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Objectives: Anticitrullinated protein antibodies (ACPA) are specifically associated with rheumatoid arthritis (RA) and produced in inflamed synovial membranes where citrullinated fibrin, their antigenic target, is abundant. We showed that immune complexes containing IgG ACPA (ACPA-IC) induce FcγR-mediated tumour necrosis factor (TNF)-α secretion in macrophages. Since IgM rheumatoid factor (RF), an autoantibody directed to the Fc fragment of IgG, is also produced and concentrated in the rheumatoid synovial tissue, we evaluated its influence on macrophage stimulation by ACPA-IC., Methods: With monocyte-derived macrophages from more than 40 healthy individuals and different human IgM cryoglobulins with RF activity, using a previously developed human in vitro model, we evaluated the effect of the incorporation of IgM RF into ACPA-IC., Results: IgM RF induced an important amplification of the TNF-α secretion. This effect was not observed in monocytes and depended on an increase in the number of IgG-engaged FcγR. It extended to the secretion of interleukin (IL)-1β and IL-6, was paralleled by IL-8 secretion and was not associated with overwhelming secretion of IL-10 or IL-1Ra. Moreover, the RF-induced increased proinflammatory bioactivity of the cytokine response to ACPA-IC was confirmed by an enhanced, not entirely TNF-dependent, capacity of the secreted cytokine cocktail to prompt IL-6 secretion by RA synoviocytes., Conclusions: By showing that it can greatly enhance the proinflammatory cytokine response induced in macrophages by the RA-specific ACPA-IC, these results highlight a previously undescribed, FcγR-dependent strong proinflammatory potential of IgM RF. They clarify the pathophysiological link between the presence of ACPA and IgM RF, and RA severity., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Systemic vasculitides constitute a heterogeneous group of diseases. Autoimmunity mediated by B lymphocytes and their humoral effector mechanisms play a major role in ANCA-associated vasculitis (AAV) as well as in non-ANCA associated primary systemic vasculitides and in the different types of autoimmune connective tissue disorders and rheumatoid arthritis. In order to detect autoantibodies in systemic vasculitides, we screened protein macroarrays of human cDNA expression libraries with sera from patients with ANCA-associated and ANCA-negative primary systemic vasculitides. This approach led to the identification of antibodies against progranulin, a 88 kDA secreted glycoprotein with strong anti-inflammatory activity in the course of disease of giant-cell arteritis/polymyalgia rheumatica (14/65), Takayasu's arteritis (4/13), classical panarteritis nodosa (4/10), Behcet's disease (2/6) and in the course of disease in granulomatosis with polyangiitis (31/75), Churg-Strauss syndrome (7/23) and in microscopic polyangiitis (7/19). In extended screenings the progranulin antibodies were also detected in other autoimmune diseases such as systemic lupus erythematosus (39/91) and rheumatoid arthritis (16/44). Progranulin antibodies were detected only in 1 of 97 healthy controls. Anti-progranulin positive patients with systemic vasculitides, systemic lupus erythematosus or rheumatoid arthritis had significant lower progranulin plasma levels, indicating a neutralizing effect. In light of the anti-inflammatory effects of progranulin, progranulin antibodies might exert pro-inflammatory effects thus contributing to the pathogenesis of the respective autoimmune diseases and might serve as a marker for disease activity. This hypothesis is supported by the fact that a positive progranulin antibody status was associated with active disease in granulomatosis with polyangiitis., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Fibroblast-like synoviocytes (FLSs) are important actors in rheumatoid arthritis (RA) pathogenesis. The autoimmune nature of RA is attributed to autoantibody production, which confers to B cells a predominant role in RA. Several arguments support an induction of class switch recombination (CSR) in RA synovium, causing--in conjunction with somatic hypermutation--the production of potentially pathogenic IgG. To determine whether RA FLSs can directly promote CSR and to analyze the role of external factors like TLR signals and BAFF (B cell activating factor) family cytokines in this FLS-B cell crosstalk, we performed cocultures of blood B cells (from normal individuals or RA patients) with RA FLSs and analyzed CSR induction by quantification of AICDA (encoding activation-induced cytidine deaminase, AID) and switch circular transcripts expression, and IgG secretion. RA FLSs--and to a lesser extent osteoarthritis or control FLSs--promoted CSR, and TLR3 stimulation potentialized it. In addition, induction of CSR by RA FLSs was totally dependent on cell-cell contact in basal conditions, and partially dependent in the case of TLR3 stimulation. Finally, we showed that the mechanism by which RA FLSs induce CSR is mostly BAFF-dependent. Our results support the hypothesis that CSR can be induced outside the ectopic lymphoid structures in RA., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Objective: Arthritis in the K/BxN mouse model results from pathogenic immunoglobulins that recognize glucose-6-phosphate isomerase (GPI), a glycolytic enzyme residing in the cytoplasm of all cells. Antibodies directed against GPI can, alone, transfer arthritis to healthy recipients. Previous experiments have revealed significant titers of anti-GPI antibodies in the serum of many patients with rheumatoid arthritis (RA). We evaluated the generality of these observations in cohorts of patients with 12 different arthritic and chronic autoimmune diseases and in population-matched healthy control subjects., Methods: Anti-GPI antibodies were assayed in 811 individual serum samples by enzyme-linked immunosorbent assay with 2 forms of GPI, recombinant and native. Results were confirmed by immunoblotting., Results: Several patients had significantly elevated anti-GPI antibody titers, but without the prevalence or the specificity reported previously. Only 15% of RA patients had anti-GPI antibodies (range 12-29% in different cohorts), with a higher prevalence in patients with active disease. Psoriatic arthritis, undifferentiated arthritis, and spondylarthropathy patients also displayed anti-GPI antibodies at similar frequencies (12-25%). Similar titers were detected in a proportion (5-10%) of control subjects or patients with Crohn's disease or sarcoidosis. Very high titers were found in rare cases of RA and systemic lupus erythematosus., Conclusion: No disease-specific pattern of antibody positivity to GPI was apparent. While the antibody-mediated mechanism at play in the mouse model may exemplify a generic mechanism for some forms of arthritis in humans, GPI itself does not appear to be a target common to the majority of RA patients.

