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.

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.

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.

Integration of signals mediated by B-cell receptor, B-cell activating factor of the tumor necrosis factor family (BAFF) and Fas (CD95).

The survival of the mature resting B cells depends on signaling from B cell receptor (BCR), and a plethora of positive and negative regulators, that maintain cellular homeostasis and ultimately determine cell's fate, i.e., survival or programmed death (apoptosis). Among these regulators we have investigated the B cell activating factor belonging to tumor necrosis factor family (BAFF) and the prototypic death receptor Fas/CD95 mediated signals. We have shown that BAFF inhibits Fas-mediated cell death, however, the BCR-driven survival signals were not strengthened by BAFF. Therefore, we propose that BAFF may function independently of the antigen specificity of BCR, thus may enhance the risk of autoimmune diseases by promoting the survival of bystander B cells in the germinal center.