Increased apoptosis of HIF-2α–deficient inflammatory neutrophils. (A-B) Hif2a^flox/flox;LysMCre^−/− (WT) and littermate Hif2a^flox/flox;LysMCre^+/− (KO) mice were challenged with nebulized LPS (3 mg). BAL was performed at 48 and 72 hours. Neutrophil apoptosis was determined by morphology. Data are mean and SEM for n = 5. (C) Wild-type (WT) C57BL/6 mice (C57) were irradiated (12 fractions of 1Gy), injected with bone marrow from Hif2a^flox/flox;LysMCre^−/− (WT) or Hif2a^flox/flox;LysMCre^+/− (KO) mice, and allowed to reconstitute for 5 weeks before challenge with nebulized LPS. BAL was performed at 48 hours, and neutrophil apoptosis was determined by morphology. Data are mean and SEM for min n = 5. (D-G) Hif2a^flox/flox;LysMCre^−/− (WT) and Hif2a^flox/flox;LysMCre^+/− (KO) mice were challenged with nebulized LPS. At 24 hours, peripheral blood neutrophils isolated by (D) negative magnetic selection and (E) inflammatory neutrophils recovered from BAL were cultured with GEA3162 (GEA) for 9 hours. Apoptosis was determined by morphology. Data are mean and SEM for n = 4. (F-G) Representative cytospin images showing (F) WT and (G) KO inflammatory neutrophils recovered from BAL following 9 hours of culture with 30 µM GEA3162. Original magnification ×1000. (H-I) WT and KO mice were challenged with nebulized LPS (3 mg). BAL was performed at 6 and 24 hours, and expression of catalase (Cat) and superoxide dismutase 2 (Sod2) in BAL cell lysates was determined by Taqman and normalized to Actb. Data are mean and SEM for minute n = 4.