Presentation of arthritogenic peptide to antigen‐specific T cells by fibroblast‐like synoviocytes

Objective

To assess the ability of rheumatoid arthritis (RA) fibroblast‐like synoviocytes (FLS) to function as antigen‐presenting cells (APCs) for arthritogenic autoantigens found within inflamed joint tissues.

Methods

Human class II major histocompatibility complex (MHC)–typed FLS were used as APCs for murine class II MHC–restricted CD4 T cell hybridomas. Interferon‐γ (IFNγ)–treated, antigen‐loaded FLS were cocultured with T cell hybridomas specific for immunodominant portions of human cartilage gp‐39 (HC gp‐39) or human type II collagen (CII). T cell hybridoma activation was measured by enzyme‐linked immunosorbent assay of culture supernatants for interleukin‐2. Both synthetic peptide and synovial fluid (SF) were used as sources of antigen. APC function in cocultures was inhibited by using blocking antibodies to human class II MHC, CD54, or CD58, or to murine CD4, CD11a, or CD2.

Results

Human FLS could present peptides from the autoantigens HC gp‐39 and human CII to antigen‐specific MHC‐restricted T cell hybridomas. This response required pretreatment of FLS with IFNγ, showed MHC restriction, and was dependent on human class II MHC and murine CD4 for effective antigen presentation. Furthermore, FLS were able to extract and present antigens found within human SF to both the HC gp‐39 and human CII T cell hybridomas in an IFNγ‐dependent and MHC‐restricted manner.

Conclusion

RA FLS can function as APCs and are able to present peptides derived from autoantigens found within joint tissues to activated T cells in vitro. In the context of inflamed synovial tissues, FLS may be an important and hitherto overlooked subset of APCs that could contribute to autoreactive immune responses.

     

Activated memory B cells may function as antigen‐presenting cells in the joints of children with juvenile idiopathic arthritis

Objective

B cells impact the perpetuation of chronic inflammatory or autoimmune diseases in multiple ways. A role of B cells as antigen‐presenting cells (APCs) in the pathogenesis of chronic arthritis in humans has been suggested; however, as of yet the presence of such B cells at the site of inflammation has not been demonstrated. This study was undertaken to investigate whether synovial B cells in patients with juvenile idiopathic arthritis (JIA) might display features of APCs.

Methods

The frequency, phenotype, and immunoglobulin repertoire of synovial B cells were studied by flow cytometry and single‐cell polymerase chain reaction (PCR). Cytokine expression by B cells was analyzed by real‐time PCR, and interaction between B cells and T cells was investigated in a mixed lymphocyte culture.

Results

CD27+IgD− and CD27−IgD− B cells accumulated in the joints of JIA patients and displayed an activated phenotype. Both B cell subsets expressed hypermutated and class‐switched immunoglobulins, indicators of memory B cells. The accumulating memory B cells expressed the costimulatory molecules CD80/CD86 and showed a higher capacity to activate allogeneic T cells and prime a Th1 phenotype than their peripheral blood counterparts.

Conclusion

Activated immunoglobulin class–switched CD27− and CD27+ memory B cells, indicating a phenotype of APCs with expression of costimulatory molecules, accumulate in the joints of patients with JIA and might be involved in the amplification of pathogenic T cell activation. These findings provide evidence that B cells play an antibody‐independent immunopathologic role in human chronic inflammatory arthritis of childhood.

     

Disruption of the interaction of T cells with antigen-presenting cells by the active leflunomide metabolite teriflunomide: involvement of impaired integrin activation and immunologic synapse formation

