Volume 114, Issue 4, Pages 675-689 (April 1998) Mice lacking secretory phospholipase A2 show altered apoptosis and differentiation with Helicobacter felis infection  Timothy C. Wang, James R. Goldenring, Charles Dangler, Susumu Ito, Annegret Mueller, Woo Kyu Jeon, Theodore J. Koh, James G. Fox  Gastroenterology  Volume 114, Issue 4, Pages 675-689 (April 1998) DOI: 10.1016/S0016-5085(98)70581-5 Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 (A–I) H. felis infection results in variable histopathologic responses in inbred mouse strains. H&E-stained sections showing progression of representative lesions in the gastric corpus of C57BL/6, BALB/c, and C3H/HeJ mice (A, D, and G) 6 weeks, (B, E, and H) 19 weeks, and (C, F, and I) 41 weeks after infection with H. felis. (A–C) C57BL/6 mice developed marked mucosal hyperplasia, mucous metaplasia, loss of oxyntic gland epithelium, and intense inflammatory cell infiltration in response to H. felis infection. (D–F) BALB/c mice and (G–I) C3H/HeJ mice infected with H. felis for a similar duration typically did not develop extensive mucosal changes or inflammation. All images are at the same magnification. Bar = 300 μm. Gastroenterology 1998 114, 675-689DOI: (10.1016/S0016-5085(98)70581-5) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 Severe inflammation and gastric mucosal hyperplasia in C57BL/6 and 129SV mouse strains in response to H. felis infection. (A) Gastric corpus of H. felis–infected C57BL/6 mouse (41 WPI) showing foveolar hyperplasia and mucous metaplasia of the glandular epithelium. Loss of oxyntic glandular epithelium was observed frequently, in tandem with lymphoplasmacytic inflammation of the submucosa and granulocytic infiltration of the periglandular and deep lamina propria. Focal invasion of mucous epithelium through the muscularis mucosa and into the submucosa (arrow) was occasionally seen at this time point in infected C57BL/6 mice. Bar = 200 μm. (B) Gastric corpus of H. felis–infected 129SV mice showing moderate mucosal hyperplasia, mucous metaplasia, and inflammation. H&E; bar = 200 μm. Gastroenterology 1998 114, 675-689DOI: (10.1016/S0016-5085(98)70581-5) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 Severe inflammation and gastric mucosal hyperplasia in C57BL/6 and 129SV mouse strains in response to H. felis infection. (A) Gastric corpus of H. felis–infected C57BL/6 mouse (41 WPI) showing foveolar hyperplasia and mucous metaplasia of the glandular epithelium. Loss of oxyntic glandular epithelium was observed frequently, in tandem with lymphoplasmacytic inflammation of the submucosa and granulocytic infiltration of the periglandular and deep lamina propria. Focal invasion of mucous epithelium through the muscularis mucosa and into the submucosa (arrow) was occasionally seen at this time point in infected C57BL/6 mice. Bar = 200 μm. (B) Gastric corpus of H. felis–infected 129SV mice showing moderate mucosal hyperplasia, mucous metaplasia, and inflammation. H&E; bar = 200 μm. Gastroenterology 1998 114, 675-689DOI: (10.1016/S0016-5085(98)70581-5) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 Increased apoptosis in C57BL/6 mice during H. felis infection. TUNEL assay of gastric corpus sections from H. felis–infected (6 weeks after infection) and uninfected C57BL/6 mice. (A) Uninfected C57BL/6 corpus showing apoptosis primarily limited to the surface mucous cells of the gastric pits. (B) Infected C57BL/6 mouse corpus showing increased apoptosis that extends throughout the neck region of the gastric glands. Gastroenterology 1998 114, 675-689DOI: (10.1016/S0016-5085(98)70581-5) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 Increased apoptosis in C57BL/6 mice during H. felis infection. TUNEL assay of gastric corpus sections from H. felis–infected (6 weeks after infection) and uninfected C57BL/6 mice. (A) Uninfected C57BL/6 corpus showing apoptosis primarily limited to the surface mucous cells of the gastric pits. (B) Infected C57BL/6 mouse corpus showing increased apoptosis that extends throughout the neck region of the gastric glands. Gastroenterology 1998 114, 675-689DOI: (10.1016/S0016-5085(98)70581-5) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 Transmission electron micrographs of H. felis–infected C57BL/6 mice. Electron micrographs of fundic mucosa from (A) H. felis–infected C57BL/6 mice and (B) uninfected C57BL/6 control mice, showing loss of parietal (P) cells and increased mucous neck (N) cells in the infected mice in A. The hyperplastic cells are clearly large, mature mucous neck cells in these electron-microscopic studies. Arrows in A show apoptotic bodies. (C) Abundant H. felis organisms (arrow) deep within the gastric