Chymotrypsin activity signals to intestinal epithelium by protease-activated receptor-dependent mechanisms.

BACKGROUND AND PURPOSE: Chymotrypsin is a pancreatic protease secreted into the lumen of the small intestine to digest food proteins. We hypothesized that chymotrypsin activity may be found close to epithelial cells and that chymotrypsin signals to them via protease-activated receptors (PARs). We deciphered molecular pharmacological mechanisms and gene expression regulation for chymotrypsin signalling in intestinal epithelial cells. EXPERIMENTAL APPROACH: The presence and activity of chymotrypsin were evaluated by Western blot and enzymatic activity tests in the luminal and mucosal compartments of murine and human gut samples. The ability of chymotrypsin to cleave the extracellular domain of PAR1 or PAR2 was assessed using cell lines expressing N-terminally tagged receptors. The cleavage site of chymotrypsin on PAR1 and PAR2 was determined by HPLC-MS analysis. The chymotrypsin signalling mechanism was investigated in CMT93 intestinal epithelial cells by calcium mobilization assays and Western blot analyses of (ERK1/2) phosphorylation. The transcriptional consequences of chymotrypsin signalling were analysed on colonic organoids. KEY RESULTS: We found that chymotrypsin was present and active in the vicinity of the colonic epithelium. Molecular pharmacological studies have shown that chymotrypsin cleaves both PAR1 and PAR2 receptors. Chymotrypsin activated calcium and ERK1/2 signalling pathways through PAR2, and this pathway promoted interleukin-10 (IL-10) up-regulation in colonic organoids. In contrast, chymotrypsin disarmed PAR1, preventing further activation by its canonical agonist, thrombin. CONCLUSION AND IMPLICATIONS: Our results highlight the ability of chymotrypsin to signal to intestinal epithelial cells via PARs, which may have important physiological consequences in gut homeostasis.

Active thrombin produced by the intestinal epithelium controls mucosal biofilms.

Proteolytic homeostasis is important at mucosal surfaces, but its actors and their precise role in physiology are poorly understood. Here we report that healthy human and mouse colon epithelia are a major source of active thrombin. We show that mucosal thrombin is directly regulated by the presence of commensal microbiota. Specific inhibition of luminal thrombin activity causes macroscopic and microscopic damage as well as transcriptomic alterations of genes involved in host-microbiota interactions. Further, luminal thrombin inhibition impairs the spatial segregation of microbiota biofilms, allowing bacteria to invade the mucus layer and to translocate across the epithelium. Thrombin cleaves the biofilm matrix of reconstituted mucosa-associated human microbiota. Our results indicate that thrombin constrains biofilms at the intestinal mucosa. Further work is needed to test whether thrombin plays similar roles in other mucosal surfaces, given that lung, bladder and skin epithelia also express thrombin.

Neutrophil extracellular traps exert both pro- and anti-inflammatory actions in rheumatoid arthritis that are modulated by C1q and LL-37.

