Circulating IgA from acute stage of childhood Henoch-Schönlein purpura can enhance endothelial interleukin (IL)-8 production through MEK/ERK signalling pathway

Abstract

Recently, sera from children with active Henoch-Schönlein purpura (HSP) have been found to enhance interleukin (IL)-8 production by human umbilical venous endothelial cells (HUVEC). To further determine the possible factor with the ability to enhance endothelial IL-8 production in sera from acute stage of HSP, 10 children with HSP at the acute stage and 10 healthy controls were enrolled. IgA antiendothelial cell antibodies (AECA) were detected by cell-based ELISA. Active sera with or without pretreatment with anti-human IgA antibody, sera of controls, and immunoglobulin A (IgA) derived from sera were used to stimulate the HUVEC. The ability of these factors to enhance endothelial IL-8 production was evaluated. Furthermore, signalling pathways were also assayed by different inhibitors, and confirmed by immunoblotting. Serum levels of IgA AECA in HPS patients at the acute stage were significantly higher than in controls (P < 0.001). The active sera could enhance endothelial IL-8 production (P = 0.004, compared with control sera), and the ability of these sera was mostly abolished when pretreated with fixed anti-human IgA antibody. The supernatant IL-8 levels of endothelial cells stimulated by IgA derived from acute stage of HSP were statistically higher than controls (P < 0.001). PD98059, an inhibitor of ERK phosphorylation, significantly reduced IgA AECA-stimulated endothelial IL-8. IgA AECA also enhanced the phosphorylation of ERK1 with a time-dependent manner. Together with these findings, it is concluded that IgA AECA derived from acute stage of HSP may bind to endothelial and enhance endothelial cells to produce IL-8 via MEK/REK signalling pathway.

Fig. 1

Cell-based ELISA assay revealed the values of serum IgA AECA (expressed as ELISA ratio (ER)) of 10 HSP patients at the acute stage and 10 healthy controls.

Fig. 2

The supernatant IL-8 levels between HUVEC cocultured with active sera, active sera pretreated by anti-human IgA antibody, and sera from healthy controls.

Fig. 3

(a) TNF-α of different concentrations (0 pg/ml, 100 pg/ml, 1 ng/ml, 10 ng/ml, 50 ng/ml) enhanced HUVEC to release IL-8 with a dose-dependent manner. *P < 0·001 versus 0 pg/ml. (b) The supernatant IL-8 levels between HUVEC cocultured with active sera pretreated by anti-human TNF-α antibody (1 µg/ml) and those cells incubated with sera without antibody pretreatment.

Fig. 4

HUVEC were stimulated by adding HSP IgA alone, or adding HSP IgA and 4 inhibitors: Helenalin (1 µM), SB203580 (20 µM), PD98059 (25 µM), and JNK inhibitor II (5 µM), respectively. PD98059 significantly inhibited the endothelial IL-8 releasing. *P = 0·001, compared with those HUVEC stimulated by HSP IgA only.

Fig. 5

Immunoblotting for intracellular total protein of ERK1 and ERK2, and the phosphorylation of ERK1 after IgA AECA stimulation at different time intervals (0 min, 10 min, 30 min, 60 min), and after further treatment by PD98059.