Detection and quantitation of eosinophils in the murine respiratory tract by flow cytometry

Abstract

Traditionally, the identification and quantification of eosinophils in inflammatory tissues and exudates has been primarily based upon morphologic criteria and manual counting. In this study, we describe a new flow cytometry-based assay to enumerate eosinophils present in murine bronchoalveolar lavage fluid (BAL) and lung parenchyma obtained from the normal/non-inflamed respiratory tract, following experimentally-induced allergic pulmonary inflammation, and during experimental infection with respiratory syncytial virus (RSV). By using a murine Siglec-F-specific antibody in combination with antibodies directed to CD45 and CD11c, we demonstrate that eosinophils can be distinguished from other cell types in the BAL fluid and lung parenchyma based upon their distinct CD45 + Siglec-F + and CD11c low/− staining profile. In the BAL fluid, this flow cytometry-based method of eosinophil identification/quantitation yields results comparable to the standard morphology-based method without the potential observer bias or staining artifacts inherent in morphology-based quantitation. Furthermore, this flow cytometry-based method can be directly adapted to enumerate eosinophils infiltrating the inflamed lung parenchyma, thereby obviating the need for quantitative morphometry of tissue sections.

CD4+ cells are required for chronic eosinophilic lung inflammation but not airway remodeling

“…CD4 depleting antibody (GK 1.5) was purified from inhouse hybridoma. GK 1.5 (750 g) or control rat IgG (Millipore) was administered IP on days 30,35,43, and 50 for one experimental protocol (Fig. 1A).…”

“…Siglec-F is a sialic acid-recognizing lectin present on the surface of mouse eosinophils and is upregulated during allergic lung inflammation (40). Other cells, including pulmonary macrophages, may also express Siglec-F but are successfully excluded by costaining with CD11c, with the CD11c-negative population of Siglec-F-positive lung cells identifying eosinophils (35). Lung Siglec-F-positive, CD11c-negative cells were reduced by more than 75% following anti-CD4 administration (Fig.…”

Fractionation of Mature Eosinophils in GATA-Reporter Transgenic Mice

“…For example, when mice are challenged with aerosolized chicken ovalbumin (OVA), the eosinophil population increases in an interleukin (IL)-5-dependent manner in the bone marrow, peripheral blood and in the BALF (Foster et al 1996;Kopf et al 1996). Detection of eosinophils has primarily been based on morphologic criteria, i.e., granulocytes with prominent eosinophil-specific basic granules (Shen et al 2003;Ishizaki et al 2006;Stevens et al 2007). This morphologic identification approach has several disadvantages, including variability in staining procedures, uniformity of inflammatory cell distribution, and reliance on the subjective interpretation of cell morphology in the quantitation of infiltrating cells.…”

“…To date, specific types of monoclonal antibodies combined with strict forward/side scatter gating are used in flow cytometry-based methods to fractionate the cells (Hansel et al 1991;Du et al 2002;Iwasaki et al 2005;Ishizaki et al 2006;Munoz and Leff 2006;Stevens et al 2007;Fukushima et al 2009;Mori et al 2009;Shen et al 2009). However, expression of multiple cell surface markers (e.g.…”

“…However, expression of multiple cell surface markers (e.g. CD16, Gr1,Mac1,etc) that are shared by other cells of hematopoietic origin and are normally present in inflammatory infiltrates, hampers the easy detection of eosinophils using flow cytometry (Hansel et al 1991;Gounni et al 2000;Stevens et al 2007;Fukushima et al 2009). To analyze the physiology and pathology of eosinophils, cytokine-induced differentiation systems of hematopoietic progenitor cells or cell lines are exploited (Clutterbuck and Sanderson 1988;Clutterbuck et al 1989;Caldenhoven et al 1998;Yamaguchi et al 1999;Querfurth et al 2000;Dyer et al 2008;Fukushima et al 2009;Qiu et al 2009).…”