ANALYSES OF LEUKOCYTE INTERACTIONS WITH IMMUNE COMPLEXES

描述(申请人提供)：该项目的长期目标是确定中性粒细胞与免疫复合体(IC)相互作用的受体和分子机制，并激活有助于细胞功能的信号通路。这些事件是生理性炎症的基础，但它们的失调是IC介导的疾病的一个定义特征。该项目的工作证明了中性粒细胞FcGammaRIII(FcgRIII)在抗肾小球基底膜肾炎(GBM)肾炎模型中，在生理性流动条件下和IC原位沉积的情况下，在IC诱导的中性粒细胞体外募集中起到了直接作用。为了提供招募过程的机制细节，开发了一种服从活体显微镜的IC沉积的小鼠模型。Fc‘yRs促进缓慢的白细胞滚动；这一步骤有利于黏附，尤其是FcgRIII，对于牢固的黏附和迁移是必不可少的。以往的研究表明，白细胞CD18整合素Mac-1(CD11b/CD18)是IC介导的黏附所必需的。FcgRIII通过凝集素-糖类相互作用在细胞表面与Mac-1物理结合，FcgRIIl-ICs相互作用导致Mac-1激活。对Mac-1缺陷(-/-)小鼠的研究揭示了Mac-1在体内FcgRIII依赖的黏附和抗体依赖的细胞毒性(ADCC)中的重要作用。转基因表达的人FcaR介导的ADCC也需要小鼠中性粒细胞表面的Mac-1。总之，我们的研究将小鼠中性粒细胞FCRs和Mac-1置于急性炎症的关键节点，并表明它们在IC介导的功能中的合作具有重要的生物学意义。FcgRIII-Mac-1协同作用的分子基础尚不清楚。初步数据表明，CD18亚单位胞外I样区的残基和Mac-1配体结合的激活促进了中性粒细胞与ICs的相互作用。目前应用的目的建立在我们正在进行的工作的基础上，该中心假设人FcgRIIIB和IIA和Mac-1是中性粒细胞在体内募集和细胞毒所必需的，FcyR-Mac-1通过它们胞外区的相互作用促进IC介导的中性粒细胞功能。其目的是：i)确定人中性粒细胞FcgRIIA和GPL连接的FcgRIIIB(在小鼠中没有遗传等价物)在ICI诱导的中性粒细胞募集和黏附中的作用，并研究Mac-1在这些人FcgRs背景下的生物学意义；以及ii)阐明FcgR-Mac-1相互作用的分子基础，重点是细胞外CD1Lb配体结合I和凝集素样域，以及CD18的I样结构域在这种相互作用中的作用。这些研究将为人类FcgR的功能及其在体内与Mac-1的协同作用提供有价值的数据，这可能具有直接的临床意义，并将为Mac-1和FcgR如何相互作用促进中性粒细胞与ICs的黏附提供新的概念。

DESCRIPTION (provided by applicant): The long-term goal of this project is to define the receptors and the molecular mechanisms by which neutrophils interact with immune complexes (ICs) and activate signaling pathways that contribute to cellular functions. These events are fundamental to physiologic inflammation, but their dysregulation is a defining feature in IC-mediated disorders. Work on this project demonstrated a direct role for neutrophil FcgammaRIII (FcgRIII) in IC-induced neutrophil recruitment in vitro under physiological flow and following in situ IC deposition in a model of anti-glomerular basement membrane (GBM) nephritis. A murine model of IC deposition that was amenable to intravital microscopy was developed to provide mechanistic details of the recruitment process. Fc'yRs promoted slow leukocyte rolling; a step that favors adhesion and FcgRIII in particular was essential for firm adhesion and transmigration. Previous studies have shown that the leukocyte CD18 integrin Mac-1 (CD11b/CD18) is required for IC-mediated adhesion. FcgRIII physically associates with Mac-1 on the cell surface through lectin-carbohydrate interactions and FcgRIIl-ICs interactions leads to Mac-1 activation. Studies in Mac-1 deficient (-/-) mice revealed an essential role for Mac-1 in FcgRIII dependent adhesion and antibody dependent cytotoxicity (ADCC) in vivo. Mac-1 on murine neutrophils was also needed for ADCC mediated by transgenically expressed human FcaR. Together our studies place murine neutrophil FcRs and Mac-1 at critical junctures in acute inflammation and suggest that their cooperation in IC-mediated functions is biologically important. The molecular basis of FcgRIII-Mac-1 cooperation remains unclear. Preliminary data suggest that a residue in the extracellular I-like domain of the CD18 subunit and activation of Mac-1 ligand binding promotes neutrophil interactions with ICs. The aims in the current application builds on our ongoing work based on the central hypothesis that human FcgRIIIB and IIA and Mac-1 are required for neutrophil recruitment and cytotoxicity in vivo, and FcyR-Mac-1 cooperate, throuqh interactions of their extracellular domains, to promote IC-mediated neutrophil functions. The aims are: I) To identify the in vivo role of human neutrophil FcgRIIA and the GPl-linked FcgRIIIB (for which there are no genetic equivalents in mice) in ICinduced neutrophil recruitment and adhesion, and examine the biological importance of Mac-1 in the context of these human FcgRs; and II) To elucidate the molecular basis for FcgR-Mac-1 cross-talk focusing on the role of the extracellular CD1 lb ligand binding I and lectin-like domains, and the I-like domain of CD18 in this cross-talk. These studies should provide valuable data on human FcgR functions and their cooperation with Mac-1 in vivo, which could have direct clinical relevance, and will provide new concepts on how Mac-1 and FcgR cross-talk to promote neutrophil adhesion to ICs.