抗FcεRⅠ自身抗体活化肥大细胞在自身免疫反应中的作用及机制研究

Autoimmune disease occurs when a specific adaptive immune response is mounted against self antigens. The mechanisms of tissue damage in autoimmune diseases are essentially the same as those that operate in protective immunity and in hypersensitivity diseases. Mast cells are classically considered as potent contributors to allergic reactions. However, recent advances in the realm of autoimmunity have made it clear that these cells are also involved in the pathogenic responses that exacerbate disease. However, there is still much to learn. We developed an ELISA system for the measurement of anti- FcεRⅠautoAbs in non-fractionated serum samples and found anti-FcεRⅠautoreactivity in 34.3% of patients with autoimmune diseases but not in healthy individuals. The anti-FcεRⅠrecognize FcεRⅠon cells and trigger histamine release from freshly isolated blood basophils. However, the latter effect requires further study. The aim of the present study was to investigate mast cell activation and degranulation by the functional autoantibodies against FcεRI; Animal experiments to further study the anti-FcεRI activation effect of tissue mast cells and inflammatory response. Thus, mast cells may function as a cellular link between autoantibodies, soluble mediators, and other effector populations in inflammatory of autoimmune response. It is suggest that autoantibodies against FcεRI may be involved in the pathogenesis of mast cell mediated autoimmune disease. The finding in our study provide evidences for the diagnosis of autoimmune diseases and provide important basis for targeted therapy.

自身免疫性疾病累及多组织多脏器，造成严重组织损伤和功能障碍，对其发病机制的研究仍在继续。新近发现在自身免疫反应局部组织中存在大量肥大细胞浸润，且肥大细胞活化、脱颗粒释放炎症介质成为触发局部炎症反应的关键环节，然而肥大细胞的活化机制尚未明确。对课题组前期收集的自身免疫病患者血清分析发现，其中34.3%存在高水平anti-FcεRⅠ自身抗体，并证实该自身抗体可特异性结合并活化肥大细胞，但其诱导肥大细胞脱颗粒效应及炎症反应作用机制尚不清楚。本项目拟将anti-FcεRⅠ作用于肥大细胞研究其调节肥大细胞脱颗粒及炎症介质进一步合成情况；动物实验进一步研究anti-FcεRⅠ对组织肥大细胞的活化效应及炎症反应的调控。阐明anti-FcεRⅠ自身抗体诱导肥大细胞活化、脱颗粒及引发局部自身免疫性炎症反应的生物学功能和可能的机制，揭示自身免疫反应发生的又一重要途径，为该病的明确诊断和靶向治疗提供重要依据。

肥大细胞是机体重要的天然免疫细胞，肥大细胞及其自身抗体参与风湿性关节炎、多发性硬化等多种自身免疫性疾病的发生、发展。本项目进行了以下研究，进一步阐释肥大细胞相关的抗FcεRⅠ与自身免疫性疾病的相关性及其作用机制。1）肥大细胞相关自身抗体及分子IgE、抗IgE、FcεRⅠα、抗FcεRⅠ与自身免疫性肝病、良性甲状腺疾病、慢性荨麻疹及湿疹的相关性分析。检测血清抗FcεRⅠ水平发现， 33例自身免疫性肝病[47.22（36.89~55.29）mg/L]、41例Graves’病[61.8（42.8~104.0）mg/L]、70例湿疹[38.64（33.10~60.32）mg/L]患者的抗FcεRⅠ水平显著高于健康对照组[35.40（24.74~44.89）mg/L]。2）采用体外培养的小鼠骨髓源性肥大细胞（BMMC）及MC/9 肥大细胞株检验抗FcεRⅠ体外活化肥大细胞的作用效果。发现小鼠来源的抗FcεRⅠ血清在体外可以激活MC/9细胞脱颗粒释放类胰蛋白酶，其中4号血清激活效率最高，类胰蛋白酶释放率达到51.24%，与单抗M-70（释放率为50.76%）作用相当。3）肥大细胞来源的外泌体（Exosomes）与淋巴细胞相互作用后对淋巴细胞增殖、分化的影响。证明Exosomes通过OX40-OX40L间相互作用促进Th2细胞分化。在健康小鼠外周淋巴结组织中发现肥大细胞与CD4+淋巴细胞存在空间上相互靠近的共定位情况。体外培养的BMMC会持续释放带有其细胞成分的Exosomes，经鉴定，此类Exosomes表面表达CD63和OX40L分子。体外将BMMC源性Exosomes与CD4+细胞共培养，可以显著促进CD4+细胞的增殖，并且表达IL-4的Th2细胞亚群与对照组相比显著增高（28% vs 13%，p<0.05）。在相互作用过程中BMMC源性Exosomes粘附于CD4+细胞表面，依赖于Exosomes表面表达的OX40L与CD4+细胞表面的OX40相互识别和结合，当培养体系中加入抗CD40L抗体阻断时，抗体组与Exosomes组及对照组相比Th2类细胞显著减少。上述结果表明，血清中抗FcεRⅠ通过活化肥大细胞，促进肥大细胞内颗粒物质释放参与自身免疫性炎症反应；活化的肥大细胞产生exosomes作用于周围淋巴细胞，促进Th2类细胞分化，增强体液免疫反应。