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. 2015 Apr 27;180(3):442–451. doi: 10.1111/cei.12598

Proportions of several types of plasma and urine microparticles are increased in patients with rheumatoid arthritis with active disease

V Viñuela-Berni *,1, L Doníz-Padilla †,1, N Figueroa-Vega *, H Portillo-Salazar †, C Abud-Mendoza *,‡, L Baranda *,‡, R González-Amaro *,†,✉
PMCID: PMC4449772  PMID: 25639560

Abstract

We analysed the proportions of different microparticles (MPs) in plasma from patients with rheumatoid arthritis (RA), and assessed their relationship with disease activity/therapy and their in-vitro effect on proinflammatory cytokine release. Blood and urine samples were obtained from 55 patients with RA (24 untreated and 31 under conventional therapy) and 20 healthy subjects. Fourteen patients with systemic lupus erythematosus (SLE) were also studied. The proportions of CD3+, CD14+, CD19+, CD41+ and CD62E+ MPs were determined by flow cytometry analysis. The in-vitro effect of plasma MPs on the release of interleukin (IL)-1, IL-6, IL-17 and tumour necrosis factor (TNF)-α was also analysed. We detected that the proportions of different types of annexin-V+ MPs were enhanced in plasma (CD3+, CD14+, CD19+, CD41+ and CD62E+ MPs) and urine (CD14+, CD3+ and CD19+ MPs) from RA patients with high disease activity (DAS28 index > 5·1). Accordingly, a significant positive correlation was observed between the levels of MPs and DAS28 score, and these levels diminished significantly at week 4 of immunosuppressive therapy. Finally, MPs isolated from patients with high disease activity induced, in vitro, an enhanced release of IL-1, IL-17 and TNF-α. In SLE, enhanced levels of different types of plasma MPs were also detected, with a tight correlation with disease activity. Our data further support that MPs have a relevant role in the pathogenesis of RA and suggest that the analysis of the proportions of these microvesicles in plasma could be useful to monitor disease activity and therapy response in patients with RA.

Keywords: immune cells, immunosuppressive therapy, microparticles, rheumatoid arthritis

Introduction

Cell-derived microparticles (MPs) or ectosomes are a heterogeneous population of extracellular vesicles ranging between 100 and 1000 nm in diameter 1. Generation and shedding of MPs occurs during different biological processes, including not only cellular activation or exposure to high shear stress, but also cellular differentiation, senescence or apoptotic cell breakdown 2. These vesicles are detected in different body fluids, including plasma, synovial fluid and urine 1, and they are released from various cell types as neutrophils, endothelial cells, dendritic cells and, mainly, platelets 2,3. Microparticles are formed by budding of cell membrane, followed by shedding, and this process is related to an increase in [Ca2+]i, which modulates the activity of enzymes involved in maintaining the structure of the cytoskeleton and cell membrane 4. MPs contain cell-surface receptors, cytosolic proteins, DNA, mRNA and microRNA derived from the cell of origin, and their composition depends on the stimulus that induces their formation. The production of MPs is accompanied not only by specific membrane changes, such as the translocation of phosphatidylserine (PS) residues to the extracellular side of membrane, but also by changes in the local concentrations of specific intracellular molecules 1,5. The mechanism of release, size and composition are characteristic of MPs, and different from other cellular-derived vesicles such as apoptotic bodies and exosomes 6.

After their first description in 1967, MPs were considered largely as cellular debris, without any biological role. However, subsequent studies have demonstrated that MPs are important mediators in the intercellular exchange of biological signals and information 2,6. Thus, MPs can transfer bioactive molecules from parent to target cells, and are able to modulate various biological phenomena such as the immune response, cell proliferation, angiogenesis or coagulation. As expected, MPs are able to transfer surface molecules to other cells, and they can exchange membrane and cytosol components. As stated above, the functional consequences of this type of intercellular communication include the amplification or modulation of the immune response 3,4. In this regard, although low to moderate levels of MPs can be detected in healthy subjects, they are increased in several prothrombotic and inflammatory disorders, including autoimmune diseases. As the composition and contents of MPs reflect the state of the cells that originate them, these subcellular structures are potential markers of disease activity and response to immunosuppressive therapy 7.

