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Human Thioredoxin-1 Ameliorates Experimental Murine Colitis in Association With Suppressed Macrophage Inhibitory Factor Production

2006; Elsevier BV; Volume: 131; Issue: 4 Linguagem: Inglês

10.1053/j.gastro.2006.08.023

ISSN

1528-0012

Autores

Hiroyuki Tamaki, Hajime Nakamura, Akiyoshi Nishio, Hiroshi Nakase, Satoru Ueno, Norimitsu Uza, Masahiro Kido, Satoko Inoue, Sakae Mikami, Masanori Asada, Keiichi Kiriya, Hiroshi Kitamura, Shinya Ohashi, Toshiro Fukui, Kimio Kawasaki, Minoru Matsuura, Yasuyuki Ishii, Kazuichi Okazaki, Junji Yodoi, Tsutomu Chiba,

Tópico(s)

Macrophage Migration Inhibitory Factor

Resumo

Background & Aims: Thioredoxin-1 (TRX) is a small multifunctional protein with antioxidative and redox-regulating functions. In this study, we investigated the significance of TRX in patients with inflammatory bowel disease (IBD) and the ability and mechanism to ameliorate experimental colitis. Methods: Serum TRX and macrophage migration inhibitory factor (MIF) levels were measured in patients with IBD. The effects of TRX were evaluated in a dextran sulfate sodium (DSS)-induced colitis model by comparing TRX-overexpressing transgenic (TRX-TG) and control mice. We further evaluated the effect of recombinant human TRX (rhTRX) administration on DSS-induced colitis and colonic inflammation of interleukin (IL)-10 knockout (IL-10 KO) mice. Colonic inflammation was examined clinically and histologically. Proinflammatory cytokine levels were examined in colonic tissues, and MIF levels were measured in colonic tissues and sera in mice. The effect of TRX on MIF production was also analyzed in vitro. Results: Serum TRX and MIF levels were significantly higher in patients with IBD than normal controls, and TRX levels correlated with disease activity. TRX significantly ameliorated DSS-induced colitis and colonic inflammation of IL-10 KO mice. Increase of tumor necrosis factor-α and interferon-γ in colonic tissues was significantly suppressed in TRX-TG mice compared with wild-type mice. MIF levels in colonic tissues and sera were significantly lower in TRX-TG mice than in wild-type mice, irrespective of DSS administration. Anti-TRX treatment exacerbated DSS-induced colitis. In vitro studies demonstrated that rhTRX suppressed MIF production in human monocyte cells. Conclusions: TRX might have a potential as a novel therapeutic agent for the treatment of IBD. Background & Aims: Thioredoxin-1 (TRX) is a small multifunctional protein with antioxidative and redox-regulating functions. In this study, we investigated the significance of TRX in patients with inflammatory bowel disease (IBD) and the ability and mechanism to ameliorate experimental colitis. Methods: Serum TRX and macrophage migration inhibitory factor (MIF) levels were measured in patients with IBD. The effects of TRX were evaluated in a dextran sulfate sodium (DSS)-induced colitis model by comparing TRX-overexpressing transgenic (TRX-TG) and control mice. We further evaluated the effect of recombinant human TRX (rhTRX) administration on DSS-induced colitis and colonic inflammation of interleukin (IL)-10 knockout (IL-10 KO) mice. Colonic inflammation was examined clinically and histologically. Proinflammatory cytokine levels were examined in colonic tissues, and MIF levels were measured in colonic tissues and sera in mice. The effect of TRX on MIF production was also analyzed in vitro. Results: Serum TRX and MIF levels were significantly higher in patients with IBD than normal controls, and TRX levels correlated with disease activity. TRX significantly ameliorated DSS-induced colitis and colonic inflammation of IL-10 KO mice. Increase of tumor necrosis factor-α and interferon-γ in colonic tissues was significantly suppressed in TRX-TG mice compared with wild-type mice. MIF levels in colonic tissues and sera were significantly lower in TRX-TG mice than in wild-type mice, irrespective of DSS administration. Anti-TRX treatment