Lipopolysaccharide and Sphingosine-1-Phosphate Cooperate To Induce Inflammatory Molecules and Leukocyte Adhesion in Endothelial Cells

Abstract

Given that TLRs and sphingosine-1-phosphate (S1P) are key players in inflammation, we explored the potential interplay between TLRs and S1P in the adhesion/inflammatory pathways in primary human endothelial cells. As determined by Western blot and flow cytometry, cells treated with LPS (a TLR4 ligand) and S1P showed significantly enhanced expression of adhesion molecules such as ICAM-1 and E-selectin compared with the effect of either ligand alone. Cell-type differences on E-selectin upregulation were observed. In contrast, no cooperation effect on ICAM-1 or E-selectin was observed with a TLR2/TLR1 ligand. Consistent with an increase in adhesion molecule expression, endothelial cell treatment with LPS plus S1P significantly enhanced adhesion of PBMCs under shear stress conditions compared with the effect of either ligand alone and exhibited comparable levels of cell adhesion strength as those after TNF-α treatment. Moreover, LPS and S1P cooperated to increase the expression of proinflammatory molecules such as IL-6, cyclooxygenase-2, and prostacyclin, as determined by ELISA and Western blot. The analysis of signaling pathways revealed the synergistic phosphorylation of ERK upon LPS plus S1P treatment of HUVEC and human aortic endothelial cells and cell-type differences on p38 and NF-κB activation. Moreover, pharmacological and small interfering RNA experiments disclosed the involvement of S1P1/3 and NF-κB in the cooperation effect and that cell origin determines the S1P receptors and signaling routes involved. Sphingosine kinase activity induction upon LPS plus S1P treatment suggests S1P– Sphingosine kinase axis involvement. In summary, LPS and S1P cooperate to increase proinflammatory molecules in endothelial cells and, in turn, to augment leukocyte adhesion, thus exacerbating S1P-mediated proadhesive/proinflammatory properties.

High-Density Lipoprotein–Associated Apolipoprotein M Limits Endothelial Inflammation by Delivering Sphingosine-1-Phosphate to the Sphingosine-1-Phosphate Receptor 1

“…43,44 As S1P, S1P1 has been linked to proinflammatory responses and anti-inflammatory responses. 10,11 However, the use of a specific aortic endothelium S1P1 knock-out mouse strongly supports the anti-inflammatory role of S1P1, 13 which is in consonance with our findings in human endothelium. Along the same line, HDL promotes the formation of endothelial adherent functions and endothelial barrier function through ApoM-bound S1P and the S1P1 receptor.…”

“…It is possible that the HDL size limits the ApoM and S1P1 interaction. S1P 0.01 µmol/L was not statistically significant for any of the treatments.Given that (1) exogenous stimulation of noncarrier-mediated S1P on endothelial cells is often described as a proinflammatory stimulus, [10][11][12] (2) most albumin molecules in plasma do not carry S1P, (3) S1P is largely bound to ApoM, and (4) ApoM-bound S1P is more potent anti-inflammatory than albumin-S1P, we conclude that ApoM takes up a prominent role in S1P biology and vascular homeostasis. …”

“…In this way, S1P is able to regulate several biological processes, such as immune cell trafficking, angiogenesis, endothelial cell migration, and endothelial barrier function. 9 The role of S1P in the regulation of vascular inflammation has been studied, and contradictory results have been obtained, for example, direct stimulation by S1P is reported to increase the abundance of adhesion molecules, whereas other studies [10][11][12] show that S1P inhibits tumor necrosis factor-α (TNF-α) induction of adhesion molecules, such as E-selectin, ICAM-1, or VCAM-1. …”

Filamin A Expression Negatively Regulates Sphingosine-1-Phosphate-Induced NF-κB Activation in Melanoma Cells by Inhibition of Akt Signaling

“…In contrast, in their counterpart A7 melanoma cells that express FLNA, S1P does not activate NF-B unless the expression of FLNA is downregulated. Several previous reports suggested that S1P can activate NF-B via S1PRs (14,15,(29)(30)(31)(32)(33)(34). Although in some studies, the S1PR involved was not identified and the concentration of S1P used was too high to substantiate the involvement of S1PR-mediated events (35), others have clearly implicated S1PR1 to -3 in different cell types.…”

“…Subsequently, specific antisense oligonucleotides were used to show that the activation of NF-B in HUVECs requires mainly S1PR3 and, to a much lesser extent, S1PR1 (15). Moreover, pharmacological and siRNA experiments substantiated the involvement of S1PR1 and S1PR3 in NF-B activation in cooperation with lipopolysaccharide (LPS) to induce inflammatory molecules and leukocyte adhesion in endothelial cells (29). Surprisingly, S1PRs have been linked not only to stimulatory pathways leading to the activation of NF-B but also to inhibitory pathways in HUVECs.…”