Attenuation of collagen arthritis and modulation of delayed-type hypersensitivity by type II collagen reactive T-cell lines

T-cell lines were established from the lymph node cells of syngeneic Louvain (LOU) rats previously immunized with native chick type II collagen (CII) emulsified in incomplete Freund's adjuvant. The CII lines proliferated in vitro to type II collagen but not to type I collagen, ovalbumin (OV), or PPD. Control lines, developed from LOU rats immunized with OV emulsified in complete Freund's adjuvant, were OV specific because they did not respond to other antigens in vitro. CII line cells could adoptively transfer delayed-type hypersensitivity (DTH) but did not induce IgG antibody production to collagen. Moreover, the intravenous administration of 2 X 10(7) CII line cells prevented the subsequent induction of collagen arthritis following immunization and suppressed DTH to collagen without affecting antibody responses in the recipients. Spleen cells, but not sera, from these resistant rats decreased CII line reactivity in vitro. OV or irradiated CII lines had no effect on clinical or immunologic parameters in this model. These findings demonstrate protection from arthritis afforded by T-cell line transfer and suggest that the phenomenon results from down-regulation of the recipients' cellular immunity to collagen.     

Suppression of collagen arthritis with antibodies to an arthritogenic, oligoclonal T cell line.

Rats immunized with type II collagen (CII) develop an immunologically mediated polyarthritis. T cells have been implicated in the pathogenesis of this model since they can adoptively transfer the disease. A CII-specific T cell line (VA), consisting of three distinct clones by Southern blot analysis, has been shown to be arthritogenic. Antibodies specific for this line were generated by immunizing rabbits. In an attempt to prevent collagen-induced arthritis (CIA), Louvain rats were injected with 1 ml of anti-VA ip on Days -1, +1, +3 and 0.5 ml on Day +5 (early treatment). To evaluate its effect on existing disease, rats received anti-VA on the day of arthritis onset and subsequently on 4 successive alternate days using the same dosage protocol (late treatment). Control rats received no therapeutic injections or were administered normal rabbit serum. All rats were immunized with CII on Day 0 to induce CIA. Rats administered antibodies using the early anti-VA treatment protocol had a significantly diminished incidence of arthritis compared to controls. Established arthritis was significantly diminished compared to controls in rats given the late anti-VA treatment. In both protocols, radiographic evidence of joint destruction was significantly reduced compared to controls. T cell phenotyping using flow cytometry analysis demonstrated that the anti-VA antibody therapy selectively eliminated a small subset of T cells since there was little difference in total T cell counts in the experimental versus control groups. Delayed type hypersensitivity and IgG antibody titers to CII were minimally decreased in the experimental versus control group. These results suggest that antibodies raised to an oligoclonal arthritogenic T cell line can suppress collagen arthritis. This may have implications with respect to 1) the size of the T cell receptor repertoire involved in the pathogenesis of collagen arthritis and 2) immunospecific protocols for CIA and other autoimmune diseases.     

Collagen type II (CII)-specific antibodies induce arthritis in the absence of T or B cells but the arthritis progression is enhanced by CII-reactive T cells

Antibodies against type II collagen (anti-CII) are arthritogenic and have a crucial role in the initiation of collagen-induced arthritis. Here, we have determined the dependence of T and B cells in collagen-antibody-induced arthritis (CAIA) during different phases of arthritis. Mice deficient for B and/or T cells were susceptible to the CAIA, showing that the antibodies induce arthritis even in the absence of an adaptive immune system. To determine whether CII-reactive T cells could have a role in enhancing arthritis development at the effector level of arthritis pathogenesis, we established a T cell line reactive with CII. This T cell line was oligoclonal and responded to different post-translational forms of the major CII epitope at position 260–270 bound to the A^q class II molecule. Importantly, it cross-reacted with the mouse peptide although it is bound with lower affinity to the A^q molecule than the corresponding rat peptide. The T cell line could not induce clinical arthritis per se in A^q-expressing mice even if these mice expressed the major heterologous CII epitope in cartilage, as in the transgenic MMC (mutated mouse collagen) mouse. However, a combined treatment with anti-CII monoclonal antibodies and CII-reactive T cells enhanced the progression of severe arthritis.     