OBJECTIVE: Leflunomide, a potent disease-modifying antirheumatic drug of the isoxazole class, exhibits antiinflammatory, antiproliferative, and immunosuppressive effects by largely unknown mechanisms, although alterations of pyrimidine synthesis have been proposed. Successful immune responsiveness requires T cell activation by interaction with antigen-presenting cells (APCs), and integrin activation and formation of an immunologic synapse (IS). In this study, we evaluated the impact of the active leflunomide metabolite teriflunomide on T cell integrin activation, evolution of the IS, and antigen-specific formation of stable T cell/APC conjugates. METHODS: Effects of pharmacologic concentrations of teriflunomide on CD3/CD28- and lymphocyte function-associated antigen 1-induced signal transduction and activation of primary human T cells were investigated. Furthermore, T cells were stimulated with superantigen- and antigen-pulsed APCs to study relocalization of molecules to the IS and T cell/APC conjugate formation. RESULTS: Teriflunomide inhibited T cell receptor (TCR)/CD3-mediated calcium mobilization, but other critical T cell signaling events, including activation of MAPK and NF-kappaB, remained unaltered. In contrast, inhibition of TCR/CD3-triggered beta1,2 integrin avidity and integrin-mediated costimulation (outside-in signaling) by teriflunomide revealed a striking interference with integrin function that was independent of altered pyrimidine synthesis. Moreover, teriflunomide abolished molecule relocalization to the IS and induction of T cell/APC conjugates. CONCLUSION: These data show that the active metabolite of leflunomide prevents the interaction of T cells with APCs to form an IS. Since IS formation is crucial for eliciting an immune response, this novel mechanism could underlie the beneficial effects of leflunomide in immune-mediated disorders such as rheumatoid arthritis.     

Activation signal transduction by β1 integrin in T cells from patients with systemic lupus erythematosus

Objective

Beta 1 integrin is a representative adhesion molecule for cell–cell and cell–extracellular matrix interactions, and it provides costimulatory signals to T cells. However, the relevance of β1 integrin to T cell activation in systemic lupus erythematosus (SLE) remains unclear. We undertook this study to perform a quantitative and functional analysis of β1 integrin–mediated signaling to T cells in patients with SLE.

Methods

Expression of cell surface molecules was assessed by flow cytometric analysis. Engagement of β1 integrins was performed by crosslinking using a specific monoclonal antibody. To assess tyrosine kinases in β1 integrin–mediated signaling, the cells were transfected with a wild‐type (WT) focal adhesion kinase (FAK), a dominant‐negative truncation of the FAK, or a WT PTEN expression plasmid via nucleofection.

Results

Beta 1 integrin expression was significantly up‐regulated on peripheral blood T cells from patients with active SLE, particularly those with the complication of World Health Organization class IV nephritis, whereas CD28 was significantly decreased in patients with active SLE compared with normal individuals. Beta 1 integrin expression closely correlated with serum hypocomplementemia. Engagement of β1 integrin on T cells from patients with active SLE, but not on those from normal individuals, induced cell proliferation as well as CD40L expression on T cells. Up‐regulation of CD40L expression and T cell proliferation, induced by β1 integrin stimulation, were completely inhibited by transfection of the dominant‐negative truncations of FAK or WT PTEN.

Conclusion

These results suggest that engagement of β1 integrins on SLE T cells could induce FAK‐mediated signaling and subsequent CD40L expression and proliferation. Thus, the β1 integrin signaling cascade might serve to enhance autoreactive T cell activation.

     

Defective costimulatory function is a striking feature of antigen‐presenting cells in an HLA–B27–transgenic rat model of spondylarthropathy

Objective

A disease resembling the human spondylarthropathies develops in HLA–B27–transgenic rats. This disease in rats is mediated by CD4+ T cells, but antigen‐presenting cells (APCs) may also play a role. Dendritic cells (DCs) have been reported to be defective in allogeneic mixed lymphocyte culture in this model. Here, we further investigated the functional defect of APCs.

Methods

DCs and B cells from nontransgenic, HLA–B27 (33–3)–transgenic, and HLA–B7 (120–4)–transgenic rats were used to stimulate T cells. Surface expression of HLA–B transgene and rat molecules on APCs and the formation of conjugates between DCs and T cells were monitored by flow cytometry.

Results

We observed a strikingly defective stimulation of allogeneic and syngeneic T lymphocytes by APCs from HLA–B27 but not HLA–B7 rats, even if stimulation was driven in the presence of anti–T cell receptor (TCR) antibody. We found no evidence that HLA–B27 DCs were immature, lacked production of some diffusible factor, or produced an inhibitory factor for T cells. When comparing the levels of expression of class II major histocompatibility complex, CD2, intercellular adhesion molecule 1, lymphocyte function–associated antigen 1, B7, and CD40 molecules at the surface of DCs from 33‐3, 120‐4, and nontransgenic rats, we found little difference. However, HLA–B27–transgenic DCs formed fewer conjugates with T cells than did nontransgenic DCs. Furthermore, the proportion of conjugates formed between DCs and T cells, as well as the difference between nontransgenic and HLA–B27–transgenic DCs, were in large part reduced by blocking CD86 on DCs.

