Collagen-induced arthritis in CD4- or CD8-deficient mice: CD8+ T cells play a role in initiation and regulate recovery phase of collagen-induced arthritis

Collagen-induced arthritis (CIA) is an experimental autoimmune disease induced by immunization with collagen type II (CII). We studied CIA in CD4- or CD8-deficient DBA/1 mice to further define the roles of CD4+ and CD8+ T cells in the disease. CD4-deficient mice developed severe arthritis, and no differences in incidence, clinical course, and severity were observed between CD4 -/- and CD4 +/- mice. Proliferative responses of lymph node T cells to CII was, however, reduced in CD4 -/- mice, and inflamed joints revealed relative accumulation of CD4-CD8-TCR(alpha)(beta)+ cells. A CII-specific T cell line generated from CD4-deficient mice responded to CII in a MHC-restricted fashion and had a CD4-CD8-TCR(alpha)(beta)+ phenotype. Disease incidence in CD8 -/- mice was significantly decreased compared with CD8 +/- mice, even though the severity of arthritis in arthritic mice was not different. These results suggests a role for CD8+ T cells in initiating CIA. Interestingly, CD8-deficient mice were more susceptible to a second induction of arthritis after remission of initial disease, pointing towards an immunoregulatory role for CD8+ T cells. CD8-deficient mice did not, however, show any defect in oral tolerance induction using CII. Taken together, our findings demonstrate that CD4-CD8-TCR(alpha)(beta) cells can trigger systemic arthritis in CD4-deficient mice and that CD8+ T cells can play dual and opposing roles, important both in initiation of CIA and in providing resistance to reinduction of CIA after recovery from initial disease.     

Down-modulation of CD2 delays deletion of superantigen-responsive T cells

Collagen-induced arthritis (CIA) is an autoimmune animal model for some types of human rheumatoid arthritis (RA). We have evaluated the effectiveness of intranasal administration of antigen in inhibiting CIA in DBA/1 mice. The intranasal administration of heat-denatured or trypsin-digested bovine type II collagen (CII) before immunization with CII strongly delayed the onset of CIA, whereas administration of native CII did not do so. The mice administered denatured or digested CII possessed much lower titers of anti-CII IgG2a than the control mice, whereas titers of anti-CII IgG1 and IgG2b were unchanged or slightly decreased. Responding to CII and peptides containing immunodominant T cell determinants, lymph node cells from mice administered denatured CII produced less IFN-gamma. These results suggest that intranasal administration of antigen downregulated preferentially Th1-type responses, whereas an enhanced Th2-type response was not observed. We demonstrate that the methods shown here are a possible treatment for rheumatoid arthritis.     

Development of a polymeric surgical paste formulation for taxol

The purpose of the study was to map the dominant T cell epitope of the CB11 sequence of CII in RTlu haplotype rats and to determine if, when used as a synthetic peptide, it would induce tolerance to protect against CIA. A dominant epitope corresponding to residues 184-198 included in the sequence of the CB11 fragment of bovine CII was identified in proliferation assay using peptides in an epitope scanning system using synthetic peptides of 15 amino acids, overlapping by 12 amino acids. This epitope is bovine-specific, but cross-reacts with the corresponding rat peptide. Minor epitopes in the bovine CB11 sequence was also autoantigenic. Use of independently synthesized and purified 184-198 peptide confirmed its dominance in the T cell responses of arthritic rats. The peptide itself was not arthritogenic. Cells from lymph nodes draining arthritic feet were particularly responsive to the dominant peptide sequence, and showed evidence of epitope spreading to include reactions to at least four subdominant epitopes. Mucosal tolerance was successfully induced by instilling CII into the nose of rats before induction of CIA: this was found to delay the onset of disease, reduce mean disease severity, shift the anti-CII antibody response to favour antibodies of the IgG1, rather than the IgG2b isiotype, and to reduce T cell reactivity to both CII and to the 184-198 peptide. The dominant 184-198 peptide itself had the same tolerogenic effects when given nasally to rats daily, on the 4 days immediately preceding the induction of CIA. Two forms of CIA with acute and delayed disease onset were each modified by pre-treatment with the peptide. This study demonstrates that mucosal tolerance to CII can be induced by delivering it nasally in a way similar to that achieved previously by oral delivery, and that the use of an immunodominant epitope contained in a synthetic peptide will also suppress the immunologic and arthritic responses to collagen.     

