Eszter Szarka

Induction of osteoclastogenesis and bone loss by human autoantibodies against citrullinated vimentin

Autoimmunity is complicated by bone loss. In human rheumatoid arthritis (RA), the most severe inflammatory joint disease, autoantibodies against citrullinated proteins are among the strongest risk factors for bone destruction. We therefore hypothesized that these autoantibodies directly influence bone metabolism. Here, we found a strong and specific association between autoantibodies against citrullinated proteins and serum markers for osteoclast-mediated bone resorption in RA patients. Moreover, human osteoclasts expressed enzymes eliciting protein citrullination, and specific N-terminal citrullination of vimentin was induced during osteoclast differentiation. Affinity-purified human autoantibodies against mutated citrullinated vimentin (MCV) not only bound to osteoclast surfaces, but also led to robust induction of osteoclastogenesis and bone-resorptive activity. Adoptive transfer of purified human MCV autoantibodies into mice induced osteopenia and increased osteoclastogenesis. This effect was based on the inducible release of TNF-α from osteoclast precursors and the subsequent increase of osteoclast precursor cell numbers with enhanced expression of activation and growth factor receptors. Our data thus suggest that autoantibody formation in response to citrullinated vimentin directly induces bone loss, providing a link between the adaptive immune system and bone.

Specific expression of PAD4 and citrullinated proteins in lung cancer is not associated with anti-CCP antibody production

Anti-citrullinated protein antibodies (ACPAs), produced against citrullinated proteins, are diagnostic and prognostic markers of rheumatoid arthritis (RA). The underlying mechanism that explains the connection of smoking, citrullination [catalyzed by peptidyl arginine deiminases (PADs)] and ACPAs is still unclarified in RA. Thus, we searched for a non-arthritic model in which an increased cell death allows the formation of autoantibodies. Data supporting that lung cancer might be a good candidate are as follows: (i) smoking plays a role in its pathogenesis, (ii) the disease is frequently accompanied by paraneoplastic syndrome, (iii) smoking increases citrullination in the lung, (iv) various types of malignancies are associated with increased citrullination and (v) lung cancer tissue shows similarities with RA synovium. Serum PAD4, rheumatoid factor (RF) and ACPA levels were measured in 42 lung cancer patients; expression of cytokeratin 7 (CK7), PAD4 and citrullinated proteins was visualized in 113 lung cancer tissues. All parameters were analyzed in correlation with smoking history. None of the patients had polyarthritis or autoimmune disease. Significantly increased RF levels were associated with higher PAD4 levels in smoker lung cancer patients compared with non-smokers. Both PAD4 and citrullination immunostaining strongly correlated with that of CK7 in lung cancer, however, did not differ according to smoking history. Two of 30 smoker lung cancer patients had high anti-cyclic citrullinated peptide levels. In conclusion, PAD4 and citrullination may be helpful in distinguishing lung cancer from healthy tissue. Smoking, abnormal serum PAD4 and RF levels may not be sufficient for the production of ACPAs and development of autoimmunity.

Recognition of new citrulline-containing peptide epitopes by autoantibodies produced in vivo and in vitro by B cells of rheumatoid arthritis patients

Anti‐citrullinated peptide/protein antibodies (ACPAs) are highly sensitive and specific markers of rheumatoid arthritis (RA). Identification of peptide epitopes that may detect different subgroups of RA patients might have diagnostic and prognostic significance. We have investigated citrulline‐ and arginine‐containing peptide pairs derived from filaggrin, collagen or vimentin, and compared this citrulline‐peptide panel with the serological assays conventionally used to detect ACPAs. Furthermore, we studied if the same citrulline‐peptides identify antibody‐secreting cells in in vitro cultures of RA B cells. Recognition of citrulline‐ and arginine‐containing filaggrin, vimentin and collagen peptide epitopes were tested by Multipin ELISA system, by indirect ELISA and by a peptide‐specific microarray. B cells were purified from blood by negative selection; antibody‐producing cells were enumerated by ELISPOT assay. The panel composed of citrulline‐peptide epitopes of filaggrin, collagen and vimentin was recognized by RA sera with a sensitivity and specificity comparable with the currently used tests. Moreover, the combined citrulline‐peptide panel including the new short epitope peptide of filaggrin, fil311‐315, also identified nearly one‐third of RA cases that were negative for antibodies against cyclic citrullinated peptides, mutated citrullinated vimentin or for rheumatoid factor. The results with the peptide‐specific microarray have shown that although most ACPAs recognizing the four citrulline peptides are IgG, some of them specifically recognizing citrulline‐containing filaggrin peptides (fil311–315 and fil306–326) are IgM, and so may be produced either by newly formed activated B cells or by unswitched B memory cells. Furthermore, the citrulline‐peptides of filaggrin and vimentin detect ACPA‐producing cells, and so could also be applied to study the B cells of RA patients.

