Original Article

Immunology and Cell Biology (2007) 85, 633–639; doi:10.1038/sj.icb.7100112; published online 4 September 2007

The correlation between proinflammatory cytokines, MAdCAM-1 and cellular infiltration in the inflamed colon from TNF-alpha gene knockout mice

Yinghua Xu1, Nicholas H Hunt1 and Shisan Bao1

1Discipline of Pathology, Bosch Institute, School of Medical Sciences, University of Sydney, Sydney, New South Wales, Australia

Correspondence: Dr S Bao, Discipline of Pathology, University of Sydney, Room 572, Blackburn Building D06, Sydney, New South Wales 2006, Australia. E-mail: bobbao@med.usyd.edu.au

Received 2 February 2007; Revised 11 July 2007; Accepted 16 July 2007; Published online 4 September 2007.

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Abstract

Tumour necrosis factor (TNF) is important in the development of inflammatory bowel disease. TNF-alpha-deficient mice show more severe colonic inflammation than wild-type (Wt) mice, but the underlying mechanism remains unclear. Using immunohistochemistry, enzyme-linked-immunosorbent assay and histopathology, we found that there was a higher level of macrophage infiltration in TNF-alpha-/- compared to Wt mice. This is consistent with higher levels of monocyte chemotactic protein-1, interleukin (IL)-6 and granulocyte monocyte colony-stimulating factor (GM-CSF) in the inflamed colon from the TNF-alpha-/- mice, compared to the Wt mice, following dextran sulphate sodium (DSS) challenge. There was close correlation between clinical observations and histopathological findings in both Wt and TNF-alpha-/- mice. The expression of mucosal addressin cell adhesion molecule 1 (MAdCAM-1) was upregulated in the colon of Wt and TNF-alpha-/- mice following DSS challenge. Interestingly, the induction of MAdCAM-1 was relatively lower in the inflamed colon of TNF-alpha-/- mice, despite the higher inflammatory cell infiltrate, compared to their Wt counterparts. On the other hand, TNF-alpha-/- mice had significantly lower baseline levels of colonic IL-4, IL-6 and GM-CSF. Furthermore, there was a reduction of both immunoglobulin A (IgA) and IgG in the gut from TNF-alpha-/- mice following DSS challenge. These data indicate that TNF-alpha deficiency alters homoeostasis of the colonic chemokine/cytokine environment and humoral immune response, resulting in an exacerbation of acute DSS-induced colitis in TNF-alpha-/- mice. These findings support the idea that TNF-alpha plays a role in the acute stage of intestinal inflammation.

Keywords:

inflammatory bowel disease, pathogenesis, tumour necrosis factor, cytokines, inflammation

Ulcerative colitis and Crohn's disease, referred to as inflammatory bowel disease (IBD), are chronic inflammatory disorders of the intestinal tract,1, 2 characterized by spontaneous relapses and remission.3 Despite extensive research, the precise mechanisms underlying IBD remain unclear. Current hypotheses suggest that the aetiology of IBD involves complex interactions between genetic predisposition, immunological and environmental factors, in particular a dysregulated immune response to the normal intestinal bacteria flora.4, 5, 6 Accumulating evidence supports a crucial role for a disturbed balance of local pro- and anti-inflammatory cytokines in the pathogenesis of IBD.7, 8

Tumour necrosis factor-alpha (TNF-alpha) is a pleiotropic cytokine with numerous biological effects including cytotoxicity, anti-infection, growth modulation and cellular differentiation.9 The action of TNF-alpha in intestinal inflammation was previously considered to be harmful, due to its well-characterized proinflammatory properties. The role of TNF-alpha in the development of IBD has been studied using TNF-alpha-/- mice.10 TNF-alpha-/- mice developed more severe colitis and had a decreased survival rate compared to wild-type (Wt) mice.10 Severity correlated with upregulation of interferon-gamma (IFNgamma) and inducible nitric oxide synthase (iNOS) mRNA. Upregulation of mRNA iNOS is consistent with NO production in the gut.

