Role of tumor necrosis factor receptor 2 (TNFR2) in colonic epithelial hyperplasia and chronic intestinal inflammation in mice

GASTROENTEROLOGY 2002;122:134-144

Role of Tumor Necrosis Factor Receptor 2 (TNFR2) in Colonic Epithelial Hyperplasia and Chronic Intestinal Inflammation in Mice EMIKO MIZOGUCHI,*,t ATSUSHI MIZOGUCHI, * , t HIDETOSHI TAKEDATSU,* ELKE CARLO,*,§ YPE P. DE JONG, *,11 CHOON JIN OO1,*,§ RAMNIK J. XAVIER,*,§ COX TERHORST,*,II DANIEL K. PODOLSKY,*,§ and ATUL K. BHAN*,* *Center for the Study of Inflammatory Bowel Disease, tDepartment of Pathology, §Division of Gastroenterology, Massachusetts General Hospital; and IIDivision of Immunology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts

Background&Aims: Tumor necrosis factor (TNF) induces multiple effects including cell proliferation and death by ligation with TNF receptor type II (TNFR2). We studied the role of TNFR2 in chronic inflammationinduced colonic epithelial alteration. Methods: TNFR2 expression in colonic epithelial cells (CECs) was assessed by ribonuclease protection assay (RPA) and immunohistochemistry (IHC) in patients with inflammatory bowel disease (IBD) and murine colitis models. TNFR2 expression was also analyzed using COL0205 cells. The role of TNFR2 in colonic epithelial homeostasis was examined by generating interleukin 6-deficient TCRaKO (odL-6DKO) or TNFR2-deficient TCR~ (oLTNFR2DKO) mice. Results: TNFR2 expression was up-regulated in CEC in both human ulcerative colitis and Crohn's disease. In vitro studies showed that TNFR2 expression was upregulated by a cooperative effect of key proinflammatory cytokines. By RPA, the increased expression of TNFR2 was detectable in TCRo~KO mice with colitis compared with TCRoLKO mice without colitis or wild-type mice. In odL-6DKO mice, TNFR2 expression, proliferation, and nuclear factor kappa B activation of CECs were markedly reduced compared with TCRoLKO mice. oLTNFR2 mice also showed significantly less colonic epithelial proliferation compared with TCRoLKO mice. Conclusions: Expression of TNFR2 is consistently increased on CECs in both murine colitis models as well as patients with IBD. TNFR2 may play an important role in colonic inflammation-associated alteration in the intestinal epithelium.

some bacterial products (i.e., intiminp play an important role in innate immune-mediated epithelial homeostasis and induction of acquired immune responses. The presence of epithelial hyperplasia has been associated with many experimental model of IBD produced by genetic manifestation, and altered epithelial turnover has been shown in and associated with human IBD. However, the in vivo mechanisms inducing colonic epithelial alteration associated with dysregulated acquired immune responses in IBD are unclear. Tumor necrosis factor (TNF) produces multiple effects including altered cell proliferation and death through distinct signaling cascades resulting from binding to members of TNF receptor family, 55 kilodaltons of TNF receptor type I (TNFR1) and 75 kilodaltons of TNFR type lI (TNFR2). 1° 14 TNF-mediated signal pathways seem to play a variety of roles in innate and adoptive immunity. Cell death, altered target gene transcription, and cytokine production is mediated by TNFRl.15"16 In contrast, engagement of TNFR2 promotes either in cooperation with or independently of TNFR1, cell proliferation and cell survival. 11 TNFR2 may have an antiapoptotic effect acting through a nuclear factor kappa B (NF-KB) pathway that results in exacerbated expression of several inflammatory mediators. TNFR2 initiates cell survival by induction of antiapoptotic molecules and inhibition of proapoptotic proteins (cIAP-1 and clAP2). lv,18 The mechanism of TNFR-mediated cell death

bservations from many laboratories indicate that intestinal epithelial cells play an active role in mucosal immune responses and participate in the pathogenesis of inflammatory bowel disease (IBD). 1-6 These cells also establish an anatomic barrier and provide an interface with luminal bacteria and their products. The polymeric immunoglobulin (Ig) A v and Toll-like receptors 8 on the epithelial cells, intestinal defensin,9 and

Abbreviations used in this paper: CEC, colonic epithelial cell; DMEM, Dulbecco's modified Eagle medium; DSS, dextran sodium sulfate; ELISA, enzyme-linked immunosorbent assay; FITC, fluorescein isothiocyanate; IL, interleukin; KO, knockout; LP, lamina propria; mAb, monoclonal antibody; NF-KB, nuclear factor kappa B; RPA, ribonuclease protection assay; STAT, signal transducer and activator of transcription; TNF, tumor necrosis factor; TNFR, tumor necrosis factor receptor. © 2002 by the American Gastroenterological Association 0016-5085/02/$35.00 doi:10.1053/gast.2002.30347

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January 2 0 0 2

and survival in colonic e p i t h e l i a l cells u n d e r chronic i n f l a m m a t o r y c o n d i t i o n s has not been defined yet. T h e spontaneous d e v e l o p m e n t o f chronic colitis in g e n e t i c a l l y m a n i p u l a t e d mice provides a means to s t u d y the pathogenesis o f h u m a n I B D i n c l u d i n g the role of cytokines and c y t o k i n e receptors. 19,2° U s i n g these mice, we have shown an i m p o r t a n t role o f T N F R 2 in the d e v e l o p m e n t of chronic colitis in association w i t h colonic e p i t h e l i a l alterations.