exacerbated DSS-induced colitis. In vitro studies demonstrated that rhTRX suppressed MIF production in human monocyte cells. Conclusions: TRX might have a potential as a novel therapeutic agent for the treatment of IBD. Inflammatory bowel diseases (IBD), such as ulcerative colitis (UC) and Crohn’s disease (CD), are serious disorders. Although several intrinsic factors such as deregulated immune responses, and environmental factors such as food, are suggested to be involved, an integrated concept explaining the pathogenesis of IBD has yet to be proposed. Nevertheless, it is strongly suggested that mediators of immunoregulation and inflammation are involved in the pathogenesis.1Fiocchi C. Inflammatory bowel disease: etiology and pathogenesis.Gastroenterology. 1998; 115: 182-205Abstract Full Text Full Text PDF PubMed Scopus (1865) Google Scholar In particular, recent studies have focused on proinflammatory cytokines such as tumor necrosis factor (TNF)-α and macrophage migration inhibitory factor (MIF), both of which have been investigated as target molecules for novel therapies.2van Dullemen H.M. van Deventer S.J. Hommes D.W. Bijl H.A. Jansen J. Tytgat G.N. Woody J. 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CD95 (APO-1/Fas)-mediated apoptosis in colon epithelial cells: a possible role in ulcerative colitis.Gastroenterology. 1997; 113: 160-167Abstract Full Text PDF PubMed Scopus (234) Google Scholar Thus, scavenging ROS is considered to be critical for regulating intestinal inflammation. Thioredoxin-1 (TRX), originally cloned as a soluble factor named adult T-cell leukemia-derived factor,11Tagaya Y. Maeda Y. Mitsui A. Kondo N. Matsui H. Hamuro J. Brown N. Arai K. Yokota T. Wakasugi H. et al.ATL-derived factor (ADF), an IL-2 receptor/Tac inducer homologous to thioredoxin; possible involvement of dithiol-reduction in the IL-2 receptor induction.EMBO J. 1989; 8: 757-764Crossref PubMed Scopus (516) Google Scholar is one of the most important molecules controlling the redox regulation system and contains a redox-active disulfide/dithiol within the conserved active site sequence Cys32-Gly-Pro-Cys35. TRX has a pivotal role in scavenging ROS with peroxiredoxins and thus prevents apoptosis of various cells, such as lymphocytes, monocytes, and epithelial cells, by inhibiting apoptosis signal-regulating kinase 1.12Saitoh M. Nishitoh H. Fujii M. Takeda K. Tobiume K. Sawada Y. Kawabata M. Miyazono K. Ichijo H. Mammalian thioredoxin is a direct inhibitor of apoptosis signal-regulating kinase (ASK) 1.EMBO J. 1998; 17: 2596-2606Crossref PubMed Scopus (2083) Google Scholar Moreover, intracellular TRX regulates DNA binding of several transcription factors including p53, nuclear factor-κB, and activator protein-1.13Powis G. Montfort W.R. Properties and biological activities of thioredoxins.Annu Rev Biophys Biomol Struct. 2001; 30: 421-455Crossref PubMed Scopus (279) Google Scholar In addition, circulating TRX inhibits neutrophil infiltration into the sites of inflammation in an air pouch model.14Nakamura H. Herzenberg L.A. Bai J. Araya S. Kondo N. Nishinaka Y. Yodoi J. Circulating thioredoxin suppresses lipopolysaccharide-induced neutrophil chemotaxis.Proc Natl Acad Sci U S A. 2001; 98: 15143-15148Crossref PubMed Scopus (201) Google Scholar, 15Nakamura H. De Rosa S.C. Yodoi J. Holmgren A. Ghezzi P. Herzenberg L.A. Chronic elevation of plasma thioredoxin: inhibition of chemotaxis and curtailment of life expectancy in AIDS.Proc Natl Acad Sci U S A. 2001; 98: 2688-2693Crossref PubMed Scopus (133) Google Scholar These results suggest that TRX has important roles, not only as an antioxidant and antiapoptotic molecule, but also as an anti-inflammatory molecule. Serum TRX levels are elevated in several diseases such as human immunodeficiency virus infection,15Nakamura H. De Rosa S.C. Yodoi J. Holmgren A. Ghezzi P. Herzenberg L.A. Chronic elevation of plasma thioredoxin: inhibition of chemotaxis and curtailment of life expectancy in AIDS.Proc Natl Acad Sci U S A. 2001; 98: 2688-2693Crossref PubMed Scopus (133) Google Scholar hepatitis C virus infection,16Sumida Y. Nakashima T. Yoh T. Nakajima Y. Ishikawa H. Mitsuyoshi H. Sakamoto Y. Okanoue T. Kashima K. Nakamura H. Yodoi J. Serum thioredoxin levels as an indicator of oxidative stress in patients with hepatitis C virus infection.J Hepatol. 