Enteric glial protein S100b up-regulation stimulates nitric oxide production in ulcerative colitis

In the central nervous system glial derived S100B protein has been associated with inflammation via nitric oxide (NO) production. Activation of enteroglial cells (EGC) has been described in celiac disease, but its role in inflammatory bowel disease has been poorly investigated in humans. We aimed to evaluate the role of S100B and its association with NO production in ulcerative colitis (UC). Basal S100B mRNA and protein expression, iNOS protein expression and NO production were evaluated in rectal biopsies from 40 controls and 35 UC patients. To verify the correlation between EGC activation and NO production, biopsies were stimulated for 24 h with exogenous S100B (0.005-5μM), either alone or in the presence of budesonide (BUD, 30μM) and receptor for advanced glycation endproducts antibody (RAGE, 1:1000). To test the involvement of EGC in experimental inflammation, S100B and iNOS expression were evaluated after a 24 h incubation of biopsies with lipopolysaccharides (LPS, 10μg/ml)+interferon-gamma (IFN-γ, 300U/mL) either in the presence or absence of BUD (30μM). Moreover, iNOS expression was evaluated after LPS/IFN-γ stimulus in the presence of anti-RAGE (1:1000) or anti-S100B (1:1000) antibodies. In biopsies from UC patients, S100B immunoreactivity, mRNA (10720±192%), protein expression (1476±186%) and release (537±218%) were significantly higher than in controls; similarly iNOS protein expression and NO levels were significantly increased (1030±272% and 268±38%). Exogenous S100B induced a significant and concentration-dependent increase of both iNOS expression (174±4; 672±7; 914±14 and 1438±12%) and NO production (90±2; 228±4; 412±10 and 766±10%), which were significantly inhibited by anti-RAGE antibody (-65±4 and -61±2%, respectively). In biopsies from controls and UC patients, incubation with LPS/IFN-γ induced a significant increase of S100B expression (1.4 and 10 fold increase vs basal) and release (0.05 and 0.31 ng/ml), which were not affected by BUD. LPS/IFN-γ also stimulated iNOS expression and NO production (2.9 and 4.1 and 1.5 and 2.5 fold increase vs basal respectively). As expected, the increase of iNOS expression and NO production were significantly reduced by BUD, both in control and UC patients (-64 and -46% respectively); a significant inhibition was also obtained by anti-RAGE and anti-S100B antibodies (-61 and -68, -47 and -63% in controls and UC, respectively). We showed that EGC activation stimulates NOproduction in UC via S100B up-regulation and via activation of RAGE in a steroid insensitive pathway. Our data highlight a pivotal role of S100B in the mechanism of NO production underlying the involvement of EGC in gut inflammation.