Conclusion

We confirmed defective stimulation of T cells by APCs in HLA–B27 rats, the mechanism of which appears to implicate APC/T cell contact, independent of TCR engagement. In addition, decreased use of the CD86 costimulatory molecule by B27 DCs was observed. Impaired costimulatory function could result in a loss of tolerance toward microbial flora in this model.

     

Selective ERK activation differentiates mouse and human tolerogenic dendritic cells,expands antigen‐specific regulatory T cells,and suppresses experimental inflammatory arthritis

Objective

Most therapeutic treatments for autoimmune arthritis rely on immunosuppressive drugs, which have side effects. Although a previous study by our group showed that specific ERK activation suppressed immune responses, its application in a therapeutic setting has never been tested. The aim of the present study was to define the ERK‐dependent immunosuppressive mechanisms and to apply selective ERK activation for the treatment of experimental inflammatory arthritis.

Methods

A constitutively active ERK activator was coexpressed with a model antigen using lentivectors. Immunosuppressive mechanisms were characterized at the level of dendritic cell (DC) function, differentiation of antigen‐specific Treg cells, and inhibition of inflammatory T cells. Administration of the ERK activator with antigen as a strategy to suppress inflammatory arthritis was tested in an experimental mouse model.

Results

Selective ERK activation induced mouse and human DCs to secrete bioactive transforming growth factor β, a process required for suppression of T cell responses and differentiation of antigen‐specific Treg cells. Treg cells strongly proliferated after antigen reencounter in inflammatory conditions, and these cells exhibited antigen‐dependent suppressive activities. Inflammatory arthritis was effectively inhibited through antigen‐specific mechanisms. Importantly, this strategy did not rely on identification of the initiating arthritogenic antigen. Equivalent mechanisms were demonstrated in human monocyte–derived DCs, setting the scene for a possible rapid translation of this approach to patients with rheumatoid arthritis.

Conclusion

This strategy of selective ERK activation resulted in an effective therapeutic protocol, with substantial advantages over DC or T cell vaccination.

     

HLA–B27–restricted antigen presentation by human chondrocytes to CD8+ T cells: Potential contribution to local immunopathologic processes in ankylosing spondylitis

Objective

Analysis of the histopathologic features of hip arthritis in patients with ankylosing spondylitis (AS) has revealed accumulation of infiltrating mononuclear cells in the bone end plate and presence of hyaline articular cartilage that is not found in areas of total cartilage destruction. This study was undertaken to assess whether chondrocytes attract lymphocytes and whether cartilage chondrocytes from patients with AS have the potential to directly stimulate T cells in an HLA‐restricted manner.

Methods

Human HLA–B27+ T cell lines, specific for the Epstein‐Barr virus–derived peptide EBNA_258–266, and autologous chondrocytes, serving as nonprofessional antigen‐presenting cells (APCs), were available for use in a model system to study chondrocyte functions in femoral head joint cartilage of patients with AS. Peptide functionality of cytotoxic T cells was assessed by flow cytometry, and cellular interactions were detected by fluorescence confocal microscopy.

Results

When maintained in an alginate matrix, chondrocytes isolated from the femoral heads of patients with AS constitutively expressed type II collagen and CD80. When pulsed with the EBNA_258–266 peptide, autologous chondrocytes functioned as APCs and, specifically, induced interferon‐γ production in CD8+ T cells. In mixed chondrocyte–T cell cultures, cell–cell contacts were dependent on the presence of the EBNA_258–266 peptide. T cells adjacent to chondrocytes produced perforin and granzyme B; both molecules were found in focal aggregates, a prerequisite for antigen‐specific lysis of target cells.

Conclusion

Antigen presentation through human chondrocytes allows the stimulation of peptide‐specific CD8+ T cells. These results indicate that human chondrocytes can act as nonprofessional APCs, and suggest that there is an interferon‐γ–triggered autocrine loop of immune cell–mediated chondrocyte activation in the already inflamed environment. Thus, local HLA‐dependent activation of peptide‐specific cytotoxic CD8+ T cells by chondrocytes might contribute to inflammatory processes in the spondylarthritides.

