| Lipopolysaccharide (LPS) is a major component of Gram-negative bacteria that can activate immune cells to produce excessive amounts of inflammatory mediators including tumor necrosis-alpha (TNF-alpha), interleukin-1 (IL-1), interleukin-2 (IL-2), and nitric oxide (NO), thereby contributing to the pathophysiological effects of LPS. This thesis outlines the potential role that cell-type specificity, extracellular nucleotides, and P2 purinergic receptors play in regulating LPS action.;We initially tested the hypothesis that varying sensitivities to LPS by different cell-types is, in part, due to a varying capacity of LPS to activate certain signaling molecules. In vitro studies on LPS action revealed that LPS potently stimulates ERK1/2 activation in RAW 264.7 macrophages but does not activate ERK1/2 in BV-2 microglia, suggesting cell type specificity in LPS signaling. Furthermore, these differences in LPS-induced responses between cell types, together with the observations that (a) BzATP, an agonist of the P2X7 receptor, promotes LPS-stimulated NO production and IL-beta release and (b) P2X7 blockade inhibits LPS-stimulated NO and TNF-alpha production in RAW 264.7 macrophages prompted an in vivo analysis of the role of P2X7 in LPS-initiated events. These studies were also driven by previous reports that had revealed that the P2X and P2Y receptor ligand, 2-MeS-ATP, can protect mice from an LPS-induced death and that this outcome correlates with reduced serum levels of TNF-alpha and IL-1. To this end, we tested the ability of agonists to specific P2X and/or P2Y receptors to protect mice from an LPS-induced death and to regulate LPS-stimulated cytokine production. These studies revealed that only agonists to both P2X and P2Y receptors can protect mice from an LPS-induced death. Furthermore, prevention of death by 2-Cl-ATP correlates with reduced serum levels of LPS-stimulated TNF-alpha, but not with changes in serum IL-2 levels. However, inhibition of TNF-alpha or IL-2 release is not sufficient to render protection from a lethal challenge of LPS, given that BzATP inhibits LPS-stimulated production of both TNF-alpha and IL-2 but is unable to protect mice from an LPS-induced death. Altogether, these data indicate that both P2X and P2Y receptors play critical immunological roles that may be linked to the control of septic shock. |
