Clostridium difficile has emerged as the important causative agent of antibiotics-associated pesudomembranous colitis; especially its toxin A is presumed to be responsible for the colitis. We examined the pathophysiological roles of IFN-γ in toxin A-induced enteritis using IFN-γ knockout (KO) mice. When toxin A of C. difficile was injected into the ileal loops of BALB/c wild-type (WT) mice, massive fluid secretion, disruption of intestinal epithelial structure, and massive neutrophil infiltration developed within 4 h after the injection. IFN-γ protein was faintly detected in some CD3-positive lymphocytes in the lamina propria and submucosa of the ileum of untreated WT mice. On the contrary, at 2 and 4 h after toxin A injection, IFN-γ protein was detected in infiltrating neutrophils and to a lesser degree in CD3-positive lymphocytes. In the ileum of WT mice, toxin A treatment markedly enhanced the gene expression of TNF-α, macrophage inflammatory protein-1α and -2, KC, and ICAM-1 >2 h after treatment. In contrast, the histopathological changes were marginal, without enhanced fluid secretion in the ileum of toxin A-treated IFN-γ KO mice. Moreover, toxin A-induced gene expression of TNF-α, neutrophil chemotactic chemokines, and ICMA-1 was remarkably attenuated in IFN-γ KO mice. Furthermore, pretreatment of WT mice with a neutralizing anti-IFN-γ Ab prevented toxin A-induced enteritis. These observations indicate that IFN-γ is the crucial mediator of toxin A-induced acute enteritis and suggest that IFN-γ is an important molecular target for the control of C. difficile-associated pseudomembranous colitis.

An extensive administration of wide-range antibiotics frequently causes pseudomembranous colitis, with an estimated annual incidence of >3 million cases in the United States alone (1, 2). Clostridium difficile, a spore-forming and Gram-positive anaerobic bacillus, has now emerged as the leading causative agent for pseudomembranous colitis. C. difficile produces two potent protein exotoxins, toxins A and B, with molecular weights of 308 and 270 kDa, respectively (3, 4). Because toxin A, but not toxin B, can produce fluid secretion and an inflammatory response in intestinal loops of experimental animals, toxin A is also referred to as the enterotoxin (4, 5).

Toxin A-induced enteritis is characterized by massive fluid secretion. Severe epithelial cell damage with massive neutrophil infiltration is the pathological hallmark of toxin A-induced enteritis (6, 7, 8). Once toxin A binds to its plasma membrane receptor, internalization begins within minutes. Internalized toxin A induces glucosylation of Rho, Rac, and Cdc 42, and eventually actin disorganization, which leads to epithelial cell damage and massive fluid secretion (9, 10). Internalized toxin A also induces mitochondrial damage and eventually activation of the transcription factor, NF-κB. NF-κB activation enhances the expression of chemokines with neutrophil chemotactic activities and adhesion molecules, thereby causing massive infiltration of neutrophils (10). Several lines of evidence suggest that some inflammatory cellular response to C. difficile toxin A may occur before and independently of Rho glucosylation (10, 11). Thus, it is necessary to elucidate the non-Rho-dependent pathway to fully understand the molecular pathological mechanisms of toxin A-induced enteritis.

IFN-γ exerts a wide variety of actions, including antiviral activity, activation of macrophages and NK cells, and up-regulation of MHC Ag expression on macrophages (12). Moreover, accumulating evidence has defined IFN-γ as a crucial regulatory mediator of Th1 immune responses. Thus, IFN-γ has been implicated as being involved in various types of chronic inflammatory diseases, particularly Th1-mediated immunopathologies, including Crohn’s disease (13, 14). Intrapleural injection of Staphylococcus aureus induced neutrophil infiltration into pleural space in a CD4- and IFN-γ-dependent manner (15). Moreover, combined administration of IL-12 and IL-18 induced acute lethal inflammation within both the small and large intestines in an IFN-γ-dependent manner (16). Furthermore, we observed that IFN-γ knockout (KO)5 mice exhibited less neutrophil infiltration and tissue damage in acute hepatic inflammation caused by Propionibacterium acnes and acetaminophen (17, 18). These observations prompted us to evaluate the roles of IFN-γ in the pathogenesis of toxin A-induced acute enteritis. In this report, we demonstrate that IFN-γ KO mice exhibit attenuated enteritis, as evidenced by reduced fluid secretion and pathological changes. We will discuss the potential roles of IFN-γ in the pathogenesis of toxin A-induced acute enteritis.

