Sulphatides trigger polymorphonuclear granulocyte spreading on collagen-coated surfaces and inhibit subsequent activation of 5-lipoxygenase

Abstract

Sulphatides are sulphate esters of galactocerebrosides that are present on the surfaces of many cell types and act as specific ligands to selectins. The present study was undertaken to investigate the effect of sulphatides on polymorphonuclear granulocyte (PMN) attachment, spreading and 5-lipoxygenase (5-LO) metabolism. Sulphatides, but not non-sulphated galactocerebrosides, dose-dependently enhanced attachment to collagen, as measured by the myeloperoxidase assay. Studies with blocking antibodies indicated that the increased attachment was mediated by CD11b/CD18 (Mac-1) β2 integrin. Scanning electron microscopy indicated that sulphatides also greatly enhanced the degree of cell spreading. In PMNs treated in suspension, sulphatides had no effect on the ionophore A23187-stimulated release of arachidonic acid and the synthesis of 5-LO metabolites. In contrast, in PMNs attached to collagen, the enzymic conversion of arachidonic acid by 5-LO was inhibited by sulphatides. Inhibition of 5-LO metabolism by sulphatides was observed even in the presence of exogenous substrate, suggesting that sulphatides directly inhibited 5-LO action. Consistent with this, sulphatides interfered with ionophore-induced translocation of the 5-LO to the nuclear envelope. Substances competing with sulphatide binding to cells, like dextran sulphate, or a strong inhibitor of cell spreading, like the actin-polymerizing agent jasplakinolide, prevented the effects of sulphatides on PMN attachment and spreading and leukotriene synthesis. We conclude that shape changes occurring in response to sulphatides specifically impair PMN leukotriene synthesis by inhibiting translocation of 5-LO.

Identification of Two Novel Nuclear Import Sequences on the 5-Lipoxygenase Protein

“…Subsequent observations have further indicated that nuclear import of 5-LO is a regulated process. Nuclear import can be triggered by adherence (2,4,11), by recruitment (2, 3), or by cytokines (5,12). Conditions that cause nuclear import of 5-LO can enhance (2,5,12) or suppress (4) LT production.…”

Activation of NF‐κB transcription factor in human neutrophils by sulphatides and L‐selectin cross‐linking

“…It is known that L-selectin cross-linking [12,21] and sulphatides [8] induce activation of L2 integrins. Do they signal in a similar way to PMN attachment onto a collagen-coated surface?…”

“…The role of sulphatides as L-selectin signalling molecules was demonstrated also by increasing cytosolic free calcium and enhancing expression of TNF and interleukin-8 genes in human neutrophils [7]. We have previously reported that sulphatides stimulated adhesion of human neutrophils to surfaces coated with collagen and ¢bronectin [8]. Since this may proceed through an activation for NF-UB we have now studied its activation in neutrophils and found it triggered by sulphatides.…”

“…In a recent publication [8] we have shown that sulphatide induced Mac-1-mediated attachment of human neutrophils to a collagen-coated surface. To determine if L-selectin was necessary for sulphatide-induced attachment, we determined PMN attachment to a collagen-coated surface after pretreatment of PMN with K-chymotrypsin, phorbol myristate acetate (PMA) and jasplakinolide, all known to induce L-selectin shedding [7,10,13^15].…”

Neutrophil Cellular Responses to Various Salmonella typhimurium LPS Chemotypes

“…PMNLs were isolated from freshly drawn EDTA-anticoagulated donor blood by standard techniques, as previously described (Sud'ina et al, 2001). Leukocyte-rich plasma was prepared by sedimentation of RBCs with 3% dextran T-500 at room temperature.…”