Monocyte chemotactic protein-1 (MCP-1), a proinflammatory marker, can be induced by TNF-alpha at inflammatory sites in gastrointestinal mucosa,11 and interleukin (IL)-6, IL-18 and IL-4 also contribute to the development of IBD.12, 13 On the other hand, granulocyte monocyte colony-stimulating factor (GM-CSF) provides protection from TNF injury by inhibiting TNF-alpha release in a model of indomethacin-induced gastric injury in rats.14 However, the production of cytokines/chemokines at the protein level in the intestinal mucosa of TNF-alpha-/- mice, particularly in colitis, was unclear, due to the complex nature of immunity. Furthermore, the infiltration of inflammatory cells and its relationship with mucosal addressin cell adhesion molecule 1 (MAdCAM-1), and the humoral response to dextran sulphate sodium (DSS)-induced IBD in Wt and TNF-alpha-/- mice, remain to be explored. Here we investigate these issues and the potential clinical relevance of our observations is discussed.

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Results

TNF-alpha-deficient mice are more susceptible to acute DSS-induced colitis

The unchallenged TNF-alpha-/- mice used in this study did not exhibit the diarrhoea, rectal bleeding and body weight loss that are normally associated with spontaneous intestinal inflammation. Moreover, histopathological observations did not reveal any evidence of epithelial injury and mucosal inflammation in TNF-alpha-/- unchallenged mice. A significant decrease (P<0.01) in body weight was observed in the TNF-alpha-/- mice when compared to the Wt mice on day 5–7 (day 7, 78.4plusminus1.1 vs 86.8plusminus0.8% of baseline body weight; Figure 1a). disease activity index (DAI) scores were significantly lower in DSS-treated Wt mice compared to the DSS-treated TNF-alpha-/- mice (P<0.01 from day 4 to day 7; Figure 1b).

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Body weight loss (a), DAI score (b), appearance of the colon (c) and histopathological score (d) in wild-type (Wt) and tumour necrosis factor (TNF)-alpha-/- mice following 2.5% dextran sulphate sodium (DSS) treatment for 7 days. (b) DAI was determined by scoring changes in body weight, presence of faecal blood and stool consistency (range from 0 to 12). (c) Paraffin sections of the ascending (A), transverse (T) and descending (D) colon of mice were stained with H&E (times 100). (d) Histopathological score was quantified by scoring changes in crypt damage and inflammatory cell infiltration (range from 0 to 8). Data are Meanplusminuss.e.m., n=8. *P<0.05 and **P<0.01.

Full figure and legend (194K)

In agreement with previous studies,15, 16 histopathology showed that the entire colon, particularly the transverse and descending portions, was inflamed following DSS challenge. The histological appearance of DSS-treated TNF-alpha-/- mouse colon showed more severe epithelial destruction, that is epithelial ulceration, crypt damage, goblet cell depletion and inflammatory cell infiltration, compared to that from the Wt mice following DSS treatment (Figure 1c). The histopathological damage score of the colon of DSS-treated TNF-alpha-/- mice was significantly higher than that of the Wt mice on day 7 (5.6plusminus0.26 vs 3.2plusminus0.36; Figure 1d).

Infiltration of IgA+ and IgG+ cells

In unchallenged mice, immunoglobulin A (IgA)+ and IgG+ cells were located mainly in the lamina propria between the crypts or at the base of crypts. The submucosa was devoid of Ig-producing cells. Surprisingly, unchallenged TNF-alpha-/- mice exhibited 2.5-fold higher numbers of IgA+ cells in the colonic lamina propria than did their Wt counterparts (Figure 2a). However, there was no significant difference in the number of IgG+ cells in the colonic lamina propria between unchallenged Wt and TNF-alpha-/- mice (Figure 2b). Administration of 2.5% DSS for 7 days resulted in a twofold reduction of IgA+ cells in the colonic lamina propria of TNF-alpha-/- mice, but colonic IgA+ cells slightly increased in the Wt mice (Figure 2a). The numbers of colonic IgG+ cells significantly increased in Wt mice, but were remarkably decreased (50% reduction) in TNF-alpha-/- mice (Figure 2b) after 7 days DSS treatment.

