GM-CSF in conditioned medium from stimulated rheumatoid arthritis synovial fibroblasts maintains neutrophil viability. Conditioned medium from rheumatoid arthritis synovial fibroblasts stimulated with IL-17 and TNFα (RASFIL-17/TNF) maintains neutrophil viability in part through the release of granulocyte–macrophage colony-stimulating factor (GM-CSF) and via phosphatidylinositol-3-kinase-dependent and NF-κB-dependent pathways. (a) Using either an irrelevant control antibody (open bars) or specific GM-CSF antibodies (filled bars) conjugated to agarose beads, serum-free conditioned medium (unstimulated or IL-17A/TNFα stimulated) was depleted of GM-CSF and added to freshly isolated peripheral blood neutrophils for 24 hours. Error bars show the mean ± standard deviation from three independent experiments. *P < 0.05. (b) The degree of depletion of GM-CSF was determined by ELISA in fibroblast-conditioned medium (FCM) from unstimulated or IL-17A/TNFα-stimulated FCM, before (open bars) and after (filled bars) depletion with anti-GM-CSF antibodies/agarose beads. A fixed dose of 100 pg/ml recombinant human (rh)GM-CSF was used as a positive control for the ELISA and to check the efficiency of GM-CSF depletion (filled bars). ND, not detectable. (c) Freshly isolated neutrophils were pretreated with vehicle control (open bars), 20 μM Ly294002 (filled bars) or 1 μM Bay 11-7085 (filled bars) before being cultured for 24 hours in FCM from unstimulated or IL-17/TNFα-stimulated fibroblasts. *P < 0.05. (d) Neutrophils that had been exposed to medium alone (cont), TNFα (as a positive control), or IL-17/TNFα-stimulated FCM were subjected to western blotting and were labelled using primary antibodies to inhibitor of NF-κB (IκB) and, as a loading control, β-actin.
Parsonage et al. Arthritis Research & Therapy 2008 10:R47 doi:10.1186/ar2406