Gene-targeted mice reveal importance of L-selectin-dependent rolling for neutrophil adhesion

It has not been determined whether L-selectin-mediated rolling can promote leukocyte adhesion in vivo independent of P- and E-selectin. We used intravital microscopy of E- and P-selectin double-mutant mice (E−/P−) stimulated with tumor necrosis factor-α for 6–8 h to investigate the importance of L-selectin-dependent rolling in cremaster muscle venules. Rolling leukocyte flux in E−/P− mice was 9 ± 2 cells/min compared with 77 ± 17 cells/min in wild-type (WT) mice. Pretreatment with the L-selectin monoclonal antibody MEL-14 significantly reduced rolling in both E−/P− (by 89%) and WT mice (by 79%). L-selectin-dependent rolling in E−/P− mice resulted in leukocyte adhesion comparable to that seen in WT mice. MEL-14 pretreatment of E−/P− mice reduced leukocyte adhesion by 50%. The majority (∼80%) of intravascular leukocytes in both WT and E−/P− mice were neutrophils. We conclude that L-selectin can mediate rolling that results in sufficient leukocyte recruitment to account for the robust inflammatory response seen in E−/P− mice at later times.

Histamine can induce leukocyte rolling in rat mesenteric venules

Rolling is a retarded movement of leukocytes that depends on the function of the selectin class of adhesion molecules. In venules of internal organs, rolling is rapidly induced on tissue exposure by unknown mediator(s). Rolling was investigated with intravital microscopy in venules of the exteriorized mesentery of anesthetized rats. Immediately after exteriorization, rolling leukocyte flux was very low (10 +/- 4 min-1) and increased rapidly to reach a peak of 76 +/- 10 min-1 at 30 min. Intraperitoneal pretreatment of rats with histamine induced near-maximal rolling in mesenteric venules immediately after exteriorization. Leukocyte rolling was significantly inhibited by intravenular microinfusion of monoclonal antibody PB1.3 that recognizes and functionally blocks human and rat P-selectin. The effect of histamine was blocked by pretreatment with ranitidine, an H2-receptor antagonist, but not with diphenhydramine, which blocks H1 receptors. However, ranitidine did not block the subsequent increase of rolling leukocyte flux, and rolling leukocyte flux was similar in all groups 20 min after exteriorization. Histological investigation revealed degranulation of mast cells, suggesting release of histamine and other mediators. These findings indicate that histamine can induce leukocyte rolling in vivo via H2 receptors, most likely by inducing endothelial expression of P-selectin. In addition, mediators other than histamine released from mast cells and/or other cells are likely to promote sustained rolling.

Fucoidin, but not yeast polyphosphomannan PPME, inhibits leukocyte rolling in venules of the rat mesentery

Leukocyte rolling in venules is inhibited by several sulfated polysaccharides, by antibodies to the leukocyte adhesion receptor L- selectin (LECAM-1), and by recombinant soluble L-selectin. The sulfated fucose polymer fucoidin and the polyphosphomannan PPME bind to L- selectin and inhibit L-selectin-mediated lymphocyte adhesion to lymph node high endothelial venules (LN-HEV). We investigated whether fucoidin and PPME also inhibit leukocyte rolling. Rolling leukocyte flux was determined by intravital microscopy in 47 venules (diameter 21 to 50 microns) of the rat mesentery with and without micro-infusion of each reagent through 8-microns glass micropipettes. Micro-infusion (1 mg/mL) or intravenous (IV) injection (25 mg/kg) of fucoidin, but not vehicle, reduced leukocyte rolling by greater than 90%. The half- effective concentration was approximately 2.5 micrograms/mL. Stroboscopic fluorescence video microscopy showed that fucoidin decreased the fraction of rolling leukocytes from 44% of all leukocytes passing the venules in control to less than 1%. PPME micro-infusion (1 mg/mL) or IV injection (14 mg/kg) did not reduce leukocyte rolling. Hence, leukocyte rolling differs from lymphocyte homing with respect to the effect of PPME. This may be related to fucoidin binding to L- selectin with greater affinity than PPME. Alternatively, inflamed venular endothelium may express a ligand for L-selectin different from that constitutively expressed on LN-HEV.

Fucoidin, but not yeast polyphosphomannan PPME, inhibits leukocyte rolling in venules of the rat mesentery

Leukocyte rolling in venules is inhibited by several sulfated polysaccharides, by antibodies to the leukocyte adhesion receptor L- selectin (LECAM-1), and by recombinant soluble L-selectin. The sulfated fucose polymer fucoidin and the polyphosphomannan PPME bind to L- selectin and inhibit L-selectin-mediated lymphocyte adhesion to lymph node high endothelial venules (LN-HEV). We investigated whether fucoidin and PPME also inhibit leukocyte rolling. Rolling leukocyte flux was determined by intravital microscopy in 47 venules (diameter 21 to 50 microns) of the rat mesentery with and without micro-infusion of each reagent through 8-microns glass micropipettes. Micro-infusion (1 mg/mL) or intravenous (IV) injection (25 mg/kg) of fucoidin, but not vehicle, reduced leukocyte rolling by greater than 90%. The half- effective concentration was approximately 2.5 micrograms/mL. Stroboscopic fluorescence video microscopy showed that fucoidin decreased the fraction of rolling leukocytes from 44% of all leukocytes passing the venules in control to less than 1%. PPME micro-infusion (1 mg/mL) or IV injection (14 mg/kg) did not reduce leukocyte rolling. Hence, leukocyte rolling differs from lymphocyte homing with respect to the effect of PPME. This may be related to fucoidin binding to L- selectin with greater affinity than PPME. Alternatively, inflamed venular endothelium may express a ligand for L-selectin different from that constitutively expressed on LN-HEV.