Involvement of testosterone in the induction of hepatic microsomal cytochrome P-450 2B1/2 (P-450 2B1/2) by 1-benzylimidazole in male and female rats: sex-differentiated induction of P-450 2B1/2 species

Collagen-induced arthritis (CIA) is an animal model for the human autoimmune disease rheumatoid arthritis (RA). CIA can be induced in several species including primates by immunization with heterologous type-II collagen (CII). Polyclonal antibodies are formed upon immunization with CII that exhibit a broad range of epitope specificities (some that cross-react with hose CII); however, only antibodies directed against certain specific epitopes on CII are arthritogenic. Recently, the importance of cognate interactions between T-cells and B-cells to the induction of CIA was demonstrated by administration of monoclonal antibodies against a T-cell surface protein, gp39. Blocking the interaction of T-cell gp39, with its receptor/ligand on the surface of B-cells (CD40), completely blocked induction of CIA in mice. A concomitant reduction in the level of anti-CII IgG produced in anti-gp39-treated animals was observed, demonstrating the crucial importance of T-cell:B-cell interactions via gp39:CD-40 binding to the primary immune response to CII in vivo and therefore to the induction of CIA. Other features of CIA are important in elucidating the condition and this article will deal with some important issues.     

Characterization of a peptide analog of a determinant of type II collagen that suppresses collagen-induced arthritis

Immunization of susceptible strains of mice with type II collagen (CII) elicits an autoimmune arthritis known as collagen-induced arthritis (CIA). One analogue peptide of the immunodominant T cell determinant, A9 (CII245-270 (I260-->A, A261-->B, F263-->N)), was previously shown to induce a profound suppression of CIA when coadministered at the time of immunization with CII. In the present study, A9 peptide was administered i.p., orally, intranasally, or i.v. 2 to 4 wk following CII immunization. We found that arthritis was significantly suppressed even when A9 was administered after disease was induced. To determine the mechanism of action of A9, cytokine responses to A9 and wild-type peptide A2 by CII-sensitized spleen cells were compared. An increase in IL-4 and IL-10, but not in IFN-gamma, was found in A9 culture supernatants. Additionally, cells obtained from A9-immunized mice produced higher amounts of IL-4 and IL-10 when cultured with CII compared with cells obtained from mice immunized with A2, which produced predominantly IFN-gamma. Suppression of arthritis could be transferred to naive mice using A9-immune splenocytes. Lastly, phosphorylation of TCRzeta was not altered in the immunoprecipitates from the lysates of cells exposed to analogue peptides (A9 and A10) together with wild-type A2 in a T cell line and two I-Aq-restricted, CII-specific T hybridomas. We conclude that analogue peptide A9 is effective in suppressing established CIA by inducing T cells to produce a Th2 cytokine pattern in response to CII.     

Involvement of human cytochromes P450 (CYP) in the reductive metabolism of AQ4N, a hypoxia activated anthraquinone di-N-oxide prodrug