M a t e r i a l s and M e t h o d s Mice TCRot knockout (KO), T N F R I KO, TNFR2 KO, and IL-6 KO mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and maintained under specific pathogen-free facilities at Massachusetts General Hospital (Boston, MA). These inbred mice were all of C57BL/6 strain (H-2b) background. To generate TCRot KO mice deficient in interleukin (IL)-6 or TNFR2, TCRot KO mice were crossed with IL-6 KO or TNFR2 KO mice. Double KO mice were obtained from the heterozygous ( T C R & + / - T N F R 2 + / or T C R o t + / - I L 6 + / - ) breeding pairs, which were obtained by crossing TCRot KO mice with either TNFR2 KO or IL-6 KO mice. Screening was performed as previously described. 2. Briefly, genomic D N A extracted from tail was subjected to polymerase chain reaction. To distinguish heterozygous and homozygous, the pairs of 3 primers were used (IL-6 forward: 5'-AACCGCT A T G A AGTTCCTCTC TCTG, IL-6 reverse: 5'-CACGATTTCCCAGAGAACATG, Neo/IL-6 forward: 5'-CATCATGG C T G A T G C A A T G for screening of IL-6, and IMR338: 5'-CCTCTCATG CTGTCCCG GAAT, IMR339: 5'-AGCTCC A G C ~ A C A A G C ~ C G G G , mfr2neo: 3'-CTTC CATTTGTCACGTCC TG for screening of TNFR2). The human CD3e transgenic (CD3eTg) mice model and CD45RB hi cell transfer model were also studied as previously described.22,23

Colonic Epithelial Cell Preparation For isolation of viable crypt cells, Bjerknes' method 24 was used with some modification. Mice were anesthetized by inhalation of isoflurane and the abdomen was opened. A 21gauge needle was inserted in the proximal part of the colon and the lumen was flushed with 10 mL of 37°C 1 mmol/L EDTA in Hank's balanced salt solution (HBSS). The thoracic cavity was opened and perfused through the left ventricle with 10 mL of 37°C 30 mmol/L EDTA in HBSS. At the end of the perfusion, the entire colon excluding cecum was removed, inverted, and placed in a cold tube with 2 mL of cold HBSS. The tube was shaken at 2500 rpm for 20 seconds at 4°C by mini beater (Biospec-Products, Bartlesville, OK). The tissue remnants were discarded and the crypts in the supernatant were allowed to settle to the bottom of the tube and then washed 3 times with HBSS. The viability and purity of colonic epithelial ceils (CECs) were determined by trypan blue staining and ribonuclease protection assay using mCD-1 mouse cell

TNFR2 EXPRESSION OF EPITHELIAL CELLS IN IBD

135

surface antigen multiprobe template sets (PharMingen, San Diego, CA) to examine concentration of immune cells. Over 95% of freshly isolated epithelial cells were viable, and contamination of CD45 + cells was less than 1%-3%.

Disease Score The disease score ( 0 - 6 ) was estimated on the basis of both gross and histologic findings. The gross score was rated as: 0, presence of normal appearance of colon; 1, absence of beaded appearance of colon; 2, focal thickened colon; and 3, marked thickness of the entire colon. The severity of colitis (0, normal; 1, mild; 2, moderate; and 3, severe colitis) in H&Estained sections was determined according to the diagnostic criteria previously described. 2°

Antibodies and Cytokine Reagents The antibodies used in this study including purified or biotin-conjugated mouse anti-human TNFR2 (clones: 897C2G9 and 911B3H10) monoclonal antibodies (mAbs) were purchased from Biosource (Camarillo, CA). Fluorescein isothiocyanate (FITC)-conjugated anti-human TNFR2 mAb (clone: 22235.311) was purchased from R&D systems (Minneapolis, MN). Unconjugated hamster anti-mouse TNFR2 (clone TR75- 54) and biotinylated hamster anti-mouse TNFR2 (clone TR75-89) mAbs were purchased from PharMingen. Recombinant human IL-I~, IL-6, and TNF-ot were purchased from R&D Systems and recombinant mouse IL-6 and TNF-ot were purchased from PharMingen.

