이은소 이중선 2009-02 9766 https://aurora.ajou.ac.kr/handle/2018.oak/17555 학위논문(박사)--아주대학교 일반대학원 :의학과,2009. 2 연구배경: T cell immunoglobulin mucin-3 (TIM-3)는 최근 1형 T세포(TH1) 특이 분자로 밝혀졌고 TH1 반응이 우세한 질병의 발병기전 및 면역 반응을 조절하는 역할을 한다고 알려졌다. 증가된 TH1 면역반응은 만성 전신정 염증성 질환인 베체트병의 주요한 발병요인 중의 하나이다. 하지만 아직까지 베체트병에서 TIM-3의 역할에 대한 연구는 되어있지 않다. 연구목적: 이 연구는 베체트병에서 TIM-3분자의 세포표면 발현, TIM-3단백질의 발현 및 mRNA의 발현을 말초혈액단핵구에서 확인하고 또한 질병활성도에 따른 발현 양상을 알아보고자 했다. 재료 및 방법: 베체트병 환자 (67명), 정상 대조군 (13명), 건선 환자 (14명)를 대상으로 하였다. 베체트병 환자는 질병의 활성도에 따라 활동성과 비활동성으로 구분하였다. 유세포분석기를 통해 한국인 베체트병의 면역학적 양상을 알아보고, 세포표면의 TIM-3, CD4, CD8, CD11b 및 CD56의 발현을 알아보았다. 단백질 수준의 TIM-3발현 분석은 웨스턴블롯팅을 이용하였다. TIM-3 발현은 환자군과 대조군에서 그리고 같은 환자에서 질병이 활동성일 때와 비활동성일 때를 비교하였다. 중합효소연쇄반응을 통하여 TIM-3 전사수준의 발현을 분석하였다. 베체트병 환자의 피부 병변에서는 면역조직화학적으로 TIM-3의 발현을 살펴보았다. 결과: 베체트병 활성지표인 적혈구침강속도와 C반응성단백은 활동성 베체트병 환자에서 비활동성 보다 높았다. 세포 표면 TIM-3 발현은 정상에 비하여 베체트병 환자에서 현저하게 높았고 비활동성 환자에서 활동성 환자보다 높은 경향을 보였다. 환자의 피부병변의 침윤된 염증세포에서 TIM-3가 발현됨을 확인하였고 연속된 조직 슬라이드에서 CD4+세포가 같은 위치에서 확인되었다. 웨스턴블랏팅에서도 단백질 수준의 TIM-3가 정상보다 환자에서 더 많이 발현되었다. 또한 같은 환자에서도 질병활성도에 따라 TIM-3 발현에 차이를 보였는데, 비활동성 질환 상태일 때 활동성 상태보다 높은 발현을 보였다. 말초혈액단핵구 중에서 CD8양성 세포만 베체트병 환자에서 정상보다 낮았으며, CD8, CD56세포에서 TIM-3의 발현은 베체트병 환자에서 정상보다 높았다. 하지만 TIM-3발현 수준은 CD4세포에서의 TIM-3 발현 정도와 가장 높은 상관관계를 보였다. 말초혈액 단핵구를 자극 후 시간에 따른 TIM-3 발현에는 변화가 없었고, CD4, CD8세포의 TIM-3발현능에도 자극 시간에 따른 변화는 보이지 않았다. 또한 TIM-3 mRNA 수준의 발현도 활동성 환자에서 정상에 비해 높게 나타났고, 같은 환자에서 질환이 비할동성일 때 보다 활동성 일 때 TIM-3 mRNA 가 높게 발현함을 확인하였다. 결론: 이 연구는 베체트병에서 TIM-3의 발현을 세포표면, 단백질 수준 및 유전자 수준에서 확인하였고 질병 활성도에 따라 TIM-3발현에 차이가 있음을 확인하였으며 이는 TIM-3가 면역학적 반응을 조절하여 베체트병의 발병에 관계가 있을 가능성을 제시한다. TABLE OF CONTENTS ABSTRACT-------------------------------------------- ⅰ TABLE OF CONTENTS--------------------------------- ⅳ LIST OF FIGURES-------------------------------------- ⅵ LIST OF TABLES---------------------------------------ⅷ I. INTRODUCTION---------------------------------------1 II. MATERIALS AND METHODS-------------------------- 8 A. Patients and samples------------------------------ 8 B. Methods-------------------------------------------8 1. Cell preparation-----------------------------------9 2. Cell culture and stimulation of PBMCs ------------- 9 3. Flow cytometric analysis--------------------------10 4. Reverse transcription polymerase chain reaction (RT-PCR)---------------------------------------- 11 5. Immunohistochemistry----------------------------12 6. Western blot analysis-----------------------------13 7. Statistical analysis------------------------------- 14 III. RESULTS-------------------------------------------15 A. Subjects characteristics--------------------------- 15 B. TIM-3 expression in PBMCs and the effect of medication --------------------------------------- 20 C. Western blot analysis of TIM-3 protein --------------22 D. Immunohistochemical analysis of TIM-3 in erythema nodosum-like skin lesions of Behçet’s diseases ---- 23 E. Correlation of TIM-3 expression with Behcet’s disease activity ------------------------------------------- 26 F. Analysis of surface markers on PBMCs and TIM-3 expression on leukocyte subpopulations-------------27 G. Correlation of TIM-3+ frequency in leukocyte subpopulation with Behcet’s disease- activity ------- 31 H. Expression of TIM-3 in stimulated PBMCs between Behçet’s patients and