中文摘要
研究背景: 類風濕關節炎是一種慢性關節炎，可導致關節的破壞及變形。發炎性細胞激素如腫瘤壞死因子α與白細胞介素6與其病理機制有高度相關。CD4與CD8 T細胞、B細胞、呼吸道上皮細胞、肥胖細胞及被細胞激素活化的纖維母細胞皆可分泌白細胞介素16來做為強力吸引T細胞的趨化激素。在明顯發炎的類風濕關節炎患者的關節液中，白細胞介素16的數值已被證實是升高的。類風濕關節炎病人血中的免疫球蛋白G會誘發其本身的纖維母細胞產生白細胞介素16，而此種現象是經由免疫球蛋白G與胰島素樣生長因子1受體結合而產生。此外，T細胞上也有胰島素樣生長因子1受體。本實驗是用來評估在類風濕關節炎與退化性關節炎的病人，血清中的白細胞介素16的數值是否有差異及類風濕關節炎病人血清中的白細胞介素16的數值是否與其疾病嚴重程度或其餘發炎性細胞激素有相關。而胰島素樣生長因子1受體在刺激T細胞分泌腫瘤壞死因子α及纖維母細胞分泌白細胞介素6與基質金屬蛋白脢3的角色也將進一步釐清。
研究方法: 我們以酶聯免疫吸附測定法來測量在39位類風濕關節炎與39退化性關節炎病人血清中的白細胞介素16的數值。在30位類風濕關節炎的病人，白細胞介素16與臨床疾病狀況的參考項目如紅血球沉降速率、C反應性蛋白、類風濕因子、疼痛關節數、腫脹關節數及DAS28，在一開始及3個月後各評估一次。白細胞介素16與臨床疾病狀況的參考項目的相關係數以橫斷及追蹤式研究來評估。橫斷式研究採用第一次檢查的結果，而追蹤式研究則分析白細胞介素16與臨床疾病狀況的參考項目三個月間的變化。我們也測量在21位類風濕關節炎的病人中的腫瘤壞死因子α及在15位類風濕關節炎的病人中的白細胞介素6與其臨床疾病狀況的參考項目，相關係數的評估方式與白細胞介素16相同。我們同時評估白細胞介素16的變化與腫瘤壞死因子α及白細胞介素6變化間的相關係數及白細胞介素16與胰島素樣生長因子1受體及其抗體結合強度間的相關係數。 我們使用流式細胞儀來收集T細胞，然後使用類風濕關節炎病人的血清予以刺激，並以抗胰島素樣生長因子1受體的阻斷性單株抗體來評估胰島素樣生長因子1受體在T細胞分泌腫瘤壞死因子α的重要性。我們也使用類風濕關節炎病人的血清來刺激纖維母細胞，測試是否能促使其製造基質金屬蛋白脢3或白細胞介素6。假如基質金屬蛋白脢3或白細胞介素6的製造會增加，我們會使用胰島素樣生長因子1受體的阻斷性單株抗體或是移除胰島素樣生長因子1受體的血清抗體來測試是否能阻斷其製造。
研究結果: 在39位類風濕關節炎與39位退化性關節炎的病人間，血清的白細胞介素16並沒有差別 (P=0.799)。在橫斷式研究中，白細胞介素16與疾病狀況的參考項目沒有相關。在追蹤式研究中，白細胞介素16的變化與紅血球沉降速率 (r=0.185, P=0.328)、 C反應性蛋白(r=0.206, P=0.274)、類風濕因子 (r=-0.129, P=0.496)、疼痛關節數 (r=0.226, P=0.221)、腫脹關節數 (r=0.068, P=0.715)及DAS28 (r=0.237, P=0.208)等的變化皆無相關。而不管在橫斷式或追蹤式的研究，腫瘤壞死因子α或白細胞介素6與所有疾病狀況的參考項目也無相關。白細胞介素16的變化與腫瘤壞死因子α有高度相關 (r=0.712, P<0.001)，而與白細胞介素6沒有相關 (r=0.104, P=0.713)。血清白細胞介素16的數值與胰島素樣生長因子1受體及其抗體結合強度無相關 (r=-1.21, P=0.621)。由類風濕關節炎病人分離出的T細胞加入類風濕關節炎病人的血清，其腫瘤壞死因子α的濃度在加入胰島素樣生長因子1受體的阻斷性單株抗體後並沒有減少。類風濕關節炎病人的血清在加入類風濕關節炎病人的纖維母細胞後，基質金屬蛋白脢3的製造並沒有增加。而類風濕關節炎病人的血清在加入類風濕關節炎病人的纖維母細胞後，白細胞介素6的濃度有明顯的增加 (P<0.001)；但是加入胰島素樣生長因子1受體的阻斷性單株抗體或把抗體從血清中移除，並不會抑制白細胞介素6的濃度。甚至使用蛋白A把免疫球蛋白G移除，對白細胞介素6的製造也沒有影響。
研究結論: 研究結果顯示，39位類風濕關節炎與39位退化性關節炎的病人間，血清的白細胞介素16並沒有差別。在橫斷與追蹤式研究中，白細胞介素16、腫瘤壞死因子α及白細胞介素6與所有疾病狀況的參考項目，包括紅血球沉降速率、C反應性蛋白、類風濕因子、疼痛關節數、腫脹關節數及DAS28皆無相關。白細胞介素16的變化與腫瘤壞死因子α有高度相關而與白細胞介素6則無。白細胞介素16與腫瘤壞死因子α間的關係需要進一步的研究。胰島素樣生長因子1受體在T細胞製造腫瘤壞死因子α方面並無角色。類風濕關節炎病人的血清在加入類風濕關節炎病人的纖維母細胞後，基質金屬蛋白脢3的製造並沒有增加。而類風濕關節炎病人的血清在加入類風濕關節炎病人的纖維母細胞後，白細胞介素6的濃度有明顯的增加，但是胰島素樣生長因子1受體在這方面仍然沒有角色。甚至，白細胞介素6的增加也與免疫球蛋白G無關。
關鍵字: 類風濕關節炎、白細胞介素16、腫瘤壞死因子α、白細胞介素6、纖維母細胞、胰島素樣生長因子1受體、基質金屬蛋白脢3、T淋巴球

Abstract