Rheumatoid arthritis is a common and debilitating autoimmune disease whose cause and mechanism remain a mystery. We recently described a T cell receptor transgenic mouse model that spontaneously develops a disease with most of the clinical, histological, and immunological features of rheumatoid arthritis in humans. Disease development in K/BxN mice is initiated by systemic T cell self-reactivity; it requires T cells, as expected, but B cells are also needed, more surprisingly. Here, we have identified the role of B cells as the secretion of arthritogenic immunoglobulins. We suggest that a similar scenario may unfold in some other arthritis models and in human patients, beginning with pervasive T cell autoreactivity and ending in immunoglobulin-provoked joint destruction.

To better understand the structural basis for rheumatoid factor [RF] activity and the origin of autoantibodies in human autoimmune diseases, we isolated the RF producing B cells from the peripheral blood and from the synovial fluid of a patient suffering from rheumatoid arthritis [RA]. We previously demonstrated that a significant fraction of these RF were derived from three V kappa III genes known to encode most of the monoclonal RF light chain variable regions. To get more insight into the actual repertoire of RF-V kappa genes during RA, we analyzed the nucleotide sequences of RF light chain variable regions of other V kappa families. Using two sets of polymerase chain reactions in order to amplify the cDNA derived from RF producing cells from the same patient KRA, we isolated only three different rearranged V kappa-J kappa complexes: slkv5, slkv7 and bkv42, all derived from V kappa I germ-line genes not previously known to be associated with RF activity; this suggests that the repertoire of VL genes coding for RF during RA is more diverse than the one involved in the generation of paraprotein RF during monoclonal lymphoid proliferations, although there remains a possible bias in favor of the V kappa III family. Moreover, each of these genes is somatically mutated with a pattern suggesting a selective pressure of the antigen. Particularly interesting is the additional proline residue at the V kappa-J kappa junction of bkv42, an unorthodox feature that we found previously in more than 50% of RF V kappa III-J kappa gene complexes. Finally, the homogeneity of some non conservative mutations suggests the existence of a restricted set of pathogenic epitopes driving the production of RF during RA.

Much interest was stirred in recent years by the evidence that rheumatoid factors (RF) variable regions are encoded by a restricted set of V genes, with little or no somatic mutations, that are often overexpressed in the fetal repertoire. This is reminiscent of what has been observed for natural autoantibodies. However, these data come from studies of monoclonal RF (mRF) isolated from patients with lymphoproliferative disorders who usually do not present autoimmune symptoms. The molecular characterization of RF during autoimmune diseases such as rheumatoid arthritis (RA) has been hampered for some time because of their polyclonality; recently using the polymerase chain reaction method, we have demonstrated that RF kappa variable regions from a patient with RA were encoded by V kappa III genes known to code for mRF but that these genes had undergone somatic mutations with a pattern suggesting an antigen-driven maturation. Because an important role of the light chain third complementarity-determining region (CDR3) in anti-IgG reactivity and idiotype expression has already been suspected for RF, we now report the molecular characterization of the junction regions of these rearranged V kappa gens. Surprisingly, our data show that in 55% of the cases there is addition of a proline and/or glycine amino acid residue at the recombination site between V kappa and J kappa. The sequence analysis of our patients' germ-line Vg and J kappa 4 genes segments and their flanking regions demonstrates that the additional codons are not readily explicable by recombination between germ-line sequences and probably result from an N addition process. Since we could not find such an additional codon in 15 previously published mRF kappa chains we suggest that "pathogenic" RF during RA and mRF derive from different, although overlapping, B cell subsets. Moreover, since additional codons at the recombination site of V kappa and J kappa seem exceptional in expressed human kappa chains and because the resulting amino acid residue is a proline in most cases, we think that RF kappa chain CDR3 is under a very strong selective pressure during RA.