Culture of Human Epithelial Cell Lines DLD-1, COLO205, SW 480, and T84 cell lines were obtained from American Type Culture Collection (Rockville, MD). DLD-1, COLO205, and SW 480 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum containing a mixture of antibiotics (amphotericin B, penicillin G, and streptomycin). These cell lines were incubated at 37°C in an atmosphere of 5% CO2 in T25 cm e culture flask or 24-well culture plate (Fisher Scientific, Pittsburgh, PA). For in vitro cytokine stimulation experiments, 7 0 % - 8 0 % confluent cells (adherent or floating conditions) were exposed to recombinant I L - I ~ (12.5 ng/mL), IL-6 (50 ng/mL), and/or TNF-ot (50 nglmL). 25,e6 T84 cells were maintained and grown in DMEM-Ham's F12 (DMEM-F12; 1:1) containing 10% fetal calf serum and penicillin, streptomycin, glutamine, and HEPES. For growth on filter membranes, cells were cultured as previously described. 27 Briefly, 1 × 106/1 mL cells on 24-mm Transwell filters (clear polystyrene, 0.4-b~m pore size; Costar, Cambridge, MA) w e r e cultured for 5 - 6 days until reached to a differentiated state as confluent polarized monolayer. Confluent monolayers were used only if their transepithelial resistance was > 1 0 0 0 • cm e. Ceils were stimulated with IL-6 and/or TNF-ot (50 ng/mL, each) for 10 hours from upper or lower compartments. After stimulation, culture supernatant of each compartment was harvested separately. Cells on the filter membrane were stained with FITC-conjugated anti-TNFR2 m A b (R&D Sys-

136 MIZOGUCHIET AL.

tems) and analyzed by fluorescence microscopy (Olympus AX70, Shinjuku, Tokyo, Japan).

BrdU Incorporation of CECs For BrdU incorporation study, 5 mg/200 btL of 5bromo-2'-deoxyuridine (BrdU; Sigma Chemical Co., St. Louis, MO) was injected intraperitoneally 1 hour before sacrifice of mice. Anti-BrdU antibody (Harlan Sera-Lab, Loughborough, England) was used to detect BrdU-incorporated cells in situ as previously described. 2°

Detection of TNFR2 Ribonuclease protection assay (RPA) was performed using 10 Dg of total RNA with the RiboQuant multi-probe RPA system (PharMingen) by following the manufacturer's specifications. In brief, RNA was hybridized overnight with the [,~/_32p] uridine triphosphate-labeled probe, which is in vitro translated by T7 RNA polymerase. After hybridization, samples were treated with ribonuclease A followed by proteinase K. After phenol/chloroform extract and ethanol precipitation, the protected fragments were resolved by electrophoresis on a 5% acrylamide/urea gel. After running the gel, it was dried at 80°C for 1.5 hours and exposed to radiograph film at -85°C. For the TNFR2 enzyme-linked immunosorbent assay (ELISA), the procedure was performed following the Cytosets instruction protocol from Biosource. Briefly, Maxisorp 96-well microtiter plates (Nunc, Naperville, IL) were coated with mouse anti-human TNFR2 mAb (Biosource), 1 I~g/mLin 100 gtL of coating buffer (0.14 mol/L NaC1, 10 mmol/L Na2HPO4, 1 mmol/L KH2PO4, and 2.6 mmol/L KC1 in dH20; pH 7.4), and incubated overnight at 4°C. The wells were washed between each incubation step with washing buffer (0.15 mol/L NaCI and 0.1% Tween-20 in dH20; pH 7.4). After washing, the plates were blocked by adding 300 btL of blocking solution (coating buffer with 0.5% bovine serum albumin; pH 7.4) for 2 hours at room temperature. After further washing, 50 DL/ well of samples were added and incubated at room temperature for 1 hour. Next, 50 DL of biotin-labeled mouse anti- human TNFR2 (Biosource), 0.4 gtg/mL in diluent/assay buffer (coating buffer with 0.1% bovine serum albumin; pH 7.4), was used as the detection antibody and the plate was incubated at room temperature for 1 hour. After washing, 100 b~L/well of streptavidin-biotin-horseradish peroxidase complex (Biosource), diluted 1:3000 in diluent/assay buffer, was added and incubated for 30 minutes at room temperature. After 10 times washing with washing buffer, 100 DL/well of tetramethylbenzidine substrate solution (PharMingen) was added; the reaction was stopped after 10 minutes with 50 gtL of 10% orthophosphoric acid. The optical density at 450 nm was read by Auto-Reader (Bio-Tec Instruments, Burlington, VT). To standardize the assay, recombinant human TNFR2 (Biosource) was used as a positive control. A standard curve was created with the optical density of 1:2 (5 p~g/mL) to 1:256 (39.0 pg/mL) dilutions of recombinant TNFR2. The quantity of soluble

GASTROENTEROLOGYVol. 122, No. 1

TNFR2 in the culture supernatant was calculated using this standard line.

Histology and Immunohistochemical Analysis Cryopreserved human colonic tissue samples were obtained from the tissue bank of the Center for the Study of Inflammatory Bowel Disease at the Massachusetts General Hospital (Boston, MA). Murine tissue samples were obtained from the distal and proximal part of colon and were fixed in 10% buffered formalin. Paraffin-embedded tissue sections were stained with H&E using standard technique. Fresh tissue samples from both human and mouse colon were frozen OCT (optimal cutting temperature) compound (Ames Company, Elkhart, IN) and stored at - 8 0 ° C until use. Frozen tissue sections (4-1~m thick) were air dried again for 20 minutes and stained by the avidinbiotin complex method as described previously. 2°