     

A thrombin–cross‐reactive anticardiolipin antibody binds to and inhibits the anticoagulant function of activated protein C

Objective

To test the hypotheses that some thrombin‐reactive anticardiolipin antibodies (aCL) may bind to protein C (PC) and/or activated PC (APC), and that some of the PC‐ and APC‐reactive aCL may inhibit PC activation and/or the function of APC.

Methods

We studied the reactivity of patient‐derived monoclonal aCL with PC and APC. We examined the effects of the reactive antibodies on PC activation and on the activity of APC in plasma coagulation.

Results

Five of 5 patient‐derived, thrombin‐reactive monoclonal aCL bound to PC and APC. In addition, 1 patient‐derived monoclonal antiprothrombin antibody (APT) that displayed aCL activity and reacted with thrombin also bound to PC and APC. Of these 6 PC‐ and APC‐reactive aCL/APT, all failed to inhibit PC activation, but 1 (CL15) shortened the plasma coagulation time in the presence of exogenous APC and thus inhibited the anticoagulant function of APC.

Conclusion

Most of the thrombin‐reactive aCL in patients with antiphospholipid syndrome may bind to PC and APC. Of the APC‐reactive aCL, some (like CL15) may inhibit the anticoagulant function of APC and are thus likely to be prothrombotic in the host.

     

Occurrence of an activated,profibrotic pattern of gene expression in lung CD8+ T cells from scleroderma patients

Objective

Pulmonary fibrosis is a major cause of death in scleroderma patients. Previous studies have shown an increase in CD8+ T cells in the lungs of scleroderma patients. In the present study, we sought to determine whether activated CD8+ T cells contribute to pulmonary fibrosis in scleroderma patients through the production and activation of profibrotic mediators.

Methods

CD8+ cells were isolated from bronchoalveolar lavage fluid obtained from 19 scleroderma patients and 7 healthy subjects. The phenotype of these cells was determined using DNA array technology. Expression of selected genes was confirmed in real‐time polymerase chain reaction and enzyme‐linked immunosorbent assay experiments.

Results

Hierarchical clustering of gene expression profiles revealed 2 groups of subjects. Group 1 consisted of 11 patients (8 with and 3 without lung inflammation). Group 2 consisted of 15 subjects (7 healthy controls and 2 patients with and 6 without lung inflammation). Gene expression in group 1 indicated T cell activation, a type 2 phenotype, production of profibrotic factors and matrix metalloproteinases, and reduced activation‐induced cell death. Increased expression of β6 integrin messenger RNA by CD8+ T cells in group 1 suggested the possibility that these T cells might induce cell‐contact–dependent activation of latent transforming growth factor β (TGFβ).

Conclusion

A subset of scleroderma patients at higher risk of progressive lung disease have activated, long‐lived CD8+ T cells in their lungs that could promote fibrosis directly, through production of profibrotic factors such as interleukin‐4 and oncostatin M, as well as indirectly, through activation of TGFβ.

     

Abatacept (CTLA‐4IG) treatment reduces the migratory capacity of monocytes in patients with rheumatoid arthritis

Objective

The binding of abatacept (CTLA‐4Ig) to the B7 ligands CD80 and CD86 prevents the engagement of CD28 on T cells and thereby prevents effector T cell activation. In addition, a direct effect of CTLA‐4Ig on antigen‐presenting cells (APCs) could contribute to the therapeutic effect. To further elucidate the mechanism of CTLA‐4Ig, we performed phenotype and functional analyses of APCs in patients with rheumatoid arthritis (RA) before and after the initiation of CTLA‐4Ig therapy.

Methods

Peripheral blood mononuclear cells were analyzed before and at 2 and 4 weeks after the initiation of CTLA‐4Ig therapy. Proportions of APCs were determined by flow cytometry. CD14+ monocytes were further analyzed for the expression of costimulatory and adhesion molecules and for their transendothelial migratory capacity in vitro. In addition, CD14+ monocytes from healthy controls were analyzed for their migratory and spreading capacity.

Results

Proportions and absolute numbers of monocytes were significantly increased in RA patients treated with CTLA‐4Ig. The expression of several adhesion molecules was significantly diminished. In addition, monocytes displayed a significant reduction in their endothelial adhesion and transendothelial migratory capacity upon treatment with CTLA‐4Ig. Likewise, isolated monocytes from healthy controls revealed a significant reduction in their migratory and spreading activity after preincubation with CTLA‐4Ig or anti‐CD80 and anti‐CD86 antibodies.