OBJECTIVE: The 65 kDa heat shock protein (HSP) chaperonin is a highly conserved intracellular protein. HSP are involved in the pathogenesis of arthritis, but are not able to induce experimental arthritis. T cell clones recognizing the 180-188 amino acid sequence of 65 kDa HSP are present in inflamed synovium of both adjuvant arthritis and rheumatoid arthritis (RA). Oral administration of bovine collagen II or co-chaperonin 10 kDa HSP has been shown to induce an immune tolerance state to collagen induced arthritis (CIA). We investigate the effect of oral gavage with 65 kDa HSP on CIA. METHODS: We immunized 6-8-week-old DBA1 male mice with bovine type II collagen. A group of 25 mice were given oral recombinant mycobacterial 65 kDa HSP before immunization (30 microg in 200 microl phosphate buffered saline (PBS) at Days -7, -5, -2) while PBS alone was administered in 27 controls. A 3rd group was fed 65 kDa HSP according to the same protocol but was not immunized with collagen II (n = 8). The clinical arthritis score was recorded 3 times/week until Day 60. Antibodies to collagen II were determined by ELISA. RESULTS: The incidence of arthritis was comparable in the 2 groups (72 vs 70%). The onset of arthritis was not delayed in mice fed HSP. However, the severity of arthritis was lower 10 days after arthritis onset in animals fed 65 kDa HSP (clinical score 1.83 +/- 0.79 vs 2.74 +/- 1.1; p < 0.0001). No animals in Group 3 had arthritis. Serum IgG anti-type II collagen levels were decreased in HSP treated mice (optical density 0.33 +/- 0.21 vs 0.46 +/- 0.21; p < 0.0001). However, the ratio of IgG1/IgG2a antitype II collagen antibody response remained unchanged in the mice fed 65 kDa HSP. CONCLUSION: These results suggest that oral administration of 65 kDa HSP may diminish collagen induced arthritis.     

Relationship between collagen-induced and adjuvant arthritis in the Lewis rat

Adjuvant arthritis (AA) and type II collagen (CII)-induced arthritis (CIA) in the rat serve as models of chronic human arthritis. Adoptive transfer of AA was observed in 21 of 25 Lewis rats given concanavalin A (Con A)-treated spleen cells prepared from animals immunized with Mycobacterium butyricum in mineral oil (complete Freund's adjuvant, CFA). No arthritic changes were noted in rats given spleen cells obtained from donors that had received incomplete Freund's adjuvant (IFA, 0/22), type I collagen in IFA (CI-IFA, 0/6) or CII-IFA (0/28). Administration of spleen cells from IFA, CI-IFA or CII-IFA-injected animals did not modify the development of CIA when these rats were subsequently challenged with CII-IFA. However, partial protection against induction of AA was provided by the transfer of spleen cells prepared from rats immunized with CII-IFA (6/11) but not by those obtained from rats injected with IFA (1/15) or CI-IFA (0/3). Rats that did not develop clinically evident arthritis following the administration of spleen cells prepared from CFA-injected rats were also resistant to AA induction by CFA. Pre-treatment of rats with a synthetic peptide, corresponding to amino acids 180-188 of the Mycobacterium 65 kD heat shock protein (65 kD HSP), significantly delayed the onset of AA, but not that of CIA. Disease-specific resistance to AA, provided by spleen cells prepared from rats injected with CII-IFA and by pre-treatment with the 65 kD HSP 180-188 peptide, may result from the induction of protective tolerance to arthritogenic epitopes present in the Mycobacterium and CII preparations.     

Epitope glycosylation plays a critical role for T cell recognition of type II collagen in collagen-induced arthritis

Immunization of mice with type II collagen (CII) leads to collagen-induced arthritis (CIA), a model for rheumatoid arthritis. T cell recognition of CII is believed to be a critical step in CIA development. We have analyzed the T cell determinants on CII and the TCR used for their recognition, using twenty-nine T cell hybridomas derived from C3H.Q and DBA/1 mice immunized with rat CII. All hybridomas were specific for the CII(256-270) segment. However, posttranslational modifications (hydroxylation and variable O-linked glycosylation) of the lysine at position 264 generated five T cell determinants that were specifically recognized by different T cell hybridoma subsets. TCR sequencing indicated that each of the five T cell epitopes selected its own TCR repertoire. The physiological relevance of this observation was shown by in vivo antibody-driven depletion of TCR Valpha2-positive T cells, which resulted in an inhibition of the T cell proliferative response in vitro towards the non-modified CII(256-270), but not towards the glycosylated epitope. Most hybridomas (20/29) specifically recognized CII(256-270) glycosylated with a monosaccharide (beta-D-galactopyranose). We conclude that this glycopeptide is immunodominant in CIA and that posttranslational modifications of CII create new T cell determinants that generate a diverse TCR repertoire.     

