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. 2006 Dec 1;119(4):479–487. doi: 10.1111/j.1365-2567.2006.02459.x

Glucocorticoid-induced tumour necrosis factor receptor family-related receptor signalling exacerbates hapten-induced colitis by CD4+ T cells

Sun K Lee 1, Beom K Choi 1, Young H Kim 1, Woo J Kang 1, Kwang H Kim 1, Shimon Sakaguchi 2, Jae H Suh 3, Tae Y Kim 1, Byoung S Kwon 1,4
PMCID: PMC2265816  PMID: 17177830

Abstract

The glucocorticoid-induced tumour necrosis factor receptor family related gene (GITR) has been reported to be expressed on the activated T and CD4+CD25+ regulatory T cells (Treg). Signalling triggered by GITR not only neutralizes the suppressive effect of Treg cells, but also augments activation, proliferation and cytokine production of effector T cells. To test the role of GITR in 2,4,6-trinitrobenzene sulphonic acid (TNBS)-induced colitis − a murine model of mucosal inflammation − TNBS-injected Balb/c mice were treated with agonistic anti-GITR monoclonal antibody (mAb). Anti-GITR treatment increased the death rate compared to rat IgG-treated mice. Typically, death occurred within 4 days after the TNBS injection when the mice were treated with anti-GITR. The mice that survived anti-GITR treatment suffered from severe inflammation in their entire intestines. CD4+ T-depletion protected the mice from colitis; even an anti-GITR effect was not apparent. In contrast, CD8+ T depletion showed less protective than did CD4+ T depletion. Stimulation of GITR enhanced the production of proinflammatory cytokines including interferon (IFN)-γ, tumour necrosis factor (TNF)-α, interleukin (IL)-6 and IL-12. It also enhanced the humoral response such as serum levels of IgG2b and IgA, which was completely dependent on CD4+ T cells. Taken together, this study demonstrated that GITR signalling on CD4+ T cells is involved in the development and progress of colitis by enhancing both T helper type 1 (Th1) and Th2 type responses.

Keywords: co-stimulation/co-stimulatory molecules, Crohn' disease, helper T cells, inflammatory bowel disease, T cells

Introduction

Crohn's disease (CD) and ulcerative colitis (UC) are the two major forms of chronic inflammatory bowel disease (IBD).1 Because mice raised in germ-free conditions did not develop IBD, impairment of the continuing innate and adaptive immune response to microbiota might result in IBD.2,3 Increasing evidence using dozens of experimental mouse models suggests that abnormal responses of mucosal T cells may have a pivotal role in the pathogenesis of IBD by secreting proinflammatory cytokines and chemokines, accumulating inflammatory cells and inducing tissue damage.4–8 In particular, CD4+ T cells reacting to the enteric microflora are the effector cells inducing IBD in most experimental models,9,10 and therefore depletion of CD4+ T cells clinically abrogates colitis.11 There are also several reports in animal models that an excessive amount of the T helper type 1 (Th1) response mediates CD and the Th2 response, such as natural killer T (NK T) cells producing interleukin (IL)-13 and Th2 CD4+ T cells, is responsible for an induction of Th2-like colitis in oxazolone-induced colitis and the T cell receptor (TCR)α-deficient model, respectively.12–15

Studies have reported that signals through co-stimulatory molecules are required to regulate T lymphocyte activation, survival and differentiation.16 Among the co-stimulatory molecules, the tumour necrosis factor (TNF) receptor and ligand family have been investigated extensively due to their regulatory functions of the immune response.16 One of the TNF receptor (TNFR) and ligand family, the glucocorticoid-induced TNFR family-related gene (GITR), was cloned from the dexamethasone-treated 3DO T cell line.17 GITR is expressed on spleen and lymph node cells including CD4+ and CD8+ T cells at a low level, and CD4+CD25+ regulatory T cells (Tregs) could be induced upon T cell activation.18 Signalling through GITR can co-stimulate the effector T cells following stimulation of the TCR/CD3 complex and also neutralize the suppressive activity of Tregs.19,20

