Prolonged, granulocyte–macrophage colony-stimulating factor-dependent, neutrophil survival following rheumatoid synovial fibroblast activation by IL-17 and TNFalpha-4

Granulocyte–macrophage colony-stimulating factor (GM-CSF) and via phosphatidylinositol-3-kinase-dependent and NF-κB-dependent pathways. 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. *< 0.05. 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. 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. *< 0.05. 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.<p><b>Copyright information:</b></p><p>Taken from "Prolonged, granulocyte–macrophage colony-stimulating factor-dependent, neutrophil survival following rheumatoid synovial fibroblast activation by IL-17 and TNFalpha"</p><p>http://arthritis-research.com/content/10/2/R47</p><p>Arthritis Research & Therapy 2008;10(2):R47-R47.</p><p>Published online 23 Apr 2008</p><p>PMCID:PMC2453767.</p><p></p

Prolonged, granulocyte–macrophage colony-stimulating factor-dependent, neutrophil survival following rheumatoid synovial fibroblast activation by IL-17 and TNFalpha-3

filter suspended above fibroblasts (Tw) for 24 hours. Error bars show the mean ± standard deviation from three independent experiments. **< 0.01, *< 0.05; ns, nonsignificant. Culture supernatant from rheumatoid arthritis synovial fibroblasts stimulated with IL-17 and TNFα was heated to 92°C for the times indicted before culture with neutrophils for 24 hours, and neutrophil survival was measured. Error bars show the mean ± standard deviation from three independent experiments<p><b>Copyright information:</b></p><p>Taken from "Prolonged, granulocyte–macrophage colony-stimulating factor-dependent, neutrophil survival following rheumatoid synovial fibroblast activation by IL-17 and TNFalpha"</p><p>http://arthritis-research.com/content/10/2/R47</p><p>Arthritis Research & Therapy 2008;10(2):R47-R47.</p><p>Published online 23 Apr 2008</p><p>PMCID:PMC2453767.</p><p></p

Prolonged, granulocyte–macrophage colony-stimulating factor-dependent, neutrophil survival following rheumatoid synovial fibroblast activation by IL-17 and TNFalpha-2

� (RASF) (closed squares), and their survival was assessed every 24 hours by flow cytometry. ***< 0.001. The ability to produce superoxide radical in response to f-Met-Leu-Phe was determined in freshly isolated neutrophils (open bars) and neutrophils cocultured with RASFfor 24 hours (filled bars). **< 0.01 versus unstimulated cells.<p><b>Copyright information:</b></p><p>Taken from "Prolonged, granulocyte–macrophage colony-stimulating factor-dependent, neutrophil survival following rheumatoid synovial fibroblast activation by IL-17 and TNFalpha"</p><p>http://arthritis-research.com/content/10/2/R47</p><p>Arthritis Research & Therapy 2008;10(2):R47-R47.</p><p>Published online 23 Apr 2008</p><p>PMCID:PMC2453767.</p><p></p

Prolonged, granulocyte–macrophage colony-stimulating factor-dependent, neutrophil survival following rheumatoid synovial fibroblast activation by IL-17 and TNFalpha-6

Ls were localised with von Willebrand factor (vWF) (green). Nuclear staining is shown in grey. IL-17 (red) expression is associated with CD4T cells (blue) but not CD8(green) T cells. Nuclear staining is shown in grey. flow cytometric analysis of peripheral blood (PB) and synovial fluid (SF) CD3T cells demonstrates that IL-17 is expressed in SF CD4T cells. PE, phycoerythrin; FITC, fluorescein isothyocyanate.<p><b>Copyright information:</b></p><p>Taken from "Prolonged, granulocyte–macrophage colony-stimulating factor-dependent, neutrophil survival following rheumatoid synovial fibroblast activation by IL-17 and TNFalpha"</p><p>http://arthritis-research.com/content/10/2/R47</p><p>Arthritis Research & Therapy 2008;10(2):R47-R47.</p><p>Published online 23 Apr 2008</p><p>PMCID:PMC2453767.</p><p></p

Prolonged, granulocyte–macrophage colony-stimulating factor-dependent, neutrophil survival following rheumatoid synovial fibroblast activation by IL-17 and TNFalpha-1

Es. Recombinant human (rh)TNFα concentrations: open circle, 0 pg/ml; open square, 1 pg/ml; open triangle, 10 pg/ml; open inverted triangle, 100 pg/ml; filled circle, 1,000 pg/ml; filled square, 10,000 pg/ml. *< 0.05 versus rhTNFα = 0 pg/ml. Peripheral blood neutrophils were cultured alone or cocultured with RASF pretreated for 24 hours with the indicated cytokines both at a concentration of 10 ng/ml. **< 0.01. Data represent mean ± standard deviation from at least five independent experiments. Absolute neutrophil survival was determined by flow cytometry using fixed volume dumping, with exclusion of apoptotic cells by gating on cells with a maintained mitochondrial membrane potential as assessed by 3,3'-dihexyloxacarbocyanine iodide staining. Neutrophil morphology was examined on cytospins after 24 hours of coculture with untreated RASF or RASF stimulated with TNFα and IL-17.<p><b>Copyright information:</b></p><p>Taken from "Prolonged, granulocyte–macrophage colony-stimulating factor-dependent, neutrophil survival following rheumatoid synovial fibroblast activation by IL-17 and TNFalpha"</p><p>http://arthritis-research.com/content/10/2/R47</p><p>Arthritis Research & Therapy 2008;10(2):R47-R47.</p><p>Published online 23 Apr 2008</p><p>PMCID:PMC2453767.</p><p></p