Toxin A was purified to homogeneity from the dialysis culture filtrate of C. difficile strain VPI 10463 using sequentially bovine thyroglobulin affinity and anion exchange chromatograph columns as described previously (19). The following mAbs and polyclonal Abs (pAbs) were used for immunohistochemical and immunofluorescence analyses: rat anti-mouse IFN-γ mAb (clone XMG 1.2; BD PharMingen, San Diego, CA); rabbit anti-myleoperoxidase (anti-MPO) pAb (Neomarkers, Fremont, CA); rat anti-mouse CD3 mAb (Dainippon Pharmaceutical, Osaka, Japan); cyanine dye 3 (cy3)-conjugated donkey anti-rabbit IgG pAb, cy3-conjugated donkey anti-rat IgG pAb, and FITC-conjugated donkey anti-rat IgG pAb (Jackson ImmunoResearch Laboratories, West Grove, PA); rat anti-mouse IFN-γ neutralizing mAb (clone R4-6A2; gift from Dr. H. Fujiwara, Osaka University School of Medicine, Osaka, Japan); and recombinant murine IFN-γ and murine TNF-α (PeproTech, London, U.K.).

Pathogen-free, 8- to 12-wk-old male BALB/c mice were obtained from Sankyo Laboratories (Tokyo, Japan) and were designated WT mice in the following experiments. Age- and sex-matched IFN-γ KO mice, which were backcrossed to BALB/c mice for more than eight generations, were used in the experiments described in this report (17, 18). The animals were housed individually in cages under specific pathogen-free conditions during the whole course of the experiments. All animal experiments complied with the standards set out in the Guidelines for the Care of Laboratory Animals of Kanazawa University.

Mice were fasted overnight, but allowed access to water ad libitum. After mice were deeply anesthetized with i.p. administration of pentobarbital (50 μg/kg), toxin A was administered as described previously (20, 21). Briefly, after a midline laparotomy, two 4-cm ileal loops were ligated and injected with 200 ng in 0.2 ml of PBS, (pH 7.2) or the same volume of PBS as a vehicle. The abdomen was closed, and the animals were allowed to regain consciousness. At the indicated time intervals after toxin A or a vehicle injection, mice were euthanized with an overdose of diethyl ether, and the ileal loops were removed for subsequent analyses. The loop length and weight were measured, and fluid secretion was assessed as the loop weight (milligrams) divided by the length (centimeters). In another series of experiments, 250 μg of neutralizing rat anti-mouse IFN-γ mAb (clone R4-6A2) or the same amount of normal rat IgG was i.p. injected 1 h before toxin A treatment.

Resected ileal loops were fixed in 4% formaldehyde buffered with PBS and then embedded with paraffin. Deparaffinized 6-μm-thick sections were stained with H&E for histological analysis, and the tissue injuries were scored by an examiner without prior knowledge of the experimental procedures, as described previously (20, 21). Immunohistochemical analyses using anti-IFN-γ mAb (clone XMG 1.2) or anti-MPO pAb were performed as described previously (22). In some experiments the anti-IFN-γ mAb (1 μg/ml) was incubated with an equal volume of recombinant murine IFN-γ or TNF-α (40 μg/ml) at 4°C overnight before use.

Double-color immunofluorescence analysis was also performed to determine the types of IFN-γ-expressing cells in the ileum of mice treated with toxin A, as described previously (23). Briefly, deparaffinized sections were incubated with PBS containing 1% normal donkey serum and 1% BSA to reduce nonspecific reactions. The sections were then incubated with anti-MPO Ab or anti-CD3 Ab, followed by incubation with cy3-conjugated secondary Ab (15 μg/ml) at room temperature for 1 h. Thereafter, the sections were further incubated with FITC-conjugated anti-IFN-γ Ab at a concentration of 1 μg/ml at 4°C overnight, followed by observation under a fluorescence microscopy.

MPO activities were measured for evaluation of neutrophil recruitment into the tissues (22). Briefly, a portion of the resected ileum was freeze-dried and homogenized in 1 ml of 50 mM potassium phosphate buffer with 0.5% hexadecyl trimethyl ammonium bromide (Sigma-Aldrich, St. Louis, MO) and 5 mM EDTA. The tissues were disrupted with sonication for 20 s and three freeze-thaw cycles and were thereafter centrifuged at 12,000 × g at 4°C. MPO activities in the resultant supernatants were determined using a SUMILON peroxidase assay kit (Sumitomo Berkuraito, Tokyo, Japan) according to the manufacturer’s instructions. Human MPO (Biogenesis, Poole, U.K.) was used as a standard, and the data were expressed as units per dry weight (grams).

