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. 2002 Nov;161(5):1903-15.
doi: 10.1016/S0002-9440(10)64466-5.

Induction of intestinal inflammation in mouse by activation of proteinase-activated receptor-2

Nicolas Cenac  1 , Anne-Marie Coelho, Cathy Nguyen, Steven Compton, Patricia Andrade-Gordon, Wallace K MacNaughton, John L Wallace, Morley D Hollenberg, Nigel W Bunnett, Rafael Garcia-Villar, Lionel Bueno, Nathalie Vergnolle
Affiliations

Induction of intestinal inflammation in mouse by activation of proteinase-activated receptor-2

Nicolas Cenac et al. Am J Pathol. 2002 Nov.

Abstract

Proteinase-activated receptor (PAR)-2, a G-protein-coupled receptor for trypsin and mast cell tryptase, is highly expressed in the intestine. Luminal trypsin and tryptase are elevated in the colon of inflammatory bowel disease patients. We hypothesized that luminal proteinases activate PAR-2 and induce colonic inflammation. Mice received intracolonically PAR-2 agonists (trypsin, tryptase, and a selective PAR-2-activating peptide) or control drugs (boiled enzymes, inactive peptide) and inflammatory parameters were followed at various times after this treatment. Colonic administration of PAR-2 agonists up-regulated PAR-2 expression and induced an inflammatory reaction characterized by granulocyte infiltration, increased wall thickness, tissue damage, and elevated T-helper cell type 1 cytokine. The inflammation was maximal between 4 and 6 hours and was resolved 48 hours after the intracolonic administration. PAR-2 activation also increased paracellular permeability of the colon and induced bacterial trans-location into peritoneal organs. These proinflammatory and pathophysiological changes observed in wild-type mice were not detected in PAR-2-deficient mice. Luminal proteinases activate PAR-2 in the mouse colon to induce inflammation and disrupt the integrity of the intestinal barrier. Because trypsin and tryptase are found at high levels in the colon lumen of patients with Crohn's disease or ulcerative colitis, our data may bear directly on the pathophysiology of human inflammatory bowel diseases.

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Figures

Figure 1.
Figure 1.
Kinetic detection of PAR-2 in mouse distal colon by RT-PCR (A, B) or immunofluorescence and confocal microscopy (C), after intracolonic administration of the PAR-2-AP SLIGRL-NH2 (100 μg/mouse), the control peptide LRGILS-NH2 (100 μg/mouse), or vehicle. PAR-2 was assessed by the amplification of a specific 549-bp PCR fragment; GAPDH was a 306-bp fragment, in colonic tissues from mice 10 hours after the intracolonic injection of LRGILS-NH2 (lane 1), or 6 hours (lane 2), 10 hours (lane 3), and 24 hours (lane 4) after the intracolonic injection of SLIGRL-NH2. In B, values are mean ± SEM, n = 8 per group; *, significantly different from LRGILS-NH2-treated group, P < 0.05. In C, PAR-2 was localized using antiserum B5 (1:250, 24 hours, 4°C; arrowheads, plasma membrane; arrow, intracellular compartments). Images are composites of 9 to 12 optical sections of 0.5 to 0.6 μm. Tissues were collected at various times after intracolonic administration of vehicle (veh), 100 μg of SLIGRL-NH2 (SL-NH2), or inactive LRGILS-NH2 (LR-NH2). Note the down-regulation of PAR-2 immunoreactivity 4 hours after SL-NH2 followed by up-regulation in crypts at 6 to 10 hours. The control shows omission of primary antibody. Scale bar, 10 μm (C).
Figure 2.
Figure 2.
Kinetic (A–E) and dose-response curves (F and G) of inflammation induced by the intracolonic administration of the PAR-2-AP SLIGRL-NH2 (100 μg/mouse for A–E) or the control peptide LRGILS-NH2 (100 μg/mouse for A–E) in two different strains of mice: C57BL6 and Swiss 3T3. Different inflammatory parameters were followed: macroscopic (A) and microscopic (D and F) damage scores, wall thickness (B) and MPO activity (C, E, and G). Values are mean ± SEM, n = 8 per group, *, Significantly different from LRGILS-NH2-treated group, P < 0.05.
Figure 3.
Figure 3.
Representative H&E-stained histological sections of colon from wild-type mice that have received an intracolonic injection of saline (A), LRGILS-NH2 (100 μg/mouse, 6-hour time point) (B), SLIGRL-NH2 (100 μg/mouse, 6-hour time point) (C), or PAR-2−/− mouse that have received an intracolonic injection of SLIGRL-NH2 (100 μg/mouse, 6-hour time point). Scale bar, 15 μm (applies to A–D).
Figure 4.
Figure 4.
Inflammation induced by the intracolonic administration of trypsin (50, 100, and 400 U/mouse) in C57BL6 and Swiss 3T3 mice and tryptase (1 μg/mouse) in C57BL6 mice. Macroscopic damage score (A), wall thickness (B), and MPO activity (C and D) were evaluated as inflammatory parameters 6 hours after the intracolonic injection. Values are mean ± SEM, n = 8 per group. *, Significantly different from boiled enzyme-treated group, P < 0.05.
Figure 5.
Figure 5.
Cytokine mRNA expression in mouse colonic tissues 4 hours after the intracolonic administration of the PAR-2-AP SLIGRL-NH2 (100 μg/mouse), the control peptide LRGILS-NH2 (100 μg/mouse), or their vehicle. Values are mean ± SEM, n = 8 per group. *, Significantly different from LRGILS-NH2-treated group, P < 0.05.
Figure 6.
Figure 6.
Changes in intestinal permeability in vivo 3 and 6 hours after the intracolonic administration of the PAR-2-AP SLIGRL-NH2 (100 μg/mouse), the control peptide LRGILS-NH2 (100 μg/mouse), or their vehicle. The passage of the macromolecule 51Cr-EDTA from the gut lumen to the blood was used as an index of intestinal permeability. Values are mean ± SEM, n = 8 per group. *, Significantly different from LRGILS-NH2-treated group, P < 0.05.
Figure 7.
Figure 7.
Inflammation (A–C), as observed by the macroscopic damage score (A), wall thickness (B), MPO activity (C), cytokine mRNA expression (D), and intestinal permeability as observed by the passage of the macromolecule 51Cr-EDTA from the gut lumen to the blood (E) after the intracolonic administration of the PAR-2-AP SLIGRL-NH2 (100 μg/mouse), in wild-type or PAR-2-deficient (PAR-2−/−) mice. Values are mean ± SEM, n = 8 per group except for E where n = 6 per group. *, Significantly different from wild type, P < 0.05.
Figure 8.
Figure 8.
Inflammation as observed by the macroscopic damage score (A), wall thickness (B), and MPO activity (C), induced after the intracolonic administration of trypsin (400 U/mouse) or tryptase (1 μg/mouse), in wild-type or PAR-2-deficient (PAR-2−/−) mice. Values are mean ± SEM, n = 8 per group. *, Significantly different from wild type, P < 0.05.
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