| Lipopolysaccharide (LPS) from Gram-negative bacteria is a powerful modulator of innate immunity by stimulating the production of pro-inflammatory mediators and cytokines that are critical for macrophage function, but can also lead to the detrimental consequences seen in septic shock. Evidence that extracellular nucleotides can influence LPS-mediated signaling events, cytokine production, and mortality via the activation of nucleotide receptors suggests that nucleotides have an important, but poorly defined, role in macrophage biology. The nucleotide receptor P2X7 is widely expressed in leukocytes and in macrophages P2X7 activity is linked to inflammatory mediator production and morphological changes. However, the molecular events associated with these processes remain unclear. Because mitogen-activated protein (MAP) kinases can influence inflammatory gene expression and cellular morphology via cytoskeletal architecture, it was hypothesized that MAP kinases contribute to nucleotide-mediated inflammatory responses.; These studies focus on the critical P2X7-dependent molecular events that contribute to macrophage function with regard to cytoskeletal reorganization and transcription of two pro-inflammatory genes, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). With respect to these mechanisms, the current studies demonstrate that P2X7 agonists promote the activation of the MAP kinases ERK1/ERK2, p38, and JNK, which are differentially regulated by intracellular calcium, protein kinase C, and production of reactive oxygen species (ROS). The activation of p38, in concert with the small molecular weight G-protein Rho, controls P2X7-dependent morphological changes, cytoskeletal reorganization, and membrane blebbing. Because the production of nitric oxide (NO) by iNOS and synthesis of prostaglandins by COX-2 contribute to LPS-mediated inflammation, the functional interaction between P2X7 and LPS-mediated pathways was examined. These studies demonstrate that LPS-stimulated ROS production, iNOS expression and NO production, and COX-2 expression are increased in the presence of P2X7 agonists. Furthermore, the activation of the transcription factors NF-κB and cyclic AMP response element binding protein (CREB) was observed, which likely contributes to expression of these genes. Although the ERK1/ERK2 pathway negatively regulates NO production, inhibition of ERK abolished P2X7-dependent CREB activation and COX-2 expression, suggesting differential regulation of inflammatory genes by MAP kinases. In conclusion, P2X7-dependent MAP kinase activation contributes to macrophage inflammatory functions and enhances macrophage activation following an LPS challenge. |
