Role for protease activity in visceral pain in irritable bowel syndrome

Abstract

Mediators involved in the generation of symptoms in patients with irritable bowel syndrome (IBS) are poorly understood. Here we show that colonic biopsy samples from IBS patients release increased levels of proteolytic activity (arginine cleavage) compared to asymptomatic controls. This was dependent on the activation of NF-kappaB. In addition, increased proteolytic activity was measured in vivo, in colonic washes from IBS compared with control patients. Trypsin and tryptase expression and release were increased in colonic biopsies from IBS patients compared with control subjects. Biopsies from IBS patients (but not controls) released mediators that sensitized murine sensory neurons in culture. Sensitization was prevented by a serine protease inhibitor and was absent in neurons lacking functional protease-activated receptor-2 (PAR2). Supernatants from colonic biopsies of IBS patients, but not controls, also caused somatic and visceral hyperalgesia and allodynia in mice, when administered into the colon. These pronociceptive effects were inhibited by serine protease inhibitors and a PAR2 antagonist and were absent in PAR2-deficient mice. Our study establishes that proteases are released in IBS and that they can directly stimulate sensory neurons and generate hypersensitivity symptoms through the activation of PAR2.

Figures

Figure 1. Proteolytic activity in human colonic biopsies and present in colonic washes.

Arginine-directed protease activity in supernatants of biopsy from controls (black squares), IBS patients (IBS-D: D, circles; IBS-C: C, white squares; or IBS-D/C: D/C, diamonds), IBD patients (ulcerative colitis [UC]: black triangles; Crohn disease [CD]: x’s) or in IBS biopsy supernatants preincubated with the serine protease inhibitor FUT-175 or supernatants from biopsies incubated with the NF-κB inhibitor BAY 11-7085. IBS biopsy supernatants were separated according to the presence (I) or not (NI) of inflammatory signs, and biopsies were harvested at the level of the ascending colon (A) or the rectum (B). All + FUT, all IBS patient supernatants (I and NI) incubated in the presence of FUT-175. (C) Protease activity in colonic washes from control (black squares) and IBS patients. Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.005 compared with control group.

Figure 2. Tryptase and trypsin expression in human colonic biopsies and culture supernatants.

(A) Dual RT-PCR of tryptase and GADPH mRNA and of trypsin and gusb from IBS and control patient biopsies quantified by densitometry analysis of the gels. (B) Western blots of trypsin and tryptase proteins from control, IBS, or IBD patient biopsy supernatants (top panel), quantified by densitometry analysis of the blots (bottom panel). Data are mean ± SEM; n = 12. *P < 0.05 compared with control group; **P < 0.01.

Figure 3. Tryptase immunoreactivity in rectal biopsies from control (A, D, and E) and IBS patients (B, C, and F).

Tryptase was mostly localized to cells found throughout the lamina propria (A–C) and around the base of rectal mucosal crypts (C). Some cells (e.g., C), had obvious processes at either the apical and/or basal poles, and often these cells displayed intense immunoreactivity compared with neighboring cells (images in B and C were taken under identical exposure conditions and reflect differences in intensity). Occasional tryptase-immunoreactive cells were found in the epithelium of both control (D) and IBS patients; these had the appearance of enteroendocrine cells, based on the obvious basolateral process. No differences were observed in the cell populations of IBS and control patients with respect to either the density of cells in rectal biopsies (G) or the intensity of immunoreactivity. Alcian blue–labeled mast cells in a control (E) and IBS patient (F). Note that the majority of tryptase-immunoreactive cells (e.g., B) have the size, shape, and cellular location of mast cells. Scale bars: 50 μm.

Figure 4. Calcium mobilization in sensory neurons exposed to human colonic biopsy supernatants.

Calcium flux in sensory neurons from wild-type (PAR2+/+) and PAR2–/– mice exposed to supernatants of biopsies from control and IBS patients or biopsy supernatants preincubated with a serine protease inhibitor (FUT-175). IBS patient supernatants were either regrouped together (all supernatants [All]) or divided into subgroups. Biopsies were collected either from the ascending colon (A) or the rectum (B). Data are mean ± SEM. *P < 0.05 compared with control group; †P < 0.05 compared with the group with all IBS supernatants tested in PAR2+/+ neurons.

Figure 5. Somatic nociception of mice in response to injection of human colonic biopsy supernatants into paws.

Graph of withdrawal latency (A) and nociceptive scores (B–D) in response to thermal (plantar test) or mechanical (von Frey filaments) stimulation, respectively, of mouse paws that had received an injection of control or IBS patient biopsy supernatants or saline solution. Wild-type mice (PAR2+/+: squares [A], black bars [B–D]) and PAR2-deficient mice (PAR2–/–: x’s [A], gray bars [B–D]) had received intraplantar injections. Three sizes of von Frey filaments were used: filament 3.61 (B), filament 3.84 (C), and filament 4.08 (D). All biopsies represented in this figure were collected from the rectum; n = 8 for controls; n = 9 for IBS. Data are mean ± SEM for withdrawal latencies and median and range (in brackets) for mechanical stimuli testing (von Frey filaments). *P < 0.05 compared with basal measures (before the intraplantar injection: time 0).

Figure 6. Visceral sensitivity in response to human colonic biopsy supernatants.

Recordings of mouse abdominal muscle contraction in response to colorectal distension before (control) or 6 hours after intracolonic administration of ascending colon biopsy supernatants from IBS patients (A and B) or control patients (filled triangles in C). (A) Representative traces of mouse abdominal muscle contractions in response to 60 mmHg colorectal distension, in PAR2+/+ or PAR2–/– mice before (control) or after intracolonic (i.c.) administration of IBS ascending colon biopsy supernatant alone or in combination with the protease inhibitor FUT or a PAR2 antagonist. (B) Abdominal contraction responses to different pressures of distension (15–60 mmHg) before (time 0) or after intracolonic administration of IBS patient ascending colon biopsy supernatants. Data are mean ± SEM; n = 8. *P < 0.05, **P < 0.01, ***P < 0.005 compared with basal (time 0) measurements. (C) Mouse abdominal contraction response to increasing pressures of distension in PAR2+/+ or PAR2–/– mice before (control distension) or 6 hours after intracolonic administration of IBS biopsy supernatants (filled squares) alone or in combination with the protease inhibitor FUT or a PAR2 antagonist or after the intracolonic administration of biopsy supernatants from control patients (filled triangles). Data are mean ± SEM; n = 12 for the biopsy supernatants from control patients; n = 18 for IBS biopsy supernatants. *P < 0.05, **P < 0.01, ***P < 0.005 compared with control distension. For all data in this figure, biopsies were collected from the ascending colon.

Figure 7. Visceral sensitivity in response to human colonic biopsy supernatants from different IBS patient subgroups.

Abdominal muscle contraction responses of mice before (white bars) and 6 hours after intracolonic administration of IBS ascending colon biopsy supernatant (gray bars). Results were analyzed in different subgroups: IBS-C, IBS-D, IBS-D/C, inflammation, and no inflammation. Data are mean ± SEM. *P < 0.05 compared with control distension.

Figure 8. Visceral sensitivity in response to different doses of trypsin.

Abdominal contraction response of mice 6 hours after intracolonic administration of trypsin (0.2, 0.5, and 1.0 U of trypsin) or its vehicle (saline) in response to different pressures of distension (15–60 mmHg). Data are mean ± SEM; n = 8 per dose of trypsin. *P < 0.05, **P < 0.01, ***P < 0.005 compared with control distension (time 0).