Volume 127, Issue 3, Pages 816-825 (September 2004) Transmembrane tumor necrosis factor is a potent inducer of colitis even in the absence of its secreted form  Nadia Corazza, Thomas Brunner, Caroline Buri, Silvia Rihs, Martin A. Imboden, Inge Seibold, Christoph Mueller  Gastroenterology  Volume 127, Issue 3, Pages 816-825 (September 2004) DOI: 10.1053/j.gastro.2004.06.036 Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 1 TmTNF induces persistent colitis and weight loss in a TNF-dependent model of colitis. Reconstitution of wtTNF RAG2−/− mice with 2 × 105 wtTNF CD4 CD45RBhi donor T cells (■) and of tmTNF tg RAG2−/− mice with tmTNF tg CD4 CD45RBhi T cells (♦) leads to progressive weight loss whereas TNF−/− RAG2−/− recipients, reconstituted with 2 × 105 TNF−/− CD4 CD45RBhi T cells (●) show no signs of weight loss. The change in body weight is expressed as percentage of the weight at the time of cell transfer. Data represent mean values ± SD of 6–12 mice per group. Gastroenterology 2004 127, 816-825DOI: (10.1053/j.gastro.2004.06.036) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 2 Histopathologic assessment of (A) individual wtTNF RAG2−/− recipients of 2 × 105 wtTNF CD4 CD45RBhi donor T cells (■) (N = 11), tmTNF tg RAG2−/− recipients of tmTNF CD4 CD45RBhi T cells (♦) (N = 6), and of TNF−/− RAG2−/− recipients of TNF−/− CD4 CD45RBhi T cells (●) (N = 12). The colitis scores represent the total of individual scores for cellularity, mucin depletion, crypt abscesses, epithelial erosion, hyperemia, and thickness of the colon, as described in the Materials and Methods section. Annotated numbers represent the mean colitis score of N mice per group. Representative tissue sections of the colon from a (B) wtTNF CD4 CD45RBhi → wtTNF RAG2−/− mouse, (C) tmTNF tg CD4 CD45RBhi → tmTNF tg RAG2−/− mouse, and (D) TNF−/− CD4 CD45RBhi → TNF−/− RAG2−/− mouse are shown (H&E staining, magnification: 12×). Gastroenterology 2004 127, 816-825DOI: (10.1053/j.gastro.2004.06.036) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 3 Distribution of TNF mRNA-expressing cells in the colon of a (A) wtTNF CD4 CD45RBhi T-cell-reconstituted wtTNF RAG2−/− mouse, (B) tmTNF CD4 CD45RBhi T-cell-reconstituted tmTNF tg RAG2−/− mouse, and (C) TNF−/− CD4 CD45RBhi T-cell-reconstituted TNF−/− RAG2−/− mouse on day 24 after adoptive cell transfer as assessed by in situ hybridization with a 35S labeled TNF antisense probe. (D) As a control, a section from the same tissue as in (A) was hybridized with a sense probe of the TNF gene. Nuclear fast red counterstaining, magnification: 50×. Gastroenterology 2004 127, 816-825DOI: (10.1053/j.gastro.2004.06.036) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 4 tmTNF produced by recipient non-T cells is sufficient to induce clinical signs of colitis on transfer of TNF−/− CD4 CD45RBhi T cells. Reconstitution of tmTNF tg RAG2−/− mice with 2 × 105 TNF−/− CD4 CD45RBhi T cells (♦) induces colitis and weight loss comparable with wtTNF RAG2−/− recipients of TNF−/− colitogenic T cells (■). As negative controls, both groups of recipient mice were injected with 2 × 105 CD4 CD45RBlo T cells (◊, □). Data are the mean ± SD of 4–14 mice per group. Gastroenterology 2004 127, 816-825DOI: (10.1053/j.gastro.2004.06.036) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 5 Histopathologic assessment of (A) individual wtTNF RAG2−/− (■, □) and tmTNF tg RAG2−/− recipients (♦, ◊) of TNF−/−CD4 CD45RBhi T cells (■, ◊) and CD4 CD45RBlo T cells (□, ◊) at the end of the observation period. Annotated numbers represent mean colitis scores of all individual mice in each group. Representative tissue sections of the colon from a (B) TNF−/− CD4 CD45RBhi → wtTNF RAG2−/− mouse, (C) a TNF−/− CD4 CD45RBhi → tmTNF tg RAG2−/− mouse, and (D) a TNF−/− CD4 CD45RBlo → tmTNF tg RAG2−/− mouse are shown (H&E staining, magnification: 12×). Gastroenterology 2004 127, 816-825DOI: (10.1053/j.gastro.2004.06.036) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 6 tmTNF tg CD4 CD45RBhi T cells, but not wtTNF CD4 CD45RBhi T cells, occasionally induce inflammatory alterations in the colons of TNF−/− RAG2−/− mice. Colitis scores were determined as described in the Materials and Methods section on paraffin sections from TNF−/− RAG2−/− recipients of CD4 CD45RBhi (●), and CD4 CD45RBlo control cells (○) from wtTNF, or tmTNF tg donor mice on day 24 after cell transfer. Annotated numbers represent mean colitis scores of all individual mice in each group. Gastroenterology 2004 127, 816-825DOI: (10.1053/j.gastro.2004.06.036) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 7 TNF and IFNγ mRNA-expressing cells show similar distribution patterns in the colons of tmTNF tg and wtTNF RAG2−/− recipients. Distribution of (A—D) TNF mRNA- and (E—H) IFNγ-expressing cells in the colons of a (A, E) wtTNF CD4 CD45RBhi T-cell-reconstituted wtTNF RAG2−/− mouse, (B, F) tmTNF tg CD4 CD45RBhi T-cell-reconstituted tmTNF tg RAG2−/− mouse, (C, G) TNF−/− CD4 CD45RBhi T-cell-reconstituted tmTNF tg RAG2−/− mouse, and (D, H) a TNF−/− CD4 CD45RBhi T-cell-reconstituted TNF−/− RAG2−/− mouse on day 24 after adoptive cell transfer as assessed by in situ hybridization with a 35S-labeled TNF and IFNγ antisense probe, respectively. l, intestinal lumen. Nuclear fast red counterstaining, magnification: 15×. Gastroenterology 2004 127, 816-825DOI: (10.1053/j.gastro.2004.06.036) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 8 Apoptotic cells are more frequent in the affected colon of (A) tmTNF and (B) wtTNF RAG2−/− recipients of CD4 CD45RBhi T cells from tmTNF tg and wtTNF donors, respectively, than in (C) TNF−/− RAG2−/− recipients of TNF−/− CD4 CD45RBhi T cells. Apoptotic cells (indicated by arrows) were identified on formamide-treated paraffin colonic tissue sections obtained from recipients on day 24 after CD4 T-cell transfer by immunostainings with a primary antibody specific for single-strand DNA. Immunoperoxidase staining, magnification: 200×. Gastroenterology 2004 127, 816-825DOI: (10.1053/j.gastro.2004.06.036) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 9 tmTNF tg and wtTNF RAG2−/− recipients of CD4 CD45RBhi T cells show increased expression of ICAM-1 and VCAM-1 in the colonic mucosa. Colonic frozen sections of wtTNF, tmTNF tg, and TNF−/− RAG2−/− recipients, obtained on day 24 after transfer of CD4 CD45RBhi T cells were stained for MAdCAM-1, ICAM-1, and VCAM-1. Immunoperoxidase staining, magnification: 75×. Gastroenterology 2004 127, 816-825DOI: (10.1053/j.gastro.2004.06.036) Copyright © 2004 American Gastroenterological Association Terms and Conditions