Induction of arthritis in HLA–DR4–humanized and HLA–DQ8–humanized mice by human cartilage proteoglycan aggrecan but only in the presence of an appropriate (non‐MHC) genetic background

Objective

To determine whether the rheumatoid arthritis (RA)–predisposing class II molecules of the major histocompatibility complex (MHC) can present cartilage proteoglycan (PG) aggrecan, and if so, to determine the epitope repertoire of the human cartilage PG in HLA‐transgenic mice and determine whether HLA‐transgenic mice develop arthritis in response to immunization with human cartilage PG.

Methods

Mice transgenic for HLA–DR2.Ab⁰, DR3.Ab⁰, DR4.Ab⁰, and DQ8.Ab⁰, lacking their own (mouse) class II antigens (Ab⁰), on the original (arthritis‐resistant) and the arthritis‐susceptible BALB/c backgrounds, were immunized with human cartilage PG. The T cell epitope repertoire presented by these class II MHC alleles was determined using a synthetic peptide library (143 peptides of the core protein of human cartilage PG), and arthritis development was monitored and compared in wild‐type and HLA‐transgenic/congenic BALB/c mice.

Results

Mice of the 4 HLA‐transgenic lines, either on the original mixed, arthritis‐resistant background or DR4.Ab⁰‐ and DQ8.Ab⁰‐transgenic/congenic mice on the arthritis‐susceptible BALB/c genetic background, responded well to PG immunization (as assessed by T cell responses and antibody and cytokine production), and a number of T cell epitopes along the core protein of human cartilage PG were identified. DR4.Ab⁰‐ and DQ8.Ab⁰‐transgenic mice immunized with human cartilage PG developed arthritis, but only when these class II MHC molecules were present on the arthritis‐susceptible (BALB/c) genetic background.

Conclusion

A number of human cartilage PG epitopes can be presented by HLA alleles that predispose to the development of RA, but the epitopes of the cartilage PG presented by HLA–DR4 or HLA–DQ8 can induce arthritis only in the presence of an appropriate genetic (non‐MHC) background.

     

Specific overexpression of rheumatoid arthritis–associated HLA–DR alleles and presentation of low‐affinity peptides

Objective

To compare levels of HLA–DR expression in rheumatoid arthritis (RA) patients and healthy controls for whom an ordered expression according to the DR alleles is demonstrated and to test the functional consequences of this expression on peptide presentation.

Methods

Using monoclonal antibodies that recognize different DRB1 alleles, DR molecules were quantitated at the surface of the peripheral blood B cells of 23 RA patients and 17 healthy subjects. The functional consequences of the level of DR surface expression was tested using a universal model of antigen presentation and mutated peptides with variable affinities for the T cell receptor.

Results

In healthy subjects, surface HLA–DR molecules were expressed at different levels according to allele (DR53, DR4, and DR11 less than DR1 less than DR7 less than DR15). In RA patients, this hierarchy was not conserved and, furthermore, the density of RA‐associated DR4 and DR1 molecules was enhanced in patients compared with the basal density in healthy individuals. We demonstrated that an increased expression of DR molecules at the surface of antigen‐presenting cells allowed a noteworthy presentation of low‐affinity peptides that under normal conditions are not efficient in generating a T cell response at physiologic surface density of the DR molecules.

Conclusion

Our results suggest that the specific overexpression of RA‐associated HLA molecules could be responsible for the presentation of low‐affinity autopeptides and therefore the activation of peripheral autoreactive T cells.

     

Dendritic cells from spondylarthritis‐prone HLA–B27–transgenic rats display altered cytoskeletal dynamics,class II major histocompatibility complex expression,and viability

Objective

Spondylarthritis (SpA) is characterized by spinal and peripheral joint inflammation, frequently combined with extraarticular manifestations. Despite the well‐established association of SpA with the class I major histocompatibility complex (MHC) allele HLA–B27, there are still different, parallel hypotheses on the relationship between HLA–B27 and disease mechanisms. The present study was undertaken to investigate several characteristics of mature dendritic cells (DCs), which are believed to be essential for triggering disease in a model of SpA in HLA–B27–transgenic rats.

