Flare-up of delayed-type hypersensitivity initially induced by murine cloned helper T cells

Delayed-type hypersensitivity (DTH) reactions were induced in mice by cloned helper T cells directed against methylated bovine serum albumin (mBSA). The DTH reactions were induced either by local injection of the helper T cells together with the antigen in the hind feet or by intravenous (iv) administration of the cloned T cells and local injection of the antigen. Local or systemic (oral or iv) administration of mBSA after waning of the DTH induced by the cloned helper T cells caused a flare-up reaction. This indicates that functional helper T cells persist at the inflammation site. The inflammations were quantified in a foot swelling assay and were examined histologically. The inflammation measured in the flare-up reaction was generally lower than in the acute reaction. Histologically the acute inflammation showed edema and a large proportion of granulocytes, whereas the flare-up reaction appeared more histiocytic and showed less edema.     

Studies on antigen-induced arthritis in mice. III. Cell and serum transfer experiments.

Antigen-induced arthritis in mice occurs after immunization and subsequent intraarticular challenge with methylated bovine serum albumin (mBSA). In adoptive transfer experiments, susceptible C57BL mice and resistant CBA mice were compared in their capacity to express delayed-type hypersensitivity (DTH) by ear assay, and to express arthritis. The expression of DTH could be transferred incrementally by lymphoid cells in C57BL mice, but not in CBA mice. Both immune lymphoid cells and, to a much lesser extent, serum transferred the capacity to develop arthritis in C57BL mice. The reactivity of transferred cells was abolished by anti-Thy-1 but enhanced by enrichment for T cells with anti-immunoglobulin columns. If this model disease can be equated with human rheumatoid synovitis, the lesions in the human disease would be an expression of a T cell-dependent activity.     

Near-infrared imaging of flare-up arthritis with native fluorochrome Cy5.5 and albumin-bound Cy5.5

The aim of the study was to visualize chronic experimental arthritis with near-infrared fluorescence imaging (NIRF) in a murine experimental arthritis model of rheumatoid arthritis (RA) (flare-up arthritis). The flare-up arthritis model is a modification of the primary antigen-induced arthritis (AIA) model. NIRF was done for two preparations of the fluorochrome Cy5.5, one native and the other albumin conjugated. Histological features of flare-up arthritis were evaluated.AIA was induced in 16 mice (strain C57/Bl6); flare-up arthritis was induced in a subgroup of eight. On day 7 after induction of flare-up arthritis, four mice received 50 nmol/kg native dye and four mice equimolar concentrations of the dye as albumin-dye conjugate intravenously. NIRF imaging was performed immediately before injection (baseline) and until 72 h thereafter. Arthritis severity was evaluated histologically for primary AIA and flare-up arthritis mice.NIRF imaging revealed higher fluorochrome uptake in all inflamed knees compared to contralateral ones. The signal intensities induced by native Cy5.5 were higher than those generated by albumin-Cy5.5 conjugate. Histological evaluation of arthritic joints showed similar abnormalities in flare-up arthritis and in primary AIA joints.Imaging of flare-up arthritis in the near-infrared range was successful for both fluorochrome preparations, but albumin conjugation prior to injection does not improve the uptake of dye in arthritic joints. Flare-up arthritis is a feasible model of chronic relapse of arthritis in human RA.     

Suppression of collagen-induced arthritis in DBA/1J mice by preimmunization with house dust mite extract.

Collagen-induced arthritis (CIA) can be induced in DBA/1J mice by immunization with bovine type II collagen (bCII) and is a model of some types of human autoimmune rheumatoid arthritis. In this study we examined whether preimmunization of the mice with various antigens could inhibit the development of CIA. Preimmunization of the mice with an extract of the house dust mite Dermatophagoides farinae (mite antigen), chicken ovalbumin, or keyhole limpet hemocyanin strongly inhibited CIA development, but hen egg lysozyme, beta-lactoglobulin from bovine milk or myelin basic protein from guinea pig brain did not substantially affect CIA development. Splenic T cells and serum antibodies specific for mite antigen did not cross-react with bCII. Preimmunization of the mice with mite antigen did not affect the IFN-gamma and proliferative response of splenic T cells to bCII, nor serum antibody responses. The most inhibitory constituent had a molecular weight between 1,000 and 10,000.     

