IL1β‐ and LPS‐induced serotonin secretion is increased in EC cells derived from Crohn’s disease

Abstract

Gut mucosal enterochromaffin (EC) cells are regarded as key regulators of intestinal motility and fluid secretion via secretion of serotonin (5HT), are increased in numbers in mucosal inflammation and located in close proximity to immune cells. We examined whether interleukin (IL)1β and Escherichia coli lipopolysaccharide (LPS) induced EC cell 5HT release through Toll-like/IL-1 (TIL) receptor activation, nuclear factor kappa B (NFκB) and mitogen-activated protein kinase (MAPK) phosphorylation and evaluated whether somatostatin could inhibit this phenomenon. Pure (>98%) human intestinal EC cells were isolated by fluorescent activated cell sorting from preparations of normal (n = 5) and Crohn's colitis (n = 6) mucosa. 5HT release was measured (ELISA), and NFκB and ERK phosphorylation quantitated (ELISA) in response to IL1β and LPS. 5HT secretion was increased by both E. coli LPS (EC50 = 5 ng mL−1) and IL1β (EC50 = 0.05 pmol L−1) >2-fold (P < 0.05) in Crohn's EC cells compared with normal EC cells. Secretion was reversible by the TLR4 antagonist, E. coli K12 LPS (IC50 = 12 ng mL−1) and the IL1β receptor antagonist (ILRA; IC50 = 3.4 ng mL−1). IL1β caused significant (P < 0.05) NFκB and MAPK phosphorylation (40–55%). The somatostatin analogue, lanreotide inhibited IL1b-stimulated secretion in Crohn's (IC50 = 0.61 nmol L−1) and normal EC cells (IC50 = 1.8 nmol L−1). Inter-leukins (IL1β) and bacterial products (E. coli LPS) stimulated 5HT secretion from Crohn's EC cells via TIL receptor activation (TLR4 and IL1β). Immune-mediated alterations in EC cell secretion of 5HT may represent a component of the pathogenesis of abnormal bowel function in Crohn's disease. Inhibition of EC cell-mediated 5HT secretion may be an alternative therapeutic strategy in the amelioration of inflammatory bowel disease symptomatology.

Differential signal pathway activation and 5-HT function: the role of gut enterochromaffin cells as oxygen sensors

“…Macroscopically "normal" tissue obtained from patients undergoing surgery for diverticulitis (n ϭ 3) or colon cancer (colon: n ϭ 4) (all tissue collected between 2009 and 2011 at Yale University, Department of Surgery and designated by the IRB as nonhuman subjects research). EC cells (Ͼ98% purity) were isolated by mucosal stripping, enzymatic digestion, and a combination of Nycodenz gradient fractionation and fluorescence-activated cell sorting as described (27). Approximately 1 ϫ 10 6 cells were obtained per mucosal sample, a quantity sufficient for real-time PCR, short-term culture, and Western blots.…”

“…Studies were performed on the continuously growing EC cell tumor-derived KRJ-I cells, a model for normal EC cells (6,28), as well as isolated, short-term cultured human ileal EC cells isolated as described (27). HIF signaling pathway activation was measured using transient transfection with Renilla luciferasehypoxia transcriptional response element (HRE) constructs in cells exposed to hypoxia, defined as 0.5% O 2 (maximally responsive effective concentration; Ref.…”

“…RNA was extracted from isolated, short-term cultured EC cells (1 ϫ 10 5 , n ϭ 4) or KRJ-I cells (1 ϫ 10 6 , n ϭ 6) using TRIZOL (Invitrogen) and cleaned (Qiagen, RNeasy kit). After conversion to cDNA (High Capacity cDNA Archive Kit, Applied Biosystems) (27), quantitative RT-PCR analysis was performed using Assays-on Demand and the ABI 7900 Sequence Detection System (25,26). Primer sets were all obtained from Applied Biosystems, and PCR mix on gels were performed to confirm presence of single bands for each primer set.…”

“…EC cells mediate gut secretion, peristalsis, and motility by the secretion of 5-HT, as well as of melatonin, substance P, and guanylin (27). 5-HT release is altered in pathological states such as IBD, irritable bowel syndrome (IBS), and Celiac disease.…”

Chlamydiae in Gastrointestinal Disease

“…At this stage, it is unclear if the altered distribution of serotonin and CgA in C. trachomatis infected EEC is induced by the bacteria themselves or is part of an innate immunity response via up-regulation of toll-like receptors. Others have reported increased LPSinduced serotonin secretion in EEC derived from patients with Crohn's disease (Kidd et al, 2009). TLR4 stimulation with its agonist LPS also caused the release of human β-defensin-2 (HBD-2) from EEC (Palazzo et al, 2007) and elevated HBD-2 levels have recently been found in patients with IBS (Langhorst et al, 2009).…”