Volume 117, Issue 5, Pages 1078-1088 (November 1999) Anti–interleukin 12 treatment regulates apoptosis of Th1 T cells in experimental colitis in mice  Ivan J. Fuss*, Thomas Marth‡, Markus F. Neurath§, Glen R. Pearlstein*, Ashish Jain*, Warren Strober*  Gastroenterology  Volume 117, Issue 5, Pages 1078-1088 (November 1999) DOI: 10.1016/S0016-5085(99)70392-6 Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 1 Weight changes of mice with TNBS-induced colitis treated with a single dose of rat IgG control antibody (2 mg IP), a single dose of anti–IL-12 antibody (2 mg IP), or multiple doses of anti–IFN-γ (2 mg IP, 3 doses) as specified in Materials and Methods. Each point represents average weight data from 40 mice studied in 8 similar independent experiments. Error bars represent SEM. TNBS-induced colitis can be reversed by administration of a single dose of anti–IL-12, whereas multiple doses of anti–IFN-γ are necessary to reverse TNBS-induced colitis. No significant weight changes were seen in ethanol-treated control mice given no antibodies (data not shown). Gastroenterology 1999 117, 1078-1088DOI: (10.1016/S0016-5085(99)70392-6) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 2 Histological grading of colon sections from ethanol-treated control mice, mice with TNBS-induced colitis treated with a single dose of rat IgG control antibody, and mice with TNBS-induced colitis treated with either a single dose of anti–IL-12 antibody or multiple doses of anti–IFN-γ antibodies. Colon specimens from mice in each group were taken 1 week after initiation of treatment and subjected to histological grading as described in Materials and Methods. Data were pooled from 8 independent experiments. Gastroenterology 1999 117, 1078-1088DOI: (10.1016/S0016-5085(99)70392-6) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 3 In vitro IFN-γ secretion by LP CD4+ T cells from mice treated with ethanol alone, mice with TNBS-induced colitis treated with a single dose of rat IgG control antibody, and mice with TNBS-induced colitis treated with either anti–IL-12 antibodies or anti–IFN-γ. See Materials and Methods for experimental details. T cells from mice treated with anti–IL-12 do not produce increased amounts of IFN-γ, whereas T cells from mice treated with anti–IFN-γ do. Gastroenterology 1999 117, 1078-1088DOI: (10.1016/S0016-5085(99)70392-6) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 4 Apoptotic cells in cryosections of splenic tissue determined by a modified TUNEL technique (original magnification 150×). (A) Mice with TNBS-induced colitis treated with a single dose of rat IgG control antibody; (B) mice with TNBS-induced colitis treated with multiple doses of anti–IFN-γ antibodies; and (C) mice with TNBS-induced colitis treated with a single dose of anti–IL-12 antibody. Gastroenterology 1999 117, 1078-1088DOI: (10.1016/S0016-5085(99)70392-6) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 5 Apoptotic cells in cryosections of LP tissue determined by a modified TUNEL technique (original magnification 150×). (A) Mice with TNBS-induced colitis treated with a single dose of rat IgG control antibody; (B) mice with TNBS-induced colitis treated with multiple doses of anti–IFN-γ antibodies; and (C) mice with TNBS-induced colitis treated with a single dose of anti–IL-12 antibody. Increased numbers of apoptotic cells are found in tissue from mice treated with anti–IL-12. Data are representative of 8 independent experiments. Gastroenterology 1999 117, 1078-1088DOI: (10.1016/S0016-5085(99)70392-6) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 6 Flow cytometric analysis of dispersed spleen cell population obtained from mice with TNBS-induced colitis. Total number of TUNEL positive cells per total number of spleen cells (%) in mice treated with ethanol alone (7.16 ± 1.3 × 107), mice with TNBS-induced colitis treated with a single dose of rat IgG control antibody (8.79 ± 0.96 × 107), mice with TNBS-induced colitis treated with multiple doses of anti–IFN-γ antibody (1.28 ± 0.19 × 108), and mice with TNBS-induced colitis treated with a single dose of anti–IL-12 antibody (1.05 ± 0.22 × 108). Each error bar represents SEM from 4 independent experiments. Numbers within the parentheses represent average spleen cell numbers for respective treatment groups. Gastroenterology 1999 117, 1078-1088DOI: (10.1016/S0016-5085(99)70392-6) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 7 In vitro IFN-γ secretion by LP CD4+ T cells extracted from MRL/MpJ or MRL/MpJ-lprfas mice with TNBS-induced colitis on day 5 after TNBS administration and mice with TNBS-induced colitis treated with anti–IL-12 antibody on day 3. See Materials and Methods for explanation. Each error bar represents SEM from 3 independent experiments. Gastroenterology 1999 117, 1078-1088DOI: (10.1016/S0016-5085(99)70392-6) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 8 Effect of murine Fas-Fc in mice treated concomitantly with anti–IL-12. Microscopic study (original magnification 100×) of (A) colons of mice with TNBS-induced colitis treated with a single dose of anti–IL-12 (no significant evidence of inflammation) and (B) colons of mice with TNBS-induced colitis treated concomitantly with murine Fas-Fc and anti–IL-12 (bowel wall thickening, loss of goblet cells, and transmural lymphocytic infiltrates). Gastroenterology 1999 117, 1078-1088DOI: (10.1016/S0016-5085(99)70392-6) Copyright © 1999 American Gastroenterological Association Terms and Conditions