Induction of IL-10-producing CD4+CD25+T cells in animal model of collagen-induced arthritis by oral administration of type II collagen
© Min et al., licensee BioMed Central Ltd. 2004
Received: 22 October 2003
Accepted: 26 February 2004
Published: 11 March 2004
Induction of oral tolerance has long been considered a promising approach to the treatment of chronic autoimmune diseases, including rheumatoid arthritis (RA). Oral administration of type II collagen (CII) has been proven to improve signs and symptoms in RA patients without troublesome toxicity. To investigate the mechanism of immune suppression mediated by orally administered antigen, we examined changes in serum IgG subtypes and T-cell proliferative responses to CII, and generation of IL-10-producing CD4+CD25+ T-cell subsets in an animal model of collagen-induced arthritis (CIA). We found that joint inflammation in CIA mice peaked at 5 weeks after primary immunization with CII, which was significantly less in mice tolerized by repeated oral feeding of CII before CIA induction. Mice that had been fed with CII also exhibited increased serum IgG1 and decreased serum IgG2a as compared with nontolerized CIA animals. The T-cell proliferative response to CII was suppressed in lymph nodes of tolerized mice also. Production of IL-10 and of transforming growth factor-β from mononuclear lymphocytes was increased in the tolerized animals, and CD4+ T cells isolated from tolerized mice did not respond with induction of IFN-γ when stimulated in vitro with CII. We also observed greater induction of IL-10-producing CD4+CD25+ subsets among CII-stimulated splenic T cells from tolerized mice. These data suggest that when these IL-10-producing CD4+CD25+ T cells encounter CII antigen in affected joints they become activated to exert an anti-inflammatory effect.
Keywordscollagen-induced arthritis IL-10 oral tolerance type II collagen
Oral tolerance is a state of absent or minimal immune responsiveness to protein antigens that were repeatedly administered by oral feeding . Induction of peripheral tolerance by oral administration of antigen has been applied to the treatment of autoimmune diseases such as rheumatoid arthritis (RA) [2, 3], multiple sclerosis, systemic sclerosis, type I diabetes and iritis , but the mechanisms by which orally administered antigen can induce peripheral tolerance have not yet been elucidated. Studies conducted in animal models have suggested that the possible mechanism may involve secretion of anti-inflammatory cytokines including IL-4, IL-10 and transforming growth factor (TGF)-β by mucosal T lymphocytes that have differentiated into T-helper (Th)2 or Th3 cells after encountering the antigen [5–7]. However, individual studies often report conflicting findings, depending on the route, dose and timing of antigen administration .
Although much of the RA pathogenesis remains to be elucidated, it has been reported that joint proteins, probably type II collagen (CII), play a key role in the instigation of T-cell mediated immune responses. Administration of CII to DBA/1 mice induces polyarthritis with pathological symptoms similar to those observed in human RA [9, 10]. In order to investigate the cellular mechanisms that underlie oral tolerance, we studied an animal model of collagen-induced arthritis (CIA), in which mice undergo repeated oral administration of CII, and monitored changes in immune cell function and factors associated with inflammation. We found that serum levels of IgG subtypes, as well as the production of IL-10, TGF-β and IFN-γ, were affected in tolerized mice. We also noticed greater proportions of IL-10-producing CD4+CD25+ T cells in the Peyer's patch, mesenteric lymph nodes and the spleen of tolerized mice. Production of these cells, exhibiting the characteristics of the Treg subset, was induced to a significant degree by lymphocytes from tolerized spleen when stimulated in vitro with CII. We hypothesize that expansion of these suppressor T cells in the periphery might have contributed to the reduced inflammation observed in affected joints.
Materials and methods
Induction of oral tolerance in DBA/1 mice
DBA/1 mice used in this study were fed either with 100 μg bovine CII (a kind gift from Prof. Andrew Kang, University of Tennessee) dissolved in 0.05 N acetic acid at 2 mg/ml (50 μl solution plus 150 μl acetic acid) or with an equal volume of phosphate-buffered saline (PBS) using an oral Zonde needle (Natsume, Japan) every 2 days for 2 weeks. All experimental procedures were examined and approved by the Animal Research Ethics Committee at The Catholic University of Korea.