2000; 33: 616-622Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 17Kato A. Odamaki M. Nakamura H. Yodoi J. Hishida A. Elevation of blood thioredoxin in hemodialysis patients with hepatitis C virus infection.Kidney Int. 2003; 63: 2262-2268Crossref PubMed Scopus (23) Google Scholar and rheumatoid arthritis.18Yoshida S. Katoh T. Tetsuka T. Uno K. Matsui N. Okamoto T. Involvement of thioredoxin in rheumatoid arthritis: its costimulatory roles in the TNF-α-induced production of IL-6 and IL-8 from cultured synovial fibroblasts.J Immunol. 1999; 163: 351-358PubMed Google Scholar Although these findings suggest that TRX can be a good marker for oxidative stress in various diseases, the reason for the increased TRX levels in such diseases is poorly understood. However, overexpression of human TRX (hTRX) in transgenic (TRX-TG) mice induced resistance to harmful conditions including thioacetamide- or lipopolysaccharide (LPS)-induced acute hepatitis,19Okuyama H. Nakamura H. Shimahara Y. Araya S. Kawada N. Yamaoka Y. Yodoi J. Overexpression of thioredoxin prevents acute hepatitis caused by thioacetamide or lipopolysaccharide in mice.Hepatology. 2003; 37: 1015-1025Crossref PubMed Scopus (83) Google Scholar adriamycin-induced cardiotoxicity,20Shioji K. Kishimoto C. Nakamura H. Masutani H. Yuan Z. Oka S. Yodoi J. Overexpression of thioredoxin-1 in transgenic mice attenuates adriamycin-induced cardiotoxicity.Circulation. 2002; 106: 1403-1409Crossref PubMed Scopus (172) Google Scholar and proinflammatory cytokine- or bleomycin-induced lung injury.21Hoshino T. Nakamura H. Okamoto M. Kato S. Araya S. Nomiyama K. Oizumi K. Young H.A. Aizawa H. Yodoi J. Redox-active protein thioredoxin prevents proinflammatory cytokine- or bleomycin-induced lung injury.Am J Respir Crit Care Med. 2003; 168: 1075-1083Crossref PubMed Scopus (147) Google Scholar Moreover, administration of recombinant hTRX (rhTRX) prevented ischemic lung injury,22Okubo K. Kosaka S. Isowa N. Hirata T. Hitomi S. Yodoi J. Nakano M. Wada H. Amelioration of ischemia-reperfusion injury by human thioredoxin in rabbit lung.J Thorac Cardiovasc Surg. 1997; 113: 1-9Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar cerebral infarction,23Hattori I. Takagi Y. Nakamura H. Nozaki K. Bai J. Kondo N. Sugino T. Nishimura M. Hashimoto N. Yodoi J. Intravenous administration of thioredoxin decreases brain damage following transient focal cerebral ischemia in mice.Antioxid Redox Signal. 2004; 6: 81-87Crossref PubMed Scopus (111) Google Scholar and myosin-induced myocarditis24Liu W. Nakamura H. Shioji K. Tanito M. Oka S. Ahsan M.K. Son A. Ishii Y. Kishimoto C. Yodoi J. Thioredoxin-1 ameliorates myosin-induced autoimmune myocarditis by suppressing chemokine expressions and leukocyte chemotaxis in mice.Circulation. 2004; 110: 1276-1283Crossref PubMed Scopus (87) Google Scholar in animal models. These findings suggest that TRX has protective effects on various diseases, possibly by its antioxidative and anti-inflammatory actions. Little is known, however, about the role of TRX in colonic inflammation. Therefore, the present study sought to elucidate the role of TRX in the pathophysiology of IBD. For this purpose, we first investigated the serum levels of TRX in patients with IBD. Next, we examined the effects of endogenous and exogenous TRX on DSS-induced colitis in TRX-TG and wild-type (WT) mice. Furthermore, we investigated the effect of TRX on chronic colonic inflammation in interleukin (IL)-10 knockout (IL-10 KO) mice. Finally, we investigated the effects of TRX on MIF production in vitro. In addition to the antioxidative and anti-inflammatory actions previously