At the indicated time intervals after toxin A challenge, ileal samples were obtained and homogenized with 0.3 ml of PBS containing Complete Protease Inhibitor Mixture (Roche, Tokyo, Japan). The homogenates were centrifuged at 12,000 × g for 15 min. IFN-γ levels in the supernatant were measured with a commercial ELISA kit (BioSource, Camarillo, CA) according to the manufacturer’s recommendation. Total protein in the supernatant was measured with a commercial kit (bicinchoninic acid PROTEIN ASSAY KIT; Pierce, Rockford, IL). The data were expressed as picograms of IFN-γ per milligram of total protein for each sample.

A semiquantitative RT-PCR was conducted as described previously (22). Total RNAs were extracted from the removed ileal tissues using ISOGENE (Nippon Gene, Toyama, Japan) according to the manufacturer’s instructions. Five micrograms of total RNA was reverse transcribed at 42°C for 1 h in 20 μl of reaction mixture containing mouse Moloney leukemia virus reverse transcriptase (Toyobo, Osaka, Japan) with oligo(dT) primers (Amersham Pharmacia Biotech Japan, Tokyo, Japan). Thereafter, the resultant cDNA was amplified together with Taq polymerase (Nippon Gene, Toyama, Japan) using the specific sets of primers shown in Table I. PCR of each molecule was conducted with the optimal numbers of cycles consisting of 94°C for 1 min, optimal annealing temperature (Table I) for 1 min, and 72°C for 1 min, followed by incubation at 72°C for 3 min. The generated PCR products did not reach a saturable level with the determined optimal cycle numbers. The amplified PCR products were fractionated on a 2% agarose gel and visualized by ethidium bromide staining. The band intensities were measured using NIH Image Analysis software version 1.61 (National Institutes of Health, Bethesda, MD), and the ratio of each band to β-actin was calculated.

Table I.

Sequences of the primers used for RT-PCRa

TranscriptSequenceAnnealing Temperature (°C)CycleProduct Size (bp)
IFN-γ (F) 5′-AGCGGCTGACTGAACTCAGATTGTAG-3′ 60 30 247 
 (R) 5′-GTCACAGTTTTCAGCTGTATAGGG-3′    
     
IL-12p35 (F) 5′-AACAAGAGGGAGCTGCCTGCC-3′ 60 36 300 
 (R) 5′-CGGGTGCTGAAGGCGTGAAGC-3′    
     
IL-12p40 (F) 5′-CGTGCTCATGGCTGGTGCAAAG-3′ 55 36 576 
 (R) 5′-GAACACATGCCCACTTGCTG-3′    
     
IL-18 (F) 5′-CGTGCTCATGGCTGGTGCAAAG-3′ 55 32 434 
 (R) 5′-GAACACATGCCCACTTGCTG-3′    
     
TNF-α (F) 5′-CAGCCTCTTCTCATTCCTGCTTGTG-3′ 62 32 511 
 (R) 5′-CTGGAAGACTCCTCCCAGGTATAT-3′    
     
MIP-2 (F) 5′-GAACAAAGGCAAGGCTAACTGA-3′ 59 34 204 
 (R) 5′-AACATAACAACATCTGGGCAAT-3′    
     
KC (F) 5′-GGATTCACCTCAAGAACATCCAGAG-3′ 62 28 454 
 (R) 5′-CACCCTTCTACTAGCACAGTGGTTG-3′    
     
MIP-1α (F) 5′-GCCCTTGCTGTTCTTCTCTGT-3′ 60 32 258 
 (R) 5′-GGCAATCAGTTCCAGGTCAGT-3′    
     
ICAM-1 (F) 5′-GGAGCAAGACTGTGAACACG-3′ 60 36 435 
 (R) 5′-GAGAACCACTGCTAGTCCAC-3′    
     
β-Actin (F) 5′-TTCTACAATGAGCTGCGTGTGGC-3′ 62 26 456 
 (R) 5′-CTCATAGCTCTTCTCCAGGGAGGA-3′    
TranscriptSequenceAnnealing Temperature (°C)CycleProduct Size (bp)
IFN-γ (F) 5′-AGCGGCTGACTGAACTCAGATTGTAG-3′ 60 30 247 
 (R) 5′-GTCACAGTTTTCAGCTGTATAGGG-3′    
     
