|Year : 2018 | Volume
| Issue : 4 | Page : 222-228
Relevance of elevated microparticles in peripheral blood and synovial fluid of patients with rheumatoid arthritis
Benita N R Michael1, Durga Prasanna Misra2, KG Chengappa1, Vir Singh Negi1
1 Department of Clinical Immunology, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry, India
2 Department of Clinical Immunology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
|Date of Web Publication||18-Nov-2018|
Dr. Vir Singh Negi
Department of Clinical Immunology, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry - 605 006
Source of Support: None, Conflict of Interest: None
Introduction: Microparticles (MPs) are submicron sized heterogeneous membrane-bound vesicles released from cells undergoing cell activation or apoptosis. Elevated platelet-derived MPs (PMPs) are reported in rheumatic diseases. We profiled Annexin-V+ MPs and CD61+ MPs in plasma and synovial fluid of patients with rheumatoid arthritis (RA) and their clinical correlates.
Methods: Twenty-three newly diagnosed disease-modifying anti-rheumatic drug naïve patients with RA, 17 with osteoarthritis (OA) and 22 healthy controls (HC) were enrolled. Five milliliters of synovial fluid from the knee joint of patients with RA and OA was collected; 5 ml of peripheral blood was collected from patients with RA, OA, and HC. Cell-free synovial fluid and platelet poor plasma samples were stained with Annexin-V-APC and Anti-CD61-BV510 antibodies followed by flow cytometry analysis (FACSAria III) (results expressed as the mean ± standard deviation of % population).
Results: Significantly greater levels of synovial fluid MPs (Annexin-V+ MPs) were observed in RA versus OA (P < 0.0001), significantly higher synovial fluid PMPs (Annexin V+ CD61+ MPs) levels in RA compared to HC (P = 0.0472). MPs other than those of platelet origin (Annexin V+ CD61− MPs) were also increased in the synovial fluid of RA patients compared to OA (P < 0.0001). Kruskal–Wallis test revealed the significant difference in the levels of plasma MPs (Annexin-V+), PMPs (Annexin V+ CD61+ MPs), and MPs other than those of platelet origin (Annexin V+ CD61− MPs) between RA, OA, and HC groups (P < 0.0001) with a higher percentage in RA group.
Conclusions: Higher levels of Annexin-V+ MPs in both plasma and synovial fluid of RA suggest a role for MPs in the pathogenesis of RA. Elevated plasma PMPs (Annexin V+ CD61+ MPs) in RA may suggest their role in systemic involvement. Furthermore, increased levels of MPs other than those of platelet origin (Annexin V+ CD61− MPs) indicate the necessity to study the MPs from other cell lineages.
Keywords: Autoimmunity, flow cytometry, microparticles, platelets, rheumatoid arthritis
|How to cite this article:|
Michael BN, Misra DP, Chengappa K G, Negi VS. Relevance of elevated microparticles in peripheral blood and synovial fluid of patients with rheumatoid arthritis. Indian J Rheumatol 2018;13:222-8
|How to cite this URL:|
Michael BN, Misra DP, Chengappa K G, Negi VS. Relevance of elevated microparticles in peripheral blood and synovial fluid of patients with rheumatoid arthritis. Indian J Rheumatol [serial online] 2018 [cited 2021 Jul 29];13:222-8. Available from: https://www.indianjrheumatol.com/text.asp?2018/13/4/222/241782
| Introduction|| |
Extracellular vesicles are membranous vesicles released by a variety of cells into the extracellular environment. There are three classes of extracellular vesicles, i.e., exosomes, microparticles (MPs), and apoptotic bodies. Among them, MPs are submicron-sized (0.1–1 μm) membrane-bound vesicles released during cell activation and apoptosis. Depending on the source of origin, they contain cellular components of their origin, such as cytoplasmic and membranous molecules, cytokines, and surface receptors, and may act as sources of autoantigens, cytokines, and cell surface receptors. The MPs have effects on various components of inflammatory disease such as thrombosis, angiogenesis, vascular reactivity, and inflammation. Rheumatoid arthritis (RA) is one of the most common inflammatory arthritides characterized by symmetric polyarthritis with progression to joint deformities if inadequately treated. Elevated levels of MPs have been found in biological fluids of patients with RA.
