Mechanisms that regulate the function of the selectins and their ligands.

Selectins are a family of three cell adhesion molecules (L-, E-, and P-selectin) specialized in capturing leukocytes from the bloodstream to the blood vessel wall. This initial cell contact is followed by the selectin-mediated rolling of leukocytes on the endothelial cell surface. This represents the first step in a cascade of molecular interactions that lead to leukocyte extravasation, enabling the processes of lymphocyte recirculation and leukocyte migration into inflamed tissue. The central importance of the selectins in these processes has been well documented in vivo by the use of adhesion-blocking antibodies as well as by studies on selectin gene-deficient mice. This review focuses on the molecular mechanisms that regulate expression and function(s) of the selectins and their ligands. Cell-surface expression of the selectins is regulated by a variety of different mechanisms. The selectins bind to carbohydrate structures on glycoproteins, glycolipids, and proteoglycans. Glycoproteins are the most likely candidates for physiologically relevant ligands. Only a few glycoproteins are appropriately glycosylated to allow strong binding to the selectins. Recently, more knowledge about the structure and the regulated expression of some of the carbohydrates on these ligands necessary for selectin binding has been accumulated. For at least one of these ligands, the physiological function is now well established. A novel and exciting aspect is the signaling function of the selectins and their ligands. Especially in the last two years, convincing data have been published supporting the idea that selectins and glycoprotein ligands of the selectins participate in the activation of leukocyte integrins.

Stimulation of P-selectin glycoprotein ligand-1 on mouse neutrophils activates beta 2-integrin mediated cell attachment to ICAM-1.

The entry of neutrophils into inflamed tissues is initiated by cell rolling on the blood vessel wall followed by arrest and transendothelial migration. Rolling is mediated by the selectins, while the two subsequent steps require activated beta 2-integrins. We have investigated whether the binding of P-selectin to mouse neutrophils could trigger the activation of beta 2-integrins. We show that cross-linking of P-selectin glycoprotein ligand-1 (PSGL-1) on mouse neutrophils with an antibody-like recombinant form of P-selectin or with monoclonal antibodies stimulated the production of reactive oxygen intermediates and enhanced neutrophil attachment to intercellular adhesion molecule 1 (ICAM-1)-expressing CHO cells. This effect was independent of whether complete antibodies or F(ab')2 fragments were used. The adhesion-stimulating effect of P-selectin could be blocked by monoclonal antibodies against PSGL-1. Increase of cell attachment was dependent on lymphocyte function-associated antigen 1 (LFA-1) and on Mac-1, since it could be blocked with antibodies against both respective integrin alpha-chains. Moreover, cell surface expression of Mac-1 increased upon cross-linking of PSGL-1. In agreement with published data, treatment of human neutrophils with P-selectin-IgG did not enhance attachment to ICAM-1. Our data suggest that ligation of PSGL-1 on mouse neutrophils, but not on human neutrophils, activates beta 2-integrin mediated cell attachment to ICAM-1.

P-selectin glycoprotein ligand-1 mediates rolling of mouse bone marrow-derived mast cells on P-selectin but not efficiently on E-selectin.

It has been shown recently that mast cells play an essential role as a source of tumor necrosis factor-alpha production during neutrophil recruitment to sites of bacterial infection. Increased numbers of mast cells are indeed noted at sites of wound healing and inflammation. These cells are either recruited from the bone marrow or proliferate locally under cytokine stimulation. Little is known about how mast cell progenitors extravasate into tissue. Using antibody-like fusion proteins of mouse E-selectin and P-selectin, we have analyzed the ability of immature mouse bone marrow-derived mast cells (BMMC) to interact with the endothelial selectins. The P-selectin glycoprotein ligand-1 (PSGL-1) was affinity-isolated from detergent extracts of surface biotinylated BMMC with both selectin-IgG fusion proteins. However, only P-selectin-IgG, but not E-selectin-IgG showed significant interaction with intact BMMC as tested by flow cytometry and cell attachment assays with the immobilized fusion proteins under flow and non-flow conditions at physiological shear stress. Thus, in spite of carrying the necessary carbohydrate modifications which enable solubilized PSGL-1 to bind avidly to E-selectin, PSGL-1 on the surface of BMMC is presented in a way that prevents it from interacting efficiently with E-selectin. Affinity-purified rabbit antibodies against mouse PSGL-1 almost completely blocked the interaction of BMMC with P-selectin-IgG in flow cytometry as well as in cell adhesion assays under static and under flow conditions. Our data reveal that PSGL-1 is the major binding site for P-selectin on mouse BMMC progenitors, but does not support efficient interactions with E-selectin.

L-selectin from human, but not from mouse neutrophils binds directly to E-selectin.

L-Selectin on neutrophils as well as inducible E- and P-selectin on endothelium are involved in the recruitment of neutrophils into inflamed tissue. Based on cell attachment assays, L-selectin was suggested to function as a carbohydrate presenting ligand for E- and P-selectin. However, previous affinity isolation experiments with an E-selectin-Ig fusion protein had failed to detect L-selectin among the isolated E-selectin ligands from mouse neutrophils. We show here that L-selectin from human neutrophils, in contrast to mouse neutrophils, can be affinity-isolated as a major ligand from total cell extracts using E-selectin-Ig as affinity probe. Binding of human L-selectin to E-selectin was direct, since purified L-selectin could be reprecipitated with E-selectin-Ig. Recognition of L-selectin was abolished by sialidase-treatment, required Ca2+, and was resistant to treatment with endoglycosidase F. Binding of L-selectin to a P-selectin-Ig fusion protein was not observed. In agreement with the biochemical data, the anti-L-selectin mAb DREG56 inhibited rolling of human neutrophils on immobilized E-selectin-Ig but not on P-selectin-Ig. No such inhibitory effect was seen with the anti-mouse L-selectin mAb MEL14 on mouse neutrophils. Rolling of E-selectin transfectants on purified and immobilized human L-selectin was inhibited by mAb DREG56. We conclude that L-selectin on human neutrophils is a major glycoprotein ligand among very few glycoproteins that can be isolated by an E-selectin affinity matrix. The clear difference between human and mouse L-selectin suggests that E-selectin-binding carbohydrate moieties are attached to different protein scaffolds in different species.