ANALYSES OF LEUKOCYTE INTERACTIONS WITH IMMUNE COMPLEXES

免疫复合物（IC）在血管内和血管周围的沉积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。已描述的抗体触发中性粒细胞积累的机制包括补体和内皮依赖性募集，随后是IC触发的中性粒细胞激活。在静态条件下，中性粒细胞粘附在ic上是由低亲和力的Fcgamma受体（Fcgamma） II和III以及β 2整合素家族成员Mac-1 （CD11b/CD18, CR3）支持的。Mac-1不直接与IgG结合，但与FcgammaRII和III结合，维持中性粒细胞与ic的粘附。我们的假设是，在免疫介导的疾病中，沉积在血管壁的ic是中性粒细胞捕获和激活的早期主要效应物，而白细胞的低亲和力FcgammaRs和Mac-1在这一过程中至关重要。为了支持这一假设，我们有初步数据表明，中性粒细胞可以在生理上相关的流体剪切应力水平下束缚并立即阻止/扩散到ic上。中性粒细胞粘附在ic上的效率与粘附在p -选择素（一种公认的内皮粘附受体）上的效率一样。这表明，在缺乏内皮细胞黏附受体、补体或血管相关信号的情况下，仅ic就足以拴住和抑制中性粒细胞。此外，通过FcgammaRIIIB、FcgammaRIIA和Mac-1转染细胞系、Mac-1缺陷中性粒细胞和相关的功能阻断抗体，我们证明了FcgammaRIIIB和Mac-1在中性粒细胞粘附到ic中的重要作用，这种作用仅在生理相关刺激、剪切应力下才被揭示出来。对粘附在ic上的Mac-1缺陷中性粒细胞的磷酸化模式的分析表明，与野生型中性粒细胞相比，不同蛋白的磷酸化存在缺陷。因此，Mac-1相关的信号通路可能在维持中性粒细胞粘附于ic中起重要作用。本研究的目的是：1)确定血流条件下白细胞黏附于ic的分子要求；2)确定Mac-1依赖的细胞内信号机制，促进中性粒细胞对ic的持续黏附；3)在体内研究ic介导的白细胞募集以及FcgammaRs和Mac-1在这一过程中的作用。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贩运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.