Induction of CIA and evaluation of arthritis
Bovine CII was dissolved in 0.05 N acetic acid at 2 mg/ml and emulsified with an equal volume of Freund's complete adjuvant (CFA). As primary immunization, 0.1 ml of the emulsion, containing 100 μg CII, was injected into the tails of DBA/1 mice (both tolerized mice and nontolerized control mice; n = 6 per group). Two weeks later, a booster injection consisting of 200 μg CII similarly dissolved and emulsified 1:1 with incomplete Freund's adjuvant was injected into a hind leg. Starting from 2.5 weeks (18 days) after primary immunization, three independent observers examined the degree of arthritis three times a week for up to 11 weeks. The severity of arthritis was represented as mean arthritic index on a 0–4 scale according to the following criteria : 0 = no oedema or swelling; 1 = slight oedema and erythema limited to the foot and/or ankle; 2 = slight oedema and erythema from the ankle to the tarsal bone; 3 = moderate oedema and erythema from the ankle to the tarsal bone; and 4 = oedema and erythema from the ankle to the entire leg. The sum of values from three legs, excluding the hind leg into which CII/incomplete Freund's adjuvant was injected, was determined at the time of the second injection. The final values presented in the Results section represent an average of the indices recorded by three independent observers. All experimental procedures were examined and approved by the Animal Research Ethics Committee at The Catholic University of Korea.
Analysis of IgG antibody subtypes
Blood samples obtained from each mouse at 3, 5 and 7 weeks after primary immunization were used to investigate IgG antibody subtype concentrations using the mouse IgG1/IgG2a ELISA quantitation kit (Bethyl Lab Co., Montgomery, TX, USA). Levels of IgG1 and IgG2a were measured in mice sera diluted 50,000- to 400,000-fold.
Determination of collagen-specific T-cell proliferative response
The draining lymph nodes and the spleen were removed from each mouse and washed twice with PBS. Tissues were minced and the cells were filtered through a cell strainer and centrifuged at 1500 rpm at 4°C for 10 min. The cell pellet was resuspended in RPMI-1640 medium to a concentration of 1 × 105 cells/ml. Cells were than plated in 96-well microtitre plates at a concentration of 2 × 105 cells/well concentration and cultured with 40 μg/well CII in 0.3 ml Click's medium supplemented with 0.5% mouse serum for 3 days. The same amount of ovalbumin was added instead of CII to control wells. Eighteen hours before the termination of culture, 0.5 μCi [3H]thymidine (NEN, Boston, MA, USA) was added to each well. Cells were harvested onto glass fibre filters and counted on a Matrix-96 direct ionization β counter (Packard Instrument Co., Downers Grove, IL, USA). The degree of T-cell proliferation is presented as the stimulation index, which is calculated by dividing the counts/min in the presence of CII by the counts/min in the presence of ovalbumin.
Analyses of cytokine production by ELISA
Mononuclear cells were isolated from the Peyer's patch and spleen, and cultured at a density of 0.5 × 106 cells/ml in flat-bottomed, 48-well tissue culture plates (Corning, Corning, NY, USA). After 2 days, culture supernatants were harvested and stored at -70°C. To determine the amount of IL-10 and TGF-β in each supernatant, 96-well ELISA plates were coated with rat antimurine IL-10 and TGF-β monoclonal antibodies (R&D Systems, Minneapolis, MN, USA) for 24 hours at 4°C. After incubating with blocking agents, the plates were incubated with previously collected supernatants for 1 hour at room temperature. The plates were then washed and incubated for 1 hour at room temperature with biotin-conjugated rat antimurine IL-10 and TGF-β monoclonal antibodies, followed by an alkaline phosphatase-conjugated goat antibiotin monoclonal antibody (R&D Systems). The fluorescent substrate for alkaline phosphatase (R&D Systems) was used for colour development, and fluorescence was measured using a microtitre plate reader (Dynex, Chantilly, VA, USA) at excitation and emission wavelengths of 450 nm. The amounts of cytokines present in test samples were determined from standard curves established with serial dilutions of recombinant murine IL-10 and TGF-β.
Fluorocytometric analysis of T cells
Single-cell suspensions were prepared from each lymphoid organ and cultured in 24-well plates at a concentration of 1 × 106/well with or without 40 μg/well CII for 3 days. Golgi Stop (Pharmingen, San Diego, CA, USA) was added 4 hours before the termination of culture. Cells were subsequently washed and resuspended in fluorescence-activated cell sorting (FACS) staining buffer (PBS plus 0.1% bovine serum albumin plus 0.09% sodium azide), and probed with anti-CD4-perCP and/or anti-CD25-FITC (Pharmingen) for 30 min at 4°C. Next, cells were fixed with cytoperm/cytofix (Pharmingen) for 20 min and probed for intracellular cytokines using phycoerythrin-labelled anti-IL-10 antibody, anti-IFN-γ antibody, or isotype control antibody (Pharmingen) for 30 min at room temperature. Finally, cells were washed with PBS and analyzed on a FACScalibur flow cytometer (Becton Dickinson, San Jose, CA, USA).
