Endothelin Receptors Expressed by Immune Cells Are Involved in Modulation of Inflammation and in Fibrosis: Relevance to the Pathogenesis of Systemic Sclerosis

Abstract

Endothelin-1 (ET-1) plays a pivotal role in vasoconstriction, fibrosis, and inflammation, the key features of systemic sclerosis (SSc). ET-1 receptors (ETA and ET(B)) are expressed on endothelial cells, smooth muscle cells, and fibroblasts, but their presence on immune cells has not been deeply investigated so far. Endothelin receptors antagonists such as bosentan have beneficial effects on vasoconstriction and fibrosis, but less is known about their potential anti-inflammatory effects. We studied the expression of ET-1 receptors on immune cells (T and B lymphocytes, monocytes, and neutrophils) and the link between ET-1 and inflammation in patients with SSc. We show here that ET-1 exerts a proinflammatory effect in CD4+ T cells, since it induces an increased IFN-γ production; preincubation with antagonists of both receptors reduces IFN-γ production. Moreover, following ET-1 stimulation, neutrophils produce proinflammatory mediators, thus amplifying the effects of activated CD4+ T cells. Our data indicate that ET-1 system is involved in the pathogenesis of inflammation and fibrosis typical of SSc, through the activation of T lymphocytes and neutrophils and the consequent release of proinflammatory and profibrotic cytokines. These findings suggest that dual ET-1 receptors antagonist therapy, besides its effect on vasculopathy, has a profound impact on the immune system favouring antiinflammatory and antifibrogenic effects.

Figure 1

ET_A and ET_B expression by cells obtained from SSc patients. The quantification of receptors expression by T (a) and B lymphocytes (b), monocytes (c), and neutrophils (d) is represented by the difference of fluorescence intensity between the sample (continuous line) and its negative control (dotted line). The profile of one of 41 SSc patients is shown. All the other patients had a similar behaviour.

Figure 2

ET_A and ET_B transcripts amplified by RT-PCR in fibroblasts, CD4+ T lymphocytes, activated CD4+ T cells, and neutrophils. ET_A corresponds to a molecular weight of 446 bp and ET_B to a molecular weight of 558 bp. (a) ET_A and ET_B transcripts amplified by RT-PCR in fibroblasts and neutrophils. Lane 1: molecular weight ladder; lane 2: negative control; lane 3: fibroblasts (ET_A); lane 4: neutrophils (ET_A), lane 5: negative control; lane 6: fibroblasts (ET_B); lane 7: neutrophils (ET_B). (b) ET_A transcripts amplified by RT-PCR in T lymphocytes, activated T cells, and fibroblasts. Lane 1: T lymphocytes; lane 2: activated T cells; lane 3: fibroblasts; lane 4: negative control, lane 5: molecular weight ladder. (c) ET_B transcripts amplified by RT-PCR in T lymphocytes, activated T lymphocytes, and fibroblasts. Lane 1: negative control; lane 2: T lymphocytes; lane 3: activated T cells; lane 4: fibroblasts; lane 5: molecular weight ladder.

Figure 3

Change in ET_A and ET_B expression on activated T CD4+ and CD8+ cells. The stimulation of cells, performed with microbeads coated by anti-CD3/CD28 antibodies, leads to a reduction of ET_A and an increase of ET_B expression, both in CD4+ (a-b; c-d) and CD8+ cells (e-f; g-h), respectively. The profile of one of ten similar experiments is shown.