Session Title: Cytokines, Mediators, and Gene Regulation
Session Type: Abstract Submissions (ACR)
Background/Purpose: Decreased sensitivity of rheumatoid arthritis (RA) synovial fibroblasts (SF) to apoptosis leads to synovial hyperplasia and destruction of joints in RA. Activation of NF-kB was shown to modulate apoptotic pathways in different target cells. Based on our recent finding that microparticles (MP) from monocytes, stimulated with Toll-like receptor 3 ligand Poly(I:C) (PIC), increase the resistance of RASF to TRAIL-induced apoptosis and enhance the production of NF-kB-dependent cytokines IL-6 and IL-8, the aim of the present study was to examine the role of NF-κB signaling in the resistance of RASF to TRAIL-induced apoptosis mediated via MP.
Methods: MP were isolated by differential centrifugation from supernatants of untreated or PIC-stimulated (16h) U937 cells or peripheral blood mononuclear cells (PBMC) and were analysed by nanoparticle tracking analysis, flow cytometry and BCA Protein Assay. RASF were treated with MP±TRAIL for 24h. To investigate direct effects of PIC on the apoptosis, RASF were stimulated with PIC±TRAIL for 24h. Apoptosis of RASF was measured by flow cytometry using Annexin V/propidium iodide staining. SC-514, a selective inhibitor of IκB kinase 2, was used to test the role of NF-kB signaling in MP actions in RASF. NF-κB activity was determined by luciferase reporter gene assay in RASF treated with MP for 6h.
Results: PIC-induced MP, but not MP from untreated U937 cells, significantly decreased TRAIL-induced apoptosis of RASF (9±3% vs TRAIL: 18±6%, p=0.003, n=8), and similar effects were observed with PIC-induced MP derived from PBMC (18.±27% vs 35±27%, n=3). In contrast, a direct stimulation with PIC alone significantly increased apoptosis of RASF (11±4% vs 6±2% in untreated RASF, p=0.03, n=6), however it did not affect TRAIL-induced apoptosis of RASF. Number (MP from untreated cells: 3.0*1010/mL vs PIC-induced MP: 3.1*1010/mL; n=2 each), size (median diameter 207 vs 199nm, n=2 each), surface annexin V binding (66±10% vs 63 ± 9%, n=3 each) and total protein content (330±50 vs 325±83 ng/mL, n=3 each) did not differ significantly between MP from untreated and PIC-stimulated U937 cells. SC-514 significantly increased TRAIL-induced apoptosis of RASF in the presence of PIC-induced MP (20±3% vs 9±3% in the absence of SC-514, p=0.002, n=8). PIC-induced MP from U937 cells led to activation of NF-κB signaling in RASF (median x-fold change: 13 vs untreated RASF, n=4).
Conclusion: Most interestingly, we could show that the activation of NF-κB plays a major role in the resistance of RASF to TRAIL-induced apoptosis mediated via PIC-induced MP. This observed effect may reflect a specific composition of PIC-induced MP. Alternatively, the effects of MP could result from small amounts of PIC associated with MP although its activity would differ from that in the free state.
M. Frank Bertoncelj,
Articulum, Masterswitch-FP7, IMI BTcure, IAR,
B. A. Michel,
R. E. Gay,
D. S. Pisetsky,
Actelion, Pfizer, Ergonex, BMS, Sanofi-Aventis, United BioSource Corporation, medac, Biovitrium, Boehringer Ingelheim Pharma, Novartis, 4 D Science, Bayer, and Active Biotec ,
IAR, Masterswitch-FP7, IMI-BTCure,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/activation-of-nf-kb-via-polyic-induced-monocyte-derived-microparticles-decreases-trail-induced-apoptosis-of-rheumatoid-arthritis-synovial-fibroblasts/