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. 2005 Sep;141(3):412–421. doi: 10.1111/j.1365-2249.2005.02877.x

Macrophage migration inhibitory factor contributes to the development of acute dextran sulphate sodium-induced colitis in Toll-like receptor 4 knockout mice

T Ohkawara *,†, H Takeda *, J Nishihira †, K Miyashita *, M Nihiwaki †, Y Ishiguro ‡, K Takeda §, S Akira , T Iwanaga **, T Sugiyama ††, M Asaka *
PMCID: PMC1809451  PMID: 16045730

Abstract

Toll-like receptor 4 (TLR4), which recognizes lipopolysaccharides, plays an important role in the innate immune response. In this study, we investigated the role of TLR4 in the development of experimental colitis with regard to the biological actions of macrophage migration inhibitory factor (MIF) using TLR4 null (−/−) mice. TLR4−/− mice were given 2% dextran sulphate sodium (DSS) in drinking water to induce colitis, which was clinically and histologically as severe as that seen in wild-type (WT) mice. The level of tumour necrosis factor (TNF)-α in colon tissues was increased in WT mice but unchanged in TLR4−/− mice. The level of myeloperoxidase (MPO) activity in colon tissues was increased by DSS administration in both TLR4−/− and WT mice. The expression of MIF was up-regulated in the colons of TLR4−/− mice with acute DSS-induced colitis. An anti-MIF antibody significantly suppressed colitis and elevation of matrix metalloproteinase-13 in TLR4−/− mice. The current results obtained from TLR4−/− mice provide evidence that MIF plays a critical role in the development of acute DSS-induced colitis.

Keywords: dextran sulphate sodium-induced colitis, inflammatory bowel disease, inhibitory factor, macrophage migration, Toll-like receptor 4

Introduction

Inflammatory bowel diseases (IBDs), including Crohn's disease (CD) and ulcerative colitis, are characterized by chronic and relapsed inflammation of the gut, but their aetiology remains unknown [1]. Recent studies have shown that various inflammatory mediators, such as tumour-necrosis factor (TNF)-α, interleukin (IL)-6 and macrophage migration inhibitory factor (MIF), are involved in the pathogenesis of IBDs [2–5]. In fact, humanized antibodies specific for TNF-α and IL-6 have been developed for treatment of patients with CD [6–8]. Recently, several studies have focused on dysfunction of the innate immune response in the pathogenesis of IBDs [1,9,10].

Toll-like receptor (TLR), the human homologue of Drosophila Toll, plays an essential role in the innate immune response. TLR belongs to the IL-1/Toll receptor family [11–14] and to a family of pattern-recognition receptors that detect conserved molecular products of microorganisms [15]. TLR4, one of the TLRs, is the receptor for lipopolysaccharides (LPS), the major component of a Gram-negative bacterial cell wall. Accordingly, lack of TLR4 abolishes LPS-induced inflammation and disorders, including endotoxin shock [16]. Various polymorphisms have been identified in genes encoding for TLR4 [17,18]. In the gastrointestinal tract, it has been reported that intestinal epithelial cells express some pattern-recognition receptors in vitro[19] and that expression of TLR4, but not TLR3, was observed in epithelial cells from the colons of patients with IBD [20]. In the mouse colon, it has been reported that TLR4 protein and mRNA expression were significantly up-regulated during dextran sulphate sodium (DSS)-induced colitis [21].

MIF is the first cytokine discovered in T lymphocytes [22,23]. This cytokine is expressed ubiquitously in various kinds of cells and has been re-evaluated as a multi-functional molecule involved in immune response [24,25]. MIF is known to be a proinflammatory cytokine released mainly by macrophages [26] and a T lymphocyte activator in immune responses [27]. MIF is essential for the development of LPS-induced disorders, and neutralization of MIF by its antibody protects mice and rats against endotoxin shock [28]. In the cytokine network, TNF-α plays an important role in the development of inflammation, in which MIF regulates expression of TNF-α[28–30].

