WATCHING TARGET CELL OXIDATION AND CYTOLYSIS

这个研究项目的长期目标是了解人类是如何 PMN是由免疫复合体触发的。我们将检验这一假设 Fc受体-补体受体3型(CR3)相互作用产生 超氧化物生成和颗粒所需的钙信号 放手。我们将首先测试物理和功能之间的关联 CR3和FcRIII。标记PMN的共振能量转移研究将 被用来测试它们的物理接近程度。的功能关联 CR3和FcRIII将使用不溶性免疫复合体和 F(ab‘)2段抗FcRIII抗体作为刺激物。抗CR3光纤 而阻止CR3-FcRIII共同封顶(或对照)的糖将是 测试它们影响跨膜钙、信号和 超氧化物生产。LAD(白细胞黏附缺陷)中性粒细胞 缺乏CR3的患者也将接受类似的检查。配基-和 第二信使依赖的跨膜连接的形成 FcRII、FcRIII和CR3以及微丝将使用 本实验室发展的微观能量转移方法。这个 钙在超氧化物和颗粒释放中的作用将被直接测试 使用细胞内钙离子螯合剂BAPTA。经BAPTA治疗的PMN将 与免疫球蛋白调理的红细胞孵育。超氧化物的输送 将使用Soret和荧光显微镜对目标进行跟踪。 BAPTA抑制超氧化物歧化和乳铁蛋白(一种特异的 颗粒标记物)靶向递送和离子霉素和 将研究用钙来解除抑制作用。我们预料到 这些研究将提供详细的免疫分子机制。 复合体介导的中性粒细胞炎症触发。获得的洞察力 这一研究方案可能会对开发合理的药物有所帮助 控制炎症性疾病的疗法。

The long range goal of this research program is to understand how human PMNs are triggered by immune complexes. We will test the hypothesis that Fc receptor-complement receptor type 3 (CR3) interactions generate a calcium signal that is required for super-oxide production and granule release. We will first test the physical and functional association of CR3 and FcRIII. Resonance energy transfer studies of labeled PMNs will be used to test their physical proximity. The functional association of CR3 and FcRIII will be tested using insoluble immune complexes and F(ab')2 fragments of anti-FcRIII antibodies as stimuli. Anti-CR3 Fabs and saccharides that block CR3-FcRIII co-capping (or controls) will be tested for their ability to affect transmembrane calcium, signaling and superoxide production. PMNs from LAD (leukocyte adhesion deficiency) patients, which lack CR3, will be similarly examined. The ligand- and second messenger-dependent formation of transmembrane linkages between FcRII, FcRIII and CR3 and microfilaments will be tested using a microscopic energy transfer method developed in this laboratory. The role of calcium in superoxide and granule release will be directly tested using the intracellular calcium chelator BAPTA. BAPTA-treated PMNs will be incubated with IgG-opsonized erythrocytes. The delivery of superoxide to the targets will be followed using Soret and fluorescence microscopy. The ability of BAPTA to inhibit superoxide and lactoferrin (a specific granule marker ) delivery to targets and the ability of ionomycin and calcium to relieve the inhibition will be studied. We anticipate that these studies will provide a detailed molecular mechanism of immune complex-mediated inflammatory triggering of PMNs. The insights gained from this research program may be useful in developing rational drug therapies to control inflammatory diseases.