Figure 5

The effect of Oncostatin M on Z-AAT expression in ex-vivo cultured BECs. ( A ) Real-time PCR analysis for AAT mRNA in ex vivo cultured BECs from patients homozygous for M-AAT or Z-AAT. Cells from four patients homozygous for Z-AAT and five patients homozygous for M-AAT were incubated in the absence (OsM-) or presence (OsM+) of Oncostatin M (50 ng/ml) for 24 hours. After treatment cells from M-AAT and Z-AAT patients were pooled and AAT mRNA levels were measured. The fold increase in expression of Oncostatin M-treated versus control cells is indicated. Results are expressed as mean ± SEM (n = 3–4). *P < 0.05 Student’s t-test. ( B ) ELISA for total AAT protein in cell lysates and ( C ) cell culture media of ex vivo cultured BECs from four Z-AAT and five M-AAT homozygous patients. Cells were incubated in the absence (OsM-) or presence (OsM+) of Oncostatin M (50 ng/ml) for 24 hours and the intracellular AAT accumulation and secretion were measured for lysates of pooled cells and 10x concentrated cell culture media, respectively. Results are expressed as mean ± SEM (n = 3–4). *P < 0.05 ANOVA followed by Bonferroni t-test, Oncostatin M-treated versus untreated cells; +P < 0.05 ANOVA followed by Bonferroni t-test, M-AAT– versus Z-AAT–expressing cells. SDS-PAGE and Western blot analysis using an antibody to detect total AAT was carried out on the cell culture media of ex vivo cultured BECs. Cells from five patients homozygous for Z-AAT ( D ) and five patients homozygous for M-AAT ( E ) were cultured in the absence (OsM-) or presence (OsM+) of 50 ng/ml of Oncostatin M. At 24 hours after treatment cell media were harvested and concentrated 10x. Typical fluorograms from three independent experiments, which gave superimposable results, are shown.