Measurement of lipocortin 1 levels in murine peripheral blood leukocytes by flow cytometry: modulation by glucocorticoids and inflammation

Abstract

1 Lipocortin 1 (LC1) immunoreactivity in murine peripheral blood leukocytes was quantified by use of a flow cytometric technique associated with a permeabilisation protocol with saponin. Using specific antisera raised against the whole protein or against its N-terminus peptide, cell-associated LC1-like immunoreactivity was easily detected in circulating neutrophils and monocytes, whereas very low levels were found in lymphocytes. Of the total protein measured 17.6% and 36% were associated with the external plasma membrane in neutrophils and monocytes, as assessed in the absence of cell permeabilisation, whereas no signal was detected on lymphocyte plasma membrane. 2 Treatment of mice with dexamethasone (Dex; 0.5-5 μg per mouse corresponding to ∼ 0.015-1.5 mg kg⁻¹) increased LC1 levels in neutrophils and monocytes. The 2–3 fold increase in LC1 levels was time-dependent with a peak at 2 h. Treatment of mice with the steroid antagonist, RU486 (two doses of 20 mg kg⁻¹ orally) decreased LC1-like immunoreactivity in all three types of circulating leukocytes by ≥50%. 3 Extravasation of blood neutrophils into inflamed tissue sites resulted in a consistent reduction (≥50%) in LC1 levels compared with circulating neutrophils. A high LC1-like immunoreactivity was also measured in resident macrophages, of which approximately one third was membrane-associated. Induction of an acute inflammatory response in the murine peritoneal cavity did not modify total LC1 levels measured in macrophages, but reduced membrane-associated LC1 to a significant extent, i.e. up to 70%. 4 In conclusion, flow cytometric analysis is a rapid and convenient method for detecting and measuring LC1 in murine leukocytes. We confirmed that LC1 protein expression is controlled by exogenous and endogenous glucocorticoids. Amongst other factor(s) influencing protein concentrations, extravasation was found to be associated with a reduced LC1 expression in the emigrated cells.