Volume 125, Issue 4, Pages 1125-1136 (October 2003) Helicobacter pylori strain-selective induction of matrix metalloproteinase-7 in vitro and within gastric mucosa  Howard C Crawford, Uma S Krishna, Dawn A Israel, Lynn M Matrisian, M.Kay Washington, Richard M Peek  Gastroenterology  Volume 125, Issue 4, Pages 1125-1136 (October 2003) DOI: 10.1016/S0016-5085(03)01206-X

Figure 1 Induction of MMP-7 by H. pylori in AGS cells is specific and requires contact with viable bacteria. (A ) AGS cells were incubated with phosphate-buffered saline (control), live H. pylori strain 60190, heat-killed H. pylori (100°C for 30 minutes), a sterile H. pylori 60190 filtrate, or medium alone. Coculture supernatants were concentrated after 24 hours of incubation and analyzed by Western blot using a rat monoclonal MMP-7-specific antibody.64 A representative blot is shown. Anti-actin blots served as normalization controls for AGS cell viability under different experimental conditions. (B) Densitometric analysis of multiple Western blot repetitions performed on at least 3 occasions. Error bars = SD. ∗P < 0.05 vs. AGS cells alone. (C ) AGS cells were incubated with medium alone (control), H. pylori strain 60190 (100:1 bacteria/epithelial cell ratio), or viable E. coli strain HB101, or C. jejuni (100:1 bacteria/epithelial cell ratio). Coculture supernatants were concentrated after 24 hours of incubation and analyzed by Western blot using a rat monoclonal MMP-7-specific antibody.64 A representative blot is shown. Anti-actin blots served as normalization controls for AGS cell viability under different experimental conditions. (D) Densitometric analysis of multiple Western blot repetitions performed on at least 3 occasions. Error bars = SD. ∗P < 0.05 vs. AGS cells alone. Gastroenterology 2003 125, 1125-1136DOI: (10.1016/S0016-5085(03)01206-X)

Figure 2 Induction of MMP-7 by H. pylori in vitro is strain specific. (A ) AGS cells were cocultured with the H. pylori cag+ toxigenic strains 60190 and B128 or the wild-type cag− nontoxigenic strain J68 at bacteria/cell ratios of 100:1. Twenty-four hours after incubation, filtered coculture supernatants were concentrated and subjected to Western blot analysis using an anti-MMP-7 antibody as previously described.64 (−), cells incubated with medium alone. A representative blot is shown. Anti-actin blots served as normalization controls for AGS cell viability under different experimental conditions. (B) Densitometric analysis of multiple Western blot repetitions performed on at least 2 occasions. Error bars = SD. ∗P < 0.05 vs. AGS cells alone. Gastroenterology 2003 125, 1125-1136DOI: (10.1016/S0016-5085(03)01206-X)

Figure 3 MMP-7 stimulation by H. pylori is dependent on specific genes within the cag pathogenicity island. (A ) AGS cells were cultured in the absence or presence of the H. pylori cag+ toxigenic strain 60190 or its isogenic cagA, cagE, or vacA null mutant derivatives at bacteria/cell ratios of 100:1. Twenty-four-hour coculture supernatants were then used for Western blot analysis as described in Materials and Methods.64 (−), cells incubated with medium alone. A representative blot is shown. Anti-actin blots served as normalization controls for AGS cell viability under different experimental conditions. (B) Densitometric analysis of multiple Western blot repetitions performed on at least 3 occasions. Error bars = SD. ∗P < 0.05 vs. AGS cells alone. Gastroenterology 2003 125, 1125-1136DOI: (10.1016/S0016-5085(03)01206-X)

Figure 4 H. pylori induction of MMP-7 in AGS cells is dependent on activation of ERK 1/2. (A ) H. pylori strain 60190 (cag+) was grown in Brucella broth with 5% fetal bovine serum for 48 hours, harvested by centrifugation, and added to AGS cells at a bacteria/cell concentration of 100:1 in the absence or presence of vehicle alone (dimethyl sulfoxide) or the ERK 1/2 inhibitor U0126 (10 μmol/L). Twenty-four-hour supernatants were concentrated and subjected to Western blot analysis using an anti-MMP-7 antibody as described.64 (−), cells incubated with medium alone. A representative blot is shown. Anti-actin blots served as normalization controls for AGS cell viability under different experimental conditions. (B) Densitometric analysis of multiple Western blot repetitions performed on at least 3 occasions. Error bars = SD. ∗P < 0.05 vs. AGS cells alone. Gastroenterology 2003 125, 1125-1136DOI: (10.1016/S0016-5085(03)01206-X)

Figure 5 H. pylori induction of MMP-7 in AGS cells is not dependent on activation of p38. (A ) H. pylori strain 60190 was grown in Brucella broth with 5% fetal bovine serum for 48 hours, harvested by centrifugation, and added to AGS cells at a bacteria/cell concentration of 100:1 in the absence or presence of vehicle (dimethyl sulfoxide) or the p38 inhibitor SB203580 (10 μmol/L). Twenty-four-hour supernatants were concentrated and subjected to Western blot analysis using an anti-MMP-7 antibody as described.64 (−), cells incubated with medium alone. A representative blot is shown. Anti-actin blots served as normalization controls for AGS cell viability under different experimental conditions. (B) Densitometric analysis of multiple Western blot repetitions performed on at least 3 occasions. Error bars = SD. ∗P < 0.05 vs. AGS cells alone. Gastroenterology 2003 125, 1125-1136DOI: (10.1016/S0016-5085(03)01206-X)

Figure 6 H. pylori does not induce IL-6 secretion and MMP-7 is not responsive to IL-8 stimulation in AGS cells. (A ) AGS cells were incubated with or without H. pylori wild-type strain 60190 or IL-1β (positive control, 200 pg/mL; R&D Systems), and IL-6 production in coculture supernatants was quantified by enzyme-linked immunosorbent assay. Results represent at least 3 independent experiments performed in triplicate and are expressed as nanograms per milliliter. Bars = SD. ∗P < 0.05 vs. AGS cells alone. (B) Recombinant IL-8 (20 ng/mL) was added to AGS cells; 24 hours later, supernatants were concentrated and subjected to Western blot analysis using an anti-MMP-7 antibody as described.64 (C ) Densitometric analysis of multiple Western blot repetitions performed on at least 3 occasions. Error bars = SD. Gastroenterology 2003 125, 1125-1136DOI: (10.1016/S0016-5085(03)01206-X)

Figure 7 Immunohistochemical staining for MMP-7 in gastric antral mucosa harvested from (A ) uninfected, (B) H. pylori cagA−-infected, and (C and D) H. pylori cagA+-infected persons. MMP-7 was detected in sections of gastric mucosa using a monoclonal anti-MMP-7 antibody as described.81 Representative cells positive for MMP-7 are identified by arrows. Staining for MMP-7 was focal and localized exclusively to gastric epithelial cells. (Original magnification A-C, 200×; D, 400×.) Gastroenterology 2003 125, 1125-1136DOI: (10.1016/S0016-5085(03)01206-X)