Gr1^highCD11b^highα₃β₁^high neutrophils have pro-inflammatory phenotypes. (A) Total bone marrow (BM) cells from C57BL/6 mice (6 hours after CLP) were stained with Ly6G and CD11b antibodies. Cells were sorted into Ly6G^high and Ly6G^low populations, as indicated, and stained with hematoxylin and eosin after cytospin. Ly6G^high cells demonstrate neutrophil morphology with a multilobed nucleus, and Ly6G^low cells have a monocyte-like appearance. (B) The gating strategy for α₃β₁^high and α₃β₁^low neutrophil populations in mice is shown. The pseudo color plots demonstrate α₃β₁ upregulation on mouse BM neutrophils (gated as Ly6G^highCD11b^high single cells) 6 hours after CLP compared with neutrophils from naive mice. (C) Neutrophils from total BM of endotoxemia-treated mice were gated as Ly6G^highCD11b^high cells and fluorescence-activated cell sorted based on their α₃β₁ expression levels into α₃β₁^high and α₃β₁^low populations, as shown in panel B. Fold changes in Il6 and Il10 gene expression 12 hours after sepsis induction, compared with neutrophils from naive mice, were quantified by reverse transcription polymerase chain reaction. Data are expressed as mean ± SD of 3 separate experiments. *P < .05 (Mann-Whitney test). (D) Fluorescence-activated cell -sorted α₃β₁^high and α₃β₁^low neutrophils isolated from endotoxemia mice 12 hours after sepsis induction were in vitro stimulated with LPS (100 ng/mL) to induce cytokine production. Culture supernatants were collected after 24 hours of stimulation and secreted IL-6 and IL-10 levels were measured by enzyme-linked immunosorbent assay (ELISA). Data are expressed as mean ± SD of 3 separate experiments. *P < .05 (Mann-Whitney test). (E) MPO activity of α₃β₁^high and α₃β₁^low BM neutrophils from CLP and endotoxemia mice was measured using bioluminescence. Sorted cells (5 × 10⁴ cells/well) were stimulated with PMA (1 μM) in the presence of luminol (1 mg/well), and the luminescence intensity was imaged. The graphs of MPO activity kinetics (left) in α₃β₁^high and α₃β₁^low cells and the representative luminescence images (right) are shown. Endotox., endotoxin.