Activation of the human neurtrophil

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DESCRIPTION (provided by applicant): Secretion of oxygen radicals by neutrophils is essential for microbicidal activity, but can also give rise to the tissue damage of inflammation in diseases such as sepsis, rheumatoid arthritis, asthma. Ligands such as tumor necrosis factor (TNFa), immune complexes and chemotactic peptides such as fMet-Leu-Phe, elicit secretion of oxygen radicals and granule contents and adherence to a surface. We hypothesize that discrete protein kinase C (PKC) isotypes play both positive and negative roles in signaling for proinflammatory responses. Proinflammatory signaling can be regulated by PKC isotypes at numerous levels including: a) the receptor, b) generation and regulation of i) second messenger Ca2+, and ii) lipid cofactors, and c) assembly of complexes such as the NADPH oxidase for O2-generation. Using antisense strategies to deplete specific PKC isotypes in differentiated HL60 (dHL60), we demonstrated positive signaling roles for both a-PKC and b-PKC for O2- generation. Conversely, b-PKC downregulates ligand-initiated Ca2+ uptake, and 5-PKC negatively regulates the p60TNF receptor (p60TNFR) function. Antisense strategies will be used to selectively deplete specific PKC isotypes, to establish roles for lipids and lipid-dependent PKC isotypes in signal transduction for proinflammatory responses. We will: I Establish selective roles for Ca2+-dependent a-PKC and b-PKC isotypes in activation of the NADPH oxidase, and phosphorylation of p47phox. II Determine selective roles for Ca2+-dependent a-PKC and b-PKC isotypes in activation of adherence and degranulation and phosphorylationof MARCKS. III Determine the PKC isotypes responsible for regulation of ligand-initiated mobilization of intracellular Ca2+ and store operated Ca2+ channels. IV Examine selective roles for Ca2+-dependent a-PKC and b-PKC isotypes in the regulation of ligand initiated signalling involving effector enzymes PLCb/PLCg, and PLD1, and enzymes involved in the regeneration of PIP2. V Establish the role of d-PKC in downregulation of signalling pathways triggered by the p60TNFR. Determine if d-PKC acts on a serine residue(s) in the juxtamembrane region or the death domain.