Immunogenicity and arthritogenicity of recombinant CB10 in B10.RIII mice

Two major T cell determinants are recognized by I-Ar-specific T cells in CII, the immunodominant CII610-618 (GPAGT AGA R) within CB10 and the subdominant CII445-453 (GPAGP AGE R) within CB8. Although the determinants differ by only two residues, CB8 is capable of inducing collagen-induced arthritis (CIA), while CB10 is not. We, therefore, investigated the structural differences between the two determinants that are critical to inducing arthritis. When the CB10 determinant was mutated to that of CB8 using recombinant techniques, the resulting mutant rCB10T614P,A617E product became arthritogenic. Conversely, when the CB8 determinant was mutated to that of CB10, the resulting mutant CB8P449T,E452A was no longer arthritogenic. Comparison of the epitope specificity of the autoantibodies induced by wild-type CB10 and mutant rCB10T614P, A617E revealed no qualitative differences. T cells from mice immunized with either CB10 or mutant rCB10 produced predominantly Th1 cytokines when cultured with the immunizing Ag. In contrast, when cultured with mouse CII, T cells from mice immunized with the nonarthritogenic CB10 produced predominantly Th2 (IL-4 and IL-10) cytokines whereas the arthritogenic mutant rCB10 induced predominantly Th1 (IFN-gamma) cytokines. We conclude that the T cell cytokine response most critical for the induction of CIA is that induced against the corresponding homologous murine T cell determinant and, further, that the structural differences between the T cell determinants in CB8 and -10 are important in breaking self tolerance and inducing autoimmune response.     

Isolation of T cell line capable of protecting mice against collagen-induced arthritis

A T cell line specific to human type II collagen (CII) was selected and propagated from DBA/1J mice immunized with human CII. The line cells were not reactive to type I or type III collagen of human origin, but they were cross-reactive to bovine, rat, and rabbit CII and they recognized both native and heat-denatured human CII. The cells were reactive to an N-terminal three-quarters fragment of human CII, produced by tadpole collagenase digestion of human CII, but not to a C-terminal one-quarter fragment of human CII. The cells showed Thy-1+, Lyt-1+, Lyt-2-, and L3T4+ phenotypes characteristic of T helper cells or delayed-type hypersensitive cells, determined by the immunofluorescence method. To clarify the role of T cells in the pathogenesis of collagen-induced arthritis, we inoculated this cell line into DBA/1J mice and found that they developed clinical arthritis, albeit at a low incidence. The cells attenuated by x-ray were capable of inducing resistance to the subsequent induction of collagen-induced arthritis of DBA/1J mice. The sera from mice protected by inoculation of the cell line exhibited anti-idiotypic antibody response against conventional and monoclonal anti-CII antibodies. Anti-T cell receptor response may be involved in the mechanism for the protective effect of the cell line against autoimmune murine arthritis.     

Chronic development of collagen-induced arthritis is associated with arthritogenic antibodies against specific epitopes on type II collagen

Antibodies against type II collagen (CII) are important in the development of collagen-induced arthritis (CIA) and possibly also in rheumatoid arthritis. We have determined the fine specificity and arthritogenicity of the antibody response to CII in chronic relapsing variants of CIA. Immunization with rat CII in B10.Q or B10.Q(BALB/c×B10.Q)F₂ mice induces a chronic relapsing CIA. The antibody response to CII was determined by using triple-helical peptides of the major B cell epitopes. Each individual mouse had a unique epitope-specific response and this epitope predominance shifted distinctly during the course of the disease. In the B10.Q mice the antibodies specific for C1 and U1, and in the B10.Q(BALB/c×B10.Q)F₂ mice the antibodies specific for C1, U1 and J1, correlated with the development of chronic arthritis. Injection of monoclonal antibodies against these epitopes induced relapses in chronic arthritic mice. The development of chronic relapsing arthritis, initially induced by CII immunization, is associated with an arthritogenic antibody response to certain CII epitopes.     

