Glycosaminoglycans Mediate Cell Surface Oligomerization of Chemokines

Abstract

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 concentrations. Cell surface heparan sulfate may sequester chemokines, increasing 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 chemokine was seen with increasing concentrations of unlabeled chemokine in the nanomolar range. Unlabeled chemokine concentrations between 0.25 and 50 microM were needed to compete the bound radioactivity. This biphasic 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 chemokine and heparin eluted from gel filtration columns with apparent molecular masses of 33-60 kDa, suggesting that chemokine multimerization had occurred. The physiological relevance of this multimerization process was seen from studies using human endothelial cells. The endothelial cell binding sites for IL-8, RANTES, and MCP-1 were deduced to be glycosaminoglycans since competition assays showed the biphasic curves and micromolar IC50 values seen in studies with immobilized heparin, and mRNA for known chemokine receptors was not detected. Furthermore, digestion of endothelial cell monolayers with glycosaminidases decreased chemokine binding by up to 80%. Glycosaminoglycans can act as modulators of the ligand binding affinity of chemokine receptor-bearing cells. Removal of glycosaminoglycans from CHO cells expressing chemokine receptors CXCR1, CCR1, or CCR2 resulted in 40-70% decreases in the binding of RANTES, MCP-1, IL-8, and MIP-1alpha. Our data show that cell surface glycosaminoglycans induce polymerization of chemokines, increasing their local concentration and therefore enhancing their effects on high-affinity receptors within the local microenvironment.

Examination of MCP-1 (CCL2) partitioning and presentation during transendothelial leukocyte migration

“…7,8 It has been proposed that cell surface GAG form a scaffold within which chemokines are assembled into multimers. 45 While MCP-1 has been found as a dimer in solution at concentrations in the physiological range and as a tetramer at higher concentrations, this appears to have no functional significance as obligate monomers show a normal activity. 35,42,44 Chemokine multimerization within the cell surface GAG layer could provide a further explanation for the focal distribution observed for MCP-1.…”

“…45 In order to investigate this possibility, an obligate monomeric MCP-1 mutant P8A was added to the basal chamber and immunoreactive protein was subsequently identified on the apical surface of the model endothelium. As for the wild-type chemokine, it was found that the mutant protein was largely concentrated into focal regions on the apical surface of 26% (23 of 87 cells examined in 29 preparations) of the cells (Figure 3e).…”

Human fractalkine mediates leukocyte adhesion but not capture under physiological shear conditions; a mechanism for selective monocyte recruitment

“…First, chemokines indirectly mediate firm adhesion by activating leukocyte integrins to bind their endothelial ligands with high affinity [4,5]. In this instance, chemokines are presented to leukocytes through immobilization on glycosaminoglycans on the luminal surface of the endothelium [6,7]. Second, the chemotactic properties of chemokines direct leukocyte migration out of the vessel lumen and into the tissue [8,9].…”

Induction of a CXCL8 binding site on endothelial syndecan‐3 in rheumatoid synovium

“…The functional importance of chemokine-HS interactions has been shown in vivo, because administration of modified chemokines that exhibit reduced affinity to HS or heparin resulted in diminished leukocyte recruitment (31,32). Other functions of glycosaminoglycan-chemokine interactions have been proposed, including the formation of immobilized chemokine gradients in the extracellular matrix and the protection of these mediators from degradation, resulting in the prolongation of their activities (15,35,36).…”