1. Volume 122, Issue 1, Pages 134-144 (January 2002)
Role of tumor necrosis factor receptor 2 (TNFR2) in colonic epithelial hyperplasia and chronic intestinal inflammation in mice 
Emiko Mizoguchi, Atsushi Mizoguchi, Hidetoshi Takedatsu, Elke Cario, Ype P. De Jong, Choon Jin Ooi, Ramnik J. Xavier, Cox Terhorst, Daniel K. Podolsky, Atul K. Bhan 
Gastroenterology 
Volume 122, Issue 1, Pages (January 2002)
DOI: /gast
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2. Fig. 1
Detection of TNFR2 in CEC with chronic colitis. (A) Single crypt units of freshly isolated CECs stained with toluidine blue stain (objective, 40×). (B) The lamina propria after the removal of epithelial cells (shown in panel A) from the mucosa (objective, 20×). (C-L) Immunohistochemical analysis of the cellular expression of TNFR2. TNFR2 is detected by staining with hamster anti-mouse TNFR2 mAb in the colonic epithelium of (E and F) 12-week-old TCRα KO but not in (C) TNFR2 KO or (D) TCRα KO colon stained with control hamster serum (C-E: objective, 20×; F: objective, 40×). The specificity of mouse anti-human TNFR2 mAb is confirmed by (G) the negative staining on DLD-1 and by (H) the positive staining on SW480 colon cancer cell lines (G and H: objective, 20×). In normal human colon, there is no staining for (J) TNFR2 but intense staining for (I) TNFR1. In contrast, colonic biopsy specimens from (K) UC and (L) CD show positive staining for TNFR2 of the CEC (I-L: objective, 20×).
Gastroenterology  , DOI: ( /gast )
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3. Fig. 2
Expression of TNFR2 and TNFR1 by RPA using freshly isolated CECs. TNFR expression in C57BL/6 WT mice (lane 1), 3-4-week-old (lane 2) and 6-8-week-old (lanes 3 and 4) TCRα KO mice without colitis, and week-old (lanes 5-7) TCRα KO mice with colitis are shown. TNFR2 expression is markedly up-regulated in TCRα KO mice with mild (lane 5), moderate (lane 6), and severe (lane 7) colitis compared with WT mice. TNFR2 expression is up-regulated in RAG-1 KO after transfer of CD45RBhi cells (lanes 9 and 10), and F1 bone marrow cells transplanted CD3ϵTg (lanes 11 and 12) compared with WT (lane 13) mice, but showed comparable expression in TCRα KO mice with severe colitis (lane 8).
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

4. Fig. 3
In vitro effect of proinflammatory cytokines in up-regulation of TNFR2. (A) RPA using mRNA extracts of COLO205 or DLD-1 (5 μg/lane) cultured for 10 hours in vitro with or without stimulation with IL-1β (12.5 ng/mL), IL-6 (50 ng/mL), and/or TNF-α (50 ng/mL). TNFR2 expression in both cell lines is significantly increased after simultaneous stimulation with TNF-α and IL-6, TNF-α, and IL-1β, or all the cytokines mixed together. The data are representative of 5 individual experiments. (B) Identification of the membrane binding form of TNFR2 in COLO205 cell line. Representative fluorescence-activated cell sorter data show COLO205 (1 × 105 cells) stained with FITC–anti-TNFR2 mAb after 10 hours of incubation of suspended cells with or without stimulation. (C) Production of soluble form of TNFR2 in the culture supernatant of the COLO205 cell line without (▴) or with IL-6 (▵), TNF-α (●), TNF-α + IL-1β (○), TNF-α + IL-6 (■), or TNF-α + IL-6 + IL-1β (2). (D) Message and protein (membranous and soluble forms) levels of TNFR2 after stimulation with IL-6, TNF-α, and/or IL-1β in COLO205 and DLD-1 cells for 10 hours. TNFR2 mRNA level was detected by RPA, and the density of each band was analyzed by densitometer with the program of Quantity One (Bio Rad, Hercules, CA) and the ratio of TNFR2 and TNFR1 (R2/R1) calculated. The membranous form of TNFR2 was analyzed by fluorescence-activated cell sorter analysis and the soluble form of TNFR2 by ELISA.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

