We used 7-week-old male MT-I/II double knockout (MT null) mice (25) and age-matched C57BL/6 wild-type mice. MT-I/II knockout mice were kindly provided by Dr Hirohisa Takano (Center for Environmental Health Sciences, National Institute for Environmental Studies, and Kyoto University, Graduate School of Engineering, Kyoto, Japan). C57BL/6 mice were purchased from Shimizu Laboratory Supplies Co., Ltd. (Kyoto, Japan). MT-I/II knockout mice were routinely bred in the vivarium of Kyoto Prefectural University of Medicine. Mice were housed in cages under a 12-h light/dark cycle, and a controlled temperature of 22°C and negative atmospheric pressure. Mice were maintained in a specific pathogen-free environment and provided with tap water and food ad libitum. Mice were fed a rodent diet (CE-2; Nihon CLEA, Tokyo, Japan) during the experiment. All experimental procedures were carried out in accordance with the National Institutes of Health (NIH) guidelines for the Care and Use of Laboratory Animals. Experimental protocols were approved by the Animal Care and Use Committee of Kyoto Prefectural University of Medicine (Kyoto, Japan).

Male wild-type mice and MT-I/II knockout mice (7 weeks old) were randomized into control and experimental groups (6–8 mice in each group). Experimental acute colitis was induced by treatment with 2.0% DSS (molecular weight 1,000–9,000, Lot SDR4219; Wako Pure Chemical Industries, Osaka, Japan) in drinking water for 7 days, as reported previously (26,27). Mice were sacrificed under anesthesia 7 days after DSS treatment, and the colons were removed for macroscopic and histological examination. Colonic specimens were also obtained for biochemical assay and RNA isolation.

DAI, colon length and histology were analyzed. DAI was determined by scoring changes in animal weight, occult blood positivity, gross bleeding and stool consistency, as described previously (26,28,29). We used 5 grades of weight loss (0, no loss or weight gain; 1, 1–5% loss; 2, 5–10% loss; 3, 10–20% loss; 4, >20% loss), 3 grades of stool consistency (0, normal; 2, loose; and 4, diarrhea), and 3 grades of occult blood (0, negative; 2, occult blood-positive; and 4, gross bleeding) based on previous studies (26,27). After determining DAI, the mice were sacrificed, and the entire colon was removed from the cecum to the anus, and colon length was measured as an indirect marker of inflammation.

Immediately after dissection, the distal colon was fixed in 10% buffered formalin for histological analysis. Sections (4-μm-thick) were prepared and stained with hematoxylin and eosin (H&E). The slides were then examined and scored in a blinded manner using a previously published grading system (30,31). Briefly, a combined score of inflammatory cell infiltration and tissue damage was determined as follows: as regards cell infiltration: score 0, occasional inflammatory cells in the lamina propria; 1, increased inflammatory infiltrate in the lamina propria predominantly at the base of crypts; 2, confluence of inflammatory infiltrate extending into the mucosa; and 3, transmural extension of inflammatory infiltrate. As regards tissue damage: score 0, no mucosal damage; 1, partial (up to 50%) loss of crypts in large areas; 2, partial to total 50–100% loss of crypts in large areas, epithelium intact; and 3, total loss of crypts in large areas and epithelium lost. The total histological score represents the sum of the cell infiltration and tissue damage scores, and thus ranges from 0 to 6.

MPO activity in the colonic mucosa, an index of polymorphonuclear leukocyte accumulation, was determined using a modification of the method described by Grisham et al (32). A total of 2 ml of mucosal homogenate was centrifuged at 20,000 × g for 15 min at 4°C to pellet the insoluble cellular debris. The pellet was then re-homogenized in an equivalent volume of 0.5% hexadecyltrimethylammonium bromide. Samples were centrifuged at 20,000 × g for 15 min at 4°C, and the supernatants were saved. MPO activity was assessed by measuring the H₂O₂-dependent oxidation of 3,3′,5,5′-tetramethylbenzidine. One unit of enzyme activity was defined as the amount of MPO that caused the absorbance to change by 1.0/min at 655 nm and 25°C.

