Endotoxin-induced leukocyte accumulation in aqueous fluid of rats is decreased by a small molecule selectin antagonist

Purpose. Selectins are a family of carbohydrate binding proteins expressed on surfaces of endothelium (E- and P-selectin), platelets (P-selectin) and leukocytes (L-selectin). Since binding of the sialyl Lewis^X oligosaccharides on leukocytes to selectins initiates cellular recruitment in response to inflammatory stimuli, these experiments investigated a possible role for selectins in leukocyte accumulation in aqueous fluid following exposure to endotoxin. Methods. A non-oligosaccharide selectin inhibitor, TBC265, was assayed for its ability to inhibit binding of HL60 cells (which express surface sialyl Lewis^X-bearing glycoproteins) to recombinant E-, P-, and L-selectin-IgG fusion proteins bound to magnetic beads. To induce uveitis, 200 μg LPS (Salmonella minnesota) was injected as a divided dose into both hind footpads of male, 175-200 g Lewis rats anesthetized with halothane. Vehicle (0.5 ml PBS) or TBC265 (30 mg/0.5 ml PBS) was injected s.c. 3, 5, and 7 hours post LPS injection, and rats were killed after 24 h. Total recoverable neutrophils (PMN) were calculated from a total cell count and differential cell determination using aqueous fluid pooled from both eyes. Results. IC₅₀ values for TBC265 inhibition of HL60 cell binding to E-, P-, and L-selectin-IgG fusion proteins was 3, 2, and 2 mM, respectively. Endotoxin increased recoverable PMN from essentially 0 in controls to 9,400 ± 1,940 (mean ± SEM; n=26); TBC265 decreased recoverable PMN 80 % to 1790 ± 516 (n=14; p<0.001). Conclusions. Results from these experiments suggest that selectin adhesion molecules play a role in the response of the anterior uvea to endotoxin, and indicate that small molecule mimetics of sialyl Lewis^X oligosaccharides may have therapeutic potential in preventing sequelae of acute inflammation.