Duffy antigen receptor for chemokines and CXCL5 are essential for the recruitment of neutrophils in a multicellular model of rheumatoid arthritis synovium
OBJECTIVE: The role of chemokines and their transporters are poorly described in rheumatoid arthritis (RA). Evidence suggests that CXCL5 plays an important role as it is abundant in RA tissue and its neutralization moderates joint damage in animal models of arthritis. The chemokine transporter, Duffy Antigen Receptor for Chemokines (DARC), is also upregulated in early RA. Here we investigate the role of CXCL5 and DARC in regulating neutrophil recruitment using an in vitro model of the RA synovium. METHODS: To model the RA synovium, rheumatoid fibroblasts (RAF) were cocultured with endothelial cells (EC) for 24h. Gene expression in cocultured cells was investigated using TaqMan gene arrays. Roles of CXCL5 and DARC were determined by incorporating cocultures into a flow-based adhesion assay, where their function was demonstrated by blocking neutrophil recruitment with neutralizing reagents. RESULTS: EC-RAF coculture induced chemokine expression in both cell types. While CXC chemokines were modestly upregulated in EC, CXCL1, CXCL5 and CXCL8 expression were greatly increased in RAF. RAF also promoted the recruitment of flowing neutrophils to EC. Anti-CXCL5 antibody abolished neutrophil recruitment by neutralizing CXCL5 expressed on EC, or when used to immuno-deplete coculture conditioned medium. DARC was also induced on EC by coculture and an anti-Fy6 antibody or siRNA targeting of DARC expression effectively abolished neutrophil recruitment. CONCLUSION: For the first time in a model of human disease, the function of DARC has been demonstrated as essential for editing the chemokine signals presented by EC and for promoting unwanted leukocyte recruitment
