Background Aberrant resistance of activated rheumatoid arthritis (RA) synovial fibroblasts (SF) to death receptor-mediated apoptosis can lead to synovial hyperplasia and joint destruction in RA. Among factors that may affect this process, cell-derived microparticles (MP) can modulate apoptotic signalling pathways in different target cells.
Objectives To investigate the effects of monocyte-derived MP on death receptor-mediated apoptosis of RASF, assessing the role of surface proteins on MP on this response.
Methods MP were isolated by differential centrifugation from supernatants of U937 cells, untreated or stimulated with Toll-like receptor 3 ligand Poly(I:C) for 16h. The number and surface protein expression on MP were analysed by FACSCalibur Flow Cytometer. RASF were treated with MP ± TNFα-Related Apoptosis Inducing Ligand (TRAIL) or Fas Ligand (FasL) for 24h. Spontaneous and TRAIL- or FasL-mediated apoptosis of RASF were measured by flow cytometry using Annexin V/propidium iodide staining. To investigate the role of surface proteins on MP in TRAIL-mediated apoptosis of RASF, MP were treated with different doses of proteinase K for 30 or 60 min. Flow cytometry was used to examine proteinase K-mediated degradation of surface proteins on MP. Additionally, RASF were stimulated with Poly(I:C) ± TRAIL for 24h to exclude that observed effects on apoptosis were due to transfer of Poly(I:C) via MP.
Results Both, untreated and Poly(I:C)-stimulated U937 cells (n=6 each) released similar numbers of MP, as detected by FACSCalibur flow cytometer, with more than 90% of MP displaying surface molecules, such as CD4 and CD29, from the cell of origin. Spontaneous apoptosis of RASF was not affected by MP. TRAIL or FasL enhanced apoptosis of RASF (x-fold±SD: 4.2±1.9, n=7, p=0.003 and 3.1±1.3, n=5, p=0.01, respectively). Poly(I:C)-induced MP significantly decreased TRAIL- and FasL- mediated apoptosis of RASF by mean±SD: 33.4±13.4% (n=7, p=0.015) and 40.2±15.5% (n=5, p=0.001). In contrast, MP derived from the same number of unstimulated cells did not affect TRAIL- or FasL- mediated apoptosis of RASF. Direct Poly(I:C) stimulation increased spontaneous (n=7), but not TRAIL-mediated apoptosis of RASF (n=6), excluding the possibility that the protective effect of MP on apoptosis resulted from direct transfer of Poly(I:C) via MP. Proteinase K efficiently degraded surface proteins on MP as detected by 80% to 90% loss of the surface CD4 expression on MP. Most interestingly, treatment of Poly(I:C)-induced MP with proteinase K impaired MP-mediated decrease in TRAIL-mediated apoptosis of RASF (mean decrease±SD: 5.9±12.5%, n=4), while Poly(I:C)-induced MP treated in the same way, but without adding proteinase K, effectively decreased TRAIL-induced apoptosis of RASF (mean decrease±SD: 26.7±6.7%, n=5, p=0.031).
Conclusions Microparticles can decrease apoptosis of RASF induced by death receptors, with the impairment of TRAIL-induced apoptosis dependent on the interaction of microparticle surface proteins with synovial fibroblasts. This interaction may represent one of the mechanisms underlying the resistance of RASF to apoptosis in the RA synovium, an important characteristic of this disease.
Disclosure of Interest None Declared
Statistics from Altmetric.com
If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.