Flow Cytometric Analysis COLO205 monolayers were removed from culture flask by trypsinization. After washing with complete DMEM medium, 4 )< 106 cells/mL were incubated for 10 hours with or without cytokines in a shaking incubator (37°C, 80 rpm) to enable regeneration of TNFR2, and the cells were transferred to a 5-mL tube in phosphate-buffered saline buffer supplemented with 1% bovine serum albumin and 0.5% sodium azide at the concentration of 1 X 105 cells/25 p~L. Fc-blocked ceils were incubated with FITC-conjugated mouse anti-human TNFR2 mAb (R&D Systems) for 45 minutes on ice. As a negative control, FITC-conjugated mouse IgG1, K (PharMingen) was used. After the incubation, cells were washed with phosphate-buffered saline buffer and resuspended in 400b~L of 2% paraformaldehyde. Cell surface expression of the TNFR2 was determined by flow cytometric analysis using 488-nm wavelength laser excitation.

Dextran Sodium Sulfate-Induced Colitis Mice were treated with dextran sodium sulfate (DSS) as previously described. 28,29 Briefly, colitis was induced by addition of 4.0% (wt/vol) DSS (mol wt, 35,000-44,000; ICN Biomedicals, Irvine, CA) in drinking water for 5 days and followed by distilled water for 7 days. The mice were killed on days 0, 4, 8, and 12. 28,29

Immunoblot Analysis Western blot analysis was performed as previously described. 29 Briefly, freshly isolated CECs were washed with HBSS. After washing, the cells were homogenized in a lysis buffer containing 50 mmol/L Tris (pH 8.0), 0.5% NP-40, 1 mmol/L EDTA, 150 mmol/L NaCI, 10% glycerol, 50 mmol/L sodium fluoride, 10 mmol/L sodium pyrophosphate, 1 mmol/L sodium orthovanadate, 1 mmol/L phenylmethylsulfonyl fluoride, and a tablet of protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Tyrosine phosphorylation of signal transducer and activator of transcription (STAT)-3 was

January 2002

detected by using anti-phospho-STAT-3 antibody (Cell Signaling Technology, Beverly, MA). After stripping anti-phospho-STAT-3 antibody by using Restore Western blot stripping buffer (Pierce, Rockford, IL), the membranes were reprobed by using anti-STAT-3-specific antibody (Santa Cruz Biotechnology, Santa Cruz, CA).

Nuclear Extracts and Assessment of NF-KB Activation by Electrophoretic Mobility-Shift Assay Nucleic extracts were prepared according to a method previously described) o Oligonucleotide probes containing consensus and mutant sequences for NF-KB were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Doublestranded oligonucleotides were labeled with [y_32p] adenosine triphosphate using T4 polynucleotide kinase (Promega, Madison, WI).

Statistical Analysis Statistical significance was evaluated by the MannWhitney U test for nonparametric data or the Student t test for parametric data.

Results TNFR2 Expression Is Increased in the CECs of TCR~ KO Mice With Colitis Colonic crypt alteration is a characteristic feature of human IBD as well as both T h l - and Th2-mediated colitis models. 2,4 Expression of selected apoptosis- and proliferation-associated molecules by freshly isolated coIonic epithelial cells (Figure 1A)24 w a s initially assessed by RPA using commercially available probes (PharMingen). Increased expression of TNFR2 was found in CECs from TCRot KO mice with colitis compared with C57BL/6 wild-type (WT) mice. Increased TNFR2 expression was proportionate to the severity of colitis (Figure 2). The semiquantification of TNFR2 expression (the density of TNFR2 messenger RNA (mRNA) divided by the density of glyceraldehyde-3-phosphate dehydrogenase mRNA) by densitometry analysis showed higher expression in TCRot KO mice with colitis (11.8 --- 1.2; n = 8) compared with TCR~x KO mice without colitis (3.1 --- 0.4; n = 7). An increase of TNFR2 m R N A expression was initially detectable in CECs by 8 - 1 2 weeks of age in TCRot KO mice (data not shown). Immunohistochemical analysis using hamster antimouse TNFR2 mAb confirmed these findings (Figure 1C-F). TNFR2 expression was observed in colonic epithelium of TCRot KO mice with colitis. However, there was no obvious TNFR2 expression by the colonic lamina propria (LP) cells and intraepithelial lymphocytes of these mice (Figure 1F). In contrast to TNFR2, TNFR1

TNFR2 EXPRESSION OF EPITHELIAL CELLS IN IBD

:1.37

m R N A was constitutively expressed in the CEC of W T and TCRoL KO mice without colitis (Figure 2). The expression of TNFR1 m R N A also increased with development of colitis in TCRo~ KO mice.