Methods

We combined different whole‐proteome approaches (2‐dimensional polyacrylamide gel electrophoresis and iTRAQ) to define the most aberrant molecular processes occurring in spleen DCs. Videomicroscopy and flow cytometry were used to confirm both cytoskeletal and class II MHC expression deficiencies.

Results

Our proteome studies provided evidence of up‐regulation of proteins involved in class I MHC loading, and unfolded protein response, along with a striking down‐regulation of several cytoskeleton‐reorganizing proteins. The latter result was corroborated by findings of deficient motility, altered morphology, and decreased immunologic synapse formation. Furthermore, class II MHC surface expression was reduced in DCs from B27‐transgenic rats, and this could be linked to differences in class II MHC–induced apoptotic sensitivity. Finally, we found reduced viability of the CD103+CD4− DC subpopulation, which likely exerts tolerogenic function.

Conclusion

Taken together, our findings have different important implications regarding the physiology of B27‐transgenic rat DCs, which have a putative role in spontaneous disease in these rats. In particular, the reduced motility and viability of putatively tolerogenic CD4+ DCs could play an important role in initiating the inflammatory process, resulting in SpA.

     

Endogenous HLA–DR–restricted presentation of the cartilage antigens human cartilage gp‐39 and melanoma inhibitory activity in the inflamed rheumatoid joint

Objective

The cartilage proteins melanoma inhibitory activity (MIA) and human cartilage gp‐39 (HC gp‐39) are candidate autoantigens in rheumatoid arthritis (RA). The present study was undertaken to investigate the endogenous HLA–DR4–restricted presentation of these self proteins, in order to seek in vivo evidence in support of their potential immunologic role.

Methods

MIA and HC gp‐39 were assessed in synovial fluid (SF) by enzyme‐linked immunosorbent assay and in synovial tissue (ST) by immunohistochemistry. Presentation by SF cells was investigated using specific, HLA–DR–restricted T cell hybridomas.

Results

MIA and HC gp‐39 were detected in RA SF and ST, as well as in specimens from patients with other forms of arthritis. When HC gp‐39–specific and MIA‐specific HLA–DR4–restricted T cell hybridomas raised in HLA–DR4–transgenic mice were incubated with RA SF cells as antigen‐presenting cells in the presence of HC gp‐39 or MIA peptides, the corresponding T cell hybridomas showed strong responses, which were blocked by anti–HLA–DR antibodies. Weaker but qualitatively similar responses were observed with exogenous protein, indicating uptake and processing of these antigens by SF cells. More importantly, without addition of peptide or protein, endogenous presentation of MIA and HC gp‐39 was detected in SF cells from 53% and 80% of HLA–DRB1*0401–positive RA patients, respectively. In addition, SF cells from 3 of 10 patients with spondylarthritis exhibited endogenous HC gp‐39 presentation.

Conclusion

These data indicate that immunodominant epitopes of MIA and HC gp‐39 are actively presented in an HLA–DR–restricted manner in the inflamed RA joint. The question remains as to whether this leads to activation of autoreactive T cells, which could play a role in either the immunopathology or the immunomodulation of arthritis.

     

Identification of type II collagen peptide 261–273‐specific T cell clones in a patient with relapsing polychondritis

Objective

To characterize and clone T cells specific for type II collagen (CII) in a patient with relapsing polychondritis (RP) and to establish whether the immunodominant epitope of CII determined in HLA transgenic mice is used in the human autoimmune response to CII.

Methods

T cell responses to CII were examined in a patient with RP, who was heterozygous for the HLA‐DR allele DRB1*0101/DRB1*0401. T cell clones were established from this patient and characterized for peptide specificity, class II restriction, cytokine production, and staining with HLA‐DRB1*0401 class II tetramers.