Immunobiological function of normal rabbit synovial cells

The ability of enzyme-dissociated primary cultures of synovial cells (Sy) to present antigen was investigated. Adult rabbits were immunized in foot pads with bovine serum albumin (BSA) in complete Freund's adjuvant (CFA) or with CFA alone. Four to six weeks later draining popliteal lymph node cells (LNC) and synovial cells were obtained. Synovial cells were cultured overnight with or without antigen. About 20% of these synovial cells had Fc receptors and 15% C3 receptors. As a positive control splenic adherent cells (SAC) were similarly treated. Next day, autologous lymph node cells were added to the extensively washed and irradiated synovial cells or splenic adherent cells. Lymphocyte proliferation was measured by [3H]thymidine uptake. Synovial cells as well as splenic adherent cells induced mixed lymphocyte reaction (MLR) and autologous mixed lymphocyte reaction (AMLR) and effectively presented antigen for specific immune response to the priming antigen. Thus, the synovium contains macrophage-like cells that can effectively interact with lymphocytes and participate in the immune phenomena in the joints of patients with rheumatoid arthritis.     

Antigen-specific T cells transduced with IL-10 ameliorate experimentally induced arthritis without impairing the systemic immune response to the antigen

For the treatment of rheumatoid arthritis, efficient drug delivery methods to the inflamed joints need to be developed. Because T cells expressing an appropriate autoantigen-specific receptor can migrate to inflamed lesions, it has been reasoned that they can be employed to deliver therapeutic agents. To examine the ability and efficiency of such T cells as a vehicle, we employed an experimentally induced model of arthritis. Splenic T cells from DO11.10 TCR transgenic mice specific for OVA were transduced with murine IL-10. Adoptive transfer of the IL-10-transduced DO11.10 splenocytes ameliorated OVA-induced arthritis despite the presence of around 95% nontransduced cells. Using green fluorescent protein as a marker for selection, the number of transferred cells needed to ameliorate the disease was able to be reduced to 10(4). Preferential accumulation of the transferred T cells was observed in the inflamed joint, and the improvement in the disease was not accompanied by impairment of the systemic immune response to the Ag, suggesting that the transferred T cells exert their anti-inflammatory task locally, mainly in the joints where the Ag exists. In addition, IL-10-transduced DO11.10 T cells ameliorated methylated BSA-induced arthritis when the arthritic joint was coinjected with OVA in addition to methylated BSA. These results suggest that T cells specific for a joint-specific Ag would be useful as a therapeutic vehicle in rheumatoid arthritis for which the arthritic autoantigen is still unknown.     

Interleukin 6 knock-out mice are resistant to antigen-induced experimental arthritis

In order to assess the potential role of IL-6 in rheumatoid arthritis (RA), we have compared IL-6 deficient (IL-6 ko) mice and their wild-type (wt) counterpart for the capacity to develop methylated bovine serum albumin (mBSA)-induced arthritis. Our data show that IL-6 ko mice are not susceptible to antigen-induced arthritis (AIA). In fact, IL-6 ko mice treated by a standard protocol of immunization with mBSA did not develop joint swelling following intra-articular mBSA injection, nor revealed the characteristic joint lesions by histological examination. Conversely, wt mice treated according to the same protocol developed arthritis about 9 days after intra-articular injection, as detected by knee joint swelling and histological confirmation. We observed that the proliferative response of splenocytes to mBSA was impaired in ko mice following arthritis induction, as compared to the strong response observed in wt mice. Furthermore, anti-mBSA IgG levels were lower in ko mice as compared to wt mice. Finally, we show that sensitivity to AIA can be reconstituted in ko mice by subcutaneous injections of recombinant human IL-6 (rhIL-6). In addition, co-administration of IL-6 with mBSA by intra-articular injection into the joint was only partially effective in conferring sensitivity to AIA, suggesting the importance of a systemic effct for IL-6, but also that an additional role for this cytokine can be envisaged in the local inflammatory reaction during establishment of AIA.     

Studies on antigen-induced arthritis in mice. II. Immunologic correlates of arthritis susceptibility in mice.