Role of N - Or C -terminal biotinylation in autoantibody recognition of citrullin containing filaggrin epitope peptides in rheumatoid arthritis

Here, we report on the synthesis, conformational analysis, and autoantibody binding properties of new sets of rheumatoid arthritis (RA) specific biotin-peptide conjugates derived from filaggrin epitope peptides. The biotin with or without a linker was attached to the Cit or Arg containing epitope core ((311)TXGRS(315)) or epitope region ((306)SHQESTXGXSXGRSGRSGS(324)) peptide (where X = Cit), through an amide bond at the N- or C-terminal of the epitopes. Antibody binding was detected by indirect enzyme-linked immunosorbent assay (ELISA) using sera from RA, Systemic lupus erythematosus (SLE) patients, as well as healthy individuals, and the secondary structure of conjugates was investigated by electronic circular dichroism (ECD). We found that autoantibodies from RA patients recognize specifically both filaggrin epitope region ((306)SHQESTXGXSXGRSGRSGS(324)) and short epitope core ((311)TXGRS(315)) peptides. Our data also indicate that the positioning of the biotin label within a peptide sequence can markedly influence the antibody binding, but the length of the linker incorporated has essentially no effect on the recognition. ECD experiments demonstrate that the Arg/Cit change does not influence the solution conformation of the peptide conjugates. However, the presence and position of the biotin moiety has a pronounced effect on the conformation of the 5-mer epitope core peptides, while it does not alter the secondary structure of the 19-mer epitope region peptides.

Bead arrays for antibody and complement profiling reveal joint contribution of antibody isotypes to C3 deposition.

The development of antigen arrays has provided researchers with great tools to identify reactivities against self or foreign antigens from body fluids. Yet, these approaches mostly do not address antibody isotypes and their effector functions even though these are key points for a more detailed understanding of disease processes. Here, we present a bead array-based assay for a multiplexed determination of antigen-specific antibody levels in parallel with their properties for complement activation. We measured the deposition of C3 fragments from serum samples to reflect the degree of complement activation via all three complement activation pathways. We utilized the assay on a bead array containing native and citrullinated peptide antigens to investigate the levels of IgG, IgM and IgA autoantibodies along with their complement activating properties in serum samples of 41 rheumatoid arthritis patients and 40 controls. Our analysis revealed significantly higher IgG reactivity against the citrullinated fibrinogen β and filaggrin peptides as well as an IgA reactivity that was exclusive for citrullinated fibrinogen β peptide and C3 deposition in rheumatoid arthritis patients. In addition, we characterized the humoral immune response against the viral EBNA-1 antigen to demonstrate the applicability of this assay beyond autoimmune conditions. We observed that particular buffer compositions were demanded for separate measurement of antibody reactivity and complement activation, as detection of antigen-antibody complexes appeared to be masked due to C3 deposition. We also found that rheumatoid factors of IgM isotype altered C3 deposition and introduced false-positive reactivities against EBNA-1 antigen. In conclusion, the presented bead-based assay setup can be utilized to profile antibody reactivities and immune-complex induced complement activation in a high-throughput manner and could facilitate the understanding and diagnosis of several diseases where complement activation plays role in the pathomechanism.

CD16/32-specific biotinylated 2.4G2 single-chain Fv complexed with avidin–FITC enhances FITC-specific humoral immune response in vivo in a CD16-dependent manner

Fcgamma receptors (FcgammaRs) play an essential role in the regulation of immune response due to their ability to bind immune complexes. Activating FcgammaRs may facilitate antigen presentation and dendritic-cell maturation, while in the late phase of the immune response, the inhibitory FcgammaRIIb may down-regulate B-cell activation upon cross-linking with activating receptors. In this study, we investigated the in vivo role of FcgammaRs on the modulation of humoral immune response. In order to get well-defined immune complexes that can bind to both the activating and the inhibitory FcgammaRs, we designed a mono-biotinylated single-chain fragment variable construct from the rat anti-mouse CD16/32 clone 2.4G2, linked to avidin-FITC, and tested its effect on the FITC-hapten-specific T-independent type 2 (TI-2) and T-dependent (TD) immune response. When injected intravenously in mice, the complex bound to a small portion of B220+, CD11b(high) and CD11c(high) cells and was localized in the spleen on marginal zone macrophages 15 min after treatment. When applied as a booster following primary immunization with TI-2 (FITC-dextran) or TD (FITC-keyhole limpet haemocyanin) antigens, the complex elevated the number of hapten-specific IgM/IgG-producing B cells. This effect was diminished in CD16KO mice, suggesting that the activating-type FcgammaRIII might be a key mediator of this mechanism.

