title: Prolonged, granulocyte-macrophage colony-stimulating factor-dependent, neutrophil survival following rheumatoid synovial fibroblast activation by IL-17 and TNFalpha.
abstract: BACKGROUND
  A surprising feature of the inflammatory infiltrate in rheumatoid arthritis is the accumulation of neutrophils within synovial fluid and at the pannus cartilage boundary. Recent findings suggest that a distinct subset of IL-17-secreting T-helper cells (TH17 cells) plays a key role in connecting the adaptive and innate arms of the immune response and in regulating neutrophil homeostasis. We therefore tested the hypothesis that synovial fibroblasts bridge the biological responses that connect TH17 cells to neutrophils by producing neutrophil survival factors following their activation with IL-17.
  
  
  METHODS
  IL-17-expressing cells in the rheumatoid synovium, and IL-17-expressing cells in the peripheral blood, and synovial fluid were examined by confocal microscopy and flow cytometry, respectively. Peripheral blood neutrophils were cocultured either with rheumatoid arthritis synovial fibroblasts (RASF) or with conditioned medium from RASF that had been pre-exposed to recombinant human IL-17, TNFalpha or a combination of the two cytokines. Neutrophils were harvested and stained with the vital mitochondrial dye 3,3'-dihexyloxacarbocyanine iodide before being enumerated by flow cytometry.
  
  
  RESULTS
  TH17-expressing CD4+ cells were found to accumulate within rheumatoid synovial tissue and in rheumatoid arthritis synovial fluid. RASF treated with IL-17 and TNFalpha (RASFIL-17/TNF) effectively doubled the functional lifespan of neutrophils in coculture. This was entirely due to soluble factors secreted from the fibroblasts. Specific depletion of granulocyte-macrophage colony-stimulating factor from RASFIL-17/TNF-conditioned medium demonstrated that this cytokine accounted for approximately one-half of the neutrophil survival activity. Inhibition of phosphatidylinositol-3-kinase and NF-kappaB pathways showed a requirement for both signalling pathways in RASFIL-17/TNF-mediated neutrophil rescue.
  
  
  CONCLUSIONS
  The increased number of neutrophils with an extended lifespan found in the rheumatoid synovial microenvironment is partly accounted for by IL-17 and TNFalpha activation of synovial fibroblasts. TH17-expressing T cells within the rheumatoid synovium are likely to contribute significantly to this effect.
authors.affiliation: Rheumatology Research Group, MRC Centre for Immune Regulation, Institute for Biomedical Research, University of Birmingham, Vincent Drive, Edgbaston, Birmingham B15 2TT, UK. g.n.parson@bham.ac.uk
authors.forename: Greg
authors.initials: G
authors.lastname: Parsonage
authors.forename: Andrew
authors.initials: A
authors.lastname: Filer
authors.forename: Magdalena
authors.initials: M
authors.lastname: Bik
authors.forename: Debbie
authors.initials: D
authors.lastname: Hardie
authors.forename: Sian
authors.initials: S
authors.lastname: Lax
authors.forename: Katherine
authors.initials: K
authors.lastname: Howlett
authors.forename: Leigh D
authors.initials: LD
authors.lastname: Church
authors.forename: Karim
authors.initials: K
authors.lastname: Raza
authors.forename: See-Heng
authors.initials: SH
authors.lastname: Wong
authors.forename: Emily
authors.initials: E
authors.lastname: Trebilcock
authors.forename: Dagmar
authors.initials: D
authors.lastname: Scheel-Toellner
authors.forename: Mike
authors.initials: M
authors.lastname: Salmon
authors.forename: Janet M
authors.initials: JM
authors.lastname: Lord
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Buckley
mesh_terms: D000328:Adult; D000368:Aged; D001172:Arthritis, Rheumatoid / Q000276:immunology* / Q000378:metabolism; D015496:CD4-Positive T-Lymphocytes / Q000378:metabolism; D002470:Cell Survival / Q000502:physiology; D005260:Female; D005347:Fibroblasts / Q000378:metabolism*; D005434:Flow Cytometry; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism*; D006801:Humans; D020381:Interleukin-17 / Q000378:metabolism*; D008297:Male; D018613:Microscopy, Confocal; D008875:Middle Aged; D009504:Neutrophils / Q000378:metabolism*; D013583:Synovial Membrane / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D016176:T-Lymphocyte Subsets / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
chemical_list: D020381:Interleukin-17; D014409:Tumor Necrosis Factor-alpha; D016178:Granulocyte-Macrophage Colony-Stimulating Factor