Collagen-Induced Arthritis Suppressed with Monoclonal Anti-Idiotypic Antibody

In foregoing work, we identified at least 5 distinct epitopes on human type II collagen (CII), using 8 murine monoclonal antibodies (mAb) against human CII, and suggested that a species-nonspecific epitope on CII recognized by anti-CII mAb termed 1-5 is an arthritogenic epitope. We also found that antibody response against a selected epitope of human CII could be induced by immunization with rabbit anti-idiotypic (Id) antibody against anti-CII mAb. The author developed and characterized monoclonal anti-Id antibodies against 1-5 mAb recognizing a putative arthritogenic epitope. The author also investigated whether the anti-Id mAb could regulate antibody response directed against a selected epitope recognized by 1-5 mAb, and the induction of collagen-induced arthritis in DBA/1J mice. DBA/1J mice intravenously preinjected with anti-Id mAb to 1-5, did not produce anti-CII antibody expressing 1-5 Id upon immunization with human CII. Furthermore, as the development of collagen-induced arthritis (CIA) in DBA/1J mice pretreated with anti-Id mAb to 1-5 was significantly suppressed, anti-Id mAb will be a useful tool for studying the regulation of antibody response to a selected epitope. This study lends support to our hypothesis that the 1-5 epitope is an arthritogenic epitope.     

An arthritogenic monoclonal antibody to type II collagen, CII-C1, impairs cartilage formation by cultured chondrocytes

Antibodies to type II collagen (CII) cause articular damage in collagen-induced arthritis (CIA) in mice as judged by passive transfer to naive animals of mAb to CII. We tested the hypothesis that mAb degrade cartilage structure by reacting with functionally important regions of the collagen molecule by examining the effects of an arthritogenic mAb to CII, CII-C1, on cultured bovine chondrocytes at high density, at days 7 and 14. The effects were compared of CII-C1, an isotype-matched control mAb, or medium alone, on chondrocyte proliferation and viability, cell morphology, matrix structure by light and electron microscopy, and matrix synthesis by metabolic labelling with 3H-proline for collagen or 35SO4 for proteoglycans. Chondrocytes in culture remained viable, proliferated, and produced an extracellular matrix in which CII was the major collagen. The addition of CII-C1, but not a control mAb, increased the synthesis of CII and proteoglycan, and caused disorganization of the extracellular matrix and thin collagen fibrils ultrastructurally. Moreover, using a cell-free assay, CII-C1 inhibited the normal self-assembly of collagen fibrils from CII in solution. The finding that the mAb to CII, CII-C1 has striking degradative effects in vitro on cartilage synthesis suggests that antibodies to collagen perpetuate the chronic phase of CIA and that, in mice at least, such antibodies are an important component of pathogenesis.     

T cell regulation of collagen-induced arthritis in mice. I. Isolation of Type II collagen-reactive T cell hybridomas with specific cytotoxic function

After immunization with native type II collagen (CII), susceptible strains of mice (H-2q) develop a polyarthritis that mimics rheumatoid arthritis. Although the underlying mechanisms are still undefined, T cells and particularly CD4+ lymphocytes seem to play a crucial role in the initiation of collagen-induced arthritis. To investigate whether CD8+ cells may participate in the pathogenesis of the disease, we have generated lines and clones of cytotoxic T cell hybridomas reactive to CII by fusion of lymph node and spleen cells from bovine native CII-primed C3H.Q (H-2q) mice and the AKR-derived thymoma cell line BW 5147. Clones were selected for their ability to lyse syngeneic macrophages pulsed with bovine native CII in an Ag-dependent manner. The two hybrid clones that were characterized, exhibited cell surface phenotypes of cytotoxic cells and reacted with CII purified from various species. However, each of them recognized different determinants on the CII molecule. P3G8 clone was specific for an epitope shared by CII and type XI collagen, whereas P2D9 clone reacted with CII and type IX collagen. Both hybridomas recognized CII-pulsed targets in association with H-2Kq molecules. These data indicate that the two CII-specific cytotoxic clones recognize different epitopes that are shared by other articular collagens and will allow us to test their influence on the development of arthritis in vivo.     

