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.
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