healthy controls -------------- 32 I. Expression of TIM-3 mRNA was analyzed by RT-PCR between Behçet’s disease group and healthy controls- ---------------------------------------------------34 J. Expression of TIM-3 mRNA was analyzed by RT-PCR after stabilization of clinical symptoms of Behçet’s disease -------------------------------------------36 IV. DISCUSSION----------------------------------------38 V. CONCLUSION---------------------------------------45 REFERENCES----------------------------------------- 46 국문요약 ---------------------------------------------- 57|LIST OF FIGURES Fig. 1. Schematic representation of human TIM protein structures--------------------------------------- 4 Fig. 2. Mechanisms by which Tim-3 might modulate immune function--------------------------------- 7 Fig. 3. The level of disease activity makers of Behçet’s disease ----------------------------------------18 Fig. 4. Cytokine profile in PBMCs from Behçet’s disease patients ex vivo ---------------------------------19 Fig. 5. Expression of TIM-3 in PBMCs -------------------20 Fig. 6. The effect of medications on the TIM-3 expression by FACS analysis -------------------------------21 Fig. 7. The representative result of expression of TIM-3 protein in PBMCs from BD patients by Western blotting----------------------------------------- 22 Fig. 8. TIM-3 expression in cutaneous lesion ------------24 Fig. 9. TIM-3 expression in CD4+ cells in EN-like lesion of Behçet’s disease-------------------------------- 25 Fig. 10. The comparison of frequcncy of TIM-3 expression between active disease and stable state in the same patient -----------------------------------26 Fig. 11. Analysis of surface markers on PBMCs and TIM-3 expression on leukocyte subpopulations -------- 29 Fig. 12. Correlation between TIM-3 frequency and TIM-3 expression in leukocyte subpopulations --------- 30 Fig. 13. The comparison of TIM-3 expression on each cellular population of PBMCs between active and stable disease state in the same patient --------- 31 Fig. 14. TIM-3 expression on stimulated PBMCs--------- 33 Fig. 15. Expression of TIM-3 in PBMCs assessed by RT- PCR ------------------------------------------ 34 Fig. 16. TIM-3 mRNA was analyzed according to the disease state ---------------------------------- 36 |LIST OF TABLES Table 1. Clinical characteristics of Behçet’s disease patients--------------------------------------- 16 Table 2. Clinical characteristics and laboratory results of Behçet’s disease patients according to disease activity-----------------------------------------17 Table 3. Pearson correlations between the expression level of TIM-3 and TIM-3 frequency in PBMCs subpopulation ---------------------------------27 eng The Graduate School, Ajou University 아주대학교 논문은 저작권에 의해 보호받습니다. 베체트병 활성도에 따른 TIM-3의 발현 양상 Joong Sun Lee Thesis 아주대학교 일반대학원 Joong Sun Lee 일반대학원 의학과 2009. 