Secretory pathways leading to A1AT transcytosis across cultured pulmonary endothelial monolayers.

<p>(A–D) Immunoblot showing effect of inhibition of classical secretory pathway with tunicamycin (A–C; at the indicated doses; 18 h) or brefeldin A (D; 1 µg/mL, 60 min) on intracellular A1AT (A, B, D) and secretion of A1AT (A, C) detected by Western blotting and quantified by densitometry, using vinculin as loading control. Secreted A1AT was measured in equal volumes of supernatants (which were concentrated 25-fold). (E) Pre-inhibition of the classical secretory pathway with tunicamycin (1 h) enhances A1AT (3 h) basolateral secretion, measured by Western blotting of concentrated supernatants, suggesting the utilization of a non-classical secretory pathway. Bands are from the same immunoblot. (F) Alternative secretion of A1AT (100 µg/mL, 2 h) by endothelial cells via microparticles release, as detected by Western blotting of A1AT in endothelial microparticles isolated from supernatants via ultracentrifugation (representative blot of n = 3). (G) Time course of basolateral EMP release from endothelial cells treated with A1AT (100 µg/mL). Bars represent mean+SEM; *p<.05, #p = .07 vs. control; n = 3.</p

A1AT trafficking across cultured pulmonary endothelial and epithelial bilayers.

<p>(A) Co-culture schematic showing pulmonary epithelial cells cultured on the bottom of the transwell membrane and pulmonary endothelial cells cultured on the top. Endothelial cells only were exposed to A1AT. (B) Phalloidin staining (top and epithelial cells) and brightfield microscopy (bottom) showing confluent monolayers of endothelial and epithelial cells seeded on transwell inserts. (C) Immunoblotting of A1AT in cell lysates showing intracellular presence of A1AT in endothelial and epithelial cells and immunoblot of secreted A1AT from concentrated bottom supernatant (representative blots of n = 3). Bands shown are from the same immunoblot. (D). Schematic showing A1AT treatment of the basolateral media or apical surface of NHBE cells differentiated at ALI. (E). Confocal microscopy of NHBE cells differentiated at ALI after 2 h incubation with fluorescently labeled A1AT (green, 20 µM) added to either the basolateral media (i) or apical surface (ii). Only the basolaterally applied A1AT was observed to enter the cells. Arrows indicate ciliated side of the epithelium (cilia stained in white, nuclei in blue). (F). Densitometric quantification of epithelial cell lysates and ASL collected at the indicated times after adding 20 µM of A1AT in either the basolateral (solid line, triangles) or the apical (solid line, circles) compartment from 3 different lung donors measured by Western blotting (mean+SEM; n = 3). Plot (dashed line, squares) showing the relative A1AT present in the ASL after the basolateral application experiment. All fold changes are relative to time 0 before the addition of A1AT. (G) Concentration of A1AT in ASL of ALI cultures after A1AT (20 µM) addition to the basolateral compartment. ASL was collected with 250 µl PBS washes. A1AT quantification was made by customized ELISA and corrected for wash dilution (n = 3 different lung donors). (H) Levels of intracellular and secreted A1AT measured by ELISA (n = 2) in NHBE cells treated with conditioned endothelial media (containing 43.4 nM endothelial-secreted A1AT, 2 h);. n = 1.</p

Polarity of A1AT trafficking across cultured pulmonary endothelial monolayers.

<p>(A) Fraction (%) of FITC-Dextran (Dex) of 20 kDa (light gray bars) or 250 kDa (dark gray bars) or of A1AT (100 µg/mL; black bars) that crossed confluent endothelial cell monolayers grown on 0.4 µm transwells. (B) Immunoblots of intracellular and basolaterally secreted A1AT from endothelial cells cultured on transwell inserts treated with exogenous A1AT (100 µg/mL; for up to 120 min). (C) Intracellular levels of A1AT quantified by densitometry after normalizing to the vinculin loading control. (D) Transcytosed levels of A1AT normalized to .5 ul of concentrated supernatant. Bars represent mean+SEM; *p = .05 vs A1AT 2 h; #p<.05 vs. A1AT 10 min; n = 3.</p

Intravital microscopy of A1AT trafficking across the lung microvascular circulation in the intact mouse.

<p>Selected frame from timelapse video of the pulmonary microcirculation following intravenous injections of rhodamine-labeled rat albumin (red) and airspaces (dark) showing no AF488-A1AT (green) prior to injection (pre-A1AT). Note that following AF488-A1AT intravenous administration there is circulating protein (orange microcirculation) as well as A1AT protein in the airspaces (green punctate signal), without evidence of lung edema (no red extravasation), suggesting active transcytosis from the circulation at 10 min and 50 min post injection.</p

Effect of cigarette smoke extract exposure on A1AT transcytosis across cultured lung endothelial monolayers.

<p>(A) Immunoblot of A1AT from concentrated supernatants showing basolaterally secreted A1AT in endothelial cells co-treated with A1AT (100 µg/mL) and CS or AC extract (2.5%) for up to 120 min (representative blot of n = 3). (B,E) Transcytosed A1AT levels in concentrated supernatants. Bars represent mean+SEM; *p<.05 vs. respective AC; n = 3. (C) Immunoblots of intracellular and basolaterally secreted A1AT in endothelial cells treated with native (Nat) or polymerized (Polym, heated at 60°, 2 h) A1AT (100 µg/mL; Baxter Healthcare). (D) Intracellular levels of A1AT (52 kDa) quantified by densitometry and normalized to the vinculin loading control. n = 2.</p