Collagen degradation and neutrophilic infiltration: a vicious circle in inflammatory bowel disease

Abstract

Objective: Proline-glycine-proline (PGP) has been shown to have chemotactic effects on neutrophils via CXCR2 in several lung diseases. PGP is derived from collagen by the combined action of matrix metalloproteinase (MMP) 8 and/or MMP9 and prolyl endopeptidase (PE). We investigated the role of PGP in inflammatory bowel disease (IBD).

Design: In intestinal tissue from patients with IBD and mice with dextran sodium sulfate (DSS)-induced colitis, MMP8, MMP9 and PE were evaluated by ELISA, immunoblot and immunohistochemistry. Peripheral blood polymorphonuclear cell (PMN) supernatants were also analysed accordingly and incubated with collagen to assess PGP generation ex vivo. PGP levels were measured by mass spectrometry, and PGP neutralisation was achieved with a PGP antagonist and PGP antibodies.

Results: In the intestine of patients with IBD, MMP8 and MMP9 levels were elevated, while PE was expressed at similar levels to control tissue. PGP levels were increased in intestinal tissue of patients with IBD. Similar results were obtained in intestine from DSS-treated mice. PMN supernatants from patients with IBD were far more capable of generating PGP from collagen ex vivo than healthy controls. Furthermore, PGP neutralisation during DSS-induced colitis led to a significant reduction in neutrophil infiltration in the intestine.

Conclusions: The proteolytic cascade that generates PGP from collagen, as well as the tripeptide itself, is present in the intestine of patients with IBD and mice with DSS-induced colitis. PGP neutralisation in DSS-treated mice showed the importance of PGP-guided neutrophilic infiltration in the intestine and indicates a vicious circle in neutrophilic inflammation in IBD.

Figure 1

Protease expression in inflammatory bowel disease (IBD). Matrix metalloproteinase 8 (MMP8) protein concentration (A) and prolyl endopeptidase (PE) activity (B) in homogenates of inflamed (infl; n=50) and non-inflamed (non-infl; n=42) intestinal tissue from patients with IBD and colorectal cancer controls (n=20). Individual values are shown, and horizontal lines represent mean values. Statistical analysis was by unpaired t test and paired t test. (C) MMP8 and PE immunoblot on control samples (C) and paired non-inflamed (N) and inflamed (I) samples of five patients with IBD, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control. (D) Immunohistochemistry of MMP8, MMP9 and PE (and negative control) in intestinal tissues of a patient with IBD. The MMP9, PE and negative control shown were on consecutive sections of the same tissue sample. Original magnification ×400.

Figure 2

Proline–glycine–proline (PGP) and N-Ac-PGP levels in inflammatory bowel disease (IBD). Intestinal N-Ac-PGP (A) and PGP (B) levels in colorectal cancer controls (n=5) and patients with IBD (inflamed (infl), n=25; non-inflamed (non-infl), n=18). Non-inflamed and inflamed values were averaged per patient resulting in one intestinal (N-Ac-)PGP value per patient (IBD, n=27). Individual values, expressed as ng (N-Ac-)PGP/g intestinal tissue, are shown, and horizontal lines represent mean values. *p<0.05, unpaired t test.

Figure 3

Protease expression and proline–glycine–proline (PGP) generation by polymorphonuclear cells (PMNs) from patients with inflammatory bowel disease (IBD). Matrix metalloproteinase 8 (MMP8) (A) and MMP9 (B) protein levels and prolyl endopeptidase (PE) activity (C) in conditioned medium of PMNs isolated from peripheral blood of patients with IBD (n=15–20) and healthy controls (n=12). N-Ac-PGP (D) and PGP (E) generation from collagen type I by PMN conditioned medium (IBD, n=12; control, n=8). Individual values are shown, and horizontal lines represent mean values. Mann–Whitney tests were used to determine significance. *Values above trendline were not used to calculate significance. AMC, 7-amido-4-methylcoumarin.

Figure 4

Proteases in dextran sodium sulfate (DSS)-induced colitis. Intestinal prolyl endopeptidase (PE) activity (A) of huCXCR2 knock-in mice treated with 2-day cycles of DSS over time confirmed by immunoblot with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control (B). Active and total matrix metalloproteinase 9 (MMP9) levels (C) were confirmed by zymography (D), and MMP8 levels (E) were confirmed by immunoblot with GAPDH as a loading control (F). All values are mean+SEM (n=3–8). *p<0.05 versus day 0 (control); **p<0.01 versus day 0. Statistical analysis was by one-way analysis of variance followed by Dunnett's multiple comparison test. AMC, 7-amido-4-methylcoumarin.

Figure 5

Protease expression and proline–glycine–proline (PGP) levels in dextran sodium sulfate (DSS)-induced colitis. (A) Immunohistochemistry of intestinal matrix metalloproteinase 8 (MMP8), MMP9 and prolyl endopeptidase (PE) (and negative control) on consecutive intestinal sections of a DSS-treated huCXCR2 knock-in mouse. Original magnification ×400. Intestinal N-Ac-PGP (B) and PGP (C) levels in DSS-induced colitis, expressed as ng (N-Ac-)PGP/g intestinal tissue. All values are mean+SEM (n=3–8). No significant differences were detected, although there was a strong trend towards increased N-Ac-PGP levels at day 8 (versus day 0, p=0.09, unpaired t-test).

Figure 6

Proline–glycine–proline (PGP) neutralisation in dextran sodium sulfate (DSS)-induced colitis. The Disease Activity Index (A) and colonic length (B) of huCXCR2 knock-in mice treated with 1.5% (w/v) DSS for 5 days, and daily intravenous injections of arginine–threonine–arginine (RTR) with phosphate-buffered saline (PBS) as control, or PGP antibody with isotype antibody as control, and non-DSS controls. (C) Histopathological scoring and (D) representative images of H&E-stained intestinal sections of all groups. (E) Representative images of neutrophil (LY-6B.2) immunohistochemistry on intestinal sections of DSS-treated animals treated with RTR, PBS, PGP antibody and isotype antibody. (F) Myeloperoxidase (MPO) levels in intestinal tissue homogenates of all groups as a measurement of neutrophil infiltration. Original magnifications ×200. All values are mean+SEM (n=8). Significance of differences determined by two-tailed Student t test: *p<0.05; **p<0.01, ***p<0.001.

Figure 7

In inflammatory bowel disease (IBD), intestinal damage by proteases indicates a vicious circle of events leading to proline–glycine–proline (PGP)-induced neutrophilic transmigration. In IBD, triggering of the epithelial layer by bacteria or their products leads to extensive release of CXCL8 by epithelial cells and subsequent CXCL8–CXCR1/2-mediated transmigration of neutrophils from the circulation (left panel). Invading neutrophils produce large amounts of matrix metalloproteinase (MMP) 8 and MMP9, which proteolytically cleave collagen into small fragments. These collagen fragments are further cleaved to the tripeptide, PGP, by epithelial- and neutrophil-derived prolyl endopeptidase (PE) (middle panel). PGP is N-terminally acetylated by an as yet unknown enzyme that is at least produced by neutrophils. (N-Ac-)PGP causes neutrophil chemotaxis and stimulates recruited neutrophils to produce CXCL8 (right panel), leading to a self-maintaining chronic situation.