Abstract 1045 Cytokines: Intracellular Signaling Platform, Tuesday, 5/4

LPS-mediated immune responses, including activation of monocytes, macrophages, and endothelial cells (EC) play an important role in the pathogenesis of gram-negative bacteria-induced sepsis syndrome. Activation of NF-κB is thought to be required for cytokine release from LPS-responsive cells, a critical step for endotoxic effects. The molecular mechanisms of the signaling cascade induced by LPS to activate NF-κB are unknown. Furthermore, the signaling LS receptor is still unidentified. Recent studies suggested that a toll like receptor (TLR) might be a signaling receptor that can be activated by LPS. Here we investigated the role and involvement of IL-1 and TNF-signal transducer molecules in LPS signaling in human dermal microvessel EC (HDMEC), and THP-1 monocytic cells. Cell were transfected with reporter genes pCMV-gal and ELAM-NF-κB-luciferase. LPS stimulation of HDMEC and THP-1 cells initiated an IL-1 receptor-like NF-κB signaling cascade (about 10-fold induction of NF-κB luciferase activity). In transient co-transfection experiments, dominant-negative mutants of the IL-1 signaling pathway, including MyD88, IRAK, IRAK2, and TRAF6 inhibited both IL-1- and LPS-induced NF-κB-luciferase activity in both cell types (mean inhibition of about 70-80%, in triplicate experiments). LPS-induced NF-κB activation was not inhibited by a dominant-negative mutant of TRAF2 which is involved in TNF signaling. LPS-induced activation of NF-κB responsive reporter gene was not inhibited by IL-1 receptor antagonist. We have also found that HDMEC and THP-1 express both TLR2 and TLR4, by immunohistochemistry, western blotting and RT-PCR. These findings show for the first time that the LPS signaling cascade uses an analogous molecular framework for signaling as IL-1 in LPS-responsive cells i.e., mononuclear phagocytes and EC, an suggest that a signal transduction molecule in the LPS receptor complex may belong to IL-1 receptor/TLR super family.

(Supported by NIH, A140275 to M.A.)