reported, the present study suggests that TRX exerts a protective effect on experimental colitis, at least in part through the suppression of MIF production. Serum samples were obtained from 10 patients (7 men, 3 women) with active CD, 11 (8 men, 3 women) with inactive CD, 11 (9 men, 2 women) with active UC, 12 (7 men, 5 women) with inactive UC, 11 (7 men, 4 women) with active ischemic colitis, and 10 (6 men, 4 women) with colon adenoma (as normal controls). All samples were centrifuged within 1 hour of collection, and sera were frozen at −80°C until the assays were performed. To determine disease activity, the Crohn’s Disease Activity Index (CDAI)25Harvey R.F. Bradshaw J.M. A simple index of Crohn’s disease activity.Lancet. 1980; 1: 514Abstract PubMed Scopus (2155) Google Scholar was used for patients with CD and the Ulcerative Colitis Disease Activity Index (UCDAI)26Sutherland L.R. Martin F. Greer S. Robinson M. Greenberger N. Saibil F. Martin T. Sparr J. Prokipchuk E. Borgen L. 5-Aminosalicylic acid enema in the treatment of distal ulcerative colitis, proctosigmoiditis, and proctitis.Gastroenterology. 1987; 92: 1894-1898Abstract PubMed Google Scholar for patients with UC. Informed consent was obtained from each patient, and the experimental designs of these studies were approved by the Kyoto University Hospital Ethics Committee. Female C57BL/6 (WT) mice (9–11 weeks of age; weighing 20–22 g) were obtained from CLEA Japan (Shizuoka, Japan). The generation and maintenance of TRX-TG mice was described previously.27Takagi Y. Mitsui A. Nishiyama A. Nozaki K. Sono H. Gon Y. Hashimoto N. Yodoi J. Overexpression of thioredoxin in transgenic mice attenuates focal ischemic brain damage.Proc Natl Acad Sci U S A. 1999; 96: 4131-4136Crossref PubMed Scopus (307) Google Scholar hTRX cDNA was inserted between the β-actin promoter and the β-actin terminator. There were no differences in the expression of Mn-superoxide dismutase, CuZn-superoxide dismutase, and glutathione peroxidase between WT and TRX-TG mice, analyzed by immunohistochemistry and Western blotting (data not shown). The presence of the TRX transgene was confirmed by reverse transcription-polymerase chain reaction analysis. C57BL/6-IL-10 KO mice (aged 4–6 weeks) were obtained from the Jackson Laboratory (Bar Harbor, ME). All mice were maintained under specific pathogen-free conditions and had free access to commercial food and water. Among them, IL-10 KO mice were transferred from specific pathogen-free to conventional housing conditions at 6 weeks of age because they spontaneously develop colitis in our conventional housing conditions by 8 weeks of age. All animal experiments were performed in accordance with our institutional guidelines, and the Review Board of Kyoto University granted ethical permission for this study. DSS (molecular weight, 36,000–50,000) was purchased from ICN Pharmaceuticals Inc. (Costa Mesa, CA). Adult sex-matched WT mice (control group: n = 10) and TRX-TG mice (TG group: n = 10) were given 3% (wt/vol) DSS in their drinking water for 7 days, and percentage weight changes were recorded throughout DSS administration (Figure 1A). Percentage weight change for each mouse was calculated as follows: percentage weight change =[(weight at specific day(day 0 to day 7)−weight on day 0)/weight on day 0] × 100. Mice were killed under ether anesthesia on day 7. To investigate the effects of TRX on colonic inflammation, we designed 3 protocols using the DSS-induced acute colitis model. In these protocols, WT mice were fed 3% (wt/vol) DSS in their drinking water from day 0 to day 5, followed by a return to normal water, and were killed on day 10. Five mg/kg of rhTRX (kindly supplied by Ajinomoto Co. Ltd, Kawasaki, Japan) dissolved in 100 μL of phosphate-buffered saline (PBS) or 100 μL of PBS alone was administered intraperitoneally from day 1 to day 9 (Figure 1B, a, each group: n = 10). Five mg/kg of rhTRX or PBS alone was administered intraperitoneally from day 3 to day 9 (Figure 1B, b, each group: n = 10). One hundred microliters of rabbit antimouse TRX antiserum or normal rabbit serum (DAKO Corp. Carpinteria, CA) was administered intraperitoneally on days 0, 1, and 3 (Figure 1B, c, each group: n = 5).24Liu W. Nakamura H. Shioji K. Tanito M. Oka S. Ahsan M.K. Son A. Ishii Y. Kishimoto C. Yodoi J. Thioredoxin-1 ameliorates myosin-induced autoimmune myocarditis by suppressing chemokine expressions and leukocyte chemotaxis in mice.Circulation. 