IL-12p35 (F) 5′-AACAAGAGGGAGCTGCCTGCC-3′ 60 36 300 
 (R) 5′-CGGGTGCTGAAGGCGTGAAGC-3′    
     
IL-12p40 (F) 5′-CGTGCTCATGGCTGGTGCAAAG-3′ 55 36 576 
 (R) 5′-GAACACATGCCCACTTGCTG-3′    
     
IL-18 (F) 5′-CGTGCTCATGGCTGGTGCAAAG-3′ 55 32 434 
 (R) 5′-GAACACATGCCCACTTGCTG-3′    
     
TNF-α (F) 5′-CAGCCTCTTCTCATTCCTGCTTGTG-3′ 62 32 511 
 (R) 5′-CTGGAAGACTCCTCCCAGGTATAT-3′    
     
MIP-2 (F) 5′-GAACAAAGGCAAGGCTAACTGA-3′ 59 34 204 
 (R) 5′-AACATAACAACATCTGGGCAAT-3′    
     
KC (F) 5′-GGATTCACCTCAAGAACATCCAGAG-3′ 62 28 454 
 (R) 5′-CACCCTTCTACTAGCACAGTGGTTG-3′    
     
MIP-1α (F) 5′-GCCCTTGCTGTTCTTCTCTGT-3′ 60 32 258 
 (R) 5′-GGCAATCAGTTCCAGGTCAGT-3′    
     
ICAM-1 (F) 5′-GGAGCAAGACTGTGAACACG-3′ 60 36 435 
 (R) 5′-GAGAACCACTGCTAGTCCAC-3′    
     
β-Actin (F) 5′-TTCTACAATGAGCTGCGTGTGGC-3′ 62 26 456 
 (R) 5′-CTCATAGCTCTTCTCCAGGGAGGA-3′    
a

(F), forward primer; (R), reverse primer.

Peritoneal neutrophils were isolated as described previously (24). Briefly, WT mice were i.p. injected with 2 ml of 3% proteose peptone (Difco, Detroit, MI) in MEM at 8 h before i.p. administration with toxin A or vehicle. Four hours after toxin A or vehicle treatment, the peritoneal cavity of mice was washed with MEM, and the abdominal lavage fluid was collected. Thereafter, polymorphonuclear neutrophils were isolated from the pellets of the abdominal lavage by gradient density centrifugation with Percoll (50/75%). The purity of the isolated neutrophils was >95%, as assessed by microscopic examination with Giemsa staining (data not shown). Gene expression of IFN-γ was examined on total RNAs extracted from the obtained neutrophils by RT-PCR, as mentioned above.

The means and SEMs were calculated for all parameters determined in this study. Statistical significance was evaluated using ANOVA or Mann-Whitney’s U test. A value of p < 0.05 was accepted as significant.

Under the conditions used, IFN-γ mRNA was faintly detected in the ileum of untreated WT mice (Fig. 1,A). Toxin A, but not vehicle, treatment markedly enhanced IFN-γ mRNA expression in the ileum by 2 h after the treatment. Moreover, the gene expression of IL-12 and IL-18 was up-regulated in the ileum of WT mice 1 and/or 2 h after toxin A treatment, but not after vehicle treatment (Fig. 1,A). Consistently, IFN-γ protein levels were significantly increased 2 h after toxin A challenge, but not after vehicle treatment, and reached the maximum level at 4 h (Fig. 1,B). At 2 h after toxin A treatment, the ileal structure was moderately disrupted, with some infiltration of polymorphonuclear leukocytes (Fig. 2,B); at 4 h after toxin A injection, the histopathological changes were more evident, with a massive infiltration of polymorphonuclear leukocytes (Fig. 2,C). Consistently, MPO activities in the ileal tissues were increased progressively after toxin A treatment (Fig. 1,C). IFN-γ protein was faintly detected immunohistochemically in the lamina propria and submucosa of untreated WT mice (Fig. 3, A and B). Later than 2 h after toxin A injection, immunoreactive IFN-γ was detected mainly in polymorphonuclear leukocytes (Fig. 3, C–F). No immunoreactivities were observed when the isotype-matched control Ab was used (data not shown). Moreover, preadsorption of the Ab with an excess amount of IFN-γ, but not TNF-α, abrogated positive signals (Fig. 3, G and H), indicating the specificity of the reactions. A double-color immunofluorescence analysis detected immunoreactive IFN-γ in some of the CD3-positive cells in the lamina propria and submucosa of the ileum of untreated WT mice (Fig. 4,A). At 4 h after toxin A treatment, IFN-γ proteins were detected in MPO-positive infiltrating neutrophils and, to a lesser degree, in CD3-positive cells within the mucosa (Fig. 4, B and C). These observations implied that neutrophils were a main cellular source of IFN-γ during the course of toxin A-induced enteritis. This idea was further supported by the observation that i.p. injection of toxin A could enhance IFN-γ mRNA expression in neutrophils (Fig. 5).

FIGURE 1.