glands are adjacent to parietal cells, and (D) on occasion, H. felis (arrow) could be detected inside the canaliculus of parietal cells. Gastroenterology 1998 114, 675-689DOI: (10.1016/S0016-5085(98)70581-5) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 Transmission electron micrographs of H. felis–infected C57BL/6 mice. Electron micrographs of fundic mucosa from (A) H. felis–infected C57BL/6 mice and (B) uninfected C57BL/6 control mice, showing loss of parietal (P) cells and increased mucous neck (N) cells in the infected mice in A. The hyperplastic cells are clearly large, mature mucous neck cells in these electron-microscopic studies. Arrows in A show apoptotic bodies. (C) Abundant H. felis organisms (arrow) deep within the gastric glands are adjacent to parietal cells, and (D) on occasion, H. felis (arrow) could be detected inside the canaliculus of parietal cells. Gastroenterology 1998 114, 675-689DOI: (10.1016/S0016-5085(98)70581-5) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 Transmission electron micrographs of H. felis–infected C57BL/6 mice. Electron micrographs of fundic mucosa from (A) H. felis–infected C57BL/6 mice and (B) uninfected C57BL/6 control mice, showing loss of parietal (P) cells and increased mucous neck (N) cells in the infected mice in A. The hyperplastic cells are clearly large, mature mucous neck cells in these electron-microscopic studies. Arrows in A show apoptotic bodies. (C) Abundant H. felis organisms (arrow) deep within the gastric glands are adjacent to parietal cells, and (D) on occasion, H. felis (arrow) could be detected inside the canaliculus of parietal cells. Gastroenterology 1998 114, 675-689DOI: (10.1016/S0016-5085(98)70581-5) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 Transmission electron micrographs of H. felis–infected C57BL/6 mice. Electron micrographs of fundic mucosa from (A) H. felis–infected C57BL/6 mice and (B) uninfected C57BL/6 control mice, showing loss of parietal (P) cells and increased mucous neck (N) cells in the infected mice in A. The hyperplastic cells are clearly large, mature mucous neck cells in these electron-microscopic studies. Arrows in A show apoptotic bodies. (C) Abundant H. felis organisms (arrow) deep within the gastric glands are adjacent to parietal cells, and (D) on occasion, H. felis (arrow) could be detected inside the canaliculus of parietal cells. Gastroenterology 1998 114, 675-689DOI: (10.1016/S0016-5085(98)70581-5) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 Distribution of parietal, chief, and mucous neck cells hyperplasia in H. felis–infected and uninfected BALB/c mice. Fundic mucosal sections from (A–C) uninfected mice and (D–F) infected (19 weeks after infection) mice were immunostained for (A and D) parietal cells (H+,K+-ATPase), (B and E) chief cells (intrinsic factor), and (C and F) mucous neck cells (SP). There is a slight increase in parietal cells in the infected BALB/c mice, but no other significant changes were observed. Gastroenterology 1998 114, 675-689DOI: (10.1016/S0016-5085(98)70581-5) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 6 Distribution of parietal, chief, and mucous neck cells hyperplasia in H. felis–infected and uninfected C3H/HeJ mice. Fundic mucosal sections from (A–C) uninfected mice and (D–F) infected (19 weeks after infection) mice were immunostained for (A and D) parietal cells (H+,K+-ATPase), (B and E) chief cells (intrinsic factor), and (C and F) mucous neck cells (SP). There is no significant change seen in the cell lineages of infected mice. Gastroenterology 1998 114, 675-689DOI: (10.1016/S0016-5085(98)70581-5) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 7 Distribution of parietal, chief, and mucous neck cells hyperplasia in H. felis–infected and uninfected C57BL/6 mice. Fundic mucosal sections from (A–C) uninfected mice and (D–F) infected (19 weeks after infection) mice were immunostained for (A and D) parietal cells (H+,K +-ATPase), (B and E) chief cells (intrinsic factor), and (C and F) mucous neck cells (SP). A marked increased in SP-positive mucous neck cells was observed in H. felis–infected C57BL/6 mice. Gastroenterology 1998 114, 675-689DOI: (10.1016/S0016-5085(98)70581-5) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 8 H. felis leads to altered gene expression in the gastric mucosa in a strain dependent fashion. Northern blots showing expression of mRNAs for pepsinogen, and H+,K+-ATPase in (A) BALB/c mice and (B) C57BL/6 mice. Northern blots showing expression of mRNAs for pS2 and SP in (C) BALB/c mice and (D) C57BL/6 mice. Also shown are ethidium bromide (EtBr)-stained gels or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) where