REGULATION OF PHAGOCYTE AND NK CELL C3 RECEPTOR FUNCTION

目标集中在两个密切相关的膜受体， CR 3和CR 4，CD 11b和CD 11 c，或Mac-1和p150，95。 这些Beta 2- 整合素分子介导多种吞噬功能， 缺乏白细胞粘附缺陷（LAD）与 慢性、危及生命的细菌感染。 尽管他们 尽管吞噬细胞的功能被公认为重要， 已知它们对NK细胞的功能或重要性。 一个有趣 其特征是它们介导细胞间粘附的可变能力 具有广谱配体的事件。 结合位点亲和力为 显然受附着在受体上的细胞骨架蛋白的调节 胞质结构域。 此外，细胞骨架还允许CR 3和 CR 4介导吞噬作用。 在当前的奖励期间，收购 介导吞噬作用或同型聚集的能力 与CR 3 β亚基CD 18的磷酸化有关。 蛋白激酶C的抑制阻断了这些CR 3依赖性功能。 拟议的具体目标是基于核心假设，即CR 3 和CR 4是识别不同配体的重要受体 由病原微生物（例如β-葡聚糖和LPS）、肿瘤 细胞（例如，旁路途径产生的固定iC 3b），或正常 组织反受体（如β-葡聚糖和LPS）。 NK细胞， 进一步假设β-葡聚糖的活化允许CR 3和 CR 4（CR 3/4）识别肿瘤并启动细胞毒性。 目标1 与未活化CR 3/4相关的细胞骨架蛋白 将中性粒细胞和单核细胞/巨噬细胞与这些蛋白质进行比较 与活化的CR 3/4相关，以确定哪些细胞骨架蛋白 负责触发激活状态。 合成肽 代表CD 18的胞质结构域将用于 溶解的吞噬细胞的亲和层析和直接结合 用纯化的细胞骨架蛋白进行的研究， 触发激活状态。 一种合成肽， CD 18的胞质结构域将用于两种亲和层析 溶解的吞噬细胞和纯化的直接结合研究 已知与β 1或β 3整联蛋白相互作用的细胞骨架蛋白。 用纯化蛋白质证明的细胞骨架蛋白缔合 将通过完整细胞的免疫荧光显微镜检查确认。 对于目标2，假设β-葡聚糖激活中性粒细胞， 单核细胞或NK细胞使CR3.4能够触发吞噬作用 和/或表达内源性配体的靶点的细胞毒性 或外源固定的iC 3b/C3 dg。 调查将确定是否 CR 3/4的活化是通过β-葡聚糖与CR 3/4的直接结合介导的， 或者是否存在独特的β-葡聚糖受体。 此外，尝试将 用于鉴定β-葡聚糖活化的CR 3配体， NK细胞对K562细胞的作用。 对于目标3，假设固定的iC 3b和 C3 dg存在于体内许多类型的人肿瘤细胞上， 乳腺癌粘蛋白的天然和诱导抗体的存在 促进C. 进一步 提出NK细胞或单核细胞CR 3/4与可溶性β- 葡聚糖将促进这种iC 3b/C3 dg靶向肿瘤的细胞毒性 细胞 以乳腺癌为重点，平行研究将检查乳腺癌 肿瘤中存在固定的C3片段和乳腺肿瘤细胞系 因为它们能够激活C并被β-葡聚糖激活的 NK细胞或单核细胞。 如果这一目标成功，它可能会导致一个新的 一种治疗乳腺癌的方法，其中可溶性β-葡聚糖 结合IL-2。

The objectives center on two closely related membrane receptors referred to as CR3 and CR4, CD11b and CD11c, or Mac-1 and p150,95. These Beta2- integrin molecules mediated a variety of phagocytic functions, and their absence in leukocyte adhesion deficiency (LAD) is associated with chronic, life-threatening, bacterial infections. Despite their recognized importance in the functions of phagocytic cells, little is known of their functions or importance on NK cells. An interesting feature is their variable ability to mediate intercellular adhesion events with a broad spectrum of ligands. Binding site affinity is apparently regulated by cytoskeletal proteins that attach to receptor cytoplasmic domains. In addition, the cytoskeleton also permits CR3 and CR4 to mediate phagocytosis. In the current award period, acquisition of the ability to mediate phagocytosis or homotypic aggregation was shown to be associated with phosphorylation of the beta-subunit of CR3, CD18. Inhibition of protein kinase C blocked these CR3-dependent functions. The proposed specific aims are based on the central hypothesis that CR3 and CR4 are important receptors for recognition of diverse ligands expressed by pathogenic microorganisms (e.g. Beta-glucan and LPS), tumor cells (e.g., fixed iC3b generated by the alternative pathway), or normal tissue counter receptors (e.g. Beta-glucan and LPS). With NK cells it is further hypothesized that activation with beta-glucan allows CR3 and CR4 (CR3/4) to recognize tumors and initiate cytotoxicity. For aim 1 cytoskeletal proteins associated with resting CR3/4 on unactivated neutrophils and monocyte/macrophages will be compared to those proteins associated with activated CR3/4 to determine which cytoskeletal proteins are responsible for triggering the activated state. A synthetic peptide representing the cytoplasmic domain of CD18 will be used for both affinity chromatography of solubilized phagocytes and for direct binding studies with purified cytoskeletal proteins are responsible for triggering the activated state. A synthetic peptide representing the cytoplasmic domain of CD18 will be used for both affinity chromatography of solubilized phagocytes and for direct binding studies with purified cytoskeletal proteins known to interact with Beta1 or Beta3 integrins. Cytoskeletal protein associations demonstrated with purified proteins will be confirmed by immunofluorescence microscopy with intact cells. For aim 2 it is hypothesized that Beta-glucan activation of neutrophils, monocytes, or NK cells make CR3.4 capable of triggering phagocytosis and/or cytotoxicity of targets that express either an endogenous ligand or exogenously fixed iC3b/C3dg. Investigations will be determine whether CR3/4 activation is mediated by direct binding of beta-glucan to CR3/4, or whether there is a distinct beta-glucan receptor. Also, attempts will be made to identify the CR3 ligand recognized by beta-glucan-activated NK cells on K562 cells. For aim 3 it is hypothesized that fixed iC3b and C3dg are present on many types of human tumor cells in vivo because of the presence of natural and induced antibodies to breast cancer mucin that promote activation of the classical pathway of C. It is further proposed that activation of NK cell or monocyte CR3/4 with soluble beta- glucan will promote the cytotoxicity of such iC3b/C3dg-targeted tumor cells. Focusing on breast cancer, parallel studies will examine breast tumors for the presence of fixed C3 fragments and breast tumor cell lines for their ability to activate C and be killed by beta-glucan-activated NK cells or monocytes. If this aim is successful, it may lead to a new form of therapy for breast cancer in which soluble Beta-glucan is combined with IL-2.