Results

A response to CII and the peptide 255–273 was present in this patient. T cells specific for the CII epitope 261–273 were cloned. Evaluation of these clones demonstrated a response to CII 261–273 in the context of both DR alleles. HLA‐DR4 CII tetramer did not demonstrate staining of either CII‐specific DRB1*0401‐restricted T cell clones or a polyclonal population of CII‐reactive T cells from this individual.

Conclusion

T cells directed against CII were present in this patient with RP. Also, T cell clones isolated from this individual were found to be specific for the CII peptide 261–273 and were restricted to either the DRB1*0101 or the DRB1*0401 allele. These findings establish that a T cell response directed against CII is present in this patient with RP and that the CII peptide 261–273 plays a role in the human immune response to CII.

     

Activation of the interferon‐γ signaling pathway in systemic lupus erythematosus peripheral blood mononuclear cells

Objective

To investigate interferon‐γ (IFNγ) signaling in peripheral blood mononuclear cells (PBMCs) from patients with systemic lupus erythematosus (SLE) by analyzing IFNγ receptor (IFNγR) expression, STAT‐1 expression and phosphorylation, and the regulation of IFNγ‐inducible genes.

Methods

Fluorocytometry was used to investigate expression of STAT‐1, pSTAT‐1, CD95, HLA–DR, class I major histocompatibility complex (MHC), IFNγ‐inducible 10‐kd protein (IP‐10), monokine induced by IFNγ (Mig), and IFNγR in PBMCs from SLE patients and healthy individuals. STAT‐1 phosphorylation was determined by fluorocytometry and Western blotting after stimulation with IFNα or IFNγ. Quantitative polymerase chain reaction was used to assess messenger RNA (mRNA) expression of the IFNγ‐inducible genes IP‐10 and Mig shortly after preparation or after stimulation with IFNγ in monocytes.

Results

STAT‐1 expression was increased in PBMCs from SLE patients and correlated significantly with disease activity and with the IFN‐inducible expression of CD95 and HLA–DR. STAT‐1 expression also showed a trend toward association with class I MHC expression. In addition, the expression of other IFNγ‐inducible genes, such as IP‐10 or Mig, was increased in SLE monocytes. While STAT‐1 phosphorylation in SLE PBMCs and PBMCs from healthy individuals was similar after IFNα stimulation, incubation with IFNγ induced STAT‐1 phosphorylation only in SLE lymphocytes. Moreover, SLE monocytes showed a considerably higher increase in pSTAT‐1 expression upon IFNγ stimulation than monocytes from healthy individuals. Increased responsiveness of SLE monocytes to IFNγ was also confirmed on the mRNA level, where expression of the IFN‐inducible, STAT‐1–dependent genes IP‐10 and Mig was more efficiently increased in SLE cells. However, IFNγR was similarly expressed on SLE lymphocytes and monocytes and those from healthy individuals.

Conclusion

In addition to supporting the role of IFNs in SLE immunopathogenesis in general, the findings of the present study support a role of IFNγ in this disease.

     

A novel human autoantigen,endothelial cell growth factor,is a target of T and B cell responses in patients with Lyme disease

Objective

Autoantigen presentation by HLA–DR molecules is thought to be a central component of many autoimmune diseases, but identifying disease‐relevant autoantigens has been a difficult challenge. In this study we aimed to identify autoantigens in patients with antibiotic‐refractory Lyme arthritis, in which infection‐induced autoimmunity is thought to play an important role.

Methods

Using tandem mass spectrometry, naturally presented HLA–DR self peptides from a patient's synovium were identified, synthesized, and reacted with his peripheral blood mononuclear cells (PBMCs). Immunoreactive peptides and their source proteins were then tested for T and B cell responses using large numbers of patient cells or sera.

Results

Of 120 HLA–DR–presented self peptides identified from one patient, one peptide derived from endothelial cell growth factor (ECGF) caused his PBMCs to proliferate. T and B cell responses to ECGF occurred systemically in ∼10–30% of patients with early or late manifestations of Lyme disease, primarily in those with refractory arthritis–associated HLA–DR alleles, such as DRB1*0101 and 0401. Compared with patients with antibiotic‐responsive arthritis, those with antibiotic‐refractory arthritis had significantly higher concentrations of ECGF in synovial fluid (P < 0.0001) and more often had ECGF antibody reactivity. Among non–antibiotic‐treated historical patients who developed arthritis, 26% had ECGF reactivity, which often developed before the onset of arthritis and was associated with significantly longer courses of arthritis.

