Examination of the Function of RANTES, MIP-1α, and MIP-1β following Interaction with Heparin-like Glycosaminoglycans

Abstract

Chemokines are a group of small proteins that have a variety of functions, including the activation and recruitment of immune cells during episodes of inflammation. In common with many cytokines, it has been observed that chemokines have the potential to bind heparin-like glycosaminoglycan molecules, which are normally expressed on proteoglycan components of the cell surface and extracellular matrix. The significance of this interaction for chemokine activity remains a subject of debate. In this study, Chinese hamster ovary cells were transfected separately with the human chemokine receptors CCR1 and CCR5, and these receptors were shown to induce an intracytoplasmic Ca 2؉ flux and cellular chemotaxis following stimulation with the natural CC chemokine ligands (MIP-1␣, RANTES (regulated on activation normal T cell expressed), and MIP-1␤). In further experiments, mutant CHO cells, with a defect in normal glycosaminoglycan (GAG) expression, were also transfected with, and shown to express similar levels of, CCR1 and CCR5. Although these receptors were functional, it was found that the mutant cells required exposure to higher concentrations of ligands than the wildtype cells in order to produce the same intracytoplasmic Ca 2؉ flux. Radioligand binding experiments demonstrated that specific chemokine receptors expressed by wild-type cells had a significantly greater affinity for MIP-1␣ than similar receptors expressed by GAG-deficient mutants. However, there was no significant difference between these cells in their affinity for RANTES or MIP-1␤. In conclusion, it has been demonstrated clearly that GAG expression is not necessary for the biological activity of the chemokines MIP-1␣, RANTES, or MIP-1␤. However, the presence of cell surface GAGs does enhance the activity of low concentrations of these chemokines by a mechanism that appears to involve sequestration onto the cell surface.

Chemokines: attractive mediators of the immune response

“…The association of chemokines with GAGs of the extracelluar matrix and on the cell surface has important implications in terms of presentation and increasing local chemokine levels. Using GAG deficient Chinese hamster ovary cells transfected with CCR1 and CCR5, Ali et al [17] demonstrated that surface GAGs increase the concentration of chemokines presented to the CCRs at the molecular level. CCL5 has been shown to bind GAGs with distinct specificity [16] suggesting that CCL5 may be crucial in mediating inflammatory responses.…”

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

“…5,47 A variety of experiments have shown that GAG are critically important for chemokine activity, 3,5,23,48 with loss of cell surface heparan sulfate severely limiting chemokine activity. 34,42,49 Soluble chemokine concentration gradients have been shown to direct immune cell migration in vitro. During inflammation in vivo, it is likely that chemokines produced by subendothelial tissues contribute to the recruitment of blood-borne leukocytes following redistribution and presentation on the apical surface of the endothelium.…”

Identification of Receptor Binding and Activation Determinants in the N-terminal and N-loop Regions of the CC Chemokine Eotaxin

“…1). The quaternary structures of chemokines vary dramatically within the superfamily, with a range of monomeric, dimeric, and tetrameric structures seen, although there is convincing evidence that the monomer is sufficient for receptor activation (19,20). The three-dimensional structures of eotaxin (21), eotaxin-2 (22), and eotaxin-3 2 have recently been determined and are all monomeric.…”