Neurotensin is a proinflammatory neuropeptide in colonic inflammation

Abstract

The neuropeptide neurotensin mediates several intestinal functions, including chloride secretion, motility, and cellular growth. However, whether this peptide participates in intestinal inflammation is not known. Toxin A, an enterotoxin from Clostridium difficile, mediates pseudomembranous colitis in humans. In animal models, toxin A causes an acute inflammatory response characterized by activation of sensory neurons and intestinal nerves and immune cells of the lamina propria. Here we show that neurotensin and its receptor are elevated in the rat colonic mucosa following toxin A administration. Pretreatment of rats with the neurotensin receptor antagonist SR-48, 692 inhibits toxin A-induced changes in colonic secretion, mucosal permeability, and histologic damage. Exposure of colonic explants to toxin A or neurotensin causes mast cell degranulation, which is inhibited by SR-48,692. Because substance P was previously shown to mediate mast cell activation, we examined whether substance P is involved in neurotensin-induced mast cell degranulation. Our results show that neurotensin-induced mast cell degranulation in colonic explants is inhibited by the substance P (neurokinin-1) receptor antagonist CP-96,345, indicating that colonic mast activation in response to neurotensin involves release of substance P. We conclude that neurotensin plays a key role in the pathogenesis of C. difficile-induced colonic inflammation and mast cell activation.

Figure 1

Toxin A administration increases NT content in rat colonic mucosa. One closed loop was prepared in the proximal colon of anesthetized rats and injected with either 5 μg of toxin A or buffer (Control). At the indicated time points, animals were sacrificed, colonic loops were excised, and the mucosa was removed and processed for measurements of NT content. Arrow shows limit of detection. Each bar represents the mean ± SEM of 6–8 loops for each experimental condition. *P < 0.05, ** P < 0.01 vs. control. NT, neurotensin.

Figure 2

Increased NTR1 mRNA expression during toxin A–induced colitis in rats. Rat colonic loops were exposed to either toxin A or buffer. After 1 h, animals were sacrificed, and colonic tissues were processed for in situ hybridization using 383-base-digoxigenin–labeled antisense riboprobe encoding for the NTR1 mRNA. Tissues were examined by confocal microscopy. (a) Section from a rat colon exposed only to buffer shows the presence of little hybridization signal in the epithelial layer and in cells of the lamina propria. (b) Colon exposed for 1 h to toxin A shows increased signal for the NTR1 mRNA primarily in intestinal epithelial cells (arrows), but also in cells of the lamina propria (arrowheads). (c) Colon from toxin A–exposed loop hybridized with a sense riboprobe encoding for the NTR1 mRNA shows absence of specific signal. Results are representative of three experiments for each experimental condition. Scale bar: 50 μm. NTR1, NT receptor-1.

Figure 3

Increased NTR1 protein expression during toxin A colitis in rats. Rat colonic loops were injected with either toxin A (5 μg) or buffer (control). Animals were sacrificed after 2 h, and colonic tissues were processed for immunohistochemical detection using a rabbit polyclonal antibody directed against the NH₂-terminal amino acids 1–28 and 50–69 of the rat NTR1 receptor. Sections were examined by confocal microscopy. (a) Buffer-exposed colon (2 h). (b) Toxin A–exposed colon (2 h). (c) Toxin A–exposed colon (2 h) incubated with the NTR1 antiserum, which was preincubated with an excess of the two NH₂-terminal peptides of the NTR1 described above. (d) Higher magnification of b. Note the presence of signal for NTR1 in the colonic mucosa, in particular the colonic epithelial cells (arrows in b and d), and in cells in the lamina propria (arrowhead). Note also the increased immunoreactivity after 2 h exposure to toxin A (b) compared with control (a). Preabsorption of the NTR1 antiserum with an excess of the receptor peptides used to generate the antibody causes complete disappearance of positive staining in toxin A–exposed colon (c). Results are representative of three experiments for each experimental condition. Scale bar: 50 μm.

Figure 4

The SP antagonist CP-96,345 inhibits NT-mediated mucosal mast cell degranulation in rat colon. Colonic explants (2 × 2 mm) were cultured at 37°C in Krebs buffer alone or in buffer containing 0.1 mM of either the SP receptor antagonist CP-96,345 or the NT receptor antagonist SR-48,692. After 1 h, either NT or SP (10^–8 M) was added to the explants, and mucosal mast cell degranulation was determined after 2 h by measuring RMCPII released into the culture media. Both SP and NT caused a significant mast cell degranulation as measured by increased levels of RMCPII in the culture media. NT-induced mast cell degranulation was completely inhibited by the SP receptor antagonist CP-96,345, but not by its inactive enantiomer, CP-96,344. Data are presented as mean ± SEM of four experiments, with triplicate determinations per group. **P < 0.01 vs. control; ⁺⁺P < 0.01 vs. NT alone. SP, substance P.