Local removal of phagocytic synovial lining cells by clodronate-liposomes decreases cartilage destruction during collagen type II arthritis

OBJECTIVE: To investigate whether local removal of phagocytic synovial lining cells (SLCs) from the knee joint before onset of collagen type II arthritis has an effect on development of cartilage destruction. METHODS: Phagocytic SLCs were selectively depleted by a single injection of clodronate laden liposomes in the knee joint seven days before induction of collagen type II arthritis (CIA). Clodronate laden liposomes were given in one knee joint either alone or in combination with a short-term oral treatment of dexamethasone. Cartilage damage including proteoglycan depletion and chondrocyte death was measured in total knee joints sections stained with safranin-o or haematoxylin. RESULTS: Local removal of phagocytic SLCs, seven days before arthritis onset, prevented cell influx for the larger part. Chondrocyte death was significantly decreased in the SLC depleted arthritic joint both at an early (6 days) and late (12 days) time point after CIA induction. However, depletion of proteoglycans from femoral and patellar cartilage layers was not prevented. If the mild acute inflammation caused by a single clodronate laden liposome injection in the left knee joint, was blocked by a short-term (on consecutive days 9, 8, 7, 6, 5 before CIA onset) oral treatment with dexamethasone, cell influx, but also proteoglycan depletion was almost completely blocked. In the contralateral control right knee joint prominent cell influx and severe cartilage damage was observed, indicating that there was no effect of dexamethasone anymore at the onset of CIA. CONCLUSIONS: This study shows that removal of phagocytic lining cells before CIA induction, particularly in the presence of a short-term treatment with dexamethasone, decreases cartilage destruction.     

Induction of T helper cell hyporesponsiveness in an experimental model of autoimmunity by using nonmitogenic anti-CD3 monoclonal antibody

Autoimmune diseases can be characterized by increases in Th cell activities, suggesting that inhibition of Th cell function might ameliorate autoimmunity. We have recently reported that administration of nonmitogenic anti-CD3 mAb (nmCD3) to nonautoimmune mice can induce long-term Th cell hyporesponsiveness, reflected by reduced IL-2 secretion upon re-exposure to Ag. This study was designed to determine the effects of nmCD3 on autoimmunity by using the murine collagen-induced arthritis model. Treatment of DBA/1 mice with nmCD3 delayed the onset and reduced the severity of arthritis in mice immunized with type II collagen (CII). This effect was not caused by depletion of T cells or modulation of TCR. The observed inhibition of arthritis was not caused by decreased Ab production, as anti-CII titers were not affected. Rather, lymph node cells from CII-immunized mice treated with nmCD3 were hyporesponsive to in vitro stimulation with CII. This hyporesponsiveness was reflected by a marked decrease in secretion of IL-2 and IFN-gamma, but not of IL-4, which suggests that nmCD3 had its principal effect on Th1 cells. The hyporesponsiveness was not Ag-specific, because IL-2 and IFN-gamma production in response to a pan-T cell mitogen was also reduced. These results demonstrate that induction of Th1 cell hyporesponsiveness with nmCD3 can significantly alter the course of CIA and suggest that IL-2 and/or IFN-gamma play a crucial role in disease pathogenesis.     

Inhibition of mouse acetylcholinesterase by fasciculin: crystal structure of the complex and mutagenesis of fasciculin

Animal models of autoimmune diseases have been instrumental in advancing our understanding of autoimmunity in humans. Collagen-induced arthritis in mice is an autoimmune disease model of rheumatoid arthritis, which is MHC class II restricted and CD4 T cell dependent. To better understand the fundamental role of T cells in arthritis, we have generated a transgenic mouse carrying the rearranged Valpha11.1 and Vbeta8.2 TCR chain genes isolated from a type II collagen (CII)-specific T cell hybridoma. Cell surface analysis indicated that Vbeta8.2 chain was expressed on the surface of nearly all peripheral T cells. Analysis of T cell subsets in transgenic mice revealed a profound skewing in peripheral T cells towards the CD4 population. Although peripheral T cells were not tolerant to CII and responded to CII stimulation in vitro, transgenic mice did not develop spontaneous arthritis. However, a rapid onset of arthritis with severe clinical signs was detected in transgenic mice after immunization with CII in complete Freund's adjuvant. Histological analysis of inflamed joints showed a great resemblance to arthritic joints in man. This unique transgenic mouse model provides valuable insights into the mechanism of arthritis and into potential specific immune interventions.     

