POS0428 PATHOGENETIC ROLE OF MICROPARTICLES IN RHEUMATOID ARTHRITIS

Abstract

BackgroundMicroparticles (MPs) are fragments of surface membranes of activated eukaryotic cells. They are characterized by different dimensions (from 0.1 to 1μm) and expression of surface antigens, depending on their origin. MPs are important mediators of cell-to-cell communication as they can be internalized in a dose-dependent manner by macrophages, endothelial cells and other cell types, influencing both functional and phenotypic characteristics of the target cells. Even if MPs formation is enhanced by cell activation or apoptosis, constitutive exocytosis is a continuous ongoing process in vivo for many cells, and MPs originating from different cells can be always found in the plasma. In various autoimmune diseases, it has been found an increased number of MPs derived from activated platelets, leukocytes, vascular endothelium cells and other cell types. In Rheumatoid Arthritis (RA) an excessive production of MPs may predispose to autoimmune manifestations. Moreover, it has been speculated that MPs can stimulate the production, secretion, and transport of inflammatory factors in RA.ObjectivesWe investigated the presence on the surface of RA-MPs of antigens derived from post-translationally modified proteins (citrullinated peptides and carbamylated peptides). We assumed that these specific antigens carried on the surface of RA-MPs could participate in RA pathogenetic process.MethodsWe enrolled 20 RA patients naïve for biological therapy fulfilling the 2010 American College of Rheumatology RA criteria and 20 healthy controls (HC), matched for age and sex. For each patient, laboratory and clinical data were recorded and clinical indexes were measured (TJ, SJ, CDAI, VAS pain, CDAI, SDAI, DAS28). A fasting blood sample, obtained from RA patients and HC, was centrifugated in order to obtain platelet-poor plasma (PPP), rich in MPs. Thereafter, MPs in RA patients and HC were measured using nanoparticle tracking analysis. Later on, MPs were incubated with unconjugated anti-citrullinated/carbamylated proteins antibodies and processed by flow cytometry and western blot to evaluate the surface expression of citrullinated/carbamylated antigens.ResultsNanoparticle tracking analysis revealed a significant increase of number of MPs in RA compared to HC. Moreover, densitometric analysis showed a significative higher expression of citrullinated antigens on MPs’ surface in RA than controls (p < 0.0001), while no substantial difference was found in the expression of carbamylated antigens. The data obtained were confirmed with the western blot which identified the cytoskeletal protein vimentin, the cytoplasmatic glycolytic enzyme alpha-enolase1 and type II collagen as the main citrullinated and carbamylated proteins carried by MPs. Finally, a relevant correlation between the expression of citrullinated and carbamylated antigens and disease activity was found (Figure 1).Figure 1.The figure shows: (A) concentration of MPs in RA patients and HC (nanoparticle tracking analysis); (B) expression of citrullinated and carbamylated antigens on MPs’ surface in RA patients (flow cytometry analysis); (C) expression of citrullinated antigens in RA patients and HC and correlation between expression of citrullinated and carbamylated antigens on MPs’ surface in RA patients and DAS28, CDAI, SDAI; (D) cytoskeletal protein vimentin, cytoplasmatic glycolytic enzyme alpha-enolase1 and collagen type II (western blot).ConclusionThe results of this study confirm an important role of MPs in the pathogenesis of RA not only as markers of disease activity but also as possible inducers of autoimmunity.Disclosure of InterestsNone declared.