Figure 2.
Figure 2 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Numbers of IgA+ (a) and IgG+ (b) cells, represented as numbers of positive cells per mm muscularis mucosa, in wild-type (Wt) and tumour necrosis factor (TNF)-alpha-/- mice transverse colon before and after dextran sulphate sodium (DSS) challenge. Data are meansplusminuss.e.m., n=5. *P<0.05 and **P<0.01.

Full figure and legend (26K)

Infiltration of neutrophils and macrophages

RB6-8C5 was used as a neutrophil marker. Few RB6-8C5+ cells were observed in the colon of the unchallenged Wt and TNF-alpha-/- mice. The numbers of infiltrating neutrophils (RB6-8C5+ cells) increased after DSS administration in both strains of mice, and were mainly localized in inflamed mucosa and submucosa. However, there was no significant difference in neutrophil infiltration in the colonic submucosa between DSS-treated TNF-alpha-/- mice and their Wt counterparts (data not shown).

F4/80 is a commonly used macrophage marker. In colons of unchallenged mice, the F4/80+ cells were localized in the lamina propria mucosa, in the subepithelial region, between crypts or at the base of crypts. The submucosa was almost devoid of F4/80+ cells. Following 7 days DSS treatment the number of macrophages (F4/80+ cells) increased significantly, particularly at the base of crypts and the submucosal layer, in both Wt and TNF-alpha-/- mice (Figure 3a). Macrophages in the colon of TNF-alpha-/- mice were 30% higher than in that of Wt mice (39plusminus2.9 vs 29plusminus1.4; Figure 3b) following DSS challenge.

Figure 3.
Figure 3 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

(a) Macrophage (F4/80+ cells) infiltration in the colonic tissues of wild-type (Wt, left) and tumour necrosis factor (TNF)-alpha-/- (right) mice with (top row) and without (lower row) DSS treatment. (b) Numbers of macrophages (F4/80+ cells) (represented as numbers of positive cells per mm muscularis mucosa) in submucosa of transverse colon before and after dextran sulphate sodium (DSS) treatment. Data are meansplusminuss.e.m., n=8. *P<0.05.

Full figure and legend (335K)

Expression of mucosal addressin cell adhesion molecule

Mucosal addressin cell adhesion molecule 1 was constitutively expressed on the vessels of mesenteric lymph node (MLN), but almost undetectable in colonic tissue in unchallenged Wt and TNF-alpha-/- mice. After 7 days DSS treatment, expression of MAdCAM-1 on the vessels in the colonic tissue and MLN was significantly increased in both strains of mice. Interestingly, expression of MAdCAM-1 in the colon (Figure 4) and MLNs (data not shown) from TNF-alpha-/- mice was weaker and more sporadic than in the same tissues from their Wt mice counterparts.

Figure 4.
Figure 4 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Numbers of mucosal addressin cell adhesion molecule 1+ (MAdCAM-1+) vessels (represented as numbers of positive vessels per mm muscularis mucosa) in wild-type (Wt) and tumour necrosis factor (TNF)-alpha-/- mice in transverse colon before and after dextran sulphate sodium (DSS) challenge. Data are meansplusminuss.e.m., n=4. *P<0.05.