The analysis of MPs is technically complex due to their heterogeneity and small size. Flow cytometry is a useful technique to detect MPs based on size, exposure of phosphatidylserine (which can be detected by annexin-V staining) and by other cell lineage-specific markers. MPs have been scarcely explored in rheumatic disorders, and their role in pathogenesis and disease progression is yet unclear. Few studies have reported higher levels of platelet-derived MPs (PMPs) in the patients with RA than healthy controls (HC)., Therefore, we aimed at assessing pathogenic role of MPs profile especially, those of platelet origin and those from cellular origin other than platelets in the pathogenesis of RA by profiling MPs bearing phosphatidylserine (Annexin V+ MPs) and Annexin V+ CD61+ MPs (PMPs) and Annexin V+ CD61− MPs (non-PMPs ) subpopulations in the peripheral blood and synovial fluid of patients with RA compared to osteoarthritis (OA – disease control) and healthy individuals.
| Methods|| |
Twenty-three patients satisfying the American College of Rheumatology 2010 criteria for RA were included in the study. Seventeen patients with OA with knee effusion and 22 age and sex-similar healthy individuals without a history of autoimmune disease were enrolled as disease controls and HC respectively.
Sample collection and processing
A volume of 5 ml of synovial fluid was collected in ethylenediaminetetraacetic acid (EDTA) tubes from the knee joints of patients with RA and OA. Cell-free synovial fluid samples were obtained by centrifugation (1550 g, 20 min, and room temperature), snap frozen in liquid nitrogen and stored at −80°C until future use. Furthermore, 5 ml of peripheral blood was collected into EDTA tubes from patients with RA, OA, and healthy individuals. A two-step centrifugation removed blood cells (at 1800 g for 10 min at 21°C, followed by 3000 g for 10 min at 21°C) to obtain platelet poor plasma. Plasma samples were also snap frozen in liquid nitrogen and stored at −80°C until further use. All samples were collected by 18 G needle to avoid hemolysis.
Flow cytometry analysis
Samples were thawed on ice before analysis, then diluted 1:10 with Annexin V binding buffer (BD Biosciences, USA), further labeled with Annexin V APC (BD Biosciences, USA) and Anti-CD61 BV510 (BD Biosciences, USA) for 15 min in the dark, resuspended in 300 μl Annexin V binding buffer and analyzed on BD FACSAria III sorter with FACSDIVA software (BD Biosciences, USA). Forward scatter (FSC) and side scatter (SSC) were set as logarithmic gain. The low threshold was set at 200 on FSC and APC channel. As internal size control, MP gating was accomplished using 0.2, 0.5, and 1 μm beads of flow cytometry submicron particle size reference kit (Thermo Fisher Scientific, USA) with segregation of background corresponding to debris present in buffers [Figure 1]. Two-micron size beads were also run to exclude events >1 μm events. Initially, these beads were run separately and identified based on green fluorescence intensity and further backgated on FSC versus SSC plot. Annexin-V only stained samples were run as a control. Only Annexin V+ events in the MP gate region were chosen as to gate the phosphatidylserine (MPs - Marker) exposing MPs and further subgrouped based on a CD61 expression. Results were expressed as mean % ± standard deviation (SD) of the percentage of total events.
|Figure 1: Microparticle gate settings: (a and b) represents internal size control beads: microparticle gate was accomplished using 0.2, 0.5, 1 μm green fluorescent beads of flow cytometry submicron particle Size Reference kit (Thermo Fisher Scientific, USA), (c) segregation of background corresponding to debris in phosphate buffered saline and microparticles gate|
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Serum rheumatoid factor (RF) and high-sensitivity C-reactive protein (hsCRP) were determined by Nephelometry BN ProSpec System (Siemens, Germany). Test values >15.9 IU/ml were considered positive for RF and Test values >3.02 g/l were considered as high hsCRP. Anti-cyclic citrullinated peptide antibodies (ACPA) status was identified by enzyme-linked immunosorbent assay (AESKULISA CCP kit, AESKU Diagnostics GmbH and Co. KG, Germany, >18 U/ml considered positive).
Demographic variables like age of patient and age at disease onset were presented as mean + SD. Categorical variables were presented as frequencies. Differences between RA and control groups were tested by Mann–Whitney U-test or by Kruskal–Wallis test followed by Dunn's multiple comparisons depending on the number of groups compared. The normality of continuous data was tested using Shapiro–Wilks test.