Rheumatoid arthritis (RA) is an autoimmune systemic inflammatory disease that affects an important proportion of human beings. T and B cells have an important role in the pathogenesis of this condition, which is characterized by chronic synovial inflammation, resulting in cartilage and bone damage, leading eventually to joint destruction 8. In these patients, Knijff-Dutmer et al. detected enhanced levels of platelet-derived MPs, which correlated with disease activity 9. However, it has also been reported that plasma from patients with RA and healthy controls contain comparable numbers of platelet-derived MPs 10. Moreover, the synovial fluid from patients with RA contains a high number of MPs 11, and Boilard and co-workers have described that platelet-derived MPs are able to amplify the inflammatory phenomenon seen in this condition 12. In addition, it has been reported that MPs derived from leucocytes are able to induce the in-vitro release of proinflammatory cytokines, angiogenic molecules and matrix metalloproteinases by synovial fibroblasts 6. Although all these data strongly suggest the involvement of MPs in the pathogenesis of the inflammatory phenomenon seen in RA, it is of interest that the levels of plasma MPs associated with complement activation are not modified significantly by intensive anti-inflammatory therapy with sulphasalazine, prednisone and methotrexate 13. In other autoimmune inflammatory disorders such as as systemic lupus erythematosus (SLE), although an early report indicated that these patients show similar levels of plasma C1q-positive MPs to healthy controls 14, Sellam et al. subsequently detected enhanced levels of plasma MPs in SLE 10. In addition, Pereira et al. showed that the number of platelet-derived MPs is increased in patients with SLE, and that this phenomenon is associated with a procoagulant state but not with disease activity 15. Furthermore, it has been reported that the composition of plasma MPs is different in patients with SLE and healthy controls, with enhanced levels of annexin V(−) MPs and diminished numbers of total and annexin V+ MPs in these patients 16. Finally, an enhanced number of plasma immunoglobulin (Ig)G-positive MPs, which is associated with the presence of different autoantibodies and decreased leucocyte counts, has been reported in SLE 17.

Although clinical and laboratory criteria are extremely useful to monitor the therapeutic effect of anti-rheumatic agents, it is evident that it would be convenient to characterize additional markers of disease remission and disease flare 8,18,19. In this regard, although different studies suggest that the absolute number of plasma MPs is associated with disease activity or tissue damage in patients with RA, the proportions of different types of MPs have not been studied in this condition. Thus, we decided to analyse the levels of different types of MPs in plasma and urine samples from patients with RA and healthy subjects. We have found that several types of MPs are increased in plasma and urine from patients with RA, with a significant association with disease activity. Accordingly, we detected that immunosuppressive therapy induces a significant diminution in the levels of MPs in these patients. In addition, MPs isolated from patients with high disease activity score (DAS)28 index induced an enhanced release of interleukin (IL)-1, IL-17 and tumour necrosis factor (TNF)-α.

Materials and methods

Patients and controls

Fifty-five patients with RA (51 females and four males, age range = 19–56 years, median age = 38·9 years) diagnosed according to the criteria of the American College of Rheumatology (ACR) 20 were included into the study. Twenty patients were untreated at the time of their inclusion in the study, and their mean DAS28 21 was 4·73. The remaining patients (n = 31, with a mean DAS28 of 4·53) were under therapy with disease-modifying anti-rheumatic drugs (DMARDs) and glucocorticoids, receiving mainly methotrexate (17·5 ± 5 mg/week), sulphasalazine (1·5 ± 0.5 g/day) and prednisone (7·5 ± 2·5 mg/day). When results were analysed, all these patients were classified according to their DAS28 values into the following categories: (1) patients in remission (n = 6, DAS28 < 2·6); (2) patients with low disease activity (n = 6, DAS28 from 2·6 to 3·1); (3) patients with moderate disease activity (n = 22, DAS28 from 3·2 to 5·1); and (4) patients with high disease activity (n = 21, DAS28 > 5·1). Fourteen female patients with SLE and a median age of 38·0 years were also studied. Nine patients had active disease [mean Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) = 7·6] and five patients had inactive disease (mean SLEDAI = 1·2). Two patients were untreated when they were included into the study (mean SLEDAI = 7·0), and 12 were under immunosuppressive therapy (mean SLEDAI = 5·0) with methotrexate (7·5–17·5 mg/week), azathioprine (25–100 mg/day), prednisone (10–15 mg/day) and mycophenolate mofetil (1.5 g/day).

