Abstract
IntroductionA 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.MethodsIL-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, TNFα 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.ResultsTH17-expressing CD4+ cells were found to accumulate within rheumatoid synovial tissue and in rheumatoid arthritis synovial fluid. RASF treated with IL-17 and TNFα (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-κB pathways showed a requirement for both signalling pathways in RASFIL-17/TNF-mediated neutrophil rescue.ConclusionThe increased number of neutrophils with an extended lifespan found in the rheumatoid synovial microenvironment is partly accounted for by IL-17 and TNFα activation of synovial fibroblasts. TH17-expressing T cells within the rheumatoid synovium are likely to contribute significantly to this effect.
Highlights
A surprising feature of the inflammatory infiltrate in rheumatoid arthritis is the accumulation of neutrophils within synovial fluid and at the pannus cartilage boundary
BSA = bovine serum albumin; ELISA = enzyme-linked immunosorbent assay; FCM = fibroblast-conditioned medium; FITC = fluorescein isothyocyanate; GM-CSF = granulocyte–macrophage colony-stimulating factor; HBSS = Hank's Buffered saline solution; IL = interleukin; IFN = interferon; MEM = Eagle's "Minimal Essential Media"; NF = nuclear factor; PBS = phosphate-buffered saline; RA = rheumatoid arthritis; RASF = rheumatoid arthritis synovial fibroblasts; RASFIL-17/TNF = rheumatoid arthritis synovial fibroblasts stimulated with TNFα and IL-17; rh = recombinant human TBST = Tris-buffered saline containing 0.1% Tween 20; TH17 cells = IL-17-secreting T-helper cells; TNF = transforming growth factor
IL-17-expressing CD4 T cells accumulate within the rheumatoid synovium We first determined whether TH17 cells are present within the rheumatoid synovium
Summary
A surprising feature of the inflammatory infiltrate in rheumatoid arthritis is the accumulation of neutrophils within synovial fluid and at the pannus cartilage boundary. With a predominantly T-helper type-1 T-cell cytokine profile [24]. This T-helper type-1 T-cell paradigm, does not adequately account for the large numbers of neutrophils that accumulate within the synovial space. During active phases of disease, large numbers of activated neutrophils are found in the synovial fluid of both very early RA and established RA patients [5,6]. For example the T-helper type-1 T-cell-associated cytokine IL-18 indirectly induces the recruitment of neutrophils in a murine model of arthritis [7]. IFNγ-stimulated neutrophils have been shown to release potent chemoattractants for T-helper type-1 T cells and NK cells [8]
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