Background Evidence suggests that B cells play a key role in the pathogenesis of Systemic Sclerosis (SSc). B cell depletion therapy has been used in the treatment of SSc with favorable results in lung function and skin fibrosis. However its mechanism of action remains to be elucidated.
Objectives We investigated the effect of RTX on T-helper 1 (Th1), T-helper 2 (Th2) and T-helper 17 (Th17) cells as well as on serum cytokines, aiming at exploring the potential mechanisms of action of RTX.
Methods We assessed 14 patients with SSc prior to and 6 months following RTX administration and 3 patients with SSc on standard treatment at the same time points. CD4 cells were separated from peripheral blood, stimulated with PMA and Ionomycin, stained for intracellular cytokines and measured using Flow Cytometry. Th1, Th2 and Th17 cells were defined as CD4 cells stained positive for Interferonγ (INFγ), Interleukin 4 (IL4) and Interleukin 17 (IL17) respectively. Serum levels of IL4 were measured by employing a quantitative ELISA method. IL4 expression in the skin was assessed using immunohistochemistry in skin biopsies obtained from 12 patients with SSc prior to and 6 months following RTX treatment and 3 control SSc patients. Immunohistochemical assessment was performed using computerized image analysis. Statistical analysis was performed by employing the paired t-test. Results were expressed as mean ± SEM.
Results We found a significant decrease in circulating Th2 cells in all 14 patients following RTX treatment (mean±SEM: 13.9±4.2% vs 9.3±3.7 prior to and following RTX administration respectively, p=0.016). RTX had no effect on Th1 (mean±SEM: 3.47±0.68 vs 2.79±0.93 prior to and following RTX administration respectively, p=NS) and Th17 cells (mean±SEM: 0.25±0.07 vs 0.19±0.04 prior to and following RTX administration respectively, p=NS). We found no changes in serum levels of IL4 in all 14 patients. The 3 patients who were not treated with RTX exhibited no differences in Th1, Th2 and Th17 cells. A decrease in IL4 expression in the skin was small but significant in all 12 patients following RTX treatment both in the papillary (mean±SEM: 1.95±0.28% vs 1.15±0.164 prior to and following RTX administration respectively, p=0.004) and the reticular dermis of the skin (mean±SEM: 1.78±0.26 vs 1.15±0.15 prior to and following RTX administration respectively, p=0.008) while no differences were found in IL4 expression in the skin of controls. Immunostaining for IL4 was almost exclusively found in skin infiltrating mononuclear cells.
Conclusions Our study shows that RTX reduces circulating Th2 cells and IL4 expression in the skin. Th2 cells are currently thought as strongly related with fibrosis. Further studies on the mechanisms of RTX action in patients with SSc are warranted.
Disclosure of Interest None Declared
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