REGULATION OF PHAGOCYTE AND NK CELL C3 RECEPTOR FUNCTION

目标集中在两个密切相关的膜受体上。 转成CR3和CR4、CD11b和CD11c或Mac-1和p150、95。这些Beta2- 整合素分子介导多种吞噬功能，其 白细胞黏附缺陷(LAD)缺失与 慢性、危及生命的细菌感染。尽管他们 承认吞噬细胞功能的重要性，很少是 已知它们在NK细胞上的功能或重要性。一个有趣的故事 特征是它们调节细胞间黏附的能力不同 具有广泛配基的事件。结合部位亲和力为 表面上受附着在受体上的细胞骨架蛋白调控 胞质结构域。此外，细胞骨架还允许CR3和 CR4介导吞噬作用。在本授权期内，收购 介导吞噬或同型聚集的能力 与CR3、CD18的β亚基的磷酸化有关。 蛋白激酶C的抑制阻断了这些依赖CR3的功能。 提出的具体目标是基于CR3的中心假设 和CR4是识别多种配体的重要受体 由致病微生物(如β-葡聚糖和脂多糖)、肿瘤表达 细胞(例如，通过替代途径产生的固定的IC3b)，或正常 组织对抗受体(如β-葡聚糖和脂多糖)。对于NK细胞来说 进一步假设用β-葡聚糖激活允许CR3和 CR4(CR3/4)识别肿瘤并启动细胞毒作用。对于目标1 与静息CR3/4相关的细胞骨架蛋白 中性粒细胞和单核/巨噬细胞将与这些蛋白质进行比较。 与激活的CR3/4相关，以确定哪些细胞骨架蛋白 负责触发激活状态。一种合成肽 代表CD18的细胞质结构域将用于这两个 溶解吞噬细胞的亲和层析及其直接结合 对纯化的细胞骨架蛋白的研究负责 触发激活状态。一种合成肽，代表 CD18的胞浆结构域将用于两种亲和层析 对溶解的吞噬细胞和与纯化的 已知与β1或β3整合素相互作用的细胞骨架蛋白。 细胞骨架蛋白与纯化蛋白的相互作用 将通过免疫荧光显微镜与完整的细胞进行确认。 对于目标2，假设中性粒细胞的β-葡聚糖激活， 单核细胞或NK细胞使CR3.4能够触发吞噬功能 和/或表达内源性配体的靶标的细胞毒性 或外源固定的IC3b/C3dg。调查将确定是否 CR3/4的激活是通过β-葡聚糖直接与CR3/4结合而介导的， 或者是否存在一种独特的β-葡聚糖受体。此外，尝试将 识别由β-葡聚糖激活的CR3配体 K562细胞上的NK细胞。对于目标3，假设固定的IC3b和 C3dG存在于体内许多类型的人类肿瘤细胞上，因为 乳腺癌粘液天然抗体和诱导抗体的存在 促进了C的经典途径的激活，它进一步 建议用可溶性β-内毒素激活NK细胞或单核细胞CR3/4。 葡聚糖将促进这种iC3b/C3dg靶向肿瘤的细胞毒作用 细胞。聚焦于乳腺癌，平行研究将检查乳房 肿瘤中存在固定的C3片段和乳腺肿瘤细胞系 因为他们有能力激活C并被β-葡聚糖激活的人杀死 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 治疗乳腺癌的一种形式，其中可溶性β-葡聚糖是 联合IL-2治疗。

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.