Vivianne Malmström

Isolation and functional characterization of regulatory CD25brightCD4+ T cells from the target organ of patients with rheumatoid arthritis

In the homeostasis of the immune system regulatory cells play a major role. Removal of one group of regulatory cells, the CD25+CD4+ T cells, leads to autoimmune manifestations in experimental animal models, and reintroduction of this population prevents disease. This study addresses the role of such regulatory T cells in humans with an autoimmune disease, where we demonstrate the presence of CD25brightCD4+ T cells in the target organ of patients with active rheumatoid arthritis. The patients displayed an enrichment of CD25brightCD4+ T cells in synovial fluid as compared to peripheral blood. These cells are functional regulatory cells, as they were able to suppress in vitro proliferation of autologous T cells, bothfrom synovial and peripheral blood origin. Although the frequency of CD25brightCD4+ T cells varied between patients, it was found to be constant over time in any one joint during each relapse. Numbers were also comparable in two inflamed knee joints of one and the same patient, emphasizing the symmetry of the disease. In summary, it is striking that in addition to all activated, potentially pathological T cells the synovial fluid from RA patients also contains CD25‐expressing CD4+ T cells with a regulatory capacity.

Autoimmunity to specific citrullinated proteins gives the first clues to the etiology of rheumatoid arthritis

Summary:  Rheumatoid arthritis (RA) is now clearly a true autoimmune disease with accumulating evidence of pathogenic disease‐specific autoimmunity to citrullinated proteins. Citrullination, also termed deimination, is a modification of arginine side chains catalyzed by peptidylarginine deiminase (PAD) enzymes. This post‐translational modification has the potential to alter the structure, antigenicity, and function of proteins. In RA, antibodies to cyclic citrullinated peptides are now well established for clinical diagnosis, though we argue that the identification of specific citrullinated antigens, as whole proteins, is necessary for exploring pathogenic mechanisms. Four citrullinated antigens, fibrinogen, vimentin, collagen type II, and α‐enolase, are now well established, with others awaiting further characterization. All four proteins are expressed in the joint, and there is evidence that antibodies to citrullinated fibrinogen and collagen type II mediate inflammation by the formation of immune complexes, both in humans and animal models. Antibodies to citrullinated proteins are associated with HLA ‘shared epitope’ alleles, and autoimmunity to at least one antigenic sequence, the CEP‐1 peptide from citrullinated α‐enolase (KIHAcitEIFDScitGNPTVE), shows a specific association with HLA‐DRB1*0401, *0404, 620W PTPN22, and smoking. Periodontitis, in which Porphyromonas gingivalis is a major pathogenic bacterium, has been linked to RA in epidemiological studies and also shares similar gene/environment associations. This is also the only bacterium identified that expresses endogenous citrullinated proteins and its own bacterial PAD enzyme, though the precise molecular mechanisms of bacterial citrullination have yet to be explored. Thus, both smoking and Porphyromonas gingivalis are attractive etiological agents for further investigation into the gene/environment/autoimmunity triad of RA.

CD25brightCD4+regulatory T cells are enriched in inflamed joints of patients with chronic rheumatic disease

CD25+CD4+ regulatory T cells participate in the regulation of immune responses. We recently demonstrated the presence of CD25brightCD4+ regulatory T cells with a capacity to control T cell proliferation in the joints of patients with rheumatoid arthritis. Here, we investigate a possible accumulation of these regulatory T cells in the inflamed joint of different rheumatic diseases including rheumatoid arthritis. The studies are also extended to analyze whether cytokine production can be suppressed by the regulatory T cells. Synovial fluid and peripheral blood samples were obtained during relapse from 36 patients with spondyloarthropathies, 21 adults with juvenile idiopathic arthritis and 135 patients with rheumatoid arthritis, and the frequency of CD25brightCD4+ T cells was determined. Of 192 patients, 182 demonstrated a higher frequency of CD25brightCD4+ T cells in synovial fluid than in peripheral blood. In comparison with healthy subjects, the patients had significantly fewer CD25brightCD4+ T cells in peripheral blood. For functional studies, synovial fluid cells from eight patients were sorted by flow cytometry, and the suppressive capacity of the CD25brightCD4+ T cells was determined in in vitro cocultures. The CD25brightCD4+ T cells suppressed the production of both type 1 and 2 cytokines including interleukin-17, as well as proliferation, independently of diagnosis. Thus, irrespective of the inflammatory joint disease investigated, CD25brightCD4+ T cells were reduced in peripheral blood and enriched in the joint, suggesting an active recruitment of regulatory T cells to the affected joint. Their capacity to suppress both proliferation and cytokine secretion might contribute to a dampening of local inflammatory processes.

Synovial fluid is a site of citrullination of autoantigens in inflammatory arthritis.