2004; 110: 1276-1283Crossref PubMed Scopus (87) Google Scholar The insulin disulfide reduction assay was performed to confirm that the anti-TRX antiserum effectively neutralized TRX reducing activity (data not shown). In these studies, percentage weight change was recorded throughout the period. Mice were killed under ether anesthesia, and the colons were removed and processed for pathologic studies as described later. Five milligrams/kilogram per day of rhTRX or PBS alone was administered intraperitoneally to IL-10 KO mice at 6 weeks of age. Fourteen days after the start of treatment, rhTRX- or PBS-treated mice were killed for histologic analysis of colonic tissue (Figure 1C). The entire colon was dissected out, and the length was recorded. In TRX-TG mice, blood content in the stool was scored using previously described criteria (Table 1).28Jeffers M. McDonald W.F. Chillakuru R.A. Yang M. Nakase H. Deegler L.L. Sylander E.D. Rittman B. Bendele A. Sartor R.B. Lichenstein H.S. A novel human fibroblast growth factor treats experimental intestinal inflammation.Gastroenterology. 2002; 123: 1151-1162Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar For histopathologic examination, 3 distal colonic regions spaced approximately 1.5 cm apart were collected into 10% neutral-buffered formalin, processed for paraffin embedding, sectioned (4 μm), and stained with H&E. Inflammation and crypt damage were assessed using a validated scoring scheme as described previously (Table 2),29Williams K.L. Fuller C.R. Dieleman L.A. DaCosta C.M. Haldeman K.M. Sartor R.B. Lund P.K. Enhanced survival and mucosal repair after dextran sodium sulfate-induced colitis in transgenic mice that overexpress growth hormone.Gastroenterology. 2001; 120: 925-937Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar and the means of the scores of each region were determined. The scores were determined in a blinded manner by 2 examiners.Table 1Scoring System for Bloody StoolScoreDescription0Normal to semisolid stool, no blood1Normal to semisolid stool, blood-tinged2Semisolid to fluid stool with definite evidence of blood3Bloody fluid Open table in a new tab Table 2Histologic Scoring System for DSS-Induced ColitisFeature scoredScoreDescriptionInflammation severity0None1Mild2Moderate3SevereInflammation extent0None1Mucosa2Mucosa and submucosa3TransmuralCrypt damage0None1Basal 1/3 damaged2Basal 2/3 damaged3Crypts lost: surface epithelium present4Cryps and surface epithelium lostPercentage involvement00%11%–25%226%–50%351%–75%475%–100% Open table in a new tab Mice were monitored for clinical signs of colitis, including diarrhea and weight loss. At necropsy, samples of the colon (transverse, distal, and proximal) and the rectum were collected and histopathologically examined as described previously. For each section, inflammation (macrophage, lymphocyte, and neutrophil infiltration in the lamina propria or submucosa) was scored for severity according to the following criteria: normal, 0; minimal, 1; mild, 2; moderate, 3; marked, 4; and severe, 5. Gland loss and epithelial hyperplasia were scored by percentage of area involved: none, 0; 1, 1%–10% of the mucosa affected; 2, 11%–25% affected; 3, 26%–50% affected; 4, 51%–75% affected; and 5, 76%–100% affected. The summed scores for inflammation (lamina propria or submucosa), gland loss, and gland hyperplasia were then determined for each animal. For each section, mucosal thickness was quantitated by measuring the distance from the muscularis mucosa to the internal epithelial border in a nontangential area showing the most representative change in severity. Selected sections were scored by a second person, with excellent agreement. Fragment culture of distal colon segments was performed following published methods.30Sellon R.K. Tonkonogy S. Schultz M. Dieleman L.A. Grenther W. Balish E. Rennick D.M. Sartor R.B. Resident enteric bacteria are necessary for development of spontaneous colitis and immune system activation in interleukin-10-deficient mice.Infect Immun. 