Distinct Expression of TNFR2 of CEC in Chronic Colitis The colitis in TCRo~ KO mice is mediated by the IL-4-dependent Th2 pathway 31,52 and shares some features with human ulcerative colitis (UC). 19 In contrast, the colitis in CD3626 transgenic mice reconstituted with bone marrow cells from W T donors (CD3eTg model), and in scid or RAG-1 KO mice reconstituted with splenic CD4 + CD45RB hi cells (CD45RB model), is mediated by signal transducers and activators of transcription (STAT)-4/IL-12 associated with the T h l pathw a y . 23'33'34 These 2 models exhibit features of Crohn's disease (CD). 19,23 Despite the apparent differences in the immunopathogenesis of these models, a significant increase of TNFR2 in the CEC was also present in both CD45RB and CD3¢Tg models compared with WT, CD3eTg control, or RAG-1 KO mice (Figure 2 and data not shown). To determine whether the increase in TNFR2 present in murine models of IBD was also present in human IBD, samples from patients with colonic disease were subjected to immunohistochemical analysis with antiTNFR2 mAb. TNFR2 expression was found in 90% of patients with CD. Staining was mostly present in crypt epithelial cells; scattered intraepithelial lymphocyte and LP cells were also stained (Figure 1L). TNFR2 staining of CEC was also present in 80% of UC patients (Figure 1K) and seemed to be more intense than in CD patients (Figure 1L). There was no T N F R 2 + cells in the colonic LP of patients with UC. In 2 of 5 cases of infectious colitis, up-regulation of TNFR2 expression was also observed in the colonic epithelium (data not shown). In contrast, there was no or faint staining of TNFR2 (Figure 1./and Table 1) in the normal colon tissue specimens. No staining was present on DLD-1 cells (Figure 1G), which do not express TNFR2, whereas positive staining was present on SW480 ceils (Figure 1H), which constitutively express TNFR2, confirming the specificity of the staining with anti-TNFR2 mAb.

In Vitro Effect of IL-I~ and IL-6 in the Induction of TNFR2 Although distinct cytokine networks initiate colitis in both T h l - and Th2-mediated experimental colitis, proinflammatory cytokines such as IL-113 and IL-6 seem to be involved in both types of colitis. 2°,32,~5 Therefore, we examined the effect of IL-113 and IL-6 together

138

MIZOGUCHI ET AL.

GASTROENTEROLOGY Vol. 122, No. 1

D

%

J'%'-~

~

K

~

i

e

.

Figure 1. Detection of TNFR2 in CEC with chronic colitis. (A) Single crypt units of freshly isolated CECs stained with toluidine blue stain (objective, 4 0 x ) . (B) The lamina propria after the removal of epithelial cells (shown in panel A) from the mucosa (objective, 20x). (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 TCRe KO but not in (C) TNFR2 KO or (D) TCR~ KO colon stained with control hamster serum (C-E: objective, 2 0 x ; F: objective, 4 0 x ) . 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, 2 0 x ) . In normal human colon, there is no staining for (J) TNFR2 but intense staining for (/) TNFRI. In contrast, colonic biopsy specimens from (K) UC and (L) CD show positive staining for TNFR2 of the CEC (/-L: objective, 20×).

g

[3 Figure 4. Polarized effect of proinflammatory cytokines in up-regulation of CEC TNFR2. (A and B) Fluorescence microscopic analysis of IL-6- and TNF-~-treated T 84 cells stained for FITC-anti-TNFR2 mAb. T84 cells stimulated for 10 hours with IL-6 (100 ng/mL) and TNF-e (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 lOOX, objective with oil, and exposure times at 2.810 seconds. (C) Soluble TNFR2 production of T84 cells detected in the culture supernatant by ELISA (see Materials and Methods). Untreated (C]) or cells treated for 10 hours with 100 ng/mL of TNF-e and/or 100 ng/mL of IL-6 added to upper (apical stimulation) ([]) or lower (basolateral stimulation) (11) 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.

C

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January 2002

TNFR2 EXPRESSIONOF EPITHELIALCELLS IN IBD 139

TCR~ KO WT no colitis colitis ....

TNFR2 TNFR1

~

e

o

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o

q P W ~ B

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L32 GAPDH 1 2 3 4 5 6 7

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9 10 11 12 13

Figure 2. Expression of TNFR2 and TNFR1 by RPA using freshly isolated CECs. TNFR expression in C57BL/6 WT mice (lane 1), 3-4week-old (lane 2) and 6-8-week-old (lanes 3 and 4) TCRe KO mice without colitis, and 20-25-week-old (lanes 5-7) TCRu KO mice with colitis are shown. TNFR2 expression is markedly up-regulated in TCRe 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 CD45RB h' cells (lanes 9 and 10), and F1 bone marrow cells transplanted CD3eTg (lanes 11 and 12) compared with WT (lane 13) mice, but showed comparable expression in TCR~ KO mice with severe colitis (lane 8).

