Research article

Susceptibility of rheumatoid arthritis synovial fibroblasts to FasL- and TRAIL-induced apoptosis is cell cycle-dependent

Noreen Pundt¹ , Marvin A Peters¹ , Christina Wunrau¹ , Simon Strietholt¹ , Carsten Fehrmann² , Katja Neugebauer¹ , Christine Seyfert³ , Frans van Valen¹ , Thomas Pap¹ and Ingmar Meinecke^1,4

¹  Institute of Experimental Musculoskeletal Medicine, University Hospital Muenster, Domagkstr. 3, Muenster, 48149, Germany

²  Institute of Medical Microbiology, University Hospital Muenster, Domagkstr. 10, Muenster, 48149, Germany

³  Department of Orthopaedic Surgery, Zeisigwaldkliniken Bethanien Chemnitz, Zeisigwaldstr. 101, Chemnitz, 09130, Germany

⁴  Department of Orthopaedic Surgery, Park-Krankenhaus Leipzig-Suedost GmBH, Struempellstr. 41, Leipzig, 04289, Germany

author email corresponding author email

Arthritis Research & Therapy 2009, 11:R16doi:10.1186/ar2607

Published:	5 February 2009

Abstract

Introduction

The rheumatoid arthritis (RA) synovium is characterised by the presence of an aggressive population of activated synovial fibroblasts (RASFs) that are prominently involved in the destruction of articular cartilage and bone. Accumulating evidence suggests that RASFs are relatively resistant to Fas-ligand (FasL)-induced apoptosis, but the data concerning tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) have been conflicting. Here, we hypothesise that the susceptibility of RASFs to receptor-mediated apoptosis depends on the proliferation status of these cells and therefore analysed the cell cycle dependency of FasL- and TRAIL-induced programmed cell death of RASFs in vitro.

Methods

Synovial fibroblasts were isolated from patients with RA by enzymatic digestion and cultured under standard conditions. Cell cycle analysis was performed using flow cytometry and staining with propidium iodide. RASFs were synchronised or arrested in various phases of the cell cycle with 0.5 mM hydroxyurea or 2.5 μg/ml nocodazol and with foetal calf serum-free insulin-transferrin-sodium selenite supplemented medium. Apoptosis was induced by stimulation with 100 ng/ml FasL or 100 ng/ml TRAIL over 18 hours. The apoptotic response was measured using the Apo-ONE^® Homogenous Caspase-3/7 Assay (Promega GmbH, Mannheim, Germany) and the Cell Death Detection (ELISA^Plus) (enzyme-linked immunosorbent assay) (Roche Diagnostics GmbH, Mannheim, Germany). Staurosporin-treated cells (1 μg/ml) served as a positive control. Expression of Fas and TRAIL receptors (TRAILR1-4) was determined by fluorescence-activated cell sorting analysis.

Results

Freshly isolated RASFs showed only low proliferation in vitro, and the rate decreased further over time, particularly when RASFs became confluent. RASFs expressed Fas, TRAIL receptor-1, and TRAIL receptor-2, and the expression levels were independent of the cell cycle. However, the proliferation rate significantly influenced the susceptibility to FasL- and TRAIL-induced apoptosis. Specifically, proliferating RASFs were less sensitive to FasL- and TRAIL-induced apoptosis than RASFs with a decreased proliferation rate. Furthermore, RASFs that were synchronised in S phase or G₂/M phase were less sensitive to TRAIL-induced apoptosis than synchronised RASFs in G₀/G₁ phase.

Conclusions

Our data indicate that the susceptibility of RASFs to FasL- and TRAIL-induced apoptosis depends on the cell cycle. These results may explain some conflicting data on the ability of RASFs to undergo FasL- and TRAIL-mediated cell death and suggest that strategies to sensitise RASFs to apoptosis may include the targeting of cell cycle-regulating genes.