5-lipoxygenase- and Glucocorticoid-dependent eosinophilia in a novel surgical model in mice

Abstract

BackgroundSubcutaneous implants of heat–coagulated egg white (egg white implants, EWI) induce intense local eosinophilia and prime for hyperreactivity following airway ovalbumin challenge. The roles of allergen sensitization, surgical trauma–induced glucocorticoids, and the 5-lipoxygenase (5-LO) pathway were hitherto unexplored in this model, in which quantitative recovery and large-scale purification of the eosinophils from the inflammatory site for functional and immunopharmacological studies are difficult to achieve. MethodsWe overcame this limitation by shifting the implantation site to the peritoneal cavity (EWIp), thereby enabling quantitative leukocyte retrieval. ResultsBy day 7 post–surgery, eosinophil counts reached ~ 30% of all leukocytes recovered. Eosinophilia was prevented by: a) induction of allergen–specific oral tolerance to ovalbumin, the main allergen in egg white; b) inactivation of the 5–lipoxygenase pathway; c) blockade of endogenous glucocorticoid signaling by pretreatment with metirapone plus mifepristone before surgery. Highly purified eosinophils (~99% pure) could be obtained from the peritoneal exudate of EWIp–carrier mice in 2 simple, antibody–free steps. Preparative-scale yields, suitable for most biochemical, pharmacological, and molecular applications, were routinely obtained, and could be further enhanced through addition of pre–or post–surgery immunization steps (active or adoptive). The recovered eosinophils were fully functional in vivo, as demonstrated by the transfer of purified eosinophils into eosinophil–deficient Δdbl–GATA–1-KO mice, which upon subsequent challenge with eotaxin-1 present secondary accumulation of neutrophils, but not of mononuclear phagocytes. ConclusionThese findings document glucocorticoid−, allergen– and 5–lipoxygenase–dependent eosinophilia, which makes EWIp carriers an abundant source of pure, nontransgenic eosinophils for immunopharmacological studies.