The mRNA expression levels of MT-I/II, TNF-α, interferon (IFN)-γ, interleukin (IL)-17 and β-actin (internal standard) were determined by real-time-PCR. Samples for mRNA isolation were removed from colonic tissue. Total RNA was isolated with the acid guanidinium phenol chloroform (AGPC) method using Isogen reagent (Nippon Gene, Toyama, Japan). RNA was stored at 70°C until it was used for reverse-transcription polymerase chain reaction (RT-PCR). A total of 1 μg of extracted RNA was reverse-transcribed into first-strand complementary DNA (cDNA) using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA). Real-time PCR for CINC-1 and β-actin was carried out with the 7300 Real-Time PCR system (Applied Biosystems) using the DNA-binding dye SYBR^®-Green for the detection of PCR products. Primers had the following sequences: MT-I sense, 5′-GCTGTGCCTGATGTGACGAA-3′ and antisense, 5′-AGGAAGACGCTGGGTTGGT-3′; MT-II sense, 5′-TGCGC TCGACCCAATACTC-3′ and antisense, 5′-TCTAGGAGCG TGATGGAGAGAAG-3′; TNF-α sense, 5′-ATCCGCGACGT GGAACTG-3′ and antisense, 5′-ACCGCCTGGAGTTCTG GAA-3′; IFN-γ sense, 5′-CCTGCGGCCTAGCTCTGA-3′ and antisense, 5′-CCATGAGGAAGAGCTGCAAAG-3′; IL-17 sense, 5′-TCATCTGTGTCTCTGATGCTGTTG-3′ and antisense, 5′-TCGCTGCTGCCTTCACTGT-3′; and β-actin sense, 5′-GAGCAAACATCCCCCAAAGTT-3′ and antisense, 5′-GCC GTGGATACTTGGAGTGACT-3′. Relative quantities of gene expression with real-time PCR data were calculated relative to β-actin.

We determined the concentrations of TNF-α, IFN-γ and IL-17 in the supernatant of colonic mucosal homogenates using an enzyme-linked immunosorbent assay (ELISA) kit for TNF-α, IFN-γ and IL-17 (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. Subtractive readings at 550 nm from the readings at 450 nm were converted to pg/ml using values obtained from standard curves generated with varying concentrations of recombinant TNF-α, IFN-γ and IL-17.

After 24-h fixation in formalin, samples were embedded in paraffin, after which 4-μm-thick sections were cut using a microtome cryostat and mounted on MAS-coated slides. We performed antigen retrieval using proteinase K solution, and the sections were rinsed with distilled water for 5 min, followed by incubation with 3% hydrogen peroxide in methanol for 30 min to block endogenous peroxidase activity. After incubation, the sections were washed in phosphate-buffered saline (PBS)-Tween-20 for 5 min. Non-specific binding was blocked by incubating the slides with Dako Cytomation protein block (Dako, Tokyo, Japan) for 30 min at room temperature. The sections were then incubated with primary antibody against MTs (Dako, Glostrup, Denmark) diluted at 1:50 and F4/80 (Novus Biologicals, Inc., Littleton, CO) diluted at 1:200 with antibody dilution (Dako) overnight at 4°C. The sections were washed 3 times in PBS-Tween-20 for 5 min and incubated with secondary antibody [Histofine Simple Stain mouse MAX PO (rabbit); Nichirei Biosciences, Inc., Tokyo, Japan] for 30 min at room temperature. Unbound antibodies were removed by 3 washes in PBS for 5 min, and bound antibodies were visualized using diaminobenzidine as a chromogen substrate reagent. Negative controls for non-specific binding incubated with secondary antibodies were confirmed to produce no signal. All sections were counterstained with hematoxylin. Sections were finally dehydrated, cleared and coverslipped.

For the immunofluorescence staining of MTs and F4/80 (diluted at 1:200; Novus Biologicals, Inc.), bound antibodies were visualized using the secondary antibodies, anti-rabbit Alexa 594 (Molecular Probes, Inc., Eugene, OR) and anti-rat Alexa 488 (Molecular Probes), respectively, diluted at 1:1,000. Negative controls used for non-specific binding and incubated with secondary antibodies were confirmed to produce no signal. Fluorescence staining was observed under an inverted fluorescence microscope (IX70-23FL/DIC-SP; Olympus, Tokyo, Japan).

We evaluated the number of F4/80-positive cells in the intestinal mucosa of wild-type and MT-I/II knockout mice following DSS-induced colitis. The number of positively stained cells in the intestinal mucosa was counted in 50 high-power fields per section under a microscope, as described previously (33).

Resident peritoneal macrophages were obtained by peritoneal lavage using 10 ml of PBS from both wild-type and MT-I/II knockout mice. Cells were plated at 5×10⁵ cells/well (24-well plate) and incubated for 2 h at 37°C in RPMI-1640 medium with 5% fetal bovine serum (FBS), 2 mM glutamine, 50 mg/ml streptomycin and 50 U/ml penicillin (all obtained from Life Technologies Corp.). The cells were washed twice with PBS to remove non-adherent cells and cultured in RPMI-1640 for 24 h at 37°C. Subsequently, the cells were stimulated with LPS (Escherichia coli 055:B5; Sigma-Aldrich Corp., St. Louis, MO) at 10 ng/ml. After a 12-h incubation with LPS, cell supernatants were collected and stored at −70°C. TNF-α, IFN-γ and IL-17 levels in culture supernatants were determined using an ELISA kit according to the manufacturer’s instructions (R&D Systems).

