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 interac tion 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 mye loperoxidase; and bf measurement of radiolabeled neutrophils and eryth rocytes 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, laborat ory study. Setting: University research laboratory. Subjects: Male Wis tar 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), Experiment al animals received 2 mg/kg body weight iv administration of a P- and E-selectin blocking monoclonal antibody, CY-1747, immediately after bu rn, Lung tissue neutrophils were estimated by measuring myeloperoxidas e in lung tissue. Neutrophil retention in lung, liver, spleen, gut, sk in, muscle, kidney, and brain tissues was determined by removing (preb urn) and differentially radiolabeling neutrophils ((111)ln) and erythr ocytes (Cr-51), reinfusing cells 4.5 hrs after burn, and measuring tis sue radioactivity 30 mins later, Edema was estimated by measuring extr avasated I-125 labeled albumin in the various tissues. Peripheral bloo d neutrophils were analyzed for intracellular hydrogen peroxide conten t, utilizing a fluorescent dye that reacts with hydrogen peroxide, cou pled with analysis of cell fluorescence by flow cytometry. Measurement s and Main Results: Myeloperoxidase concentration was increased in lun gs 5 hrs after burn (p <.05), indicating neutrophil deposition. Radioi sotope studies demonstrated significant (p <.05) leukosequestration in to the lung, gut, kidney, skin, and brain tissues at 5 hrs after burn, Flow cytometry showed increased intracellular hydrogen peroxide conte nt in peripheral blood neutrophils 5 hrs after burn, Tissue edema, man ifested by radiolabeled albumin retention, was not seen in any tissues , Postburn neutrophil deposition in lungs and liver was blocked (p< .0 5) by administration of CY-1747 after burn, but maximal neutrophil hyd rogen peroxide content was unaffected. Conclusion: Burn injury in rats results in accumulation of neutrophils in multiple tissues, Neutrophi l deposition in the lungs and liver is blocked by administration of th e E/P-selectin blocking antibody, CY-1747. Since sequestration of meta bolically active neutrophils may induce tissue injury, therapies that block postburn leukosequestration may improve clinical outcomes by lim iting remote tissue injury.