5. Fig. 4
Polarized effect of proinflammatory cytokines in up-regulation of CEC TNFR2. (A and B) Fluorescence microscopic analysis of IL-6- and TNF-α-treated T 84 cells stained for FITC-anti-TNFR2 mAb. T84 cells stimulated for 10 hours with IL-6 (100 ng/mL) and TNF-α (100 ng/mL) added to the (A) upper compartment (apical aspect) or (B) lower compartment (basolateral aspect) were stained as described in Materials and Methods and analyzed with a fluorescence microscope. Original magnification at 100×, objective with oil, and exposure times at seconds. (C) Soluble TNFR2 production of T84 cells detected in the culture supernatant by ELISA (see Materials and Methods). Untreated (2) or cells treated for 10 hours with 100 ng/mL of TNF-α and/or 100 ng/mL of IL-6 added to upper (apical stimulation) (▨) or lower (basolateral stimulation) (■) compartments. T84 cells produced an increased amount of sTNFR2 after basolateral stimulation compared with untreated or apical stimulation. Data represent mean value ± SEM. P value was calculated for comparison “with” and “without” stimulation in each compartment; n = 6.
Gastroenterology  , DOI: ( /gast )
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6. Fig. 5
Effect of IL-6 deficiency on development of colitis and CEC TNFR2 expression in TCRα KO mice. (A) Disease score (0-6) of colitis in weeks of TCRα+/− (n = 11), TCRα KO (n = 27), and αIL-6 DKO (n = 24) mice as assessed by both gross and histologic findings. αIL-6 DKO mice exhibited milder colitis compared with TCRα KO mice. (B) In vivo BrdU incorporation index in week-old WT (n = 5; ■), IL-6 KO (n = 8; 2), TCRα KO (n = 6; ▨), and αIL-6 DKO (n = 6; ●) mice. The data represent mean values ± SEM. *P < 0.1; **P < vs. WT mice. (C) Left: RPA for the detection of TNFR2 using extracts from CEC in 20-week-old WT (lanes 1 and 2), TCRα KO (lane 3: grade 4 colitis; lane 4: grade 1 colitis), and IL-6-deficient TCRα KO (αIL-6 DKO, lane 5: grade 1 colitis; lane 6: grade 2 colitis) mice. Right: The expression ratio of TNFR2/TNFR1 mRNA evaluated by densitometry analysis (Quantity One; Bio Rad) is compared in 20-week-old αIL-6 DKO mice (n = 12, grade 1 colitis) with the age-matched TCRα KO mice (n = 15, grade 1 colitis). (D) Electrophoretic mobility shift assay using nuclear extracts of freshly isolated CECs from 20-week-old WT, TCRα KO, and αIL-6 DKO mice. The radiolabeled (γ-32P) NF-κB consensus DNA-protein complex is shown by an arrowhead. (E-G) Representative histology of the distal part of colons in 20-week-old (E) WT, (F) αIL-6 DKO, and (G) TCRα KO mice (objective, 20×). Although there is some cellular infiltration in the colonic lamina propria both of αIL-6 DKO and TCRα KO mice (arrowhead) compared with WT mice, there is no obvious crypt elongation in αIL-6 DKO mice compared with TCRα KO mice.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

7. Fig. 6
Activation of STAT3 and induction of TNFR2 mRNA after 4% DSS treatment in WT mice. (A) C57BL/6 WT mice (n = 4 for each time point) were treated with 4% DSS in drinking water for 5 days, changed to normal water after day 5, and examined at days 0, 4, 8, and 12. (B) CEC isolated from the mice treated with DSS were lysed and 15 μg of lysates was subjected to immunoblot with anti–phospho-STAT3 (PY-STAT3). After stripping the antibodies, the membrane was reprobed with anti–STAT-3 antibodies. (C) mRNA was extracted from the CECs and TNFR2 expression was analyzed by RPA.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

8. Fig. 7
Decreased incidence of colitis and colonic epithelial proliferation in αTNFR2 DKO mice. (A) Disease score (0-6) of colitis in week-old TCRα KO (n = 25), αTNFR2 DKO (n = 25), and TNFR2 KO (n = 7) mice as assessed by both gross and histologic findings. (B) In vivo BrdU incorporation index of CECs in the distal part of the colon of 18-week-old WT (n = 4), αTNFR2 DKO (n = 5), and TCRα KO (n = 4) mice.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions