Regulationof Chemoattractant Activation of Neutrophils

Montana State University - Bozeman, Bozeman MT

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Abstract

The long term goal of the proposed work is to understand the molecular basis of chemoattractant activation of human neutrophils. Neutrophil invasion is the hallmark of inflammation and an understanding of how these cells manage to find their way from the blood to a site of injury or infection without releasing their arsenal of microbicidal products until arrival, remains unknown. Our efforts have suggested that functional plasma membrane domains play a role in this process, because in such domains, N-formyl peptide chemoattractant receptors (FPR) are exposed to signal transduction partners and regulators of their function and interaction. These surface microdomains remain uncharacterized. The working hypothesis of this proposal is that plasma membrane domains play an essential role in the regulation of neutrophil activation by providing local environments that are compositionally rich in signal transduction partners, adaptors, and regulators of FPR function. This environment controls access of FPR to FPR-interactive partners and thus FPR function. To test this hypothesis, the protein and lipid composition of up to 4 plasma membrane domains will be determined by modern methods of proteomics and lipid analysis utilizing HPLC and mass spectrometry. The compositions of the domains will be examined for four defined activation states mediated by occupancy of FPR by formyl peptide fMLF and compared to Triton X-100-insoluble membrane rafts produced from the same fractions. The functional state and molecular associations of FPR in each of its resident domains will also be determined. Residency in these domains will be correlated to location in the cell as determined by immunofluorescence microscopy, using FPR-specific monoclonal antibodies (Mab), sensitive to activation state of the receptor. Additionally, the location and state of the receptors during migration up simple chemoattractant gradients in Zigmond chambers and or across epithelial monolayers grown on transwell chambers will be determined by confocal immunofluorescence microscopy. Finally, new methods for FPR purification will be used to generate new Mab probes of FPR. Understanding, how this complex regulation occurs should provide insight about how host defense might be enhanced or how inflammatory damage might be mitigated.