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ObjectivesAnticitrullinated 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.MethodsWith 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.ResultsIgM 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.ConclusionsBy 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.

ObjectivesIn the inflamed synovium of patients with rheumatoid arthritis (RA), autoantibodies to citrullinated proteins (ACPA) probably form immune complexes (IC) on deposits of citrullinated fibrin. We showed that in vitro such ACPA-IC activate a pro-inflammatory cytokine response in M-CSF-differentiated macrophages. Our objective was to evaluate how macrophage polarisation influences this response.MethodsCD14-positive monocytes from healthy donors were cultured in the presence of M-CSF, IFN-γ, interleukin (IL)-4 or IL-10. Expression of markers specific for polarised macrophages was analysed by flow cytometry. Their cytokine secretion was prompted by in vitro generated autoantibodies to citrullinated proteins immune complexes (ACPA-IC) and assayed in the culture supernatants.ResultsIFN-γ-polarised cells exhibited high levels of CD64 and CD80. Low expression of CD14 and high expression of CD206 characterised the IL-4-polarised cells. Exposure to IL-10 or M-CSF raised the expression of CD14, CD32 and CD163. The two cell types lacked CD80 and exhibited similar expression of CD64, CD200R and CD206. In response to ACPA-IC, the secretion of IL-1β, IL-6 and IL-8 was similar among cells exposed to IFN-γ, IL-4 or IL-10. However, the later cells were associated with the highest IL-1Ra:IL-1β ratio and the lowest tumour necrosis factor (TNF)-α:IL-10 ratio. Conversely, M-CSF-exposed cells secreted the highest levels of pro-inflammatory cytokines, exhibited a high TNF-α:IL-10 ratio and the lowest IL-1Ra:IL-1β ratio.ConclusionsDespite their phenotypic similarity, IL-10-polarised and M-CSF-polarised macrophages clearly differ in their cytokine response to ACPA-IC. M-CSF-polarised cells exhibit the highest pro-inflammatory potential. Since M-CSF is abundant in the RA synovium, therein it probably drives macrophages towards a strong pro-inflammatory cytokine response to the locally formed ACPA-IC.

Objective To analyse Fcγ receptor (FcγR) expression on monocytes and macrophages from rheumatoid arthritis (RA) patients versus healthy controls (HC), and to compare their responses to immune complexes containing RA-specific anti-citrullinated proteins auto antibodies (ACPA). Methods Monocytes and monocyte-derived macrophages were obtained from the peripheral blood of 34 RA patients and 69 HC. FcγR expression was studied by flow cytometry. Cells were stimulated with ACPA-containing immune complexes, and tumour necrosis factor alpha (TNFα) was assayed in culture supernatants. Results Variations distinguished RA from HC monocytes, corresponding to a 5% and 6% decrease in the percentages of monocytes expressing FcγRI and FcγRII, respectively, and a 7% increase in the proportion of FcγRIII-positive monocytes. Although in both HC and RA patients macrophage differentiation was accompanied by a dramatic increase in the percentage of FcγRIII-expressing cells (72% vs 74.5%), the parallel decline in the proportion of FcγRI-positive cells was markedly smaller in RA (7% vs 43%). Monocytes and macrophages from patients were responsive to ACPA-containing immune complexes but TNFα production in both cell types neither differed from that observed with the corresponding cells from HC, nor correlated with FcγR expression or clinical or biological data. In RA as in HC, ACPA-containing immune complexes induced secretions of more TNFα in macrophages than in paired monocytes (ninefold). Finally, the proinflammatory potential of ACPA-containing immune complexes was confirmed in CD14-positive monocyte macrophages from the synovial fluid of four RA patients. Conclusions ACPA-containing immune complexes induce TNFα secretion by blood and synovial fluid-derived macrophages from RA patients, fitting with their probable involvement in RA pathophysiology.

