Pathogenesis of Systemic Sclerosis: pro-inflammatory role of ET-1 receptors

Endothelin-1 (ET-1) plays a pivotal role in vasoconstriction, fibrosis and inflammation, the three major aspects in the pathogenesis of Systemic Sclerosis (SSc). ET-1 receptors are ETA and ETB. The receptors are expressed on the majority of cells involved in SSc, such as endothelial cells, smooth muscle cells and fibroblasts. Little is known about the expression of ET-1 receptors on leukocytes, except for macrophages and monocytes; there is no information about the expression of ETA and ETB on lymphocytes, neutrophils and other cells involved in the innate and acquired immune response. Endothelin receptors antagonists are used in the treatment of scleroderma patients with recurrent ischemic digital ulcers and/or pulmonary arterial hypertension. They have beneficial effects on vasoconstriction and fibrosis, but less is known about their anti-inflammatory role. We aimed at studying the link between ET-1 and inflammation in SSc. Since T and B cells, monocytes and neutrophils are among the most important cells in inflammatory responses in SSc, we studied ETA and ETB expression on these cells with flow cytometry, and also ETA- and ETB-coding mRNA expression in T CD4+ cells and neutrophils by RT-PCR. We have studied the different expression of receptors on T and B cell and monocytes between patients and controls, the correlations of the expression with the cutaneous form of disease, with Bosentan therapy, and ischemic digital ulcers, pulmonary arterial hypertension and interstitial lung disease. We also studied the receptors modulation on activated T cells by flow cytometry. In order to evaluate the pro-inflammatory effects of ET-1 and the anti-inflammatory role of Bosentan, we studied how ET-1 influences IFN-γ and IL-4 production by T CD4+ cells, with or without receptors blockage. We also studied the expression of IFN-γ-, IL-4- IL-6-, IL-10- and IL-17-coding mRNA in T CD4+ cells by Real Time PCR at different times in order to understand the timing of T CD4+ cells response to stimulation with ET-1. We previously described that T cells and monocytes express both ETA and ETB receptors. We have validated our data in a larger cohort of patients and controls. We confirmed that not only T cells and monocytes but also B lymphocytes and neutrophils express ETA and ETB on their surface. Moreover, the expression of ETA was greater than ETB both in patients and controls in T cells and monocytes, while for B cells there was not difference between ETA and ETB expression. Interesting, neutrophils express both ETA and ETB. Neutrophils participate in the early stages of SSc and contribute to endothelial damages, by production of reactive oxygen species, fibroblasts activation and recruitment T and B cells. ET-1, through ETA and ETB, can contribute to trigger neutrophils activation, that lead to vascular damage. Considering that ETB expression was lower in dSSc- rather than lSSc-patients, ETA signal seemed to be important in the cutaneous profibrotic effects of ET-1. Since a lower ETB expression on monocytes correlated with PAH and a lower ETA expression on T cells correlates with ILD, we can hypothesize that a different pattern of receptors expression is associated with a different response of T cells or monocytes in the preferential induction of PAH or ILD. Therefore ETA or ETB signalling may lead to different clinical features. Considering that ETB expression is increased on activated T cells, ETB signal probably played a major role in inflammation. We also show that activation of ETA and ETB receptors plays an immunomodulatory role, since the production of cytokines changes over time in relation to the stimulation by ET-1 in the presence or absence of the selective blockade of one or both receptors. Furthermore these results support the hypothesis that ET-1 system has a role not only on vasoconstriction and fibrosis but also on inflammation.