In this study, we attempted to clarify further the mechanism underlying the development of DSS-induced colitis with regard to the cytokine network using TLR4 null (−/−) mice, and particularly with respect to the relationship between MIF and TNF-α. We found that MIF was up-regulated during DSS-induced colitis independently of TNF-α through TLR4 signalling. Our results provide novel evidence that MIF plays a critical role in the development of intestinal inflammation independently of TNF-α.

Materials and methods

Materials

Nitrocellulose membrane filters were obtained from Millipore (Bedford, MA, USA). The ECL Western blotting detection system was from Amersham Bioscience (Piscataway, NJ, USA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody and Micro BCA protein assay kits were from Pierce (Rockford, IL, USA). DSS (molecular mass, 40 kilodaltons) and rabbit IgG fraction were from ICN Biomedicals (Aurora, OH, USA), and anti-β-actin antibody was from Sigma-Aldrich Co. (Temecula, CA, USA). Enzyme-linked immunosorbent assay (ELISA) development kits for mouse TNF-α were from Genzyme Techne (Minneapolis, MN, USA). Recombinant mouse MIF and a polyclonal anti-MIF antibody were prepared as described previously [31,32]. All other chemicals used were of analytical grade.

Mice

Specific pathogen-free C57BL6 male mice 8–10 weeks of age were purchased from Japan Charles River Co. (Shizuoka, Japan). TLR4−/− mice (background: C57BL) were established as described previously [16]. Mice were inbred under a light : dark cycle every 12 h at room temperature and fed food and water ad libitum. The experimental protocol adhered to the Declaration of Helsinki and was in accordance with the Animal Experiment Ethics Committee of the Graduate School of Medicine of Hokkaido University.

Induction and general assessment of DSS colitis

To induce colitis, mice were given 2% DSS in distilled water ad libitum. Mice were weighed daily and inspected visually for rectal bleeding and diarrhoea. Mice were anaesthetized by intraperitoneal injection of thiopental on day 7 after the first administration of DSS and killed for histological evaluation and molecular analysis. The colons were removed, collected and stored at −80°C until use for molecular analysis. For histological evaluation, the samples of colons were opened longitudinally and fixed with 10% neutral buffered formalin. The disease activity index (DAI) was used to assess the grade of colitis based on a previously published scoring system by Cooper et al. [33]. The DAI score correlates well with tissue damage scores and with inflammatory mediators such as myeloperoxidase (MPO) activity. To exclude bias, the DAI score and body weight were determined in a blinded fashion by an examiner.

Histological examination and immunohistochemistry

The colon tissues fixed with 10% neutral buffered formalin were embedded in paraffin. Sections (4 µm) were stained with haematoxylin and eosin (H&E). For histological evaluation of colitis, specimens were quantified microscopically as described previously [5]. The colocecal damage was categorized into six grades: grade 0, normal mucosa; grade 1, infiltration of inflammatory cells; grade 2, shortening of the crypt by less than half; grade 3, shortening of the crypt by more than half; grade 4, crypt loss; and grade 5, destruction of epithelial cells (ulceration and erosion). In addition, we evaluated the extent of inflammatory lesions. The extent of lesions in the total colon was classified into six grades: grade 0, 0%; grade 1, 1–20%; grade 2, 21–40%; grade 3, 41–61%; grade 4, 61–80%; and grade 5, 81–100%. To exclude bias, the histological score was determined in a blinded fashion by two pathologists.

For assessment of immunohistochemistry for MIF, the sections of tissues (5 µm) were stained with polyclonal anti-MIF antibody (diluted 100 : 1) as described previously [5].

Enzyme-linked immunosorbent assay

Colon tissue samples in phosphate buffered saline (PBS) containing a cocktail of protease inhibitors (1 µl to 20 mg of tissue according to the manufacturer's protocol) were homogenized with a Polytron homogenizer (Kinematica, Luzern, Switzerland) and centrifuged at 12 000 g for 10 min. The supernatants were subjected to the assay. TNF-α contents in tissues were measured using ELISA kits in accordance with the manufacturer's protocol.