BackgroundAutoantibodies to citrullinated proteins (ACPA) are specifically associated to rheumatoid arthritis (RA) and produced in the inflamed synovium where citrullinated fibrin, their main antigenic target, is abundant. Using a human in vitro model we showed that macrophages generated by differentiation of blood monocytes from healthy individuals or patients with RA secrete TNF- alpha in response to immune complexes formed by ACPA and citrullinated fibrinogen (ACPA-IC). Moreover while in both healthy individuals and RA patients the TNF- alpha response of macrophages was much higher than that of their monocyte precursors, the TNF- alpha production of blood monocytes and monocyte-derived macrophages from RA patients did not differ from that observed with the healthy controls.ObjectivesTo further assess the impact of ACPA-IC on joint inflammation, we evaluated the TNF- alpha response they prompt in monocyte-macrophages isolated from the synovial fluid (SF) of patients with RA and with other arthritides.Materials and MethodsSF samples were obtained from 7 patients with RA (4 ACPA-positive, 3 ACPA-negative) and 8 ACPA-negative control patients with various arthritides. Polymorphonuclear cells were eliminated by centrifugation over Ficoll or capture with CD15-conjugated magnetic beads then monocyte-macrophages further purified using CD14-beads (median purity: 95%). The purified cells were stimulated with IC generated by capture of ACPA from IgG fractions prepared from RA sera, on immobilised citrullinated fibrinogen, as described (Clavel, Arthritis Rheum, 2008).ResultsWith SF monocyte-macrophages from both RA and control patients, secretion of TNF- alpha sometimes occurred in the absence of any stimulation. However in RA patients, irrespective of their ACPA status, TNF- alpha secretion increased when the cells were cultured on ACPA-IC (median (range) = 16 (2-110) pg/ml). Such activation was also observed with the SF monocyte-macrophages from control patients (184 (33-638) pg/ml). In the whole series of SF samples, the increase in TNF- alpha secretion was found to be highly significant (p < 0.001).ConclusionsIn contrast with our previous observations on CD14-positive blood monocytes and on derived macrophages, it appears that CD14-positive monocyte-macrophages can be pre-activated in the SF and secrete TNF- alpha spontaneously, but that they can nevertheless be further activated by ACPA-IC. These properties are not restricted to RA patients and seem to be characteristic for the SF CD14-positive monocyte-macrophages.Since citrullinated fibrin and ACPA have been described in the SF of RA patients, it is highly probable that ACPA-IC play a direct pro-inflammatory role in the SF by inducing or enhancing TNF- alpha secretion by the SF monocyte-macrophages.

Background Autoantibodies to citrullinated proteins (ACPA) are specifically associated to rheumatoid arthritis (RA) and produced in the inflamed synovium where citrullinated fibrin, their main antigenic target, is abundant. Using a human in vitro model we showed that macrophages generated by differentiation of blood monocytes from healthy individuals or patients with RA secrete TNF-α in response to immune complexes formed by ACPA and citrullinated fibrinogen (ACPA-IC). Moreover while in both healthy individuals and RA patients the TNF-α response of macrophages was much higher than that of their monocyte precursors, the TNF-α production of blood monocytes and monocyte-derived macrophages from RA patients did not differ from that observed with the healthy controls. Objectives To further assess the impact of ACPA-IC on joint inflammation, we evaluated the TNF-α response they prompt in monocyte-macrophages isolated from the synovial fluid (SF) of patients with RA and with other arthritides. Materials and Methods SF samples were obtained from 7 patients with RA (4 ACPA-positive, 3 ACPA-negative) and 8 ACPA-negative control patients with various arthritides. Polymorphonuclear cells were eliminated by centrifugation over Ficoll or capture with CD15-conjugated magnetic beads then monocyte-macrophages further purified using CD14-beads (median purity: 95%). The purified cells were stimulated with IC generated by capture of ACPA from IgG fractions prepared from RA sera, on immobilised citrullinated fibrinogen, as described (Clavel, Arthritis Rheum, 2008). Results With SF monocyte-macrophages from both RA and control patients, secretion of TNF-α sometimes occurred in the absence of any stimulation. However in RA patients, irrespective of their ACPA status, TNF-α secretion increased when the cells were cultured on ACPA-IC (median (range) = 16 (2–110) pg/ml). Such activation was also observed with the SF monocyte-macrophages from control patients (184 (33–638) pg/ml). In the whole series of SF samples, the increase in TNF-α secretion was found to be highly significant (p < 0.001). Conclusions In contrast with our previous observations on CD14-positive blood monocytes and on derived macrophages, it appears that CD14-positive monocyte-macrophages can be pre-activated in the SF and secrete TNF-α spontaneously, but that they can nevertheless be further activated by ACPA-IC. These properties are not restricted to RA patients and seem to be characteristic for the SF CD14-positive monocyte-macrophages. Since citrullinated fibrin and ACPA have been described in the SF of RA patients, it is highly probable that ACPA-IC play a direct pro-inflammatory role in the SF by inducing or enhancing TNF-α secretion by the SF monocyte-macrophages.