Conclusion

We describe direct effects of CTLA‐4Ig therapy on phenotype and functional characteristics of monocytes in RA patients that might interfere with the migration of monocytes to the synovial tissue. This additional mechanism of CTLA‐4Ig might contribute to the beneficial effects of CTLA‐4Ig treatment in RA patients.

     

The antirheumatic drug leflunomide inhibits osteoclastogenesis by interfering with receptor activator of NF‐κB ligand–stimulated induction of nuclear factor of activated T cells c1

Objective

Suppression of bone destruction is required as part of an effective therapeutic strategy for autoimmune arthritis. Although numerous antirheumatic drugs are in clinical use, little is known about whether they inhibit bone destruction by acting on activated T cells or other cell types, such as bone‐resorbing osteoclasts. This study was undertaken to determine whether leflunomide has a direct action on the osteoclast lineage and to gain insights into the molecular basis for the bone‐protective effect of leflunomide.

Methods

The direct effect of leflunomide on osteoclast differentiation was investigated using an in vitro culture system of bone marrow monocyte/macrophages stimulated with receptor activator of NF‐κB ligand (RANKL) and macrophage colony‐stimulating factor. The molecular mechanism of the inhibition was analyzed by genome‐wide screening. The T cell–independent effect of leflunomide was examined in rag‐2^−/− mice.

Results

Leflunomide blocked de novo pyrimidine synthesis and RANKL‐induced calcium signaling in osteoclast precursor cells in vitro; hence, the induction of nuclear factor of activated T cells c1 (NF‐ATc1) was strongly inhibited. The inhibition of this pathway is central to the action of leflunomide, since the inhibition was overcome by ectopic expression of NF‐ATc1 in the precursor cells. Leflunomide suppressed endotoxin‐induced inflammatory bone destruction even in rag‐2^−/− mice.

Conclusion

Leflunomide has a direct inhibitory effect on RANKL‐mediated osteoclast differentiation by inhibiting the induction of NF‐ATc1, the master switch regulator for osteoclast differentiation. Our study suggests that the direct inhibitory action of leflunomide on osteoclast differentiation constitutes an important aspect in the amelioration of bone destruction, and that the RANKL‐dependent NF‐ATc1 induction pathway is a promising target for pharmacologic intervention in arthritic bone destruction.

     

Regulation of pulmonary inflammation and fibrosis through expression of integrins αVβ3 and αVβ5 on pulmonary T lymphocytes

Objective

Pulmonary diseases associated with fibrosis, including scleroderma lung disease, are characterized by the accumulation of T cells in the lungs. These cells are thought to facilitate lung fibrosis, but the exact mechanisms of their profibrotic action are not clear. Several αV‐containing integrins, including αVβ3 and αVβ5, have been shown to directly activate transforming growth factor β (TGFβ) and promote collagen accumulation. The aim of this study was to investigate whether pulmonary T cells express profibrotic integrins and regulate collagen accumulation.

Methods

Expression of integrins was assessed by immunohistochemical analysis of lung tissue, by flow cytometry using bronchoalveolar lavage fluid from patients with systemic sclerosis (SSc), and in a CCL18 overexpression animal model of pulmonary T cell infiltration. Experiments in cell cultures were performed to determine whether integrin‐expressing T cells are profibrotic in cocultures with pulmonary fibroblasts and, if so, through what possible mechanism.

Results

Lymphocytes and integrin‐positive cells were present in the lungs, and pulmonary T cells expressed integrins αVβ3 and αVβ5 in patients with SSc and in the animal model. Systemic administration of neutralizing anti–integrin αV antibody or a genetic deficiency of integrin β3 in the CCL18 overexpression model significantly attenuated CCL18‐driven pulmonary lymphocytic infiltration and collagen accumulation. Jurkat T cells overexpressing integrin αVβ3 or integrin αVβ5 in cocultures with primary pulmonary fibroblasts stimulated collagen accumulation and Smad2 nuclear translocation. Neutralizing anti‐TGFβ antibody attenuated the profibrotic effect of integrin‐expressing T cells.

Conclusion

Pulmonary infiltrating T lymphocytes may express integrins αVβ3 and αVβ5 that are necessary for lymphocytic infiltration and T cell–associated TGFβ activation and collagen accumulation.

