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Abstract
The homeostatic lung protective effects of alpha-1 antitrypsin (A1AT) may require the transport of circulating proteinase inhibitor across an intact lung endothelial barrier. We hypothesized that uninjured pulmonary endothelial cells transport A1AT to lung epithelial cells. Purified human A1AT was rapidly taken up by confluent primary rat pulmonary endothelial cell monolayers, was secreted extracellularly, both apically and basolaterally, and was taken up by adjacent rat lung epithelial cells co-cultured on polarized transwells. Similarly, polarized primary human lung epithelial cells took up basolaterally-, but not apically-supplied A1AT, followed by apical secretion. Evidence of A1AT transcytosis across lung microcirculation was confirmed in vivo by two-photon intravital microscopy in mice. Time-lapse confocal microscopy indicated that A1AT co-localized with Golgi in the endothelium whilst inhibition of the classical secretory pathway with tunicamycin significantly increased intracellular retention of A1AT. However, inhibition of Golgi secretion promoted non-classical A1AT secretion, associated with microparticle release. Polymerized A1AT or A1AT supplied to endothelial cells exposed to soluble cigarette smoke extract had decreased transcytosis. These results suggest previously unappreciated pathways of A1AT bidirectional uptake and secretion from lung endothelial cells towards the alveolar epithelium and airspaces. A1AT trafficking may determine its functional bioavailablity in the lung, which could be impaired in individuals exposed to smoking or in those with A1AT deficiency.
Highlights
Alpha-1 antitrypsin (A1AT) is a glycoprotein serine protease inhibitor that is produced and secreted from hepatocytes into the systemic circulation
We and others have shown that A1AT is taken up primarily by clathrin, and via caveolae-dependent endocytosis, both of which have been implicated in transcytosis of molecules across the endothelium [3,7]
Previous work from our laboratory demonstrated that pulmonary endothelial cells, which do not synthesize A1AT, internalize the serpin through an active mechanism of endocytosis [3]
Summary
Alpha-1 antitrypsin (A1AT) is a glycoprotein serine protease inhibitor that is produced and secreted from hepatocytes into the systemic circulation. A1AT undergoes N-linked glycosylation and is released through the classical secretory pathway, via processing through the ER and the Golgi apparatus [1,2]. Certain intracellular proteins can be handled through non-classical secretory pathways, via lysosomes, exosomes formed from multiple vesicular bodies, direct transport from the cytosol to the extracellular space, or by plasma membrane blebbing and vesicle shedding [5]. It is not known which if any of these mechanisms handles A1AT trafficking or transcytosis across the capillaryalveolar membrane. No studies of A1AT transcytosis have been described, to the best of our knowledge
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