Affinity Purification and Comparative Biosensor Analysis of Citrulline-Peptide-Specific Antibodies in Rheumatoid Arthritis

Background: In rheumatoid arthritis (RA), anti-citrullinated protein/peptide antibodies (ACPAs) are responsible for disease onset and progression, however, our knowledge is limited on ligand binding affinities of autoantibodies with different citrulline-peptide specificity. Methods: Citrulline-peptide-specific ACPA IgGs were affinity purified and tested by ELISA. Binding affinities of ACPA IgGs and serum antibodies were compared by surface plasmon resonance (SPR) analysis. Bifunctional nanoparticles harboring a multi-epitope citrulline-peptide and a complement-activating peptide were used to induce selective depletion of ACPA-producing B cells. Results: KD values of affinity-purified ACPA IgGs varied between 10−6 and 10−8 M and inversely correlated with disease activity. Based on their cross-reaction with citrulline-peptides, we designed a novel multi-epitope peptide, containing Cit-Gly and Ala-Cit motifs in two–two copies, separated with a short, neutral spacer. This peptide detected antibodies in RA sera with 66% sensitivity and 98% specificity in ELISA and was recognized by 90% of RA sera, while none of the healthy samples in SPR. When coupled to nanoparticles, the multi-epitope peptide specifically targeted and depleted ACPA-producing B cells ex vivo. Conclusions: The unique multi-epitope peptide designed based on ACPA cross-reactivity might be suitable to develop better diagnostics and novel therapies for RA.

Identification of a new citrullinated epitope on filaggrin for the early diagnosis of rheumatoid arthritis

Anti-citrullinated protein antibodies (ACPA) are sensitive and specific markers for diagnosis and prognosis in rheumatoid arthritis (RA). Citrullination is a post-translational modification of arginine by deimination, induced by peptidylarginine deiminase. It is a physiologically occurring phenomena during apoptosis, inflammation or keratinisation. Citrullination has been observed in different synovial proteins, including fibrinogen, vimentin and collagen. Antibodies specific for cyclic citrullinated filaggrin peptides (CCP) were detected in RA sera and anti-CCP positivity is widely used for diagnostic purposes. However, to determine …

A5.11 Detection of ACPA Producing B-Cells by a Citrulline Peptide Panel

Background and Objectives Anti-citrullinated protein/peptide antibodies (ACPAs) are the most sensitive and specific serological markers of RA. To identify the optimal epitopes that detect different subgroups of RA patients with high sensitivity and specificity, we have investigated citrulline and arginine containing peptides derived from filaggrin, collagen or vimentin. We have identified a citrulline-containing peptide panel that was recognised by RA sera with high specificity. Our aim was to compare this peptide panel with the conventionally used serological assays and to detect peptide-specific ACPA producing B-cells in in vitro cultures. Materials and Methods Previously selected citrulline- and arginine-containing filaggrin, vimentin and collagen peptide epitopes were investigated. We compared the recognition of these peptides by RA and control sera using indirect ELISA. B-cells were purified from peripheral blood by negative selection, IgG production was stimulated by B-cell activators (R848 and recombinant human IL-2) provided with the human ELISPOT kit. Antibody producing cells were enumerated after 4 days culture by using peptide-specific ELISPOT assay. Results Sera samples from 247 RA and 148 age-matched (57 ± 14 years) healthy controls were collected. The citrulline peptide panel detected approximately 80% of RA patients, including 20% of seronegative/CCP negative patients as well. Individual peptides detected different subgroups of RA patients. The more peptides recognised by a particular RA serum sample, the more severe the disease of the patient was. In vitro cultured B-cells from selected RA patients synthesised multiple citrulline-containing peptide-specific antibodies after polyclonal stimulation, while B-cells from healthy blood donors did not. Conclusions The citrulline peptide panel can detect 20% of ACPA negative RA patients thus may have a prognostic value. Furthermore, the panel is suitable to detect citrulline peptidespecific antibody producing cells, thus enables us to study ACPA producing B-cells of RA patients.

Exacerbation of collagen induced arthritis by Fcγ receptor targeted collagen peptide due to enhanced inflammatory chemokine and cytokine production

Antibodies specific for bovine type II collagen (CII) and Fcγ receptors play a major role in collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis (RA). Our aim was to clarify the mechanism of immune complex-mediated inflammation and modulation of the disease. CII pre-immunized DBA/1 mice were intravenously boosted with extravidin coupled biotinylated monomeric CII-peptide epitope (ARGLTGRPGDA) and its complexes with biotinylated FcγRII/III specific single chain Fv (scFv) fragment. Disease scores were monitored, antibody titers and cytokines were determined by ELISA, and binding of complexes was detected by flow cytometry and immune histochemistry. Cytokine and chemokine secretion was monitored by protein profiler microarray. When intravenously administered into collagen-primed DBA/1 mice, both CII-peptide and its complex with 2.4G2 scFv significantly accelerated CIA and increased the severity of the disease, whereas the monomeric peptide and monomeric 2.4G2 scFv had no effect. FcγRII/III targeted CII-peptide complexes bound to marginal zone macrophages and dendritic cells, and significantly elevated the synthesis of peptide-specific IgG2a. Furthermore, CII-peptide containing complexes augmented the in vivo secretion of cytokines, including IL-10, IL-12, IL-17, IL-23, and chemokines (CXCL13, MIP-1, MIP-2). These data indicate that complexes formed by the CII-peptide epitope aggravate CIA by inducing the secretion of chemokines and the IL-12/23 family of pro-inflammatory cytokines. Taken together, these results suggest that the in vivo emerging immune complexes formed with autoantigen(s) may trigger the IL-12/23 dependent pathways, escalating the inflammation in RA. Thus blockade of these cytokines may be beneficial to downregulate immune complex-induced inflammation in autoimmune arthritis.