Isolation and characterization of eosinophils in bovine blood and small intestine

Abstract

An effective method to isolate functional eosinophils from blood and tissues is required to analyze the multiple roles eosinophils play in innate immunity and tissue homeostasis. Highspeed cell sorting was used to isolate bovine eosinophils from blood polymorphonuclear (PMN) cells and from small intestine intraepithelial leukocytes. Eosinophils and neutrophils were purified from bovine blood with highspeed cell sorting after gating on autofluorescence (FL1) high and low PMN subpopulations. Highspeed sorting of intestinal eosinophils was accomplished by using a combination of positive (CD45+, CD11cLow, side scatterHigh) and negative (CD3−) selection parameters. Eosinophils sorted from blood PMNs were 88.6 ± 5.8 % (mean + 1 SD; n = 4) pure and yielded significantly (p < 0.05) more RNA than purified neutrophils. Analysis of Toll-like receptor (TLR) gene expression and TLR ligand-induced pro-inflammatory cytokine (IL-1, IL-6, IL-8, and TNFα) gene expression demonstrated significant (p < 0.01) functional differences between blood eosinophils and neutrophils. Eosinophils varied between 14.7 % to 29.3 % of CD45+ IELs and purity of sorted intestinal eosinophils was 95 + 3.5 % (mean + 1SD; n = 5). A comparison of mucosal and blood eosinophils revealed significant (p < 0.01) differences in TLR gene expression, supporting the hypothesis that functionally distinct eosinophil populations are present in blood and tissues. In conclusion, highspeed cell sorting provides an effective method to isolate viable eosinophils from blood and tissues that can then be used for transcriptome analyses and in vitro function assays.