HEMIN MODIFIED LPS EFFECTS ON PMN FUNCTION

Lipopolysaccharides (LPS) are among the most potent cell agonists known: LPS can act at picomolar concentrations to trigger the release of toxic oxygen radicals and cytokines from inflammatory cells. The LPS from the opportunistic oral pathogen, Porphyromonas gingivalis (Pg), possess little proinflammatory activity according to some reports, while other reports contradict these findings. Our published studies indicate that the level of hemin in the growth media can have a profound effect on the LPS of Pg. Hemin replete growth conditions (H+)result in specific morphologic and antigenic modifications of Pg LPS; moreover, hemin-replete LPS from Pg (H+LPS) is more biologically active when compared with LPS from hemin- depleted Pg (H-LPS). This activity includes increased PMN priming and increased binding to LPS-binding protein (LBP). Interestingly, H+Pg whole cells are more readily phagocytosed than H-Pg, but are not killed by PMN intracellularly, suggesting that fundamental changes to the outer membrane of Pg are induced under H+ conditions. We propose to test the following hypotheses: Hypothesis 1: Under hemin-replete growth conditions, Pg produces an LPS moiety (H+LPS) that is different structurally from that produced under hemin-depleted growth conditions (H-LPS) Hypothesis 2: The increased biological activity of H+LPS is dependent on its binding affinity for LPS-binding protein (LBP) and its effects on CD14 expression on PMN> We propose the following specific aims to test these hypotheses: Specific aim 1: To characterize H+LPS and H-LPS from Pg structurally and biochemically. Specific aim 2: To determine the roles for LBP affinity and/or increased PMN CD14 expression in activation of PMNs by H+LPS from Pg. It is anticipated that the additional data and publications generated in these studies will help the principal investigator in preparing a successful F.I.R.S.T. award application.