OBJECTIVE: Neutrophil extracellular traps (NET), produced by activated polymorphonuclear neutrophils (PMN), are supposed to play a role in the pathogenesis of rheumatoid arthritis (RA), a chronic inflammatory autoimmune disease characterized by anti-citrullinated protein antibodies (ACPA). Indeed, NET contain citrullinated autoantigens and some RA autoantibodies recognize NET. However, the mechanisms by which NET trigger or perpetuate the inflammatory process in RA are hitherto not elucidated. We hypothesized that, in addition to citrullination, NET might also contain stimulatory proteins and directly activate inflammatory target cells, as PMN and macrophages. METHODS: NET antigenic and inflammatory properties were analyzed in 157 healthy donors (HD) and RA patients, the largest analysis reported so far. Primary PMN and monocyte-derived macrophages were isolated and immunoglobulin G (IgG) purified. NET were induced (NETosis), isolated and quantified. NET antigenicity was analyzed by fluorescence microscopy. PMN and macrophages were stimulated with NET with/without ACPA, C1q, LL-37 or lipopolysaccharide (LPS) and cell activation was estimated by flow cytometry and ELISA. RESULTS: PMN from RA patients produced more NET than HD PMN. We next dissected how NET mechanistically affect inflammatory cells. Particularly, we show for the first time that RA and HD NET activated both resting macrophages and PMN, but importantly RA NET were more stimulatory, leading to secretion of inflammatory cytokines and up-regulation of HLA/CD86/CD11b. IgG from ACPA-positive RA patients specifically recognized RA and even HD NET. Nevertheless, NET-induced cell activation occurs independently of immune complex formation with ACPA. Likewise, endosomal acidification was not required. Notably, we also report that complement C1q increased the NET stimulatory activity on macrophages only, due to higher expression of C1q receptors, which was further supported by the LL-37 antimicrobial peptide. In contrast, NET specifically inhibited interleukin (IL)-6 secretion by LPS-activated macrophages and not PMN, especially with C1q/LL-37. This inhibition was not mediated by NET-derived proteases or LPS neutralization and was associated with the simultaneous induction of IL-10 secretion. CONCLUSION: We show that NET possess both pro- and anti-inflammatory properties depending on target cells, their activation levels and C1q/LL-37. Thus, independently of ACPA, NET modulate RA chronic inflammation via this new dual activity we identified. In addition, NET may trigger autoimmunity in RA as ACPA recognize NET antigens but not non-activated PMN. Therefore, we conclude that excess of NETosis together with enhanced NET activity participate to RA pathogenesis at different levels.

Seropositivity and Antibody Profiling of Patients Are Dramatically Impacted by the Features of Peptides Used as Immunosorbents: A Lesson from Anti-Citrullinated Protein/Peptide Antibody.

Quantification of Abs toward a single epitope is critical to understanding immunobiological processes. In autoimmunity, the prognostic value of the serological profiles of patients draws much attention, but the detection of Abs toward a single epitope is not well controlled. Particularly, the rheumatoid arthritis (RA)-specific anti-citrullinated protein/peptide Abs (ACPA) are specific to a two-atom change on arginyl residues and are considered a heterogeneous family of Abs. As a model, we studied ACPA to decipher how peptide features used as immunosorbent impact Ab detection. We synthesized 30 peptides encompassing immunodominant epitopes of citrullinated fibrin differing by their length and biotin location and tested them using ELISA with 120 sera from RA and non-RA rheumatic disease controls, generating over 3000 experimental measurements. We showed that minor molecular changes in peptide chemical structure had dramatic consequences. Even when peptides exhibited the same epitope, measured Ab titers were extremely variable, and patients' seropositivity was discordant in up to 50% of cases. The distance between epitope and biotin was the most critical parameter for efficient Ab detection irrespective of biotin position or peptide length. Finally, we identified a 15-mer peptide bearing a single citrullinated epitope detecting almost all ACPA-positive sera, thus revealing a high degree of homogeneity in RA autoimmune response. This integrative analysis deciphers the dramatic impact of the molecular design of peptide-based technologies for epitope-specific Ab quantification. It provides a model for assay development and highlights that the studies using such technologies can give a wrong perception of biological processes and therefore that medical use of data must be cautious.

Among human macrophages polarised to different phenotypes, the M-CSF-oriented cells present the highest pro-inflammatory response to the rheumatoid arthritis-specific immune complexes containing ACPA.

OBJECTIVES: In 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. METHODS: CD14-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. RESULTS: IFN-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-1ß, 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-1ß 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-1ß ratio. CONCLUSIONS: Despite 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.

IgM and IgA rheumatoid factors purified from rheumatoid arthritis sera boost the Fc receptor- and complement-dependent effector functions of the disease-specific anti-citrullinated protein autoantibodies.