We report the first molecular characterization of V kappa regions of the main human autoantibodies occurring during rheumatoid arthritis, the polyclonal rheumatoid factors. Using two sets of polymerase chain reactions in order to amplify the cDNA derived from both peripheral blood and synovial fluid rheumatoid factor-secreting cells, nucleotide analysis of the V kappa III family usage shows the following: (a) at least three different V kappa III genes are used to encode polyclonal rheumatoid factors in a single patient, (b) each one of these genes seems more or less somatically mutated (from 1 to 14 mutations), (c) the mutation process preferentially affects the complementarity determining regions suggesting a selective pressure of antigen and (d) there is no clear difference between the mutation rates affecting the synovial fluid and peripheral blood rheumatoid factor-secreting cells. These results are able to explain some of the known idiotypic differences between monoclonal and polyclonal rheumatoid factors in humans. They also provide evidence that polyclonal autoantibodies arising during an autoimmune disease can be the products of multiple somatically mutated genes and suggest that this process is antigen driven, whether this antigen is the Fc piece of IgG or another unknown antigen.

Using the technic of Pillot and Grangeot (précipitation by PEG 6,000 at 2.5%, followed radial immuno-diffusion), the authors have detected circulating immune complexes (CIC) 38 times in a group of 55 RA. The presence of CIC is significantly more frequent in sero-positive RA, but without correlation between the concentration of CIC and the positivity rate of Waaler-Rose reaction. There is no correlation between the evolutive state of RA and the presence or the concentration of CIC. The sedimentation rate is, in mean, significantly higher in RA with CIC; but, among these, there is no correlation between sedimentation rate and the concentration of CIC. In the group studied, the CIC are present in all cases of RA with vasculitis or visceral symptoms.

The idiotypic determinants on IgM rheumatoid factor (RF) from a single family have been analyzed. Rabbit Fab'2 antiidiotypic antibody was prepared against purified IgM-RF from a patient with rheumatoid arthritis. As measured by radioimmunoassay, the antiidiotype reacted with at least 90% of the patient's RF, but not with non-RF immunoglobulins from the same serum, nor with 10 of 11 polyclonal and monoclonal RF from unrelated individuals. Cross-reacting idiotypes were detected on RF in four of the patients' first degree relatives, spanning three generations, without apparent relation of HLA type or clinical rheumatoid arthritis. These results suggest that IgM-RF associated idiotypes were inherited in this family.

In order to explain long term seronegativity in certain patients with rheumatoid arthritis (RA), we looked for the presence of anti-idiotypic antibodies against rheumatoid factors (RFs). From a patient's serum with classical but seronegative RA, we isolated a low quantity of the IgG fraction which partially recognized polyclonal RF idiotypes. The purified Fab'2 anti-idiotypic antibodies were able to inhibit up to 48% of in vitro RF production by pokeweed mitogen stimulated lymphocytes from a patient's peripheral blood with seropositive RA. The two main conclusions of this study are: (1) this single patient with seronegative RA has serum antibodies directed against RF idiotypes and (2) these anti-idiotypic antibodies could be implicated in the generation of seronegativity.

Different human IgM rheumatoid factor (IgM RF) idiotypes have been described defined by polyclonal rabbit anti-idiotypic antibodies. These antisera do not allow clear genetic analysis of the idiotypic determinants, be they cross-reactive or private. Therefore, we tried to obtain a set of monoclonal anti-idiotypic antibodies directed against RF idiotypes. Purified IgM RF serum from a patient with classical rheumatoid arthritis was used to immunize BALB/c mice. The spleen cells were fused with Sp 2/0 Ag 14, a nonsecreting mouse myeloma cell line, and a hybrid producing monoclonal anti-idiotypic antibody was selected. The mouse antibody, an IgG1 kappa, reacts with an identical or similar determinant located on (or close to) the binding site of all tested monoclonal or polyclonal IgM RF from totally unrelated patients with Waldenströms's macroglobulinemias or rheumatoid arthritis. The monoclonal antibody also reacts with 2 rheumatoid arthritis patients' IgG RF and with a low proportion of normal polyclonal IgM without detectable RF activity. An hypothesis is proposed to explain the existence of a such highly conserved determinant on RF idiotypes.

Prior studies have shown that in vitro infection with the Epstein Barr virus (EBV) is able to induce IgM rheumatoid factor production by normal lymphocytes, with a higher degree of production by seropositive rheumatoid arthritis lymphocytes. The present investigation demonstrates that EBV-infected lymphocytes from patients with seronegative rheumatoid arthritis produce in vitro significantly less IgM rheumatoid factor than do normal lymphocytes. The results suggest that the peripheral blood of seronegative patients is deficient in the rheumatoid factor precursor B cells responsive to stimulation by Epstein Barr virus.