The results in this study are presented as the means ± standard error of the mean (SEM). Overall differences between the groups were determined by one-way analysis of variance (ANOVA). If the results from one-way ANOVA were significant, the differences between individual groups were analyzed by Bonferroni’s multiple comparisons test. P-values <0.05 were considered to indicate statistically significant differences. All analyses were performed using GraphPad Prism 5 software (GraphPad Software, San Diego, CA) on a Windows-based computer.

In order to investigate MT-I/II expression in the inflamed mucosa following the administration of DSS, we examined the expression of colonic MT-I/II mRNA using real-time PCR analysis. The results confirmed that the expression of MT-I/II was significantly higher in the inflamed colonic mucosa in the model of DSS-induced colitis (Fig. 1A).

The intake of DSS solution was monitored throughout the experiments and was found to be similar among the experimental groups (data not shown). The mice exposed to 2.0% DSS developed symptoms of acute colitis, with diarrhea accompanied by rectal bleeding and body weight loss. Decreases in body weight were significantly greater in the MT-I/II knockout mice in comparison to the wild-type mice (Fig. 1B). DAI scores in the DSS-treated mice, determined by weight loss, stool consistency and blood in stool, were significantly increased in the MT-I/II knockout mice compared to the wild-type mice (Fig. 1C).

Moreover, colon length is known to be reduced in DSS colitis along with the exacerbation of intestinal inflammation. In this study, MT-I/II knockout mice showed marked colon shortening 7 days after DSS administration (Fig. 1D). Colon length following DSS administration was significantly decreased in the MT-I/II knockout mice compared to the wild-type mice (Fig. 1E). These data suggest that MTs play a crucial role in the development of intestinal inflammation.

The protective effects of MTs were also confirmed by histological analysis. Fig. 2A shows the typical histological appearance of wild-type and MT-I/II knockout mice. In the wild-type mice, the administration of 2% DSS for 7 days resulted in colonic ulceration associated with large areas of epithelial crypt loss and inflammatory cell infiltration throughout the mucosa. On the other hand, MT deficiency resulted in larger erosions with a high degree of inflammatory cell infiltration. The histological scores reflected these findings (Fig. 2B).

Tissue-associated MPO activity (as an index of polymorphonuclear leukocyte accumulation) in the colonic mucosa increased from 0.130±0.079 mU/mg protein (basal concentration) to 0.903±0.114 mU/mg protein in the wild-type mice 7 days following the administration of DSS (Fig. 2B). MPO activity increased in the wild-type mice following DSS administration, and was significantly elevated in the DSS-treated MT-I/II knockout mice (Fig. 2B).

The expression of inflammatory cytokines in the colonic mucosa following DSS treatment was determined using real-time PCR and ELISA. mRNA expression levels of inflammatory cytokines (TNF-α, IFN-γ and IL-17) were significantly increased in the colonic mucosal tissue of the MT-I/II knockout mice compared with the wild-type mice following the administration of DSS (Fig. 3). In agreement with these results, the colonic mucosal concentrations of these inflammatory cytokines (TNF-α, IFN-γ and IL-17) in the DSS-treated MT-I/II knockout mice were significantly higher, as compared with the DSS-treated wild-type mice (Fig. 4).

MTs are mainly localized in mononuclear cells in the lamina propria and submucosal layer, and these MT-positive cells were elevated in the DSS-treated wild-type mice. By contrast, MT-positive cells were not detected in the colons of MT-I/II knockout mice before and after DSS administration (Fig. 5A).

In addition, in order to investigate the detailed localization of MT expression in the colonic mucosa, we conducted immunofluorescence staining of the colonic mucosa. MT-positive cells largely corresponded with F4/80-positive macrophages (Fig. 5B).

F4/80-positive cells were mainly localized in the lamina propria and submucosal layer in wild-type and MT-I/II knockout mice after DSS administration (Fig. 6A). Furthermore, we evaluated the number of the F4/80-positive cells in the intestinal mucosa of wild-type mice and MT-I/II knockout mice. No significant differences were observed in the number of F4/80-positive cells between the DSS-treated wild-type and MT-I/II knockout mice (Fig. 6B).

In order to determine the production of inflammatory cytokines (TNF-α, IFN-γ and IL-17) in murine peritoneal macrophages following treatment with LPS, we measured the levels of these cytokines in the supernatant of cultured cells using ELISA. The concentrations of the inflammatory cytokines (TNF-α, IFN-γ and IL-17) in the LPS-treated macrophages isolated from the MT-I/II knockout mice were significantly higher as compared with the LPS-treated macrophages derived from the wild-type mice (Fig. 7).