     

Antigen‐specific immunomodulation of collagen‐induced arthritis with tumor necrosis factor–stimulated dendritic cells

Objective

Dendritic cells (DCs) are crucial for the initiation of T cell immunity and therefore play an important role in the initiation and regulation of immune responses in arthritis. Full mobilization of effector T cells depends on the proper maturation of DCs. Current evidence indicates that the type of T cell response induced is crucially dependent on the activation status of the DCs. In this study, we explored the immunologic effects of differentially matured DCs on the development of collagen‐induced arthritis (CIA).

Methods

Bone marrow–derived DCs were cultured in the presence of granulocyte–macrophage colony‐stimulating factor (GM‐CSF). Before immunization with bovine type II collagen (CII) protein, mice were repeatedly injected with DCs that had been pulsed with CII. Immature, semimature, or fully mature DCs were injected. Mice were boosted on day 21 after CII immunization, and the disease course was monitored.

Results

While vaccination with immature or lipopolysaccharide‐activated DCs had no significant effect on the disease course, administration of antigen‐loaded, tumor necrosis factor (TNF)–modulated DCs propagated in GM‐CSF with or without interleukin‐4 resulted in a delayed onset of arthritis and a lower clinical score. The response was antigen‐specific, since TNF‐treated DCs pulsed with a control antigen did not modify the disease course. A specific decrease in the collagen‐specific “Th1‐associated” IgG2a response was observed, whereas IgG1 titers were unaffected.

Conclusion

CIA can be prevented through vaccination with TNF‐matured DCs in an antigen‐specific manner. These findings provide a rationale for immunotherapy using DCs in rheumatoid arthritis.

     

CD28‐dependent differentiation into the effector/memory phenotype is essential for induction of arthritis in interleukin‐1 receptor antagonist–deficient mice

Objective

Interleukin‐1 receptor antagonist (IL‐1Ra)–deficient mice on a BALB/c background spontaneously develop a chronic inflammatory polyarthropathy closely resembling that of rheumatoid arthritis in humans. To elucidate the role of CD28 costimulatory signals in the development of this disease, we studied IL‐1Ra/CD28–double‐deficient mice.

Methods

We crossed IL‐1Ra–deficient mice with CD28–deficient mice and observed the incidence and severity of arthritis. To investigate functions of IL‐1Ra/CD28–double‐deficient T cells, cells were stimulated with CD3 monoclonal antibody or allogeneic antigen‐presenting cells (APCs) and their proliferative responses and levels of cytokine production were measured.

Results

Disease severity was lower in IL‐1Ra/CD28–double‐deficient mice than in mice that were deficient only in IL‐1Ra, although incidence of arthritis was not affected by the presence or absence of CD28. When pathogenic IL‐1Ra–KO T cells were transferred into nude mice, severe arthritis developed. Even though T cells from double‐deficient mice showed the same diminished proliferative capacity as was seen in T cells from CD28–single‐deficient animals, nude mice into which double‐deficient T cells were transferred never developed arthritis.

Conclusion

These findings indicate that IL‐1Ra/CD28–double‐deficient T cells can be activated by IL‐1Ra–deficient activated APCs, resulting in induction of arthritis; however, these T cells did not induce the disease under normal conditions, because they did not differentiate into effector/memory phenotype.

     

Effector CD8+ T cells in systemic sclerosis patients produce abnormally high levels of interleukin‐13 associated with increased skin fibrosis

Objective

T lymphocytes play an important role in systemic sclerosis (SSc), a connective tissue disease characterized by inflammation, fibrosis, and vascular damage. While their precise role and antigen specificity are unclear, T cell–derived cytokines likely contribute to the induction of fibrosis. The aim of this study was to establish the role of cytokine dysregulation by T cells in the pathogenesis of SSc.

Methods

To identify relationships between a specific cytokine, T cell subset, and the disease course, we studied a large cohort of patients with diffuse cutaneous SSc (dcSSc) or limited cutaneous SSc (lcSSc). Using Luminex analysis and intracellular cytokine staining, we analyzed the intrinsic ability of CD4+ and CD8+ T cell subsets to produce cytokines following in vitro activation.

Results

High levels of the profibrotic type 2 cytokine interleukin‐13 (IL‐13) were produced following activation of peripheral blood effector CD8+ T cells from SSc patients as compared with normal controls or with patients with rheumatoid arthritis. In contrast, CD4+ T cells showed a lower and more variable level of IL‐13 production. This abnormality correlated with the extent of fibrosis and was more pronounced in dcSSc patients than in lcSSc patients.

