Figure 1
Lipopolysaccharide (LPS) administration increases the frequency of CD11b+Gr1+F4/80+ cells in the lung. Lung cells were isolated by enzymatic digestion of lung tissue. The cells were stained with anti-CD11b and anti-Gr1 monoclonal antibodies and were analyzed by flow cytometry. The forward vs. side light scatter pattern revealed a distinct non-lymphocytic population of cells in which CD11b-expressing and Gr1-expressing cells were concentrated (data not shown). This population of cells was gated upon for subsequent analyses. (a) Expression of CD11b and Gr1 on lung cells measured by flow cytometry with or without LPS treatment of mice (upper panels). The numbers of CD11b+ cells expressing high (hi) or intermediate (int) levels of Gr1 were determined in response to increasing concentrations of LPS per treatment (lower panel). (b) Two populations of CD11b+ cells from the lungs of LPS-treated mice were sorted based upon expression of intermediate (Gr1int) or high (Gr1hi) levels of Gr1. The purity of Grint and Grhi populations was >95%. (c) Levels of myleoperoxidase (MPO) in the supernatants of Grint and Grhi populations were detected using an enzyme-linked immunosorbent assay (ELISA) kit for MPO. *P<0.05. (d) Expression of F4/80, CD11c, CD124, CD115, CD62L, CD34, and CD206 on the LPS-induced CD11b+Grint cells by flow cytometry. Cursors are placed based on staining with isotype control antibodies. (e) CD11b+ cells isolated from the lungs of LPS-treated mice were sorted based upon expression of Gr1 and F4/80 (upper panels). The purity of CD11b+Gr1intF4/80+ and CD11b+Gr1hiF4/80− cells was >94%. Cytospin slides were prepared and stained using the three-step stain kit (Richard-Allan Scientific, Waltham, MA). CD11b+/Gr1hi/F4/80− cells were identified as neutrophils with characteristic lobular-shaped nuclei. The CD11b+Gr1int/F4/80+ cells seemed to be a heterogeneous population with the majority containing ring-shaped nuclei (inset) (lower panels). The data are representative of at least three independent experiments.