Rheumatoid factors (RF) and the disease-specific anti-citrullinated protein autoantibodies (ACPA) coexist in the joints of rheumatoid arthritis (RA) patients where they probably contribute to synovitis. We investigated the influence of IgM and IgA RF on the FcR- and complement-dependent effects of ACPA immune complexes (ACPA-IC). When stimulated by ACPA-IC formed in the presence of IgM RF or IgA RF fractions purified from RA serum pools, M-CSF-generated macrophages skewed their cytokine response toward inflammation, with increases in the TNF-α/IL-10 ratio and in IL-6 and IL-8 secretion, and decreases in the IL-1Ra/IL-1ß ratio. In the IgM RF-mediated amplification of the inflammatory response of macrophages, the participation of an IgM receptor was excluded, notably by showing that they did not express any established receptor for IgM. Rather, this amplification depended on the IgM RF-mediated recruitment of more IgG into the ACPA-IC. However, the macrophages expressed FcαRI and blocking its interaction with IgA inhibited the IgA RF-mediated amplification of TNF-α secretion induced by ACPA-IC, showing its major implication in the effects of RF of the IgA class. LPS further amplified the TNF-α response of macrophages to RF-containing ACPA-IC. Lastly, the presence of IgM or IgA RF increased the capacity of ACPA-IC to activate the complement cascade. Therefore, specifically using autoantibodies from RA patients, the strong FcR-mediated or complement-dependent pathogenic potential of IC including both ACPA and IgM or IgA RF was established. Simultaneous FcR triggering by these RF-containing ACPA-IC and TLR4 ligation possibly makes a major contribution to RA synovitis.

IgM rheumatoid factor amplifies the inflammatory response of macrophages induced by the rheumatoid arthritis-specific immune complexes containing anticitrullinated protein antibodies.

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)-1ß 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.

Contact with stimulated T cells up-regulates expression of peptidylarginine deiminase 2 and 4 by human monocytes.

OBJECTIVE: The antigenic targets of the rheumatoid arthritis (RA)-associated autoantibodies to "citrullinated" proteins are generated following citrullination by a peptidylarginine deiminase (PAD). Of the five PAD isotypes, two - PAD2 and PAD4 - are expressed in RA synovial tissue. Within this tissue, the activation of macrophages and fibroblasts mediated by T-cell contact or driven by cytokines plays a prominent part in the pathogenesis. We wanted to determine whether cytokine stimulation and contact with T cells could play a role in PAD expression by peripheral blood monocytes and fibroblastic synoviocytes. METHODS: Human monocytes and T lymphocytes were derived from the blood of healthy donors. HUT-78 cells and T lymphocytes were stimulated with PHA and PMA. Human synovial fibroblasts were isolated after surgical synoviectomy. The expression of PAD was determined by real-time PCR and immunoblot. RESULTS: In monocytes, the PADI2 and PADI4 mRNAs were transiently up-regulated by contact with stimulated HUT-78 and/or T lymphocytes. Positive modulation of the PAD2 and PAD4 proteins were also observed upon contact with stimulated HUT-78 T cells. Stimulation by IL-1ß or IFN-ß did not modify the PADI2 and PADI4 mRNAs, but enhanced PAD4 protein expression. No isotype of PAD was detected at the mRNA or protein level in resting or stimulated synovial fibroblasts. CONCLUSION: Contact between stimulated T cells and monocyte-macrophages or cytokine-activated monocyte-macrophages constitutes a highly likely source of PAD2 and PAD4, which are observed in inflamed synovial tissues. In contrast, it is most unlikely that fibroblastic synoviocytes contribute to PAD expression in rheumatoid synovial membranes.

The antigen specificity of the rheumatoid arthritis-associated ACPA directed to citrullinated fibrin is very closely restricted.

