Mireille Sebbag | Université Toulouse III (original) (raw)

Papers by Mireille Sebbag

Research paper thumbnail of Epitopes of human fibrin recognized by the rheumatoid arthritis-specific autoantibodies to citrullinated proteins

European journal of immunology, 2006

Formation of the epitopes recognized by the rheumatoid arthritis (RA)-specific autoantibodies to ... more 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 l...

Research paper thumbnail of Autoimmune Response to Post-Translationally Modified (Citrullinated) Proteins: Prime Suspect in the Pathophysiology of Rheumatoid Arthritis

Zouali/The Epigenetics of Autoimmune Diseases, 2009

ABSTRACT Deimination (citrullination) corresponds to the post-translational conversion of arginyl... more ABSTRACT Deimination (citrullination) corresponds to the post-translational conversion of arginyl residues of peptides into citrullyl residues. This modification is essential in the formation of the epitopes recognized by autoantibodies to citrullinated proteins (ACPA), which are IgGs specifically found in the serum of patients with rheumatoid arthritis (RA), a frequent autoimmune disease associated with chronic and destructive inflammation of synovial joints. Here we make an account of the research that led from the initially described RA-associated anti-perinuclear factor and anti-keratin antibodies to ACPA, and to the recognition that B cell autoreactivity to citrullinated proteins occurs in the synovial membrane and probably plays a major role in RA synovitis. Moreover, the nature of the antigen targeted by ACPA in the RA synovium and the stimuli at play in the induction of the autoimmune response to citrullinated proteins are discussed.

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

Annals of the Rheumatic Diseases, 2014

Anticitrullinated protein antibodies (ACPA) are specifically associated with rheumatoid arthritis... more 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. 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. 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. 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.

Research paper thumbnail of Autoanticorps anti-protéines “citrullinées” dans la polyarthrite rhumatoïde

Résumé En 1964, RLF Nienhuis et coll. [1] décrivaient, dans plus de deux tiers des sérums de pat... more Résumé En 1964, RLF Nienhuis et coll. [1] décrivaient, dans plus de deux tiers des sérums de patients atteints de polyarthrite rhumatoïde (PR), des anticorps marquant en immunofluorescence indirecte des granules périnucléaires présents dans les cellules les plus superficielles de la muqueuse jugale humaine. Ces anticorps étaient appelés “facteur anti-périnucléaire” (antiperinuclear factor: APF) par analogie avec le “facteur” rhumatoïde (FR) qui

Research paper thumbnail of Deimination and expression of peptidylarginine deiminases during cutaneous wound healing in mice

European journal of dermatology : EJD

Deimination, the conversion of protein-bound arginines into citrullines, is a post-translational ... more 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 ...

Research paper thumbnail of Intérêt clinique et rôle physiopathologique de la réponse auto-immune contre les protéines citrullinées dans la polyarthrite rhumatoïde

Revue du Rhumatisme, 2004

Disponible sur internet le 17 septembre 2004

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

European cytokine network, 2012

Objective: The antigenic targets of the rheumatoid arthritis (RA)-associated autoantibodies to &q... more 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-regula...

Research paper thumbnail of The cytokeratin filament-aggregating protein filaggrin is the target of the so-called "antikeratin antibodies," autoantibodies specific for rheumatoid arthritis

Journal of Clinical Investigation, 1993

In rheumatoid arthritis (RA), the high diagnostic value of serum antibodies to the stratum corneu... more In rheumatoid arthritis (RA), the high diagnostic value of serum antibodies to the stratum corneum of rat esophagus epithelium has been widely reported. These so-called "antikeratin antibodies," detected by indirect immunofluorescence, were found to be autoantibodies since they also labeled human epidermis. Despite their name, the actual target of these autoantibodies was not known. In this study, a 40-kD protein (designated as 40K), extracted from human epidermis and specifically immunodetected by 75% of RA sera, was purified and identified as a neutral/acidic isoform of basic filaggrin, a cytokeratin filament-aggregating protein, by peptide mapping studies and by the following evidences: (a) mAbs specific for filaggrin reacted with the 40K protein; (b) the autoantibodies, affinity-purified from RA sera on the 40K protein, immunodetected purified filaggrin; (c) the reactivity of RA sera to the 40K protein was abolished after immunoadsorption with purified filaggrin; (d) the 40K protein and filaggrin had similar amino acid compositions. Furthermore, autoantibodies against the 40K protein and the so-called "antikeratin antibodies" were shown, by immunoadsorption experiments, to be largely the same. The identification of filaggrin as a RA-specific autoantigen could contribute to the understanding of the pathogenesis of this disease and, ultimately, to the development of methods for preventing the autoimmune response. (J. Clin. Invest. 1993.