A, The gene expression of IFN-γ, IL-12p35, IL-12p40, and IL-18 in the ileum of WT mice. RT-PCR was performed on total RNAs extracted from the ileum 1, 2, and 4 h after toxin A challenge as described in Materials and Methods. Representative results from six independent experiments are shown. The ratio of each cytokine to β-actin was calculated. Each value represents the mean ± SEM (n = 6 animals). ∗, p < 0.05; ∗∗, p < 0.01 (vs nontreated). B, The protein levels of IFN-γ in the ileum of WT mice were determined as described in Materials and Methods. Each value represents the mean ± SEM (n = 6 animals). ∗∗, p < 0.01 (vs nontreated). C, MPO activity in the ileum 2 and 4 h after toxin A treatment. Each value represents the mean ± SEM (n = 6 animals). ∗∗, p < 0.01 (vs nontreated).

FIGURE 1.

A, The gene expression of IFN-γ, IL-12p35, IL-12p40, and IL-18 in the ileum of WT mice. RT-PCR was performed on total RNAs extracted from the ileum 1, 2, and 4 h after toxin A challenge as described in Materials and Methods. Representative results from six independent experiments are shown. The ratio of each cytokine to β-actin was calculated. Each value represents the mean ± SEM (n = 6 animals). ∗, p < 0.05; ∗∗, p < 0.01 (vs nontreated). B, The protein levels of IFN-γ in the ileum of WT mice were determined as described in Materials and Methods. Each value represents the mean ± SEM (n = 6 animals). ∗∗, p < 0.01 (vs nontreated). C, MPO activity in the ileum 2 and 4 h after toxin A treatment. Each value represents the mean ± SEM (n = 6 animals). ∗∗, p < 0.01 (vs nontreated).

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FIGURE 2.

Histopathological (A–C and E; H&E staining) and immunohistochemical (D and F; anti-MPO pAb) observations of the ileum (magnification, ×100) from WT (A–D) and IFN-γ KO (E and F) mice at 0 h (A), 2 h (B), and 4 h after toxin A (C–F). Representative results from six animals are shown. Histopathological severity, including epithelial damage (G) and congestion and edema (H), was scored in WT (□) and IFN-γ KO (▪) mice as described previously (2021 ). I, MPO activity was measured in WT (□) and IFN-γ KO mice (▪) at the indicated time points after toxin A challenge, as described in Materials and Methods. All values represent the mean ± SEM (n = 6 animals). ∗∗, p < 0.01 (vs toxin A-treated WT mice).

FIGURE 2.

Histopathological (A–C and E; H&E staining) and immunohistochemical (D and F; anti-MPO pAb) observations of the ileum (magnification, ×100) from WT (A–D) and IFN-γ KO (E and F) mice at 0 h (A), 2 h (B), and 4 h after toxin A (C–F). Representative results from six animals are shown. Histopathological severity, including epithelial damage (G) and congestion and edema (H), was scored in WT (□) and IFN-γ KO (▪) mice as described previously (2021 ). I, MPO activity was measured in WT (□) and IFN-γ KO mice (▪) at the indicated time points after toxin A challenge, as described in Materials and Methods. All values represent the mean ± SEM (n = 6 animals). ∗∗, p < 0.01 (vs toxin A-treated WT mice).

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FIGURE 3.

Immunohistochemical analysis using an anti-IFN-γ mAb in the ileum of WT mice at 0 h (A and B), 2 h (C and D), and 4 h (E–H) after toxin A challenge. Representative results from six independent experiments are shown (magnification: A, C, and E, ×100; B, D, and F, ×400). In the nontreated ileum, mononuclear cells in the lamina propria and submucosa were faintly immunostained with anti-IFN-γ mAb. Polymorphonuclear leukocytes showed immunopositive reactions for IFN-γ 2 and 4 h after toxin A treatment. G and H, A preadsorption test was performed to confirm the specificity of the anti-IFN-γ mAb in the serial sections of the ileum 4 h after toxin A treatment (magnification, ×100). After the adsorption with recombinant murine IFN-γ, immunopositive reactions were abolished (H), compared with adsorption with murine TNF-α (G).

FIGURE 3.

Immunohistochemical analysis using an anti-IFN-γ mAb in the ileum of WT mice at 0 h (A and B), 2 h (C and D), and 4 h (E–H) after toxin A challenge. Representative results from six independent experiments are shown (magnification: A, C, and E, ×100; B, D, and F, ×400). In the nontreated ileum, mononuclear cells in the lamina propria and submucosa were faintly immunostained with anti-IFN-γ mAb. Polymorphonuclear leukocytes showed immunopositive reactions for IFN-γ 2 and 4 h after toxin A treatment. G and H, A preadsorption test was performed to confirm the specificity of the anti-IFN-γ mAb in the serial sections of the ileum 4 h after toxin A treatment (magnification, ×100). After the adsorption with recombinant murine IFN-γ, immunopositive reactions were abolished (H), compared with adsorption with murine TNF-α (G).