The major targets of the disease-specific autoantibodies to citrullinated proteins (ACPA) in synovium of rheumatoid arthritis (RA) patients are borne by the citrullinated α- and ß-chains of fibrin. We demonstrated that ACPA target a limited set of citrullinated fibrin peptides and particularly four multicitrullinated peptides which present the major epitopes. In this study, we established the clear immunodominance of the peptides α36-50Cit(38,42) and ß60-74Cit(60,72,74) which were recognised by 51/81 (63%) and 61/81 (75%) of ACPA-positive patients, respectively, more than 90% recognising one, the other or both peptides. We also identified the citrullyl residues αCit(42), ßCit(72) and ßCit(74) as essential for antigenicity, and at a lesser degree αCit(38). Then, we assayed on overlapping 7-mer peptides encompassing the sequences of the two peptides, 3 series of sera recognising either α36-50Cit(38,42) or ß60-74Cit(60,72,74) or both peptides. In each series, the reactivity profiles of the sera, largely superimposable, allowed identification of the two 4/5-mer overlapping epitopes (α: VECit(42)HQ and α': Cit(38)VVE), and the single 5-mer epitope (ß: GYCit(72)ACit(74)), all located to a flexible globular domain of fibrin on a topological 3D model. In conclusion, we demonstrated that only 3 immunodominant epitopes are targeted by ACPA on citrullinated fibrin stressing their actual oligoclonality. However, the reactivity to the 3 epitopes distinguishes three subgroups of patients. The closely restricted antigen specificity suggests that the autoimmune reaction to citrullinated fibrin is antigen-driven. The accessibility of the epitopes reinforces the hypothesis of a pathogenic role for ACPA via immune complexe formation in the synovial tissue.

Deimination and expression of peptidylarginine deiminases during cutaneous wound healing in mice.

Deimination, the conversion of protein-bound arginines into citrullines, is a post-translational modification catalyzed by a peptidylarginine deiminase (Pad). In the epidermis, three Pads are expressed, namely Pad1, 2 and 3, and the major deiminated protein is filaggrin. Deimination of fibrin has been observed in various pathological inflammatory conditions. Here, we analyzed the expression of Pads and citrullination of proteins during cutaneous wound healing, i.e. in a physiological inflammatory condition. Full-thickness punches were performed on adult mouse back skin, and wound recovery was analyzed over 10 days by immunohistology and western blotting. Pad1 was immunodetected in all the neo-epidermis. Pad3, normally expressed in the stratum granulosum, was not detected in the hyperproliferative tongue of the neo-epidermis, but was shown to be co-localized with (pro)filaggrin in a large number of keratinocyte layers in its differentiating part. Deiminated proteins were detected in the stratum corneum of the neo-epidermis in the late phase of re-epithelialization, and in the clot and the clot-derived scab. In the clot where we only detected Pad4, one of the deiminated proteins was shown to be fibrin. Deimination of the clot proteins, and more generally wound healing and keratinocyte differentiation, seemed to be Pad2-independent, as shown using Padi2(-/-) mice.

Fcγ receptor profile of monocytes and macrophages from rheumatoid arthritis patients and their response to immune complexes formed with autoantibodies to citrullinated proteins.

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.

Induction of macrophage secretion of tumor necrosis factor alpha through Fcgamma receptor IIa engagement by rheumatoid arthritis-specific autoantibodies to citrullinated proteins complexed with fibrinogen.

OBJECTIVE: Macrophage-derived tumor necrosis factor alpha (TNFalpha) is a dominant mediator of synovitis in rheumatoid arthritis (RA). This study was undertaken to assess whether and how immune complexes (ICs) formed by the interaction of disease-specific autoantibodies to citrullinated proteins (ACPAs) with their main synovial target antigen, citrullinated fibrin, contribute to TNFalpha production by macrophages. METHODS: An in vitro human model was developed in which monocyte-derived macrophages were stimulated with ACPA-containing ICs that were generated by capturing ACPAs from RA sera on immobilized citrullinated fibrinogen. Cellular activation was evaluated by TNFalpha assay in culture supernatants. Selective blockade of IC interactions with the 3 classes of Fcgamma receptors (FcgammaR) was used to assess the contribution of each receptor to macrophage activation. In addition, 2 citrullinated fibrin-derived peptides bearing major ACPA epitopes were tested for their capacity to inhibit formation of macrophage-activating ACPA-containing ICs. RESULTS: ACPA-containing ICs induced a dose-dependent TNFalpha secretion by macrophages from 14 of 20 healthy donors. The macrophage response was systematically higher than that of the paired monocyte precursors. TNFalpha secretion was not reduced by blockade of FcgammaRI or FcgammaRIII, but was strongly repressed when interaction of ICs with FcgammaRII was prevented. The 2 citrullinated peptides significantly inhibited ACPA reactivity to citrullinated fibrinogen and, when tested together, almost completely abolished formation of macrophage-activating ICs, thereby diminishing the secreted TNFalpha levels. CONCLUSION: Our model demonstrates the inflammatory potential of ACPA-containing ICs via engagement of FcgammaRIIa at the surface of macrophages, strongly supporting their pathophysiologic involvement. Continuing dissection of these molecular pathways could open the way to new therapeutic approaches in patients with RA.