Measurement of myeloperoxidase activity

Tissue MPO activity was determined by a standard enzymatic procedure as described previously [34], with minor modifications. Briefly, after the samples had been weighed, a tissue sample (approximately 300 mg) was homogenized in a buffer (0·5% hexadecyltrimethylammonium bromide in 50 m m potassium phosphate buffer, pH 6·0) using a Polytron-type homogenizer three times for 30 s each on ice. The sample was centrifuged at 20 000 g for 20 min at 4°C and the supernatant was collected. The supernatant (100 µl) was then added to 2·9 ml of 50 mM phosphate buffer (pH 6·0) containing 0·167 mg/ml O-dianisidine hydrochloride and 0·0005% hydrogen peroxide, and absorbances were measured using a spectrometer at 25°C. The protein concentration of the supernatant was determined using a Bradford assay kit (Bio-Rad Laboratories, Hercules, CA, USA) for calibration, and the values were standardized using MPO purified from human leucocytes (Sigma, St Louis, MO, USA).

Western blot analysis

Western blot analysis for MIF was performed in accordance with a previous report [31]. Briefly, colon tissue was disrupted with a Polytron homogenizer (Kinematica, Lucerne, Switzerland). The protein concentrations of the tissue homogenates were quantified using a Micro BCA protein assay reagent kit. Equal amounts of proteins were dissolved in 20 µl of Tris-HCL, 50 mM (pH 6·8), containing 2-mercaptoethanol (1%), sodium dodecyl sulphate (SDS) (2%), glycerol (20%) and bromophenol blue (0·04%), and the samples were heated at 100°C for 5 min. The samples were then subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred electrophoretically onto nitrocellulose membranes. The membranes were blocked with 5% non-fat dry milk in PBS, probed with a polyclonal anti-MIF antibody (diluted 2000 : 1) and reacted with a goat anti-rabbit IgG antibody coupled with horseradish peroxidase (HRP). Western blot analysis of matrix metalloproteinase (MMP)-13 was performed in a similar manner using an anti-MMP-13 antibody (diluted 5000 : 1; Chemicon, Temecula, CA, USA). The resultant complexes were processed for detection by an ECL Western blotting detection system according to the manufacturer's protocol. The proteins were visualized with a Konica HRP-1000 immunostaining kit in accordance with the manufacturer's protocol.

Treatment with anti-MIF antibody

Polyclonal anti-MIF antibody (0·4 mg/mouse) or non-immune rabbit lgG (0·4 mg/mouse in PBS) was injected intraperitoneally on 2, 4 and 6 days after the first DSS treatment. DAI and histology were assessed to evaluate the effect of anti-MIF antibody on DSS-induced colitis in TLR4−/− mice. DAI and histological scores were quantified by two pathologists in a blind fashion using a scoring system as previously described [5].

Statistics

Data are presented as the means ± standard error (SE). The results were analysed statistically using the unpaired Student's t-test and one-way anova with a post hoc test (StatView; SAS Institute, Cary, NC, USA). P-values < 0·05 were considered to be statistically significant.

Results

Development of DSS-induced colitis in both TLR4−/− and WT mice

We assessed clinical symptoms using scoring systems that have been reported to be reliable indicators of pathological changes. The disease activity index, which reflects the frequencies of diarrhea, rectal bleeding and body weight loss, had increased in both TLR4−/− and WT mice 7 days after DSS treatment (3·5 ± 0·1 and 3·2 ± 0·3, respectively) (Fig. 1a). The colon length, which is shortened by DSS treatment, has often been used as a morphological parameter for the degree of colitis induced by DSS. Under normal conditions, no significant difference in colon length was found between TLR4−/− and WT mice (8·0 ± 0·1 and 8·1 ± 0·3, respectively). However, when mice were treated with 2% DSS for 7 days, the colon length was significantly shortened in TLR4−/− mice (6·1 ± 0·3 cm) as well as in WT mice (5·5 ± 0·2 cm) (Fig. 1b).

Fig. 1.