Conclusion

Dysregulated IL‐13 production by effector CD8+ T cells is important in the pathogenesis of SSc and is critical in the predisposition to more severe forms of cutaneous disease. Our study is the first to identify a specific T cell phenotype that correlates with disease severity in SSc and can be used as a marker of immune dysfunction in SSc and as a novel therapeutic target.

     

The critical role of kinase activity of interleukin‐1 receptor–associated kinase 4 in animal models of joint inflammation

Objective

We have previously reported that the kinase activity of interleukin‐1 receptor–associated kinase 4 (IRAK‐4) is important for Toll‐like receptor and interleukin‐1 receptor signaling in vitro. Using mice devoid of IRAK‐4 kinase activity (IRAK‐4 KD mice), we undertook this study to determine the importance of IRAK‐4 kinase function in complex disease models of joint inflammation.

Methods

IRAK‐4 KD mice were subjected to serum transfer–induced (K/BxN) arthritis, and migration of transferred spleen lymphocytes into joints and cartilage and bone degradation were assessed. T cell response in vivo was tested in antigen‐induced arthritis (AIA) by measuring the T cell–dependent antigen‐specific IgG production and frequency of antigen‐specific T cells in the spleen and lymph nodes. T cell allogeneic response was tested in vitro by mixed lymphocyte reaction (MLR).

Results

Lipopolysaccharide‐induced local neutrophil influx into subcutaneous air pouches was impaired in IRAK‐4 KD mice. These mice were also protected from inflammation in the K/BxN and AIA models, as shown by reduced swelling of joints. Histologic analysis of joints of K/BxN serum–injected mice revealed that bone erosion, osteoclast formation, and cartilage matrix proteoglycan loss were reduced in IRAK‐4 KD mice. Assessment of T cell response by MLR, by frequency of antigen‐specific clones, and by production of antigen‐specific IgG did not reveal substantial differences between IRAK‐4 KD and wild‐type mice.

Conclusion

These results demonstrate that IRAK‐4 is a key component for the development of proarthritis inflammation, but that it is not crucial for T cell activation. Therefore, the kinase function of IRAK‐4 appears to be an attractive therapeutic target in chronic inflammation.

     

Constitutive expression of BCL‐XL in the T lineage attenuates collagen‐induced arthritis in Bcl‐XL transgenic mice

Objective

To determine if inhibition of T cell apoptosis through constitutive expression of Bcl‐X _L in the T lineage influences inflammatory arthritis in the mouse collagen‐induced arthritis (CIA) model.

Methods

The incidence and severity of arthritis were quantified in Bcl‐X _L transgenic mice and nontransgenic littermates after immunization with type II collagen (CII). To correlate T cell responses with disease phenotype, antigen‐specific T cell proliferation was measured by ³H‐thymidine incorporation. Apoptosis and cell cycle progression were analyzed by flow cytometry using propidium iodide. Production of CII‐specific interferon‐γ (IFNγ), interleukin‐5 (IL‐5), and IL‐10 was determined by enzyme‐linked immunosorbent assay.

Results

Disease severity in CIA was significantly attenuated in Bcl‐X _L transgenic mice compared with their nontransgenic littermates. Inhibition of CIA was associated with decreased T cell apoptosis, delayed cell cycle progression, and reduced IFNγ production.

Conclusion

Rather than promoting inflammation, inhibition of apoptosis by expression of the Bcl‐X _L protein in the T lineage attenuates disease progression in CIA, probably through inhibition of IFNγ production.

     

Reduction of the incidence and severity of collagen‐induced arthritis by constitutive Nur77 expression in the T cell lineage

Objective

To test the hypothesis that constitutive expression of Nur77 in the T cell lineage will suppress the development and pathogenesis of collagen‐induced arthritis (CIA) and to understand the mechanisms by which Nur77 overexpression influences the arthritogenic response to type II collagen (CII).

Methods

Nur77‐transgenic and wild‐type C57BL/6 mice were immunized with CII and were monitored for the development and severity of arthritis. Pathologic changes were examined by histology and radiography. The effects of Nur77 overexpression on immune responses were evaluated by cytokine production in vitro and serum levels of CII‐specific antibodies. Sensitivity of T cells to apoptosis induction was analyzed in vitro following stimulation with anti‐CD3 monoclonal antibody or glucocorticoid.