with TNF-ot on TNFR2 expression using human colon cancer cell lines. The COLO205 cell line, which spontaneously expresses low levels of TNFR2, 36 was initially used. Addition of any of the cytokines, IL-I~, IL-6, or TNF-ot, failed to induce an increase in TNFR2 expression in COLO205 cells (3, 6, 10, 12, and 24 hours) (Figure 3A and data not shown). In contrast, IL-1]3 or IL-6 in combination with TNF-ot increased COLO205 TNFR2 expression. Highest TNFR2 expression was achieved in COLO205 cells when stimulated with the combination of all 3 cytokines (IL-6, IL-1[3, and TNF-o0 (Figure 3A and D). Interestingly, this combination of cytokines also induced TNFR2 expression in DLD-1 cells, which only express TNFR1 in normal culture conditions 36 (Figure 3A and D). Flow cytometric analysis also showed that COLO205 constitutively expressed cell surface TNFR2 and expression increased after stimulation with the combination of IL-6, IL-I[3, and TNF-o~ (Figure 3B). Cell surface TNFR2 is proteolytically cleaved and subsequently released as soluble fragments) 7 Analysis of culture me-

dium by ELISA showed that the soluble form of TNFR2 was also increased in the supernatants of COLO205 after stimulation with the combination of cytokines (IL-6, IL-I~, and TNF-o0 (Figure 3C and D). These in vitro studies indicate that the expression of TNFR2 requires cooperation of cytokines such as IL-6 and IL-l[3 with TNF-cx. The apical surface of CEC is constitutively exposed to the luminal contents, whereas the basal portion is exposed to factors in the lamina propria. To determine whether apical or basolateral stimulation mediates induction and secretion of TNFR2 in CEC, confluent polarized T84 monolayers were exposed to TNF-ot and IL-6 either from the apical or basolateral compartments for 10 hours. Apical stimulation with IL-6 and TNF-ot did not increase TNFR2 expression (Figure 4A). In contrast, after basolateral stimulation, a marked increase of TNFR2 expression was observed (Figure 4B) and a 4-6-fold increase in secretion of soluble TNFR2 was present in the lower compartment (Figure 4C). In Vivo

Effects of IL-6 on TNFR2 Expression

Because increased IL-6 production also has been shown in TCRoL KO mice with colitis, 32 IL-6-deficient TCRoL KO double mutant (odL-6 DKO) mice were generated to assess the role of IL-6 in requiring CEC TNFR2 expression in vivo. The lack of IL-6 led to suppression of chronic colitis in TCRoL KO mice (Figure 5A). TNFR2 expression and BrdU-incorporated cell number of CECs was markedly reduced in cilL-6 D K O mice (10.6 + 0.3, mean --- SEM; SD = 2.61), even in the presence of mild colitis compared with age-matched TCRot KO mice (22.2 _+ 0.8, SD = 6.48) (Figure 5B and C). Although an increased number of infiltrating

Table 1. Immunohistochemical Analysis of TNFR2 Expression by CECs

No. of subjects Negativeb Weakly positive b Strongly positive b

Controls"

UC

CD

11 8 / 1 1 (72.8%)

13 2 / 1 3 (15.3%)

10 1 / 1 0 (10%)

3 / 1 1 (27.2%)

3 / 1 3 (23.1%)

5 / 1 0 (50%)

0 / 1 1 (0%)

8/13 (61.5%)

4 / 1 0 (40%)

aThe colonic specimens from control patients had normal histologic appearance. ~ h e degree of staining was assessed by the intensity and the number of positive epithelial cells. Negative represents no definite staining of CECs. Strongly positive represents intense diffuse staining of CECs; the staining pattern between negative and strongly positive represents weakly positive. The mouse IgG1 used as a negative control for mouse anti-human TNFR2 mAb showed no positive staining.

140

MIZOGUCHI El" AL.

GASTROENTEROLOGY Vol. 122, No. 1

A DLD-1

COLO-205

~

IL-1RII TNFR2 TNFR1

IL-IRII TNFR2 TNFR1 gp130

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Soluble Form

R2/R1

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0.012

0

0.020

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IL-1IS+ TNF¢t i

0.151

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0.016

TNF(! + IL-6

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0.184

IL-IIS+ TNFo+ IL-6 i i

0.072

IL-1IS+ TNF(z ~

0,283

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0,302

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0.969

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260

olo

17.84 28o

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11.94 19.37

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mononuclear ceils was found in the colonic LP of both oLIL-6DKO mice (Figure 5F, arrowhead)and TCRoL KO mice with mild colitis (grade 1) (Figure 5G, arrowhead) compared with W T mice (Figure 5E), crypt elongation

1000

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Figure 3, In vitro effect of proinflammatory cytokines in up-regulation of TNFR2. (A) RPA using mRNA extracts of C O L 0 2 0 5 or DLD-1 (5 i~g/lane) cultured for 10 hours in vitro with or without stimulation with IL-113 (12.5 ng/mL), 1l--6 (50 ng/mL), a n d / o r TNF-~ (50 n g / mL). TNFR2 expression in both cell lines is significantly increased after simultaneous stimulation with TNF-~ and IL-6, TNF-~, and IL-I~, or all the cytokines mixed together. The data are representative of 5 individual experiments. (B) Identification of the membrane binding form of TNFR2 in C 0 L 0 2 0 5 cell line. Representative fluorescence-activated cell sorter data show C 0 L 0 2 0 5 (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 C 0 L 0 2 0 5 cell line without (A) or with IL-6 (A), TNF-~ (e), TNF+ IL-1!3 (©), TNF-~ + IL-6 ( i ) , or TNF-~ + IL-6 + IL-115 ([3). (D) Message and protein (membranous and soluble forms) levels o f TNFR2 after stimulation with IL-6, TNF-~, a n d / o r 11-118 in C 0 L 0 2 0 5 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 ( R 2 / R1) calculated. The membranous form of TNFR2 was analyzed by fluorescence-activated cell sorter analysis and the soluble form of TNFR2 by ELISA.