Background: Rheumatoid arthritis (RA) is a chronic inflammatory arthritis, which could result in joint destruction and deformity. Inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), are highly involved in pathogenesis. Interleukin-16 (IL-16) could be secreted by CD4+ and CD8+ T lymphocytes, B lymphocytes, airway epithelium, mast cells and cytokine-activated fibroblasts and functions as a potent chemoattractant for T lymphocytes. IL-16 has been demonstrated in the synovial fluid of patients with active RA. Synovial fibroblasts from patients with RA could produce high levels of IL-16 in response to immunoglobulin G of RA (RA-IgG). This induction necessarily involves through the insulin-like growth factor-1 receptor (IGF-1R). Besides, IGF-1R is also expressed on T lymphocytes. This study is designed to evaluate the difference of IL-16 between RA and osteoarthritis (OA) patients and the correlation between IL-16 and disease status and other inflammatory cytokines in RA patients. The roles of IGF-1R in production of TNF-α by T lymphocytes and IL-6 or matrix metalloproteinase 3 (MMP3) by fibroblasts in RA patients would also be delineated.
Methods: We checked the seral level of IL-16 with ELISA in 39 RA and 39 OA patients. IL-16 and disease status parameters of RA, such as erythrocyte sediment rate (ESR), C-reactive protein (CRP), rheumatoid factor (RF), tender joint count (TJC), swollen joint count (SJC) and DAS 28 in 30 patients were evaluated initially and 3 months later. The correlation coefficient of IL-16 and each disease parameter was disclosed with the cross-section and follow-up studies. The data of the first examination was adopted in the cross-section study and the difference of IL-16 or each parameter of disease status during the 3 months was analyzed in the follow-up study. We also checked TNF-α and disease parameters in 21 patients, and IL-6 and parameters of disease status in 15 patients. The correlations of TNF-α or IL-6 with disease activity were measured as that of IL-16. The correlation coefficients between difference of IL-16 and change of TNF-α or IL-6 were evaluated. Besides, the correlation of seral level of IL-16 and the binding optic density of IGF-1R and its antibody was also revealed. We collected T lymphocytes by flow cytometry and used sera of RA patients to stimulate T lymphocytes. Blocking monoclonal antibody against IGF-1R was used to evaluate the significance of IGF-1R in TNF-α production. The sera of RA patients were also used to stimulate fibroblasts to see if MMP3 or IL-6 expression could be up-regulated. In case of increased production of MMP3 or IL-6, blocking monoclonal antibody against IGF-1R or removal of seral antibody of IGF-1R would be used to evaluate if the process could be blocked.
