Selectin-mediated rolling of neutrophils on immobilized platelets

Abstract

Interaction between neutrophils and platelets at the site of vascular damage or in ischaemic tissue may promote thrombosis and/or vascular occlusion. To study this interaction, we have developed a novel technique that allows visualization of adhesion of flowing neutrophils to immobilized, activated platelets. The total number of adherent neutrophils decreased with increasing wall shear stress in the range 0.05 to 0.4 Pa. Although a proportion of the adherent neutrophils were stationary, most were rolling with a velocity greater than 0.4 micron/s. The percentage of rolling cells increased with increasing wall shear stress, but the mean rolling cell velocity was nearly independent of shear stress. Adhesion of neutrophils was nearly abolished by treatment of the platelets with antibody to P-selectin, or by treatment of neutrophils with either neuraminidase, dextran sulfate, or EDTA. Studies with a series of antibodies to L-selectin (TQ-1, Dreg- 56, LAM1–3, and LAM1–10) suggested that this molecule was one neutrophil ligand for rolling adhesion. Thus, sialylated carbohydrate on neutrophils appears essential for P-selectin-mediated adhesion, and a proportion of this ligand may be presented by L-selectin. Treatment of the neutrophils with N-formyl-methionyl-leucyl-phenylalanine decreased the number of rolling cells, and increased the rolling velocity, possibly due to shedding of neutrophil ligand(s) and/or cell shape change. In vivo, immobilized platelets could play an important role in promoting attachment of neutrophils to vessel walls, eg, by slowing neutrophils so that integrin-mediated immobilization could occur.

Differing mechanisms of leukocyte recruitment and sensitivity to conditioning by shear stress for endothelial cells treated with tumour necrosis factor‐α or interleukin‐1β

“…Our inability to block adhesion to IL‐1B‐treated HUVEC with antibodies against selectins is puzzling, even though there was clear upregulation of E‐selectin mRNA and surface expression as expected. The same antibodies against selectins have been effective in our previous studies in other adhesion models (Buttrum et al ., 1993; Rainger et al ., 1995; Bahra et al ., 1998; Rainger et al ., 1998). Others showed that blockade of L‐selectin or E‐selectin caused partial inhibition of adhesion of neutrophils to IL‐1B‐treated HUVEC in static or flow‐based assays (Bevilacqua et al ., 1987; Kishimoto et al ., 1991; Smith et al ., 1991; Abbassi et al ., 1993; Jones et al ., 1996), although the effects were not additive (Kishimoto et al ., 1991).…”

“…In some experiments, neutrophils were treated with the platelet‐activating factor receptor (PAF‐R) antagonist YM264 (10 −4 M; Yamanouchi Pharmaceutical Co. Ltd, Tsukuba, Japan) for 3 min before assay. These agents have been effective in blocking function in our previous studies (Buttrum et al ., 1993; Rainger et al ., 1995; 1998; Bahra et al ., 1998).…”

“…In chosen experiments, monoclonal antibodies (mAb) against P‐selectin (mAb G1, 50 μ g ml −1 ; gift of Rodger McEver, University of Oklamhoma, U.S.A.) or E‐selectin (ENA2 F(ab’)2, 1 μ g ml −1 ; Bradshaw Biologicals, Chepshet, U.K.) were added to HUVEC with the cytokines for the last 20 min before adhesion assay. These antibodies have been effective in blocking adhesion in our previous studies (Buttrum et al ., 1993; Rainger et al ., 1996; Bahra et al ., 1998). In other experiments, nordihydroguaiaretic acid (NDGA; 5 μ M ) and indomethacin (10 μ M ; both Sigma) were added to HUVEC cultures along with IL‐1B, to inhibit lipoxygenase and cycloxygenase pathways (Ibe et al ., 1989).…”

Binding of activated platelets to WBCs in vivo after transfusion

“…Lacto‐N‐fucopentose (Lewis x , CD15) and sialyl‐Lewis x (CD15s) are also known to be possible ligands 30,38 . Differences in the degree of coaggregation of monocytes to neutrophil granulocytes may be explained by the different configuration of CD15 and CD15s, 25,39 and by the low affinity of the two antigens to CD62p 40 . In contrast to L‐selectin of neutrophil granulocytes, L‐selectin of lymphocytes does not express Le x and thus cannot serve as a receptor for P‐selectin 41 .…”

The role of E-selectin in two murine models of acute inflammation

“…Flowing neutrophils can roll on surfaces coated with purified P-selectin, histaminestimulated endothelial cells, or immobilized, activated platelets providing in vitro evidence for P-selectin-mediated neutrophil rolling (33,223,224) In addition, Table 4 summarizes these studies. leukocytes/slide using a light microscope at 630x oil immersion.…”