CRF induces intestinal epithelial barrier injury via the release of mast cell proteases and TNF-α - PubMed (original) (raw)
CRF induces intestinal epithelial barrier injury via the release of mast cell proteases and TNF-α
Elizabeth L Overman et al. PLoS One. 2012.
Abstract
Background and aims: Psychological stress is a predisposing factor in the onset and exacerbation of important gastrointestinal diseases including irritable bowel syndrome (IBS) and the inflammatory bowel diseases (IBD). The pathophysiology of stress-induced intestinal disturbances is known to be mediated by corticotropin releasing factor (CRF) but the precise signaling pathways remain poorly understood. Utilizing a porcine ex vivo intestinal model, the aim of this study was to investigate the mechanisms by which CRF mediates intestinal epithelial barrier disturbances.
Methodology: Ileum was harvested from 6-8 week-old pigs, mounted on Ussing Chambers, and exposed to CRF in the presence or absence of various pharmacologic inhibitors of CRF-mediated signaling pathways. Mucosal-to-serosal flux of 4 kDa-FITC dextran (FD4) and transepithelial electrical resistance (TER) were recorded as indices of intestinal epithelial barrier function.
Results: Exposure of porcine ileum to 0.05-0.5 µM CRF increased (p<0.05) paracellular flux compared with vehicle controls. CRF treatment had no deleterious effects on ileal TER. The effects of CRF on FD4 flux were inhibited with pre-treatment of tissue with the non-selective CRF(1/2) receptor antagonist Astressin B and the mast cell stabilizer sodium cromolyn (10(-4) M). Furthermore, anti-TNF-α neutralizing antibody (p<0.01), protease inhibitors (p<0.01) and the neural blocker tetrodotoxin (TTX) inhibited CRF-mediated intestinal barrier dysfunction.
Conclusion: These data demonstrate that CRF triggers increases in intestinal paracellular permeability via mast cell dependent release of TNF-α and proteases. Furthermore, CRF-mast cell signaling pathways and increases in intestinal permeability require critical input from the enteric nervous system. Therefore, blocking the deleterious effects of CRF may address the enteric signaling of mast cell degranulation, TNFα release, and protease secretion, hallmarks of IBS and IBD.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Figures
Figure 1. CRF induces elevations in FD4 flux in porcine ileum.
Porcine ileum was placed on Ussing Chambers and treated with CRF at increasing concentrations (0.05, 0.1, and 0.5 µM) and the rate of FD4 flux was measured over a 180-minute period. CRF at all concentrations induced elevations in FD4 flux (p<0.01) with the highest FD4 flux rates observed with 0.5 µM CRF. Data for each experimental treatment are expressed as means ± SE for n = 6−8 pigs. Symbols (*,†) differ from other treatments by p<0.05; ANOVA.
Figure 2. CRF-induced FD4 flux is Prevented with Astressin B.
CRF (0.5 µM) induced elevations in the rate of FD4 flux across the porcine ileum mounted on Ussing chambers. Pre-treatment of ileal mucosa with Astressin B (1 µM) 30 minutes prior to CRF exposure prevented CRF-mediated increases in FD4 flux rates. Data for each experimental treatment are expressed as means ± SE for n = 6−8 pigs. Symbols (*,†) differ from other treatments by p<0.05; ANOVA.
Figure 3. Histological appearance of CRF-treated porcine ileal mucosa on Ussing chambers.
Ileal histology (20× magnification) of hematoxylin and eosin-stained porcine ileal sections after 180 minutes on Ussing chamber following treatment with vehicle control (A) or 0.5 µM CRF (B). Images are representative of ileal sections from n = 6 pigs/treatment.
Figure 4. Occludin localization in porcine ileum exposed to CRF.
In control (vehicle-treated) tissues (A), occludin (green) was localized predominantly to the interepithelial tight junctions as demonstrated by the epithelial membrane staining patterns whereas CRF-treated tissues (B) exhibited a disrupted occluding staining pattern. Images are representative of tissues from n = 3 pigs.
Figure 5. Histologic analysis of CRF-mediated intestinal mast cell degranulation.
Treatment of porcine ileal tissues mounted on Ussing chambers with CRF caused and increase in mast cell degranulation as determined by toluidine blue staining. Arrows indicate the release of mast cell granules into surrounding tissues. Sodium cromolyn, a mast cell stabilizing agent, prevented CRF-induced mast cell degranulation. Figures are representative of ileal tissue sections from n = 4 pigs.
Figure 6. The role of mast cell activation in CRF-mediated changes in FD4 flux.
Treatment of porcine ileal tissues mounted on Ussing chambers with CRF increased mast cell degranulation indicated by the increased percentage of degranulated mast cells (C) and the increased release of mast cell tryptase (A) and TNF-α (B). Treatment of porcine ileal tissues with cromolyn sodium 30 minutes prior to exposure to 0.5 µM CRF blocked mast cell degranulation (C) and TNF-α release (B) and prevented CRF-mediated increases in FD4 permeability (D). c48/80 (5 µg/mL) induced marked degranulation of mast cells (C) and increases in FD4 flux (D) that were inhibited with sodium cromolyn. Data for each FD4 flux experiments are expressed as means ± SE for n = 6−8 pigs. Symbols (*,†) differ from other treatments by p<0.05; ANOVA.
Figure 7. Neutralization of TNF-α and protease activity inhibits CRF-induced increases in FD4 flux in porcine ileum.
Pretreatment of porcine ileum mounted on Ussing chambers with neutralizing anti-TNF-α antibody prevented CRF –induced increased in FD4 flux. Pre-treatment of porcine ileum with a protease inhibitor (PI) cocktail reduced baseline FD4 flux values and CRF-induced increases in FD4 flux. Values represent means SE; n = 6−8 animals. Symbols (*,†) differ from other treatments by p<0.05.
Figure 8. The Role of the enteric nervous system in CRF-mediated mast cell activation and FD4 flux.
Toluidine blue staining of histological sections (100 X magnification) (A) and quantitation of the % of degranulated tissue mast cells (B) in porcine ileum revealed increased degranulation of mast cells in CRF–treated tissues. Treatment of ileal tissues with tetrodotoxin (TTX) inhibited mast cell degranulation and FD4 flux (C) under basal and CRF-stimulated conditions and inhibited. For histological analysis, values represent means ± SE; n = 6 animals and are presented as the percentage of total cell count per treatment at 20× magnification. FD4 flux values represent means ± SE; n = 6−8 animals Symbols (*,†) differs significantly by p<0.05 from vehicle control.
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