Prevention of murine collagen-induced arthritis in the knee and ipsilateral paw by local expression of human interleukin-1 receptor antagonist protein in the knee

OBJECTIVE: To determine the efficacy of local human interleukin-1 receptor antagonist (HuIL-1Ra) gene therapy in murine collagen-induced arthritis (CIA). METHODS: DBA/1 mice were immunized against bovine type II collagen. Before the onset of arthritis, NIH/3T3 fibroblasts transfected with pMFG-IRAP were transplanted into the knee cavity. Normal NIH/3T3 cells served as controls. Paws were evaluated macroscopically for redness, swelling, and deformities during the course of arthritis. Swelling of the knee joints was measured by external gamma counting of 99mtechnetium accumulation in the joint. Paws and knee joints were dissected and processed for histologic studies to evaluate inflammation and cartilage destruction. RESULTS: The NIH/3T3 fibroblasts survived in the joint cavity of DBA mice for at least 7 days. The transduced cells expressed immunoreactive and bioactive HuIL-1Ra in the knee joint, and produced sufficient amounts to block the effect of 1 ng of recombinant murine IL-1alpha on chondrocyte proteoglycan synthesis. The onset of CIA was almost completely prevented in knee joints containing HuIL-1Ra-producing cells, whereas joints containing normal cells showed severe inflammation and destruction of cartilage. Moreover, onset of CIA in the draining joints (ipsilateral paws) of the HuIL-1Ra gene-bearing knees was also prevented. CONCLUSION: Local production of HuIL-1Ra in the knee was able to ameliorate the effects of IL-1 on cartilage and could prevent the onset of CIA not only in that knee, but also in the "draining" paw. This indicates the feasibility of gene transfer as a therapeutic approach to modulating arthritis.     

The nuclear factor-kappa B RelA transcription factor is constitutively activated in human pancreatic adenocarcinoma cells

To assess the efficiency of nasally administered cartilage-specific collagens as vaccination against development of arthritis and to ameliorate already established chronic arthritis, experimental models which develop chronic arthritis, pristane-induced arthritis (PIA), and homologous collagen-induced arthritis (CIA) in the rat were selected. Cartilage-specific collagens type IX (CIX) and type II (CII) were used for vaccination intranasally. A single dose of 250 microg CII instilled intranasally in rats with established PIA ameliorated the disease. For the prevention of disease, the same dose given before immunization was found to be most effective. Most importantly, the disease was more severe if this dose was given three times. For treatment of PIA, CIX was found to be more effective than CII, whereas for treatment of CIA only CII was effective. The amelioration of CIA was associated with a marked suppression of delayed type hypersensitivity and the flare reaction to CII and lower levels of IgG2b anti-CII antibodies in serum, i.e., with suppression of the TH1 rather than the TH2 response to CII. These findings, that cartilage proteins, if given intranasally, can both prevent and ameliorate established chronic arthritis in rats, are of significant importance for possible use in rheumatoid arthritis. The identification of two different cartilage-specific proteins (CII and CIX) effective against a disease induced with a well-defined nonimmunogenic adjuvant such as pristane will be of value for enhancing the effectiveness of the treatment.     

Therapeutic effects of monoclonal antibodies to alpha beta TCR but not to CD4 on collagen-induced arthritis in the rat

The role which T cells play in the pathogenesis of the collagen-induced arthritis (CIA) model is not yet fully understood. Although CIA is most likely dependent on the activity of class II-restricted CD4+ T cells, only prophylactic but not therapeutic anti-CD4 treatments have been successful. The lack of therapeutically effective anti-T cell monoclonal antibody treatments has questioned the importance of T cells in ongoing CIA. However, recently we found that ongoing CIA in DA rats induced with homologous CII can be suppressed by injections with an anti-alpha beta TCR antibody. Having a CIA model where ongoing disease was clearly dependent on T cells, we addressed in the present work whether also an anti-CD4 treatment could suppress ongoing arthritis in this model. Although no CD4hi lymph node cells were seen after an anti-CD4 injection, the arthritis was suppressed only after treatment at immunization but not after treatment just before onset of disease. In comparison, the anti-TCR treatment at the time of onset was clearly suppressive even though a large fraction of the T cells was not depleted. This indicates that the different outcome of the anti-TCR and anti-CD4 treatment was not due to a different capacity to deplete T cells in vivo.     

