Sphingosine-1-phosphate (S1P) enhances glomerular endothelial cells activation mediated by anti-myeloperoxidase antibody-positive IgG

Abstract

Cumulating evidences suggested an important role of sphingosine-1-phosphate (S1P) and its receptors in regulating endothelial barrier integrity. Our previous study revealed that the circulating S1P levels and renal expression of S1PRs correlated with disease activity and renal damage in patients with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). This study investigated the role of S1P and its receptors in myeloperoxidase (MPO)-ANCA-positive IgG-mediated glomerular endothelial cell (GEnC) activation. The effect of S1P on morphological alteration of GEnCs in the presence of MPO-ANCA-positive IgG was observed. Permeability assay was performed to determine endothelial monolayer activation in quantity. Both membrane-bound and soluble ICAM-1 and VCAM-1 levels were measured. Furthermore, antagonists and/or agonists of various S1PRs were employed to determine the role of different S1PRs. S1P enhanced MPO-ANCA-positive IgG-induced disruption of tight junction and disorganization of cytoskeleton in GEnCs. S1P induced further increase in monolayer permeability of GEnC monolayers in the presence of MPO-ANCA-positive IgG. S1P enhanced MPO-ANCA-positive IgG-induced membrane-bound and soluble ICAM-1/VCAM-1 up-regulation of GEnCs. Soluble ICAM-1 levels in the supernatants of GEnCs stimulated by S1P and MPO-ANCA-positive IgG increased upon pre-incubation of S1PR1 antagonist, while pre-incubation of GEnCs with the S1PR1 agonist down-regulated sICAM-1 level. Blocking S1PR2-4 reduced sICAM-1 levels in the supernatants of GEnCs stimulated by S1P and MPO-ANCA-positive IgG. Pre-incubation with S1PR5 agonist could increase sICAM-1 level in the supernatants of GEnC stimulated by S1P and MPO-ANCA-positive IgG. S1P can enhance MPO-ANCA-positive IgG-mediated GEnC activation through S1PR2-5.

Keywords: ANCA; sphingosine-1-phosphate; vasculitis.

Figure 1

S1P could induce alterations in cellular morphology of GEnCs in the presence of MPO‐ANCA‐positive IgG.

Figure 2

S1P could induce increased endothelial permeability of GEnC monolayers in the presence of MPO‐ANCA‐positive IgG. Bars represent mean ± S.D. of repeated measurements of five independent experiments or donors.

Figure 3

S1P could up‐regulate membrane‐bound adhesion molecule expression levels of GEnC in the presence of MPO‐ANCA‐positive IgG. A. S1P could up‐regulate mICAM‐1 expression on GEnC in the presence of MPO‐ANCA‐positive IgG. B. S1P could up‐regulate mVCAM‐1 expression on GEnC in the presence of MPO‐ANCA‐positive IgG. Bars represent mean ± S.D. of repeated measurements of five independent experiments or donors.

Figure 4

S1P could up‐regulate soluble adhesion molecule expression levels in the supernatants of GEnC in the presence of MPO‐ANCA‐positive IgG. A. S1P could up‐regulate sICAM‐1 level in the supernatants of GEnC in the presence of MPO‐ANCA‐positive IgG. B. S1P could up‐regulate sVCAM‐1 level in the supernatants of GEnC in the presence of MPO‐ANCA‐positive IgG. Bars represent mean ± S.D. of repeated measurements of five independent experiments or donors.

Figure 5

Differential activation of S1PRs mediated the S1P‐induced sICAM‐1 up‐regulation of GEnCs in the presence of MPO‐ANCA‐positive IgG. A. S1PR1 antagonist W146 significantly increased sICAM‐1 level in the supernatants of GEnC stimulated by S1P plus MPO‐ANCA‐positive IgG. B. S1PR1 agonist SEW significantly reduced sICAM‐1 level in the supernatants of GEnC stimulated by S1P plus MPO‐ANCA‐positive IgG. C. S1PR2 antagonist JTE significantly reduced sICAM‐1 level in the supernatants of GEnC stimulated by S1P plus MPO‐ANCA‐positive IgG. D. S1PR3 antagonist TY significantly reduced sICAM‐1 level in the supernatants of GEnC stimulated by S1P plus MPO‐ANCA‐positive IgG. E. S1PR4 antagonist CYM significantly reduced sICAM‐1 level in the supernatants of GEnC stimulated by S1P plus MPO‐ANCA‐positive IgG. F. S1PR5 agonist A97 significantly increased sICAM‐1 level in the supernatants of GEnC stimulated by S1P plus MPO‐ANCA‐positive IgG. Bars represent mean ± S.D. of repeated measurements of five independent experiments or donors.