indicated. Gastroenterology 1998 114, 675-689DOI: (10.1016/S0016-5085(98)70581-5) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 8 H. felis leads to altered gene expression in the gastric mucosa in a strain dependent fashion. Northern blots showing expression of mRNAs for pepsinogen, and H+,K+-ATPase in (A) BALB/c mice and (B) C57BL/6 mice. Northern blots showing expression of mRNAs for pS2 and SP in (C) BALB/c mice and (D) C57BL/6 mice. Also shown are ethidium bromide (EtBr)-stained gels or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) where indicated. Gastroenterology 1998 114, 675-689DOI: (10.1016/S0016-5085(98)70581-5) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 8 H. felis leads to altered gene expression in the gastric mucosa in a strain dependent fashion. Northern blots showing expression of mRNAs for pepsinogen, and H+,K+-ATPase in (A) BALB/c mice and (B) C57BL/6 mice. Northern blots showing expression of mRNAs for pS2 and SP in (C) BALB/c mice and (D) C57BL/6 mice. Also shown are ethidium bromide (EtBr)-stained gels or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) where indicated. Gastroenterology 1998 114, 675-689DOI: (10.1016/S0016-5085(98)70581-5) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 8 H. felis leads to altered gene expression in the gastric mucosa in a strain dependent fashion. Northern blots showing expression of mRNAs for pepsinogen, and H+,K+-ATPase in (A) BALB/c mice and (B) C57BL/6 mice. Northern blots showing expression of mRNAs for pS2 and SP in (C) BALB/c mice and (D) C57BL/6 mice. Also shown are ethidium bromide (EtBr)-stained gels or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) where indicated. Gastroenterology 1998 114, 675-689DOI: (10.1016/S0016-5085(98)70581-5) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 9 Toluidine blue thin sections show expansion of mucous neck cells and loss of parietal cells in C57BL/6 mice during H. felis infection. Toluidine blue–stained sections of fundic mucosa from (A) control and (B) H. felis–infected C57BL/6 mice. In A, parietal cells (large arrows) are prominent, whereas in B, parietal cells are markedly decreased in number and instead numerous mucous neck cells (small arrow) are evident. In addition, H. felis–like organisms can be seen at the base of the glands (large arrow). Gastroenterology 1998 114, 675-689DOI: (10.1016/S0016-5085(98)70581-5) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 9 Toluidine blue thin sections show expansion of mucous neck cells and loss of parietal cells in C57BL/6 mice during H. felis infection. Toluidine blue–stained sections of fundic mucosa from (A) control and (B) H. felis–infected C57BL/6 mice. In A, parietal cells (large arrows) are prominent, whereas in B, parietal cells are markedly decreased in number and instead numerous mucous neck cells (small arrow) are evident. In addition, H. felis–like organisms can be seen at the base of the glands (large arrow). Gastroenterology 1998 114, 675-689DOI: (10.1016/S0016-5085(98)70581-5) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 10 H. felis infection leads to increased expression of sPLA2 mRNA in BALB/c and C3H/HeJ mice, but not in C57BL/6 mice. Northern blot showing expression of sPLA2 mRNA in three different mouse strains (BALB/c, C3H/HeJ, and C57BL/6) with or without H. felis infection (19 WPI). BALB/c lanes 1 and 2 are mice without infection; lanes 3 and 4 are mice with H. felis infection. C3H/HeJ lanes 5 and 6 are mice without infection; lanes 7 and 8 are mice with H. felis infection. C57BL/6 lanes 9 and 10 are mice without infection; lanes 11 and 12 are mice with H. felis infection. The ethidium bromide (EtBr)-stained gel, and the same blot probed for glyceraldehyde-3-phosphate dehydrogenase, are shown at the bottom. Gastroenterology 1998 114, 675-689DOI: (10.1016/S0016-5085(98)70581-5) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 11 H. felis infection leads to increased expression of sPLA2 peptide in BALB/c mice, but not C57BL/6 mice. Western blot showing up-regulation of sPLA2 protein in the gastric mucosa of BALB/c mice, but not C57BL/6 mice, after H. felis infection. The (+) positive control represents 10 μg of protein extract from BALB/c ileum, whereas the (−) control represents 10 μg of protein extract from C57BL/6 ileum. The arrow shows the 14-kilodalton band representing sPLA2. Lane 1, uninfected BALB/c mouse; lane 2, infected BALB/c mouse; lane 3, uninfected C57BL/6 mouse; lane 4, infected C57BL/6 mouse. Size markers are shown on the left. Gastroenterology 1998 114, 675-689DOI: (10.1016/S0016-5085(98)70581-5) Copyright © 1998 American Gastroenterological Association Terms and Conditions