheum 1998; 41: 657-62. 10. Tan EM, Cohen AS, Fries JP, Masi AT, McShane DJ, Rothfield NF, et al. The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 1982; 25: 1271-7. 11. Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS, et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988; 31: 37-43. 12. Berke G. The Fas-based mechanism of lymphocytotoxicity. Human Immunol 1997; 54: 1-7. 13. Nagata S and Golstein P. The Fas death factor. Science 1995; 267: 1449-56. 14. Smith CA, Farrah T, Goodwin RG. The TNF receptor superfamily of cellular and viral proteins: Activation, costimulation, and death. Cell 1994; 76: 959-62. 15. Suda T, Takahashi T, Golstein P, Nagata S. Molecular cloning and expression of the Fas ligand: A novel member of the tumor necrosis factor family. Cell 1993; 75: 1169-78. 16. Trauth BC, Klas C, Peters AMJ, Matzuku S, Moller P, Falk W, et al. Monoclonal antibody-mediated tumor regression by induction of apoptosis. Science 1989; 245 :301-5. 17. Yonehara S, Ishii A, Yonehara M. A cell-killing monoclonal antibody (anti-fas) to a cell surface antigen co-down regulated with the receptor of tumor necrosis factor. J Exp Med 1989; 148: 1274-9. 18. Lee SH, Kim SY, Lee JY, Shin MS, Dong SM, Na EY, et al. Detection of soluble Fas mrna using in situ reverse transcription-polymerase chain reaction. Lab Invest 1998; 78: 453-9. 19. Bombardier C, Gladman DD, Urowitz MB, Caron D, Chang CH, and the Committee on Prognosis Studies in SLE: Derivation of the SLEDAI: a disease activity index for lupus patients. Arthritis Rheum 1992; 35: 630-40. 20. Rose LM, Latchman DS, Isenberg DA. Elevated soluble fas production in SLE correlates with HLA status not with disease activity. Lupus 1997; 6: 717-22. 21. Tokano Y, Miyake S, Kayagaki N, Nozawa K, Morimoto S, Azuma M, et al. Soluble Fas molecule in the serum of patients with systemic lupus erythematosus. J Clin Immunol 1996; 16: 261-5. 22. Nozawa K, Kayagaki N, Tokano Y, Yagita H, Okumura K, Hashimoto H, et al. Soluble Fas (APO-1, CD95) and soluble Fas ligand in rheumatic diseases. Arthritis Rheum 1997; 40: 1126-9. 23. Goel N, Ulrich DT, Clair EW, Fleming JA, Lynch DH, Seldin MF. Lack of correlation between serum soluble Fas/APO-1 levels and autoimmune disease. Arthritis Rheum 1995; 38: 1738-43. 24. Knipping E, Krammer PH, Onel KB, Lehman TJ, Mysler E, Elkon
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