1998; 66: 5224-5231Crossref PubMed Google Scholar Briefly, colon segments were washed in PBS containing 100 μg/mL streptomycin (Sigma Chemical Co., St. Louis, MO) and 100 μg/mL penicillin (Sigma Chemical Co.) and then kept in cold serum-free media (RPMI-1640 medium [Gibco BRL, Eggenstein, Germany] with streptomycin and penicillin). After the dry weight was measured, the organs were cut into small pieces in a Petri dish containing fresh media, and 100 mg of tissue fragments were incubated at 37°C in 1 mL fresh media for 18 hours. Culture supernatants were collected and stored at −80°C and assayed for TNF-α, interferon (IFN)-γ, and MIF secretion by enzyme-linked immunosorbent assay (ELISA). The human monocytic leukemia cell line THP-131Tsuchiya S. Yamabe M. Yamaguchi Y. Kobayashi Y. Konno T. Tada K. Establishment and characterization of a human acute monocytic leukemia cell line (THP-1).Int J Cancer. 1980; 26: 171-176Crossref PubMed Scopus (1681) Google Scholar was obtained from the RIKEN Bioresource Center (No. RCB1189; Tsukuba, Japan) and maintained at 37°C and 5% CO2 in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 μg/mL streptomycin, 100 μg/mL penicillin, and 2 mmol/L glutamine (RPMI complete medium). Cells were cultured in 12-well plates (1 × 106 cells/well) in the presence of 16 nmol/L of phorbol-12-myristate13-acetate (Sigma Chemical Co.) for 12 hours, washed twice with PBS, and incubated with RPMI complete medium in the presence or absence of rhTRX (0.01–1 nmol/L) for an additional 24 hours. Cells were then washed twice with PBS and harvested 12 hours after stimulation with 1 μg/mL of Escherichia coli O55:B5 LPS (Sigma Chemical Co.) and 10 ng/mL of IFN-γ (Strathmann Biotec AG, Hamburg, Germany) in RPMI complete medium. The serum levels of human TRX were determined by a sandwich ELISA Kit (Redox Bioscience Inc., Kyoto, Japan) as previously described.15Nakamura H. De Rosa S.C. Yodoi J. Holmgren A. Ghezzi P. Herzenberg L.A. Chronic elevation of plasma thioredoxin: inhibition of chemotaxis and curtailment of life expectancy in AIDS.Proc Natl Acad Sci U S A. 2001; 98: 2688-2693Crossref PubMed Scopus (133) Google Scholar, 32Kondo N. Ishii Y. Son A. Sakakura-Nishiyama J. Kwon Y.W. Tanito M. Nishinaka Y. Matsuo Y. Nakayama T. Taniguchi M. Yodoi J. Cysteine-dependent immune regulation by TRX and MIF/GIF family proteins.Immunol Lett. 2004; 92: 143-147Crossref PubMed Scopus (37) Google Scholar The serum levels of human MIF were determined by ELISA using the human MIF ELISA kit (Sapporo I.D.L, Sapporo, Japan).33Mizue Y. Nishihira J. Miyazaki T. Fujiwara S. Chida M. Nakamura K. Kikuchi K. Mukai M. Quantitation of macrophage migration inhibitory factor (MIF) using the one-step sandwich enzyme immunosorbent assay: elevated serum MIF concentrations in patients with autoimmune diseases and identification of MIF in erythrocytes.Int J Mol Med. 2000; 5: 397-403PubMed Google Scholar Mouse TNF-α, IFN-γ, and MIF concentrations in culture supernatants of colon fragments were determined by ELISA using the mouse TNF-α ELISA kit (R&D Systems Inc., Minneapolis, MN), mouse IFN-γ ELISA set (BD Biosciences, San Diego, CA), and mouse MIF ELISA kit (Sapporo I.D.L.). MIF concentrations in supernatants from an in vitro study were determined by the human MIF ELISA kit (Sapporo I.D.L.). For preparation of colon tissue samples, tissues were lysed in RIPA buffer (1% Triton X-100, 1% Na-deoxydolate, 0.1% sodium dodecyl sulfate [SDS], 20 mmol/L Tris-HCl [pH 7.4], 