Effects of E-selectin and P-selectin blockade on neutrophil sequestration in tissues and neutrophil oxidative burst in burned rats

Objective: Neutrophil deposition in tissues (leukosequestration) after shock may produce local tissue injury from proteases and high-energy oxygen species released from sequestered neutrophils. The initial step in the binding of neutrophils to capillary endothelium is the interaction of adhesion molecule (selectin) receptors between neutrophils and endothelial cells. We quantified leukosequestration in the tissues of burned rats using two methods of analysis: a) measurement of lung myeloperoxidase; and bf measurement of radiolabeled neutrophils and erythrocytes deposited in multiple tissues. We then determined the ability of a selectin receptor blocking agent to affect neutrophil deposition in tissues after burn injury. Design: Prospective, controlled, laboratory study. Setting: University research laboratory. Subjects: Male Wistar rats (200 to 300 g). Interventions: After tracheostomy and venous cannulation, rats received 17% total body surface area full thickness contact burns and were resuscitated with saline (20 mL ip), Experimental animals received 2 mg/kg body weight iv administration of a P- and E-selectin blocking monoclonal antibody, CY-1747, immediately after burn, Lung tissue neutrophils were estimated by measuring myeloperoxidase in lung tissue. Neutrophil retention in lung, liver, spleen, gut, skin, muscle, kidney, and brain tissues was determined by removing (preburn) and differentially radiolabeling neutrophils ((111)ln) and erythrocytes (Cr-51), reinfusing cells 4.5 hrs after burn, and measuring tissue radioactivity 30 mins later, Edema was estimated by measuring extravasated I-125 labeled albumin in the various tissues. Peripheral blood neutrophils were analyzed for intracellular hydrogen peroxide content, utilizing a fluorescent dye that reacts with hydrogen peroxide, coupled with analysis of cell fluorescence by flow cytometry. Measurements and Main Results: Myeloperoxidase concentration was increased in lungs 5 hrs after burn (p <.05), indicating neutrophil deposition. Radioisotope studies demonstrated significant (p <.05) leukosequestration into the lung, gut, kidney, skin, and brain tissues at 5 hrs after burn, Flow cytometry showed increased intracellular hydrogen peroxide content in peripheral blood neutrophils 5 hrs after burn, Tissue edema, manifested by radiolabeled albumin retention, was not seen in any tissues, Postburn neutrophil deposition in lungs and liver was blocked (p< .05) by administration of CY-1747 after burn, but maximal neutrophil hydrogen peroxide content was unaffected. Conclusion: Burn injury in rats results in accumulation of neutrophils in multiple tissues, Neutrophil deposition in the lungs and liver is blocked by administration of the E/P-selectin blocking antibody, CY-1747. Since sequestration of metabolically active neutrophils may induce tissue injury, therapies that block postburn leukosequestration may improve clinical outcomes by limiting remote tissue injury.