Research paper thumbnail of The antiperinuclear factor and the so-called antikeratin antibodies are the same rheumatoid arthritis-specific autoantibodies

Journal of Clinical Investigation, 1995

The so-called antikeratin antibodies (AKA) and the antiperinuclear factor (APF) are the most spec... more The so-called antikeratin antibodies (AKA) and the antiperinuclear factor (APF) are the most specific serological markers of RA. Using indirect immunofluorescence, AKA label the stratum corneum of various cornified epithelia and APF the keratohyalin granules of human buccal mucosa epithelium. We recently demonstrated that AKA recognize human epidermal filaggrin. Here, we report the identification of the major APF antigen as a diffuse protein band of 200-400 kD. This protein is seen to be closely related to human epidermal (pro) filaggrin since it was recognized by four antifilaggrin mAbs specific for different epitopes, and since the APF titers of RA sera were found to be correlated to their AKA titers and to their immunoblotting reactivities to filaggrin. Immunoabsorption of RA sera on purified epidermal filaggrin abolished their reactivities to the granules of buccal epithelial cells and to the 200-400-kD antigen. Moreover, antifilaggrin autoantibodies, i.e., AKA, affinity purified from RA sera, were shown to immunodetect the 200-400-kD antigen and to stain these granules. These results indicate that AKA and APF are largely the same autoantibodies. They recognize human epidermal filaggrin and (pro) filaggrin-related proteins of buccal epithelial cells. Identification of the epitopes recognized by these autoantibodies, which we propose to name antifilaggrin autoantibodies, will certainly open new paths of research into the pathophysiology of RA.

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

Journal of Autoimmunity, 2011

The major targets of the disease-specific autoantibodies to citrullinated proteins (ACPA) in syno... more The major targets of the disease-specific autoantibodies to citrullinated proteins (ACPA) in synovium of rheumatoid arthritis (RA) patients are borne by the citrullinated aand b-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.

Research paper thumbnail of Clinical and pathophysiological significance of the autoimmune response to citrullinated proteins in rheumatoid arthritis

Joint Bone Spine, 2004

Rheumatoid arthritis (RA) is the most frequent human autoimmune disease, affecting about 1% of th... more 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 aand b-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 selfmaintenance 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.

Research paper thumbnail of Analysis of the peptidylarginine deiminase V gene in rheumatoid arthritis

Arthritis Research & Therapy, 2003

Research paper thumbnail of Epitopes of human fibrin recognized by the rheumatoid arthritis-specific autoantibodies to citrullinated proteins

European Journal of Immunology, 2006

Formation of the epitopes recognized by the rheumatoid arthritis (RA)-specific autoantibodies to ... more Formation of the epitopes recognized by the rheumatoid arthritis (RA)-specific autoantibodies to citrullinated proteins (ACPA) on filaggrin and on the aand b-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 aand b-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.

Research paper thumbnail of Induction of macrophage secretion of tumor necrosis factor α through Fcγ receptor IIa engagement by rheumatoid arthritis–specific autoantibodies to citrullinated proteins complexed with fibrinogen

Arthritis & Rheumatism, 2008

Objective. Macrophage-derived tumor necrosis factor ␣ (TNF␣) is a dominant mediator of synovitis ... more Objective. Macrophage-derived tumor necrosis factor ␣ (TNF␣) 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 TNF␣ production by macrophages.

