Rapid development of colitis in NSAID-treated IL-10–deficient mice Daniel J. Berg, Juan Zhang, Joel V. Weinstock, Hanan F. Ismail, Keith A. Earle, Hector Alila, Rifat Pamukcu, Steven Moore, Richard G. Lynch  Gastroenterology  Volume 123, Issue 5, Pages 1527-1542 (November 2002) DOI: 10.1053/gast.2002.1231527 Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 1 Pathology of acute NSAID-induced colitis. (A) Colon from a control 6-week-old IL-10−/− mouse (10×). There is minimal cellular infiltration in the lamina propria. (B) Acute colitis in a sulindac-treated IL-10−/− mouse (300 ppm sulindac in the diet for 2 weeks, 6 weeks of age). Note the prominent mononuclear infiltrate in the lamina propria. The submucosa is not involved. (C) Low-power view of a colon from a 6-week-old IL-10−/− mouse treated with piroxicam for 2 weeks (200 ppm piroxicam in the diet for 2 weeks) (4×). Note the prominent inflammation within the wall of the colon. (D) Colon from a 6-week-old IL-10−/− mouse treated with piroxicam for 2 weeks (10×). Note the transmural infiltrate of mononuclear cells. (E) Colon from a 6-week-old IL-10−/− mouse treated with sulindac (300 ppm in the diet) for 2 weeks (4×). Note the presence of an ulceration and associated inflammation. (F) Colon from a 6-week-old IL-10−/− mouse after 2 weeks of treatment with sulindac (10×). Note the prominent lamina propria infiltration as well as infiltration of mononuclear cells into the submucosa. Gastroenterology 2002 123, 1527-1542DOI: (10.1053/gast.2002.1231527) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 2 Pathology of colitis 4–6 weeks after discontinuation of NSAID treatment. Four-week-old IL-10−/− mice were treated with NSAID (piroxicam 200 ppm in the diet) for 2 weeks. The animals subsequently were placed on standard rodent diet for 4–6 weeks before assessment of colitis. (A) Cellular atypia in an epithelial gland from an IL-10−/− mouse 4–6 weeks after discontinuation of NSAID treatment (40×). Mild nuclear and cytologic pleomorphism are present as well as loss of basal orientation of the epithelial nuclei. (B) Invasive epithelium in an IL-10−/− mouse 4–6 weeks after discontinuation of NSAID treatment (4×). The epithelium extends almost to the serosa. (C) Extensive area of invasive epithelium in an IL-10−/− mouse 4–6 weeks after discontinuation of NSAID treatment (10×). Gastroenterology 2002 123, 1527-1542DOI: (10.1053/gast.2002.1231527) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 3 Immunohistochemical staining of colon tissue from control and piroxicam-treated IL-10−/− mice. (A) CD4+ T cells in control (6 wk old) IL-10−/− colon. A small number of T cells are noted in the lymphoid aggregate; rare CD4+ T cells are noted in the lamina propria (10×). (B) CD4+ T cells in piroxicam-treated (6 wk old) IL-10−/− colon. Extensive infiltration of the lamina propria by CD4+ T cells can be seen (20×). (C) F4/80+ macrophages in control (6 wk old) IL-10−/− colon (10×). (D) F4/80+ macrophages in piroxicam-treated (6 wk old) IL-10−/− colon (20×). Note the marked infiltration of macrophages in both the lamina propria and submucosa. (E) B220+ B cells in control (6 wk old) IL-10−/− colon (10×). (F) B220+ B cells in piroxicam-treated (6 wk old) IL-10−/− colon (10×). Increased numbers of B220+ cells can be seen in the lamina propria of NSAID-treated IL-10−/− mice. Gastroenterology 2002 123, 1527-1542DOI: (10.1053/gast.2002.1231527) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 4 (A) RNase protection analysis of the effect of NSAID treatment on cytokine mRNA expression in the colon. Four-week-old IL-10−/− mice were maintained on a standard diet (control) or placed on a diet containing piroxicam (200 ppm in the diet). After 2 weeks, total colon mRNA was isolated and cytokine mRNA expression was