The study was approved by the Institutional Ethics Committee. Informed written consent was obtained from all participants prior to their enrollment in this study.
| Results|| |
Demographical, clinical, and serological assessment
Twenty-three RA patients (21 females and 2 males) with a mean age of 42.96 ± 11.36 years were enrolled in the study. Seventeen patients with OA (15 females and 2 males) had a mean age of 52.47 ± 7.47 years, and 22 HC (18 females and 4 males) had a mean age of 44.63 ± 6.95 years. Clinical characteristics such as age at onset, disease duration, the presence of deformities, extra-articular manifestations, disease activity based on disease activity score using 28-joint counts modified by erythrocyte sedimentation rate (DAS28 ESR), RF positivity, ACPA positivity, and inflammatory markers such as CRP were assessed in patients with RA [Table 1].
|Table 1: Clinical and serological features of patients with rheumatoid arthritis (n=23)|
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Synovial fluid microparticles in patients with rheumatoid arthritis
We initially profiled the synovial fluid MPs (Annexin-V+) by flow cytometry. These MPs were further subclassified into Annexin V+ CD61+ (PMPs ) and Annexin V+ CD61− (Non-PMPs ) subpopulations in patients with RA and OA. Synovial fluid MPs (Annexin-V+) in RA (mean % ± SD = 2.81% ± 1.84%; n = 21) were significantly higher than those in OA (mean % ± SD = 0.86% ± 0.62; n = 17) (P < 0.0001). On further characterization of the MPs based on CD61 expression, we found that PMPs (Annexin V+ CD61+ MPs) were significantly higher in patients with RA (mean % ± SD = 0.15% ±0.14%; n = 21) as compared to OA (mean % ± SD = 0.08% ± 0.17%; n = 17); (P = 0.0472). Non-PMPs were also elevated in a similar fashion (Annexin V+ CD61- MPs) in RA (mean % ± SD = 2.61% ± 1.80%; n = 21) as compared to OA (mean % ± SD = 0.75% ± 0.51; n = 17 [P < 0.0001] [Figure 2]a, [Figure 2]b, [Figure 2]c and [Figure 3], respectively).
|Figure 2: Annexin-V+ Microparticles and CD61+ or CD61− subpopulations in rheumatoid arthritis, (a-c) synovial fluid levels of Annexin-V+ Microparticles and CD61+ or CD61− subpopulations in rheumatoid arthritis versus osteoarthritis. (d-f) plasma levels of Annexin-V+ Microparticles and CD61+ or CD61− subpopulations among rheumatoid arthritis, healthy control, and osteoarthritis groups. Intergroup differences were evaluated by Mann–Whitney's U-test or Kruskal–Wallis test with Dunn's multiple comparisons (if more than two groups). Horizontal lines indicate mean ± standard deviation. ****P < 0.0001, *P < 0.05, **P < 0.01, ***P < 0.001|
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|Figure 3: Synovial Fluid MPs gate, Annexin-V+ Microparticles, PMPs (Annexin V+ CD61+) and non PMPs (Annexin V+ CD61-) subpopulations in patients with Rheumatoid Arthritis (a-c) and Osteoarthritis (d-f), Annexin-V+ MPs were subgrouped based on CD61 expression and expressed as mean ± SD of the percentage of total events|
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Plasma microparticles in patients with rheumatoid arthritis
MPs (Annexin-V+), PMPs (Annexin V+ CD61+), and Non- PMPs (Annexin V+ CD61−) subpopulations were profiled from the plasma of patients with RA, OA, and HC [representative images – [Figure 2]d, [Figure 2]e, [Figure 2]f and [Figure 4]. Kruskal–Wallis test revealed a significant difference in the levels of Annexin-V+ MPs in RA, OA, and HC groups (P < 0.0001). Dunn's multiple comparisons test showed that Annexin-V+ MPs are significantly higher in RA (mean % ± SD = 3.42% ± 2.69%; n = 20; P < 0.0001) and OA (mean % ± SD = 1.5% ± 0.82%; n = 17; P < 0.01) compared to HC group (mean % ± SD = 0.58 ± 0.46; n = 22).
|Figure 4: Plasma MPs gate, Annexin-V+ Microparticles, PMPs (Annexin-V+ CD61+) and Non PMPs (Annexin V+ CD61-)subpopulations in patients with Rheumatoid Arthritis (a-c, healthy control (d-f), and osteoarthritis (g-i), Annexin-V+ Microparticles were subgrouped based on CD61 expression and expressed as mean ± standard deviation of the percentage of total events|
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On evaluation of levels of PMPs (Annexin V+ CD61+ MPs) in plasma, we found significant difference between RA, OA, and HC groups (P < 0.0001). There was a significantly higher level of PMPs (Annexin V+ CD61+ MPs) in RA (mean% ± SD = 1.83% ±2%; n = 20; P < 0.0001) and OA (mean % ± SD = 0.49% ± 0.531%; n = 17; P < 0.01) plasma as compared to HC (mean % ± SD = 0.09% ± 0.19%; n = 22).
