IL1beta- 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)1beta and Escherichia coli lipopolysaccharide (LPS) induced EC cell 5HT release through Toll-like/IL-1 (TIL) receptor activation, nuclear factor kappa B (NFkappaB) 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 NFkappaB and ERK phosphorylation quantitated (ELISA) in response to IL1beta and LPS. 5HT secretion was increased by both E. coli LPS (EC(50) = 5 ng mL(-1)) and IL1beta (EC(50) = 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 (IC(50) = 12 ng mL(-1)) and the IL1beta receptor antagonist (ILRA; IC(50) = 3.4 ng mL(-1)). IL1beta caused significant (P < 0.05) NFkappaB and MAPK phosphorylation (40-55%). The somatostatin analogue, lanreotide inhibited IL1beta-stimulated secretion in Crohn's (IC(50) = 0.61 nmol L(-1)) and normal EC cells (IC(50) = 1.8 nmol L(-1)). Interleukins (IL1beta) and bacterial products (E. coli LPS) stimulated 5HT secretion from Crohn's EC cells via TIL receptor activation (TLR4 and IL1beta). 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.

Figures

Figure 1

Serotonin (5HT) and active form IL1β mucosal levels in normal colon mucosa and in Crohn's tissue. Levels of 5HT were ~7-fold higher level in grossly affected Crohn's (IBD mucosa) tissue compared with normal mucosa (A) while levels of the activated cytokine were significantly increased ~3-fold in Crohn's tissue compared with normal mucosa. Mean ± SEM, n = 6 (normal mucosa), n = 9 (Crohn's tissue). *P < 0.05.

Figure 2

Isolation methodology for enterochromaffin (EC) cells from normal and Crohn's mucosa. (A) Histogram showing the shift to the right (P4) in FITC-labelled EC cells after staining of live preparations with acridine orange (0.02 μmol L−1). Marker P4 was set to collect positive cells. P5 reflects poorly-positive cells. Doublet and debris discrimination were performed prior to sorting using standard procedures (data not shown). (B) Dual immunostaining of colonic EC cells (stained with Cy5 anti-Tph-1 – red) and acridine orange (green). Dual staining (vesicles – yellow) signifies overlapping Tph-1 and acidic vesicle localization. 98 ± 1.4% of FACS-sorted cells were Tph-1 positive. These EC-enriched cell preparations were PCR positive for Tph-1, the serotonin transporter SERT, substance P and guanylin further confirming their EC cell origin. The 1% contamination is due to neurotensin-producing endocrine cells. (C) Expression of Tph-1 transcripts (normalized using geNorm and three house-keeping genes: ALG9, TFCP2 and ZNF410 – GeNormATZ− was significantly increased ~2-fold in Crohn's-derived EC cells compared with normal EC cells. (D) SERT transcripts were not significantly altered. Mean ± SEM, n = 4 (normal EC cells), n = 5 (Crohn's EC cells). *P < 0.03.

Figure 3

TLR4, IL1β and somatostatin receptor expression in normal and Crohn's-derived enterochromaffin (EC) cells by real-time PCR (normalized as described in Fig. 2). (A) Expression of TLR4 was significantly increased ( ~5-fold) in Crohn's-derived EC cells (IBD; n = 5) compared with normal EC cells (N; n = 4). (B) IL1R was significantly increased ( ~10-fold) in Crohn's-derived EC cells. (C) Expression of somatostatin subtypes 2 (SSTR2) and 5 (SSTR5) were identified in normal EC cells. Crohn's-derived EC cells, in addition, expressed transcript for types 1 and 3. Type 4 was not evident in either EC cell phenotype. Significantly elevated transcripts were noted for SSTR1, SSTR3 and SSTR5 in Crohn's EC cells. Mean ± SEM, n = 4 (normal EC cells), n = 5 (Crohn's EC cells). *P < 0.03.

Figure 4

Serotonin (5HT) secretion from isolated normal enterochromaffin (EC) cells and Crohn's EC cells stimulated with lipopolysaccharide (LPS) or IL1β. (A) LPS had no significant effect on normal EC cells (O) but increased 5HT secretion >2-fold in Crohn's-derived EC cells (•), half-maximal effect was seen at 4.5 ng mL−1. (B) The half-maximal effect for IL1β on normal EC cells was 0.03 pmol L−1 (0.56 pg mL−1) and 0.05 pmol L−1 (0.83 pg mL−1) for Crohn's-derived EC cells. (C) The TLR4 antagonist, Escherichia coli K12 LPS, inhibited LPS-mediated secretion with a half-maximal effect of 12 ng mL−1. (D) The half-maximal inhibitory effect of IL1RA was 3.4 ng mL−1 on IL1β (0.05 pmol L−1)-stimulated 5HT secretion. Mean ± SEM, n = 3. *P < 0.05 vs normal EC cells.

Figure 5

Effect of the somatostatin receptor analogue, lanreotide, on IL1β-stimulated serotonin (5HT) release in normal enterochromaffin (EC) cells (•) and Crohn's-derived EC cells (□). The half-maximal inhibitory effect on normal cells was 1.8 × 10−9 and 6 × 10−10 mol L−1 for Crohn's-derived EC cells. Mean ± SEM, n = 3.

Figure 6

Effect of IL1β on NFκB (A) and MAPK (ERK) (B) phosphorylation in isolated normal enterochromaffin (EC) cells (A/B) and Crohn's-derived EC cells (C/D) and effect of inhibiting these signalling pathways on serotonin (5-HT) release (normal EC cells: E; Crohn's derived EC cells: F). (A) IL1β (0.03 pmol L−1/0.5 pg mL−1) significantly (P < 0.05) stimulated NFκB phosphorylation by 30% in normal EC cells. This was completely reversed by ILRA (25 ng mL−1) and the specific NFκB inhibitor SN50 (10 μmol L−1). (B) IL1β (0.03 pmol L−1/0.5 pg mL−1) significantly (P < 0.05) stimulated MAPK phosphorylation by 25% which was inhibited by ILRA (25 ng mL−1) and the MEK inhibitor, PD98059 (1 μmol L−1). (C) IL1β (0.05 pmol L−1/0.8 pg mL−1) significantly (P < 0.01) stimulated NFκB phosphorylation by 45% in Crohn's-derived EC cells. This was completely reversed by ILRA (3 ng mL−1) and SN50 (10 μmol L−1). (D) IL1β (0.05 pmol L−1/0.8 pg mL−1) significantly (P < 0.01) stimulated MAPK phosphorylation by 55% which was inhibited by ILRA (3 ng mL−1) and PD98059 (1 μmol L−1). (E) IL1β (0.03 pmol L−1/0.5 pg mL−1)-stimulated 5-HT release was significantly inhibited (P < 0.05) by ILRA (25 ng mL−1), SN50 (10 μmol L−1) and PD98059 (1 μmol L−1). (F) IL1β (0.05 pmol L−1/0.8 pg mL−1)-stimulated 5-HT release was significantly inhibited (P < 0.05) by ILRA (3 ng mL−1), SN50 (10 μmol L−1) and PD98059 (1 μmol L−1). NFκB and MAPK phosphorylation in response to IL1β stimulation were both significantly (P < 0.05) elevated in Crohn's EC cells compared with normal EC cells. Mean ± SEM, n = 4. *P < 0.05 vs unstimulated cells, **P < 0.01 vs unstimulated cells, P < 0.05 vs IL1β-stimulated normal EC cells, ##P < 0.05 vs IL1β-stimulated Crohn's EC cells.