The sepsis syndrome and sepsis induced Acute Respiratory Distress Syndrome (ARDS), associated with expo- sure to LPS, are important clinical entities without available specific therapies. The recent identification of the Toll- like receptors (TLRs), in particular TLR2 and TLR4 as LPS receptors has advanced our understanding of the initiation of signaling after LPS exposure. We hypothesize that TLR4 is the predominant receptor responsible for LPS induced NF-KB and/or p38 activation in the neutrophil with the signaling pathway involving IRAK 2 and M, syk and Rac2. Although, alternatively, LPS signaling may occur through TLR2 with less avidity and with involvement of other IRAK sub-species, tyrosine kinases, or small G proteins. We show here that human neutro- phils and PLB-985 cells express mRNA for TLRI-6 and express TLR2 protein. In addition, we show here in PLB-985 cells, that IRAK, the tyrosine kinase syk, and the small protein Rac2 associate with TLR2 at baseline and after LPS exposure, suggesting their involvement in LPS signaling through TLR2. We propose to investigate in neutrophils, both human and murine, and the PLB- 985 cell line: 1. the role of TLR2 and TLR4 in NF-KB and p38 activation, including their interdependence, 2. the macromolecular complex which associate with TLR2 or TLR4 after LPS exposure, and 3. the role and activation of IRAK 2 & M, the tyrosine kinases syk and lyn, and the small G proteins Rac2 and Cdc42 in LPS signaling. To accomplish these goals, we will develop inducible antisense retroviral techniques for the creation of antisense TLR2, TLR4, and IRAK ex- pressing cell lines, dominant negatives for TLR2, TLR4, and IRAK M, and novel immunoprecipitation techniques for 2D gel electrophoresis to examine the macromolecular complexes associated with TLR2, TLR4, and the IRAK subspecies. I will also develop techniques in 2D gel electrophoresis and protein identification by mass spec- trometry which will benefit future investigations into signaling pathways. An improved understanding of the recognition of LPS, and the signaling pathways initiated by LPS, in the neu- trophil are important to improve the understanding of the underlying pathophysiology of sepsis syndrome and sepsis induced ARDS.
