Accumulation of PTEN-null neutrophils in inflamed lungs was enhanced during E coli pneumonia. Mice were intratracheally instilled with 10⁶ CFU of E coli and killed at each indicated time point. (A) Neutrophils in BALF. The total number of cells in the lungs was counted by hemocytometer. Differential cell counts were conducted on cytospin preparations stained with a modified Wright-Giemsa stain (Volu-Sol, Salt Lake City, UT). Neutrophils were recognized by their lobular or segmented nuclei. The percentage of pulmonary neutrophils (PMNs) in the whole population was determined accordingly. Total number of PMNs recruited was calculated as follows: number of PMNs = cell density × volume × % PMN. All data are presented as mean (± SD); n ≥ 4 mice in each group. *P < .05, **P < .01 versus wild type. (B) Staining of histologic lung sections shows emigrated neutrophils and polymerized fibrin in the pulmonary parenchyma. Lungs were fixed with Bouin solution at 23 cm H₂O pressure. Tissues were embedded in paraffin, and 6-μm–thick sections were stained with hematoxylin and eosin (H&E). (C) Emigrated neutrophils in alveolar air spaces were quantified as volume fraction of the alveolar air space using standard point-counting morphometric techniques.⁶³  The relative volumes of the parenchymal regions occupied by emigrated neutrophils were calculated by investigators blinded to the identities of the mice and were expressed as the percentage of the total parenchymal region volume (including both tissue and air space). (D) PtdIns(3,4,5)P₃ signaling is specifically elevated in PTEN-null neutrophils. Neutrophils, primary spleen cells, and brain cells collected from WT and myeloid-specific PTEN KO mice were stimulated with 25 ng/mL MIP-2 and lysed at 0 and 10 minutes. Phosphorylated and total Akt were detected by Western blotting using anti–phospho-Akt (Ser473; 1:1000) and anti-Akt (1:1000) antibodies (Cell Signaling Technology, Beverly, MA), respectively. Densitometry of the blots was performed using the ImageJ software, Gel Analyzer plug-in. Phospho-Akt levels were normalized based on loading (total Akt level). (E) Pulmonary edema formation was quantified as the percentage of edema area in the total parenchymal region using IPlab imaging software. (F) BALF total protein. Protein accumulated in the inflamed lung was measured using a Bio-Rad (Hercules, CA) protein assay kit. The standard curve was constructed using bovine serum albumin (BSA). (G) Rate of mortality due to E coli–induced pneumonia in WT and myeloid-specific PTEN KO mice. Age- and sex-matched WT and PTEN KO mice were intratracheally challenged with 10⁶ live E coli and monitored for 7 days. The Kaplan-Meier and log-rank methods were used to analyze survival rates. *P < .01 versus WT.