Regulation of CD32A in neutrophils

描述（由申请人提供）：在许多自身免疫性疾病如关节炎和狼疮中，组织损伤是由炎性细胞如嗜中性粒细胞与沉积在组织上的免疫复合物（IC）的相互作用引起的。这种组织损伤还导致肾小球肾炎，导致这些疾病状态下的肾衰竭和死亡。最近对gone敲除小鼠的研究已经清楚地证明Fc γ受体（Fc γ R）在IC介导的自身免疫性疾病中起主要作用。因此，了解功能Fc γ R的调节具有重要的治疗意义。在人类中，两种类型的对IgG的低亲和力Fc γ R（CD 32A和CD 16 B）在嗜中性粒细胞上共表达。两者都以重叠的特异性结合配体，然而，只有CD 32A能够递送用于吞噬作用的信号。我们实验室最近的研究表明，CD 32A在静息中性粒细胞中功能不活跃。然而，一旦中性粒细胞被fMLP（一种细菌趋化肽）激活，CD 32A就被转化为功能活性状态，并且可以有效地结合配体。相反，用PMA（一种中性粒细胞活化佛波酯）活化中性粒细胞完全消除了CD 32A与抗体包被的红细胞的结合。有趣的是，在培养的细胞系上表达的中性粒细胞是组成型活性的。这些结果表明，亲合力调节是细胞类型和激活信号特异性的，配体结合的调节可能是人中性粒细胞调节CD 32A功能的机制之一。我们假设中性粒细胞活化过程中发生的分子变化改变了CD 32A的功能状态。在这项授权中，我们建议确定这种信号特异性和细胞类型依赖性调节CD 32A功能状态的分子基础。具体来说，我们将：1。确定嗜中性粒细胞激活剂是否通过影响CD 32A的2D和3D亲和力来改变CD 32A依赖性EA结合; 3、使用免疫印迹、2D氨基酸分析和CD 32A胞质结构域突变研究来确定CD 32A的ITAM基序的磷酸化是否通过细胞活化而改变并且与配体结合的变化相关; 3.分析中性粒细胞活化是否改变了CD 32A的细胞骨架相互作用、受体聚集和CD 32A侧向移动性的变化。由于在感染性和自身免疫性疾病期间体内发生嗜中性粒细胞活化，因此可以假设体内发生CD 32A功能状态的失调，导致高亲合力CD 32A的表达，这使得嗜中性粒细胞能够有效地结合IC，从而导致这些疾病中的组织损伤。从拟议的研究中获得的数据将有助于理解和设计治疗方法，以抑制IC介导的自身免疫性和感染性疾病的组织损伤。

DESCRIPTION (provided by applicant): In many autoimmune diseases such as arthritis, and lupus the tissue injury is caused by the interaction of inflammatory cells such as neutrophils with the immune complexes (IC) deposited on tissues. Such a tissue injury also results in glomerulonephritis leading to kidney failure and death in these disease states. Recent studies with gone knock out mice have clearly demonstrated that Fc gamma receptors (FcgammaRs) play a major role in IC mediated autoimmune diseases. Therefore understanding the regulation of function FcgammaRs has important therapeutic implications. In humans, two types of low affinity FcgammaRs for IgG, CD32A and CD16B, are coexpressed on neutrophils. Both bind ligands with overlapping specificity, however, only CD32A is capable delivering signal for phagocytosis. Recent studies from our laboratory have demonstrated that CD32A is functionally inactive in resting neutrophils. However, once neutrophils are activated by fMLP, a bacterial chemoattractant peptide, CD32A is converted to a functionally active state and can bind ligand efficiently. On the contrary, activation of neutrophils with PMA, a neutrophil activating phorbol ester, completely abolished CD32A binding to antibody-coated erythrocytes. Interestingly, the neutrophils expressed on cultured cell lines are constitutively active. These results suggest that the avidity modulation is cell type and activation signal specific and the regulation of ligand binding may be one of the mechanisms by which human neutrophil regulates CD32A function. We hypothesize that the molecular changes that occur during neutrophil activation alter the functional state of CD32A. In this grant we propose to determine the molecular basis for this signal specific and cell type dependent regulation of CD32A functional state. Specifically, we will: 1. determine whether the neutrophil activators alter the CD32A-dependent EA binding by influencing the 2D and 3D affinity of CD32A; 3, determine whether phosphorylation of ITAM motif of CD32A is altered by cell activation and correlates with change in ligand binding using immunoblotting, 2D amino acid analysis, and CD32A cytoplasmic domain mutation studies; 3. analyze whether neutrophil activation alters the cytoskeleton interaction of CD32A, receptor clustering, and change in the lateral mobility of CD32A. Since neutrophil activation occurs in vivo during infectious and autoimmune diseases it can be hypothesized that dysregulation of CD32A functional state occurs in vivo leading to the expression of high avidity CD32A which enables the neutrophils bind IC efficiently resulting in tissue injury in these diseases. The data obtained from the proposed research will be useful in understanding and designing therapies to inhibit IC mediated tissue injury in autoimmune and infectious diseases.