Full figure and legend (13K)

Production of chemokine and cytokines in colonic tissue

Colonic MCP-1 was almost undetectable in unchallenged mice; however, it was markedly increased in Wt and TNF-alpha-/- mice following 7 days of DSS challenge. DSS-treated TNF-alpha-/- mice had a significantly higher colonic level of MCP-1 than their Wt counterparts (Figure 5a). In the unchallenged group, TNF-alpha-/- mice had significantly lower colonic IL-6 (P<0.01; Figure 5b), IL-4 (P<0.02; Figure 5c) and GM-CSF (P<0.01; Figure 5d) levels, but a slightly increased IFN-gamma level (data not shown), compared to Wt mice. After 7 days administration, both Wt and TNF-alpha-/- mice had a significantly increased IL-6 level in colonic tissue (P<0.05). DSS-treated TNF-alpha-/- mice had 3-fold higher levels of IL-6 than water-treated TNF-alpha-/- mice, while only a 1.5-fold change occurred in Wt mice (Figure 5b). No significant increase of GM-CSF in the colon was observed between DSS challenged and unchallenged Wt mice. However, in TNF-alpha-/- mice, the colonic GM-CSF level was markedly increased (up to sixfold of basal level) following DSS challenge compared to their Wt counterparts (Figure 5d). Colonic levels of IL-4, IL-17, IL-18 and IFN-gamma did not change significantly in Wt or TNF-alpha-/- mice following 7 days DSS challenge (data not shown).

Figure 5.
Figure 5 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Colonic levels of inflammatory cytokines (a, MCP-1), (b, IL-6), (c, IL-4), (d, GM-CSF) (represented as pg ml-1 tissue extracts) in wild-type (Wt) and tumour necrosis factor (TNF)-alpha-/- mice, before and after 7 days dextran sulphate sodium (DSS) challenge. Data are meansplusminuss.e.m., n=4. *P<0.05 and **P<0.01.

Full figure and legend (45K)

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Discussion

In this study, we demonstrated that more severe colitis was induced by DSS in the TNF-alpha-/- mice compared to Wt mice at the acute stage, which is consistent with the findings of Naito et al.10 TNF-alpha-/- mice showed greater body weight loss, higher disease activity score, aggravated tissue damage and inflammatory cell infiltration in TNF-alpha-/- mice when compared to Wt mice (Figure 1).

More importantly, our data demonstrated that TNF-alpha-/- mice displayed an impaired humoral immune response and a disruption of colonic chemokine/cytokine homoeostasis following DSS challenge. However, TNF-alpha deficiency per se did not affect the integrity of the normal mouse intestinal mucosa, and there were no clinical or histological signs of spontaneous colitis in unchallenged TNF-alpha-/- mice.

Previous studies have revealed the critical involvement of TNF-alpha in regulating the development and organization of splenic follicular architecture and maturation of the humoral immune response. However, TNF-alpha-/- mice displayed normal Ig levels in the steady state.17, 18 Surprisingly, our current study showed that there are higher numbers of colonic IgA+ cells, but similar numbers of IgG+ cells, in unchallenged TNF-alpha-/- mice, compared to unchallenged Wt mice (Figures 2a and b). IgA is the predominant immunoglobulin of the normal colon, and provides the first line of defence against pathogens in the intestinal mucosa.19, 20 The increased colonic IgA+ cells may be a compensatory mechanism that protects TNF-alpha-/- mice from an altered colonic microenvironment such as increased luminal bacterial load and altered colonic cytokine pattern.21

TNF-alpha-/- mice may have had an impaired humoral immune response, especially IgG production, compared to Wt mice following DSS treatment. Both colonic IgA+ and IgG+ cell numbers were reduced twofold in these mice following DSS administration. Compared to DSS-treated Wt mice, a significantly lower number of colonic IgG+ cells were found in TNF-alpha-/- mice (Figure 2b). IgG has been found to reduce the occurrence of DSS-induced colitis by suppressing the recruitment of immunocompetent cells into colitis lesions in vivo,22 and by inhibiting pathogenic T-cell proliferation in vitro.22 Additionally, defective mucosal secretory IgA production and decreased numbers of IgA-containing cells in IBD patients' tissue have been reported in clinical studies.23, 24 Although it remains unclear how the decrease of IgA+ and IgG+ cells modifies the outcome of acute DSS colitis in TNF-alpha-/- mice, it has been suggested that IgA- and IgG-containing cells play an important role in DSS-induced colitis.25 Nevertheless, further investigations are required to determine the precise mechanism for the effects of IgA and IgG on the induction and progression of DSS-induced colitis in TNF-alpha-/- mice.

