Effect of Proinflammatory Stimuli On Cellular Activation and Nitric Oxide Production in Primary Cultures of Human Enteric Glia

Background: Enteric glial cells (EGC) physiologically express S100B protein and are involved in intestinal inflammation. While the role of EGC during inflammatory processes has been reported in animal models, in humans, the lack of suitable In Vitro model, has hampered the study of EGC and intestinal inflammation relationships. Aim: 1) To set up a new method to isolate, characterize and purify a primary culture of human enteric glia and 2) to study EGC activation and responses' to pro-inflammatory stimuli evaluating S100B expression and NO production. Methods: Myenteric plexus preparations were isolated from human ileum of 15 patients undergoing surgery for carcinoma of large bowel and enzimatically dissociated. Ganglia were plated and cell cultures were grown to subconfluence. After 21 days, EGC were purified of contaminating cells by incubation with the anti-Thy-1.1 ab-coated magnetic beads and separation using a Dynal Magnet®. Mixed EGC cultures and purified EGC cell line were immunocytochemically characterized with anti-S100B, anti-glial fibrillary acidic protein (GFAP) and anti-alpha-smooth muscle actin antibodies to identify EGC and fibroblasts, respectively. To study the effects of inflammatory stimuli on EGC activation, cells were incubated with lipopolysaccharide (LPS, 1μg/mL), Tumor Necrosis Factor-alpha (TNF-α, 5ng/ml), interferon-gamma (IFN-γ, 100U/mL) for 32 hours alone or in combination. A double labeling immunofluorescence using antibodies to S100B or GFAP was performed to identify EGC expressing c-fos in the nucleus. Similarly, S100B mRNA, protein expression and secretion and iNOS expression and NO production were respectively assessed. Results: In mixed EGC cultures, immunocytochemically analysis displayed that 40% of cultured cells were EGC (S100B and GFAP positives) and that 50% were fibroblasts (anti-α-SMA positives). Neurons were virtually absent. In contrast, in purified EGC cell line, the presence of contaminating cells (i.e. fibroblasts) was significantly reduced by treatments with Dynabeads ® and the majority of the cells were anti-GFAP and anti-S100B positives. Incubation with LPS and IFN-γ alone, or in combination with TNF-α, resulted in a consistent EGC's activation (c-fos positive nuclei in S100B/GFAP positive cells), and significantly increased S100B expression, iNOS protein expression and NO production. Conclusions: We characterized a new method to isolate and purify EGC from human myenteric ganglia. We also demonstrated that, in this cell population pro-inflammatory cytokines directly stimulate the release of nitric oxide production via S100B release.