No patients (RA or SLE) under therapy with biological agents were included in the study. Twenty female healthy individuals with a median age of 35.7 years were included as controls. This study was approved by the local University Ethics Committee and all subjects signed an informed consent.

Isolation of microparticles

Thirty ml of venous blood were collected in heparinized syringes and 20 ml of urine were collected in sterile bottles from all patients and controls. Immediately after collection, cells were removed by centrifugation (1000 g for 10 min), followed by removal of platelets by additional centrifugation at 1600 g for 20 min. The platelet-free plasma was then transferred to a new tube, and in order to pellet MPs samples were centrifuged at 20 000 g for 20 min, and the supernatant was discarded. Then, MPs were resuspended, washed in 500 μl of annexin V binding buffer and again resuspended in 500 μl of annexin V binding buffer. Eighty μl of MP suspension of each tube were immunolabelled and analysed by flow cytometry.

Flow cytometry analysis

Microparticles were stained with annexin V-fluorescein isothiocyanate (FITC) plus one of the following monoclonal antibodies (mAbs) labelled with phycoerythrin (PE): anti-CD3, -CD14, -CD19, -CD41a and -CD62E (all from BD Biosciences, San Jose, CA, USA) for 30 min in darkness. Finally, microparticles were resuspended in 300 μl of annexin V binding buffer and analysed in a fluorescence activated cell sorter (FACS)Calibur flow cytometer (Becton Dickinson, San Jose, CA, USA) using CellQuest software (Becton Dickinson). To set the gate of the MPs to be analysed, polystyrene beads of 0·2 and 1·0 μm (Sigma-Aldrich, St Louis, MO, USA) were employed in a forward- versus side-scatter dot-plot. In all cases, 50 000 events were acquired, and those particles included in the corresponding gate were analysed for their simultaneous binding to FITC-annexin-V and the indicated PE-labelled antibodies. MPs incubated with irrelevant mAbs labelled with FITC and PE were employed as the negative control. Results were expressed as the arithmetic mean ± standard deviation (s.d.) of the percentage of annexin V-positive MPs expressing the cell lineage marker indicated.

In-vitro effect of plasma MPs on cytokine release

MPs were isolated from plasma, as stated above, but at the end of the procedure MPs were resuspended in 200 µl of RPMI-1640 tissue culture medium supplemented with 10% fetal bovine serum, L-glutamine and penicillin–streptomycin. In addition, peripheral blood mononuclear cells (PBMCs) were isolated from the same blood samples by Ficoll-Hypaque gradient centrifugation, platelets were removed by resuspension of the PBMC in 50 ml of Hanks's balanced salt solution (HBSS), centrifuged at 200 g for 15 min and careful removal of the entire supernatant, and monocytes were then purified by using anti-CD14 microbeads (Miltenyi Biotec, San Diego, CA, USA). Thereafter, 1 × 106 PBMC or 1 × 105 monocytes were cultured in 1·0 ml of RPMI-1640 supplemented culture medium for 48 h at 37 °C, 5% CO2, in the presence or absence of autologous MPs isolated from 1·0 ml of plasma. Finally, cell culture supernatants were obtained and the concentrations of IL-1, IL-6, IL-17 and TNF-α were determined by enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN, USA).

Statistical analysis

Statistical analysis was performed with the GraphPad InStat version 5·0 software. Parametric analysis was performed with the Student's t-test and one-way analysis of variance (anova). The Mann–Whitney U-test and Kruskal–Wallis with post-hoc analyses were performed when data were not normally distributed. Before-and-after measurements were tested with the Wilcoxon sum rank test. Association between two variables was determined with the Pearson's correlation test. Values of P < 0·05 were considered significant.

Results

The strategy for MP detection by flow cytometry analysis employed in this work is depicted in Fig. 1a. According to previous reports 23, we were able to analyse the percentages of annexin-V+ MPs derived from different cellular sources in both healthy controls and patients with RA (Fig. 1). As there are no previous reports regarding the presence of MPs derived from endothelium or leucocytes in urine, we first analysed at least two aliquots from the same urine sample from three different healthy individuals; in addition, we analysed at least two samples from these individuals obtained on consecutive days. We have detected similar results in the two aliquots from each sample, and we have also obtained similar results in the two samples obtained from each individual (data not shown).