was observed in TCRo~ KO mice but not in oLIL-6DKO mice (Figure 5F). In addition, the CEC TNFR2 expression in odL-6 DKO mice with mild colitis was significantly less than that in TCRc~KO mice with mild colitis

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TNFR2 EXPRESSION OF EPITHELIAL CELLS IN IBD

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(P ~< 0.001); the average ratio of TNFR2/TNFR1 judged by mRNA expression was 0.523 + 0.052 (n = 15) in TCRcx KO and 0.147 + 0.049 (n = 12) in otlL-6 DKO mice (Figure 5C). Because NF-KB activation is a critical factor for cell proliferation and survival, nuclear extracts from freshly isolated CECs were analyzed by a gel mobility shift assay to determine whether the reduction of TNFR2 expression and CEC proliferation was associated with activation ofNF-KB. NF-KB DNA binding activity was significantly reduced in oLIL-6 DKO mice compared with TCRo~ KO mice (Figure 5D).

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Figure 5. Effect of IL-6 deficiency on development of colitis and CEC TNFR2 expression in TCRe KO mice. (A) Disease score (0-6) of colitis in 2 0 - 2 5 weeks of T C R e + / - (n = 11), TCRe KO (n = 27), and elL-6 DKO (n = 24) mice as assessed by both gross and histologic findings. elL-6 DKO mice exhibited milder colitis compared with TCRc~ KO mice. (B) In vivo BrdU incorporation index in 18-20-week-old WT (n = 5; B), IL-6 KO (n = 8; Iq), TCRe KO (n = 6; []), and elL-6 DKO (n = 6; []) mice. The data represent mean values _+ SEM. *P < 0.1; * * P < 0.001 vs. WT mice. (C) Left: RPA for the detection of TNFR2 using extracts from CEC in 20-week-old WT (lanes 1 and 2), TCRe KO (lane 3: grade 4 colitis; lane 4: grade I colitis), and IL-6-deficient TCRc~ KO (elL-6 DKO, lane 5: grade i 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 elL-6 DKO mice (n = 12, grade 1 colitis) with the age-matched TCRcx KO mice (n = 15, grade 1 colitis). (D) Electrophoretic mobility shift assay using nuclear extracts of freshly isolated CECs from 20-week-old WT, TCRe KO, and elL-6 DKO mice. The radiolabeled (,y_32p) NF-KB 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) ~IL-6 DKO, and (G) TCRe KO mice (objective, 20×). Although there is some cellular infiltration in the colonic lamina propria both of elL-6 DKO and TCRe KO mice (arrowhead) compared with WT mice, there is no obvious crypt elongation in elL-6 DKO mice compared with TCR{x KO mice.

STAT-3 Activation Followed by TNFR2 Induction in DSS-Induced Colitis IL-6-associated cascades activate STAT-3, and IL-6-induced STAT-3 activation (phosphorylation of thyrosine residue of STAT-3) has been found in the DSS-induced colitis, e9 We examined the time course of STAT-3 phosphorylation and TNFR2 expression in 4% DSS-induced colitis (Figure 6A), because the onset of inflammation can be easily identified in DSS-induced colitis compared with spontaneously developed colitis. STAT-3 phosphorylation in the CEC as detected by RPA was increased on day 4 and subsequently reduced on days 8 and 12 after the DSS administration (Figure 6B). In contrast, up-regulation of TNFR2 was detectable on day 8, but not on day 4. Furthermore, TNFR2 expression was greater on day 12 than on day 8 after the DSS administration. These findings indicate that IL-6/STAT3 signaling cascades are induced before up-regulation of TNFR2 expression on the CEC. Interestingly, elongation of colonic crypts was present on day 12, 7 days after termination of DSS administration (data not shown) as shown previously.28 The findings obtained from the DSSinduced colitis model are consistent with the contention that TNFR2 up-regulation by IL-6/STAT3 signaling cascades activated during inflammatory conditions may be involved in the induction and/or enhancement of epithelial crypt elongation.

Decreased Rate of BrdU Incorporation of CEC in TCRoL KO Mice Is Dependent on TNFR2 Expression To determine the direct effect of TNFR2 on the development of colitis and CEC proliferation, TNFR2deficient TCR(x double KO ((xTNFR2 DKO) mice were generated. Although the lack of TNFR2 led to suppression of chronic colitis in TCR(x KO mice at 18 weeks of age, about 44% of mice developed a mild to moderate form of colitis at 22-25 weeks of age (Figure 7A). BrdU-incorporation by CEC of 18-week-old WT, TCR(x

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KO, and ciTNFR2 D K O mice was determined. The BrdU incorporation index of o~TNFR2 D K O mice (11.2 + 0.4, SD = 3.04) was significantly (P < 0.001) less than those of TCR(x K O mice (22.0 + 0.8, SD = 5.39) but higher than control W T mice (8.2 + 0.2, SD = 1.62) (Figure 7B). These findings support the notion that T N F R 2 participates in colonic crypt elongation under inflammatory conditions, and T N F R 2 has a role in the pathogenesis of Th2-mediated colitis in TCRci KO mice.

