Volume 129, Issue 3, Pages 1005-1018 (September 2005) Differential Regulation of Gastric Tumor Growth by Cytokines That Signal Exclusively Through the Coreceptor gp130  Meegan Howlett, Louise M. Judd, Brendan Jenkins, Nicole L. La Gruta, Dianne Grail, Matthias Ernst, Andrew S. Giraud  Gastroenterology  Volume 129, Issue 3, Pages 1005-1018 (September 2005) DOI: 10.1053/j.gastro.2005.06.068 Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 1 (A) Immunohistochemical localization of basal levels of pYSTAT3 in the antrum of (i) wild-type, (ii and v–viii) gp130757F/F, and (iii) gp130757F/FIL-6−/− mice. Basal levels of STAT3 and pYSTAT3 were measured in the antral stomach by immunoblotting; quantitation densitometry was performed, and pYSTAT3 was expressed as a proportion of total STAT3 (B). Gastroenterology 2005 129, 1005-1018DOI: (10.1053/j.gastro.2005.06.068) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 2 mRNA levels of IL-6 in (A) 10–12-week-old mice and (B) 20–25-week-old mice and IL-11 in (C) 10–12-week-old mice and (D) 20–25-week-old mice were measured by reverse-transcription PCR. Intensity of the bands was determined by densitometry and standardized against the housekeeping gene GAPDH. *Mean value is significantly different (P < .05) from wild-type controls; **mean value is significantly different from all other groups. Gastroenterology 2005 129, 1005-1018DOI: (10.1053/j.gastro.2005.06.068) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 3 (A) The degree and type of gastric inflammation between the forestomach-fundic and antral-duodenal junctions was assessed semiquantitatively. The infiltrate was separated into (i) PMN cell infiltrate, (ii) lymphoplasmacytic (mononuclear) cell infiltrate, (iii) submucosal lymphoid follicle development, and (iv) subserosal leukocyte aggregation and scored from 0 to 4 (0, normal; 1, minimal; 2, moderate; 3, marked; and 4, severe). Each point represents the mean score from an individual mouse, and the bar indicates the mean value for the group. (B) Differential expression of Ig molecules in antral stomachs was assessed by immunoblot. Intensity of the bands was measured by densitometry and standardized against a single band on Coomassie-stained gels. *Mean value is significantly different (P < .05) from the mean value of all other groups. (C–E) Apoptotic leukocytes were assessed in stomach sections by colorimetric, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling of DNA. Representative images of stained sections are shown from (C) gp130757F/F and (D) gp130757F/FIL-6−/− mice. Point counting of apoptotic leukocytes (arrows) was performed. (E) The proportion of apoptotic cells per total leukocytes calculated and expressed graphically. *Mean value is significantly different (P < .05) from the mean value of all other groups. Gastroenterology 2005 129, 1005-1018DOI: (10.1053/j.gastro.2005.06.068) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 4 Examples of submucosal invasion (black arrows) included epithelial cells that either (A) formed glands that compromised the muscularis mucosa (white arrows) or (B–D) were entirely disseminated from the glandular tissue and separated from the mucosa by the muscularis mucosa, often encapsulating luminal spaces (Lu). (Scale bar = 100 μm.) (E) In tumorous stomachs, invasive profiles were quantitated and expressed as total intercepts per length of muscularis mucosa. (F and G) To confirm the presence of epithelial cells within blood vessels, serial sections were stained for (F) von Willebrand factor or (G) cytokeratin. (Scale bar = 200 μm.) (H) Vascularization of the stomach was assessed immunohistochemically and expressed as number of vessels per unit length. *Mean value is significantly different (P < .05) from controls. Gastroenterology 2005 129, 1005-1018DOI: (10.1053/j.gastro.2005.06.068) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 5 mRNA levels of (A) MMP-13 and (B) MMP-9 were assessed by real-time PCR, standardized against the housekeeping gene GAPDH, and expressed as fold change relative to expression in wild-type stomach. Cells expressing (C) MMP-13 or (D) MMP-9 were shown by in situ hybridization or immunohistochemistry, respectively. MMP-13 was only expressed in (C) tumor stomal cells; however, MMP-9 was expressed by (D [i and ii]) tumor stromal cells, (iii) a subset of tumor epithelial cells, and (iv) PMN cells. Gastroenterology 2005 129, 1005-1018DOI: (10.1053/j.gastro.2005.06.068) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 6 Wild-type mice were treated with a single dose of recombinant IL-11, and (A) pYSTAT3 and STAT3 protein levels were assessed by immunoblot; in addition, mRNA levels of (B) MMP-13, (C) MMP-9, (D) interferon gamma, (E) IL-6, and (F) IL-11 were measured by either (A and B) real-time PCR or (D–F) reverse-transcription PCR. Expression was standardized against the housekeeping gene GAPDH. *Mean value of the wild-type treated mice is significantly different (P < .05) from that of untreated controls. Gastroenterology 2005 129, 1005-1018DOI: (10.1053/j.gastro.2005.06.068) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 7 (A) The gp130757F/F mouse. The gp130757F/F mutation causes ablation of signaling through the Ras/ERK/AP-1 pathway and constitutive up-regulation of the JAK-STAT1/3 pathway emanating from the coreceptor gp130. This results in augmented endogenous expression of pYSTAT3 in the antrum, which directly correlates with tumor growth. Tumors have increased vascularization, likely due to stimulation of vascular endothelial growth factor expression by pYSTAT3. They also exhibit increased IL-6 and IL-11 expression, both of which lead to increased MMP expression, subsequent alteration to the ECM, and recruitment of mononuclear infiltrate resulting in chronic gastritis. (B) The gp130757F/FIL-6−/− mouse. IL-6 is dispensable for pYSTAT3 activation, vascularization, and tumor growth, as all were unchanged in the gp130757F/FIL-6−/− mouse. Regulation of IL-6 and IL-11 expression is cross-regulated, as IL-11 is further increased in gp130757F/FIL-6−/− mice. IL-6 is important for the apoptosis and controlled clearance of PMN cells, which allows for subsequent attraction of mononuclear cells into the tissue mucosa and elevated production of IgG and IgA in gp130757F/F mice. IL-11 up-regulates transcription of MMP-3, MMP-9, and MMP-13, which leads to degradation of the ECM. In the absence of IL-6, when IL-11 expression is further increased, transcription of MMP-3, MMP-9, and MMP-13 is up-regulated and leads to further degradation of the ECM, resulting in decreased stromal integrity and increased submucosal invasion. Gastroenterology 2005 129, 1005-1018DOI: (10.1053/j.gastro.2005.06.068) Copyright © 2005 American Gastroenterological Association Terms and Conditions