Fig 1.

Fig 1

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Flow cytometry analysis of levels of microparticles (MPs) in patients with rheumatoid arthritis (RA) and healthy controls. Plasma and urine MPs were labelled with annexin V and the indicated monoclonal antibody (mAb), and analysed in a fluorescence activated cell sorter (FACS)Calibur flow cytometer, as indicated in Materials and methods. (a) The size of particles to be analysed was defined in a forward- versus side-scatter plot (gate R4), taking as reference polystyrene microbeads of 0·2 and 1·0 μm (gates R2 and R3, respectively). (b–i) Representative flow cytometry dot-plots of MPs isolated from plasma of a patient with RA (b–e) and a healthy control (f–i), and derived from monocytes (CD14+), platelets (CD41+), endothelium (CD62E) and B cells (CD19+). (j–o) Representative flow cytometry dot-plots of MPs isolated from urine of a patient with RA (j–l) and a healthy control (m–o), and derived from monocytes (CD14+), T lymphocytes (CD3+) and B cells (CD19+). Numbers correspond to the percentage of MPs positive for annexin V and the indicated mAb.

Then we comparatively analysed the levels of MPs derived from various cell types in plasma and urine samples from patients with RA, SLE and healthy subjects. We detected that the levels of annexin-V-positive MPs derived from monocytes (CD14+), platelets (CD41a+), endothelial cells (CD62E+) and B lymphocytes (CD19+) were enhanced significantly in the plasma from RA patients with a DAS28 index greater than 5·1 compared with healthy controls (P < 0·001 in all cases, Fig. 2a–d). In addition, those patients with a DAS28 index between 3·2 and 5·0 also showed enhanced levels of MPs derived from monocytes and endothelium (P < 0·05 in both cases, Fig. 2a,c). However, similar levels of MPs were found in healthy controls and RA patients with a DAS28 index lower than 3·2 (P > 0·05 in all cases, Fig. 2a–d). Moreover, although the urine levels of CD14+ and CD41+ MPs tended to be higher in patients with RA with high activity compared to healthy controls, no significant differences were observed in these cases as well as in the other types of MPs detected (P > 0·05 in all cases, Fig. 2e–i). However, significant differences were observed between patients with low and high activity (DAS28 < 2·6 and DAS28 > 5·1, respectively) in the case of MPs derived from monocytes as well as T and B lymphocytes (Fig. 2e,h,i).

Fig 2.

Fig 2

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Analysis of levels of microparticles (MPs) in patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), grouped according to disease activity, and healthy controls. Levels of MPs derived from monocytes (CD14+), platelets (CD41+), endothelium (CD62E), T lymphocytes (CD3+) and B cells (CD19+) were analysed by flow cytometry in plasma and urine samples from 45 patients with RA, 14 patients with SLE and 15 healthy controls. (a–d) Analyses of plasma MPs in patients with RA and healthy controls. (e–i) Analyses of urine MPs in patients with RA and healthy controls. (j–l) Analyses of plasma MPs in patients with SLE and healthy controls. *P < 0·05; **P < 0·01 (one-way analysis of variance, with post- hoc analysis).

According to the above results, we analysed the possible association between disease activity and levels of MPs in patients with RA. We found a highly significant correlation between the plasma levels of all types of MPs detected and the values of the DAS28 index (Fig. 3a–e). Similarly, a significant association between disease activity and the levels of the different types of MPs detected in urine was also observed (P < 0·05 in all cases, Fig. 3f, and data not shown). Furthermore, when patients were grouped on the basis of time disease duration, we observed that those patients with 1–3 years of disease evolution showed higher levels of CD62E+ and CD19+ MPs compared to controls and patients with >3 years of disease evolution (P < 0·05 in all cases, data not shown). However, no apparent association was detected between the levels of the different MPs analysed in urine samples and albuminuria or haematuria, which were detected in six and nine patients (of 39 analysed), respectively.

Fig 3.