Conclusion

T and B cell responses to ECGF occur in a subset of patients with Lyme disease, particularly in those with antibiotic‐refractory arthritis, providing the first direct evidence of autoimmune T and B cell responses in this illness.

     

Treatment of murine collagen‐induced arthritis by the stress protein BiP via interleukin‐4–producing regulatory T cells: A novel function for an ancient protein

Objective

Following the demonstration that the stress protein, BiP, prevented induction of collagen‐induced arthritis (CIA) in HLA–DRB*0101^+/+ (HLA–DR1^+/+) mice, we investigated the immunotherapeutic ability of BiP to suppress disease during the active phase of CIA in HLA–DR1^+/+ and DBA/1 mice.

Methods

BiP was administered either subcutaneously or intravenously to DBA/1, HLA–DR1^+/+, or interleukin‐4 (IL‐4)–knockout mice at the onset of arthritis. Immune cells were used in adoptive transfer studies or were restimulated in culture with BiP or type II collagen (CII). Proliferation and cytokine release were measured. In addition, serum anti‐CII antibodies were measured by enzyme‐linked immunosorbent assay. Disease progression was scored using a visual analog scale.

Results

BiP was successful in suppressing established CIA in HLA–DR1^+/+ and DBA/1 mice. Serum levels of anticollagen IgG antibodies were reduced in BiP‐treated mice. T cells from BiP‐immunized mice produced Th2 cytokines, in particular, IL‐4. Treatment with BiP was also shown to increase the production of CII‐specific IL‐5, IL‐10, and interferon‐γ at the termination of the study. Development of severe CIA was prevented by the intravenous transfer of BiP‐specific cells at the time of CIA induction in HLA–DR1^+/+ mice or by transferring BiP‐specific cells to DBA/1 mice at the onset of disease. BiP failed to ameliorate the development of CIA in IL‐4^−/−, HLA–DR1^+/+ mice.

Conclusion

These novel results show that BiP can suppress active CIA by the induction of regulatory cells that act predominantly via IL‐4. Thus, BiP is a potential immunotherapeutic agent for the treatment of patients with rheumatoid arthritis.

     

Epstein‐Barr virus load in the peripheral blood of patients with rheumatoid arthritis: Accurate quantification using real‐time polymerase chain reaction

Objective

To determine whether patients with rheumatoid arthritis (RA) have elevated Epstein‐Barr virus (EBV) load in their peripheral blood mononuclear cells (PBMCs) and whether it is correlated with the HLA–DR genes they express, we developed an accurate EBV DNA quantitative assay using real‐time polymerase chain reaction (PCR) with fluorescent probes.

Methods

We studied the EBV DNA load in the PBMCs of 84 patients with RA, 69 normal controls, and 22 patients with rheumatic conditions other than RA. A 214‐bp segment from the long internal repeat of EBV was amplified from 500 ng of PBMC DNA (150,000 cells) and quantified by real‐time PCR with fluorescent probes.

Results

We demonstrated that in patients with RA, the EBV DNA load in PBMCs is increased almost 10‐fold compared with that in normal controls. The EBV load is stable over time and is not obviously influenced by disease‐modifying antirheumatic drugs or HLA–DR.

Conclusion

Patients with RA have elevated EBV load in their peripheral blood.

     

CTLA‐4Ig blocks the development and progression of citrullinated fibrinogen–induced arthritis in DR4‐transgenic mice

Objective

To assess the role of T cells in the mouse model of citrullinated human fibrinogen–induced rheumatoid arthritis (RA) using CTLA‐4Ig, an agent that blocks T cell costimulation, which is required for T cell activation.