2 Master http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000009766 Behçet’s disease Disease activity T cell immunoglobulin and mucin (TIM)-3 Background: T cell immunoglobulin mucin-3 (TIM-3) is recently described as a TH1-associated cell surface molecule that regulates TH1 responses and promotes tolerance in mice. Increased TH1 immune response has been known as one possible pathogenesis of a chronic inflammatory multisystemic disorder, Behçet's disease (BD). However, TIM-3 expression and function in BD has not been investigated. Purpose: In this study, its was examined that the surface expression of TIM-3, the expression of TIM-3 protein and the TIM-3 mRNA in peripheral blood mononuclear cells from Behçet's disease patients, and evaluated the TIM-3 expression pattern according to clinical disease activity. Methods: Behçet's patients (n=67), healthy control (n=13) and psoriasis patients (n=14) were involved. Immunologic profile of Korean patients, especially in relation to T cell immunity was examined by flow cytometry. The expression of TIM-3 as well as CD4, CD8, CD11b and CD56 in peripheral blood mononuclear cells (PBMC) was assessed by flow cytometry. BD group was divided according to the disease activity. TIM-3 expression was also compared between BD in active state and BD in remission state. Western blot analysis was performed to evaluated TIM-3 protein. TIM-3 expression after simulation of PBMCs was analyzed by flow cytometry. At the transcript level, the expression of TIM-3 was assessed by quantitative RT-PCR. The immunohistochemical analysis was done in cutaneous lesion with anti-TIM-3 antibody and anti-CD4 antibody. Results: Disease activity markers, ESR and CRP, were elevated in the blood samples from active BD compared to inactive BD. The surface expression of TIM-3 molecule was significantly upregulated in Behçet's disease patients compared with healthy controls by flow cytometry and the stable BD had a tendency to show higher TIM-3 than the active BD. The immunohistochemical study in cutaneous lesion of BD showed the co-localization of TIM-3+ cells and CD4+ cells in inflammatory site. Western blot analysis also showed the upregulated expression of TIM-3 protein in BD compared to control. Even in each patient, according to his disease activity, the expression of TIM-3 was changed, showing a higher TIM-3 expression in remission state than in active state. In leukocytes subpopulation, there were no significant differences in CD4, CD11b, and CD56 cells except CD8 cells decreased in BD and psoriasis. The mean expression of TIM-3 molecule in CD8+ and CD56+ cells was significantly increased in BD compared with controls. However the correlation between TIM-3 frequency and TIM-3 expression in CD4 cells was highest among leukocyte subpopulations. The stimulation of PBMC with anti-CD3, CD28 antibody in presence of IL-2, could not up-regulate the TIM-3 expression in BD patients with simulation time and TIM-3 expression in CD4+ and CD8+ cells were not changed after stimulation, too. The transcription level analysis of PBMCs from BD patients revealed significantly higher mRNA expression of TIM-3 compared with controls. Moreover, in the same patient, TIM-3 mRNA expression was significantly altered according to disease activity, increasing TIM-3 mRNA in active state compared with remission state. Conclusion: This study may imply the differential expression of human TIM-3 molecules by the PBMCs of TH1-driven Behçet's disease according to disease activity and suggest that there were altered kinetics in the expression of TIM-3 molecule and TIM-3 mRNA in PBMCs that might modulate immunologic response in Behçet’s disease.