Fig 3

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Analysis of the association between disease activity and levels of plasma microparticles (MPs) in patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). The relationship between the levels of the indicated types of plasma MPs and the values of the disease activity score (DAS)28 (patients with RA, n = 45) or Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) (patients with SLE, n = 14) indexes was assessed, as stated in Materials and methods. The r- and P-values are indicated (Pearson's correlation test).

Then, we analysed the possible effect of immunosuppressive therapy on the levels of MPs in patients with RA. No significant differences were detected in the proportion of the five different types of MPs studied when treated and untreated patients were analysed (data not shown). In contrast, when eight RA patients were studied before and after the administration of immunosuppressive therapy, (methotrexate 16·5 ± 3·7 mg/week; sulphasalazine 1·7 ± 0·3 g/day; and prednisone 9·4 ± 2·9 mg/day), we observed a significant diminution in the plasma levels of all types of MPs detected at week 4 of therapy (P < 0·05 in all cases, Fig. 4a–e). In the case of urine MPs, we also observed a significant diminution in the case of CD14+, CD41+ and CD3+, but not CD62E and CD19 MPs, after starting the immunosuppressive therapy (Fig. 4f–i, and data not shown).

Fig 4.

Fig 4

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Effect of immunosuppressive therapy on the levels of microparticles (MPs) in patients with rheumatoid arthritis (RA). The levels of the indicated types of MPs were determined by flow cytometry in eight patients with RA before (T0) and after (4 weeks, T1) starting immunosuppressive therapy. (a–e) Plasma MPs. (f–i) Urine MPs. P-values of are indicated (Wilcoxon's sum rank test).

Finally, we analysed the in-vitro effect of plasma MPs on the release of proinflammatory cytokines by autologous mononuclear cells (MNC) or monocytes. Plasma MPs from healthy individuals did not show any significant effect on the release of the four cytokines tested (Fig. 5). However, although no significant differences were reached, MPs isolated from patients with RA tended to induce the release of IL-1, IL-17 and TNF-α, when data from patients with high DAS28 (>5·1) were analysed separately, a significant effect was observed on the release of these cytokines, but not IL-6 (Fig. 5). In addition, a significant association between the release of IL-1 or TNF-α and DAS28 score was observed (r = 0·48, P < 0·05 and r = 0·43, P < 0·05, respectively).

Fig 5.

Fig 5

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Microparticles from patients with active rheumatoid arthritis (RA) induce in vitro the release of proinflammatory cytokines. Plasma microparticles (MPs) were isolated from healthy individuals (empty bars) and patients (filled bars) with a high disease activity score (DAS) index (>5·1) and added or not to autologous monocytes (a,b,d) or mononuclear cells (MNC) (c), and then cells were cultured for 24 h in the presence or not of lipopolysaccharide (LPS) or a phorbol ester (phorbol myristate acetate) plus ionomycin (Io). Then, cell culture supernatants were obtained and the concentration of the indicated cytokines was measured by enzyme-linked immunosorbent assay (ELISA). * P < 0·05; n.s. = not significant; Ctrl = control.

In the case of SLE patients, we also detected significant enhanced percentages of all types of MPs in plasma compared to healthy controls (Fig. 2j–l, and data not shown). In addition, the levels of CD14+ MPs were significantly higher in patients with active disease (SLEDAI > 4·0) compared to inactive patients (P < 0·05, Fig. 2j). Although the levels of other types of MPs tended to be higher in active compared to inactive patients, no significant differences were reached (Fig. 2k,l, and data not shown), due probably to the small number of patients studied. The correlation analysis between MP plasma levels and disease activity showed a tight correlation between the SLEDAI value and the percentage of all types of MPs studied (P < 0·05 in all cases, Fig. 3g,h, and data not shown). In contrast, in the case of the levels of MPs detected in urine, we did not observe any significant difference between patients and controls or an apparent association with disease activity or renal involvement (P > 0·05 in all cases, data not shown).