Research paper thumbnail of Detection of antibodies to deiminated recombinant rat filaggrin by enzyme-linked immunosorbent assay: A highly effective test for the diagnosis of rheumatoid arthritis

Arthritis & Rheumatism, 2002

To assay antifilaggrin autoantibodies, we developed an enzyme-linked immunosorbent assay (ELISA) ... more 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).

Research paper thumbnail of Influence of HLA-DR genes on the production of rheumatoid arthritis-specific autoantibodies to citrullinated fibrinogen

Arthritis & Rheumatism, 2005

Objective. Antibodies directed against citrullinated fibrinogen are highly specific for rheumatoi... more 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.

Research paper thumbnail of A5.7 Autoantibodies to the Fibrin-Derived Citrullinated Peptides  36-50 and  60-74 are Two Distinct Non-Overlapping Subfamilies of ACPA that together almost Summarise their Reactivity to Citrullinated Fibrinogen and to CCP2 Antigens

Annals of the Rheumatic Diseases, 2013

Research paper thumbnail of The fibrin-derived citrullinated peptide  60-74Cit60,72,74 bears the major ACPA epitope recognised by the rheumatoid arthritis-specific anticitrullinated fibrinogen autoantibodies and anti-CCP2 antibodies

Annals of the Rheumatic Diseases, 2013

To evaluate the proportions of rheumatoid arthritis (RA) sera containing anticitrullinated protei... more To evaluate the proportions of rheumatoid arthritis (RA) sera containing anticitrullinated proteins autoantibodies (ACPA) reactive to α36-50Cit₃₈,₄₂ and/or β60-74Cit₆₀,₇₂,₇₄, two peptides identified as bearing the immunodominant epitopes of their major target, citrullinated fibrin. To analyse the relationships of anti-α36-50Cit₃₈,₄₂ and anti-β60-74Cit₆₀,₇₂,₇₄ autoantibodies with autoantibodies reactive to the complete citrullinated human fibrinogen molecule (AhFibA) and with anti-CCP2 antibodies. 617 sera from 181 patients with established RA and 436 with non-RA rheumatic diseases were tested by ELISA for AhFibA, anti-CCP2, anti-α36-50Cit₃₈,₄₂, anti-β60-74Cit₆₀,₇₂,₇₄ autoantibodies, and by nephelometry for rheumatoid factor (RF). Diagnostic indexes, correlations and concordances between tests were analysed. Crossreactivity of anti-α36-50Cit₃₈,₄₂ and anti-β60-74Cit₆₀,₇₂,₇₄ autoantibodies was assessed in competition experiments. At a diagnostic specificity of 95%, the diagnostic sensitivity of AhFibA (83%) was significantly higher than that of all other tests. The diagnostic sensitivity of anti-β60-74Cit₆₀,₇₂,₇₄ (71%) was significantly higher than that of anti-α36-50Cit₃₈,₄₂ autoantibodies (51%) but similar to that of anti-CCP2 (74%). Titres of RF, anti-α36-50Cit₃₈,₄₂ and anti-β60-74Cit₆₀,₇₂,₇₄ autoantibodies were weakly correlated with each other, whereas titres of anti-β60-74Cit₆₀,₇₂,₇₄ were strongly correlated with those of AhFibA (r=0.633) and anti-CCP2 (r=0.634). Anti-α36-50Cit₃₈,₄₂ and anti-β60-74Cit₆₀,₇₂,₇₄ mainly corresponded to two non-crossreactive subfamilies of ACPA. More than 90% of AhFibA-positive or anti-CCP2-positive sera recognised the α36-50Cit₃₈,₄₂ and/or the β60-74Cit₆₀,₇₂,₇₄ peptide. Autoantibodies reactive to α36-50Cit₃₈,₄₂ and β60-74Cit₆₀,₇₂,₇₄ form two distinct, non-overlapping subfamilies of ACPA that, together, cover practically all the ACPA reactivity to citrullinated fibrinogen and to CCP2 antigens. In established RA, anti-β60-74Cit₆₀,₇₂,₇₄ autoantibodies show diagnostic indexes similar to those of anti-CCP2.

