GLYCOSAMINOGLYCANS MEDIATE CELL-SURFACE OLIGOMERIZATION OF CHEMOKINES

Chemokines are 8-10 kDa proteins involved in the control of leukocyte trafficking and activation. In free solution, chemokines are monomers at physiologic concentrations, although many multimerize at higher con centrations. Cell surface heparan sulfate may sequester chemokines, in creasing their local concentrations and facilitating their binding to receptors expressed on leukocytes. In competitive binding assays using immobilized heparin, a 2-3-fold increase in the bound radiolabeled ch emokine was seen with increasing concentrations of unlabeled chemokine in the nanomolar range. Unlabeled chemokine concentrations between 0. 25 and 50 mu M were needed to compete the bound radioactivity. This bi phasic competition curve was not seen for N-methyl-L25 IL-8, a variant of IL-8 which is unable to dimerize. In addition, complexes of chemok ine and heparin eluted from gel filtration columns with apparent molec ular masses of 33-60 kDa, suggesting that chemokine multimerization ha d occurred. The physiological relevance of this multimerization proces s was seen from studies using human endothelial cells. The endothelial cell binding sites for IL-8, RANTES, and MCP-1 were deduced to be gly cosaminoglycans since competition assays showed the biphasic curves an d micromolar IC50 values seen in studies with immobilized heparin, and mRNA for known chemokine receptors was not detected. Furthermore, dig estion of endothelial cell monolayers with glycosaminidases decreased chemokine binding by up to 80%. Glycosaminoglycans can act as modulato rs of the ligand binding affinity of chemokine receptor-bearing cells. Removal of glycosaminoglycans from CHO cells expressing chemokine rec eptors CXCR1, CCR1, or CCR2 resulted in 40-70% decreases in the bindin g of RANTES, MCP-1, IL-8, and MIP-1 alpha. Our data show that cell sur face glycosaminoglycans induce polymerization of chemokines, increasin g their local concentration and therefore enhancing their effects on h igh-affinity receptors within the local microenvironment.