The role of citrullination of an immunodominant proteoglycan (PG) aggrecan T cell epitope in BALB/c mice with PG-induced arthritis.

The P70-84 peptide (also called 5/4E8 epitope) of the human cartilage proteoglycan (PG) aggrecan is the dominant/arthritogenic epitope in both humans and arthritis-prone BALB/c mice (PG-induced arthritis, PGIA). An elevated T cell reactivity was demonstrated to a citrullinated version of the P70-84 epitope in most of the patients with rheumatoid arthritis (RA). The goal of this study was to understand better how a T cell epitope, if citrullinated, may affect antigenicity/arthritogenicity in PGIA, a murine model of RA. T cell reactivity to differentially citrullinated versions of either the human PG aggrecan P70-84 peptide or the corresponding mouse sequence was assessed in peptide or aggrecan-immunized and arthritic BALB/c mice as well as in T cell receptor transgenic mice specific for peptide P70-84 sequence. Peripheral T cell responses were induced by priming BALB/c mice with either the human wild-type or its citrullinated versions. Unexpectedly, priming with the citrullinated self-peptide induced a higher T cell response compared to the wild-type sequence (p<0.001), and the citrullination of the human peptide abolished T cell reactivity in PGIA. Our data suggest that T cells reactive to the citrullinated P70-84 peptide escaped thymic selection and are present in the peripheral T cell repertoire. Results of this study provide evidence that citrullination of an immunodominant T cell epitope may substantially alter, either increase or abolish, T cell recognition at the periphery in an experimental model of arthritis.

The recently identified hexosaminidase D enzyme substantially contributes to the elevated hexosaminidase activity in rheumatoid arthritis.

Since the 1970s, numerous reports have described elevated hexosaminidase activities in rheumatoid arthritis. However, due to the overlapping substrate specificities of different hexosaminidases, identification of the exact enzyme(s) responsible for the elevated activity remains incomplete. In this work we tested if the recently described enzyme, hexosaminidase D was expressed in human arthritic joints, and could contribute to the elevated hexosaminidase activity in rheumatoid arthritis. Thermostable ß-d-N-acetyl-galactosaminidase (hexosaminidase D) activities were determined in synovial fluid samples, synovial membranes, synovial fibroblast cell strains and synovial fibroblast-derived extracellular vesicles of patients with rheumatoid arthritis and osteoarthritis using chromogenic substrates. Expression of the HEXDC gene was detected both in steady state and in TGF-ß treated synovial fibroblasts by real time PCR. Strikingly, hexosaminidase D accounted for approximately 50% of the total ß-N-acetyl-galactosaminidase activity in synovial membranes and synovial fibroblasts, and it was responsible for the vast majority of the ß-d-N-acetyl-galactosaminidase activity in synovial fluid samples. TGF-ß downregulated the expression of hexosaminidase D in synovial fibroblasts dose-dependently. Of note, significant activity of hexosaminidase D was also found in association with extracellular vesicles released by synovial fibroblasts. This first study that describes the expression and disease relevance of the HEXDC gene in humans demonstrates the expression of this novel enzyme within the joints, and suggests that its activity may significantly contribute to the overall local exoglycosidase activity.

Proteomic characterization of thymocyte-derived microvesicles and apoptotic bodies in BALB/c mice.

Several studies have characterized exosomes derived from different cell sources. In this work we set the goal of proteomic characterization of two less studied populations of membrane vesicles, microvesicles (100-800 nm) and apoptotic bodies (> 800 nm) released by thymus cells of BALB/c mice. The vesicles were isolated by the combination of differential centrifugation and gravity driven multistep filtration of the supernatant of thymus cell cultures. The size distribution of vesicle preparations was determined by transmission electron microscopy. Proteins were released from the vesicles, digested in solution, and analyzed using nano-HPLC/MS(MS). Ingenuity pathway analysis was used to identify functions related to membrane vesicle proteins. In apoptotic bodies and microvesicles we have identified 142 and 195 proteins, respectively. A striking overlap was detected between the proteomic compositions of the two subcellular structures as 108 proteins were detected in both preparations. Identified proteins included autoantigens implicated in human autoimmune diseases, key regulators of T-cell activation, molecules involved in known immune functions or in leukocyte rolling and transendothelial transmigration. The presence and abundance of proteins with high immunological relevance within thymocyte-derived apoptotic bodies and microvesicles raise the possibility that these subcellular structures may substantially modulate T-cell maturation processes within the thymus.

Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles.

Release of membrane vesicles, a process conserved in both prokaryotes and eukaryotes, represents an evolutionary link, and suggests essential functions of a dynamic extracellular vesicular compartment (including exosomes, microparticles or microvesicles and apoptotic bodies). Compelling evidence supports the significance of this compartment in a broad range of physiological and pathological processes. However, classification of membrane vesicles, protocols of their isolation and detection, molecular details of vesicular release, clearance and biological functions are still under intense investigation. Here, we give a comprehensive overview of extracellular vesicles. After discussing the technical pitfalls and potential artifacts of the rapidly emerging field, we compare results from meta-analyses of published proteomic studies on membrane vesicles. We also summarize clinical implications of membrane vesicles. Lessons from this compartment challenge current paradigms concerning the mechanisms of intercellular communication and immune regulation. Furthermore, its clinical implementation may open new perspectives in translational medicine both in diagnostics and therapy.

Detection and isolation of cell-derived microparticles are compromised by protein complexes resulting from shared biophysical parameters.

Numerous diseases, recently reported to associate with elevated microvesicle/microparticle (MP) counts, have also long been known to be characterized by accelerated immune complex (IC) formation. The goal of this study was to investigate the potential overlap between parameters of protein complexes (eg, ICs or avidin-biotin complexes) and MPs, which might perturb detection and/or isolation of MPs. In this work, after comprehensive characterization of MPs by electron microscopy, atomic force microscopy, dynamic light-scattering analysis, and flow cytometry, for the first time, we drive attention to the fact that protein complexes, especially insoluble ICs, overlap in biophysical properties (size, light scattering, and sedimentation) with MPs. This, in turn, affects MP quantification by flow cytometry and purification by differential centrifugation, especially in diseases in which IC formation is common, including not only autoimmune diseases, but also hematologic disorders, infections, and cancer. These data may necessitate reevaluation of certain published data on patient-derived MPs and contribute to correct the clinical laboratory assessment of the presence and biologic functions of MPs in health and disease.

Natural autoantibodies reactive with glycosaminoglycans in rheumatoid arthritis.

INTRODUCTION: Although natural autoantibodies make up the majority of circulating immunoglobulins and are also present in high numbers in therapeutically used intravenous immunoglobulin preparations, they have received little attention and their precise role remains largely unknown. An increasing awareness of the importance of posttranslational autoantigen modifications and glycobiology led us to explore carbohydrate-reactive natural autoantibodies in patients with rheumatoid arthritis. This study examined systematic antibodies reactive to glycosaminoglycans (GAGs), the carbohydrate components of proteoglycans that are released in large amounts from degrading cartilage. METHODS: To measure antibodies reactive to six different types of GAGs, a specialised ELISA was used in which the carbohydrates were covalently linked to the plastic surface through a 2 nm spacer. Sera from rheumatoid arthritis patients (n = 66), umbilical cord serum samples (n = 11) and adult controls (n = 54) were studied. In order to explore cross-reactivity with microbial antigens, bacterial peptidoglycans and fungal polysaccharides were used. Sera and synovial fluid samples were also tested using a GlycoChip carbohydrate array to characterise individual carbohydrate recognition patterns. We followed a multistep statistical screening strategy for screening GAG-reactive antibodies as predictive disease markers. RESULTS: While anti-GAG antibodies were absent in the umbilical cord sera, they were readily detectable in adult controls and were significantly elevated in patients with rheumatoid arthritis (p < 0.001). Anti-GAG antibodies showed significant cross-reactivity among different types of GAGs. They also reacted with bacterial peptidoglycans and fungal polysaccharides. Interestingly, anti-chondroitin sulphate C IgM antibody levels showed inverse correlation both with the Disease Activity Score (DAS) 28 scores and C-reactive protein (CRP) levels in rheumatoid arthritis. CONCLUSION: The highly abundant and cross-reactive, GAG-specific natural autoantibodies in serum may serve as novel disease-state markers in patients with rheumatoid arthritis.