1. Volume 130, Issue 1, Pages 127-136 (January 2006)
Myofibroblast Matrix Metalloproteinases Activate the Neutrophil Chemoattractant CXCL7 From Intestinal Epithelial Cells 
Laurens Kruidenier, Thomas T. MacDonald, Jane E. Collins, Sylvia L.F. Pender, Ian R. Sanderson 
Gastroenterology 
Volume 130, Issue 1, Pages (January 2006)
DOI: /j.gastro
Copyright © 2006 American Gastroenterological Association Terms and Conditions

2. Figure 1
MMP activity induces an epithelial chemoattractant response to neutrophils. (A) Neutrophil and (B) monocyte chemoattractant capability of conditioned basolateral media of Caco-2 cells basolaterally exposed to the catalytic domain of MMP-3 in the absence or presence of IL-1β. Data represent the mean ± SD of at least 3 independent experiments. *P < .01 vs no MMP-3 and vs no IL-1β. (C) Caco-2 transepithelial electrical resistance in the absence or presence of IL-1β and basolateral MMP-3 (50 nmol/L). Data represent the mean ± SD of 3 independent experiments.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Terms and Conditions

3. Figure 2
Colonic myofibroblast MMPs induce a chemoattractant response from Caco-2 cells. (A) Total MMP activity by thiopeptolide cleavage assay in conditioned media of CCD-18co and Caco-2 cells, in the absence or presence of IL-1β, and IL-1β with the MMP inhibitors doxycycline, CT1847, or CT1399. Data represent the mean ± SD of at least 3 independent experiments. *P < .01 vs unstimulated, #P < .01 vs IL-1β alone. (B) (Upper panel) Reverse-transcription polymerase chain reaction analysis of various secreted MMPs in untreated or IL-1β–stimulated Caco-2 and CCD-18co cells. w, water control; b, β-actin loading control; m, 100–base pair marker. (Lower panel) Western blot analysis showing latent and active MMP-3 in conditioned media of (a) IL-1β– but not (b) unstimulated CCD-18co cells. (C) Neutrophil chemoattractant capability of conditioned media from Caco-2/CCD-18co cocultures, in the absence or presence of IL-1β, and IL-1β with doxycycline, CT1847, or CT1399. Data represent the mean ± SD of 3 independent experiments. *P < .05 vs IL-1β alone. (D) Neutrophil chemoattractant capability of conditioned media of Caco-2 cells exposed to conditioned media of confluent untreated or IL-1β–stimulated CCD-18co cells. Data represent the mean ± SD of 2 independent experiments.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Terms and Conditions

4. Figure 3
CXCL7 (NAP-2) is the principal mediator of the MMP-dependent Caco-2 chemotactic response. (A) Neutrophil chemoattractant capability of conditioned media from Caco-2/CCD-18co cocultures, and of 30 ng/mL human recombinant CXCL7 and 100 ng/mL human recombinant CXCL8 (IL-8), using peripheral blood neutrophils that had been preincubated with neutralizing anti-CXCR1 and/or anti-CXCR2 IgG antibodies. Data represent the mean ± SD of 2 independent experiments. *P < .01 vs no antibodies. (B) Neutrophil chemoattractant capability of conditioned media from Caco-2/CCD-18co cocultures, in the absence or presence of IL-1β, and IL-1β with neutralizing anti-CXCL7 IgG antibodies. An isotype-matched control antibody (anti-CXCR1 in A) did not affect neutrophil chemotaxis. Data represent the mean ± SD of 3 independent experiments. *P < .01 vs IL-1β alone. (C) Reverse-transcription polymerase chain reaction analysis of PBP expression in untreated or IL-1β–stimulated Caco-2 and CCD-18co cells. −RT, polymerase chain reaction of RNA without prior reverse transcription. (D) Enzyme-linked immunosorbent assay of CXCL7 protein and its precursor peptides in conditioned media from untreated or IL-1β–stimulated Caco-2 and CCD-18co cells. Data represent the mean ± SD of 4 independent experiments.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Terms and Conditions

5. Figure 4
Protein levels of PBP/CXCL7 (NAP-2) are elevated in patients with ulcerative colitis. (A) Enzyme-linked immunosorbent assay of CXCL7 protein and its precursor peptides in normal mucosa (n = 5) and in paired noninflamed and inflamed whole mucosa resection specimens from patients with Crohn’s disease (cd, n = 5) or ulcerative colitis (uc, n = 5). *P < .05 vs active Crohn’s disease. n, normal control mucosa; i, inactive; a, active. (B) Representative Western blot of CXCL7 protein expression in a normal mucosa sample and in paired noninflamed and inflamed whole mucosa resection specimens from a patient with Crohn’s disease and a patient with ulcerative colitis. s, recombinant human CXCL7 standard. (C) Immunohistochemical staining of PBP/CXCL7 in a whole mucosa resection specimen of a patient with ulcerative colitis. Staining is predominantly present in the intestinal epithelium (1), while the inflammatory infiltrate is mostly negative (see detail in 3). Omission of the primary antibody deleted all staining (2). (Original magnification: 1, 100×; 2 and 3, 400×.) (D) Proposed model of MMP-mediated epithelial immune activation through PBP and CXCL7. Intestinal epithelial cells under proinflammatory conditions synthesize and secrete PBP, which in itself has no chemotactic activity. However, when MMPs are released by activated subepithelial myofibroblasts, epithelial PBP is processed into chemotactically active CXCL7, resulting in the recruitment of neutrophils to the site of inflammation.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Terms and Conditions