Research paper thumbnail of Epitopes of human fibrin recognized by the rheumatoid arthritis-specific autoantibodies to citrullinated proteins

European journal of immunology, 2006

Formation of the epitopes recognized by the rheumatoid arthritis (RA)-specific autoantibodies to ... more 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 l...

Research paper thumbnail of Autoimmune Response to Post-Translationally Modified (Citrullinated) Proteins: Prime Suspect in the Pathophysiology of Rheumatoid Arthritis

Zouali/The Epigenetics of Autoimmune Diseases, 2009

ABSTRACT Deimination (citrullination) corresponds to the post-translational conversion of arginyl... more ABSTRACT Deimination (citrullination) corresponds to the post-translational conversion of arginyl residues of peptides into citrullyl residues. This modification is essential in the formation of the epitopes recognized by autoantibodies to citrullinated proteins (ACPA), which are IgGs specifically found in the serum of patients with rheumatoid arthritis (RA), a frequent autoimmune disease associated with chronic and destructive inflammation of synovial joints. Here we make an account of the research that led from the initially described RA-associated anti-perinuclear factor and anti-keratin antibodies to ACPA, and to the recognition that B cell autoreactivity to citrullinated proteins occurs in the synovial membrane and probably plays a major role in RA synovitis. Moreover, the nature of the antigen targeted by ACPA in the RA synovium and the stimuli at play in the induction of the autoimmune response to citrullinated proteins are discussed.

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

Annals of the Rheumatic Diseases, 2014

Anticitrullinated protein antibodies (ACPA) are specifically associated with rheumatoid arthritis... more 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. 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. 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. 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.

Research paper thumbnail of Autoanticorps anti-protéines “citrullinées” dans la polyarthrite rhumatoïde

Résumé En 1964, RLF Nienhuis et coll. [1] décrivaient, dans plus de deux tiers des sérums de pat... more Résumé En 1964, RLF Nienhuis et coll. [1] décrivaient, dans plus de deux tiers des sérums de patients atteints de polyarthrite rhumatoïde (PR), des anticorps marquant en immunofluorescence indirecte des granules périnucléaires présents dans les cellules les plus superficielles de la muqueuse jugale humaine. Ces anticorps étaient appelés “facteur anti-périnucléaire” (antiperinuclear factor: APF) par analogie avec le “facteur” rhumatoïde (FR) qui

Research paper thumbnail of Deimination and expression of peptidylarginine deiminases during cutaneous wound healing in mice

European journal of dermatology : EJD

Deimination, the conversion of protein-bound arginines into citrullines, is a post-translational ... more 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 ...

Research paper thumbnail of Intérêt clinique et rôle physiopathologique de la réponse auto-immune contre les protéines citrullinées dans la polyarthrite rhumatoïde

Revue du Rhumatisme, 2004

Disponible sur internet le 17 septembre 2004

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

European cytokine network, 2012

Objective: The antigenic targets of the rheumatoid arthritis (RA)-associated autoantibodies to &q... more 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-regula...

Research paper thumbnail of The cytokeratin filament-aggregating protein filaggrin is the target of the so-called "antikeratin antibodies," autoantibodies specific for rheumatoid arthritis

Journal of Clinical Investigation, 1993

In rheumatoid arthritis (RA), the high diagnostic value of serum antibodies to the stratum corneu... more In rheumatoid arthritis (RA), the high diagnostic value of serum antibodies to the stratum corneum of rat esophagus epithelium has been widely reported. These so-called "antikeratin antibodies," detected by indirect immunofluorescence, were found to be autoantibodies since they also labeled human epidermis. Despite their name, the actual target of these autoantibodies was not known. In this study, a 40-kD protein (designated as 40K), extracted from human epidermis and specifically immunodetected by 75% of RA sera, was purified and identified as a neutral/acidic isoform of basic filaggrin, a cytokeratin filament-aggregating protein, by peptide mapping studies and by the following evidences: (a) mAbs specific for filaggrin reacted with the 40K protein; (b) the autoantibodies, affinity-purified from RA sera on the 40K protein, immunodetected purified filaggrin; (c) the reactivity of RA sera to the 40K protein was abolished after immunoadsorption with purified filaggrin; (d) the 40K protein and filaggrin had similar amino acid compositions. Furthermore, autoantibodies against the 40K protein and the so-called "antikeratin antibodies" were shown, by immunoadsorption experiments, to be largely the same. The identification of filaggrin as a RA-specific autoantigen could contribute to the understanding of the pathogenesis of this disease and, ultimately, to the development of methods for preventing the autoimmune response. (J. Clin. Invest. 1993.

