Session Information
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.
Disclosure:
M. Frank Bertoncelj,
Articulum, Masterswitch-FP7, IMI BTcure, IAR,
2;
B. Rozman,
None;
B. A. Michel,
None;
R. E. Gay,
Masterswitch-PF7 ,
2;
D. S. Pisetsky,
Pfizer Inc,
5,
Bio-Rad,
5;
O. Distler,
Actelion, Pfizer, Ergonex, BMS, Sanofi-Aventis, United BioSource Corporation, medac, Biovitrium, Boehringer Ingelheim Pharma, Novartis, 4 D Science, Bayer, and Active Biotec ,
2;
S. Gay,
IAR ,
2;
A. Juengel,
IAR, Masterswitch-FP7, IMI-BTCure,
2.
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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/