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. 2001 Nov 20;98(24):13936-41.
doi: 10.1073/pnas.241377298.

Proteinase-activated receptor 2 is an anti-inflammatory signal for colonic lamina propria lymphocytes in a mouse model of colitis

S Fiorucci  1 , A Mencarelli, B Palazzetti, E Distrutti, N Vergnolle, M D Hollenberg, J L Wallace, A Morelli, G Cirino
Affiliations

Proteinase-activated receptor 2 is an anti-inflammatory signal for colonic lamina propria lymphocytes in a mouse model of colitis

S Fiorucci et al. Proc Natl Acad Sci U S A. .

Abstract

The proteinase-activated receptor 2 (PAR-2) is a member of a family of G protein-coupled receptors for proteases. Proteases cleave PARs within the extracellular N-terminal domains to expose tethered ligands that bind to and activate the cleaved receptors. PAR-2 is highly expressed in colon in epithelial and neuronal elements. In this study we show that PAR-2 activation prevents the development and induces healing of T helper cell type 1-mediated experimental colitis induced by intrarectal administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS) in mice. A role for PAR-2 in the protection against colon inflammation was explored by the use of SLIGRL-NH(2), a synthetic peptide that corresponds to the mouse tethered ligand exposed after PAR-2 cleavage. TNBS-induced colitis was dose-dependently reduced by the administration of SLIGRL-NH(2), whereas the scramble control peptide, LSIGRL-NH(2), was uneffective. This beneficial effect was reflected by increased survival rates, improvement of macroscopic and histologic scores, decrease in mucosal content of T helper cell type 1 cytokines, protein, and mRNA, and a diminished myeloperoxidase activity. SLIGRL-NH(2), but not the scramble peptide, directly inhibited IFN-gamma secretion and CD44 expression on lamina propria T lymphocytes. Protection exerted by PAR-2 in TNBS-treated mice was reverted by injecting mice with a truncated form of calcitonin gene-related peptide and by sensory neurons ablation with the neurotoxin capsaicin. Collectively, these studies show that PAR-2 is an anti-inflammatory receptor in the colon and suggest that PAR-2 ligands might be effective in the treatment of inflammatory bowel diseases.

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Figures

Figure 1
Figure 1
(A) Effect of PAR-2 activation on survival rate of mice given TNBS enema. BALB/c mice were given 2.5 mg/mouse TNBS intracolonically on days 0 and then treated with saline (TNBS alone) or 1.5 mg/kg per day SLIGRL-NH2 or LSIGRL-NH2 for 7 days. Each group contained 12–16 mice. *, P < 0.01 versus mice treated with TNBS alone or TNBS plus LRGILS-NH2. (B) Time course of wasting disease in mice with TNBS colitis. Intrarectal administration of 1.5 mg/mouse TNBS alone induces bloody diarrhea and wasting disease. Shown are weight changes over a 7-day period occurring in BALB/c control mice treated with 50% ethanol alone (○), mice treated with TNBS alone (●), mice treated with 1.5 mg/kg SLIGRL-NH2 (■), or LSIGRL-NH2, (□). Each point represents average weight data pooled from 8–12 mice. *, P < 0.01 versus TNBS-treated mice. (C and D) Injection of SLIGRL-NH2 protects against development of TNBS colitis in a dose-dependently manner. Each bar is the mean ± SE of 8–12 mice. *, P < 0.01 versus TNBS alone or LSIGRL-NH2. (E–H) Histologic analysis (hematoxylin and eosin staining) of the colons from BALB/c mice with hapten-induced colitis. (E) Transparietal colon section of a vehicle-treated mouse (×200). (F) Colon section 7 days after the induction of colitis by TNBS, showing thickening of the colon wall and inflammatory infiltrate in the lamina propria (×200). (G) Transparietal colon section of a mouse, which received 1.5 mg/kg per day SLIGRL-NH2. Shown is subepithelial edema with no inflammatory infiltrate in the mucosa and submucosa (×200). (H) Transparietal colonic section of mice treated with PAR-2 reverse peptide (×200).
Figure 2
Figure 2
PAR-2 activation prevents formation of proinflammatory mediators in the TNBS model of colitis. M, molecular masses; −, negative control (water); +, positive control (cytokine-positive cDNA); lane 1 colon for control (ethanol-treated) mice; lane 2, colon from a mouse treated with TNBS alone: lane 3, colon from a mouse treated with TNBS plus 1.5 mg/kg per day SLIGRL-NH2; and lane 4, colon from a mouse treated with TNBS plus LSIGRL-NH2. COX-2, cyclooxygenase 2.
Figure 3
Figure 3
(A and B) In vitro cytokine production and proliferation of stimulated and unstimulated LPT. LPT were isolated 7 days after TNBS from control (ethanol-treated) mice and mice treated with TNBS alone or in combination with 1.5 mg/kg per day SLIGRL-NH2 or LRGILS-NH2. Data shown represent pooled values from four independent experiments. Standard errors are indicated. *, P < 0.001 versus control; **, P < 0.001 versus TNBS alone or TNBS plus LSIGRL-NH2. (C) IFN-γ production from LPT is inhibited by SLIGRL-NH2. Data are mean ± SE of six experiments carried out in duplicate. *, P < 0.001 versus LSIGRL-NH2.
Figure 4
Figure 4
CGRP antagonist and sensory neuron ablation reverts protection exerted in vivo by PAR-2 activation in the TNBS model of colitis. (A) Effect of a CGRP receptor antagonist (CGRP8–37) on the macroscopic damage score. *, P < 0.01 versus ethanol treated groups. **, P < 0.01 versus mice treated with SLIGRL-NH2. Each group contained 8–12 mice. (B) Reverse transcription–PCR analysis of Th-1 cytokine mRNA expression 1 week after TNBS admnistration. M, molecular masses; −, negative control (water); +, positive control (cytokine-positive cDNA); lane 1, control (ethanol-treated) mice; lane 2, TNBS alone; lane 3, TNBS plus SLIGRL-NH2, 1.5 mg/kg per day; and lane 4, TNBS plus SLIGRL-NH2 in combination with CGRP8–37. (C) Cytokine production from LPT in TNBS colitis. LPT were isolated from control (ethanol-treated) mice and mice treated with TNBS alone or in combination with 1.5 mg/kg per day SLIGRL-NH2 and CGRP8–37. Data shown represent pooled values from four independent experiments. Standard errors are indicated. *, P < 0.001 versus control; **, P < 0.001 versus TNBS alone; ○, P < 0.01 versus TNBS plus SLIGRL-NH2.
Figure 5
Figure 5
TNBS administration increase CD44 expression on LPT cell surface. (A) Increased CD44 expression in LPT obtained from TNBS-treated mice. Increased CD44 expression is indicated by a left shift in fluorescence. (B) PAR-2 activation reverts CD44 up-regulation induced by TNBS. (C) Exposure to 100 μM SLIGRL-NH2, but no to LSIGRL-NH2, in vitro directly down-regulates CD44 expression on LPT obtrained from TNBS-treated mice. (D) Western blot analysis of SLIGRL-NH2-induced CD44 cleavage. Lane 1, control; lane 2, lysates obtained from LPL prepared from TNBS-treated mice; lanes 3 and 4, lysates obtained from LPL prepared from TNBS-treated mice and incubated in vitro with 10 μM SLIGRL-NH2 or LSIGRL-NH2, respectively. The results shown are representative of three independent experiments. Molecular masses of CD44 are shown.
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