Peptidyl arginine deiminase type 2 (PAD-2) and PAD-4 but not PAD-1, PAD-3, and PAD-6 are expressed in rheumatoid arthritis synovium in close association with tissue inflammation.

OBJECTIVE: Autoantibodies to citrullinated proteins (ACPAs) are specific for rheumatoid arthritis (RA) and probably are involved in its pathophysiology. Citrullyl residues, posttranslationally generated by peptidyl arginine deiminase (PAD), are indispensable components of ACPA-targeted epitopes. The aim of this study was to identify which PAD isotypes are expressed in the synovial tissue (ST) of patients with RA and are involved in the citrullination of fibrin, the major synovial target of ACPAs. METHODS: Expression of all PAD isotypes, including the recently described PAD type 6 (PAD-6), was explored by reverse transcription-polymerase chain reaction and immunoblotting, first in blood-derived mononuclear leukocytes from healthy donors, then in ST samples from 16 patients with RA and 11 control patients (4 with other arthritides and 7 with osteoarthritis [OA]). In ST samples from patients with RA, PADs were localized by immunohistochemistry. RESULTS: In lymphocytic and monocytic cells and, similarly, in ST samples from patients with RA, the PAD-2, PAD-4, and PAD-6 genes were found to be transcribed, but only PAD-2 and PAD-4 enzymes were detected. PAD-2 was also expressed in ST from control patients, including those with OA, while PAD-4 was preferentially expressed in ST from patients with other arthritides. In RA, the expression levels of PAD-2 and PAD-4 were correlated with the intensity of inflammation (cell infiltration, hypervascularization, and synovial lining hyperplasia), and both enzymes were demonstrable within or in the vicinity of citrullinated fibrin deposits. CONCLUSION: PAD-2 and PAD-4 are the only PAD isotypes expressed in the ST of patients with RA and those with other arthritides. Inflammatory cells are a major source, but PAD-4 also comes from hyperplastic synoviocytes. Both isotypes are probably involved in the citrullination of fibrin.

Epitopes of human fibrin recognized by the rheumatoid arthritis-specific autoantibodies to citrullinated proteins.

Formation of the epitopes recognized by the rheumatoid arthritis (RA)-specific autoantibodies to citrullinated proteins (ACPA) on filaggrin and on the alpha- and beta-chains of fibrin, their synovial target, requires conversion of their arginyl residues into citrullyl residues, but is also affected by their amino-acyl environment. Using competition with five citrullinated filaggrin-derived peptides bearing major ACPA epitopes, we confirmed the close cross-reactivity between filaggrin and citrullinated fibrin. To identify the sequential epitopes recognized on fibrin by ACPA, 71 citrullinated 15-mer peptides derived from all the sites of the alpha- and beta-chains of fibrin harboring arginyl residues were tested by ELISA using ACPA-positive RA sera exhibiting different reactivity profiles to the five filaggrin peptides. We identified 18 fibrin-derived peptides bearing ACPA epitopes. Regarding the ability of fibrinogen arginyl residues to be citrullinated in vitro, 11 of the peptides likely correspond to in vivo targeted epitopes. Two out of them bear major epitopes and are located in the central globular domain of the protein. In the synovial tissue, fibrin citrullination and ACPA binding could impair fibrin degradation by plasmin. The immunological conflict between ACPA and fibrin could therefore sustain synovial inflammation not only via pro-inflammatory effector mechanisms but also via impairment of fibrinolysis.