Activation of GITR signalling by anti-GITR monoclonal antibody (mAb) exacerbated murine asthma and collagen-induced arthritis (CIA) and markedly enhanced the induction of both Th1 and Th2 cytokine production.21 Therefore, GITR seemed to play proinflammatory roles in both Th1- and Th2-mediated inflammatory diseases. However, due to the unique properties of the immune system in the intestine, it was not clear whether GITR signalling would exert similar responses during intestinal inflammation. To clarify the role of GITR in intestinal inflammation, Balb/c mice were injected intrarectally with 2,4,6-trinitrobenzene sulphonic acid (TNBS) to induce Th1-type colitis and were treated with agonistic anti-GITR mAb. To determine the function of GITR on CD4+ and CD8+ T cells in vivo, colitis-induced mice were treated with depleting anti-CD4 or anti-CD8 mAb as well as anti-GITR mAb. We suggest here that GITR signalling deteriorates TNBS-induced colitis by enhancing both Th1- and Th2-type responses, which is mediated mainly through CD4+ T cells.

Materials and methods

Mice and hybridoma

Female BALB/c mice were obtained from Jackson Laboratory (Bar Harbor, ME). All mice were maintained under specific pathogen-free conditions in the animal facility of the Immunomodulation Research Center (University of Ulsan) and were used at 12–14 weeks of age. Anti-GITR (DTA-1), anti-CD4 [GK1·5; American Type Culture Collection (ATCC), Manassas, VA] and anti-CD8 (2·43; ATCC) mAbs were purified from the culture supernatant by protein G column (Sigma-Aldrich, St Louis, MO).

Induction of colitis

Mice were given a solution of TNBS (Sigma-Aldrich) dissolved in a mixture of phosphate-buffered saline (PBS), pH 7·2, and then mixed with an equal volume of ethanol for a final concentration of 2% TNBS in 50% ethanol. TNBS was administered intrarectally (i.r.) to the anesthetized mice via a 3·5 French (F) catheter equipped with a 1 ml syringe. The catheter was advanced into the rectum until the tip was 4 cm proximal to the anal verge, at which time TNBS was administered at a total volume of 100 µl (40 µg TNBS/g body weight). Mice were held in a vertical position for 30 seconds after the intrarectal injection to ensure equal distribution of TNBS within the overall colon and caecum.

Treatment with monoclonal antibody and depletion antibody

Mice were injected intraperitoneally with 500 µg anti-GITR mAb or with control rat IgG on post-injection (PI) days 0 and 3. To deplete CD4+ or CD8+ T cells, mice were injected with 400 µg of anti-CD4 mAb or anti-CD8 mAb on days − 1, 4 and 8 after TNBS administration.

Flow cytometry of GITR

To determine GITR expression on CD4+ and CD8+ T cells in vitro, lymph node (LN) cells were isolated from Balb/c mice and stimulated with 0·5 µg of anti-CD3 (145–2C11; PharMingen, San Diego, CA). The activated cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-GITR and phycoerythrin (PE)-conjugated anti-CD4 or anti-CD8 mAb at 0, 24 and 48 hr. In a separate experiment, lymphocytes were isolated from LN or lamina propria as described previously,22 before or 5 days after the TNBS injection, and stained with FITC-conjugated anti-GITR, phycoerythrin-cychrome5 (PE-Cy5)-conjugated anti-CD4 and PE-conjugated anti-CD25 or anti-CD62L mAb. All samples were analysed subsequently on a fluorescence activated cell sorter (FACS)Caliber (Becton Dickinson, San Jose, CA).

Histological analysis

The colon was snap-frozen in isopentane (2-methylbutane, Sigma-Aldrich) and embedded in Tissue-Tek optimal cutting temperature (OCT) compound (Sakura Finetek USA, Inc., Torrance, CA) on liquid nitrogen. Six-mm cryostat sections were prepared, stained with haematoxylin and eosin and mounted in glycerol vinyl alcohol aqueous (GVA) mounting solution (Zymed, San Francisco, CA). Histological grading was performed blindly according to the previous report.23 In brief, crypt distortion was graded as none (0), focal (1) and diffuse (2). Crypt with loss of goblet cells was graded as none (0), a few crypts; < 3 crypts with loss of goblet cells per 10–12 crypts in a field (1), many crypts; > 3 crypts with loss of goblet cells per 10–12 crypts in a field (2). Chronic inflammation (infiltration of mononuclear cells in lamina propria) was graded as none (0), focal (1) and multifocal (2). These three histological gradings were performed 10 days after TNBS injection. Acute inflammation (ulcers with infiltration of polymorphonuclear leucocytes) was graded as none (0), superficial (1) and beyond the layer of muscularis mucosae (2) 5 days after TNBS injection. A maximum score of 8 indicated severe colitis with acute ulcers and an overall diffuse pattern of chronic changes.