Results: The difference of seral IL-16 in 39 RA and 39 OA patients was not significant (P=0.799). In the cross-section study, IL-16 was not correlated with the disease parameters. The follow-up study revealed that the difference of IL-16 was still not correlated with change of ESR (r=0.185, P=0.328), CRP (r=0.206, P=0.274), RF (r=-0.129, P=0.496), TJC (r=0.226, P=0.221), SJC (r=0.068, P=0.715) and DAS 28 (r=0.237, P=0.208). In the cross-section and follow-up studies, TNF-α or IL-6 had no correlation with all the parameters of disease status. The difference of IL-16 was markedly correlated with change of TNF-α (r=0.712, P<0.001) but not correlated with change of IL-6 (r=0.104; P=0.713). IL-16 had no correlation with the binding intensity of IGF-1R and its antibody (r=-1.21, P=0.621). After treatment with sera of RA patients, the levels of TNF-αby T lymphocytes isolated from RA patients were not decreased in the samples added with blocking monoclonal antibody against IGF-1R than those without loading of antibody. MMP3 production was not increased than baseline seral level after fibroblasts obtained from RA patients are added. The levels of IL-6 were markedly increased (P<0.001) after fibroblasts were cultured with sera of RA patients. However, IL-6 production couldn’t be blocked with blocking monoclonal antibody against IGF-1R or removal of antibody against IGF-1R. Even removal of immunoglobulin G with protein A had no impact in production of IL-6.
Conclusion: In our study, the seral levels of IL-16 are not different between 39 RA and 39 OA patients. In the cross-section and follow-up studies, IL-16, TNF-α and IL-6 are not correlated with all the disease parameters of RA, including ESR, CRP, RF, tender joint count, swollen joint count and DAS 28. IL-16 has markedly positive correlation with TNF-α but not IL-6. The association between IL-16 and TNF-α needs to be further studied. IGF-1R plays no role in production of TNF-α by T lymphocytes. The levels of MMP3 secreted from fibroblasts are not elevated after mixed with sera of RA patients. The levels of IL-6 are markedly increased after fibroblasts are cultured with sera of RA patients, but IGF-1R still plays no role. The increased production of IL-6 is even IgG-independent.
