Effect of enteroglial-derived S100B protein on human peripheral and mucosal immune cells’ functions

Background and Aim: Emerging evidence suggests that glial cells in the gut participate to the local immune response triggered by a variety of insults. In humans, during gut inflammation, over-expression of glial-derived S100B protein amplifies nitric oxide mucosal production via receptor for advanced glycation endproducts (RAGE) interaction. Whether S100B is directly affecting the responses of peripheral and mucosal immune cells is still unknown. We aimed to investigate the ability of S100B protein in mediating human immune cells' proliferation and responses. Material and Methods: Mucosal immune cells (MIC) were isolated from rectal mucosal biopsies of 15 control subjects (mean age 53; 6 male and 9 female) with Medimachine system and then characterized by Flow cytometry technique. In the same subjects, peripheral blood mononuclear cells (PBMC) were isolated. Both MIC and PBMC were stimulated with exogenous S100B protein (0.05-5μM), in the presence of anti-RAGE neutralizing antibody (1:10000-1:1000, dil v/v), to evaluate cell proliferation (by methylthiazolydiphenyl- tetrazolium bromide conversion assay, after 72h) and responses [by measuring nitrite level, inducibile nitric oxide synthase (iNOS) and tumor necrosis factor-alpha (TNF- α) expression, each after 24h]. Results: In PBMC, S100B exposure induced a significant and concentration-dependent cell proliferation (S100B 5μM: +73% vs basal; p<0.05). This finding was associated with a significant increase of nitrite level, iNOS and TNF-α expression (S100B 5μM: +538%; +507%; +426% vs basal, respectively; all p<0.01), which were totally abolished by pre-incubation with anti-RAGE antibody (1:1000: -45%, -35% and -22% vs stimulated, respectively; all p<0.01). In MIC, similarly to PBMC, S100B induced a significant and concentration-dependent cell proliferation (S100B 5μM: +132% vs basal; p<0.01), but, differently, it failed to stimulate nitric oxide production, iNOS and TNF-α expression (1.9 vs 1.8; 0.09 vs 0.1; 0.40 vs 0.35, respectively; all p not significant). Conclusions: We show, for the first time, that glial-derived S100B protein modulates the functions of both peripheral and mucosal immune cells. The lack of responses in MIC is probably due to their localization (in close proximity with glial cells and thus continually ‘bathed' with physiological amounts of S100B) and their different subset, compared to PBMC.