Experimental findings are presented as mean ± standard deviation. Statistical significance was determined by Student's t-test using the SPSS program (version 10.0; SPSS Inc, Chicago, IL, USA). P < 0.05 was considered statistically significant.
Inhibition of arthritis development in tolerized CIA mice
Changes in the serum level of IgG subtypes in tolerized mice
Proliferation response to CII by T cells isolated from the draining lymph nodes and spleens of tolerized mice
Changes in the production of IL-10, TGF-β and IFN-γ by lymphocytes of orally tolerized mice
IFN-γ production by CD4+ T cells in lymph nodes and the spleen
% IFN-γ-positive cells
Without CII stimulation
With CII stimulation
3.4 ± 0.22
26.6 ± 0.6
4.6 ± 0.1
8.9 ± 0.2
Mesentric lymph node
3.3 ± 0.4
34 ± 0.2
2.3 ± 0.5
5.6 ± 0.8
5.4 ± 0.01
4.5 ± 0.2
5.3 ± 0.02
Induction of IL-10-producing CD4+CD25+ population from splenic T cells of orally tolerized mice by in vitroCII stimulation
Proportion of IL-10 producing CD4+CD25+ T cells in lymph nodes and spleen
% CD4+CD25+ IL-10-positive cells
Without CII stimulation
With CII stimulation
0.17 ± 0.12
0.14 ± 0.06
1.6 ± 0.7
3.6 ± 0.2
2.4 ± 1.6
6.8 ± 1.2
Mesentric lymph node
0.35 ± 0.35
0.5 ± 0.28
0.85 ± 0.35
1.8 ± 0.5
2.95 ± 1.06
7.9 ± 1.48
0.13 ± 0.04
0.25 ± 1.3
0.15 ± 0.07
3.85 ± 0.63
2.95 ± 1.62
15.5 ± 4.5
Treatment of autoimmune diseases by induction of oral tolerance is attractive because of the few side effects and easy clinical implementation of this approach. Although protein antigens administered orally are degraded quickly in the digestive system and their effects do not last long, we previously overcame these drawbacks by encapsulating CII – the major cartilage autoantigen in RA – in a polymer complex . In the present study we attempted to improve our basic understanding of the immune cell functions that participate in the process of induction of oral tolerance. Because most of the previous studies that addressed the mechanism underlying oral tolerance were conducted in transgenic animal models that overexpress T-cell clones that recognize experimental antigens such as ovalbumin, it is difficult to relate their findings to the clinical situation . In the present study we examined the changes in immune function that are involved in the induction of peripheral tolerance in an orally tolerized mouse model of arthritis.
Our findings indicate that the serum levels of proinflammatory and anti-inflammatory IgG subtypes are differentially modulated in mice that have undergone repeated oral administration of CII. We also found that, although the production of IFN-γ by CD4+ T cells from tolerized mice on CII stimulation is reduced, the production of IL-10 and TGF-β is increased not only in cells from the site of initial contact with the oral antigen (i.e. the Peyer's patch) but also in cells from lymph nodes and the spleen. That in vitro restimulation with CII generated significant enrichment in IL-10-producing CD4+CD25+ T cells among lymphocytes from tolerized spleen is particularly interesting. We hypothesize that by the time these IL-10-producing cells have reached the spleen through peripheral blood, they have also been transferred to the affected joints where CII is accumulated from cartilage degradation and have begun to alleviate inflammation.
Many recent studies have reported that IL-10, a major anti-inflammatory cytokine secreted by Th2 lymphocytes, contributes to the process of peripheral tolerance [1, 6]. Our data suggest that the establishment of CII-mediated tolerance to arthritic inflammation involved, among other mechanisms of immunosuppression, participation of CD4+CD25+ T cells and their production of IL-10 in peripheral lymphoid organs. However, roles for Th3 cells (known for their production of TGF-β [17, 18]) and for Tr1 cells (which are characterized by low proliferation and high production of IL-10 [19–21]) are equally implicated in the process of induction of peripheral tolerance. We observed elevated production of TGF-β from tolerized lymphocytes as well, and it would therefore be very interesting to investigate the role of Th3 cells. In order to further define the characteristics of cells that exert immunosuppressive functions, we are in the process of examining the expression of CD62L and CD45RB, which are accepted as additional phenotypic markers for regulatory T cells [22, 23], and the effect of transferring T cells into nontolerized CIA mice.