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FIGURE 4.

A double-color immunofluorescence analysis was performed for the determination of the IFN-γ-expressing cell types, as described in Materials and Methods. Representative results are shown. A, Untreated ileum (magnification, ×200); B and C, 4 h after toxin A treatment (magnification, ×100).

FIGURE 4.

A double-color immunofluorescence analysis was performed for the determination of the IFN-γ-expressing cell types, as described in Materials and Methods. Representative results are shown. A, Untreated ileum (magnification, ×200); B and C, 4 h after toxin A treatment (magnification, ×100).

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FIGURE 5.

Gene expression of IFN-γ in peritoneal neutrophils after toxin A treatment. Neutrophils were isolated as described in Materials and Methods. IFN-γ mRNA was significantly enhanced in the neutrophils of WT mice, which were i.p. administered with toxin A.

FIGURE 5.

Gene expression of IFN-γ in peritoneal neutrophils after toxin A treatment. Neutrophils were isolated as described in Materials and Methods. IFN-γ mRNA was significantly enhanced in the neutrophils of WT mice, which were i.p. administered with toxin A.

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To elucidate the pathophysiological roles of IFN-γ in toxin A-induced enteritis, we administered toxin A to WT and IFN-γ KO mice. There were no significant changes in terms of fluid accumulation up to 4 h after treatment between vehicle-treated WT and IFN-γ KO mice (Fig. 6). However, at 4 h after toxin A treatment, severe epithelial erosion and loss of crypt architecture were observed in WT mice (Fig. 2,C). Concomitantly, fluid accumulation was increased, with enhanced epithelial damage and edema, in WT mice (Figs. 2,C and 6). Moreover, a large number of infiltrating neutrophils were detected, as evidenced by histological analysis as well as an increase in MPO activity (Fig. 2,D), but F4/80-positive macrophages were scarcely detected in the ileum of WT mice during the whole course of the experiment (data not shown). In contrast, the increase in fluid accumulation was marginal in IFN-γ KO mice even 4 h after toxin A treatment (Fig. 6). Moreover, epithelial damage, congestion, edema, and neutrophil infiltration were markedly attenuated in IFN-γ KO mice compared with WT mice (Fig. 2, E–H). Consistent with histopathological changes, the increase in MPO activity was remarkably reduced in KO mice compared with WT mice 4 h after toxin A injection (Fig. 2 I). These observations implied that IFN-γ deficiency attenuated toxin A-induced pathological changes in ileal loops.

FIGURE 6.

Fluid accumulation in the ileal loops of WT and IFN-γ KO mice injected with toxin A or vehicle. Ileal loops were made, injected with toxin A or vehicle as described in Materials and Methods, and removed 4 h after the injection. Fluid accumulation was measured as the weight-to-length ratio (□, WT; ▪, IFN-γ KO). All values represent the mean ± SEM (n = 6 animals). ∗, p < 0.05 vs toxin A-treated WT mice.

FIGURE 6.

Fluid accumulation in the ileal loops of WT and IFN-γ KO mice injected with toxin A or vehicle. Ileal loops were made, injected with toxin A or vehicle as described in Materials and Methods, and removed 4 h after the injection. Fluid accumulation was measured as the weight-to-length ratio (□, WT; ▪, IFN-γ KO). All values represent the mean ± SEM (n = 6 animals). ∗, p < 0.05 vs toxin A-treated WT mice.

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We next examined the gene expression of TNF-α, chemokines, and ICAM-1, which are presumed to be involved in neutrophil infiltration, in the ileum of both WT and IFN-γ KO mice after toxin A treatment. Under the conditions used, the gene expression of TNF-α, macrophage inflammatory protein-1α (MIP-1α), MIP-2, KC, and ICAM-1 was only faintly detected in the ileum of both WT and IFN-γ KO mice (Fig. 7). Vehicle treatment failed to enhance the expression of these genes in both WT and IFN-γ KO mice (data not shown). The gene expression of these molecules was markedly enhanced in the ileum of WT mice >2 h after toxin A treatment (Fig. 7). This enhanced gene expression was remarkably attenuated in IFN-γ KO mice compared with WT mice (Fig. 7). These results indicate that the absence of IFN-γ leads to diminished expression of these molecules, thereby reducing leukocyte infiltration.