Serum cytokines

Serum was collected from six groups of mice 5 days after TNBS injection. The amount of cytokines was quantified using the cytometric bead array kit (CBA; BD Biosciences, San Diego, CA) on a FACSCaliber cytometer equipped with CellQuestPro and CBA software according to manufacturer's instructions (BD Biosciences).

Measurement of serum IgG subclasses and faecal IgA

Serum and faecal extracts were collected from each group of mice 10 days after TNBS injection, and anti-trinitrophenyl residue (TNP) antibodies were assessed by an end-point dilution method. Enzyme-linked immunosorbent assay (ELISA) microtitre plates were coated with 10 µg/well TNP–bovine serum albumin (BSA) overnight at 4° and blocked by the addition of 100 µl/well of 4% BSA for 1 hr. For IgG subclasses, serum was diluted twofold and added to the microtitre plate. For IgA, faecal extracts were dissolved in PBS at 500 mg/ml of concentration, diluted twofold and added to the plate. Mouse IgG subclasses and IgA were detected by using an Immunopure Monoclonal Antibody Isotyping Kit I (Pierce, Rockford, IL). End-point titres were expressed as the reciprocal log2 of the final dilution that showed an optical density comparable with that of the serum or faecal extracts obtained from naive mice.

Statistical analysis

All data were analysed using a statistical program, Prism 4·0 GraphPad (San Diego, CA). Student's t-test was used to determine statistical significance between groups.

Results

GITR expression on CD4+ T cells of lymph nodes and lamina propria before and after TNBS administration

GITR is known to be expressed at low levels on naive CD4+ and CD8+ T cells and constitutively on naive CD4+CD25+ regulatory Tregs.18 Upon activation, CD4+ and CD8+ T cells up-regulate GITR expression on their surface and play proinflammatory roles in both Th1- and Th2-mediated inflammatory diseases.20,21 Before we assessed the role of GITR in the development of TNBS-induced colitis, we first determined the expression level of GITR on CD4+ and CD8+ T cells following TCR/CD3 complex ligation. As shown in Fig. 1(a), CD4+ and CD8+ T cells expressed GITR at a low level and induced on their surface following activation. GITR expression was relatively higher on activated CD4+ T cells compared to CD8+ T cells.

Figure 1.

Figure 1

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Glucocorticoid-induced tumour necrosis factor receptor family-related gene (GITR) induction on CD4+ T cells of mesenteric lymph nodes and lamina propria after 2,4,6-trinitrobenzene sulphonic acid (TNBS) administration. (a) Lymph node (LN) cells were prepared from naive Balb/c mice and stimulated with 0·5 µg/ml anti-CD3 monoclonal antibody (mAb). Cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-GITR and phycoerythrin (PE)-conjugated anti-CD4 or anti-CD8 mAb at the indicated time. (b, c) Lymphocytes were isolated from mesenteric LNs and lamina propria of Balb/c mice before (b) or 5 days after (c) TNBS injection, and stained with FITC-conjugated anti-GITR, PE-Cy5-conjugated anti-CD4, and PE-conjugated anti-CD25 or anti-CD62L. CD4+ T cells were gated and analysed for the expression of CD25 and GITR.

Because the immune response in the intestines is different from the peripheral immune system,24 we tested GITR expression of CD4+ T cells from mesenteric lymph nodes (MLNs) and lamina propria (LP) before and after TNBS administration. To discriminate Tregs from normal CD4+ T cells, MLNs and LP cells were stained with anti-CD25 or anti-CD62L mAb along with anti-GITR and anti-CD4 mAb. CD4+ T cells were gated and analysed for GITR as well as for CD25 or CD62L expression. GITR expression was restricted strictly to CD4+CD25+ T cells in MLNs, while there were many CD25–GITR+ CD4+ T cells in LP (Fig. 1b). In addition, most GITR+CD4+ T cells showed the CD62Lhigh phenotype in MLNs, but CD62low in LP (Fig. 1b). Naive Treg cells had CD62Lhigh phenotype, and activated CD4+ or Treg cells became CD62Llow.25 Therefore, although we observed different GITR expression on CD4+ T cells between MLNs and LP, they were different only in activation status, which was not due to the intrinsic difference in GITR expression. In supporting this idea, most of the CD4+ T cells in MLNs and LP induced GITR expression following TNBS injection irrelevant to CD25 or CD62L expression (Fig. 1c).

