Figure 2.

Rheumatoid arthritis synovial fibroblasts stimulated with TNFα and IL-17 efficiently extend neutrophil survival. (a) Peripheral blood neutrophils were cocultured with rheumatoid arthritis synovial fibroblasts (RASF) pretreated for 24 hours with the indicated concentrations of cytokines. 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. *P < 0.05 versus rhTNFα = 0 pg/ml. (b) 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. **P < 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 (c) untreated RASF or (d) RASF stimulated with TNFα and IL-17.