Antigen-induced arthritis was developed in mice as a model of human rheumatoid arthritis by using methylated bovine serum albumin (mBSA) as antigen. It was found that most strains were susceptible, whereas CBA mice were resistant. We therefore investigated the humoral and cell-mediated immune responses to mBSA in resistant mice (CBA) and susceptible mice (exemplified by C57BL) to determine whether these were associated with susceptibility to arthritis. The resistant strain (CBA) differed from the suceptible strains in the following respects. First, there was a lower humoral immune response to mBSA as measured by passive hemagglutination, but this could be overcome by a larger immunogenic dose. Secondly, there were differences in response to low doses of DNP-mBSA after mBSA carrier preimmunization. Thirdly, there were striking differences in delayed-type hypersensitivity (DTH) to mBSA as determined by a radioisotopic assay in vivo; the response of CBA mice occurred early, at 5 days, declined quickly, and was weaker, whereas that of C57BL mice developed later and was long sustained. Genetic studies of the DTH response with hybrids and backcrosses showed an oligogenic control of immune responsiveness, with one gene being linked to the H-2b allele of the susceptible C57BL mice, and another being independent of the H-2 complex. Our findings indicate that in mice, susceptibility to antigen-induced arthritis with mBSA correlates with a higher responder state to this antigen, and that T cells are the major if not the only determinant of the high responder state.     

The model of arthritis induced by superantigen in mice

Subcutaneous injection of Staphylococcal enterotoxine B (SEB) produced by Staphylococcus aureus, caused severe arthritis in DBA/1J mice which had been previously immunizated with bovine type II collagen. The severity of this arthritis was dose dependent and prolonged joint inflammation with erosion of bone was observed. Anti-type II collagen antibodies were detected in the serum of arthritic mice. Effector T cells against type II collagen were also detected by means of delayed type hypersensitivity in the skin. Moreover, a significant decrease in the ratio between T cells and B cells and an increase in the ratio between CD4⁺ cells and CD8⁺ cells was observed in spleen cells from arthritic mice. Prednisolone supresses the induction and development of clinical signs of arthritis in mice. This evidence suggests that this experimental arthritis model may provide a means to examine the role of superantigens and the efficacy of pharmacological agents for the treatment of rheumatoid arthritis.     

Intraocular Ia expression in vivo induced in the absence of immune T cells

The expression of Ia antigen on the ciliary body, retinal pigment epithelium, and retinal vascular endothelium was investigated using two models of ocular inflammation. Active systemic immunization with bovine serum albumin with subsequent ocular challenge resulted in increased Ia expression in the ciliary body and retinal pigment epithelium. This was compared with passive transfer of hyperimmune serum to bovine serum albumin followed by ocular challenge when Ia expression was found to occur only in the ciliary body. An intraocular T-cell infiltrate occurred only in the actively immunized animals and was not present after passive transfer, suggesting that the Ia induction in this latter situation occurred in the absence of T cells.     

The role of regulatory T cells in antigen-induced arthritis: aggravation of arthritis after depletion and amelioration after transfer of CD4<Superscript>+</Superscript>CD25<Superscript>+</Superscript>T cells

It is now generally accepted that CD4⁺CD25⁺ T_reg cells play a major role in the prevention of autoimmunity and pathological immune responses. Their involvement in the pathogenesis of chronic arthritis is controversial, however, and so we examined their role in experimental antigen-induced arthritis in mice. Depletion of CD25-expressing cells in immunized animals before arthritis induction led to increased cellular and humoral immune responses to the inducing antigen (methylated bovine serum albumin; mBSA) and autoantigens, and to an exacerbation of arthritis, as indicated by clinical (knee joint swelling) and histological scores. Transfer of CD4⁺CD25⁺ cells into immunized mice at the time of induction of antigen-induced arthritis decreased the severity of disease but was not able to cure established arthritis. No significant changes in mBSA-specific immune responses were detected. In vivo migration studies showed a preferential accumulation of CD4⁺CD25⁺ cells in the inflamed joint as compared with CD4⁺CD25^- cells. These data imply a significant role for CD4⁺CD25⁺ T_reg cells in the control of chronic arthritis. However, transferred T_reg cells appear to be unable to counteract established acute or chronic inflammation. This is of considerable importance for the timing of T_reg cell transfer in potential therapeutic applications.     

Citrullinated proteins have increased immunogenicity and arthritogenicity and their presence in arthritic joints correlates with disease severity

Autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in patients with rheumatoid arthritis and have been suggested to be involved in the disease pathogenesis. The targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deiminase (PAD), which converts positively charged arginine to polar but uncharged citrulline. The aim of this study was to explore the effects of citrullination on the immunogenicity of autoantigens as well as on potential arthritogenicity. Thus, immune responses to citrullinated rat serum albumin (Cit-RSA) and to unmodified rat serum albumin (RSA) were examined as well as arthritis development induced by immunisation with citrullinated rat collagen type II (Cit-CII) or unmodified CII. In addition, to correlate the presence of citrullinated proteins and the enzyme PAD4 with different stages of arthritis, synovial tissues obtained at different time points from rats with collagen-induced arthritis were examined immunohistochemically. Our results demonstrate that citrullination of the endogenous antigen RSA broke immunological tolerance, as was evident by the generation of antibodies directed against the modified protein and cross-reacting with the native protein. Furthermore we could demonstrate that Cit-CII induced arthritis with higher incidence and earlier onset than did the native counterpart. Finally, this study reveals that clinical signs of arthritis precede the presence of citrullinated proteins and the enzyme PAD4. As disease progressed into a more severe and chronic state, products of citrullination appeared specifically in the joints. Citrullinated proteins were detected mainly in extracellular deposits but could also be found in infiltrating cells and on the cartilage surface. PAD4 was detected in the cytoplasm of infiltrating mononuclear cells, from day 21 after immunisation and onwards. In conclusion, our data reveal the potency of citrullination to break tolerance against the self antigen RSA and to increase the arthritogenic properties of the cartilage antigen CII. We also show that citrullinated proteins and the enzyme PAD4 are not detectable in healthy joints, and that the appearance and amounts in arthritic joints of experimental animals are correlated with the severity of inflammation.     

Pathological study of Streptococcus faecalis antigen-induced arthritis in New Zealand white rabbits

The pathological examination of the rabbit knee joint with antigen-induced arthritis produced by heat-killed Streptococcus faecalis (Str. faec.) as antigen was carried out. Macroscopically, there were findings of acute inflammation about five hours after the injection. Histopathologically, very remarkable acute exudative inflammation was seen 48 hours later. This supported the picture of Arthus reaction. The Arthus reaction disappeared with time, and this supported the view of delayed-type hypersensitivity three weeks later. After that, an obvious chronic inflammation was admitted in 10 weeks. This resembled the histopathological feature of rheumatoid arthritis (RA) when these findings are summarized. It was suggested that arthritis produced by Str. faec. progresses from an acute condition to a one with time. As mentioned above, it thought that Arthus reaction of both immediate hypersensitivity and delayed-type hypersensitivity are necessary in the occurrence of this 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.     

Tumor Necrosis Factor,but Not Neutrophils,Alters the Metabolic Profile in Acute Experimental Arthritis

Metabolic alterations are associated with arthritis apart from obesity. However, it is still unclear which is the underlying process behind these metabolic changes. Here, we investigate the role of tumor necrosis factor (TNF) in this process in an acute model of antigen-induced arthritis (AIA). Immunized male BALB/c mice received an intra-articular injection of PBS (control) or methylated bovine serum albumin (mBSA) into their knees, and were also pre-treated with different drugs: Etanercept, an anti-TNF drug, DF2156A, a CXCR1/2 receptor antagonist, or a monoclonal antibody RB6-8C5 to deplete neutrophils. Local challenge with mBSA evoked an acute neutrophil influx into the knee joint, and enhanced the joint nociception, along with a transient systemic metabolic alteration (higher levels of glucose and lipids, and altered adipocytokines). Pre-treatment with the conventional biological Etanercept, an inhibitor of TNF action, ameliorated the nociception and the acute joint inflammation dominated by neutrophils, and markedly improved many of the altered systemic metabolites (glucose and lipids), adipocytokines and PTX3. However, the lessening of metabolic changes was not due to diminished accumulation of neutrophils in the joint by Etanercept. Reduction of neutrophil recruitment by pre-treating AIA mice with DF2156A, or even the depletion of these cells by using RB6-8C5 reduced all of the inflammatory parameters and hypernociception developed after AIA challenge, but could not prevent the metabolic changes. Therefore, the induction of joint inflammation provoked acute metabolic alterations which were involved with TNF. We suggest that the role of TNF in arthritis-associated metabolic changes is not due to local neutrophils, which are the major cells present in this model, but rather due to cytokines.     

