Fig. 7. Adam8 increases leukocyte apoptosis but does not regulate clearance of apoptotic leukocytes by macrophages in the airways of mice with AAI.

In A–C, BALB/c strain WT and Adam8^−/− mice were shamsensitized with PBS or sensitized with a low dose (10 μg) of OVA along with alum by the i.p. route, and then challenged with aerosolized PBS or OVA. BAL leukocytes were isolated 24 h after the last PBS or OVA challenge. BAL leukocytes were permeabilized and immunostained with an antibody to active caspase-3 or with a non-immune control antibody (in A), or loss of mitochondrial membrane potential was measured in intact cells (in B). Intracellular levels of active caspase-3 and the percentage of cells that had lost mitochondrial membrane potential (apoptotic cells) were quantified in all leukocytes (WBC), granulocytes (Granulo), macrophages (Macs), and lymphocytes (Lymphs) by flow cytometry, as described in Methods. Data are mean ± SEM; n = 16–17 mice/group in A and n = 7–9 mice in B. In A, * indicates p = 0.03 and ** indicates p ≤ 0.006. In B, * indicates p = 0.023 and ** p ≤ 0.014. In C, BAL macrophages were isolated from mice 24 h after the last OVA (n = 11–12 mice/group) or PBS (n = 4 mice/group) challenge. Apoptotic cells that had been ingested by BAL macrophages were counted in 300 macrophages per genotype. Data are mean ± SEM apoptotic cells per BAL macrophage. Asterisk indicates p = 0.042 and ** p ≤ 0.017. In D, macrophages isolated from unchallenged BALB/c WT and BALB/c Adam8^−/− mice were incubated for 1 h at 37°C with equal numbers of apoptotic PMNs isolated from unchallenged BALB/c WT or BALB/c Adam8^−/− mice. The mean ± SEM number of apoptotic cells ingested per macrophage was counted in 300 macrophages per group (n = 3 separate experiments).