Inhibition of macropinocytosis blocks antigen presentation of type II collagen in vitro and in vivo in HLA-DR1 transgenic mice

Professional antigen-presenting cells, such as dendritic cells, macrophages and B cells have been implicated in the pathogenesis of rheumatoid arthritis, constituting a possible target for antigen-specific immunotherapy. We addressed the possibility of blocking antigen presentation of the type II collagen (CII)-derived immunodominant arthritogenic epitope CII_259–273 to specific CD4 T cells by inhibition of antigen uptake in HLA-DR1-transgenic mice in vitro and in vivo. Electron microscopy, confocal microscopy, subcellular fractionation and antigen presentation assays were used to establish the mechanisms of uptake, intracellular localization and antigen presentation of CII by dendritic cells and macrophages. We show that CII accumulated in membrane fractions of intermediate density corresponding to late endosomes. Treatment of dendritic cells and macrophages with cytochalasin D or amiloride prevented the intracellular appearance of CII and blocked antigen presentation of CII_259–273 to HLA-DR1-restricted T cell hybridomas. The data suggest that CII was taken up by dendritic cells and macrophages predominantly via macropinocytosis. Administration of amiloride in vivo prevented activation of CII-specific polyclonal T cells in the draining popliteal lymph nodes. This study suggests that selective targeting of CII internalization in professional antigen-presenting cells prevents activation of autoimmune T cells, constituting a novel therapeutic strategy for the immunotherapy of rheumatoid arthritis.     

Characterization of a tolerogenic T cell epitope of type II collagen and its relevance to collagen-induced arthritis.

A synthetic peptide representing sequences of type II collagen, (CII 245-270), has previously been used to induce tolerance and suppress arthritis in DBA/1 mice. To determine important residues, a series of peptides, each containing one or two site-directed substitutions, was generated. Mononuclear cells from DBA/1 mice immunized with CII were cultured in the presence of each peptide and the T cell response determined by measuring IFN-gamma in culture supernatant fluids. Substitutions within the region CII 260-270 led to significant decreases in IFN-gamma responses, identifying this sequence as a T cell epitope. To determine the effects of substitutions within this epitope on arthritis, substituted peptides were administered to neonatal mice as tolerogens. Five site-directed substitutions, four of which included the insertion of a residue found in type I collagen to replace its type II counterpart, abrogated the ability of the peptides to induce tolerance and suppress arthritis. These substitutions were located at residues 260, 261, 263, 264, and 266. Two patterns of T cell reactivity were observed. Peptides containing individual substitutions at positions 261, 264, or 266 were capable of generating a significant T lymphokine response, although those containing substitutions at residues 260 or 263 were ineffective Ag. Systematic analysis of the fine structures of T cell determinants important for autoimmune arthritis can lead to strategies for therapeutic intervention.     

Destructive effects of murine arthritogenic antibodies to type II collagen on cartilage explants <Emphasis Type="Italic">in vitro</Emphasis>

Certain monoclonal antibodies (mAbs) to type II collagen (CII) induce arthritis in vivo after passive transfer and have adverse effects on chondrocyte cultures and inhibit self assembly of collagen fibrils in vitro. We have examined whether such mAbs have detrimental effects on pre-existing cartilage. Bovine cartilage explants were cultured over 21 days in the presence of two arthritogenic mAbs to CII (CIIC1 or M2139), a non-arthritogenic mAb to CII (CIIF4) or a control mAb (GAD6). Penetration of cartilage by mAb was determined by immunofluorescence on frozen sections and correlated with changes to the extracellular matrix and chondrocytes by morphometric analysis of sections stained with toluidine blue. The effects of mAbs on matrix components were examined by Fourier transform infrared microspectroscopy (FTIRM). A possible role of Fc-binding was investigated using F(ab)₂ from CIIC1. All three mAbs to CII penetrated the cartilage explants and CIIC1 and M2139, but not CIIF4, had adverse effects that included proteoglycan loss correlating with mAb penetration, the later development in cultures of an abnormal superficial cellular layer, and an increased proportion of empty chondrons. FTIRM showed depletion and denaturation of CII at the explant surface in the presence of CIIC1 or M2139, which paralleled proteoglycan loss. The effects of F(ab)₂ were greater than those of intact CIIC1. Our results indicate that mAbs to CII can adversely affect preformed cartilage, and that the specific epitope on CII recognised by the mAb determines both arthritogenicity in vivo and adverse effects in vitro. We conclude that antibodies to CII can have pathogenic effects that are independent of inflammatory mediators or Fc-binding.     