Research paper thumbnail of The antiperinuclear factor and the so-called antikeratin antibodies are the same rheumatoid arthritis-specific autoantibodies

Journal of Clinical Investigation, 1995

The so-called antikeratin antibodies (AKA) and the antiperinuclear factor (APF) are the most spec... more The so-called antikeratin antibodies (AKA) and the antiperinuclear factor (APF) are the most specific serological markers of RA. Using indirect immunofluorescence, AKA label the stratum corneum of various cornified epithelia and APF the keratohyalin granules of human buccal mucosa epithelium. We recently demonstrated that AKA recognize human epidermal filaggrin. Here, we report the identification of the major APF antigen as a diffuse protein band of 200-400 kD. This protein is seen to be closely related to human epidermal (pro) filaggrin since it was recognized by four antifilaggrin mAbs specific for different epitopes, and since the APF titers of RA sera were found to be correlated to their AKA titers and to their immunoblotting reactivities to filaggrin. Immunoabsorption of RA sera on purified epidermal filaggrin abolished their reactivities to the granules of buccal epithelial cells and to the 200-400-kD antigen. Moreover, antifilaggrin autoantibodies, i.e., AKA, affinity purified from RA sera, were shown to immunodetect the 200-400-kD antigen and to stain these granules. These results indicate that AKA and APF are largely the same autoantibodies. They recognize human epidermal filaggrin and (pro) filaggrin-related proteins of buccal epithelial cells. Identification of the epitopes recognized by these autoantibodies, which we propose to name antifilaggrin autoantibodies, will certainly open new paths of research into the pathophysiology of RA.

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

Journal of Autoimmunity, 2011

The major targets of the disease-specific autoantibodies to citrullinated proteins (ACPA) in syno... more The major targets of the disease-specific autoantibodies to citrullinated proteins (ACPA) in synovium of rheumatoid arthritis (RA) patients are borne by the citrullinated aand b-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.

Research paper thumbnail of Clinical and pathophysiological significance of the autoimmune response to citrullinated proteins in rheumatoid arthritis

Joint Bone Spine, 2004

Rheumatoid arthritis (RA) is the most frequent human autoimmune disease, affecting about 1% of th... more 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 aand b-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 selfmaintenance 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.

Research paper thumbnail of Analysis of the peptidylarginine deiminase V gene in rheumatoid arthritis

Arthritis Research & Therapy, 2003

Research paper thumbnail of Epitopes of human fibrin recognized by the rheumatoid arthritis-specific autoantibodies to citrullinated proteins

European Journal of Immunology, 2006

Formation of the epitopes recognized by the rheumatoid arthritis (RA)-specific autoantibodies to ... more Formation of the epitopes recognized by the rheumatoid arthritis (RA)-specific autoantibodies to citrullinated proteins (ACPA) on filaggrin and on the aand b-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 aand b-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.

Research paper thumbnail of Induction of macrophage secretion of tumor necrosis factor α through Fcγ receptor IIa engagement by rheumatoid arthritis–specific autoantibodies to citrullinated proteins complexed with fibrinogen

Arthritis & Rheumatism, 2008

Objective. Macrophage-derived tumor necrosis factor ␣ (TNF␣) is a dominant mediator of synovitis ... more Objective. Macrophage-derived tumor necrosis factor ␣ (TNF␣) 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 TNF␣ production by macrophages.