We observed a greater proportion of IL-10-producing CD4+CD25+ T cells in mice that had undergone repeated oral administration of CII. Together with increased production of IL-10 and TGF-β in peripheral lymphoid organs, induction of this suppressor population is believed to block the induction of IFN-γ from CII-stimulated CD4+ cells. Because little is known about the cellular mechanisms that underlie orally induced tolerance in non-transgenic systems, our findings provide important basic information on the generation of peripheral immune tolerance. Once merged with findings on possible inter-cellular cooperation between CII-specific T cells and other participants such as dendritic cells, these data will provide a basis for more effective application of oral tolerance induction in RA patients.
Freund's complete adjuvant
type II collagen
enzyme-linked immunosorbent assay
fluorescence-activated cell sorting
transforming growth factor
This study was supported by the 2002 Specialized Research Center (SRC) grant from the Korean Science and Engineering Foundation (KOSEF) to the Rheumatism Research Center (RhRC) at The Catholic University of Korea.
- Weiner HL, Friedman A, Miller A, Khoury SJ, al-Sabbagh A, Santos L, Sayegh M, Nussenblatt RB, Trentham DE, Hafler DA: Oral tolerance: immunologic mechanisms and treatment of animal and human organ-specific autoimmune diseases by oral administration of autoantigens. Annu Rev Immunol. 1994, 12: 809-837. 10.1146/annurev.iy.12.040194.004113.View ArticlePubMedGoogle Scholar
- Trentham DE, Dynesius-Trentham RA, Orav EJ, Combitchi D, Lorenzo C, Sewell KL, Hafler DA, Weiner HL: Effect of oral administration of type II collagen on rheumatoid arthritis. Science. 1993, 261: 1727-1730.View ArticlePubMedGoogle Scholar
- Barnett ML, Kremer JM, St Clair EW, Clegg DO, Furst D, Weisman M, Fletcher MJ, Chasan-Taber S, Finger E, Morales A, Le CH, Trentham DE: Treatment of rheumatoid arthritis with oral type II collagen. Results of a multicenter, double-blind, placebo-controlled trial. Arthritis Rheum. 1998, 41: 290-297. 10.1002/1529-0131(199802)41:2<290::AID-ART13>3.3.CO;2-I.View ArticlePubMedGoogle Scholar
- Strobel S, Mowat AM: Immune responses to dietary antigens: oral tolerance. Immunol Today. 1998, 19: 173-181. 10.1016/S0167-5699(97)01239-5.View ArticlePubMedGoogle Scholar
- Fishman-Lobell J, Freidman A, Weiner HL: Different kinetic patterns of cytokine gene expression in vivo in orally tolerant mice. Eur J Immunol. 1994, 24: 2720-2724.View ArticlePubMedGoogle Scholar
- Tsuji NM, Mizumachi K, Kurisaki J: Interleukin-10 secreting Peyer's patch cells are responsible for active suppression in low-dose oral tolerance. Immunology. 2001, 103: 458-464. 10.1046/j.1365-2567.2001.01265.x.PubMed CentralView ArticlePubMedGoogle Scholar
- Weiner HL: Oral tolerance: immune mechanisms and treatment of autoimmune diseases. Immunol Today. 1997, 18: 335-343. 10.1016/S0167-5699(97)01053-0.View ArticlePubMedGoogle Scholar
- Friedman A, Weiner HL: Induction of anergy or active suppression following oral tolerance is determined by antigen dosage. Proc Natl Acad Sci USA. 1994, 91: 6688-6692.PubMed CentralView ArticlePubMedGoogle Scholar
- Trentham DE, Townes AS, Kang AH: Autoimmunity to type II collagen: an experimental model of arthritis. J Exp Med. 1977, 146: 857-868. 10.1084/jem.146.3.857.View ArticlePubMedGoogle Scholar
- Stuart JM, Huffstutter EH, Townes AS, Kang AH: Incidence and specificity of antibodies to type I, II, III, IV, and V collagen in rheumatoid arthritis and other rheumatic disease as measured by 125I-radioimmunoassay. Arthritis Rheum. 1983, 26: 832-840.View ArticlePubMedGoogle Scholar