Encephalitogenic T cells in the B10.PL model of experimental allergic encephalomyelitis (EAE) are of the Th-1 lymphokine subtype

T helper cells reactive to myelin basic protein are clearly implicated in the pathogenesis of murine EAE. We have developed a T cell line, BML-1 that (1) is reactive to the encephalitogenic amino terminal nonapeptide (1-9NAC) of MBP, (2) is I-Au restricted, and (3) induces relapsing EAE in B10.PL (H-2u) mice. Measurement of the lymphokine profile of BML-1 revealed secretion of IL-2, interferon-gamma and lymphotoxin but not IL-4. This profile is consistent with the Th1/DTH subtype. Coculture of BML-1 with MBP-primed B cells shows that BML-1 does not provide significant helper function in vitro. In addition, BML-1 secretion of interferon-gamma was found to inhibit LPS-induced anti-MBP antibody responses. This suggested that anti-MBP antibodies may not be necessary for induction of EAE. Sera from mice, in which severe disease was induced with the 1-9NAC peptide and Bordetella pertussis, showed no development of serum antibodies to MBP. These data show that MBP-reactive Th cells of the Th-1/DTH subtype can induce EAE and do not provide Th function for anti-MBP responses and that serum anti-MBP antibodies are not found in peptide 1-9NAC-induced disease. T cell lines specific for encephalitogenic epitopes and characterized for lymphokine secretion will provide a useful tool for understanding the role of T cells in the induction of EAE.     

Type IX collagen deficiency enhances the binding of cartilage-specific antibodies and arthritis severity

Joint cartilage is attacked in both autoimmune inflammatory and osteoarthritic processes. Type IX collagen (CIX) is a protein of importance for cartilage integrity and stability. In this study we have backcrossed a transgenic disruption of the col9a1 gene, which leads to an absence of CIX, into two different inbred mouse strains, DBA/1 and B10.Q. None of the CIX-deficient mice developed observable clinical or microscopic osteoarthritis, but DBA/1 male mice had more pronounced enthesopathic arthritis, the so-called stress-induced arthritis. Both DBA/1 and B10.Q strains are susceptible to the induction of collagen-induced arthritis, and CIX deficiency in both strains led to the development of a more severe arthritis than in the controls. Induction of arthritis with monoclonal antibodies against type II collagen (CII) led to an earlier arthritis in the paws that also involved the knee joints. The antibodies used, which were specific for the J1 and the C1^I epitopes of CII, initiate their arthritogenic attack by binding to cartilage. The C1^I-specific antibodies bound to cartilage better in CIX-deficient mice than in wild-type animals, demonstrating that the lack of CIX in cartilage leads to an increased accessibility of structures for antibody binding and thus making the joints more vulnerable to inflammatory attack. These findings accentuate the importance of cartilage stability; cartilage disrupted as a result of genetic disorders could be more accessible and vulnerable to an autoimmune attack by pathogenic antibodies.     

Antigenic properties associated with Vinca alkaloid resistance in ovarian cancer cells: identification of a 92,000 Da protein.

In order to characterize the membrane changes related to Vinca alkaloid resistance, we raised monoclonal antibodies (mAbs) against a Vincristine resistant subline (OV1/VCR) derived from a human ovarian adenocarcinoma cell line (OV1/p). Among three monoclonal antibodies selected for a higher binding to OV1/VCR than to OV1/p cells, one designated OVR09, recognized a Mr 92,000 protein. This protein appears to be gradually overexpressed along the drug resistance establishment in vitro, and to decrease slowly in absence of drug. Further, mAb OVR09 showed a much higher binding to the vinblastine resistant epidermoid tumor cell line KbV1 than to its parental counterpart. The Mr 92,000 protein was also detected in various tumor cell lines and in an ovarian carcinoma surgical sample.     