Key words: rheumatoid arthritis, IL-16, TNF-α, IL-6, fibroblast, IGF-1R, MMP3, T lymphocyte

References
1. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 31:315-24, 1988
2. Pruijn GJ, Wiik A, et al. The use of citrullinated peptides and proteins for the diagnosis of rheumatoid arthritis. Arthritis Research & Therapy 12(1):203, 2010
3. Klimiuk PA, Goronzy JJ, et al. Tissue cytokine patterns distinguish variants of rheumatoid synovitis. Am J Pathol 151:1311-19, 1997
4. Nam JL, Winthrop KL, et al. Current evidence for the management of rheumatoid arthritis with biological disease-modifying antirheumatic drugs: a systematic literature review informing the EULAR recommendations for the management of RA. Annals of the Rheumatic Diseases 69(6):976-86, 2010
5. Van Zeben D, Hazes JM, et al. Association of HLA-DR4 with a more progressive disease course in patients with rheumatoid arthritis. Arthritis Rheum 34:822-30, 1991
6. Knevel R, Schoels M, et al. Current evidence for a strategic approach to the management of rheumatoid arthritis with disease-modifying antirheumatic drugs: a systematic literature review informing the EULAR recommendations for the management of rheumatoid arthritis. Annals of the Rheumatic Diseases 69(6):987-94, 2010
7. Cruikshank W, Little F, et al. lnterleukin-16: the ins and outs of regulating T-cell activation. Critical Reviews in Immunology 28(6):467-83, 2008
8. Wilson KC, Center DM, et al. The effect of interleukin-16 and its precursor on T lymphocyte activation and growth. Growth Factors 22(2):97-104, 2004
9. Conti P, Kempuraj D, et al. Interleukin-16 network in inflammation and allergy. Allergy & Asthma Proceedings 23(2):103-8, 2002
10. Kornfeld H, Cruikshank WW, et al. Prospects for IL-16 in the treatment of AIDS. Expert Opinion on Biological Therapy 1(3):425-32, 2001
11. Center DM, Kornfeld H, et al. Interleukin-16. International Journal of Biochemistry & Cell Biology 29(11):1231-4, 1997
12. Conti P, Kempuraj D, et al. Interleukin-16 network in inflammation and allergy. Allergy & Asthma Proceedings 23(2):103-8, 2002
13. Little FF, Cruikshank WW, et al. Interleukin-16 and peptide derivatives as immunomodulatory therapy in allergic lung disease. Expert Opinion on Biological Therapy 4(6):837-46, 2004
14. Glass WG, Sarisky RT, et al. Not-so-sweet sixteen: the role of IL-16 in infectious and immune-mediated inflammatory diseases. Journal of Interferon & Cytokine Research 26(8):511-20, 2006
15. Jane Pritchard, Noah Horst, et al. Igs from patients with Graves’ disease induce the expression of T cell chemoattractants in their fibroblasts. The Journal of Immunology 168:942-50, 2002
16. Jane Pritchard, Rui Han, et al. Immunoglobulin activation of T cell chemoattractant expression in fibroblasts from patients with Graves’ disease is mediated through the insulin-like growth factor I receptor pathway. The Journal of Immunology 170:6348-54, 2003
17. Jane Pritchard, Shanli Tsui, et al. Synovial fibroblasts from patients with rheumatoid arthritis, like fibroblasts from Graves’ disease, express high levels of IL-16 when treated with Igs against insulin-like growth factor-1 receptor. The Journal of Immunolog 173:3564-69, 2004
18. Kaufmann J, Franke S, et al. Correlation of circulating interleukin 16 with proinflammatory cytokines in patients with rheumatoid arthritis. Rheumatology 40(4):474-5, 2001
19. Kaufmann J, Franke S, et al. Correlation of circulating interleukin 16 with proinflammatory cytokines in patients with rheumatoid arthritis. Rheumatology 40(4):474-5, 2001
20. Smith TJ, et al. Insulin-like growth factor-I regulation of immune function: a potential therapeutic target in autoimmune diseases?. Pharmacological Reviews 62(2):199-236, 2010
21. Clemmons DR, et al. Role of IGF-I in skeletal muscle mass maintenance. Trends in Endocrinology & Metabolism 20(7):349-56, 2009
22. Gomez JM, et al. The role of insulin-like growth factor I components in the regulation of vitamin D. Current Pharmaceutical Biotechnology 7(2):125-32, 2006
23. Kooijman R, et al. Regulation of apoptosis by insulin-like growth factor (IGF)-I. Cytokine & Growth Factor Reviews 17(4):305-23, 2006