5 mmol/L ethylenediamine-N,N,N′,N′-tetra-acetic acid [EDTA], 150 mmol/L NaCl, 1 μg/mL aprotinin, 100 μg/mL phenylmethylsulfonyl fluoride. Insoluble materials were removed by centrifugation at 12,000g for 10 minutes at 4°C. Supernatants boiled with sample buffer (0.05 mol/L Tris-HCl, 2% SDS, 6% β-mercaptoethanol, 10% glycerol, 1.25% bromophenol blue) were subjected to the assay. For preparation of cell samples, isolated cells were lysed in reducing Laemmli buffer (0.125 mol/L Tris-HCl/SDS, pH 6.8, 4% SDS, 20% glycerol, 10% β-mercaptoethanol, 2.5% bromophenol blue) and boiled for 5 minutes. After determination of protein concentration with the BCA protein assay kit (Pierce Chemical, Rockford, IL), the solubilized lysates were subjected to Western blot analysis by separation of 10 or 30 μg protein per lane on Tris-glycine gels prior to transfer onto polyvinylidine fluoride membranes (PALL Corporation, Pensacola, FL) using standard protocols. Tris-buffered saline with 0.5% Tween-20 (TBS-T) and 5% skim milk was used to block nonspecific binding to the membrane. Antibodies recognizing TRX (Redox Bio Science Inc), MIF (Santa Cruz Biotechnology, Santa Cruz, CA), or β-actin (Abcam Ltd., Cambridge, England) were added at a dilution of 1:1000, and the membranes were incubated overnight. The membranes were washed 3 times in TBS-T, followed by incubation in TBS-T with 5% skim milk containing anti-rabbit IgG antibody conjugated with horseradish peroxidase (Amersham Pharmacia Biotech, Buckinghamshire, England) at a 1:2000 dilution. Immunoreactive bands were visualized using ECL peroxidase developing solution (Amersham Pharmacia Biotech) and recorded on autoradiographic film (Amersham Pharmacia Biotech). All results were expressed as means ± standard error (SE). Pearson correlation coefficient analysis was used to examine the relationship between serum TRX and CDAI or UCDAI. Parametric data were analyzed by the Student t test. A 2-tailed P value of less than .05 was used to indicate statistical significance. Serum levels of TRX were significantly higher in patients with active CD and with active UC than in controls, whereas those in patients with inactive CD and with inactive UC were not significantly different from those in controls (Figure 2A). Patients with ischemic colitis also had significantly higher serum TRX levels than those of controls, although their levels tended to be lower than those of active CD and UC. Furthermore, there were significant correlations between serum TRX levels and CDAI in active CD patients, and between serum TRX levels and UCDAI in active UC patients, indicating that TRX levels correlate with disease activity (Figure 2B and C). To investigate the relationship between TRX and MIF, we measured serum levels of MIF in the same patients with active CD and with active UC. Serum levels of MIF were also significantly higher in patients with active CD and with active UC than in controls (Figure 2D). There was a significant correlation between serum levels of TRX and MIF in patients with active CD (Figure 2E), whereas no such correlation was found in patients with active UC (Figure 2F). To investigate whether overexpression of TRX ameliorates colonic inflammation in DSS-induced colitis, we used TRX-TG and WT mice (Figure 1A). During DSS administration, the body weight of the mice gradually decreased. However, the percentage loss of body weight in TRX-TG mice was significantly lower at day 6 and day 7 than that of WT mice (Figure 3A). There were significant differences in colonic length and bloody stool score between WT and TRX-TG mice at the end of DSS administration (Figure 3B). DSS administration for 7 days in WT mice produced an acute colonic inflammation. Histologic analysis of distal colonic sections from DSS-treated WT mice revealed multifocal inflammatory cell infiltration and edema with crypt and epithelial cell loss and ulceration. In contrast, very little mucosal

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