*4.1. Rheumatoid Arthritis*

An increase in circulating MPs secretion is closely linked to an increase in cytokine production and appears to be a significant factor which affects inflammation development within the synovial membrane in rheumatoid arthritis [46]. In one of the first studies assessing the relationship between MPs and rheumatoid arthritis in a group of 19 patients, Knifjj-Dutmer et al. observed an increased number of circulating PMPs compared to a group of healthy individuals, and a significant relationship between the number of circulating microparticles of platelet origin and the disease activity assessed by the DAS28 score [49]. Moreover, researchers suggested a possible effect of PMPs on the development of cardiovascular diseases in patients with RA, leading to increased mortality linked to vascular complications, compared to the general population [49]. Viñuela-Berni et al. observed an increased number of MPs with CD3, CD14, CD19, CD41, and CD63E antigens in plasma of RA patients with high disease activity (DAS 28 > 5.1) [52]. The link between the intensity of inflammation in RA and the number and activity of released microparticles of endothelial origin was confirmed by Barbati et al. The total pool of circulating MPs and endothelial MPs (EMPs) initially increased, then decreased after four months of anti-TNFα therapy [58]. The microparticles with CD3, CD14, CD19, CD41, and CD63E antigens also stimulated secretion of TNFα and IL-1, IL-17 by monocytes in vitro [52]. The potential role of microparticles in the pathogenesis of rheumatoid arthritis appears to be very complex (Table 3).


**Table 3.** Potential role of microparticles in the development of rheumatoid arthritis.

C—complement component; CCL—C-C motif chemokine ligand; CXC—CXC chemokines; FLS—fibroblast-like synoviocyte; IL—interleukin; MCP—monocyte chemoattractant protein; MMP—metalloproteinase; MPs—microparticles; RA—rheumatoid factor; TNF—tumor necrosis factor; VEGF—vascular endothelium growth factor.

Apart from serum, microparticles have been detected in other body fluids of the patients. Viñuela-Berni et al. observed an increased number of MPs with CD3, CD14, and CD19 antigens in the urine of RA patients with high disease activity [52]. Boilard et al. [8] analyzed samples of synovial fluid

in RA patients and found it to contain large numbers of PMPs (slightly less than 2 × 105 CD41+ MPs/μL). Synovial fluid of RA patients contained (much less than PMPs) MPs with surface antigens of neutrophils, monocytes, and T-cells. It was an interesting observation to determine a group of neutrophils present in rheumatoid synovial fluid, with simultaneously present leukocyte CD45 antigen and a platelet CD41 antigen. The immunofluorescence signal was a result of attaching to neutrophils of the bodies corresponding to PMPs rather than whole platelets [8]. Interestingly, the number of PMPs determined in rheumatoid fluid in this study was much higher than in serum of RA patients in the study by Knijff et al. [49], where it was 600 per μL. Michael et al. [54] determined the number of MPs in synovial fluid in RA patients and found a considerably larger number of MPs derived from granulocytes, monocytes, and T-cells than in patients with degenerative joint disease and in the control group. Moreover, the number of granulocyte-derived MPs in RA patients was significantly larger in the patients with aCCP antibodies.

It is not completely clear how platelets infiltrate synovial fluid, although undamaged platelets, platelet aggregates, and platelets attached to leukocytes have been found in studies with RA patients for many years [66–68]. It is probable that collagen, fibrinogen, proteolytic enzymes, inflammatory cytokines, and shear forces in a joint could stimulate the production of platelet-derived MPs. It is also possible that, owing to their size, microparticles can penetrate synovial fluid and synovial membranes because the number of platelet microparticles in synovial fluid in RA patients is considerably larger than the number of PMPs in peripheral blood, which may suggest locally increased microparticle release from activated platelets in vessels near joints. Platelets can be activated locally by stimulation through collagen, a specific platelet receptor containing glycoprotein VI (GPVI) [69]. In a study by Boilard et al., platelet activation mediated by the GPVI, conducted in vivo on an animal model with transgenic K/BxN mice, induced release of microparticles containing both forms of interleukin 1 (IL-1): IL-1α and IL-β, stimulating production and secretion of IL-6 and IL-8 by fibroblast-like synoviocytes (FLS). The findings of the study suggest the pro-inflammatory potential of PMPs and their active participation in pathogenesis of rheumatoid arthritis [8]. Activation of platelet receptor, GPVI, leads to activation of spleen tyrosine kinase (SYK) in platelets and B-cells, further activating Bruton's tyrosine kinase (BTK), which plays a key role in the activation of B-cells, which is essential in their proper function and development. Bruton's kinase inhibition with the BTK selective inhibitor was examined by Hsu et al. and found to reduce collagen-induced production of PMPs [70]. In another study, BTK blockade in activated platelet culture also resulted in a decrease in production of microparticles and inhibition of production and release of IL-6 and IL-8 [71,72].

Apart from platelet-derived microparticles, synovial fluid in RA patients was found to contain MPs from monocytes, granulocytes, T- and B-cells, and erythrocytes [50,73,74]. Monocyte-derived MPs, as determined by Headland et al., were present in a much larger amount in synovial fluid than in plasma of RA patients. Researchers obtained some interesting findings on an animal model, where they observed a protective effect of monocyte-derived microparticles on articular cartilage, which is associated with decreasing secretion of IL-8 and prostaglandin E2 [53].

