Through a unique form of innate immunity, keratinocytes from humans and mice respond to the bacterial product, lipopolysaccharide (LPS), present in the cell walls of Gram-negative bacteria by secreting inflammatory mediators. The overall objective of the proposed research herein is to define the keratinocyte LPS receptors, LPS-associated functions and signaling mechanisms that occur in this innate keratinocyte anti-bacterial defense system so that patients, who develop a functional loss of this skin-defense system leading up to recurrent bacterial skin injections, may be helped to avoid progressions to bacterial cellulitis and sepsis. We propose: (1) To identify LPS receptors on human and murine keratinocytes in vitro, in situ and in vivo and to determine what agents modulate their expression. Preliminary data shown that the membrane- bound LPS receptors, CD143, CD11a/CD18, CD11b/CD18 and CD11c/CD18 are expressed on human and mouse keratinocytes similar to macrophages. We will use primary keratinocytes and cell lines to further characterize and define the control of the expression of various LPS receptors with several agents and cytokines. We will induce and modulate the expression of LPS receptors in skin organ cultures and in live skin. Keratinocytes from different LPS receptor knock-out mice will be used for comparison. (2) To characterize cellular functions induced by LPS activation of human and murine keratinocytes by determining what cytokines by determining what cytokines and inflammatory mediators are specifically made in response to bacterial product and to define how these functions can be controlled with agents that modulate the LPS receptors or affect their interactions with LPS. With various methods, we will define the array of inflammatory cytokines and mediators, such as nitric oxide, that are produced following keratinocyte activation with LPS. Keratinocyte functions will be monitored following cell treatments that affect LPS receptor expression to help evaluate which LPS receptors are the most important for the gene promoter transcriptional activators, NF- kappaB and NF-IL-6, and up-regulation of CD14-associated type-2 Toll- like receptors following LPS receptor triggering in human and murine keratinocytes and how these events can be controlled through LPS receptor modulation. Cell activation by LPS leads to induction of calcium mobilization across the cell membrane, up-regulation of Toll-like receptor 2 (TLR2) molecules which trigger nuclear translocation of transcriptional activators, such as NF-kappaB and NF-IL-6, which in turn bind cytokine gene promoters. We will attempt to show with gel-shift assays. Western blots and quantitative RT-PCR that these signaling agents play an important role in LPS-induced activation of keratinocytes.
