探討別嘌呤醇引起藥物過敏反應中人類白血球抗原B型58:01與藥物專一性T細胞之間的免疫和分子機制

Abstract

別嘌呤醇 (Allopurinol)被用來治療痛風已經持續了近半個世紀，且到現今還是最常用的藥物之ㄧ，但是在某些服用這個藥物的病人身上會產生嚴重性皮膚不良反應 (SCAR)，包括了過敏症候群 (Hypersensitivity Syndrome)、史蒂文生氏-強生症候群 (Steven-Johnson Syndrome)與毒性表皮壞死溶解症 (Toxic Epidermal Necrolysis)。在我們先前的研究團隊中發現在這些別嘌呤醇藥物過敏病人的體內帶有人類白血球抗原B型58:01(HLA-B*58:01)這一個共同的基因標記，但是別嘌呤醇所引起嚴重皮膚過敏反應的致病機轉仍未釐清。為了探討別嘌呤醇所引起過敏反應的致病機轉，我們研究別嘌呤醇和它的代謝物氧化嘌呤醇(Oxypurinol)與人類白血球抗原B型58:01分子之間的交互作用。本論文共分析14個別嘌呤醇所引起過敏反應的病人，並研究這些病人的周邊血液單核細胞對別嘌呤醇和代謝物氧化嘌呤醇的免疫反應。我們用病人周邊血液單核細胞進行體外細胞培養，分析免疫細胞分型(cell phenotype)，並且使用檢測細胞增生(cell proliferation assay)、細胞毒性分析(T cell cytotoxic assay) 、表面電漿共振技術(surface plasmon resonance, SPR)和點突變(site-directed mutagenesis)等方法，來探討藥物和人類白血球抗原B型58:01分子之間的交互作用。我們使用這個系統將14個別嘌呤醇過敏病人血液中藥物專一性的細胞活化出來。這些藥物專一性的細胞同時對別嘌呤醇和氧化嘌呤醇有交叉反應(cross-reactivity)，並且活化出CD4+ 和CD8+的免疫細胞。這些活化後的免疫細胞對帶有人類白血球抗原B型58:01的抗原呈現細胞(Antigen-presenting cells)有專一性。這些免疫細胞的增生能力不會受到抗原呈現細胞固定(pre-fixation)的影響，因次我們認為可能在活化免疫細胞過程中，藥物的呈現不需要經過抗原呈現細胞的加工處理。利用表面電漿共振技術來分析人類白血球抗原B型58:01和藥物別嘌呤醇和氧化嘌呤醇之間的結合能力。在人類白血球抗原B型58:01中胺基酸Arg97是和藥物結合的重要位置。總而言之，這一篇研究探討人類白血球抗原(HLA)參與在別嘌呤醇或氧化嘌呤醇之間的交互作用，證實人類白血球抗原B型58:01引起嚴重皮膚過敏反應的免疫分子之間的致病機轉。

Allopurinol is a commonly prescribed drug for symptomatic hyperuricemia and its complication, gout. The major limitation of allopurinol is that it is a frequent cause of severe cutaneous adverse reactions (SCARs), including hypersensitivity syndrome (HSS), Stevens–Johnson syndrome (SJS), and toxic epidermal necrolysis (TEN). Our previous study showed that susceptibility to these life-threatening reactions induced by allopurinol is genetically determined and is strongly associated with HLA-B*58:01. The underlying mechanism remains unclear, however. To investigate the pathogenesis of allopurinol-SCAR, we aimed to study the interactions between the allopurinol or oxypurinol and HLA-B*58:01 molecules. We enrolled 14 patients with allopurinol-induced SCAR and studied the response of their cultured peripheral blood mononuclear cells (PBMCs) to allopurinol or oxypurinol. In vitro-expanded T lymphocytes from the patients were evaluated for cell phenotype, and their interaction with HLA-B*58:01 and the drug analyzed, based on proliferation ability, cytotoxic T lymphocyte (CTL) response, surface plasmon resonance (SPR) and site-directed mutagenesis. T cell lines from 14 allopurinol-SCAR patients in the presence of allopurinol or oxypurinol were generated. These T-cell lines (TCLs) displayed cross-reactivity between allopurinol and oxypurinol, and both CD4+ and CD8+ T cells were present. The expanded T Cells were activated and restricted by antigen-presenting cells expressing HLA-B*58:01 in the presence of drug. The proliferation of T cells was not altered by the pre-fixation of antigen presenting cells (APC), suggesting the involvement of an antigen-processing-independent pathway. Using SPR analysis, HLA-B*58:01 was shown to exhibit specific binding affinity to allopurinol and oxypurinol. Arg97 between the E and C pocket of HLA-B*58:01 is a key residue involved in drug presentation. This study demonstrates an interaction between HLA and allopurinol or oxypurinol, and provides a molecular-level understanding of allopurinol-induced SCARs associated with HLA-B*58:01.