Extrathymic differentiation of resident T cells in the joints of mice with collagen-induced arthritis

Murine collagen-induced arthritis (CIA) is known as a T cell-mediated autoimmune disease, although autoantibodies are also suspected to be associated with the onset of the disease. To determine the origin of such T cells in the joints of mice with CIA, their phenotypic properties as well as those of T cells in other immune organs were examined in DBA/1 mice. Since a significant number of mononuclear cells (MNC) was also yielded by the joints of normal DBA/1 mice, the properties of these T cells were examined in parallel. When CIA was induced by an intradermal injection of type II collagen at the base of the tail, the numbers of MNC yielded by the regional lymph nodes and the foot joints were doubled. Interestingly, regardless of the onset of CIA, the joints were always comprised of unique T cell populations, including IL-2(R)alpha- beta+ T cells, gammadelta T cells, CD8alpha+ beta- cells, and CD44+ L-selectin- cells. All these properties coincide with those of extrathymic T cells in liver and intestine. In the case of gammadelta T cells in joints, Vgamma and Vdelta usages were unique and different from those in the other organs. More importantly, Vgamma and Vdelta usages in gammadelta T cells in the joints of normal mice and in those of mice with CIA were essentially the same. Taken together with the expression of recombination-activating gene-1 and -2 mRNAs by MNC in mice with CIA, these findings raise the possibility that the joints have their own resident T cells that are extrathymically generated in situ.     

Induction of autoimmune arthritis in HLA-DR4 (DRB1*0401) transgenic mice by immunization with human and bovine type II collagen

Although associations between the expression of particular HLA genes and the susceptibility to specific autoimmune diseases has been known for some time, the role that these HLA molecules play in the autoimmune response is unclear. Through the establishment of a chimeric HLA-DR/I-E transgene, we have examined the function of the rheumatoid arthritis (RA) susceptibility allele HLA-DR4 (DRB1*0401) in presenting antigenic peptides derived from the model Ag, type II collagen (CII), and in mediating an autoimmune response. As a transgene, the chimeric DR4 molecule conferred susceptibility to an autoimmune arthritis induced by immunization with human CII or bovine CII. These mice developed an inflammatory, autoimmune arthritis that was similar both histologically and in severity to that previously described for the collagen-induced arthritis model. The DR4-mediated autoimmune arthritis was accompanied by T cell and B cell responses to both the immunogen and the autoantigen, murine CII. The DR4-restricted T cell response to human CII was focused on an immunodominant determinant within CII263-270 and a minor determinant within CII286-300, the same CII determinants recently identified for yet another RA susceptibility allele, HLA-DR1 (DRB1*0101). Thus these data demonstrate that, like HLA-DR1, HLA-DR4 is capable of binding peptides derived from human CII and therefore probably plays a role in the autoimmune response to human CII observed in RA patients.     

Dual role of IL-12 in early and late stages of murine collagen type II arthritis

IL-12 can promote Th1 responses, and early administration of IL-12 during immunization was shown to enhance expression of autoimmune collagen-induced arthritis (CIA). We now studied the impact of IL-12 at the stage of disease expression and during established CIA in DBA-1 mice. Accelerated onset and enhanced severity were provoked when i.p. injections of 100 ng of murine IL-12 (mIL-12) were given around the time of arthritis onset. Moreover, the onset of CIA could be ameliorated with anti-mIL-12 Abs, indicating that IL-12 is a pivotal mediator in the expression of CIA. In addition, the effect of anti-mIL-12 treatment was analyzed in established CIA. Continued treatment did not suppress established arthritis. Instead, these mice showed an impressive exacerbation of arthritis shortly after cessation of anti-mIL-12 treatment, indicative of impairment of endogenous control. Exaggerated disease was characterized by massive granulocyte influx and enhanced expression of IL-1 beta and TNF-alpha mRNA in the synovial tissue. Subsequently, we treated established collagen arthritis with recombinant mIL-12 for 7 days. Profound suppression of the arthritis score was noted, including reduced influx of cells and diminished cartilage damage. Tenfold enhanced levels of IL-10 were detected in sera of mIL-12-treated mice, and up-regulated mRNA levels of IL-10, IFN-gamma, and IL-12 were measured in synovial tissue. Finally, the anti-inflammatory effect of IL-12 on CIA could be reversed by coadministration of anti-IL-10 Abs. This study indicates that IL-12 has a stimulatory role in early arthritis expression, whereas it has a suppressive role in the established phase of collagen arthritis.     