Microparticles exhibit high pro-coagulation activity mediated by the TF/VII factor, thereby stimulating thrombin production. They can also contribute to developing proatherogenic vasculitis and to the formation of "rice bodies" within joints as a manifestation of local coagulation processes [64]. In their study with RA patients, concerning non-differentiated arthritis, Berckmans et al. found incubation of MPs obtained from synovial fluid in the presence of FLS obtained by the biopsy of the synovial membrane to result in an increase in production and release by synoviocytes of IL-6, IL-8, monocyte chemoattractant protein 1 (MPC-1), RANTES (regulation on activation normal T-cells expressed and secreted) chemokine, and vascular endothelium growth factor (VEGF) [63]. A local increase in VEGF secretion can stimulate angiogenesis within articular tissues in RA, especially at its early stages [74]. Stimulation of angiogenesis in joints affected by RA can also be mediated by chemokines. Reich et al. observed a stimulating effect of leukocyte-derived MPs on production and release by rheumatoid synoviocytes of proangiogenic CXC chemokine with an ELR motive—a sequence

of three amino acids: Glutamic acid-leucine-arginine (Glu-Leu-Arg). In their study, the authors observed increasing mRNA expression for ligands of chemokine CXC ELR+: CXCL1, CXCL2, CXCL3, CXCL5, and CXCL6 [65].

Matrix metalloproteinases (MMP) are responsible for processes of extracellular matrix component transformation and degradation. Distler et al. demonstrated that MPs derived from B-cells and monocytes can stimulate rheumatoid FLS additionally to synthesis of metalloproteinases 1, 3, 9, and 13, which participate in the destruction of the extracellular matrix of cartilaginous and bone tissue in RA. In this study, the microparticles under study stimulated the production of IL-6, IL-8, MCP-1, and MCP-2 by fibroblasts [62].

The complement system, comprising approximately 40 proteins, plays an important role in the regulation of innate immune response by stimulating phagocytosis and intensification of an inflammatory reaction. The system is activated in a cascade manner. Removing apoptotic and necrotic cells is one of the functions of the complement system [75]. These cells activate the complement system mainly through a conventional pathway, as a result of a reaction of the C1q component with the CH2 domain of the antibody Fc fragment [76–78]. Microparticles with membranous features of apoptotic and necrotic cells (with exposed phosphatidylserine, phosphatidylethynolamine, oxidated phospholipids) can participate in the development of inflammation in RA by activating the complement cascade. It has been shown that MPs formed from apoptotic Jurkat leukemia cell [79] and stimulated neutrophils [80,81] can bind the C1q and, thereby, activate the complement in vitro through a conventional activation pathway. Biro et al. found C1q, C4, and/or C3 components on microparticles isolated from synovial fluid and—to a lesser extent—on microparticles obtained from serum [60]. It seems that microparticles, especially those derived from platelets, exposing the CD41 antigen, can also take part in the formation of immune complexes (IC), usually described as structures containing antibodies, antigens, and complement components. Cloutier et al. used high resolution flow cytometry and transmission electron microscopy and detected MPs in IC present in synovial fluid in RA patients, forming specific mpIC [61].

Microparticles in rheumatoid synovial fluid can undergo the process of protein citrullination—posttranslational deimination of arginine residues catalyzed by peptidylarginine deiminase (PAD), which results in the production of antibodies against cyclic citrullinated peptides (aCCP). For platelet microparticles described in the study by Cloutier et al., with expression of the FcγRIIa receptor, mpIC were formed not by connecting antibodies to this receptor, but by binding MPs to citrullinated proteins, such as fibrinogen and vimentin. The researchers confirmed that platelet MPs can react with aCCP antibodies in a mechanism which follows citrullination of PMPs surface proteins and by binding microparticles and citrullinated proteins. The paper also describes a stimulating effect of platelet mpIC on the production of leukotrienes by neutrophils. The researchers made an interesting observation by comparing the amount of mpIC in synovial fluid collected from RA and PsA patients. Despite the presence of MPs and immunoglobulins in synovial fluid of PsA patients, the amount of detected mpIC was nearly twenty times smaller than in RA patients (2000 ± 900 mpICs/μL vs 39,400 ± 9400 mpICs/μL) [82]. Unlike the previous study, two other studies found no proof of any relationship between the amount of circulating microparticles and immune complexes containing MPs in RA patients and the indices conventionally used to assess the disease activity [49,82]. This may indicate a highly effective vascular and reticular-endothelial mechanism of IC elimination or local formation of mpIC in joints affected by inflammation.

A very important role in pathogenesis of RA is played by activated B-cells. Data from the study by Messer et al. suggest the role of microparticles in synovial fluid in inducing the release of B-cell activating factor (BAFF), thymic stromal lymphopoietin (TSLP), and antileukoprotease (SLPI) by fibroblast-like synoviocytes. MPs present in synovial fluid stimulated the secretion of BAFF to the same extent as IFN-γ used as a control. The effect was observed both among the RA patients—study participants—and in patients with degenerative joint disease, which indicated the activity of MPs in stimulating B-cells, regardless of the disease type. The main difference in this study was quantitative. The number of MPs in synovial fluid in RA patients was considerably larger than in individuals with no joint inflammation. The study assessed the ability of MPs from THP-1 monocytic–macrophagic cell line and from the CEM lymphocyte line to synthesize and release BAFF, TLSP, and SLPI by activated FLS. Microparticles of monocytic–macrophagic origin had high inflammatory activity, which indicates the important participation of monocytes in initiation of an inflammatory response. MPs from activated T-cells which, in turn, stimulate the secretion of IL-6 and IL-8, did not stimulate the release of BAFF, but of TLSP and SLPI by synoviocytes. Those same MPs, did not affect the release of BAFF, TLSP, or SLPI after being treated with actinomycin D, which suggested no effect of MPs from apoptotic T-cells on activation of B-cells [59].