Methods

Humanized HLA–DRβ1*0401–transgenic (DR4‐Tg) mice were immunized with Cit–human fibrinogen to induce arthritis. Prior to, and at the onset or peak of, arthritis, the DR4‐Tg mice were treated with CTLA‐4Ig or control human IgG1 or were left untreated. Arthritis development and progression were monitored by measuring ankle swelling with calipers and by assessing histopathologic changes. The immune responses to the citrullinated antigens and the corresponding unmodified antigens, as well as the arthritogenicity of lymphocytes from these mice, were examined. The latter was performed using lymphocyte transfers from CTLA‐4Ig–treated or control mice via intraperitoneal injection into naive DR4‐Tg mice. Recipient mice also received an intraarticular injection of Cit–human fibrinogen, unmodified human fibrinogen, or vehicle.

Results

CTLA‐4Ig–treated, but not human IgG1‐treated, arthritic mice had significantly reduced ankle swelling and pathologic joint damage. Treatment with CTLA‐4Ig, but not human IgG1, suppressed Cit–human fibrinogen–induced T cell activation, including citrulline‐specific T cell activation, when given prior to disease onset. Transfer of splenic lymphocytes from untreated or human IgG1–treated arthritic mice caused arthritis in recipients, and this occurred when Cit–human fibrinogen, but not unmodified fibrinogen, was deposited into the joint. Splenocytes from CTLA‐4Ig–treated mice were unable to transfer arthritis.

Conclusion

Activated citrulline‐specific T cells play a direct role in the development and progression of arthritis in this model of Cit–human fibrinogen–induced RA.

     

Identification and functional characterization of T cells reactive to citrullinated vimentin in HLA–DRB1*0401–positive humanized mice and rheumatoid arthritis patients

Objective

Antibodies toward the citrullinated form of the synovial antigen vimentin are specific for rheumatoid arthritis (RA) and are associated with HLA–DRB1*0401. This suggests that T cells specific for peptides derived from citrullinated vimentin presented in the context of HLA–DRB1*0401 may contribute to the etiopathogenesis of RA. The aim of this study was to identify immunodominant epitopes from citrullinated vimentin presented by HLA–DRB1*0401 and to characterize the resulting T cell responses.

Methods

We first predicted an HLA‐binding T cell epitope from citrullinated vimentin based on the binding motif of HLA–DRB1*0401 and then confirmed its affinity. A class II major histocompatibility complex (MHC) tetramer loaded with the citrullinated form of vimentin aa 59–78 (cit‐vimentin aa 59–78) was constructed and used to screen for specific T cells in HLA–DRB1*0401–transgenic mice, patients with RA, and healthy control subjects. Additionally, the cytokine output following cit‐vimentin aa 59–78 challenge was analyzed in patients and healthy control subjects by multicolor flow cytometry and Luminex‐based analysis.

Results

The citrullinated form of vimentin aa 59–78 bound to HLA–DRB1*0401, but the native form could not. Subsequently, cit‐vimentin aa 59–78–specific T cells were detected in immunized mice and in the periphery of both HLA–DR*0401–positive healthy control subjects and HLA–DR*0401–positive patients with RA, using class II MHC tetramers, CD154 up‐regulation, and intracellular cytokine measurements. As demonstrated in cell culture supernatants, the production of cytokines (predominantly interferon‐γ) in response to cit‐vimentin aa 59–78 was significantly higher in patients compared with controls.

Conclusion

Here, we describe a posttranslational modification of an RA candidate autoantigen toward which HLA–DRB1*0401–restricted T cells can be detected in both patients with RA and healthy controls but for which a proinflammatory response is observed uniquely in patients with RA.

     

Interleukin‐1β induces differentiation of human mesenchymal stem cells into osteoblasts via the Wnt‐5a/receptor tyrosine kinase–like orphan receptor 2 pathway

Objective

Mesenchymal stem cells (MSCs) are considered to be a novel tool for the treatment of rheumatoid arthritis (RA) because of their multipotency to differentiate into osteoblasts and chondrocytes, their immunosuppressive effects, and availability. The aim of this study was to assess the mechanisms of human MSC differentiation into osteoblasts under inflammatory conditions.