Therefore, we concluded that there were no intrinsic differences in the GITR expression of CD4+ T cells from MLNs and LP and CD4+ T cells induced GITR following TNBS administration.

GITR signalling exacerbates TNBS-induced colitis

To induce TNBS colitis, we injected i.r. 40 µg of TNBS/g body weight into Balb/c mice. As a control, we administered phosphate-buffered saline (PBS) or 50% ethanol (EtOH). The mice given 40 µg/g TNBS showed 50–60% mortality, but not the mice that received PBS or EtOH (Fig. 2a). For GITR stimulation, TNBS-treated mice were injected intraperitoneally (i.p.) with agonistic anti-GITR mAb or rat IgG as a control. Anti-GITR administration increased mortality to > 80% and also resulted in the shortening of the colon length (Fig. 2a,b). In particular, the majority of deaths by anti-GITR treatment occurred within 4 days after TNBS injection (data not shown). To determine the cause of death in anti-GITR-treated mice, we examined the colon of rat IgG- or anti-GITR-treated mice 5 days after the TNBS injection. Most mice that died within 5 days after the anti-GITR treatment showed a severe ulcer in the colon. Histologically, GITR triggering exacerbated TNBS-induced colitis by increasing the loss of goblet cells, infiltration of mononuclear and polymorphonuclear leucocytes, focal ulcers and the distortion of crypts (Fig. 2c). Although the increase of goblet loss and crypt distortion was not statistically significant, histological scores of acute and chronic inflammation were statistically significant (Fig. 2d).

Figure 2.

Figure 2

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Anti-glucocorticoid-induced tumour necrosis factor receptor family-related gene (GITR) stimulation deteriorates 2,4,6-trinitrobenzene sulphonic acid (TNBS)-induced colitis. Balb/c mice were received phosphate-buffered saline (PBS) intrarectally, 50% ethanol, or 40 µg TNBS/1 g body weight, and treated intraperitoneally with 500 µg anti-GITR monoclonal antibody (mAb) or rat IgG as a control on PI days 0 and 3. (a) Survival rate of TNBS colitis-induced mice on PI day 10. The plotted data are mean ± standard deviation from four separate experiments [10 mice per group for rat IgG- and anti-GITR-treated mice, three mice per group for PBS- or 50% ethanol (EtOH)-treated mice]. The numbers of the survived mice over the number of mice tested are shown in the graph. (b) Gross photographs of large intestine of rat IgG- and anti-GITR-treated mice on day 10 after TNBS administration. (c) Haematoxylin and eosin staining of the frozen sections of colon in each group on PI day 10. (d) Histological scores of rat IgG- or anti-GITR-treated mice.

Therefore, we concluded that GITR triggering increased the mortality of TNBS-injected mice by increasing both acute and chronic inflammation.

CD4+ T cells are responsible for anti-GITR-mediated exacerbation of colitis

TNBS-induced colitis has been shown to be a Th1-type of inflammation in SJL/J mice,26 while in Balb/c mice the Th1 responses were involved in acute focal lesions, and the Th2 response played a more important role in diffuse atrophic changes of crypts.27 CD4+ T cells were primarily responsible for the development of TNBS-induced colitis, but CD8+ T cells had a negligible role in TNBS-induced colitis.28 A recent report also showed that anti-GITR treatment could exacerbate significantly the severity and onset of both collagen-induced arthritis (CIA) and murine asthma.21 To examine the roles of CD4+ T and CD8+ T cells in anti-GITR-mediated exacerbation of the colitis, TNBS-induced mice were treated i.p. with anti-GITR mAb along with depleting anti-CD4 or anti-CD8 mAb. CD4+ T depletion (dCD4) increased slightly the survival rate of colitis-induced mice and CD8+ T depletion (dCD8) was also comparable with that of anti-CD4 treatment (Fig. 3a). Anti-GITR treatment exacerbated the TNBS-induced colitis in non-depleted or anti-CD8-treated mice, but not in anti-CD4-treated mice (Fig. 3a). Macroscopic examination of the intestines was also consistent with survival rate. The colon from anti-GITR- or anti-CD8 plus anti-GITR-treated mice was relatively shorter than that of other groups of mice and showed severe focal ulcers (data not shown).

