树突状细胞DC-SIGN在免疫介导实验性肾炎肾小管间质损伤中的作用及抗P选择素功能域单抗的干预调节

Role of DC-SIGN in renal tubulointerstitial lesions of immune-mediated experimental nephritis and intervention regulation by anti-P-selectin domain monoclonal antibody

目的 探讨树突状细胞（DC）表面特异的胞间黏附分子3捕获非整合素（DC-SIGN）在免疫介导肾毒血清件肾炎（NTN）肾小管间质损伤中的作用,以及抗P选择素功能域单抗（PsL-EGFmAb）的干预调节.方法 WKY大鼠随机分为正常对照组、模型组及PsL-EGFmAb干预组.模型组注射预制的兔抗大鼠肾毒血清1 nd/kg;PsL-EGFmAb组在注射肾毒血清同时及注射后2 h,注入PsL-EGFmAb 2 mg/kg;正常对照组则注射等量生理盐水.随后于实验第4、7、14天,分别观察大鼠肾功能及肾组织病理变化,并采用免疫荧光法检测肾组织DC-SIGN＋DC分布;实时定量PCR检测P选择素、T细胞表达和分泌因子（RANTES）、肿瘤坏死因子α（TNF-α）、白细胞介素（IL）-10、干扰素（IFN）-γ、IL-4的mRNA表达;流式细胞仪检测肾脏分离DC表面主要组织相容性复合体Ⅱ（MHC Ⅱ）、DC-SIGN、CD80表达;细胞迁移试验及混合淋巴细胞反应（MLR）检测DC迁移与刺激T细胞的能力;ELISA法测定MLR上清中IFN-γ、IL-4含量.结果 NTH大鼠第4天起,未成熟DC-SIGN＋DC即以肾间质为主浸润并于14 d成熟,且迁移及刺激T细胞增殖能力增强,其肾内分布与新月体形成、肾小管间质损伤程度及肾功能改变呈正相关.此外,大鼠第4天起肾内趋化因子及促炎因子RANTES、TNF-α mRNA表达持续上调,而抗炎因子IL-10 mRNA于第4天明显增强随后呈下调趋势;至14 d时IFN-γ/IL-4 mRNA比值增高,与DC成熟状况呈正相关.经PsL-EGFmAb干预,伴随Dc表面DC-SIGN及相应共刺激分子CD80表达下降,DC成熟、迁移及刺激T细胞增殖能力受抑,肾内促炎因子下降而抗炎因子上调,Th1/Th2偏移受到抑制.同时大鼠肾内新月体形成减少,肾小管间质损伤程度减轻,且肾功能改善.结论 DC-SIGN介导了DC肾间质浸润,并可能是局部免疫反应失衡以及肾小管间质病变的重要调控因素.PsL-EGFmAb在抑制DC迁移的同时可通过靶向DC-SIGN调抑DC成熟及功能,进而发挥防治效应.

Objective To explore the role of dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin （DC-SIGN） in the tubulointerstitial lesions of immune-mediated nephrotoxic nephritis （NTN） and the intervention regulation by anti-P-selectin lectin-EGF domain monoclonal antibody （PsL-EGFmAb）. Methods WKY rats were randomly divided into control,NTN and PsL-EGFmAb-treated groups. The mrs in NTN group were injected with 1 ml nephrotoxic rabbit serum per kilogram of rat body weight; the ones in PsL-EGFmAb-treated group were injected with 2 mg PsL-EGFmAb per kilogram of rat body weight simultaneously and 2 h later after nephrotoxic rabbit serum injection; and those in control group were injected with equal volume of 0.9% saline. Renal function and pathology were observed at day 4, 7 and 14 after the induction of NTN. Distribution of DC-SIGN ＋ dendritic cells （DCs） in renal tissues was measured by immunofluorescence. Real-time PCR was performed to examine the expression of P-selectin,RANTES, TNF-α, IL-10, IFN-γ and IL-4. Expression of MHC Ⅱ , CD80 and DC-SIGN on dendritic cells was analyzed by flow cytometry. Transendothelial migration was used to detect the ability of DCs migration. DCs ability to activate T cells was determined by mixed lymphocyte reaction （MLR）. ELISA was used to detect the concentration of IFN-γ and IL-4 in the supernatant of MLR. Results At day 4, immature DC-SIGN＋ DCs infiltrated the rat renal tubulointerstium of NTN group, matured at day 14, and enhanced the ability to migrate and activate T cells. The distribution of DC-SIGN ＋ DCs was significantly related to the form of crescent, tubulointerstial lesions and renal function. In addition, expression of chemokine RANTES and proinflammatory cytokine TNF-α continuously augmented since day 4, while anti-inflammatory eytokine IL-10 decreased after markedly increased at day 4. At day 14, IFN-γ/IL-4 mRNA increased, which was obviously related to DCs maturation. The intervention of PsL-EGFmAb supressed the expression of DC-SIGN and CD80 on DCs, depressed DCs maturation, migration and ability to activate T cells,down-regulated proinflammatory cytokines and up-regulated anti-inflammatory cytokines in kidney,and thus regulated Th1/Th2 bias. At the same time, kidneys showed the decrease of crescents,improvement of tnbulointerstium damage and renal function. Conclusions DC-SIGN may mediate DCs tubulointerstitial infiltration. It may be also a potent regulator of local immune reaction imbalance and pathology of tubulointerstium. PsL-EGFmAb may depress DCs migration and downregulate DCs maturation and function through DC-SIGN, and thus having a role in prevention and treatment.