Fig. 1

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Development of dextran sulphate sodium (DSS)-induced colitis in Toll-like receptor 4 null (−/−) mice. Mice were given 2% DSS in drinking water for 7 days. (a) The disease activity index (DAI) on day 7 is expressed as the mean ± standard error (SE) of 10 mice in each group. (b) Colon length was measured 7 days after the first DSS treatment. Data are shown as the means ± SE of 10 mice in each group. *P < 0·05 compared with non-treated wild-type mice.

Histological findings in the colons of DSS-treated mice were examined to evaluate the severity of tissue damage using H&E staining. In the colons of TLR4−/− mice, inflammatory infiltration was minimal before DSS treatment but had increased on day 7 (Fig. 2c,d, respectively). The findings in the colons of WT mice without or with DSS-induced colitis were similar to those in TLR4−/− mice (Fig. 2a,b, respectively). Histological scores for tissue damage and extent of lesion in TLR4−/− mice were as severe as those in WT mice on day 7 (tissue damage: 4·2 ± 0·2 and 4·4 ± 0·3, respectively; extent of lesion: 2·6 ± 0·3 and 3·0 ± 0·3, respectively) (Fig. 3).

Fig. 2.

Fig. 2

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Representative microphotograph of the histological appearance of the colon from TLR4−/− and wild-type (WT) mice with or without dextran sulphate sodium (DSS) colitis. (a) Normal colon in WT mice; (b) inflamed colon in WT mice with DSS-induced colitis; (c) colon in TLR4−/− mice without DSS treatment; (d) inflamed colon in TLR4−/− mice with DSS-induced colitis. After 7 days of DSS treatment, severe infiltrating inflammatory cells and tissue destruction occurred in the colon of both TLR4 and WT mice. Similar appearances were obtained from the colon tissues of other mice. Original magnification ×200.

Fig. 3.

Fig. 3

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Histological evaluation in the colons of mice with dextran sulphate sodium (DSS)-induced colitis. Mice were given 2% DSS in drinking water for 7 days. Histological scores of tissue damage and extent of lesion in colon tissues. Histological scores are microscopically quantified by the method described in Materials and methods. Data are expressed as the means ± SE of five mice in each group.

The TNF-α level was not increased in the colons of TLR4-deficient mice treated with DSS

ELISA was used to determine the level of TNF-α in the colons of TLR4−/− mice. The levels of TNF-α in WT and TLR4−/− mice before DSS treatment were similar (5·4 ± 0·9 and 2·8 ± 0·6, respectively). This level was significantly higher in the colons of WT mice with DSS colitis than in the colons of non-treated WT mice (16·7 ± 2·4) (Fig. 4). In contrast, the level of TNF-α did not increase in TLR4−/− mice even when colitis was induced by DSS treatment for 7 days (3·3 ± 0·4) (Fig. 4).

Fig. 4.

Fig. 4

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Tumour necrosis factor (TNF)-α contents in the colons of mice with dextran sulphate sodium (DSS)-induced colitis. The levels of TNF-α in the colons of TLR4−/− mice (n = 5) and wild-type (WT) mice (n = 5) before and 7 days after DSS treatment were measured by enzyme-linked immunosorbent assay (ELISA). Data are expressed as the means ± SE of five mice in each group. *P < 0·05 compared with non-treated WT mice.

Myeloperoxidase activity increased in the colons of TLR4 −/− mice with DSS colitis

MPO activity is a marker of neutrophil contents and is up-regulated in the tissue under inflammatory conditions. We measured MPO activities in the colons of the mice. In both WT and TLR4−/− mice, the level of MPO activity was low before DSS treatment (1·6 ± 0·6 and 1·9 ± 0·9 U/g protein, respectively) (Fig. 5). Seven days after the first DSS treatment, the level of MPO activity in the colon was remarkably increased in both WT and TLR4−/− mice (6·2 ± 0·5 and 5·8 ± 0·7 U/g protein, respectively) (Fig. 5). There was no statistically significant difference between the levels of MPO activity in WT and TLR4−/− mice treated with DSS.

Fig. 5.