Background Autoantibodies to citrullinated proteins (ACPA) are specifically associated to rheumatoid arthritis (RA) and produced in the inflamed synovium where citrullinated fibrin, their main antigenic target, is abundant. Using a human in vitro model we showed that macrophages generated by differentiation of blood monocytes from healthy individuals or patients with RA secrete TNF-α in response to immune complexes formed by ACPA and citrullinated fibrinogen (ACPA-IC). Moreover while in both healthy individuals and RA patients the TNF-α response of macrophages was much higher than that of their monocyte precursors, the TNF-α production of blood monocytes and monocyte-derived macrophages from RA patients did not differ from that observed with the healthy controls. Objectives To further assess the impact of ACPA-IC on joint inflammation, we evaluated the TNF-α response they prompt in monocyte-macrophages isolated from the synovial fluid (SF) of patients with RA and with other arthritides. Materials and Methods SF samples were obtained from 7 patients with RA (4 ACPA-positive, 3 ACPA-negative) and 8 ACPA-negative control patients with various arthritides. Polymorphonuclear cells were eliminated by centrifugation over Ficoll or capture with CD15-conjugated magnetic beads then monocyte-macrophages further purified using CD14-beads (median purity: 95%). The purified cells were stimulated with IC generated by capture of ACPA from IgG fractions prepared from RA sera, on immobilised citrullinated fibrinogen, as described (Clavel, Arthritis Rheum, 2008). Results With SF monocyte-macrophages from both RA and control patients, secretion of TNF-α sometimes occurred in the absence of any stimulation. However in RA patients, irrespective of their ACPA status, TNF-α secretion increased when the cells were cultured on ACPA-IC (median (range) = 16 (2-110) pg/ml). Such activation was also observed with the SF monocyte-macrophages from control patients (184 (33-638) pg/ml). In the whole series of SF samples, the increase in TNF-α secretion was found to be highly significant (p < 0.001). Conclusions In contrast with our previous observations on CD14-positive blood monocytes and on derived macrophages, it appears that CD14-positive monocyte-macrophages can be pre-activated in the SF and secrete TNF-α spontaneously, but that they can nevertheless be further activated by ACPA-IC. These properties are not restricted to RA patients and seem to be characteristic for the SF CD14-positive monocyte-macrophages. Since citrullinated fibrin and ACPA have been described in the SF of RA patients, it is highly probable that ACPA-IC play a direct pro-inflammatory role in the SF by inducing or enhancing TNF-α secretion by the SF monocyte-macrophages.

Background Autoantibodies to citrullinated proteins (ACPA) are suspected to play a central role in the pathophysiology of rheumatoid arthritis (RA). By stimulating monocyte-derived macrophages from healthy donors with ACPA-containing immune complexes (ACPA-IC), the authors previously established the inflammatory potential of ACPA through FcγRIIa engagement. Furthermore, the authors evidenced a major enhancement of tumour necrosis factor α (TNFα) secretion when these IC also included monoclonal IgM rheumatoid factors (RFs) derived from patients with cryoglobulinemia (ACPA/RF-IC). Objectives To further investigate the inflammatory effect of ACPA-IC including or not RF, the authors evaluated the response of blood monocytes to ACPA/RF-IC and specified the cytokine profile induced in macrophages by ACPA-IC and ACPA/RF-IC. Materials and methods Three monoclonal IgM RF were purified from the serum of patients with mixed cryoglobulinemia. Monocyte purification, macrophage differentiation and macrophage stimulation by IC in the absence of complement were performed as previously described (Clavel et al, Arthritis Rheum, 2008). In culture supernatants, TNFα was assayed by ELISA, and interleukin 1β (IL-1β), IL-6, IL-8, IL-10 and IL-1Ra were measured in a multiplexed flow cytometric assay. Results Stimulation by ACPA-IC and ACPA/RF-IC was compared in monocyte and macrophage pairs derived from three donors. While, as expected, in macrophages, TNFα secretions were higher after stimulation by ACPA/RF-IC, in monocytes only very low TNFα secretions were detected in both conditions. Stimulations with immobilised and IgG-conjugated RF in four monocyte and macrophage pairs confirmed these results. In macrophages from eight blood donors, in addition to inducing TNFα, ACPA-IC also importantly raised IL-8 secretion while no significant secretions of IL-1β, IL-6 or IL-10 were detected. Secretion of IL-1Ra was found to be enhanced in two out of two donors tested. For three of the eight donors the macrophages were additionally stimulated with ACPA/RF-IC. In all these donors, compared to stimulation with ACPA-IC, IL-10 secretion was enhanced. However, this was compensated by an important upregulation of TNFα, by the induction of IL-1β and IL-6 secretion, and persistence of high IL-8 levels. Moreover, IL-1Ra was down modulated in two out of two tested donors. Conclusions Monocytes are refractory to stimulation by IgM RF-containing IC, suggesting that circulating IgM RF-containing IC remain fairly innocuous as far as FcR engagement of circulating monocytes is concerned. In addition, this data clearly confirm the identification of RF as a potent amplifier of the FcR-dependent inflammatory reaction induced in macrophages by the tightly RA-associated ACPA-IC. They clarify the link between disease aggressiveness and both RF and ACPA.

