Reactive oxygen species (ROS) formed by the NADPH oxidase of phagocytic cells play a primary role in host defense and inflammation. A central feature of leukocyte activation upon encountering microbial products is a two-phased response, leading at first to a pre-activated, primed state, which generates a vastly enhanced response upon a secondary, activating stimulus. The molecular patterns associated with microbial pathogens are recognized by Toll-like receptors (TLRs). This recognition leads to TLR activation and signaling events that prime neutrophils for subsequent stimulation by chemoattractants. To understand the molecular basis for this coordinated neutrophil response in respect to ROS generation, we will investigate the signaling pathways from the receptors involved to the final sequence of NADPH oxidase activation. ? ? We have shown that oxidase activity is regulated by the GTPase Rac2 and the effector p21-activated kinase (Pak). Our studies so far point to a role of Pak in several aspects of the fMLP-induced respiratory burst including multiple phosphorylation events and association with cytochrome b558. The involvement of Pak in NADPH oxidase assembly at the membrane will be investigated in detail. Studies with Pak-null or transgenic mice harboring a Pak kinase inhibitory fragment will define Pak function in vivo. ? ? The signaling sequences emanating from activated TLRs seem to diverge between monocytes and neutrophils, leading to ROS generation versus priming. We hypothesize that Rac-regulated pathways are key elements for understanding how different innate immune cells regulate bacterial killing and we will investigate the molecular mechanisms involved. A common trait connecting TLR2 activation, priming and chemoattractant-induced ROS generation is the dependence on tyrosine phosphorylations. We will elucidate potential associations of TLRs or formyl peptide receptors with tyrosine kinase receptors and evaluate how these complexes relay their signals in neutrophils and microglia. These studies will yield novel insights into the overall control of oxidant formation by innate immune cells.
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