Non-PMPs (Annexin-V+ CD61− MPs) in plasma were also significantly different between RA, OA, and HC groups (P < 0.0001). These MPs (Annexin-V+ CD61− MPs) were significantly higher in both RA (mean % ± SD = 1.58% ± 0.92%; n = 20; P < 0.0001) and OA (mean % ± SD = 0.49% ± 0.53%; n = 17; P < 0.01) plasma as compared to HC (mean % ± SD = 0.46% ± 0.33%; n = 22). Although MPs (Annexin-V+), and Annexin V+ CD61+ (PMPs) and Annexin V+ CD61− (Non-PMPs ) subpopulations were at higher levels in RA plasma compared to OA plasma, the results did not attain statistical significance.
| Discussion|| |
RA is a chronic inflammatory condition primarily affecting synovial joints with progressive joint destruction. Various cells and their byproducts participate in the inflammation that follows the immune insult. MPs by virtue of their varied composition are one of the novel players recognized to participate in the pathogenesis of RA, and other autoimmune disorders.,,,
In the present study, we identified elevated numbers of Annexin V+ MPs patients with RA and OA as compared to HC in the circulation as well as in the affected joints of patients with RA compared to OA, which probably suggests an important role played by these MPs in driving the pathogenesis of RA. Various immune cells involved in the joint inflammation probably use the MPs to communicate with other cells. The content of MPs differ according to the environment they are in and may range from cytokines, cell receptors, intracellular components to autoantigens, which in turn may result in chronic immune stimulation., Although the plasma MPs were not significantly elevated in RA as compared to OA, the high synovial MPs in RA probably reflects the robust immune activation in RA as compared to OA. Few studies have also reported MP-associated immune complexes, and complement components on their surface,, in RA and systemic lupus erythematosus.
On further subclassification of MPs, we showed a significant elevation in the amount of PMPs in RA synovium than in OA synovium. This probably reflects the presence of high levels of angiogenesis in RA as compared to OA, which propels the local inflammatory response and synovial hypertrophy in RA. This finding, however, could not be replicated in the plasma of the two disease groups, which possibly can be attributed to a smaller sample size in our study. Besides, plasma PMPs may potentially serve as a biomarker of systemic attributes of RA and may reflect disease activity.,,
Although MPs were initially discovered as “platelet dust,” later, they were found to be produced by several cells and have numerous potential activities. Our study additionally found that non-PMPs (Annexin V+ CD61− MPs) are also richly present in the synovial fluid as well as in the plasma of patients with RA. This observation suggests that majority of the MPs in RA synovial fluid of affected joints may also be derived from cells other than platelets that are present locally or may be derived from the plasma due to systemic immune deregulation. Similarly, another study reported high numbers of leukocyte-derived MPs in synovial fluid from inflamed joints both in patients with RA and other arthritic disorders.
In our study given the small sample size, we could not study the distribution of MPs based on the disease and immunological phenotypes. Besides this, the non-platelet MPs were not further classified to understand their source of origin. Since identifying the definite features of MPs released during physiological as well as in pathological conditions and establishing their physiological reference range were necessary, and this study was a design to achieve that goal. Further studies are necessary to profile the MPs based on the additional cell lineage markers and disease-specific markers on their surface, which may help us identify the disease-specific MPs signatures.
Higher levels of Annexin-V+ MPs in plasma as well as in synovial fluid of the patients with RA suggest a role for MPs in the pathogenesis of RA. Elevated levels of Plasma PMPs (Annexin-V+ CD61+) in RA may indicate their role in systemic involvement. Further, increased plasma and synovial fluid non-PMPs (Annexin-V+ CD61−) MPs in RA indicate the necessity to study other cell lineage markers that may be present on the MPs.
Financial support and sponsorship
The work was supported by the Indian Council of Medical Research, India (IRIS ID No.: 2012-2279) and JIPMER Intramural Research Fund (JIP/Res/Intra-PhD/01/2014 and JIP/Res/Intra-PhD/02/2015-16).
Conflicts of interest
There are no conflicts of interest.
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