Immune complex (IC) deposition in and around blood vessels is pathogenic in a number of immune diseases, and is accompanied by neutrophil accumulation and tissue damage. Described mechanisms of antibody triggered neutrophil accumulation include complement and endothelial dependent recruitment followed by IC triggered neutrophil activation. Under static conditions, neutrophil adhesion to ICs is supported by the low affinity Fcgamma receptors (FcgammaR) II and III, and Mac-1 (CD11b/CD18, CR3), a member of the beta2 integrin family. Mac-1 does not directly bind IgG but associates with FcgammaRII and III and sustains neutrophil adhesion to ICs. Our hypothesis is that ICs deposited in the vessel wall are early, primary effectors of both neutrophil capture and activation in immune mediated diseases and that low affinity FcgammaRs and Mac-1 on leukocytes are critical in this process. In support of this hypothesis, we have preliminary data that neutrophils can tether and immediately arrest/spread on ICs under physiologically relevant levels of fluid shear stress. Neutrophil tethering to ICs was as efficient as that to P-selectin, a well-established endothelial tethering receptor. This suggests that ICs alone are sufficient for tethering and arresting neutrophils in the absence of endothelial cell based adhesion receptors, complement or vessel associated signals. Furthermore, using FcgammaRIIIB, FcgammaRIIA and Mac-1 transfected cell lines, Mac-1 deficient neutrophils and relevant functional blocking antibodies, we demonstrate essential roles for FcgammaRIIIB and Mac-1 in neutrophil tethering to ICs which were revealed only under a physiologically relevant stimulus, shear stress. Analysis of the phosphorylation pattern of Mac-1 deficient neutrophils adherent to ICs suggests a defect in the phosphorylation of distinct proteins when compared with wild-type neutrophils. Thus Mac-1 associated signaling pathways may be important in sustaining neutrophil adhesion to ICs.
The aims of this proposal are to 1) Determine the molecular requirements for leukocyte tethering and adhesion to ICs under flow 2) Define Mac- 1 dependent intracellular signaling mechanisms that promote sustained neutrophil adhesion to ICs and 3) Examine IC-mediated leukocyte recruitment and the role of FcgammaRs and Mac-1 in this process, in vivo. Information gained from the proposed studies should contribute to our basic understanding of fundamental mechanisms modulating leukocyte trafficking in immune mediated disease.
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