Methods

Human MSCs were cultured in commercialized osteogenic induction medium with inflammatory cytokines for up to 10 days. Osteoblast differentiation was detected by alkaline phosphatase staining and messenger RNA (mRNA) expression of multiple osteoblast markers. Mineralization was assessed by alizarin red S staining.

Results

Among the various cytokines tested, interleukin‐1β (IL‐1β) induced differentiation of human MSCs into osteoblasts, which was confirmed by alkaline phosphatase activity, expression of RUNX2 mRNA, and strong alizarin red S staining. Among various molecules of the Wnt family, Wnt‐5a and receptor tyrosine kinase–like orphan receptor 2 (Ror2), a major receptor of Wnt‐5a, were significantly induced in human MSCs by IL‐1β. Silencing of either WNT5A or ROR2 by small interfering RNA with 2 different sequences reduced alkaline phosphatase activity, RUNX2 expression, and alizarin red S staining of human MSCs induced by IL‐1β.

Conclusion

IL‐1β effectively and rapidly induced human MSC differentiation into osteoblasts and mineralization, mainly through the noncanonical Wnt‐5a/Ror2 pathway. These results suggest potential benefits of IL‐1β–treated human MSCs in the treatment of damaged bone as well as in the induction of self‐renewal and self‐repair of damaged tissue, including osseous tissue.

     

MicroRNA‐140 is expressed in differentiated human articular chondrocytes and modulates interleukin‐1 responses

Objective

MicroRNA (miRNA) are a class of noncoding small RNAs that act as negative regulators of gene expression. MiRNA exhibit tissue‐specific expression patterns, and changes in their expression may contribute to pathogenesis. The objectives of this study were to identify miRNA expressed in articular chondrocytes, to determine changes in osteoarthritic (OA) cartilage, and to address the function of miRNA‐140 (miR‐140).

Methods

To identify miRNA specifically expressed in chondrocytes, we performed gene expression profiling using miRNA microarrays and quantitative polymerase chain reaction with human articular chondrocytes compared with human mesenchymal stem cells (MSCs). The expression pattern of miR‐140 was monitored during chondrogenic differentiation of human MSCs in pellet cultures and in human articular cartilage from normal and OA knee joints. We tested the effects of interleukin‐1β (IL‐1β) on miR‐140 expression. Double‐stranded miR‐140 (ds–miR‐140) was transfected into chondrocytes to analyze changes in the expression of genes associated with OA.

Results

Microarray analysis showed that miR‐140 had the largest difference in expression between chondrocytes and MSCs. During chondrogenesis, miR‐140 expression in MSC cultures increased in parallel with the expression of SOX9 and COL2A1. Normal human articular cartilage expressed miR‐140, and this expression was significantly reduced in OA tissue. In vitro treatment of chondrocytes with IL‐1β suppressed miR‐140 expression. Transfection of chondrocytes with ds–miR‐140 down‐regulated IL‐1β–induced ADAMTS5 expression and rescued the IL‐1β–dependent repression of AGGRECAN gene expression.

Conclusion

This study shows that miR‐140 has a chondrocyte differentiation–related expression pattern. The reduction in miR‐140 expression in OA cartilage and in response to IL‐1β may contribute to the abnormal gene expression pattern characteristic of OA.

     

The effect of HLA–DR on susceptibility to rheumatoid arthritis is influenced by the associated lymphotoxin α–tumor necrosis factor haplotype

Objective

HLA–DRB1, a major genetic determinant of susceptibility to rheumatoid arthritis (RA), is located within 1,000 kb of the gene encoding tumor necrosis factor (TNF). Because certain HLA–DRB1*04 subtypes increase susceptibility to RA, investigation of the role of the TNF gene is complicated by linkage disequilibrium (LD) between TNF and DRB1 alleles. By adequately controlling for this LD, we aimed to investigate the presence of additional major histocompatibility complex (MHC) susceptibility genes.

