3rd Annual BMRP Investigator Meeting - Abstract
Enteric Glia in Intestinal Inflammation
Anne Rühl and Sebastian Hoff
Department of Human Biology, Technical University of Munich (Munich, Germany)
The etiology of human inflammatory bowel diseases (IBD) is as yet unknown. It has been repeatedly demonstrated that the enteric nervous system (ENS) is affected in IBD and, in turn, the ENS has been implicated in the pathogenesis of inflammatory diseases of the gut. Enteric glia (EGC) are recognized as the most abundant cell type of the ENS. Recently, it has been suggested that EGC may have a role in preventing disruption of the mucosal barrier (Cell 1998; PNAS 2001) – a process that has been repeatedly demonstrated as part of the pathogenesis of IBD. Therefore, this project aimed at investigating glial-epithelial interactions in the human intestinal mucosa, employing in vitro and in situ techniques, and assessing the contribution of these interactions to the etiopathogenesis of IBD.
To reliably identify and quantify all EGC in the human ENS, we have systematically evaluated putative glial markers (S100, GFAP, p75NGFR, Sox 10 and B-FABP) in whole-mount preparations of human myenteric, submucous and mucous plexus from ileum and all colonic segments. Neuronal counterstaining was performed with anti-HuC/D and anti-NSE antibodies. Immunoreactivity for S100, p75NGFR and Sox was detectable in all ganglionic HuC/D- and NSE-negative cells and in the interganglionic nerve strands of all plexus from all studied regions, whereas GFAP appeared to selectively label glial subpopulations. S100- and p75NGFR-IR displayed a cytoplasmic pattern while Sox-IR was strictly localized to the nucleus, allowing for identification and, thus, quantification of individual glial cells. Specific B-FABP-IR was not detectable in any layer of the human ENS.
In parallel, human EGC were isolated, purified and characterized. Employing immunohistochemistry and RT-PCR, we have excluded cellular contaminants in our cultures and established that cultured EGC express a marker profile that is identical to the in situ situation. In a transwell coculture-system with Caco-2 cells, the effects of EGC on transepithelial resistance and macromolecular permeability of Caco-2 monolayers were quantified.
Overall, we have established immunohistochemical procedures to reliably detect and quantify all EGC in the human ENS. These procedures will allow for the assessment of changes in EGC numbers associated with IBD. In addition, we have established a unique in vitro system for detailed studies of glial-epithelial interactions. Our preliminary findings of EGC-induced changes in the functional properties of intestinal epithelial cells, support the hypothesis that EGC are involved in the regulation of mucosal defence mechanisms, which is likely to contribute to the etiopathogenesis of IBD.
aPrincipal Investigator