Figure 3.

Figure 3

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CD4+ T cell depletion abolishes the effect of anti-glucocorticoid-induced tumour necrosis factor receptor family-related gene (GITR) treatment. To deplete CD4+ or CD8+ T cells, Balb/c mice were injected intraperitoneally with 400 µg anti-CD4 (dCD4) or anti-CD8 monoclonal antibody (mAb) (dCD8) on post-injection (PI) days − 1, 4 and 8. The mice were also received 40 µg 2,4,6-trinitrobenzene sulphonic acid (TNBS)/g body weight and treated intraperitoneally with rat IgG or anti-GITR mAb on PI day 0 and 3. (a) Survival rate of each group of mice on PI day 10. The plotted data are mean ± standard deviation from three separate experiments (seven mice per group for rat IgG- and anti-GITR-treated mice, 10 mice per group for anti-CD4- or anti-CD8-treated mice). The numbers of the survived mice over the number of mice tested are shown in the graph. (b) Histological scores of each group of mice. Grading was performed according to criteria described in method section.

For histological examination, five mice were selected randomly from each group of mice 10 days after TNBS injection and frozen sections were prepared from three randomly selected parts of each colon. Following haematoxylin and eosin (H&E) staining, the severity of TNBS-induced colitis in each colon was graded histologically, as described in the Methods section.23 Among the four parameters, which included crypt distortion, loss of goblet cells, acute inflammation and chronic inflammation, anti-GITR treatment notably increased acute inflammation in non-depleted and CD8+ T-depleted mice (Fig. 3c). CD4+ T-depletion again showed a low severity score even in anti-GITR-treated mice. In addition, the mice that survived anti-GITR treatment showed more severe chronic inflammation. We therefore concluded that anti-GITR treatment led to the increase of acute and chronic inflammation, and CD4+ T cells were primarily responsible for the anti-GITR-mediated deterioration of colitis.

GITR stimulation increases proinflammatory cytokines in CD4+ T-dependent manner

As GITR triggering deteriorated TNBS-induced colitis, we tested whether the proinflammatory cytokines were increased in anti-GITR-treated mice. Balb/c mice were given PBS, anti-CD4- or anti-CD8 mAb 1 day before TNBS injection as well as anti-GITR or rat IgG as a control. By collecting serum from each group of mice 5 days after TNBS injection, the amount of serum cytokines, including IL-6, IL-10, IFN-γ, TNF-α, IL-12p70 and monocyte chemotactic protein (MCP)-1, was determined by using a CBA kit (BD Biosciences).

GITR triggering increased the production of IL-6, IFN-γ, TNF-α and IL-12p70. CD8 T cell depletion did not significantly alter the anti-GITR-mediated induction of these cytokines, while CD4+ T cell depletion completely reversed cytokine production (Fig. 4). Because IL-6, TNF-α and IL-12p70 were reported as cytokines that could exacerbate TNBS-induced colitis,29–31 this result explained, at least partially, how GITR stimulation worsened TNBS-induced colitis. In addition, this result was consistent with the previous report that GITR stimulation could enhance both Th1- and Th2-type immune responses,21 because we also found an increase of Th1-type cytokines such as IFN-γ and IL-12p70 and Th2-type cytokines such as IL-6 by GITR triggering.32 Therefore, we concluded that anti-GITR treatment might promote the development and progress of TNBS-induced colitis by co-stimulating CD4+ T cells to increase the proinflammatory cytokines.

Figure 4.