Increased infiltration of neutrophils and macrophages is distinguishing feature of human IBD26 and the DSS-colitis model.15, 16 TNF-alpha has been shown to stimulate the recruitment and activation of neutrophils and monocytes to sites of inflammation.16 TNF-alpha deficiency did not affect the numbers of macrophages or neutrophils in colonic tissue without DSS challenge in the present study. In contrast, a markedly increased macrophage infiltration correlated with colitis severity and colonic MCP-1 production (Figure 5a) in DSS-treated TNF-alpha-/- mice compared to DSS-treated Wt mice. MCP-1 is a pivotal chemokine in the recruitment and activation of monocytes during inflammation.27 Watanabe et al.11 demonstrated that MCP-1 was induced by TNF-alpha in the gastric mucosa, 4 h post-injection of TNF-alpha. Further, anti-MCP-1 antibody inhibited macrophage infiltration.11 Interestingly, our results showed that there was a higher MCP-1 production in the inflamed colon of TNF-alpha-/- mice than in that of Wt mice (Figure 5a). Upregulation of MCP-1 correlated with the increased infiltration of macrophages. These findings, combined with those from Watanabe et al.11 suggest that MCP-1 and monocytes/macrophages may interact in a paracrine and/or autocrine fashion. Furthermore, our findings suggest that production of colonic MCP-1 is not affected by the absence of TNF-alpha, and that the upregulated colonic MCP-1 in TNF-alpha-/- mice after DSS challenge resulted in an enhanced macrophage infiltration. This subsequently leads to more tissue damage and induction of other proinflammatory cytokines.

Mucosal addressin cell adhesion molecule 1 is an adhesion molecule from an immunoglobulin superfamily that mediates recruitment of lymphocytes into intestinal mucosa.28 In line with previous studies,28, 29, 30 our results confirmed a constitutive expression of MAdCAM-1 in MLN and colonic tissues, and that enhanced expression of MAdCAM-1 is associated with intestinal inflammation. The importance of TNF-alpha in the induction of MAdCAM-131, 32can also be inferred, with expression of MAdCAM-1 in the TNF-alpha-/- mice being consistently weaker and more sporadic than in Wt mice under the same conditions. Kato et al.29 previously have demonstrated that anti-MAdCAM-1 treatment ameliorates chronic DSS colitis. However, the same authors also pointed out that an anti-MAdCAM-1 antibody does not attenuate acute DSS-induced colitis.29 This is further supported by anti-TNF-alpha treatment, which showed that TNF-alpha is protective for intestinal inflammation at the acute stage, but is harmful at the chronic stage.33 These two reports indicate that outcomes of anti-TNF-alpha or MAdCAM-1 treatment for colitis are stage dependent, that is TNF-alpha/MAdCAM-1 is beneficial during acute intestinal inflammation, whereas TNF-alpha/MAdCAM-1 is harmful in chronic inflammation.29 Furthermore, MAdCAM-1 is important for T-cell recruitment via its ligand alpha4beta7. We attempted to stain for T cells without much success, highlighting the need for future research. Moreover, studies have revealed that severe combined immunodeficiency mice are more susceptible to DSS-induced colitis, suggesting that the presence of T or B cells may be protective in this model.34 Therefore, we speculate that the downregulated expression of MAdCAM-1 in TNF-alpha-/- mice may decrease recruitment of circulating lymphocytes into gut-associated lymphoid tissue and intestinal tissue. This may result in the decreased IgA and IgG B cells (Figure 2) in TNF-alpha-/- mice following DSS administration.

