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

Abstract

Previous studies showed that P2 receptors are involved in neutrophil migration via stimulation of chemokine release and by facilitating chemoattractant gradient sensing. Here, we have investigated whether these receptors are involved in LPS-induced neutrophil transendothelial migration (TEM) using a Boyden chamber where neutrophils migrated through a layer of lipopolysaccharide (LPS)-stimulated human umbilical vein endothelial cells (HUVECs). In line with a role of P2 receptors, neutrophil TEM was inhibited by the P2 receptor antagonists suramin and reactive blue 2 (RB-2) acting on the basolateral, but not luminal, HUVECs' P2 receptors. HUVECs express P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 and P2Y 11 . The involvement of P2Y 4 was unlikely as this receptor is insensitive to suramin while P2Y 1 , P2Y 6 and P2Y 11 were excluded with available selective antagonists, leaving P2Y 2 as the only candidate. Indeed, the P2Y 2 knockdown in HUVECs inhibited neutrophil TEM compared to control HUVECs transfected with scrambled siRNA. Moreover, UTP, a P2Y 2 ligand, markedly potentiated LPS-induced TEM. Interestingly, IL-8 and ICAM-1 had a modest effect on neutrophil TEM in this 3 h assay which was significantly diminished by the inhibition of Rho kinase in HUVECs with Y27632. In summary, endothelial P2Y 2 receptors control the early LPSinduced neutrophil TEM in vitro via Rho kinase activation.

Neuroprotective roles of the P2Y2 receptor

“…Since both P2Y 2 Rmediated transactivation of growth factor receptors and integrins contribute to cell migration [21,24,91], it is intriguing to postulate that FLNa binding to the SH3-binding domains of the P2Y 2 R links nucleotide-induced EGFR transactivation to the RGD-dependent integrin signaling pathway that regulates cytoskeletal rearrangements required to increase cell motility. In other studies, P2Y 2 R-mediated monocyte diapedesis (i.e., transendothelial migration) has been shown to occur by disruption of intercellular adherens junctions, suggesting that cytoskeletal rearrangements promoted by the P2Y 2 R also can affect cell polarization [89,93,95,[101][102][103]. Therefore, these data suggest a mechanism whereby the tropism of monocytic cells (e.g., microglia) into damaged areas of the CNS can be induced by activation of P2Y 2 Rs.…”

“…However, mechanisms by which endothelial cells promote leukocyte diapedesis are less clear. In a recent in vitro diapedesis study, expression of the endothelial P2Y 2 R was found to be important for the transendothelial migration of neutrophils toward lipopolysaccharide (LPS), a chemoattractive component of gramnegative bacteria [102]. This study also demonstrated that WT and P2Y 2 R −/− mouse primary microglial cells were treated with oAβ (1 μM), incubated for 24 h, and supernatants were collected and analyzed for TNF-α and IL-1β by ELISA.…”

“…The process of leukocyte recruitment involves several steps: the emigration of leukocytes from bone marrow into the circulation, adhesion of circulating leukocytes to vascular endothelial cells, and diapedesis of leukocytes towards chemoattractants released at the site of injury or infection. Although the leukocyte P2Y 2 R is important for controlling the leukocyte migration step [143,239,240], other studies indicate that the endothelial P2Y 2 R promotes both the leukocyte adhesion step, by increasing the expression of VCAM-1 in endothelial cells [22,89,92] and the diapedesis step [102]. A postulated pathway for the regulation of leukocyte diapedesis by the P2Y 2 R is shown in Fig.…”

“…Our previous studies provide strong evidence that P2Y 2 Rs in endothelial cells regulate the binding and the transendothelial migration (i.e., diapedesis) of monocytic cells. As described above, P2Y 2 Rs mediate the Src-dependent transactivation of the vascular endothelial growth factor receptor-2 (VEGFR-2) in endothelial cells that promotes upregulation of monocyte-binding proteins (e.g., VCAM-1) and a decrease in endothelial adherens junction integrity [22,89,92,101,102]. Other studies have shown that microglia are attracted to and surround Aβ plaques in both human AD brain and rodent transgenic models that develop AD-like symptoms [228][229][230][231][232][233][234][235][236][237].…”

“…This study also demonstrated that WT and P2Y 2 R −/− mouse primary microglial cells were treated with oAβ (1 μM), incubated for 24 h, and supernatants were collected and analyzed for TNF-α and IL-1β by ELISA. Data represent means ± SEM (n03) **p<0.01 indicates a significant difference from untreated control diapedesis of neutrophils toward LPS required Rho kinase activity and was potentiated by treatment with UTP [102]. Nucleotides are released from leukocytes migrating toward chemoattractants [239], including Aβ 1-42 [224], and increased expression of P2Y 2 Rs in vascular endothelium of damaged tissue [244] regulates nucleotide-induced increases in [Ca 2+ ] i , phosphorylation of myosin light chain, and activation of Rho kinases [24,95].…”