Discussion

Different groups have reported that the number of MPs in plasma is increased in patients with several conditions, including inflammatory rheumatic diseases 4,6. In addition, the pathogenic role of MPs derived from different cellular sources, mainly platelet-derived MPs in patients with RA 12 and SLE patients with vascular risk, has been demonstrated 15. However, most studies regarding the detection of MPs in inflammatory diseases have been performed only in plasma samples, and the absolute number of MPs rather than their proportions have been analysed 4,6,9,10,13,15,16. In this regard, although it has been proposed as a standardized technique for the quantification of MPs by flow cytometry in patients with cardiovascular risk 7, most studies in patients with inflammatory rheumatic diseases have been performed employing different laboratory protocols, which could account for inconclusive or contradictory results. In this work, we have employed a different approach from previous studies, and analysed the proportions of different types of annexin-V+ MPs in plasma and urine samples from patients with RA and healthy controls.

We have observed that the proportions of plasma MPs derived from monocytes (CD14+), platelets (CD41a+), endothelial cells (CD62E+) and B lymphocytes (CD19+) were enhanced significantly in patients with RA, mainly in those with high disease activity. In this regard, it has been shown that monocytes, platelets and B cells have a relevant role in the pathogenesis of the inflammatory phenomenon observed in this condition 8. Similarly, endothelial cells have a key role in the extravasation of leucocytes towards inflamed tissues 22. Thus, it is very likely that the enhanced activation of leucocytes, platelets and endothelium observed in patients with active RA results in an increased release of MPs from these cells, raising their levels in plasma. In agreement with this possibility, we have detected a highly significant association between the percentages of different types of MP and disease activity. Therefore, it is feasible that the proportion of the different types of MPs detected, mainly those derived from monocytes and endothelium, could be a reliable marker of the severity of inflammation in RA. In this regard, it is of interest that the administration of a combined therapy of glucocorticoids and DMARDs to eight patients, which induced an ACR50 response in most of them, resulted in a significant diminution in the proportions of all MPs tested. The significant in-vitro effect of plasma MPs on the release of IL-1, IL-17 and TNF-α, and the positive association between the levels of cytokine release and disease activity, is also of interest. We consider that these data further support that MPs, at the same concentrations detected in vivo, may participate in the induction and perpetuation of the inflammatory process observed in patients with RA. Similarly, it is tempting to speculate that a similar phenomenon may occur in other chronic or acute inflammatory conditions. In this regard, we have also observed in this study that patients with SLE show enhanced proportions of different MPs in plasma with a tight correlation with disease activity. Unfortunately, we studied only two untreated patients with SLE, and we were thus unable to assess the possible effect of immunosuppressive therapy on the proportions of plasma MPs in this condition. Therefore, the measurement of MPs in SLE patients before and after starting conventional immunosuppressive therapy or the administration of different biological agents remains an interesting point to be studied.

Although it has been reported that plasma from patients with RA contains important amounts of annexin-V-non-binding MPs 17, we decided to study only the annexin-V-positive MPs in this work. However, we consider that it would be interesting to perform additional studies in samples from patients with RA in order to analyse the proportions of annexin-V-negative MPs as well as the possible association of these levels with clinical or laboratory parameters.

There have been few studies on MPs in urine, and they have focused on the detection and characterization of microvesicles derived from renal tubular cells 23. In this work, we have hypothesized that MPs derived from endothelial cells could be detected in the urine from patients with SLE and RA. Unexpectedly, we detected all types of the MPs tested in urine samples from both healthy controls and patients with RA and SLE. Interestingly, although no significant differences in the proportions of the urine MPs were observed in patients and controls, when RA patients with high and low activity were compared significant differences were observed in the case of CD14+, CD3+ and CD19+ MPs. It is also of interest that, as in the case of plasma MPs, the levels of these vesicles in urine showed a significant correlation with disease activity. Whether or not the MPs detected in the urine of these patients are derived from plasma or from the urinary tract remains an interesting question for further study. In the first case, the putative mechanism of transfer of MPs from blood to the glomerular filtrate would be also a relevant point to be addressed through future studies.

In summary, our data indicate that, in addition to the absolute number of plasma MPs reported previously, the proportions of different subsets of these microvesicles in patients with RA and SLE are tightly associated with disease activity and probably to the response to anti-inflammatory and immunosuppressive therapy. In addition, the presence and the enhanced levels of endothelial and leucocyte-derived MPs in urine from these patients is an interesting and unexpected finding, which requires further investigation. Finally, our data further support that MPs are involved in the pathogenesis of the inflammatory phenomenon observed in patients with autoimmune diseases, mainly RA.