Methods

We identified 274 HLA–DRB1*04–positive cases of RA and 271 HLA–DRB1*04–positive population controls. Each subject was typed for 6 single‐nucleotide polymorphisms within a 4.5‐kb region encompassing TNF and lymphotoxin α (LTA). LTA–TNF haplotypes in these unrelated individuals were determined using a combination of family data and the PHASE software program.

Results

Significant differences in LTA–TNF haplotype frequencies were observed between different subtypes of HLA–DRB1*04. The LTA–TNF haplotypes observed were very restricted, with only 4 haplotypes constituting 81% of all haplotypes present. Among individuals carrying DRB1*0401, the LTA–TNF 2 haplotype was significantly underrepresented in cases compared with controls (odds ratio 0.5 [95% confidence interval 0.3–0.8], P = 0.007), while in those with DRB1*0404, the opposite effect was observed (P = 0.007).

Conclusion

These findings suggest that the MHC contains genetic elements outside the LTA–TNF region that modify the effect of HLA–DRB1 on susceptibility to RA.

     

A new model for an etiology of rheumatoid arthritis: Smoking may trigger HLA–DR (shared epitope)–restricted immune reactions to autoantigens modified by citrullination

Objective

To investigate whether smoking and HLA–DR shared epitope (SE) genes may interact in triggering immune reactions to citrulline‐modified proteins.

Methods

In a case–control study involving patients with recent‐onset rheumatoid arthritis (RA), we studied interactions between a major environmental risk factor (smoking), major susceptibility genes included in the SE of HLA–DR, and the presence of the most specific autoimmunity known for RA (i.e., antibodies to proteins modified by citrullination). Immunostaining for citrullinated proteins in cells from bronchoalveolar lavage fluid was used to investigate whether smoking is associated with citrullination in the lungs.

Results

Previous smoking was dose‐dependently associated with occurrence of anticitrulline antibodies in RA patients. The presence of SE genes was a risk factor only for anticitrulline‐positive RA, and not for anticitrulline‐negative RA. A major gene–environment interaction between smoking and HLA–DR SE genes was evident for anticitrulline‐positive RA, but not for anticitrulline‐negative RA, and the combination of smoking history and the presence of double copies of HLA–DR SE genes increased the risk for RA 21‐fold compared with the risk among nonsmokers carrying no SE genes. Positive immunostaining for citrullinated proteins was recorded in bronchoalveolar lavage cells from smokers but not in those from nonsmokers.

Conclusion

We identified an environmental factor, smoking, that in the context of HLA–DR SE genes may trigger RA‐specific immune reactions to citrullinated proteins. These data thus suggest an etiology involving a specific genotype, an environmental provocation, and the induction of specific autoimmunity, all restricted to a distinct subset of RA.

     

Influence of HLA–DR genes on the production of rheumatoid arthritis–specific autoantibodies to citrullinated fibrinogen

Objective

Antibodies directed against citrullinated fibrinogen are highly specific for rheumatoid arthritis (RA). This study was undertaken to test whether RA‐associated HLA–DR alleles are associated with anti–citrullinated fibrinogen in RA patient sera and whether replacement of arginyl by citrullyl residues on fibrinogen peptides modifies their binding to HLA–DR molecules and their recognition by T cells.

Methods

Antikeratin, antifilaggrin, and anti–citrullinated fibrinogen antibodies were assayed in RA patients who had undergone HLA–DR typing. Direct assays were performed to investigate binding of citrullinated or native fibrinogen peptides (encompassing the entire α‐ and β‐chains of fibrinogen) to purified HLA–DR molecules. T cell proliferative responses to citrullinated or native fibrinogen peptides were measured in RA patients and controls.

Results

HLA–DRB1*0404 was associated with anti–citrullinated fibrinogen in RA sera (P = 0.002). For the RA‐associated alleles HLA–DRB1*0401 and HLA–DR1, there was a nonsignificant trend toward association (P = 0.07). Multiple peptides from the α‐ and β‐chains of fibrinogen bound many HLA–DR alleles; DRB1*0404 was the best fibrinogen peptide binder. Citrullination did not influence fibrinogen peptide binding to HLA–DR or fibrinogen peptide recognition by T cells. Peripheral blood T cells that recognized native or citrullinated fibrinogen peptides were common in RA patients but not in healthy controls.