Figure 4

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Enhanced cytokine production by anti-glucocorticoid-induced tumour necrosis factor receptor family-related gene (GITR) treatment in CD4+ T cells. Six groups of 2,4,6-trinitrobenzene sulphonic acid (TNBS)-injected mice were prepared by treating rat IgG or anti-GITR along with a depleting anti-CD4 or anti-CD8 monoclonal antibody (mAb) as described above. Serum were collected from each group of mice on post-injection day 5 and the amount of cytokines including interleukin (IL)-6, IL-10, monocyte chemoattractant protein-1, interferon-γ, tumour necrosis factor-α and IL-12-p70 was quantified using the cytometric bead array kit (CBA; BD Biosciences) on a fluorescence activated cell sorter (FACS)Caliber cytometer according to the manufacturer's instructions. The plotted data are mean ± standard deviation; n = 5 mice per group (*P < 0·05; **P < 0·01). The results are representative of two independent experiments.

Anti-GITR treatment enhances CD4+ T-dependent and antigen-specific humoral responses

Studies have reported that GITR triggering promotes both Th1- and Th2-type responses, and that the Th1-type response in TNBS-induced colitis is involved in acute inflammation and the Th2-type response plays a more important role in diffuse atrophic changes of crypts.21 As the humoral response is also involved in the progress of colitis,33 we questioned whether anti-GITR treatment enhanced the humoral immune response against TNBS. In particular, as IL-6 is able to promote Th2 differentiation by inducing IL-4 from naive CD4+ T cells,32 we measured the amount of trinitrophenyl (TNP)-specific serum IgG subclass and faecal IgA to characterize the humoral response against the given antigen. Six groups of mice were prepared as described above, and serum and faeces were collected from each group of mice 10 days after TNBS injection. The amount of antigen-specific IgG subclass and IgA was measured by using ELISA with a TNP–BSA-coated plate. Anti-GITR treatment did not significantly change TNP-specific IgG1 and IgG2a, but did increase TNP-specific IgG2b and IgA (Fig. 5a–d). Again, CD4+ T depletion completely reduced the expression of TNP-specific IgG1, IgG2b and IgA. CD4+ T depletion, however, marginally decreased IgG2a production, which implied that TNP-specific humoral responses were dependent on CD4+ T cells. CD8+ T depletion did not influence the effect of TNBS alone or anti-GITR treatment on the humoral response (Fig. 5a–d).

Figure 5.

Figure 5

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Enhanced humoral response by anti-glucocorticoid-induced tumour necrosis factor receptor family-related gene (GITR) treatment. Six groups of 2,4,6-trinitrobenzene sulphonic acid (TNBS)-injected mice were prepared by treating rat IgG or anti-GITR along with a depleting CD4+ T or CD8+ T cells as described above. Serum and faecal extracts were collected from each group of mice on post-injection day 10. Mouse IgG subclasses and IgA were determined by end-point dilution and expressed as the reciprocal log2 of the end-point titre. The plotted data are mean ± standard deviation; n = 5 mice per group (*P < 0·05; **P < 0·01). The results are representative of two independent experiments.

Taken together, we showed that the increased mortality of TNBS-injected mice by anti-GITR treatment was correlated with the enhanced production of TNP-specific IgG2b and IgA as well as IL-6, IFN-γ, TNF and IL-12p70, which appeared to be dependent on CD4+ T cells (Figs 4 and 5).

Discussion

Several lines of evidence have suggested that GITR plays a role in promoting Th1 and Th2 responses.19–21 In particular, it has been demonstrated clearly that agonistic anti-GITR treatment markedly enhances the expression of key Th1 (T-bet) and Th2 (GATA3) transcription factors in CD25–CD4+ T cells in vitro.21 Moreover, anti-GITR treatment exacerbates the murine model of arthritis and allergic airways inflammation, which implies that activation of GITR has profound effects on both Th1- and Th2-mediated disease in vivo.21 However, because a variety of cells express GITR, including Tregs, activated CD4+ and CD8+ T and NK T cells,18,34 and are spatially and timely different in GITR expression, the network of GITR–GITRL interactions becomes more complicated.