- Rosloniec EF, Cremer M, Kang A, Myers LK: Collagen-induced arthritis. In: Current Protocols in Immunology. Edited by: Coligan JE, Kruisbeek AM, Margulies DH, Shevach EM, Strober W. 1996, New York: Wiley, 3: 15.5.1-15.5.19.Google Scholar
- Seetharaman R, Mora AL, Nabozny G, Boothby M, Chen J: Essential role of T-cell NF-kappa B activation in collagen-induced arthritis. J Immunol. 1999, 163: 1577-1583.PubMedGoogle Scholar
- Butler DM, Malfait AM, Maini RN, Brennan FM, Feldmann M: Anti-IL-12 and anti-TNF antibodies synergistically suppress the progression of murine collagen-induced arthritis. Eur J Immunol. 1999, 29: 2205-2212. 10.1002/(SICI)1521-4141(199907)29:07<2205::AID-IMMU2205>3.0.CO;2-Z.View ArticlePubMedGoogle Scholar
- Piccirillo CA, Letterio JJ, Thornton AM, McHugh RS, Mamura M, Mizuhara H, Shevach EM: CD4+ CD25+ regulatory T-cells can mediate suppressor function in the absence of transforming growth factor 1 production and responsiveness. J Exp Med. 2002, 196: 237-245. 10.1084/jem.20020590.PubMed CentralView ArticlePubMedGoogle Scholar
- Kim WU, Park JS, Ryoo JW, Kim J, Youn J, Kim SH, Min SY, Bae EY, Lee WK, Hwang SY, Park SH, Cho CS, Kim HY: Suppression of collagen induced arthritis by single administration of poly(lactic-co-glycolic acid) nanoparticles entrapping CII: a novel therapeutic strategy for oral tolerance. Arthritis Rheum. 2002, 46: 1109-1120. 10.1002/art.10198.View ArticlePubMedGoogle Scholar
- Gonnella PA, Chen Y, Inobe J, Komagata Y, Quartulli M, Weiner HL: In situ immune response in gut-associated lymphoid tissue (GALT) following oral antigen in TCR-transgenic mice. J Immunol. 1998, 160: 4708-4718.PubMedGoogle Scholar
- Shi HN, Grusby MJ, Nagler-Anderson C: Orally induced peripheral nonresponsiveness is maintained in the absence of functional Th1 or Th2 cells. J Immunol. 1999, 162: 5143-5148.PubMedGoogle Scholar
- Oldenhove G, de Heusch M, Urbain-Vansanten G, Urbain J, Maliszewski C, Leo O, Moser M: CD4+CD25+ regulatory T cells control T helper cell type 1 responses to foreign antigens induced by mature dendritic cells in vivo. J Exp Med. 2003, 198: 259-266. 10.1084/jem.20030654.PubMed CentralView ArticlePubMedGoogle Scholar
- Wakkach A, Fournier N, Brun V, Breittmayer JP, Cottrez F, Groux H: Characterization of dendritic cells that induce tolerance and T regulatory 1 cell differentiation in vivo. Immunity. 2003, 18: 605-617. 10.1016/S1074-7613(03)00113-4.View ArticlePubMedGoogle Scholar
- Levings MK, Bacchetta R, Schulz U, Roncarolo MG: The role of IL-10 and TGF-beta in the differentiation and effector function of T regulatory cells. Int Arch Allergy Immunol. 2002, 129: 263-276. 10.1159/000067596.View ArticlePubMedGoogle Scholar
- Mallat Z, Gojova A, Brun V, Esposito B, Fournier N, Cottrez F, Tedgui A, Groux H: Induction of a regulatory T cell type 1 response reduces the development of atherosclerosis in apolipoprotein E-knockout mice. Circulation. 2003, 108: 1232-1237. 10.1161/01.CIR.0000089083.61317.A1.View ArticlePubMedGoogle Scholar
- Annacker O, Burlen-Defranoux O, Pimenta-Araujo R, Cumano A, Bandeira A: Regulatory CD4 T cells control the size of the peripheral activated/memory CD4 T cell compartment. J Immunol. 2000, 164: 3573-3580.View ArticlePubMedGoogle Scholar
- Jump RL, Levine AD: Murine Peyer's patches favor development of an IL-10 secreting, regulatory T cell population. J Immunol. 2002, 168: 6113-6119.View ArticlePubMedGoogle Scholar
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