Microparticles from inflammatory cells are strong inducers of matrix metalloproteinases and inflammatory cytokines in synovial fibroblasts

Microparticles (MPs) are membrane-bound vesicles that are released from cells during cellular activation and apoptosis. Synovial fluids from patients with rheumatoid arthritis contain high numbers of MPs. Here we address the effects of monocyte-derived and T-cell-derived MPs on rheumatoid arthritis synovial fibroblasts (RASF).

Apoptosis of Jurkat T cells, U937 macrophages and primary cells was induced by FasL, actinomycin-D, tumour necrosis factor alpha and staurosporine. Cells were also stimulated with IL-2, concanavalin A, anti-CD3 and lipopolysaccharide. Isolation of MPs was performed by differential centrifugation. The presence of MPs was confirmed by FACS analysis for annexin V and CD3 or CD14. Normal synovial fibroblasts, osteoarthritis synovial fibroblasts and RASF were incubated with MPs for 6–36 hours. Differentially expressed genes were analyzed by real-time PCR and confirmed on the protein level.

The release of MPs was induced in apoptotic as well as stimulated cells. Morphological analysis of MPs by electron microscopy showed a population of circular membrane blebs ranging from 200 to 700 μm. MPs from all cell types induced strongly the synthesis of mRNA for MMP-1 (72 ± 13-fold), MMP-3 (80 ± 10-fold), MMP-9 (18 ± 4-fold) and MMP-13 (37 ± 2-fold) in RASF in a dose-dependent manner. Similar effects were seen on osteoarthritis synovial fibroblasts and normal synovial fibroblasts. The induction of matrix metalloproteinases (MMPs) was time dependent, with effects primarily seen after 36 hours. The dose-dependent upregulation of biologically active MMPs was also observed on the protein level. The induction was specific for the aforementioned MMPs with no induction seen for MMP-2, MMP-14 or TIMP-1, TIMP-2 and TIMP-3. MPs also increased the synthesis of mRNA for IL-6 (27 ± 6-fold), IL-8 (35 ± 3-fold), MCP-1 (5 ± 1-fold) and MCP-2 (27 ± 3-fold) in synovial fibroblasts. No differences in cell viability were observed between fibroblasts stimulated by MPs and controls. In Iκ-B-transfected RASF, MMPs were 50–75% less inducible by MPs compared with wild-type synovial fibroblasts. The stimulation of NF-κB-dependent pathways by MPs was confirmed by EMSA.

By showing induction of various MMPs and key inflammatory cytokines by MPs, these data provide a novel link between activated and apoptotic inflammatory cells and the invasive potential of synovial fibroblasts in rheumatoid arthritis.

Affiliations

1. Center of Experimental Rheumatology, University Hospital Zurich, Switzerland
   • JHW Distler
   • , A Jüngel
   • , LC Huber
   • , A Seemayer
   • , RE Gay
   • , BA Michel
   • , S Gay
   •  & O Distler
2. Medizinische Klinik III, University Erlangen–Nuremberg, Germany
   • JHW Distler
   •  & JR Kalden
3. Division of Rheumatology, Duke University Medical Center, Durham, North Carolina, USA
   • DS Pisetsky

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