OBJECTIVE To examine synovial fluid as a site for generating citrullinated antigens, including the candidate autoantigen citrullinated alpha-enolase, in rheumatoid arthritis (RA). METHODS Synovial fluid was obtained from 20 patients with RA, 20 patients with spondylarthritides (SpA), and 20 patients with osteoarthritis (OA). Samples were resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by staining with Coomassie blue and immunoblotting for citrullinated proteins, alpha-enolase, and the deiminating enzymes peptidylarginine deiminase type 2 (PAD-2) and PAD-4. Proteins from an RA synovial fluid sample were separated by 2-dimensional electrophoresis, and each protein was identified by immunoblotting and mass spectrometry. Antibodies to citrullinated alpha-enolase peptide 1 (CEP-1) and cyclic citrullinated peptide 2 were measured by enzyme-linked immunosorbent assay. RESULTS Citrullinated polypeptides were detected in the synovial fluid from patients with RA and patients with SpA, but not in OA samples. Alpha-enolase was detected in all of the samples, with mean levels of 6.4 ng/microl in RA samples, 4.3 ng/microl in SpA samples, and <0.9 ng/microl in OA samples. Two-dimensional electrophoresis provided evidence that the alpha-enolase was citrullinated in RA synovial fluid. The citrullinating enzyme PAD-4 was detected in samples from all 3 disease groups. PAD-2 was detected in 18 of the RA samples, in 16 of the SpA samples, and in none of the OA samples. Antibodies to CEP-1 were found in 12 of the RA samples (60%), in none of the SpA samples, and in 1 OA sample. CONCLUSION These results highlight the importance of synovial fluid for the expression of citrullinated autoantigens in inflammatory arthritis. Whereas the expression of citrullinated proteins is a product of inflammation, the antibody response remains specific for RA.

Antibodies to several citrullinated antigens are enriched in the joints of rheumatoid arthritis patients.

OBJECTIVE High titers of specific anti-citrullinated protein antibodies (ACPAs) are frequently present in the serum of rheumatoid arthritis (RA) patients, but their presence in synovial fluid is less well characterized. The purpose of this study was to compare the levels of antibody to 4 well-defined citrullinated candidate RA autoantigens in serum and synovial fluid and to determine whether antibodies to one citrullinated antigen are dominant over another. Furthermore, we studied their relationships with mutated citrullinated vimentin (MCV), a newly identified RA-specific serum assay, and the classic cyclic citrullinated peptide (CCP) in the synovial fluid of well-defined HLA-DR groups. METHODS Paired serum and synovial fluid samples from 290 RA patients and serum samples from 100 age- and sex-matched healthy controls were analyzed for the presence of anti-MCV and anti-CCP antibodies and for reactivity to citrullinated fibrinogen, alpha-enolase, type II collagen, and vimentin. A total of 219 of the 290 patients were genotyped for the HLA-DR shared epitope alleles. RESULTS Significantly higher proportions of antibodies against all RA-associated citrullinated antigens were found in synovial fluid as compared with serum. This was also true for the MCV and CCP responses but not for non-RA-associated anti-tetanus toxoid antibodies. As expected, we found a high correlation between citrullinated vimentin and MCV responses. All synovial fluid ACPAs were predominantly associated with HLA-DRB1*04 alleles and were confined to the CCP+/MCV+ subset of patients. CONCLUSION MCV and CCP positivity represent a similar subset of RA patients, whereas ACPAs with different fine specificities fall into subgroups of anti-CCP+/anti-MCV+ patients. The levels of all specific ACPAs were elevated in synovial fluid, suggesting that there is local antibody production and/or retention of ACPAs at the site of inflammation governed by RA-predisposing genes.

Multiple antibody reactivities to citrullinated antigens in sera from patients with rheumatoid arthritis: association with HLA-DRB1 alleles

Background: Autoantibodies to cyclic citrullinated peptides (anti-CCP) are present in most patients with rheumatoid arthritis (RA), and associate with HLA-DRB1 shared epitope (SE) alleles. Objective: To investigate reactivities of anti-CCP to various citrullinated proteins/peptides, which represent potential autoantigens in RA, and to examine the relationship between such antibodies, and their association with genetic variants within HLA-DRB1 SE alleles. Methods: Serum samples from 291 patients with established RA and 100 sex- and age-matched healthy subjects were included in this study. Sera were first analysed for presence of anti-CCP antibodies and further for IgG and IgA antibodies towards candidate autoantigens in both their native and citrullinated form including: fibrinogen, α-enolase peptide-1 and the C1-epitope of type II collagen (C1III). Antibody specificity was confirmed by cross-reactivity tests. HLA-DR genotyping was performed. Results: 72% of patients with RA were anti-CCP positive. Among the candidate autoantigens examined, IgG antibodies to citrullinated fibrinogen were found in 66% of patients’ sera and in 41% for both citrullinated α-enolase peptide-1 and citrullinated C1III. These antibodies were mainly seen in the anti-CCP-positive patient group; they were specific for their respective antigen and displayed limited cross reactivity. IgA responses were also detected, but less frequently than IgG. Anti-CCP and anti-citrullinated protein antibodies were associated with HLA-DRB1*04 rather than with HLA-DRB1*01 alleles. Conclusions: Antibodies directed against several citrullinated antigens are present in CCP-positive RA, with many patients displaying multireactivity. All specific reactivities were primarily associated with the HLA-DRB1*04 alleles, suggesting common pathways of anti-citrulline immunity.