Research paper thumbnail of Detection of antibodies to deiminated recombinant rat filaggrin by enzyme-linked immunosorbent assay: A highly effective test for the diagnosis of rheumatoid arthritis

Arthritis & Rheumatism, 2002

To assay antifilaggrin autoantibodies, we developed an enzyme-linked immunosorbent assay (ELISA) ... more 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).

Research paper thumbnail of Influence of HLA-DR genes on the production of rheumatoid arthritis-specific autoantibodies to citrullinated fibrinogen

Arthritis & Rheumatism, 2005

Objective. Antibodies directed against citrullinated fibrinogen are highly specific for rheumatoi... more 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.

Research paper thumbnail of A5.7 Autoantibodies to the Fibrin-Derived Citrullinated Peptides  36-50 and  60-74 are Two Distinct Non-Overlapping Subfamilies of ACPA that together almost Summarise their Reactivity to Citrullinated Fibrinogen and to CCP2 Antigens

Annals of the Rheumatic Diseases, 2013

Research paper thumbnail of The fibrin-derived citrullinated peptide  60-74Cit60,72,74 bears the major ACPA epitope recognised by the rheumatoid arthritis-specific anticitrullinated fibrinogen autoantibodies and anti-CCP2 antibodies

Annals of the Rheumatic Diseases, 2013

To evaluate the proportions of rheumatoid arthritis (RA) sera containing anticitrullinated protei... more To evaluate the proportions of rheumatoid arthritis (RA) sera containing anticitrullinated proteins autoantibodies (ACPA) reactive to α36-50Cit₃₈,₄₂ and/or β60-74Cit₆₀,₇₂,₇₄, two peptides identified as bearing the immunodominant epitopes of their major target, citrullinated fibrin. To analyse the relationships of anti-α36-50Cit₃₈,₄₂ and anti-β60-74Cit₆₀,₇₂,₇₄ autoantibodies with autoantibodies reactive to the complete citrullinated human fibrinogen molecule (AhFibA) and with anti-CCP2 antibodies. 617 sera from 181 patients with established RA and 436 with non-RA rheumatic diseases were tested by ELISA for AhFibA, anti-CCP2, anti-α36-50Cit₃₈,₄₂, anti-β60-74Cit₆₀,₇₂,₇₄ autoantibodies, and by nephelometry for rheumatoid factor (RF). Diagnostic indexes, correlations and concordances between tests were analysed. Crossreactivity of anti-α36-50Cit₃₈,₄₂ and anti-β60-74Cit₆₀,₇₂,₇₄ autoantibodies was assessed in competition experiments. At a diagnostic specificity of 95%, the diagnostic sensitivity of AhFibA (83%) was significantly higher than that of all other tests. The diagnostic sensitivity of anti-β60-74Cit₆₀,₇₂,₇₄ (71%) was significantly higher than that of anti-α36-50Cit₃₈,₄₂ autoantibodies (51%) but similar to that of anti-CCP2 (74%). Titres of RF, anti-α36-50Cit₃₈,₄₂ and anti-β60-74Cit₆₀,₇₂,₇₄ autoantibodies were weakly correlated with each other, whereas titres of anti-β60-74Cit₆₀,₇₂,₇₄ were strongly correlated with those of AhFibA (r=0.633) and anti-CCP2 (r=0.634). Anti-α36-50Cit₃₈,₄₂ and anti-β60-74Cit₆₀,₇₂,₇₄ mainly corresponded to two non-crossreactive subfamilies of ACPA. More than 90% of AhFibA-positive or anti-CCP2-positive sera recognised the α36-50Cit₃₈,₄₂ and/or the β60-74Cit₆₀,₇₂,₇₄ peptide. Autoantibodies reactive to α36-50Cit₃₈,₄₂ and β60-74Cit₆₀,₇₂,₇₄ form two distinct, non-overlapping subfamilies of ACPA that, together, cover practically all the ACPA reactivity to citrullinated fibrinogen and to CCP2 antigens. In established RA, anti-β60-74Cit₆₀,₇₂,₇₄ autoantibodies show diagnostic indexes similar to those of anti-CCP2.