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 alpha- and beta-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 alpha- and beta-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.

Autoantibodies to citrullinated proteins: ACPA.

Anti-perinuclear factor and anti-keratin antibodies have long been known to be specifically associated with rheumatoid arthritis (RA). They were first demonstrated to target various forms of (pro)filaggrin, a protein of stratified epithelia. Then, they were found to belong to a single family of autoantibodies targeting proteins that bear peptidic epitopes centered by a citrullyl residue: the anti-citrullinated protein autoantibodies (ACPA). The main targets of ACPA in the synovial tissue were demonstrated to be citrullinated forms of the a- and beta-chains of fibrin. A chronic conflict between locally produced ACPA and deposits of citrullinated fibrin is probably responsible for self-maintaining of RA synovial inflammation. Various tests for the detection of ACPA have been developed: recent ELISAs confirm their high diagnostic specificity and improve their diagnostic sensitivity. Since ACPA appear very early in the course of the disease, their detection is of major interest to identify RA among recent arthritides. Moreover, their prognostic value may lead to start early 'aggressive' treatments to prevent irreversible joint damage.

Fibrin deimination in synovial tissue is not specific for rheumatoid arthritis but commonly occurs during synovitides.

Autoantibodies to deiminated (citrullinated) proteins are the most specific serological markers of rheumatoid arthritis (RA). Deimination is critical in generating the peptidic epitopes they recognize. In the synovial tissue (ST), deiminated forms of the alpha- and beta-chains of fibrin are their major autoantigenic targets (anti-human fibrin(ogen) autoantibodies (AhFibA)). We investigated whether the presence of deiminated fibrin in the ST was specific for RA, because this could explain why AhFibA are RA specific. In 13 patients with RA and 19 patients with various other rheumatological disorders, knee ST biopsies were collected in macroscopically inflamed areas identified under arthroscopy. Synovitis was histopathologically confirmed in all of the biopsies. By immunoblotting, using antisera to fibrin, Abs to citrullyl residues, and AhFibA purified from RA sera, deiminated fibrin was evidenced in ST extracts from all of the patients. Moreover, variations in the degree of fibrin deimination were observed that were not related to the disease. Immunohistochemical analysis, using Abs to citrullyl residues and an antiserum to fibrin on adjacent serial sections of ST, confirmed the results because deiminated proteins colocalized with fibrin in RA as well as in control patients. Therefore, fibrin deimination in the ST is a general phenomenon associated to any synovitis, which does not necessarily induce a B autoimmune response with production of AhFibA.

Clinical and pathophysiological significance of the autoimmune response to citrullinated proteins in rheumatoid arthritis.

Rheumatoid arthritis (RA) is the most frequent human autoimmune disease, affecting about 1% of the adult population worldwide. A better knowledge of the autoimmune mechanisms involved is essential. We identified the epithelial targets of various autoantibodies specifically associated to RA, as variants of (pro)filaggrin. We also showed that these targets correspond to deiminated ("citrullinated") proteins, of which arginyl residues have been posttranslationally transformed into citrullyl residues by a peptidylarginine deiminase (PAD). Moreover, we and others established that citrullyl residues are indispensable elements of the epitopes recognized by these autoantibodies but only in the context of specific aminoacid sequences. We also demonstrated that these autoantibodies to citrullinated proteins (ACPA) are secreted by plasma cells of the synovial tissue and that their major targets correspond to citrullinated forms of the alpha- and beta-chains of fibrin, abundant in the tissue. These results have allowed the development of new efficient immunochemical methods for the detection of ACPA. Some of them are already commercially available. These new methods have permitted the high diagnostic value of ACPA which are present very early in the course of the disease, and also their prognostic value, to be confirmed. ACPA detection should therefore prove to be also a very valuable tool to guide the choice of therapeutic strategies, from the earliest stages of the disease. The synthesis of ACPA in the rheumatoid synovial tissue and the existence therein of a specific antigenic target constitute a strong argument for the involvement of this specific immunological conflict in the pathophysiology of RA. Indeed, it could lead to activation of effector mechanisms with pro-inflammatory effects, thus to formation in the tissue of new fibrin deposits, secondarily citrullinated. We therefore, propose a new pathophysiological model accounting for the self-maintenance and chronicity of rheumatoid inflammation. Numerous questions about the pathophysiological significance of the autoimmune response to deiminated proteins in RA remain to be answered to confirm this model.