Monoclonal IgG antibodies generated from joint-derived B cells of RA patients have a strong bias toward citrullinated autoantigen recognition

Synovial IgG-expressing B cells from patients with rheumatoid arthritis show specificity for citrullinated autoantigens.

Smoking, citrullination and genetic variability in the immunopathogenesis of rheumatoid arthritis.

This review describes how studies on interactions between genetic variants, and environmental factors, mainly smoking, contribute to the understanding of how autoimmunity to post-translationally (citrullinated) proteins/peptides may occur and potentially contribute to certain subsets of rheumatoid arthritis. A main message is that studies on specific immune mechanisms in a complex and heterogeneous disease like RA should be undertaken with the help of results from genetic epidemiology. By those means, it may be possible to identify subsets of RA in a way that in the end allows development and testing of precise and subset-specific interventions against environment as well as genetically defined molecular pathways, in particular those that regulate specific immune responses.

Mucosal tolerance and suppression of collagen-induced arthritis (CIA) induced by nasal inhalation of synthetic peptide 184-198 of bovine type II collagen (CII) expressing a dominant T cell epitope

The purpose of the study was to map the dominant T cell epitope of the CB11 sequence of CII in RT1u haplotype rats and to determine if, when used as a synthetic peptide, it would induce tolerance to protect against CIA. A dominant epitope corresponding to residues 184‐198 included in the sequence of the CB11 fragment of bovine CII was identified in proliferation assay using peptides in an epitope scanning system using synthetic peptides of 15 amino acids, overlapping by 12 amino acids. This epitope is bovine‐specific, but cross‐reacts with the corresponding rat peptide. Minor epitopes in the bovine CB11 sequence were also autoantigenic. Use of independently synthesized and purified 184‐198 peptide confirmed its dominance in the T cell responses of arthritic rats. The peptide itself was not arthritogenic. Cells from lymph nodes draining arthritic feet were particularly responsive to the dominant peptide sequence, and showed evidence of epitope spreading to include reactions to at least four subdominant epitopes. Mucosal tolerance was successfully induced by instilling CII into the nose of rats before induction of CIA; this was found to delay the onset of disease, reduce mean disease severity, shift the anti‐CII antibody response to favour antibodies of the IgGl, rather than the IgG2b isiotype, and to reduce T cell reactivity to both CII and to the 184‐198 peptide. The dominant 184‐198 peptide itself had the same tolerogenic effects when given nasally to rats daily, on the 4 days immediately preceding the induction of CIA. Two forms of CIA with acute and delayed disease onset were each modified by pre‐treatment with the peptide. This study demonstrates that mucosal tolerance to CII can be induced by delivering it nasally in a way similar to that achieved previously by oral delivery, and that the use of an immunodominant epitope contained in a synthetic peptide will also suppress the immunologic and arthritic responses to collagen.

Differential effects on BAFF and APRIL levels in rituximab-treated patients with systemic lupus erythematosus and rheumatoid arthritis

The objective of this study was to investigate the interaction between levels of BAFF (B-cell activation factor of the tumour necrosis factor [TNF] family) and APRIL (a proliferation-inducing ligand) and B-cell frequencies in patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) treated with the B-cell-depleting agent rituximab. Ten patients with SLE were treated with rituximab in combination with cyclophosphamide and corticosteroids. They were followed longitudinally up to 6 months after B-cell repopulation. Nine patients with RA, resistant or intolerant to anti-TNF therapy, treated with rituximab plus methotrexate were investigated up to 6 months after treatment. The B-cell frequency was determined by flow cytometry, and serum levels of BAFF and APRIL were measured by enzyme-linked immunosorbent assays. BAFF levels rose significantly during B-cell depletion in both patient groups, and in patients with SLE the BAFF levels declined close to pre-treatment levels upon B-cell repopulation. Patients with SLE had normal levels of APRIL at baseline, and during depletion there was a significant decrease. In contrast, patients with RA had APRIL levels 10-fold higher than normal, which did not change during depletion. At baseline, correlations between levels of B cells and APRIL, and DAS28 (disease activity score using 28 joint counts) and BAFF were observed in patients with RA. In summary, increased BAFF levels were observed during absence of circulating B cells in our SLE and RA patient cohorts. In spite of the limited number of patients, our data suggest that BAFF and APRIL are differentially regulated in different autoimmune diseases and, in addition, differently affected by rituximab treatment.