Although it has been proposed that the main functional effect of MPs is the down-modulation of the immune response and the inflammatory phenomenon 24, there are also reports regarding the pathogenic role of these microvesicles in inflammatory conditions 11,12. In this work, we have detected that the proportions of different cell-derived MPs are increased in plasma from patients with RA, with a significant association with disease activity and immunosuppressive therapy. These data, as well as the significant in-vitro effect of plasma MPs on the in-vitro release of IL-1, IL-17 and TNF-α by autologous immune cells, further support the role of this type of microvesicles in the pathogenesis of RA and probably SLE.

Disclosures

The authors declare that they have no conflicts of interest.

Author contributions

V. V.-N., L. D.-P. and N. F.-V. performed the experiments, H. P.-S. performed the analysis of flow cytometry data, C. A.-M. and L. B. contributed to the study design and selection and follow-up of the patients, R. G.-A. designed the study and wrote the manuscript.

References

  1. Williams JC, Mackman N. MPs or ICs? Blood. 2011;117:1101–2. doi: 10.1182/blood-2010-11-318691. [DOI] [PubMed] [Google Scholar]
  2. Mause SF, Weber C. Microparticles: protagonists of a novel communication network for intercellular information exchange. Circ Res. 2010;107:1047–57. doi: 10.1161/CIRCRESAHA.110.226456. [DOI] [PubMed] [Google Scholar]
  3. Thery C, Ostrowski M, Segura E. Membrane vesicles as conveyors of immune responses. Nat Rev Immunol. 2009;9:581–93. doi: 10.1038/nri2567. [DOI] [PubMed] [Google Scholar]
  4. Prati C, Racadot E, Wendling D. Microparticles and inflammatory joint disease. Joint Bone Spine. 2010;77:496–8. doi: 10.1016/j.jbspin.2010.07.011. [DOI] [PubMed] [Google Scholar]
  5. Montoro-Garcia S, Shantsila E, Marín F, Blann A, Lip GY. Circulating microparticles: new insights into the biochemical basis of microparticle release and activity. Basic Res Cardiol. 2011;106:911–23. doi: 10.1007/s00395-011-0198-4. [DOI] [PubMed] [Google Scholar]
  6. Distler JH, Distler O. Inflammation: microparticles and their roles in inflammatory arthritides. Nat Rev Rheumatol. 2010;6:385–6. doi: 10.1038/nrrheum.2010.87. [DOI] [PubMed] [Google Scholar]
  7. Robert S, Poncelet P, Lacroix R. Standardization of platelet-derived microparticle counting using calibrated beads and a Cytomics FC500 routine flow cytometer: a first step towards multicenter studies? J Thromb Haemost. 2009;7:190–7. doi: 10.1111/j.1538-7836.2008.03200.x. [DOI] [PubMed] [Google Scholar]
  8. McInnes IB, Schett G. The pathogenesis of rheumatoid arthritis. N Engl J Med. 2011;365:2205–19. doi: 10.1056/NEJMra1004965. [DOI] [PubMed] [Google Scholar]
  9. Knijff-Dutmer EA, Koerts J, Nieuwland R, Kalsbeek-Batenburg M, van de Laar MA. Elevated levels of platelet microparticles are associated with disease activity in rheumatoid arthritis. Arthritis Rheum. 2002;46:1498–503. doi: 10.1002/art.10312. [DOI] [PubMed] [Google Scholar]
  10. Sellam J, Proulle V, Jüngel A, et al. Increased levels of circulating microparticles in primary Sjogren's syndrome, systemic lupus erythematosus and rheumatoid arthritis and relation with disease activity. Arthritis Res Ther. 2009;11:R156. doi: 10.1186/ar2833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Berckmans RJ, Nieuwland R, Tak PP, et al. Cell-derived microparticles in synovial fluid from inflamed arthritic joints support coagulation exclusively via a factor VII-dependent mechanism. Arthritis Rheum. 2002;46:2857–66. doi: 10.1002/art.10587. [DOI] [PubMed] [Google Scholar]
