IL-17A induces endothelial inflammation in systemic sclerosis via the ERK signaling pathway

Abstract

Recent reports have demonstrated that endothelial cells are involved in vascular inflammatory injury in systemic sclerosis (SSc) and interleukin-17A (IL-17A) plays a crucial role in the pathogenesis of SSC. However, little is known about the effects of IL-17A on endothelial cell inflammation in SSC. The aim of our study was to investigate the role of IL-17A in endothelial inflammation. Here, we showed that IL-17A mRNA and protein levels were augmented in the peripheral blood and more IL-17⁺ lymphocytes infiltrated in the perivascular areas in the involved skin of SSC patients. SSC patient serum induced chemokine and adhesion molecule expression in HUVECs, which was blocked by IL-17A neutralization. IL-17A alone induced chemokine and adhesion molecule expression and promoted T cell-HUVEC adhesion. Extracellular signal-regulated kinase (ERK) inhibition and IL-17A neutralization prominently inhibited chemokine and adhesion molecule expression and blocked T cell-HUVEC adhesion. IL-17A derived from SSC patient serum mediated endothelial cells inflammation by up-regulating chemokines and adhesion molecules, which was blocked by ERK inhibition. These data imply that ERK signal pathway might play a key role in the progression of endothelial injury induced by IL-17A in SSC.

Figure 1. Increased expression of IL-17 in SSc patients.

(A) The concentration of IL-17A in the sera of SSc patients (n=20) and healthy controls (n=16) was detected by ELISA. The ELISAs were repeated three times, and the results represent the mean ± S.D. (*P<0.05). (B) Total RNA from PBMCs in SSc patients (n=20) and healthy controls (n=16) was extracted, and real-time RT-PCR analysis was performed for IL-17A. The results of real-time RT-PCR were averaged from three separate experiments (n = 3) and are presented as relative gene expression ±S.D. (*P<0.05). (C) H&E stain of skin in SSc. (D) Expression of IL-17A was detected by immunohistochemistry in the dermis layer of SSc patients. (E) Expression of IL-17A was detected by immunohistochemistry in the subcutaneous tissue of SSc patients. (F) H&E stain of skin in healthy controls. (G) Expression of IL-17A was detected by immunohistochemistry in the dermis layer of healthy controls. (H) The expression of IL-17A was detected by immunohistochemistry in subcutaneous tissue of healthy controls. Arrows show typical positive cells. Scale bars =100 μm.

Figure 2. Serum from SSc patients and healthy individuals stimulates the expression of adhesion molecules and chemokines in HUVECs.

HUVECs were treated with the serum of SSc patients and healthy individuals for 24 h. Increased expression of CCL-20, CXCR-4, VCAM-1, and ICAM-1 was detected by western blot. The up-regulation of these proteins was partly blocked with IL-17-neutralizing antibody.

Figure 3. IL-17A promotes the expression of adhesion molecules and chemokines in HUVECs and HMEC-1.

(A, D) HUVECs and HMEC-1 were treated with IL-17A at a range of concentrations for 12 h, and mRNA expression of CCL-20 and CXCR-4 was measured by real-time RT-PCR (Comparing 0 ng/ml IL-17A and 20ng/ml IL-17A, *, P<0.05). (B, E) HUVECs and HMEC-1 were treated with IL-17A at a range of concentrations for 12 h, and mRNA expression of adhesion molecules VCAM-1 and ICAM-1 was measured by real-time RT-PCR (Comparing 0 ng/ml IL-17A and 20ng/ml IL-17A, *, P<0.05). (C, F) HUVECs and HMEC-1 were treated with IL-17A at a range of concentrations for 24 h, and protein expression of CCL-20, CXCR-4, VCAM-1, and ICAM-1 was measured by western blot. All experiments were conducted three separate times, and representative data are presented.

Figure 4. Effects of IL-17A on activation of JNK, p38-MAPK, ERK1/2, and AKT in HUVECs.

(A) HUVECs were treated with IL-17A for 10, 20, and 30 min, and the phosphorylation of JNK, p38-MAPK, ERK1/2, and AKT were detected by western blot. (B) HUVECs were exposed to serum from SSc patients and healthy controls for 20 min, the phosphorylation of ERK1/2 and p38 MAPK were detected by western blot. GAPDH was used as a loading control.

Figure 5. ERK signaling is necessary for IL-17A function in HUVECs.

(A) HUVECs were seeded in six-well plates for 12 h and then pre-treated with the different concentrations of PD98059 for 2 h before incubation with IL-17A at 20 ng/ml for 24 h. The expression of chemokines CCL-20 and CXCR-4 and adhesion molecules VCAM-1 and ICAM-1 was detected by western blot. (B) HUVECs were seeded in six-well plates and then pre-treated with PD98059 (10μM/ml) for 2 h, and incubated with serum of SSc patients and healthy controls for 24 h. The expression of chemokines and adhesion molecules were detected by western blot. GAPDH was used as a loading control.

Figure 6. ERK signaling is necessary for IL-17A–induced T cell–HUVEC adherence.

(A) Jurkat cells were co-cultured with HUVECs in the presence or absence of IL-17A, IL-17A-neutralizing antibody, or ERK inhibitor (PD98059) for 24 hours, representative picture of Jurkat cells adhering to endothelial cells (Arrows show the adhering Jurkat cells). Scale bar = 100 μm. (B) The number of adherent T cells. The experiment was repeated three times, and the data are presented as mean ± S.D. (*, P<0.05).