Antibodies against the CB10 fragment of type II collagen in rheumatoid arthritis

Antibodies against intact type II collagen (CII) are a feature of rheumatoid arthritis (RA) but have limited diagnostic value. Here we assess whether either of the two major cyanogen bromide fragments of CII, namely CB10 or CB11, are more sensitive substrates for the detection of antibodies in RA. Cleavage of bovine CII with cyanogen bromide yielded CB10 and CB11; these were purified by column chromatography for use in an enzyme-linked immunosorbent assay. Serum antibodies were measured in patients with RA, psoriatic arthritis (PsA), osteoarthritis (OA) and blood donors. Results were compared with those using intact CII. Antibodies against CB10 were found in as many as 88% of 96 patients with long-standing RA, but only 12% of 33 patients with PsA, 6% of 34 patients with OA and 3% of 93 control sera. Lower frequencies for these diseases were obtained on testing for antibodies against CB11: 50%, 6%, 21% and 2%, respectively. The sensitivity of detection in RA of antibodies against CB10 compared with antibodies against intact CII (88% versus 24%) was not at the expense of specificity, which remained high at 94%. The much higher frequency of antibodies against CB10 in RA than in other rheumatic diseases or control sera indicates that CB10 is clearly a more sensitive substrate than the intact collagen molecule and, combined with other assays (rheumatoid factor, anti-cyclic citrullinated peptide [anti-CCP]), might comprise a panel with a highly reliable predictive value. Moreover, our findings should encourage renewed interest in the role of collagen autoimmunity in the pathogenesis of RA.     

Detection of early changes in autoimmune T cell phenotype and function following intravenous administration of type II collagen in a TCR-transgenic model.

To study the phenotypic and functional changes in naive type II collagen (CII)-specific autoimmune T cells following a tolerogenic signal, a TCR-transgenic (Tg) mouse model of collagen-induced arthritis was developed. These Tg mice express an I-A(q)-restricted CII (260-267)-specific TCR that confers severe accelerated autoimmune arthritis following immunization with CII. Despite the fact that >90% of the alphabeta T cells express the Tg, these mice can be rendered completely tolerant to the induction of arthritis by i.v. administration of 200 microg of CII. As early as 24 h after CII administration, CII-specific T cells demonstrated a decreased ability to proliferate in response to the CII immunodominant peptide and phenotypically altered the expression of L-selectin to CD62L(low) and of phagocytic glycoprotein-1 to CD44(high), expression levels consistent with the phenotype of memory T cells. In addition, they up-regulated the expression of the activation markers CD71 and CD69. Functionally, following tolerogenic stimulation, the CII-specific T cells produced similar levels of IL-2 in comparison to controls when challenged with CII peptide, however, by 48 h after exposure to tolerogen, IL-2 production dropped and was replaced by high levels of IL-10 and IL-4. Based on their production of Th2 cytokines, these data suggest that T regulatory cells expressing activation and memory markers are induced by the tolerogen and may exert their influence via cytokines to protect the animals from the induction of arthritis.     

Induction of IL-10-producing CD4+CD25+ T cells in animal model of collagen-induced arthritis by oral administration of type II collagen

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-beta from mononuclear lymphocytes was increased in the tolerized animals, and CD4+ T cells isolated from tolerized mice did not respond with induction of IFN-gamma 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.     

The N-methyl-d-aspartic acid receptor antagonist memantine ameliorates and delays the development of arthritis by enhancing regulatory T cells

The neuroendocrine impact on rheumatoid arthritis is not yet fully described although numerous neurotransmitters are shown to act as inflammatory modulators. One of these is the excitatory transmitter glutamate (Glu). In this study, the influence of the Glu receptor (GluR)-mediated effects on collagen-induced arthritis (CIA) was investigated. CIA was induced in DBA/1 mice by immunization with chicken collagen type II (CII). Mice were exposed to the following GluR antagonists: group 1, the N-methyl-D-aspartic acid (NMDA) receptor channel blocker memantine; group 2, the metabotropic GluR antagonist AIDA, and group 3, the excitatory amino acid receptor antagonist kynurenic acid (KA). Arthritis was evaluated clinically and histologically and compared to PBS-treated controls. The effects of treatment on T cell populations and the levels of anti-CII and anti-citrullinated peptide antibodies were evaluated. Memantine treatment significantly improved the course of CIA, reducing synovitis (p = 0.007) and the frequency of erosions (p = 0.007). Memantine treatment up-regulated the expression of Foxp3 in spleen CD4+ T cells followed by an increase in CD4+CD25+ regulatory T cells. The other GluR antagonists, AIDA and KA, had no effect on CIA. These results demonstrate that blockade of the NMDA receptor channel with memantine delays and attenuates the development of arthritis, probably by promoting the development of regulatory T lymphocytes.