Keith E. Norman

Transit time of leukocytes rolling through venules controls cytokine-induced inflammatory cell recruitment in vivo.

Leukocyte recruitment requires leukocyte rolling, activation, firm adhesion, and transmigration. Injection of the proinflammatory cytokine TNF-alpha induces expression of E-selectin, interleukin-8, and other adhesion molecules and chemoattractants on the endothelial surface. TNF-alpha- treated CD18 null mouse cremaster muscle venules show increased leukocyte rolling velocity and reduced leukocyte recruitment efficiency. Leukocyte recruitment in CD18 null but not wild-type mice is significantly blocked by an mAb to E-selectin. To understand this overlap between adhesion events previously considered separate, we introduce a quantitative analysis of the efficiency of induction of rolling, conversion of rolling to adhesion, and of adhesion to transmigration. We find that CD18 and E-selectin cooperate to control the time a leukocyte needs to roll through an inflamed area and to convert rolling to firm adhesion. Leukocyte rolling time, defined as the time it takes for a rolling leukocyte to pass through a defined length of a vessel segment, emerges as a unifying parameter determining the efficiency of inducing firm adhesion, which is a rate-limiting step controlling leukocyte recruitment in inflammation. We conclude that leukocytes integrate chemoattractant signals while rolling along the endothelial surface until they reach a critical level of activation and become firmly adherent.

Polymers bearing sLex-mimetics are superior inhibitors of E-selectin-dependent leukocyte rolling in vivo

Selectins mediate leukocyte rolling and may represent good anti‐inflammatory drug targets. Detailed knowledge regarding the structure of selectin ligands has permitted development of selectin antagonists with varying specificities and activity. Efficacy of monovalent selectin antagonists may be increased by presenting them on a polymer backbone. We have synthesized a range of multivalent selectin antagonists and characterized their activity by using intravital microscopy of the mouse cremaster muscle. The monovalent inhibitor CGP77175A inhibited E‐selectin‐dependent leukocyte rolling at a dose of 3 mg/kg. Multivalent presentation of CGP77175A on a modified polylysine backbone (degree of polymerization = 1200; 50% of the polylysines carry the inhibitor) greatly enhanced in vivo activity giving an inhibitor that produced an equivalent effect at 0.1 mg/kg. The polylysine conjugate was also longer acting than the monovalent antagonist. In spite of greatly enhanced activity against E‐selectin compared with monovalent inhibitor, the multivalent inhibitor had no measurable effect on P‐ or L‐selectin‐dependent leukocyte rolling.

Glycosulfopeptides modeled on P-selectin glycoprotein ligand 1 inhibit P-selectin-dependent leukocyte rolling in vivo

Leukocytic inflammation can be limited by inhibiting selectin‐dependent leukocyte rolling. In spite of intensive efforts to develop small molecule selectin inhibitors with defined structure‐activity profiles, inhibition of P‐selectin‐dependent leukocyte rolling in vivo by such a compound has yet to be described. We recently reported that glycosulfopeptides (GSP), modeled on the high affinity selectin ligand PSGL‐1, inhibit leukocyte binding to P‐selectin in vitro. Here, we have used intravital microscopy to investigate whether GSP can inhibit P‐selectin‐dependent leukocyte rolling in vivo. Surgical preparation of the mouse cremaster muscle for intravital microscopy induced P‐selectin‐dependent leukocyte rolling. Baseline rolling was recorded for 1 min followed by i.v. injection of GSP. 2‐GSP‐6 and 4‐GSP‐6 substantially reversed P‐selectindependent leukocyte rolling, whereas control GSP, which are not fully glycosylated, did not. Inhibition of leukocyte rolling by 2‐ and 4‐GSP‐6 lasted 2–4 min. Clearance studies with 125Ilabeled 4‐GSP‐6 demonstrated rapid reduction in its circulating levels concurrent with accumulation in urine. These data represent the first demonstration that a precisely defined structure based on a natural P‐selectin ligand can inhibit P‐selectin‐dependent leukocyte rolling in vivo.