Fig. 5

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Myeloperoxidase (MPO) activity in the colons of mice given 2% dextran sulphate sodium (DSS) for 7 days. Mice were given 2% DSS in drinking water for 7 days. The levels of MPO activities were measured in colon tissues from mice on day 7. Data are expressed as the means ± SE of five mice in each group. *P < 0·05.

MIF expression was up-regulated in the colons of TLR4−/− mice with DSS colitis

To clarify the reason why DSS-induced colitis occurred in TLR4−/− mice, Western blot analysis for macrophage migration inhibitory factor (MIF) in the colon was performed. We have previously demonstrated up-regulation and localization of MIF in mice with DSS-induced colitis [5]. In the present study, MIF was expressed in the colons of WT and TLR4−/− mice without DSS treatment (Fig. 6). On the other hand, the expression of MIF was enhanced in the colons of both TLR4−/− and WT mice when mice were given DSS for 7 days (Fig. 6). To assess the localization of MIF protein in the colon, immunohistochemical analysis for MIF in the colons of TLR4−/− mice was carried out. Faint MIF-positive staining spots were seen in epithelial and mononuclear cells of mice without DSS colitis (Fig. 7b). This staining was observed clearly in infiltrating inflammatory cells in the colons of mice with DSS colitis (Fig. 7c). This staining was similar to that in WT mice (data not shown).

Fig. 6.

Fig. 6

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Up-regulation of macrophage migration inhibitory factor (MIF) expression in the colons of TLR4−/− mice given dextran sulphate sodium (DSS) for 7 days. The samples of colon tissues were removed from wild-type and TLR4−/− mice before and 7 days after the first DSS treatment. The expression of MIF in the colons was assessed by Western blot analysis. β-actin is used as a loading control. Similar results were obtained from the three experiments.

Fig. 7.

Fig. 7

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Localization of macrophage migration inhibitory factor (MIF)-positive cells in the colon. Sections of colons from TLR4−/− mice were stained with anti-MIF antibody (diluted 100 : 1). (a) A section stained with rabbit IgG as the primary antibody (negative control). (b) A section of the colon from non -treated mice. (c) A section of the colon from mice with colitis induced by DSS for 7 days. Original magnification ×200. The sections shown are typical of the other samples.

Anti-MIF antibody ameliorates DSS-induced colitis in TLR4−/− mice

To assess further the contribution of MIF to inflammation in TLR4−/− mice, we investigated whether blockade of MIF using an anti-MIF antibody would ameliorate colitis induced by DSS in TLR4−/− mice. Treatment with an anti-MIF antibody resulted in a significant reduction in DAI on day 7 (P < 0·01, 1·1 ± 0·1 and 2·3 ± 0·3) (Fig. 8a) and in the histological score in TLR4−/− mice given DSS for 7 days compared with treatment with non-immune IgG (tissue damage: P < 0·05, 1·8 ± 0·6 and 4·0 ± 0·5, respectively; extent of lesion: P < 0·05, 1·2 ± 0·2 and 2·6 ± 0·4, respectively) (Fig. 8b).

Fig. 8.

Fig. 8

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Ameliorating effect of anti-macrophage migration inhibitory factor (MIF) antibody on dextran sulphate sodium (DSS)-induced colitis in TLR4−/− mice. TLR4−/− mice were treated with anti-MIF antibody or non-immune IgG (0·4 mg/time/mouse) 2, 4 and 6 days after the first DSS treatment. (a) DAI values on day 7 are expressed as the means ± SE of five mice in each group. *P < 0·05 compared with non-immune IgG-treated mice. (b) Histological scores of tissue damage and extent of lesion in the colon. Data are expressed as the means ± SE of five mice in each group. *P < 0·05 compared with non-immune IgG-treated mice.