There is considerable evidence from both animal models and clinical investigations to support the notion that an imbalance of pro- and anti-inflammatory cytokines plays a pivotal role in IBD.35, 36, 37 It is well known that TNF-alpha plays a central role in the initiation and regulation of the cytokine cascade. However, a previous study in TNF-deficient mice demonstrated that systemic cytokine production induced by lipopolysaccharide is essentially intact, with the exception of reduced colony-stimulating factor activity.18 In the present study, we found that colonic IL-4, IL-6 and GM-CSF levels were significantly lower in unchallenged TNF-alpha-/- mice compared to their Wt counterparts. Therefore, it seems that local colonic cytokine production is more likely to be influenced by TNF-alpha deficiency than production of cytokines systemically. However, this observation may also be due to the different experimental protocol and/or different inducing agents used in these studies. It is worthy of note that colonic GM-CSF was increased only in DSS-treated TNF-alpha-/- mice but not in their Wt counterparts. The increased production of GM-CSF in TNF-alpha-/- mice may be a compensation for TNF-alpha deficiency, as GM-CSF has many proinflammatory actions that are similar to those of TNF-alpha.38 Elevated colonic GM-CSF may also partially contribute to the increased colonic macrophage number in DSS-treated TNF-alpha-/- mice.

The lower colonic level of IL-4 in TNF-alpha-/- mice compared to their Wt counterparts is correlated with the findings concerning Ig-secreting cells, since IL-4 is a cytokine known to stimulate B-cell proliferation and induce antibody secretion. In contrast to a previous study,10 we found no significant changes in colonic IL-4 and IFN-gamma (data not shown) in either strain of mouse following DSS treatment. Our data and other reports suggest that tissue damage in the acute DSS colitis model is mediated primarily by macrophages rather than T-cell subsets.39, 40 Enhanced production of proinflammatory chemokine/cytokines by colonic macrophages, neutrophils and intestinal epithelial cells may further perpetuate the inflammatory response.7

In conclusion, we have shown that TNF-alpha is not absolutely necessary for the maintenance of intestinal mucosal homoeostasis in the absence of inflammatory stimuli. However, TNF-alpha deficiency alters the homoeostasis of the colonic chemokine/cytokine environment and humoral immune response, resulting in an exacerbation of acute DSS-induced colitis in TNF-alpha-/- mice. These findings support the notion that TNF-alpha plays a protective role in the acute stage of intestinal inflammation. However, the locality and stage specificity of TNF-induced inflammation may also determine the ensuing beneficial or deleterious nature of the immunological response. Our data from TNFalpha-/- mice suggest that lack of TNF could modify host immunity and influence the outcome of DSS-induced colitis. Hence, optimal design of TNF target therapy needs to consider the critical effects mediated by TNF-alpha at distinct stages of the inflammatory process as well as potential side effects such as increasing the risk of opportunistic infections during long-term treatment.

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Methods

Mice

C57BL/6 Wt mice and TNF-alpha gene knockout C57BL/6 mice (TNF-alpha-/-)41 were obtained from the University of Sydney Blackburn Animal House. Both Wt and TNF-alpha-/- mice were 8–10 weeks of age, sex-matched and weighed 19–22 g. All animals were housed with environmental enrichment in a conventional laboratory with free access to food and water. Experiments and procedures were approved by the University of Sydney Animal Ethics Committee.

Induction of acute colitis

Acute colitis was induced by feeding mice 2.5% (w/v) DSS (ICN Biomedicals Australasia, Sydney, NSW, Australia) dissolved in drinking water for 7 days. The DSS solutions were freshly prepared daily and grossly examined for turbidity. The control mice were given water only.

