ANALYSES OF LEUKOCYTE INTERACTIONS WITH IMMUNE COMPLEXES

免疫复合物（IC）沉积在血管内和血管周围是许多免疫性疾病的致病因素，并伴有中性粒细胞积聚和组织损伤。 所描述的抗体触发的中性粒细胞蓄积的机制包括补体和内皮依赖性募集，随后是IC触发的中性粒细胞活化。 在静态条件下，低亲和力Fc γ受体（Fc γ R）II和III以及Mac-1（CD 11b/CD 18，CR 3）（β 2整联蛋白家族成员）支持中性粒细胞与IC的粘附。 Mac-1不直接结合IgG，但与Fc γ RII和III结合，并维持中性粒细胞粘附于IC。 我们的假设是，沉积在血管壁中的IC是免疫介导的疾病中中性粒细胞捕获和激活的早期主要效应物，并且白细胞上的低亲和力Fc γ R和Mac-1在该过程中是关键的。 为了支持这一假设，我们有初步的数据，中性粒细胞可以拴系和立即逮捕/传播的IC下生理相关水平的流体剪切应力。 中性粒细胞对IC的束缚与对P-选择素的束缚一样有效，P-选择素是一种成熟的内皮束缚受体。这表明在不存在基于内皮细胞的粘附受体、补体或血管相关信号的情况下，单独的IC足以束缚和阻止中性粒细胞。 此外，使用Fc γ RIIIB，Fc γ RIIA和Mac-1转染的细胞系，Mac-1缺陷的中性粒细胞和相关的功能阻断抗体，我们证明了Fc γ RIIIB和Mac-1在中性粒细胞拴系到IC中的重要作用，这仅在生理相关的刺激，剪切应力下显示。 Mac-1缺陷的中性粒细胞粘附到IC的磷酸化模式的分析表明，与野生型中性粒细胞相比，不同的蛋白质的磷酸化缺陷。 因此，Mac-1相关的信号通路可能是重要的维持中性粒细胞粘附IC。 本提案的目的是：1）确定流程中白细胞束缚和粘附至IC的分子要求; 2）定义促进中性粒细胞持续粘附至IC的Mac- 1依赖性细胞内信号传导机制; 3）在体内检查IC介导的白细胞募集以及Fc γ R和Mac-1在该过程中的作用。 从拟议的研究中获得的信息应有助于我们对免疫介导疾病中调节白细胞运输的基本机制的基本理解。

Immune complex (IC) deposition in and around blood vessels is pathogenic in a number of immune diseases, and is accompanied by neutrophil accumulation and tissue damage. Described mechanisms of antibody triggered neutrophil accumulation include complement and endothelial dependent recruitment followed by IC triggered neutrophil activation. Under static conditions, neutrophil adhesion to ICs is supported by the low affinity Fcgamma receptors (FcgammaR) II and III, and Mac-1 (CD11b/CD18, CR3), a member of the beta2 integrin family. Mac-1 does not directly bind IgG but associates with FcgammaRII and III and sustains neutrophil adhesion to ICs. Our hypothesis is that ICs deposited in the vessel wall are early, primary effectors of both neutrophil capture and activation in immune mediated diseases and that low affinity FcgammaRs and Mac-1 on leukocytes are critical in this process. In support of this hypothesis, we have preliminary data that neutrophils can tether and immediately arrest/spread on ICs under physiologically relevant levels of fluid shear stress. Neutrophil tethering to ICs was as efficient as that to P-selectin, a well-established endothelial tethering receptor. This suggests that ICs alone are sufficient for tethering and arresting neutrophils in the absence of endothelial cell based adhesion receptors, complement or vessel associated signals. Furthermore, using FcgammaRIIIB, FcgammaRIIA and Mac-1 transfected cell lines, Mac-1 deficient neutrophils and relevant functional blocking antibodies, we demonstrate essential roles for FcgammaRIIIB and Mac-1 in neutrophil tethering to ICs which were revealed only under a physiologically relevant stimulus, shear stress. Analysis of the phosphorylation pattern of Mac-1 deficient neutrophils adherent to ICs suggests a defect in the phosphorylation of distinct proteins when compared with wild-type neutrophils. Thus Mac-1 associated signaling pathways may be important in sustaining neutrophil adhesion to ICs. The aims of this proposal are to 1) Determine the molecular requirements for leukocyte tethering and adhesion to ICs under flow 2) Define Mac- 1 dependent intracellular signaling mechanisms that promote sustained neutrophil adhesion to ICs and 3) Examine IC-mediated leukocyte recruitment and the role of FcgammaRs and Mac-1 in this process, in vivo. Information gained from the proposed studies should contribute to our basic understanding of fundamental mechanisms modulating leukocyte trafficking in immune mediated disease.