Study on the Molecular Basis of Anti-Citrullinated Peptide Antibodies in the Immunopathogenesis of Rheumatoid Arthritis

類風濕性關節炎是免疫風濕科領域相當重要的疾病，主要以慢性對稱性多關節炎為其表現，約佔總人口的1%。關節持續的發炎腫脹，常常會造成患者關節變型，甚至導致患者的死亡率上升。雖然之前已經有相當多的研究來探討其致病機轉，但是對於真正引起關節慢性發炎的分子機轉仍然不清楚。近年來，抗瓜氨酸化胜肽抗體(anti-citrullinated peptide antibodies; ACPAs)的發現為新的里程碑。它可以非常專一的出現在類風濕性關節炎的血清中，尤其是在疾病表徵尚未產生之前就已經出現了。而且患者血清中抗瓜氨酸化胜肽抗體的效價和其疾病活動性密切相關。直到目前學界的研究結論認為，抗瓜氨酸化胜肽抗體在類風濕關節炎的致病機轉中，扮演重要的角色。然而確切的分子機轉仍然沒有合理的解釋。在本研究中分為臨床及基礎研究兩部分，首先在臨床方面，我們比較抗瓜氨酸化胜肽抗體，類風濕因子及抗無乳酸免疫球蛋白抗體在台灣臨床上鑑別診斷類風濕性關節炎及其相仿者的優劣。在基礎方面，我們假設單核白血球細胞表面上有自體抗原可以被抗瓜氨酸化胜肽抗體所認識。而抗瓜氨酸化胜肽抗體可以藉由結合這些細胞表面分子直接引起發炎反應。 在實驗方法上，首先收集類風濕性關節炎，原發性修格連氏症，慢性B型肝炎，慢性C型肝炎及健康志願者的血清，以濁度分析法偵測類風濕因子，以酵素免疫分析法偵測無乳酸免疫球蛋白抗體和抗瓜氨酸化胜肽抗體，並比較其在鑑別診斷類風濕性關節炎及其相仿者的敏感度及特異度。之後取ACPAs(+)類風濕性關節炎患者的血清利用親合性管柱結合環狀瓜氨酸化胜肽，純化出抗瓜氨酸化胜肽抗體。接著和健康人的單核性白血球或細胞株U937一起培養。以酵素免疫分析法偵測TNF-α的釋放及轉錄因子NF-κB的活化。再來以ACPAs當偵測抗體，利用西方點墨法找出作用的自體抗原，最後取出並送蛋白質鑑定。 在臨床上的研究結果顯示，在抗瓜氨酸化胜肽抗體、類風濕因子及抗無乳酸免疫球蛋白抗體之中，抗瓜氨酸化胜肽抗體對於鑑別診斷類風濕性關節炎及其相仿者的敏感度及特異度最高。在基礎研究方面，以親合性管柱純化出的抗瓜氨酸化胜肽抗體和U937細胞共同培養，可以促進TNF-α的釋放及轉錄因子NF-κB的活化。相對的，以去除掉抗瓜氨酸化胜肽抗體的類風濕關節炎患者血清，則無此作用。我們分離出U937細胞膜上的蛋白質，以抗瓜氨酸化胜肽抗體為第一抗體，用西方點墨法發現抗瓜氨酸化胜肽抗體可以和膜上的蛋白質結合，最後以蛋白質體學的方法，證實glucose-regulated protein of 78 kDa (GRP78)為其對應抗原。我們並發現，以基因合成的GRP78並不會跟抗瓜氨酸化胜肽抗體結合，但是從U937細胞所分離出來的膜蛋白，再以免疫沉澱法純化的GRP78則可以，乃因U937細胞膜上GRP78的有瓜氨酸化的現象，而基因合成的GRP78則沒有。將基因合成的GRP78給予瓜氨酸化後，可以和抗瓜氨酸化胜肽抗體結合，並且抑制抗瓜氨酸化胜肽抗體引起的U937細胞TNF-α釋放。最後我們驗證，健康人血液中的單核性白血球上有GRP78的表現，而且抗瓜氨酸化胜肽抗體也可以促進其TNF-α釋放。 本研究的結論是，抗瓜氨酸化胜肽抗體不單只是一個相當有用的類風濕性關節炎診斷上的血清學指標，其更可以藉由直接結合在細胞膜上瓜氨酸化的GRP78，促進單核性白血球轉錄因子NF-κB的活化及TNF-α的釋放，引起發炎反應。我們的實驗證實了ACPAs直接參與了類風濕性關節炎的免疫病理機轉。Rheumatoid arthritis (RA) is an important disease in rheumatology and its prevalence rate is around 1%. It is characterized by chronic symmetric polyarticular synovitis. Persistent joint swelling and inflammation not only caused the deformity of joints but also increased the mortality in RA patients. Althouth there were many studies investigating the pathogenesis of RA, the definite molecular mechanism of chronic synovitis remained to be elucidated. In recent years, the discovery of anti-citrullinated peptide antibodies (ACPAs) was a milestone in RA. Currently ACPAs was the most specific marker autoantibody in RA patients correlating with its disease activity. In addition, the most unique nature of ACPAs is that the autoantibody may appear even before the onset of arthritis. It is conceivable that ACPAs were involved in the pathogenesis of RA; however the definite molecular pathogenesis has not been elucidated. My thesis contains both clinical and basic dimension about ACPAs in the pathogenesis of RA. In clinical setting, we evaluated the usefulness of anti-agalactosyl IgG antibodies〔anti-Gal(0) IgG〕, rheumatoid factors (RF) and ACPAs in the differential diagnosis of different rheumatic disorders mimicking RA. In basic research, we hypothesized that ACPAs may directly bind to cell surface citrullinated proteins to stimulate mononuclear cells to elicite inflammatory reaction. ACPAs were purified from pooled ACPAs (+) RA sera by cyclic citrullinated peptide (CCP)-conjugated affinity column. After co-culture of U937 cells with ACPAs, the tumor necrosis factor (TNF)-α production and nuclear factor (NF)-κB DNA binding activity of the cells were measured by ELISA. The cognate antigen(s) of ACPAs on the U937 cell surface was probed by ACPAs, and the reactive bands were identified by proteomic analysis. In clinical setting, ACPAs was the most specific and sensitive biomarker for diagnosis of RA from its mimic. In basic research, ACPAs specifically enhanced TNF-α production and increased DNA binding activity of NF-κB in U937 cells. In contrast, ACPA-depleted RA sera failed to activate U937 cells. Proteomic analysis revealed that glucose-regulated protein 78 (GRP78, 72 kDa) was one of the cognate antigens for ACPAs. GST-tagged recombinant GRP78 could not recognize by ACPAs due to lack of citrullination. In addition, we found that GRP78 was citrullinated on U937 cell surface that rendered its binding activity with ACPAs. The citrullinated GRP78 inhibited ACPA-induced TNF-α production by U937. ACPAs also bound to human monocytes to promote TNF-α production. In conclusion, the presence of ACPAs was a very useful biomarker for diagnosis of RA. Furthermore, we clearly demonstrated that ACPAs enhanced the NF-κB activity and TNF-α production in monocytes/macrophages via binding to surface-expressed citrullinated GRP78 that involved in rheumatoid pathogenesis