Contribution of the putative heparan sulfate-binding motif BBXB of RANTES to transendothelial migration

Abstract

The chemokines are a family of small chemoattractant proteins that have a range of functions, including activation and promotion of vectorial migration of leukocytes. Regulation on activation, normal T cell expressed and secreted (RANTES; CCL5), a member of the CC-chemokine subfamily, has been implicated in a variety of immune responses. In addition to the interaction of CC-chemokines with their cognate cell-surface receptors, it is known that they also bind to glycosaminoglycans (GAGs), including heparan sulfate. This potential for binding to GAG components of proteoglycans on the cell surface or within the extracellular matrix might allow formation of the stable chemokine concentration gradients necessary for leukocyte chemotaxis. In this study, we created a panel of mutant RANTES molecules containing neutral amino acid substitutions within putative, basic GAG-binding domains. Despite showing reduced binding to GAGs, it was found that each mutant containing a single amino acid substitution induced a similar leukocyte chemotactic response within a concentration gradient generated by free solute diffusion. However, we found that the mutant K45A had a significantly reduced potential to stimulate chemotaxis across a monolayer of microvascular endothelial cells. Significantly, this mutant bound to the CCR5 receptor and showed a potential to mobilize Ca(2+) with an affinity similar to the wild-type protein. These results show that the interaction between RANTES and GAGs is not necessary for specific receptor engagement, signal transduction, or leukocyte migration. However, this interaction is required for the induction of efficient chemotaxis through the extracellular matrix between confluent endothelial cells.

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

“…As seen for RANTES (CCL5), apically bound chemokine was sufficient to promote leukocyte migration, although to a smaller extent to that observed under standard assay conditions. 34 These observations are consistent with a recent study that has shown that, at least for lymphocytes, a transendothelial concentration gradient is not necessary for transendothelial migration, with apically bound chemokine providing a sufficient cue. 30 In this study, we demonstrate that MCP-1 is rapidly redistributed from the basal to the apical surface of a model endothelium leaving no evidence of a haptotactic concentration gradient between the cells constituting the monolayer.…”

“…in serum-free DMEM was applied to an EA.hy926 monolayer for 1 h at 371C. 34 Monolayers were then washed twice for 5 min in serum free DMEM at room temperature prior to transfer to the standard migration assay system. Migration was for 90 min at 371C to allow direct comparison to the standard assay described above.…”

“…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.…”

Post-Transplant Renal Tubulitis: The Recruitment, Differentiation and Persistence of Intra-Epithelial T Cells

“…These gradients can provide the motivation for vectorial cell migration, resulting in selective recruitment of leukocytes from the subendothelial to the tubular compartments. This model was recently supported by data from our group showing that a mutant variant of RANTES with a reduced potential to bind heparan sulphate but normal affinity for the specific chemokine receptor CCR5 induced a smaller chemotactic response than the wild-type chemokine (33).…”

Endothelial inflammation: the role of differential expression of N-deacetylase/N-sulphotransferase enzymes in alteration of the immunological properties of heparan sulphate

“…This interaction has a number of important functions, including protection, concentration and presentation of cytokines which are produced in small quantities by a small number of cells within the microenvironment. This process is believed to be of particular importance for presentation of chemokines on the surface of endothelial cells (Tanaka et al, 1996) and for stabilisation of the concentration gradients necessary for leukocyte chemotaxis (Adams and Lloyd, 1997;Ali et al, 2002;Appay and Rowland-Jones, 2001;Patel et al, 2001;Witt and Lander, 1994).…”