FIGURE 7.

Gene expression for TNF-α, chemokines, and ICAM-1 in the ileum of WT and IFN-γ KO mice. RT-PCR was performed on total RNAs extracted from the ileum at the indicated time intervals after toxin A or vehicle treatment as described in Materials and Methods. Representative results from six independent experiments are shown here. The ratios of TNF-α, MIP-2, KC, MIP-1α, and ICAM-1 to β-actin of WT (□) and IFN-γ KO (▪) mice were calculated. All values represent the mean ± SEM (n = 6 animals). ∗, p < 0.05; ∗∗, p < 0.01 (vs toxin A-treated WT mice).

FIGURE 7.

Gene expression for TNF-α, chemokines, and ICAM-1 in the ileum of WT and IFN-γ KO mice. RT-PCR was performed on total RNAs extracted from the ileum at the indicated time intervals after toxin A or vehicle treatment as described in Materials and Methods. Representative results from six independent experiments are shown here. The ratios of TNF-α, MIP-2, KC, MIP-1α, and ICAM-1 to β-actin of WT (□) and IFN-γ KO (▪) mice were calculated. All values represent the mean ± SEM (n = 6 animals). ∗, p < 0.05; ∗∗, p < 0.01 (vs toxin A-treated WT mice).

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To exclude the possibility that secondary defects arising from IFN-γ gene deficiency account for the observed phenotypes in this model, we examined the effects of an anti-IFN-γ mAb on toxin A-induced enteritis. When WT mice were administered an anti-IFN-γ mAb 1 h before toxin A treatment, fluid accumulation was markedly reduced, with attenuated histopathological changes, compared with mice treated with control IgG (Fig. 8). These observations implied that the lack of IFN-γ accounted for the attenuated enteritis in toxin A-induced IFN-γ KO mice.

FIGURE 8.

A, Fluid accumulation in the ileal loops of WT mice treated with control IgG (□) or anti-IFN-γ Ab (▪) at 4 h after toxin A challenge. All values represent the mean ± SEM (n = 6 animals). ∗, p, < 0.05, mice treated with anti-IFN-γ Ab vs control mice. Histopathological views are shown of WT mice treated with control IgG (B) or anti-IFN-γ Ab (C) 4 h after toxin A challenge. Representative results from six independent experiments are presented (magnification, ×100).

FIGURE 8.

A, Fluid accumulation in the ileal loops of WT mice treated with control IgG (□) or anti-IFN-γ Ab (▪) at 4 h after toxin A challenge. All values represent the mean ± SEM (n = 6 animals). ∗, p, < 0.05, mice treated with anti-IFN-γ Ab vs control mice. Histopathological views are shown of WT mice treated with control IgG (B) or anti-IFN-γ Ab (C) 4 h after toxin A challenge. Representative results from six independent experiments are presented (magnification, ×100).

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Our current understanding of antibiotic-associated pseudomembranous colitis is that antibiotic therapy can disturb the normal colonic microflora to allow colonization by pathogenic bacteria, particularly C. difficile, which produces two toxins, A and B (1, 2, 3, 4). When injected into animal ileal loops, toxin A elicits fluid secretion, increased mucosal permeability, and a marked inflammatory response, whereas toxin B does not cause intestinal inflammation or alter intestinal permeability (4, 5). Toxin A-induced enteritis develops with massive neutrophil infiltration that occurs within a few hours after it is injected into ileal loops (10). Moreover, fluid secretion and ileal inflammation was less evident in rats, which were rendered neutropenic, compared with untreated ones (25). Thus, toxin A-induced enteritis represents neutrophil-dependent acute inflammation.

Accumulating evidence indicates that IFN-γ has a central role in the Th1 immune response and, eventually, in Th1-mediated immune diseases (12). Several lines of evidence suggest the potential role of IFN-γ in neutrophil-dependent acute inflammation models, including S. aureus-induced acute empyema, P. acnes-induced hepatic granuloma formation, and acetaminophen-induced acute liver injury (15, 17, 18). Consistent with these observations, IFN-γ gene deficiency or deletion of IFN-γ activity by a neutralizing Ab reduced neutrophil infiltration and ileal tissue damage even in this acute inflammation model.