cDNA cloning, gene organization and expression analysis of human peptidylarginine deiminase type I.

Peptidylarginine deiminases (PADs) catalyse a post-translational modification of proteins through the conversion of arginine residues into citrullines. The existence of four isoforms of PAD (types I, II, III and IV) encoded by four different genes, which are distinct in their substrate specificities and tissue-specific expression, was reported in rodents. In the present study, starting from epidermis polyadenylated RNA, we cloned by reverse transcriptase-PCR a full-length cDNA encoding human PAD type I. The cDNA was 2711 bp in length and encoded a 663-amino-acid sequence. The predicted protein shares 75% identity with the rat PAD type I sequence, but displays only 50-57% identity with the three other known human isoforms. We have described the organization of the human PAD type I gene on chromosome 1p36. A recombinant PAD type I was produced in Escherichia coli and shown to be enzymically active. Human PAD type I mRNAs were detected by reverse transcriptase-PCR not only in the epidermis, but also in various organs, including prostate, testis, placenta, spleen and thymus. In human epidermis extracts analysed by Western blotting, PAD type I was detected as a 70 kDa polypeptide, in agreement with its predicted molecular mass. As shown by immunohistochemistry, the enzyme was expressed in all the living layers of human epidermis, with the labelling being increased in the granular layer. This is the first description of the human PAD type I gene and the first demonstration of its expression in epidermis.

Detection of antibodies to deiminated recombinant rat filaggrin by enzyme-linked immunosorbent assay: a highly effective test for the diagnosis of rheumatoid arthritis.

OBJECTIVE: To assay antifilaggrin autoantibodies, we developed an enzyme-linked immunosorbent assay (ELISA) using a "citrullinated" recombinant rat filaggrin. Our objectives were to assess its value for diagnosing rheumatoid arthritis (RA) and to compare the results with those obtained using 4 other reference methods for detection of antifilaggrin autoantibodies, including the commercially available ELISA that uses a modified "citrullinated" synthetic peptide derived from the sequence of human filaggrin (CCP-ELISA). METHODS: We analyzed 711 sera from patients with well-characterized rheumatic diseases, including 240 patients with RA. Antifilaggrin autoantibodies were detected by an ELISA using a recombinant rat filaggrin deiminated in vitro as immunosorbent (ArFA-ELISA). The results considered were the differences between the optical densities obtained on deiminated and nondeiminated proteins. Antibodies to rat esophagus epithelium were detected by indirect immunofluorescence, while antibodies to human filaggrin were detected by immunoblotting and by a recently described ELISA using a deiminated recombinant human filaggrin. Finally, CCP-ELISA was performed according to the manufacturer's recommendations. RESULTS: At the titer thresholds allowing diagnostic specificities of 0.95, 0.985, and 0.99 to be reached, the diagnostic sensitivities of the ArFA-ELISA were 0.76, 0.67, and 0.65, respectively. At these 3 thresholds, the sensitivities were significantly higher than those of the 4 other tests. Despite incomplete overlapping of the 5 tests, the high diagnostic performance of the ArFA-ELISA allows us to propose this test to replace all the other methods for antifilaggrin autoantibody detection. CONCLUSION: ArFA-ELISA appears to be the most efficient test among those available for the detection of antifilaggrin autoantibodies, in terms of diagnostic accuracy for RA. Its diagnostic performance in early RA and its prognostic value are currently under evaluation.