MONOCYTE PATHOGENESIS--RESPIRATORY BURST OXIDASE

Cleveland Clinic Foundation, Cleveland OH

Investigators

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Abstract

Monocytes are critical for normal immune function, both for innate and specific immunity. They are also important components of inflammatory responses. Monocytes can be activated by a variety of stimuli to produce superoxide anion (O2-). Superoxide anion production appears to be critical for normal and pathologic contributions of this leukocyte and is generated by a multi- component enzyme complex, the respiratory burst oxidase (RB0). In this application, we propose to study in detail the regulation of the RB0 in activated human monocytes. Little is known about the regulation of this complex in monocytes, other than the regulation appears to be substantially different from that observed in neutrophils, the common model used for studies of this enzyme complex. We have recently shown that O2-production by activated monocytes is essential for monocytes to oxidize LDL, a process believed to have important pathologic roles in inflammation and particularly in atherogenesis. We have also shown that activation-induced PKC and cPLA2 activities are required for O2-production. Here we propose to rigorously evaluate the mechanisms for the regulation of this critical RBO complex by these important signal-transducing enzymes. In Aim 1 we will test the hypothesis that cPLA2 (through arachidonic acid) regulates phosphorylation, translocation and/or interaction of RBO components. In Aim 2 we will identify the PKCalpha-dependent phosphorylation site on cPLA2 that regulates cPLA2 activity and ultimately controls the activity of the RBO. Finally, in Aim 3 we will test the hypothesis that an isoform of PKC, other than PKCalpha or PKCbeta, is involved in regulating RBO activity. These studies will elucidate important regulatory pathways that control this critical enzyme complex. The novelty of these studies is derived from our unique opportunity to specifically dissect the roles of these pathways in intact cells by virtue of the fact that monocytes are very conducive to antisense ODN manipulation of specific protein expression. These studies will substantially influence our appreciation of the modes of regulation of the RB0 complex and will likely suggest specific and selective means for modulating its activity.