In the present study, we examined the function of GITR on TNBS-induced colitis by treating agonistic anti-GITR mAb. When we assessed GITR expression on Tregs and CD4+CD25– T cells before and after TNBS injection, we found that GITR was up-regulated on CD4+CD25– T cells following TNBS injection (Fig. 1c). CD8+ T cells of MLNs and intestinal epithelial lymphocytes also increased GITR expression, but it was a little lower than that of CD4+ T cells (data not shown). Therefore, we expected that anti-GITR mAb would stimulate GITR on the activated T cells. Consistent with previous reports,21 the same anti-GITR treatment resulted in a deterioration of TNBS-induced colitis (Fig. 2a). We noted that anti-GITR treatment increased swiftly the mortality of TNBS-injected mice within 4 days after TNBS injection, and it seemed to be due to the severe ulcers (Fig. 3c). Even after the mice survived the acute response to TNBS plus anti-GITR treatment, the surviving mice still showed a diffused chronic inflammation over the whole colon (Fig. 3c). Colitis induced by TNBS was known to be mediated by a Th1-type of inflammation in SJL/J mice.26 However, further study revealed that both Th1- and Th2-type cells contributed to the TNBS-induced colitis,6,33 in which Th1-like cytokines induced fatal, acute transmural and focal types of lesions, whereas Th2-like cytokines played a significant role in the diffuse atrophic changes in crypts and the mucosal layers. Therefore, we suspected that anti-GITR treatment might induce Th1- and Th2- responses.

In support of this hypothesis, the in vivo proliferation assay showed that anti-GITR treatment similarly promoted the proliferation of CD4+ and CD8+ T and B cells, although the proliferating rate was higher in CD4+ T and B cells compared to CD8+ T cells (data not shown). We also tested whether CD4+ T cells were more responsible than CD8+ T cells for the deterioration of colitis by depleting CD4+ or CD8+ T cells. CD4+ or CD8+ T depletion alone slightly increased the survival rate of colitis-induced mice, and the effect of anti-GITR treatment was abolished completely by depleting CD4+ T cells, but not by depleting CD8+ T (Fig. 3a). Because CD4+ T cells were known to mediate TNBS-induced colitis, it was reasonable to assume that the effects of anti-GITR treatment were dependent on CD4+ T cells. We administered anti-CD25 mAb to determine the role of Tregs, but the result was not conclusive, which seemed to be due to the co-elimination of activated CD4+ T cells expressing CD25 (data not shown). GITR signalling effectively neutralized the suppressive activity of naive Tregs, but not that of activated Tregs.35,36 Therefore, although GITR on naive Tregs might be involved in the initiation of TNBS-induced colitis, the activated Tregs following administration of TNBS appeared to have a limited role in the anti-GITR-mediated exacerbation of colitis.

Anti-GITR treatment increased the cytokine production that was involved in promoting the development and progress of TNBS-induced colitis such as IL-6, IFN-γ, TNF-α and IL-12p70, and these cytokines were reduced completely by depleting CD4+ T cells (Fig. 4). Anti-GITR treatment specifically enhanced the production of antigen-specific IgG2b and IgA, but not IgG1 or IgG2a, and the increased IgG2b and IgA production was also dependent on CD4+ T cells (Fig. 5). Previous studies have shown that cytokines such as transforming growth factor (TGF)-β, IL-5 and IL-6 are important factors for the development of IgA-producing B cells.37–39 Furthermore, CD11b+ Peyer's patch dendritic cells preferentially induced higher levels of IgA secretion from naive B cells in the dendritic cell (DC)–T–B cell co-culture system in vitro which was dependent on IL-6 production.39 Therefore, we suspected that the IL-6 production increased by anti-GITR treatment might be involved in enhanced IgA production. However, as IL-6 and IL-12p70 were known to be expressed from myeloid cells, including DCs and monocytes/macrophages, it remained to be elucidated whether anti-GITR treatment stimulated directly or activated indirectly myeloid cells to produce IL-6 or IL-12p70.

In summary, our study of experimental colitis with anti-GITR mAb shows that, by enhancing both Th1- and Th2-type responses, GITR activation is involved in the development and progress of colitis, which is mediated by CD4+ T cells. These results may contribute to future immunological interventions in the pathogenesis of Crohn's disease and ulcerative colitis. Alternatively, these results imply that the activation of GITR will be useful as an adjuvant to boost the effect of a vaccine that is designed to induce cellular and humoral immune responses.

Acknowledgments

This study was supported in part by NIH grant R01EY013325 (BSK), KRF-2005–201-E00008, KRF-2005–084-E00001, Korean Health R&D 21 no. A050260 and the SRC Fund to the Immunomodulation Research Center at the University of Ulsan from KOSEF and the Korean Ministry of Science and Technology.

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