Discussion Although alteration of colonic epithelial homeostasis is a characteristic feature of chronic intestinal inflammation in both human IBD (UC and CD) and experimental colitis, the mechanisms leading to colonic crypt elongation have not been fully defined. Here, we have investigated the role of T N F R 2 in the colonic crypt alteration in association with chronic inflammatory conditions. The members of the T N F receptor superfamily are critical regulators of biological responses. 11,16 Among this family, T N F R 2 may play an important role in abnormal conditions, but is thought to have a few effects in normal conditions31 Our results show increased expression of T N F R 2 on CEC in Th2-mediated colitis of TCRo~ KO mice and Thl-mediated colitis of CD3~Tg and CD45RB hi cell transfer models. Furthermore, T N F R 2 expression is also up-regulated in the CECs of patients with UC and CD as well as in infectious colitis. Therefore, up-regulation of T N F R 2 expression on CEC seems to be a feature of intestinal inflammatory conditions including IBD and may be involved in the perpetuation of inflammatory process and altered epithelial cell functions. Interestingly, TNFR2-positive cells were not detected in the colonic LP of UC and in TCRoL K O mice. In contrast, some scattered T N F R 2 positive cells were present in the LP of CD. Therefore, T N F R 2 expressed by LP cells may not have an important role in human UC or colitis of TCRci K O mice. Because increased production of proinflammatory cytokines is present in the lamina propria of both UC and CD as well as in murine colitis models, t9,38 we initially examined the role of IL-6, TNF-cl, and IL-I~ in regu-

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January 2002

lating of TNFR2 expression in CEC using human cancer cell lines in vitro. Although TNFo~ alone had no effect on TNFR2 expression, the combination of TNFo~ with IL-6 was able to up-regulate TNFR2. The expression of TNFR2 on the T84 cell line was induced by the stimulation provided from the basolateral but not apical side. Therefore, TNFR2-mediated CEC alteration seems ~o be regulated by IL-6 in the presence of TNF-ct. IL-6 exerts a variety of biological effects including mediating the generation of complicated loops of proinflammatory cytokines including TNF-oL and IL-I[~. 39 Recently, the IL-6 trans-signaling cascade was found to affect CD4 + T-cell survival in a Thl-mediated chemically induced experimental colitis model and human CD. 4° The suppression of colitis in odL-6 DKO mice in the present study suggests that IL-6 also plays an important role in Th2-mediated colitis of TCRct KO mice. There was significantly less up-regulation of TNFR2 expression in CECs of odL-6 DKO mice compared with TCRot KO mice even in the presence of mild colitis in both groups of mice. Furthermore, in the DSS-induced colitis model, the TNFR2 up-regulation in CECs is detectable only after the activation of the IL-6/STAT3mediated pathway. TNFRl-associated intracellular molecules contain a death domain, whereas TNFR2-associated molecules lack a death domain. 11 The interaction between TNFR1 and TNFR2 signaling pathways is thought to play a crucial role in regulation of cellular homeostasis?L41,42 Recent studies suggest that the functional differences between TNFR1 and TNFR2 are not absolute, and the effects produced by these receptors may depend on the cell types. 14 Our studies support that TNFR2 is involved in induction and/or facilitation of crypt elongation under inflammatory conditions. Dysregulated immune responses initiated by microbial-host interactions contribute to development and perpetuation of IBD. * In this process, intestinal epithelial cells play an important role linking innate and acquired immune responses.2,4,6,43 TCRot KO mice do not develop colitis when maintained in a germ-free environment. 19,21,44 The proinflammatory cytokine network generated by chronic inflammatory conditions caused by dysregulated immune response to luminal bacterial antigens may lead to TNFR2 expression in CEC. Blockade of TNF/TNFRs inducing signaling has been shown to be an effective therapeutic strategy for CD 45,46 as well as rheumatoid arthritis. 4v The study of the TNFR family is likely to reveal novel roles for these receptors in epithelial cell function in normal and diseased states including chronic colitis.

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References

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Received April 19, 2001. Accepted September 17, 2001. Address requests for reprints to: Atsushi Mizoguchi, M.D., Immunopathology Unit, Warren 501C, Massachusetts General Hospital, 55 Fruit Street, Boston, Massachusetts 02114. e-mail: [email protected]; fax: (617) 726-2365. Supported by National institutes of Health (NIH) grant DK47677 (to A.K.B.), by the Center for the Study of Inflammatory Bowel Disease at the Massachusetts General Hospital (NIH DK43351 to D.K.P., C.T., A.K.B., and E.M.), by a Career Development Award (to A.M.), by a research fellowship from the Crohn's and Colitis Foundation of America (to Y.P.J.), and by the Deutsche Forschungsgemeinscbaft (ca 226/ 2-1) (to E.C.). The authors thank Drs. Markus F. Neurath, Scott B. Snapper, and Deanna D. Nguyen for critical review of the manuscript, and Drs. Yasuhisa Imai, Akira Hokama, and Yousuke Tanaka for their assistance in performing the experiments.