Pro-inflammatory role of P2Y6 receptor signalling during vascular inflammation

Abstract

During a systemic inflammatory response endothelial-expressed surface molecules have been strongly implicated in orchestrating immune responses. Previous studies have shown enhanced extracellular nucleotide release during acute inflammatory conditions. Therefore, we hypothesized that endothelial nucleotide receptors could play a role in vascular inflammation. To address this hypothesis, we performed screening experiments and exposed human microvascular endothelia to inflammatory stimuli, followed by measurements of P2Y or P2X transcriptional responses. These studies showed a selective induction of the P2Y(6) receptor (> 4-fold at 24 hours). Moreover, studies that used real-time reverse transcription-polymerase chain reaction, Western blot analysis, or immunofluorescence confirmed time- and dose-dependent induction of P2Y(6) with tumor necrosis factor alpha or Lipopolysaccharide (LPS) stimulation in vitro and in vivo. Studies that used MRS 2578 as P2Y(6) receptor antagonist showed attenuated nuclear factor alphaB reporter activity and proinflammatory gene expression in human microvascular endothelial cells in vitro. Moreover, pharmacologic or genetic in vivo studies showed attenuated inflammatory responses in P2Y(6)(-/-) mice or after P2Y(6) antagonist treatment during LPS-induced vascular inflammation. These studies show an important contribution of P2Y(6) signaling in enhancing vascular inflammation during systemic LPS challenge and implicate the P2Y(6) receptor as a therapeutic target during systemic inflammatory responses.

Nukleotid Signaluebertragung spielt eine wichtige Rolle waehrend vaskulaerer Entzuendungsreaktionen. In dieser Arbeit haben wir Veraenderungen im Expressionsmuster endothelialer Nukleotid-Rezeptoren (P2-Rezeptoren) untersucht und konnten eine selektive Hochregulation des P2Y6-Rezeptors zeigen. Wir konnten ebenfalls zeigen, dass verschiedene infammatorische Stimuli (TNF-alpha, LPS) zu einer signifikanten Hochregulation der P2Y6-Rezeptor mRNA in vitro und in vivo fuehren. Darueber hinaus konnten wir durch Antagonisten-Studien zeigen, dass die Signaluebertragung durch den P2Y6-Rezeptor relevant fuer die Amplifikation der durch TNF-alpha induzierten NF-alphaB Aktivierung ist. Zusammengefasst deuten unsere Daten darauf hin, dass der P2Y6-Rezeptor eine wichtige Rolle bei der Signalamplifikation in Entzuendungsreaktionen spielt.