analyzed by RNase protection by using specific probes for murine cytokines. Lanes 1–4, colon RNA from control IL-10−/− mice. Lanes 5–14, colon RNA from piroxicam-treated IL-10−/− mice. Data are representative of 3 independent experiments. (B) Quantitation of cytokine mRNA expression was assessed by image analysis. Cytokine mRNA levels were normalized to the level of mRNA expression of the housekeeping gene, L32. Data are representative of 3 independent experiments. Mean ± SD, *P < 0.05, piroxicam-treated IL-10−/− mice vs. control IL-10−/− mice. □, Control IL-10−/−; ■, NSAID-treated IL-10−/−. Gastroenterology 2002 123, 1527-1542DOI: (10.1053/gast.2002.1231527) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 5 (A) Colonic cultures of piroxicam-treated IL-10−/− mice produce increased amounts of IL-12 and IFNγ. Colon explants from control IL-10−/− mice or piroxicam-treated IL-10−/− mice were cultured in medium for 36 hours. Supernatants were subsequently harvested and analyzed for cytokine content by ELISA. Ten mice per group were analyzed. Data are representative of 2 independent experiments. Mean ± SD, *P < 0.05, piroxicam-treated IL-10−/− mice vs. control IL-10−/− mice. (B) Purified lamina propria T cells from the colons of piroxicam-treated IL-10−/− mice produce IFNγ. T cells were plated at 1 × 106 cells/mL in anti-CD3 monoclonal antibody–coated plates as described in the Materials and Methods section. After 24 hours of culture, supernatants were harvested and analyzed for cytokine content (IFNγ and IL-4) by ELISA. No IL-4 or IL-5 was detected. Gastroenterology 2002 123, 1527-1542DOI: (10.1053/gast.2002.1231527) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 6 PGE2 concentration in colonic tissues from 4-week-old wild-type or IL-10−/− mice maintained on either control diet or diet containing piroxicam (200 ppm) for 5 days. Tissue PGE2 concentrations were measured by enzyme immunoassay in snap-frozen tissues homogenized in indomethacin-containing buffer. Mean ± SD, n = 5/group. *P < 0.05 control vs. piroxicam-treated mice. Gastroenterology 2002 123, 1527-1542DOI: (10.1053/gast.2002.1231527) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 7 Western blot analysis of COX expression. (A) COX-1 is expressed in colons of wild-type and control IL-10−/− mice. Western blot analysis was performed on colonic protein isolated from 4-week-old wild-type or IL-10−/− mice. Equivalent amounts of protein (25 μg/lane) were separated on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and COX-1 expression was assessed by Western blot analysis. c, control protein from COX-1 expressing 3T3 fibroblast cell line. (B) COX-2 is expressed in control and piroxicam-treated IL-10−/− mice. Western blot analysis was performed on colonic protein isolated from 4-week-old IL-10−/− mice or piroxicam-treated IL-10−/− mice. Equivalent amounts of protein (25 μg/lane) were separated on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and COX-2 expression was assessed by Western blot analysis. c, control protein from LPS-stimulated RAW 264.7 macrophage cell line. Data are representative of 3 independent experiments. (C) COX-1 expression is unchanged in control and piroxicam-treated IL-10−/− mice. Western blot analysis was performed on colonic protein isolated from 4-week-old IL-10−/− mice or 6-week-old piroxicam-treated IL-10−/− mice. Equivalent amounts of protein (25 μg/lane) were separated on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and COX-1 expression was assessed by Western blot analysis. Data are representative of 2 independent experiments. c, control protein from COX-1 expressing 3T3 fibroblast cell line. Gastroenterology 2002 123, 1527-1542DOI: (10.1053/gast.2002.1231527) Copyright © 2002 American Gastroenterological Association Terms and Conditions