Gastroenterology

Gastroenterology

Volume 130, Issue 1, January 2006, Pages 127-136
Gastroenterology

Basic–alimentary tract
Myofibroblast Matrix Metalloproteinases Activate the Neutrophil Chemoattractant CXCL7 From Intestinal Epithelial Cells

https://doi.org/10.1053/j.gastro.2005.09.032Get rights and content

Background & Aims: The up-regulation of matrix metalloproteinases (MMPs) in the inflamed gut has mainly been associated with mucosal degradation and ulceration. However, their in vitro capacity to specifically cleave inflammatory mediators indicates that MMPs may have a profound immunoregulatory impact. We hypothesized that MMPs proteolytically modify intestinal epithelial chemokine signaling.

Methods: Interleukin-1β–stimulated Caco-2 cells were exposed basolaterally to nanomolar concentrations of activated MMP-3 or cocultured with interleukin-1β–stimulated, MMP-producing, colonic myofibroblasts (CCD-18co). The conditioned media were subjected to chemotaxis assays. In addition, epithelial cells from patients with colitis were examined by real-time polymerase chain reaction, immunoblotting, and immunohistochemistry.

Results: MMP-3 dose-dependently induced the neutrophil (up to 5-fold) but not monocyte chemoattractant capacity of Caco-2 cells. A similar Caco-2 chemotactic response was obtained in the Caco-2/CCD-18co cocultures. The principal mediator of these protease-related effects was identified as the potent neutrophil chemokine CXCL7 (neutrophil activating peptide 2), a proteolytic cleavage product of chemotactically inert platelet basic protein (PBP), not previously identified in the intestine. Antibodies against CXCL7 inhibited the MMP-induced chemotactic response by 84%, and PBP mRNA and protein were detected in stimulated Caco-2 but not in CCD-18co cells. Furthermore, PBP transcript and protein levels were low in the mucosa and in isolated epithelial cells from patients with Crohn’s disease and from normal intestine but increased up to 13-fold in patients with ulcerative colitis.

Conclusions: These findings identify a novel proinflammatory action of MMPs in inflammation and suggest that lamina propria myofibroblasts are required to achieve maximal intestinal epithelial immune activation.

Section snippets

Cell Culture

Caco-2 human colon carcinoma cells (passages 30–35) were maintained in Dulbecco’s modified Eagle medium (Invitrogen, Paisley, Scotland) supplemented with 10% fetal calf serum (Invitrogen) as previously described.10 Caco-2 cells were seeded at a density of 3 × 105 on polycarbonate culture plate inserts (0.4 μm pore size; Millipore, Watford, England), which were placed in 6-well plates and incubated in 2 mL of apical medium and 2 mL of basolateral medium. Monolayer formation was followed by

MMP Activity Induces Caco-2 Cells to Attract Neutrophils

We first addressed whether MMPs could modulate the ability of intestinal epithelial cell supernatants to attract leukocytes. Caco-2 epithelial cells, a recognized model of the intestinal epithelium,10 were grown for 21–28 days on filters and then basolaterally exposed to 0.5–50 nmol/L of active recombinant human MMP-3 in the absence or presence of 1 ng/mL IL-1β. After 24 hours, the conditioned basolateral media were tested in chemotaxis assays (Figure 1A and B). MMP-3 induced a dose-dependent,

Discussion

This work identifies a novel, proinflammatory action of MMP activity in mucosal immune activation. Subepithelial myofibroblast MMPs are directly involved in local leukocyte recruitment by eliciting a functional chemokine response from the intestinal epithelium, mediated by the neutrophil chemokine CXCL7 (NAP-2).

CXCL7, not previously identified in the intestine, is the only known neutrophil chemokine that is produced as a chemotactically inactive precursor protein, in this case PBP.18 PBP (94

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Supported by grants from the Medical Research Council and the St. Bartholomew’s and the Royal London Charitable Foundation.

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