MMP-13 expression was modulated by MIF in the colons of TLR4−/− mice with DSS-induced colitis

MMP is thought to be an important molecule in tissue destruction and remodelling. Our previous study showed that MIF modulated the expression of MMP-13 mRNA and that blockade of MIF bioactivity reduced the expression of MMP-13 mRNA. Regulation of MMP by MIF has been reported in several experimental diseases, including models of arthritis and colitis [5,35]. Western blot analysis revealed that the expression of MMP-13 was remarkably (more than twofold density in blotted bands) increased in colons of TLR4−/− mice treated with DSS for 7 days (Fig. 9). Moreover, treatment with an anti-MIF antibody markedly (less than half of density in blotted band) suppressed the expression of MMP-13 in the colons of TLR4−/− mice given 2% DSS (Fig. 9).

Fig. 9.

Fig. 9

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Suppressive effect of anti-macrophage migration inhibitory factor (MIF) antibody on up-regulation of matrix metalloproteinase (MMP)-13 expression in the colons of TLR4−/− mice with dextran sulphate sodium (DSS)-induced colitis. Mice were treated with anti-MIF antibody or non-immune IgG (0·4 mg/time/mouse) 2, 4 and 6 days after the first administration of 2% DSS. Colon tissue samples were removed from non-treated, non-immune IgG- and DSS-treated, and anti-MIF antibody- and DSS-treated mice on day 7. The expression of MMP-13 in the colons was assessed by Western blot analysis. β-actin was used as a loading control. Similar results were confirmed in more than three experiments.

Discussion

An animal model of DSS-induced colitis established by Okayasu et al. [36] has been used widely for investigation of the pathogenesis of IBD and for assessment of novel treatments. Several inflammatory mediators such as cytokines have been shown to play an important role in the development of DSS-induced colitis [5,37–39].

In this study, we found that acute DSS-induced colitis occurred in TLR4−/− mice as well as in WT mice. During the course of acute DSS-induced colitis, the levels of MIF expressions were remarkably up-regulated in the colons obtained from WT and TLR4−/− mice. On the other hand, the production of TNF-α in colon tissues from WT mice was significantly increased in response to DSS, whereas that in TLR4−/− mice was minimal.

In innate immune response, TLR has recently been thought to play a central role [15], and innate immunity has been reported to play an important role in the development of DSS-induced colitis [4,40]. In the colon, Ortega-Cava et al. [21] have reported the expression and localization of TLR4 in the mouse gastrointestinal tract. The expression of TLR4 was observed in epithelial cells and lamina propria mononuclear cells in the normal mouse gut, including the small intestine and colon. The level of TLR4 mRNA expression was higher in the colon than in other tissues in WT mice. These findings suggested the possibility that mice lacking TLR4 would show different features of DSS-induced colitis, which prompted us investigate the DSS-induced colitis in TLR4−/− mice.

In this study, however, we found that severe colitis occurred in TLR4−/− mice treated with DSS. In addition to clinical and histological severity, the level of MPO activity, as a parameter of neutrophil accumulation, was increased in colons of TLR4−/− mice with DSS-induced colitis. In contrast, the increase of TNF-α level was essentially unchanged in the colons of TLR4−/− mice by DSS administration. The C3H/HeJ mouse strain, which is characterized by hyporesponsiveness to LPS and mutation in the TLR4 gene, showed severe colitis in response to DSS stimulation [41]. Very recently, it has been reported that MyD88 knockout mice show severe colitis induced by DSS, suggesting that TLR signalling is not essential in the development of DSS-induced colitis [42]. MyD88 is the adaptor protein associated with TLR signalling. Lack of MyD88 increased infiltration of macrophage and T cells in the colon of mice with DSS-induced colitis. Consistent with these findings, our results also indicate that TLR4 may not play a central role in the development of DSS-induced colitis. Furthermore, our data suggest that MIF induces inflammation under conditions of lacking TLR4.