Blockade of chemokine receptor CXCR3 inhibits T cell recruitment to inflamed joints and decreases the severity of adjuvant arthritis

T lymphocytes expressing the chemokine receptors, CCR2, CCR5, CXCR3, and CXCR6 are increased in inflamed tissues in rheumatoid arthritis. The role of CXCR3 in autoimmune arthritis induced in Lewis rats was investigated. CXCR3+ T cells migrated 2- to 3-fold more than CXCR3- T cells to inflamed joints in arthritic animals. CXCR3-expressing in vivo Ag-activated T lymphoblasts and in vitro-activated lymph node cells from arthritic animals were strongly recruited to the arthritic joints, and treatment with anti-CXCR3 mAb significantly inhibited this T cell recruitment by 40-60%. Immune T cells from the spleen and lymph nodes of actively immunized arthritic donors adoptively transferred arthritis to naive rats. Treatment with anti-CXCR3 mAb delayed the onset of arthritis and significantly reduced the severity of joint inflammation with a >50% decrease in the clinical arthritis score. Blockade of CXCR3 also significantly reduced the weight loss in the arthritic animals and inhibited neutrophil accumulation in the joints by 50-60%. There was a marked reduction in the leukocyte infiltration of the synovium in the presence of CXCR3 blockade and a decrease in the loss of articular cartilage of the joints. In conclusion, CXCR3 on T cells has an essential role in T cell recruitment to inflamed joints and the development of joint inflammation in adjuvant arthritis.     

MHC class II derived recombinant T cell receptor ligands protect DBA/1LacJ mice from collagen-induced arthritis

We previously demonstrated the therapeutic effects of MHC class II derived recombinant T cell receptor ligands (RTL), single-chain two domain complexes of the alpha1 and beta1 domains of MHC class II molecules genetically linked with an immunodominant peptide, in experimental autoimmune encephalomyelitis. In the current study, we produced a monomeric murine I-Aq-derived RTL construct covalently linked with bovine collagen type II peptide (bCII257-270) suitable for use in DBA/1LacJ mice that develop collagen-induced arthritis (CIA), an animal model of human rheumatoid arthritis, after immunization with bCII protein in CFA. In this study, we demonstrate that the I-Aq-derived RTLs reduced the incidence of the disease, suppressed the clinical and histological signs of CIA and induced long-term modulation of T cells specific for arthritogenic Ags. Our results showed that the I-Aq/bCII257-270 molecule could systemically reduce proinflammatory IL-17 and IFN-gamma production and significantly increase anti-inflammatory IL-10, IL-13, and FoxP3 gene expression in splenocytes. Moreover, I-Aq/bCII257-270 molecule could also selectively inhibit IL-1beta, IL-6, and IL-23 expression in local joint tissue. This is the first report demonstrating effective prevention of joint inflammation and clinical signs of CIA with an I-Aq-derived RTL, thus supporting the possible clinical use of this approach for treating rheumatoid arthritis in humans.     

Temporospatial expression of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases in mouse antigen-induced arthritis

Several lines of evidence speak for an important role of matrix metalloproteinases (MMPs) in the development of progressive joint destruction. To better understand the role of MMPs and their tissue inhibitors (TIMPs) in this process, we have used the antigen-induced arthritis model to study the temporospatial expression of several MMPs and TIMPs during the progression of arthritis. Arthritis was induced by a single intra-articular injection of methylated bovine serum albumin (mBSA) into one or both knee joints of adult mice previously immunised against mBSA. Samples were collected at 3, 7, 21 and 42 days after induction of arthritis for histology and RNA extraction, and analysed by Northern hybridisation, histochemistry and immunohistochemistry for production of several MMPs and TIMPs −1, −2 and −3. A systematic analysis of MMP and TIMP mRNA levels in mouse knee joints demonstrated a general upregulation of both MMPs and TIMPs during progression of arthritis. Upregulation of MMP-9, −13 and −14 coincided with the advancement of cartilage degeneration, but the expression patterns of MMP-9 and −13 also followed the course of synovial inflammation. TIMPs were steadily upregulated throughout the examination period. Immunohistochemical localisation of MMPs and TIMPs suggested the synovium to be the major source of MMP and TIMP production in arthritis, although articular cartilage chondrocytes also showed an increased production of both MMPs and TIMPs.     

Antibody to the nonapeptide Glu-Glu-Glu-Glu-Tyr-Met-Pro-Met-Glu is specific for polyoma middle T antigen and inhibits in vitro kinase activity

Middle T antigen of polyoma virus has an associated tyrosine kinase activity which phosphorylates tyrosine residue 315 on middle T in immunoprecipitates. A peptide representing the sequence of middle T from residue 311 to 319 has been synthesized. This peptide acts as a weak inhibitor of the kinase reaction. An antiserum has been raised against this peptide after conjugation to bovine serum albumin. The antibody is middle T-specific. Middle T antigen precipitated by this serum is largely inactive in the kinase reaction. Dissociation of the immune complex with peptide releases middle T in a kinase-active form.