Clinical score

Daily clinical assessment of the progression of colitis in DSS-treated mice included measurement of body weight, evaluation of stool consistency and the presence of faecal blood. A previously validated15 clinical DAI was calculated from the following parameters: stool consistency (0–4), presence or absence of faecal blood (0–4) and weight loss (0–4). The maximum possible score is 12. Mice were killed at day 7 of the experiments; colon, spleen and MLNs were removed and stored for further study.

Histology

Each colon was divided into the proximal, middle and distal portions, and fixed in cold ethanol as described.42 Fixed tissues were embedded in paraffin, and 5 mum thick sections were stained with haematoxylin and eosin (H&E). Histological damage was observed only in animals with colitis. The average score of acute colitis in the middle and distal part of the colon was calculated from observation of 20 different fields of H&E-stained longitudinal sections of colon from each animal.43 The histological scoring was performed in a double-blind fashion. A method modified from a previously validated histopathological score grading system was used to evaluate the degree of colitis.44, 45 Two independent parameters46 were measured: the extent of inflammation (0, none; 1, slight; 2, moderate; 3, severe; 4, massive) and the extent of crypt damage (0, none; 1, the basal one-third portion damaged; 2, the basal two-thirds portion damaged; 3, the entire crypt damaged but the surface epithelium intact; 4, the entire crypt and epithelium lost). The maximum possible score is 8.

Immunohistochemistry and quantitativeness of infiltrating cells

Colon, spleen and MLNs were fixed in cold ethanol,42 embedded in paraffin and sectioned at 5 mum. Immunohistochemistry was performed as described previously.42 Monoclonal primary antibodies were used in the immunostaining for detection of MAdCAM-1, macrophage/monocyte marker (F4/80), granulocyte/neutrophil marker (RB6-8C5) (Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia). Primary antibodies used in the immunostaining for IgA and IgG detection were biotin-goat anti-mouse (Zymed, Sydney, NSW, Australia). In brief, sections were incubated with a diluted primary antibody (1:200 except MAdCAM-1 (1:100) and IgG (1:300)) for 1 h at room temperature. Then the sections were washed three times in Tris-buffered saline for 3 min, and incubated with biotin-conjugated secondary antibody (for MAdCAM-1, F4/80 and RB68C5 staining only, Dako, Australia) for 1 h at room temperature, followed by avidin-biotin-peroxidase complex (ABC kit, Vector, Sydney, NSW, Australia). The peroxidase activity was visualized using diaminobenzidine. The numbers of IgA+, IgG+ cells, F4/80+ cells, RB68C5+ cells and MAdCAM-1+ vessels in the transverse colons were counted per high power field (times 400) and averaged in proportion to the length of muscularis mucosa.29 Specificities of staining for all the antibodies used were confirmed by isotype-matched controls. The numbers of infiltrating cells were expressed per millimetre of muscularis mucosa.

Colonic chemokine and cytokine measurements

The levels of MCP-1, IL-4, IL-6, IL-17, IL-18, GM-CSF and IFN-gamma in colon tissue were measured by enzyme-linked-immunosorbent assay using commercial BD OptEIA Set kits (BD Biosciences, Australia). Briefly, the colons were collected after washing in cold phosphate-buffered saline, and then homogenized in extraction buffer (EB) containing 1 protease inhibitor tablet (Roche, Sydney, NSW, Australia) in 50 ml, 100 mM phosphate buffer (100 mg tissue per ml EB), using a tissue tearer. The homogenized colon tissue was centrifuged on 2000 r.p.m. at 4 °C for 15 min. Chemokine and cytokine concentrations were determined in the supernate according to the manufacturer's instructions.

Statistics

The experiment was repeated twice with 4–5 mice per group. All data are presented as meansplusminuss.e.m. The unpaired two-tailed Student's t-test was performed for statistical analysis. Differences with P-values <0.05 were regarded as statistically significant.

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Acknowledgements

We thank the assistance of the histopathology laboratory in the Discipline of Pathology, University of Sydney.