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

Greg Parsonage , Andrew Filer , Magdalena Bik , Debbie Hardie , Sian Lax , Katherine Howlett , Leigh D Church , Karim Raza , See-Heng Wong , Emily Trebilcock , Dagmar Scheel-Toellner , Mike Salmon , Janet M Lord and Christopher D Buckley

Arthritis Research & Therapy 2008, 10:R47doi:10.1186/ar2406


Published:	23 April 2008

Abstract (provisional)

Introduction

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) 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 peripheral blood and synovial fluid IL-17 expressing cells were examined by confocal microscopy and flow cytometry respectively. Peripheral blood neutrophils were co-cultured with either rheumatoid synovial fibroblasts (RASF), or conditioned medium from RASF that had been pre-exposed to rhIL-17, TNF-alpha or a combination of the two cytokines. Neutrophils were harvested and stained with the vital mitochondrial dye DiOC6 before being enumerated by flow cytometry.

Results

TH17 expressing CD4+ cells were found to accumulate within rheumatoid synovial tissue and in RA synovial fluid. RASF treated with IL-17 and TNF-alpha(RASFIL17/TNF) effectively doubled the functional lifespan of neutrophils in co-culture. This was entirely due to soluble factors secreted from the fibroblasts. Specific depletion of GM-CSF from RASFIL17/TNF conditioned medium demonstrated that this cytokine accounted for approximately half of the neutrophil survival activity. Inhibition of phosphatidylinositol-3-kinase (PI3K) and NF-kB pathways showed a requirement for both signalling pathways in RASFIL-17/TNF mediated neutrophil rescue.

Conclusions

We conclude that the increased number of neutrophils with an extended life span found in the rheumatoid synovial microenvironment is partly accounted for by IL-17 and TNF-alpha activation of synovial fibroblasts. TH17 expressing T cells within the rheumatoid synovium are likely to contribute significantly to this effect.