Results

The incidence and severity of CIA was significantly decreased in Nur77‐transgenic mice compared with wild‐type controls. Attenuation of the disease was associated with increased apoptosis induction in transgenic T cells and decreased production of CII‐specific IgG2a antibodies in transgenic mice. Overexpression of Nur77 in the T cell compartment did not affect Th1/Th2 cytokine production or balance.

Conclusion

Nur77 overexpression in the T cell lineage attenuates the development and progression of CIA, probably by promoting activation‐induced T cell apoptosis and by inhibiting CII‐specific antibody production.

     

Treatment of experimental arthritis by inducing immune tolerance with human adipose‐derived mesenchymal stem cells

Objective

Rheumatoid arthritis (RA) is a chronic autoimmune disease caused by loss of immunologic self tolerance and characterized by chronic joint inflammation. Adult mesenchymal stem cells (MSCs) were recently found to suppress effector T cell responses and to have beneficial effects in various immune disorders. The purpose of this study was to examine a new therapeutic strategy for RA based on the administration of human adipose‐derived MSCs (AD‐MSCs).

Methods

DBA/1 mice with collagen‐induced arthritis were treated with human AD‐MSCs after disease onset, and clinical scores were determined. Inflammatory response was determined by measuring the levels of different mediators of inflammation in the joints and serum. The Th1‐mediated autoreactive response was evaluated by determining the proliferative response and cytokine profile of draining lymph node cells stimulated with the autoantigen. The number of Treg cells and the suppressive capacity on self‐reactive Th1 cells were also determined.

Results

Systemic infusion of human AD‐MSCs significantly reduced the incidence and severity of experimental arthritis. This therapeutic effect was mediated by down‐regulating the 2 deleterious disease components: the Th1‐driven autoimmune and inflammatory responses. Human AD‐MSCs decreased the production of various inflammatory cytokines and chemokines, decreased antigen‐specific Th1/Th17 cell expansion, and induced the production of antiinflammatory interleukin‐10 in lymph nodes and joints. Human AD‐MSCs also induced de novo generation of antigen‐specific CD4+CD25+FoxP3+ Treg cells with the capacity to suppress self‐reactive T effector responses.

Conclusion

Human AD‐MSCs emerge as key regulators of immune tolerance by inducing the generation/activation of Treg cells and are thus attractive candidates for a cell‐based therapy for RA.

     

Induction of costimulation of human CD4 T cells by tumor necrosis factor–related apoptosis‐inducing ligand: Possible role in T cell activation in systemic lupus erythematosus

Objective

Tumor necrosis factor–related apoptosis‐inducing ligand (TRAIL) has recently been shown to induce costimulation of mouse T cells in conjunction with signals from the T cell receptor. This study was undertaken to investigate TRAIL‐induced costimulation of human T cells in order to determine the role of TRAIL‐induced T cell activation in human systemic lupus erythematosus (SLE).

Methods

An in vitro T cell stimulation system with immobilized anti‐CD3 and recombinant TRAIL receptor DR4‐Fc proteins was used to activate human T cells purified from healthy individuals and from patients with SLE. The T cells were stimulated in vitro to assay their proliferation response by ³H‐thymidine incorporation, and their cytokine production by enzyme‐linked immunosorbent assay. Activation of p38 MAPK after TRAIL stimulation was detected with specific anti–phospho‐p38 MAPK monoclonal antibodies in Western blots.

Results

Enhanced T cell proliferation and increased interleukin‐2 and interferon‐γ (IFNγ) production were demonstrated in human T cells after stimulation with immobilized DR4‐Fc and anti‐CD3 in vitro. TRAIL engagement selectively activated human CD4, rather than CD8, T cells and augmented IFNγ production. Activation of p38 MAPK was detected after TRAIL‐induced T cell activation. T cells isolated from patients with SLE demonstrated a stronger response to TRAIL‐induced costimulation, in terms of proliferation and increased up‐regulation of CD25 after activation, when compared with T cells from healthy subjects.

Conclusion

TRAIL engagement induces costimulation of human CD4 T cells via a p38 MAPK–dependent pathway. The results suggest that enhanced reactivity of T cells to autoantigens as a result of TRAIL‐induced costimulation may play a role in the development of human autoimmune diseases.