Accumulating evidence demonstrated that IFN-γ could be produced by cells other than lymphocytes and NK cells (26). We observed that i.p. injection of toxin A could augment IFN-γ mRNA in peritoneal neutrophils, suggesting that neutrophils have the capacity to produce IFN-γ. Moreover, the combined stimulation of IL-12 and IL-18 can induce IFN-γ production in macrophages (27, 28, 29), and TNF-α can induce human neutrophils to produce IFN-γ in vitro (30). Furthermore, the gene expression of IL-12 and IL-18 was enhanced in the ileum after toxin A treatment, preceding the enhancement of IFN-γ gene expression. Collectively, early in toxin A-induced enteritis, IFN-γ might be derived from CD3-positive lamina propria cells, but with the development of enteritis, infiltrated neutrophils started to produce a massive amount of IFN-γ, probably in the presence of IL-12 and IL-18.

Toxin A treatment induced the production of IL-8, MIP-2, and MIP-1α, chemokines with potent neutrophil chemotactic activities, by enterocytes or macrophages (11, 20, 21, 31, 32). Moreover, several lines of evidence identified MIP-2 and MIP-1α as chemokines crucially involved in neutrophil infiltration and eventual tissue destruction in toxin A-induced enteritis models (20, 21). In the ileum of toxin A-challenged IFN-γ KO mice, the gene expression of MIP-2, MIP-1α, and KC was significantly reduced compared with that in WT mice. Several lines of evidence suggest that IFN-γ can inhibit the production of neutrophil chemotactic chemokines, including MIP-2, IL-8, and MIP-1α (33, 34, 35). However, a number of independent groups, including ours, reported that IFN-γ and TNF-α synergistically induced IL-8 or KC production by gastric or colonic epithelial cell lines (36, 37, 38). In this toxin A-induced enteritis, TNF-α expression was up-regulated in the ileum of WT mice, consistent with previous reports. Moreover, the enhanced TNF-α gene expression was attenuated in IFN-γ KO mice challenged with toxin A. Thus, in this toxin A-induced enteritis, locally produced IFN-γ and TNF-α synergistically augmented the expression of these chemokines and eventually induced neutrophil infiltration.

The interaction between CD18 and ICAM-1 has been shown to be essential for neutrophil infiltration in toxin A-induced enteritis, as evidenced by the effects of Abs to these adhesion molecules on this enteritis model (39, 40). Moreover, several lines of evidence have demonstrated that IFN-γ enhances ICAM-1 expression on intestinal epithelial cells (41, 42, 43, 44, 45). In line with this observation, we observed that ICAM-1 expression was enhanced in the ileum of WT, but not IFN-γ KO, mice treated with toxin A. Thus, IFN-γ may enhance the expression of ICAM-1, thereby inducing neutrophil transmigration. Furthermore, IFN-γ can augment neutrophil transmigration through a monolayer of the human intestinal cell line, T84, in vitro (46). Taken collectively, locally produced IFN-γ may enhance neutrophil transmigration directly and/or indirectly by enhancing chemokine production and ICAM-1 expression.

Increased intestinal epithelial permeability is a functional hallmark of toxin A-induced enteritis (10). In vitro, IFN-γ can modulate intestinal epithelial permeability by loosening tight junctions while not causing epithelial cell necrosis (47). In line with these previous in vitro observations, IFN-γ KO mice did not exhibit increased fluid secretion after toxin A treatment. IFN-γ can enhance inducible NO synthase (iNOS) expression in various types of cells and eventually NO production (48). Although NO is a potent vasodilator, the effect of NO on intestinal epithelial permeability is still controversial (49, 50). Toxin A augmented iNOS expression in WT and IFN-γ KO mice to similar levels (data not shown). Thus, it is probable that IFN-γ may modulate intestinal epithelial permeability directly, but not indirectly, through iNOS-NO pathway.

In this study, we provided evidence on the essential roles of IFN-γ in the pathogenesis of toxin A-induced enteritis. We have further demonstrated that the administration of a neutralizing anti-IFN-γ Ab prevented the development of toxin A-induced enteritis. Thus, IFN-γ might be a good molecular target to prevent and/or treat C. difficile-associated colitis.

We express our gratitude to Dr. Howard Young (National Cancer Institute, Frederick, MD) for his thoughtful review of the manuscript, and to Dr. H. Fujiwara (Department of Oncology, Biomedical Research Center, Osaka University Graduate School of Medicine, Osaka, Japan) for his kind gift of neutralizing rat anti-murine IFN-γ mAb. We also thank Dr. A. Matsukawa (Department of Pathology, Kumamoto University School of Medicine, Kumamoto, Japan) for his instructive advice on the isolation of murine neutrophils.

1

This work was supported in part by grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and the Yakult Bio-Science Foundation.

5

Abbreviations used in this paper: KO, knockout; cy3, cyanine dye 3; iNOS, inducible NO synthase; MIP, macrophage inflammatory protein; MPO, myeloperoxidase; pAb, polyclonal Ab; WT, wild-type.

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