Regulation of CD32A in neutrophils

描述（由申请人提供）：在许多自身免疫性疾病中，如关节炎和狼疮，组织损伤是由炎症细胞如中性粒细胞与沉积在组织上的免疫复合物（IC）相互作用引起的。这种组织损伤也会导致肾小球肾炎，在这些疾病状态下导致肾衰竭和死亡。最近对去敲除小鼠的研究清楚地表明，Fc γ受体（FcgammaRs）在IC介导的自身免疫性疾病中发挥重要作用。因此，了解FcgammaRs的功能调控具有重要的治疗意义。在人类中，两种对IgG低亲和力的FcgammaRs， CD32A和CD16B，在中性粒细胞上共表达。两者结合配体具有重叠特异性，但只有CD32A能够传递吞噬信号。我们实验室最近的研究表明，CD32A在静止中性粒细胞中功能失活。然而，一旦中性粒细胞被细菌化学引诱肽fMLP激活，CD32A就会转化为功能活性状态，并能有效地结合配体。相反，中性粒细胞被PMA（一种中性粒细胞活化的酚酯）激活后，可以完全消除CD32A与抗体包被红细胞的结合。有趣的是，在培养细胞系上表达的中性粒细胞具有组成性活性。这些结果表明，嗜中性粒细胞对CD32A功能的调节具有细胞类型和激活信号特异性，对配体结合的调节可能是人中性粒细胞调节CD32A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CD32A的功能状态。在这项资助中，我们建议确定CD32A功能状态的信号特异性和细胞类型依赖性调节的分子基础。具体来说，我们将：1。确定中性粒细胞激活剂是否通过影响CD32A的2D和3D亲和力来改变依赖CD32A的EA结合；3、通过免疫印迹、二维氨基酸分析和CD32A细胞质结构域突变研究，确定细胞活化是否改变了CD32A ITAM基序的磷酸化，并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CD32A的细胞骨架相互作用、受体聚集和CD32A横向迁移的变化。由于中性粒细胞激活在感染性和自身免疫性疾病中发生在体内，因此可以假设体内发生CD32A功能状态失调，导致CD32A的高表达，使中性粒细胞有效结合IC，从而导致这些疾病的组织损伤。从拟议的研究中获得的数据将有助于理解和设计抑制自身免疫性和感染性疾病中IC介导的组织损伤的治疗方法。

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.