Objective To analyse Fc[GAMMA] receptor (Fc[GAMMA]R) expression on monocytes and macrophages from rheumatoid arthritis (RA) patients versus healthy controls (HC), and to compare their responses to immune complexes containing RA-specific anti-citrullinated proteins auto antibodies (ACPA). Methods Monocytes and monocyte-derived macrophages were obtained from the peripheral blood of 34 RA patients and 69 HC. Fc[GAMMA]R expression was studied by flow cytometry. Cells were stimulated with ACPA-containing immune complexes, and tumour necrosis factor alpha (TNFα) was assayed in culture supernatants. Results Variations distinguished RA from HC monocytes, corresponding to a 5% and 6% decrease in the percentages of monocytes expressing Fc[GAMMA]RI and Fc[GAMMA]RII, respectively, and a 7% increase in the proportion of Fc[GAMMA]RIII-positive monocytes. Although in both HC and RA patients macrophage differentiation was accompanied by a dramatic increase in the percentage of Fc[GAMMA]RIII-expressing cells (72% vs 74.5%), the parallel decline in the proportion of Fc[GAMMA]RI-positive cells was markedly smaller in RA (7% vs 43%). Monocytes and macrophages from patients were responsive to ACPA-containing immune complexes but TNFα production in both cell types neither differed from that observed with the corresponding cells from HC, nor correlated with Fc[GAMMA]R expression or clinical or biological data. In RA as in HC, ACPA-containing immune complexes induced secretions of more TNFα in macrophages than in paired monocytes (ninefold). Finally, the proinflammatory potential of ACPA-containing immune complexes was confirmed in CD14-positive monocyte macrophages from the synovial fluid of four RA patients. Conclusions ACPA-containing immune complexes induce TNFα secretion by blood and synovial fluid-derived macrophages from RA patients, fitting with their probable involvement in RA pathophysiology.

The expression of PADs was time-dependent as substantiated by the kinetics of PADI mRNA expression which revealed that after 4 h of contact PADI mRNAs were increased but declined rapidly to levels below baseline after 12 h of co-culture, whereas the production of PAD2 and PAD4 enzymes increased at 24 and 48 h of culture.

OBJECTIVE Antiperinuclear factor (APF), “antikeratin antibodies” (“AKA”), and antibodies to human epidermis filaggrin (AFA), are highly specific serological markers of rheumatoid arthritis (RA), which recognise epitopes on various isoforms of (pro)filaggrin. It was proposed that these antibodies are globally named antifilaggrin autoantibodies. Here the diagnostic value of the detection of each one is compared and the overlap between the three tests evaluated. METHODS 492 serum samples were tested, including 279 RA serum samples, taken from patients in France and Belgium. APF and “AKA” titres were estimated by indirect immunofluorescence, and AFA titres by immunoblotting on filaggrin enriched human epidermis extracts. RESULTS By a convenient choice of the positivity thresholds, the diagnostic sensitivity and specificity of the tests were shown to be similar (0.52 and 0.97, respectively). Although the antibody titres were strongly correlated, the associations APF-AFA or AFA-“AKA” permitted more than 52% or 55% of RA to be diagnosed, with a specificity of 0.99. CONCLUSION APF, “AKA”, and AFA detection have a similar diagnostic value. However, because the three tests do not totally overlap, associating APF with “AKA” or AFA with “AKA” can improve diagnostic sensitivity. None of the three antigens used bear all the epitopes recognised by anti-filaggrin autoantibodies.