Extracellular nucleotides mediate LPS-induced neutrophil migration in vitro and in vivo

Abstract

Extracellular nucleotides are emerging as important inflammatory mediators. Here, we demonstrate that these molecules mediate LPS-induced neutrophil migration in vitro and in vivo. Apyrase, a nucleotide scavenger, reduced the ability of LPS-stimulated monocytes to recruit neutrophils, as assayed using a modified Boyden chamber. This effect resulted from the inhibition of IL-8 release from monocytes. Furthermore, LPS-induced IL-8 release by monocytes was attenuated significantly by P2Y 6 receptor antagonists, RB-2 and MRS2578. Reciprocally, UDP, the selective P2Y 6 agonist, induced IL-8 release by monocytes. As for LPS, the media of UDPstimulated monocytes were chemotactic for neutrophils; IL-8 accounted for ~50% of neutrophil migration induced by the media of LPS-or UDP-treated monocytes in transendothelial migration assays. It is important that in the murine air-pouch model, extracellular nucleotides were instrumental in LPS-induced neutrophil migration. Altogether, these data imply that LPS induces the release of nucleotides from monocytes and that by autocrine stimulation, the latter molecules regulate neutrophil migration caused by Gram-negative bacteria, suggesting a proinflammatory role of extracellular nucleotides in innate immunity.

The P2 receptor antagonist PPADS abrogates LPS-induced neutrophil migration in the murine air pouch via inhibition of MIP-2 and KC production

“…In keeping with these data, mice deficient in ATP-dependent P2Y 2 receptor exhibit an impaired neutrophil recruitment in sepsis (Inoue et al, 2008). Nucleotides can also induce neutrophil migration via activation of IL-8 secretion by human monocytes Kukulski et al, 2007).…”

“…Air pouches were pre-formed on the dorsum of female CD-1 mice (8-12 week old, Charles River) as before (Kukulski et al, 2007). The tail vein injection (iv) of P2 receptor antagonists (PPADS, suramin or RB-2; 200 μM in 0.2 ml) or apyrase (6 U in 0.2 ml) was performed ~2-3 min prior to the induction of inflammation by subcutaneous (sc) injection of LPS, Pam 3 CSK 4 , flagellin (10 ng in 1 ml) or mouse recombinant MIP-2 (2 ng in 1 ml) in the air pouch, as indicated.…”

“…These nucleotides are natural agonists of P2Y 2 (UTP) and P2Y 6 (in mouse activated by either UDP or UTP; Bar et al, 2008;Kauffenstein et al, in press) receptors that have been shown to control neutrophil migration in vivo and in vitro Chen et al, 2006;Inoue et al, 2008;Kukulski et al, 2007). UTP is also a ligand of P2Y 4 receptor which is highly sensitive to PPADS.…”

Endothelial P2Y2 receptor regulates LPS-induced neutrophil transendothelial migration in vitro

“…Distinct P2Y receptors have been implicated in LPS-induced inflammation. For example, we have previously demonstrated that in human monocytes, LPS-induced IL-8 release is mediated via activation of P2Y 6 receptors (Warny et al, 2001;Kukulski et al, 2007). As endothelial cells such as human umbilical vein endothelial cells (HUVECs) express various P2Y receptor subtypes (P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , and P2Y 11 ) and release large quantities of IL-8 in response to LPS (Beck et al, 1999;Kukulski et al, 2007), we hypothesized that these receptors might trigger neutrophil TEM via IL-8 release.…”

“…For example, we have previously demonstrated that in human monocytes, LPS-induced IL-8 release is mediated via activation of P2Y 6 receptors (Warny et al, 2001;Kukulski et al, 2007). As endothelial cells such as human umbilical vein endothelial cells (HUVECs) express various P2Y receptor subtypes (P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , and P2Y 11 ) and release large quantities of IL-8 in response to LPS (Beck et al, 1999;Kukulski et al, 2007), we hypothesized that these receptors might trigger neutrophil TEM via IL-8 release. We indeed found that endothelial nucleotide receptors are instrumental for LPSinduced neutrophil TEM in vitro, but interestingly, this migration was regulated mainly by endothelial Rho kinase and not by IL-8 which was not secreted in significant amounts during the course of the TEM assay performed in this work (3 h).…”