Conclusion

The RA‐associated HLA–DRB1*0404 allele is also associated with production of antibodies to citrullinated fibrinogen. DRB1*0401 and DRB1*01 tend to be associated with anti–citrullinated fibrinogen, but this is not statistically significant. Citrullination of fibrinogen peptide does not influence peptide–DR–T cell interaction. Finally, T cell proliferation in response to citrullinated or uncitrullinated fibrinogen peptides is frequent in RA patients and very infrequent in controls.

     

Autoimmunity and inflammation are independent of class II transactivator type PIV–dependent class II major histocompatibility complex expression in peripheral tissues during collagen‐induced arthritis

Objective

To determine the regulation of class II major histocompatibility complex (MHC) expression in fibroblast‐like synoviocytes (FLS) in order to investigate their role as nonprofessional antigen‐presenting cells in collagen‐induced arthritis (CIA).

Methods

Expression of class II MHC, class II MHC transactivator (CIITA), and Ciita isoforms PI, PIII, and PIV was examined by real‐time quantitative polymerase chain reaction, immunohistochemistry, and flow cytometry in human synovial tissues, arthritic mouse joints, and human and murine FLS. CIA was induced in mice in which isoform PIV of Ciita was knocked out (PIV^−/−), in PIV^−/− mice transgenic for CIITA in the thymus (K14 CIITA), and in their control littermates.

Results

HLA–DRA, total CIITA, and CIITA PIII messenger RNA levels were significantly increased in synovial tissue samples from patients with rheumatoid arthritis compared with the levels in tissue from patients with osteoarthritis. Human FLS expressed surface class II MHC via CIITA PIII and PIV, while class II MHC expression in murine FLS was entirely mediated by PIV. Mice with a targeted deletion of CIITA PIV lack CD4+ T cells and were protected against CIA. The expression of CIITA was restored in the thymus of PIV^−/− K14 CIITA–transgenic mice, which had a normal CD4+ T cell repertoire and normal surface levels of class II MHC on professional antigen‐presenting cells, but did not induce class II MHC on FLS. Synovial inflammation and immune responses against type II collagen were similar in PIV^−/− K14 CIITA–transgenic mice and control mice with CIA, but bone erosion was significantly reduced in the absence of PIV.

Conclusion

Overexpression of class II MHC is tightly correlated with CIITA expression in arthritic synovium and in FLS. Selective targeting of Ciita PIV in peripheral tissues abrogates class II MHC expression by murine FLS but does not protect against inflammation and autoimmune responses in CIA.

     

Cell therapy using allogeneic bone marrow mesenchymal stem cells prevents tissue damage in collagen‐induced arthritis

Objective

Mesenchymal stem cells (MSCs) are precursors of tissue of mesenchymal origin, but they also have the capacity to regulate the immune response by suppressing T and B lymphocyte proliferation in a non–major histocompatibility complex–restricted manner. Use of MSCs as immunosuppressant agents in autoimmune diseases has been proposed and successfully tested in animal models. We explored the feasibility of using allogeneic MSCs as therapy for collagen‐induced arthritis, a mouse model for human rheumatoid arthritis.

Methods

DBA/1 mice were immunized with type II collagen in Freund's complete adjuvant, and some of the animals received an intraperitoneal injection of allogeneic MSCs.

Results

A single injection of MSCs prevented the occurrence of severe, irreversible damage to bone and cartilage. MSCs induced hyporesponsiveness of T lymphocytes as evidenced by a reduction in active proliferation, and modulated the expression of inflammatory cytokines. In particular, the serum concentration of tumor necrosis factor α was significantly decreased. MSCs exerted their immunomodulatory function by educating antigen‐specific Tregs.

Conclusion

Our results suggest an effective new therapeutic approach to target the pathogenic mechanism of autoimmune arthritis using allogeneic MSCs. However, further studies are required before these results can be translated to clinical settings.