Microparticle-induced release of B-lymphocyte regulators by rheumatoid synoviocytes

Abstract

Introduction: In the present study, we investigated the ability of microparticles isolated from synovial fluids from patients with rheumatoid arthritis or osteoarthritis to induce the synthesis and release of key cytokines of B-lymphocyte modulation such as B cell-activating factor, thymic stroma lymphopoietin, and secretory leukocyte protease inhibitor by rheumatoid fibroblast-like synoviocytes.

Methods: Microparticles were analyzed in synovial fluids from patients with rheumatoid arthritis, osteoarthritis, microcristalline arthritis, and reactive arthritis. In addition, microparticle release after activation from various cell lines (CEM lymphocyte and THP-1 cells) was assessed. Microparticles were isolated by differential centrifugation, and quantitative determinations were carried out by prothrombinase assay after capture on immobilized annexin V. B cell-activating factor, thymic stroma lymphopoietin, and secretory leukocyte protease inhibitor release was evaluated by enzyme-linked immunosorbent assay.

Results: Microparticles isolated from synovial fluids obtained from rheumatoid arthritis and osteoarthritis patients or microparticles derived from activated THP-1 cells were able to induce B cell-activating factor, thymic stroma lymphopoietin, and secretory leukocyte protease inhibitor release by rheumatoid arthritis fibroblast-like synoviocytes. Conversely, CEM-lymphocytes-derived microparticles generated by treatment with a combination of PHA, PMA and Adt-D did not promote the release of B cell-activating factor but favored the secretion of thymic stroma lymphopoietin and secretory leukocyte protease inhibitor by rheumatoid arthritis fibrobast-like synoviocytes. However, microparticles isolated from actinomycin D-treated CEM lymphocytes were not able to induce B cell-activating factor, thymic stroma lymphopoietin, or secretory leukocyte protease inhibitor release, indicating that microparticles derived from apoptotic T cells do not function as effectors in B-cell activation.

Conclusions: These results demonstrate that microparticles are signalling structures that may act as specific conveyors in the triggered induction and amplification of autoimmunity. This study also indicates that microparticles have differential effects in the crosstalk between B lymphocytes and target cells of autoimmunity regarding the parental cells from which they derive.

Figure 1

Microparticle (MP) assessment in synovial fluids from arthritic patients and cell supernatants. (a) Concentrations of MPs in synovial fluids from patients with rheumatoid arthritis (RA) (n = 7), osteoarthritis (OA) (n = 5), microcristalline arthritis (MC) (n = 3), and reactive arthritis (AR) (n = 5) were determined by a solid-phase capture assay on immobilized annexin V by use of a prothrombinase assay (nM PhtdSer Eq). (b) Concentrations of MPs isolated from THP-1 cells stimulated with lipopolysaccharide (LPS) (15 μg/mL) for 18 hours were determined by a solid-phase capture assay on immobilized annexin V or on immobilized CD14 antibody by use of a prothrombinase assay (nM PhtdSer Eq). The control (C) corresponded to untreated cells. Data are expressed as the mean of triplicate samples ± standard deviation and are representative of three independent experiments. *P < 0.05. PhtdSer Eq, phosphatidylserine equivalents.

Figure 2

Induction of interleukin (IL)-6, IL-8, and B cell-activating factor (BAFF) synthesis by microparticles (MPs) isolated from synovial fluids. Rheumatoid arthritis (RA) fibroblast-like synoviocytes were stimulated with 40 nM phosphatidylserine equivalents of MPs isolated from synovial fluids of osteoarthritis (OA) and RA patients for 24 and 72 hours. IL-6 (a), IL-8 (b), and BAFF (c) release was determined by enzyme-linked immunosorbent assay. Data are expressed as the mean of triplicate samples ± standard deviation and are representative of three independent experiments. C, control medium; IFN-γ, interferon-gamma; SN, control supernatants.

Figure 3

Induction of interleukin (IL)-6, IL-8, and B cell-activating factor (BAFF) synthesis by microparticles (MPs) isolated from CEM lymphocytes. Rheumatoid arthritis fibroblast-like synoviocytes were stimulated with 40 and 400 nM phosphatidylserine equivalents of MPs isolated from CEM lymphocytes treated either with actinomycin D (ActD) alone (0.5 μg/mL) for 18 hours or with a combination of PHA, PMA and Adt-D (PPA) (phytohemagglutinin) (5 μg/mL) for 72 hours followed by ActD (0.5 μg/mL) and phorbolmyristate acetate (20 ng/mL) for an additional 18-hour incubation period. Culture supernatants were harvested 24 hours after stimulation for IL-6 (a) and IL-8 (b) determination and 72 hours after stimulation for BAFF evaluation (c) by enzyme-linked immunosorbent assay. Lipopolysaccharide (IL-6 and IL-8) and interferon-gamma (IFN-γ) (BAFF) stimulation was used as a positive control. Data are expressed as the mean of triplicate samples ± standard deviation and are representative of three independent experiments. **P < 0.01. Act, actinomycin; C, control medium; Pser, phosphatidylserine; TMP, control Hanks' balanced saline solution.

Figure 4

Induction of interleukin (IL)-6, IL-8, and B cell-activating factor (BAFF) synthesis by microparticles (MPs) isolated from lipopolysaccharide (LPS)-activated THP-1 cells. THP-1 cells were treated with LPS (15 μg/mL) for 18 hours, and MPs were isolated as described in Materials and methods. Rheumatoid arthritis (RA) FLSs were stimulated with 40 nM phosphatidylserine equivalents of MPs isolated either from THP-1 cells (MP THP-1) or from LPS-activated THP-1 cells (MP THP-1 LPS). Culture supernatants were harvested 24 hours after stimulation for IL-6 (a) and IL-8 (b) determination and 72 hours after stimulation for BAFF secretion (c) by enzyme-linked immunosorbent assay. LPS (IL-6 and IL-8) and interferon-gamma (IFN-γ) (BAFF) were used as positive controls. Data are expressed as the mean of triplicate samples ± standard deviation and are representative of three independent experiments. C, control medium; TMP, control Hanks' balanced saline solution.

Figure 5

Induction of thymic stroma lymphopoietin (TSLP) and secretory leukocyte protease inhibitor (SLPI) synthesis by activated rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs). TSLP (a) and SLPI (b) release was determined by enzyme-linked immunosorbent assay in supernatants of RA FLSs stimulated 48 hours (TSLP) and 72 hours (SLPI) with microparticles (MPs) (40 nM phosphatidylserine equivalents) isolated from RA synovial fluids, lipopolysaccharide (LPS)-treated THP-1 cells, and PHA, PMA and Adt-D (PPA)-treated CCRF-CEM cells. Data are expressed as the mean of triplicate samples ± standard deviation and are representative of three independent experiments. *P < 0.05. C, control medium; IFN-γ, interferon-gamma; SN, control supernatants; TMP, control Hanks' balanced saline solution.