  12. Boilard E, Nigrovic PA, Larabee K, et al. Platelets amplify inflammation in arthritis via collagen-dependent microparticle production. Science. 2010;327:580–3. doi: 10.1126/science.1181928. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. van Eijk IC, Tushuizen ME, Sturk A, et al. Circulating microparticles remain associated with complement activation despite intensive anti-inflammatory therapy in early rheumatoid arthritis. Ann Rheum Dis. 2010;69:1378–82. doi: 10.1136/ard.2009.118372. [DOI] [PubMed] [Google Scholar]
  14. Nauta AJ, Trouw LA, Daha MR, et al. Direct binding of C1q to apoptotic cells and cell blebs induces complement activation. Eur J Immunol. 2002;32:1726–36. doi: 10.1002/1521-4141(200206)32:6<1726::AID-IMMU1726>3.0.CO;2-R. [DOI] [PubMed] [Google Scholar]
  15. Pereira J, Alfaro G, Goycoolea M, et al. Circulating platelet-derived microparticles in systemic lupus erythematosus. Association with increased thrombin generation and procoagulant state. Thromb Haemost. 2006;95:94–9. [PubMed] [Google Scholar]
  16. Nielsen CT, Ostergaard O, Johnsen C, Jacobsen S, Heegaard NH. Distinct features of circulating microparticles and their relationship to clinical manifestations in systemic lupus erythematosus. Arthritis Rheum. 2011;63:3067–77. doi: 10.1002/art.30499. [DOI] [PubMed] [Google Scholar]
  17. Nielsen CT, Ostergaard O, Stener L, et al. Increased IgG on cell-derived plasma microparticles in systemic lupus erythematosus is associated with autoantibodies and complement activation. Arthritis Rheum. 2012;64:1227–36. doi: 10.1002/art.34381. [DOI] [PubMed] [Google Scholar]
  18. Jurgens MS, Jacobs JW, Bijlsma JW. The use of conventional disease-modifying anti-rheumatic drugs in established RA. Best Pract Res Clin Rheumatol. 2011;25:523–33. doi: 10.1016/j.berh.2011.10.006. [DOI] [PubMed] [Google Scholar]
  19. Singh JA, Furst DE, Bharat A, et al. 2012 update of the 2008 American College of Rheumatology recommendations for the use of disease-modifying antirheumatic drugs and biologic agents in the treatment of rheumatoid arthritis. Arthritis Care Res. 2012;64:625–39. doi: 10.1002/acr.21641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Arnett FC, Edworthy SM, Bloch DA, et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum. 1988;31:315–24. doi: 10.1002/art.1780310302. [DOI] [PubMed] [Google Scholar]
  21. Anderson JK, Zimmerman L, Caplan L, Michaud K. Measures of rheumatoid arthritis disease activity: patient (PtGA) and provider (PrGA) global assessment of disease activity, disease activity score (DAS) and disease activity score with 28-joint counts (DAS28), simplified disease activity index (SDAI), clinical disease activity index (CDAI), patient activity score (PAS) and patient activity score-II (PASII), routine assessment of patient index data (RAPID), rheumatoid arthritis disease activity index (RADAI) and rheumatoid arthritis disease activity index-5 (RADAI-5), chronic arthritis systemic index (CASI), patient-based disease activity score with ESR (PDAS1) and patient-based disease activity score without ESR (PDAS2), and mean overall index for rheumatoid arthritis (MOI-RA) Arthritis Care Res. 2011;63(Suppl. 11):S14–36. doi: 10.1002/acr.20621. [DOI] [PubMed] [Google Scholar]
  22. Barreiro O, Martín P, González-Amaro R, Sánchez-Madrid F. Molecular cues guiding inflammatory responses. Cardiovasc Res. 2010;86:174–82. doi: 10.1093/cvr/cvq001. [DOI] [PubMed] [Google Scholar]
  23. Rood IM, Deegens JK, Merchant ML, et al. Comparison of three methods for isolation of urinary microvesicles to identify biomarkers of nephrotic syndrome. Kidney Int. 2010;78:810–6. doi: 10.1038/ki.2010.262. [DOI] [PubMed] [Google Scholar]
  24. Sadallah S, Eken C, Schifferli JA. Ectosomes as modulators of inflammation and immunity. Clin Exp Immunol. 2011;163:26–32. doi: 10.1111/j.1365-2249.2010.04271.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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