24. Chitnis MM, Yuen JS, et al. The type 1 insulin-like growth factor receptor pathway. Clinical Cancer Research 14(20):6364-70, 2008
25. Kriegler M, Perez C, et al. A novel form of TNF/cachectin is a cell surface cytotoxic transmembrane protein: ramifications for the complex physiology of TNF. Cell 53 (1): 45–53, 1988
26. Tang P, Hung M-C, et al. Human pro-tumor necrosis factor is a homotrimer. Biochemistry 35 (25): 8216–25, 1996
27. Black RA, Rauch CT, et al. A metalloproteinase disintegrin that releases tumor-necrosis factor-alpha from cells. Nature 385(6618): 729–33, 1997
28. Castro-Rueda H, Kavanaugh A, et al. Biologic therapy for early rheumatoid arthritis: the latest evidence. Current Opinion in Rheumatology 20(3):314-9, 2008
29. Furst DE, et al. Development of TNF inhibitor therapies for the treatment of rheumatoid arthritis. Clinical & Experimental Rheumatology. 28(3 Suppl 59):S5-12, 2010
30. Barnabe C, Hanley DA, et al. Effect of tumor necrosis factor alpha inhibition on bone density and turnover markers in patients with rheumatoid arthritis and spondyloarthropathy. Seminars in Arthritis & Rheumatism 39(2):116-22, 2009
31. Leandro MJ, et al. Anti-tumour necrosis factor therapy and B cells in rheumatoid arthritis. Arthritis Research & Therapy 11(5):128, 2009
32. Ramos-Casals M, Brito-Zeron P, et al. Autoimmune diseases induced by TNF-targeted therapies. Best Practice & Research in Clinical Rheumatology 22(5):847-61, 2008
33. Statkute L, Ruderman EM, et al. Novel TNF antagonists for the treatment of rheumatoid arthritis. Expert Opinion on Investigational Drugs 19(1):105-15, 2010
34. Papagoras C, Voulgari PV, et al. Strategies after the failure of the first anti-tumor necrosis factor alpha agent in rheumatoid arthritis. Autoimmunity Reviews 9(8):574-82, 2010
35. Furst DE, et al. The risk of infections with biologic therapies for rheumatoid arthritis. Seminars in Arthritis & Rheumatism 39(5):327-46, 2010
36. Ohsugi Y, et al. Recent advances in immunopathophysiology of interleukin-6: an innovative therapeutic drug, tocilizumab (recombinant humanized anti-human interleukin-6 receptor antibody), unveils the mysterious etiology of immune-mediated inflammatory diseases. Biological & Pharmaceutical Bulletin 30(11):2001-6, 2007
37. Fonseca JE, Santos MJ, et al. Interleukin-6 as a key player in systemic inflammation and joint destruction. Autoimmunity Reviews 8(7):538-42, 2009
38. Gabay C, et al. Interleukin-6 and chronic inflammation. Arthritis Research & Therapy 8 Suppl 2:S3, 2006
39. Rose-John S, Waetzig GH, et al. The IL-6/sIL-6R complex as a novel target for therapeutic approaches. Expert Opinion on Therapeutic Targets 11(5):613-24, 2007
40. Le NT, Xue M, et al. The dual personalities of matrix metalloproteinases in inflammation. Frontiers in Bioscience 12:1475-87, 2007
41. Burrage PS, Mix KS, et al. Matrix metalloproteinases: role in arthritis. Frontiers in Bioscience 11:529-43, 2006
42. Leroy R Lard, Bart O Roep, et al. Enhanced concentrations of interleukin 16 are associated with joint destruction in patients with rheumatoid arthritis. J Rheumtol 31:35-9, 2004
43. N. L. Mathy, W. Scheuer, et al. Interleukin-16 stimulates the expression and production of pro-inflammatory cytokines by human monocytes. Immunology 100:63-9, 2000
44. Michaela Weis-Klemm, Dorothea Alexander, et al. Synovial fibroblasts from rheumatoid arthritis patients differ in their regulation of IL-16 gene activity in comparison to osteoarthritis fibroblasts. Cell Physiol Biochem 14:293-300, 2004
45. Kooijman R. Scholtens Le, et al. Type 1 insulin-like growth factor receptor expression in different developmental stages of human thymocytes. Journal of Endocrinology 147(2):203-9, 1995
46. Kooijman RK, Scholtens LE, et al. Differential expression of type I insulin-like growth factor receptors in different stages of human T cells. European Journal of Immunology 30(4):1010-8, 2000
47. Yuji Yamamura, Raj Gupta, et al. Effector Function of Resting T Cells: Activation of Synovial Fibroblasts. The Journal of Immunology 166: 2270-75, 2001