In general, TNF-α plays an important role in inflammation and immune response. Several studies have shown the role of TNF-α in human IBD and experimental colitis [1–3,42]. On the other hand, MIF and TNF-α have been shown to have a close relationship in various events in inflammatory processes in vivo and in vitro[5,28–30,43]. In our previous study, we found that WT mice with colitis induced by 7 days of DSS treatment showed increases in both TNF-α and MIF expression levels in the colon [5]. For example, Kobayashi et al. also demonstrated that up-regulation of the MIF level occurred earlier than that of the TNF-α level in a model of LPS-induced hepatitis [29]. Roger et al. also have shown that MIF mainly regulates TNF-α through TLR4 pathway in vitro[44]. In this study, TNF-α levels in colon tissues from TLR4−/− mice were not increased during acute DSS-induced colitis. Our results also suggest the possibility that MIF regulates TNF-α expression through the TLR4 signalling pathway in this model. However, it has been reported that chronic but not acute DSS-induced colitis model is known to be associated with TNF-α[46]. Moreover, it has been reported previously that the absence of TNF-α did not suppress the severity of acute DSS-induced colitis [45]. In this study, we did not investigate the expression of TNF-α in the colon in chronic DSS-induced colitis. Thus, our current results did not completely provide the evidence that the development of DSS-induced colitis is independent of TNF-α expression. Further study is needed for elucidation of the precise role of TNF-α in DSS-induced colitis.

We have demonstrated previously that MIF plays an important role in the development of DSS-induced colitis in mice [5,39]. MIF is expressed constitutively in the gastrointestinal tissues in vivo and in vitro[4,5,47–49]. de Yong et al. have focused on the pathological role of MIF in chronic colitis, revealing its pathogenic role in the disease [4]. In this study, we first investigated the expression of MIF in the colon of TLR4−/− mice. Interestingly, the expression of MIF was significantly up-regulated in the colon of TLR4−/− mice with DSS-induced colitis. This result indicates that MIF is a critical component in the colon during DSS-induced colitis, independently of TLR4 signalling.

Several studies have revealed the localization of MIF-positive staining in various tissues, such as immune cells and epithelial cells in skin with dermatitis [5,31,32,50]. Similar to the findings in skin [31], we previously observed MIF expression in epithelial cells and infiltrating immune cells of the mouse colon [5]. Consistent with these findings, we here observed weak MIF expression in mucosal intestinal epithelial cells and mononuclear cells of the colon from TLR4−/− mice under normal conditions. MIF-positive staining was greatly enhanced in numerous infiltrating inflammatory cells in the colonic mucosa of TLR4−/− mice with DSS-induced colitis. These facts provide additional evidence that local MIF expression contributes to inflammatory responses in TLR4−/− mice with DSS-induced colitis.

To examine further the role of MIF in the development of DSS-induced colitis in TLR4−/− mice, we evaluated MPO as a parameter of neutrophil accumulation. Makita et al. reported an increase of MPO activity and up-regulation of MIF expression in alveoli from mice with experimental acute lung distress syndrome [51]. In this study, the level of MPO activity was increased markedly in both WT and TLR4−/− mice with DSS-induced colitis. Thus, it is conceivable that MIF enhances the accumulation and activation of neutrophils in colons of mice with DSS-induced colitis.

Alternatively, MIF is known to up-regulate expression of MMP in cells and tissues [35]. MMP is an important molecule in tissue destruction and remodelling. We have demonstrated previously that neutralization of MIF by anti-MIF antibody suppressed the MMP-13 mRNA level, which was up-regulated in the colons of mice with DSS-induced colitis [5]. Consistent with these findings, we here found that TLR4−/− mice given DSS showed up-regulation of MIF and MMP-13 expressions in the colon. Moreover, anti-MIF antibody remarkably suppressed the up-regulation of MMP-13 expression in the colons of TLR4−/− mice with DSS-induced colitis. These facts indicate that expression of MMP-13 is modulated by MIF, which promotes DSS-induced colitis even in TLR4−/− mice. That is, MIF may enhance tissue damage through up-regulation of MMP-13 in the colons of TLR4−/− mice with DSS-induced colitis.

In conclusion, we have demonstrated that colitis could be induced by DSS in TLR4−/− mice. Our current results indicate that MIF may play a more crucial role than previously thought in the development of DSS-induced colitis independently of activation of innate immune responses mediated by the TLR4-signalling pathway.

Acknowledgments

This work was supported in part by a Grant-in-aid from the Japanese Ministry of Health and Welfare.

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