Antibodies to citrulline-modified proteins have a high diagnostic value in rheumatoid arthritis (RA). However, their biological role in disease development is still unclear. To obtain insight into this question, a panel of mouse monoclonal antibodies was generated against a major triple helical collagen type II (CII) epitope (position 359–369; ARGLTGRPGDA) with or without arginines modified by citrullination. These antibodies bind cartilage and synovial tissue, and mediate arthritis in mice. Detection of citrullinated CII from RA patients' synovial fluid demonstrates that cartilage-derived CII is indeed citrullinated in vivo. The structure determination of a Fab fragment of one of these antibodies in complex with a citrullinated peptide showed a surprising β-turn conformation of the peptide and provided information on citrulline recognition. Based on these findings, we propose that autoimmunity to CII, leading to the production of antibodies specific for both native and citrullinated CII, is an important pathogenic factor in the development of RA.

Collagen type II (CII) is a relevant joint‐specific autoantigen in the pathogenesis of rheumatoid arthritis (RA). Whereas the reasons for the breakage of self tolerance to this major cartilage component are still enigmatic, T cell responses to glycosylated CII determinants in RA patients indicate that post‐translational modifications play a role. Since the conversion of arginine into citrulline by peptidylarginine deiminases (PAD) in some non‐joint‐specific antigens such as filaggrin or fibrin has been shown to give rise to RA‐specific humoral immune responses, we investigated whether PAD modification of cartilage‐specific CII might affect its recognition by circulating autoantibodies in early RA. In vitro treatment with purified PAD led to arginine deimination of native CII or of synthetic CII peptides as evidenced by amino acid analysis. The citrullination resulted in modified recognition of the immunodominant CII epitope C1III (amino acid residues 359–369) by murine and human antibodies. In a cohort of early RA patients (n=286), IgG antibodies directed toward a synthetic citrullinated C1III peptide (citC1III‐P) were detectable with a prevalence of 40.4%. The partial autoantibody cross‐reactivity between citC1III‐P and citrullinated peptides mimicking epitopes of the cytoskeletal autoantigen filaggrin suggests that autoimmunity to cartilage‐specific modified self might be a critical intermediate bridging recognition of PAD‐modified extra‐articular autoantigens with the disruption of tolerance to native cartilage constituents.

Activation of the nuclear transcription factor κB (NF-κB) regulates the expression of inflammatory genes crucially involved in the pathogenesis of inflammatory diseases. NF-κB governs the expression of adhesion molecules that play a pivotal role in leukocyteendothelium interactions. We uncovered the crucial role of NF-κB activation within endothelial cells in models of immune-mediated diseases using a "sneaking ligand construct" (SLC) selectively inhibiting NF-κB in the activated endothelium. The recombinant SLC1 consists of three modules: (i) an E-selectin targeting domain, (ii ) a Pseudomonas exotoxin A translocation domain, and (iii) a NF-κB Essential Modifier-binding effector domain interfering with NF-κB activation. The E-selectin-specific SLC1 inhibited NF-κB by interfering with endothelial IκB kinase 2 activity in vitro and in vivo. In murine experimental peritonitis, the application of SLC1 drastically reduced the extravasation of inflammatory cells. Furthermore, SLC1 treatment significantly ameliorated the disease course in murine models of rheumatoid arthritis. Our data establish that endothelial NF-κB activation is critically involved in the pathogenesis of arthritis and can be selectively inhibited in a cell type-and activation stage-dependent manner by the SLC approach. Moreover, our strategy is applicable to delineating other pathogenic signaling pathways in a cell type-specific manner and enables selective targeting of distinct cell populations to improve effectiveness and riskbenefit ratios of therapeutic interventions.cell targeting | intracellular signaling | autoimmune disorders | mouse models | inhibit inflammation A critical step in the effector phase of pathogenic immune responses is the extravasation of circulating leukocytes from the vasculature and their migration along gradients of chemoattractants into the target tissues (1). In inflammation, endothelial activation represents a multistep cascade of events leading to increased vascular permeability for plasma proteins; the expression of proinflammatory cytokines, chemokines, and enzymes; and an up-regulation of adhesion molecules that are regulated by the nuclear transcription factor κB (NF-κB) (2-4).Activation of NF-κB via the IκB kinase (IKK) complex is regarded as the classical NF-κB pathway. The IKK complex contains the kinases IKK1 and IKK2 and the regulatory subunit NF-κB Essential Modifier (NEMO) (5). Activation of the classical NF-κB pathway requires association of NEMO with IKK2 (5). The expression of multiple proinflammatory genes including the adhesion molecules ICAM-1, VCAM-1, E-selectin, and chemokines, like MCP-1 and IL-8, contributes to the inflammatory endothelial cell response and is initiated through activation of the classical NF-κB pathway (6).

Objective. Colony-stimulating factor 1 receptor (CSF-1R) essentially modulates monocyte proliferation, migration, and activation, which are considered important for the pathogenesis of rheumatoid arthritis (RA). We undertook this study to determine CSF-1R expression in human RA as well as the efficacy of a specific anti-CSF-1R monoclonal antibody (AFS98) in 2 different animal models of RA.Methods. CSF-1R expression was examined in blood, synovium, and bone samples from RA patients, osteoarthritis (OA) patients, and healthy subjects. The efficacy of AFS98 was examined by clinical assessment, histology, and bone histomorphometry in collageninduced arthritis (CIA) and serum-transfer arthritis.Results. CSF-1R expression was increased in the synovium of RA patients compared to OA patients and healthy controls in fibroblast-like synoviocytes, follicular dendritic cells, macrophages, and osteoclasts. Circulating RA monocytes and neutrophils but not lymphocytes were CSF-1R؉. In mice, blockade of CSF-1R abrogated cartilage damage, bone erosion, and systemic bone loss, and this was associated with the depletion of osteoclasts in both models. While blockade of CSF-1R did not affect inflammation in passive serum-transfer arthritis, it significantly reduced inflammation in CIA, and this was associated with the absence of synovial macrophages and reduced splenic CD11b؉Gr-1؊ monocytes.Conclusion. CSF-1R was broadly expressed in human RA. Blockade of CSF-1R protected against bone and cartilage destruction in both mouse models and also showed significant antiinflammatory effects in the CIA model. These data provide evidence for CSF-1R as a therapeutic target in RA.Joint destruction in rheumatoid arthritis (RA) is mediated by inflammatory synovial tissue and subsequent osteoclastic bone resorption (1). This process is guided by inflammatory cytokines, which induce the expression of key factors involved in osteoclast differentiation. While numerous studies have shown the importance of RANKL in osteoclast-mediated bone resorption in both experimental inflammatory arthritis and human RA, data have been very limited to date concerning the role of the second essential factor for osteoclast differentiation, colony-stimulating factor 1 (CSF-1).The human CSF-1 receptor (CSF-1R; c-Fms) is a Drs. Toh, Bonnefoy, Haegel, Preville, Guillen, and Ancian and Ms Cochin, Mr. De Meyer, and Ms Thioudellet own stock or stock options in Transgene SA. Dr. Haegel and Ms Thioudellet are inventors of the anti-colony-stimulating factor 1 receptor monoclonal antibody CXIIG6 and its derivative, patents for which are assigned to Transgene SA.