Duffy antigen receptor for chemokines and CXCL5 are essential for the recruitment of neutrophils in a multicellular model of rheumatoid arthritis synovium

Abstract

Objective: The role of chemokines and their transporters in rheumatoid arthritis (RA) is poorly described. Evidence suggests that CXCL5 plays an important role, because it is abundant in RA tissue, and its neutralization moderates joint damage in animal models of arthritis. Expression of the chemokine transporter Duffy antigen receptor for chemokines (DARC) is also up-regulated in early RA. The aim of this study was to investigate the role of CXCL5 and DARC in regulating neutrophil recruitment, using an in vitro model of RA synovium.

Methods: To model RA synovium, RA synovial fibroblasts (RASFs) were cocultured with endothelial cells (ECs) for 24 hours. Gene expression in cocultured cells was investigated using TaqMan gene arrays. The roles of CXCL5 and DARC were determined by incorporating cocultures into a flow-based adhesion assay, in which their function was demonstrated by blocking neutrophil recruitment with neutralizing reagents.

Results: EC-RASF coculture induced chemokine expression in both cell types. Although the expression of CXC chemokines was modestly up-regulated in ECs, the expression of CXCL1, CXCL5, and CXCL8 was greatly increased in RASFs. RASFs also promoted the recruitment of flowing neutrophils to ECs. Anti-CXCL5 antibody abolished neutrophil recruitment by neutralizing CXCL5 expressed on ECs or when used to immunodeplete coculture-conditioned medium. DARC was also induced on ECs by coculture, and anti-Fy6 antibody or small interfering RNA targeting of DARC expression effectively abolished neutrophil recruitment.

Conclusion: This study is the first to demonstrate, in a model of human disease, that the function of DARC is essential for editing the chemokine signals presented by ECs and for promoting unwanted leukocyte recruitment.

Figure 1

Effects of EC-RAF coculture on CXCL1, CXCL5, CXCL8 and DARC message in A) EC or B) SkF and RAF from the RA joint. Data are expressed as mean ± SEM of 4 experiments using different fibroblast explants and EC cultures. * = P<0.05; ** = P<0.01 for comparison of cocultured cells against monocultured calibrator samples.

Figure 2

A) The effect of incubating monolayers of EC with coculture conditioned medium before or after immuno-depletion using an anti-CXCL5 or control antibody; data are mean ± SEM of 4 experiments; * = P <0.05 for comparison of non depleted and antibody depleted conditioned media; B) A representative histogram of the expression of DARC protein on EC cultured alone or after coculture with RAF assessed by immunofluorimetry; C) Quantification of EC surface expression of DARC, data are mean ± SEM of 4 experiments; * = P <0.05 for comparison of EC monocultures and EC/RAF cocultures. D) The effect of inhibiting the binding of chemokines to DARC using an anti-FY6 monoclonal antibody; data are mean ± SEM of 4-12 experiments; * = P <0.05 for comparison of untreated and antibody treated cells. E) The effect of siRNA targeting of DARC in EC in coculture with RAF, data are mean ± SEM of 2 experiments.