Background Low grade inflammation is an increasingly recognized feature in patients suffering from systemic sclerosis (SSc), with substantial infiltration and activation of lymphocytes occurring in SSc affected tissue. However, the transition from inflammation into fibrosis, the hallmark of SSc, has not been clarified conclusively. The role of tumor necrosis factor (TNF)-alpha in SSc remains controversial, despite several reports indicating the pro-fibrotic effects of TNF, notably mediated via TNF-receptor (TNF-R) 2.
Objectives To investigate cytokine expression in lymphocytes from SSc patients and healthy controls after CD3/28 activation and co-stimulation with selective TNF-R 1 or 2 agonists. To study TNF mediated IL-6 and IL-13 expression on collagen expression by fibroblasts, and a potential T-reg capacity by IL-10 production. For in vivo studies, we assessed TNF-R1 or -R2 positive lymphocytes in the dermis of SSc patients vs. healthy controls.
Methods CD3+ lymphocytes were isolated from peripheral blood of SSc patients, activated with or without CD3/28 beads and co-stimulated with cysTNF mutants, which are selective for TNF-1 or 2, as well as soluble TNF (sTNF). IL-6, IL-6 receptor, IL-10 and IL-13 expression was detected by ELISA and qPCR. Healthy fibroblasts were incubated with supernatants from TNF stimulated lymphocytes and type-1 collagen expression detected by qPCR. TNF-R expression in peripheral blood and dermis was assessed by flow cytometry in different patient subsets.
Results After CD3/28 activation, CD3+ lymphocytes isolated from peripheral blood of SSc patients produced significantly higher levels of IL-6, IL-6 receptor and IL-13 compared to lymphocytes form healthy individuals. Co-stimulation with a selective TNF-R1 agonist and sTNF further increased IL-6 expression whereas stimulation of TNF-R2 led to a higher expression of IL-6 receptor. IL-10 expression, which mainly occurred after TNF-R2 co-stimulation, was impaired in T-cells from SSc patients, indicating a deficient suppressive capacity. Supernatants of CD3/28 stimulated lymphocytes from SSc patients induced a significantly higher type-1 collagen expression in heathly fibroblasts compared to CD3/28 stimulated lymphocytes derived from healthy controls. This effect was further increased by selective TNF-R2 co-stimulation. Dual inhibition of IL-6 and IL-13 in the supernatant reversed collagen over-expression. In vivo, we found an upregulation of TNF-R1 and 2 expression on dermal lymphocytes. TNF-R2 but not TNF-R1 expression in the dermis of SSc patients correlated with skin thickening (p=0.02). In one patient, TNF-R expression on lymphocytes was reversible after autologous HSCT.
Conclusions T-cells from SSc patients express higher levels of profibrotic cytokines and induce collagen expression in fibroblasts. TNF co-stimulation via TNF-R1 further increases IL6 and IL-13 expression whereas TNF-R2 co-stimulation enhances IL-6R expression. The excess of profibrotic cytokines after TNF-costimulation seems to occur on cost of an impaired capacity to express the anti-inflammatory cytokine IL-10 via TNF-R2.
Disclosure of Interest None Declared
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