Lack of local suppression in orally tolerant CD8-deficient mice reveals a critical regulatory role of CD8+ T cells in the normal gut mucosa

We found that feeding keyhole limpet hemocyanin (KLH) to CD8-deficient (CD8-/-) mice induced oral tolerance that was comparable in both magnitude and quality to that induced in wild-type (wt) mice. The tolerance was dose dependent, and only higher doses of KLH caused significant reduction in specific Ab and T cell responses. Both Th1 and Th2 CD4+ T cell functions were affected. Feeding KLH together with cholera toxin (CT) adjuvant, however, abrogated the induction of oral tolerance equally well in CD8-/- and wt mice. On the contrary, CT adjuvant was unable to abrogate already established oral tolerance in both CD8-/- and wt mice. Most importantly, whereas Ag feeding induced hyporesponsiveness in systemic as well as in local gut IgA responses in wt mice, a lack of local suppression was evident in orally tolerant CD8-/- mice following oral immunizations. Thus, contrary to the situation in wt mice, Ag feeding induces systemic, but not local, gut IgA hyporesponsiveness in CD8-/- mice, suggesting that CD8+ T cells in the normal gut mucosa exert an important down-regulatory function. In wt mice the local suppression extended to an unrelated Ag, OVA, given together with KLH and CT adjuvant, i.e., bystander suppression. Based on these results we propose that tolerance induced by feeding Ag is highly compartmentalized, requiring CD8+ T cells for local suppression of IgA responses, whereas systemic tolerance may affect CD4+ T cells of both Th1 and Th2 types independently of CD8+ T cells. Finally, the adjuvant effect of CT abrogates induction, but not established, oral tolerance through a mechanism that does not require CD8+ T cells.     

Methotrexate specifically modulates cytokine production by T cells and macrophages in murine collagen-induced arthritis (CIA): a mechanism for methotrexate-mediated immunosuppression

Immunosuppressive therapy with methotrexate (MTX) has been established as effective treatment for patients with rheumatoid arthritis. To analyse the therapeutic potential and mechanisms of action of MTX, we determined serum cytokine levels and cytokine production by splenic T cells and macrophages in untreated and MTX-treated mice. Furthermore, we assessed the role of MTX in a murine model of experimental arthritis induced by collagen type II (CIA). MTX reduced spontaneous and IL-15-induced tumour necrosis factor (TNF) production by splenic T cells but not by macrophages from healthy mice in vitro in a dose-dependent manner. In contrast, interferon-gamma (IFN-gamma) production was less strikingly reduced and IL-4 production was virtually unaffected. In addition, treatment of healthy mice with MTX in vivo led to reduced TNF serum levels and diminished TNF production by splenic T cells and macrophages. Intraperitoneal administration of MTX prior to the onset of arthritis completely prevented clinical and pathological signs of CIA. This was associated with a striking reduction of TNF production by spleen cells from MTX-treated mice. The role of TNF in MTX-mediated effects on cytokine production was further underlined by the finding that MTX effects on IFN-gamma production were augmented in TNF-transgenic mice but abrogated in mice in which the TNF-alpha gene had been inactivated by homologous recombination. Thus, MTX specifically modulates spontaneous and IL-15-induced TNF-alpha production in mice and prevents experimental murine CIA. These data suggest that TNF production by T cells is an important target of MTX and may serve as a basis to understand and further analyse MTX-mediated mechanisms of immunosuppression in patients with RA.