Alpha1-Antitrypsin: New insights into the biological role in vitro and in vivo

Sammanfattning: Alpha1-Antitrypsin (AAT), an acute-phase glycoprotein produced predominantly in the liver by hepatocytes, but also by macrophages, pulmonary alveolar cells and intestinal epithelial cells, is a member of the SERPIN superfamily (SERine Protease INhibitors), and is one of the major inhibitors of neutrophil elastase in human plasma. Although AAT plays a critical role in regulating inflammation by inhibiting neutrophil serine proteases, recent studies suggest that AAT exhibits much broader anti-inflammatory activity. We have shown that Prolastin, a preparation of purified, pooled human plasma AAT used for augmentation therapy, inhibits bacterial endotoxin-induced pro-inflammatory responses in vitro and in vivo (Paper 1). We also discovered that two key activities of AAT in vitro, namely inhibition of lipopolysaccharide (LPS)-stimulated tumor necrosis factor alpha (TNFalpha) and enhancement of interleukin-10 (IL-10) in human monocytes, are mediated by an elevation of cAMP and activation of cAMP-dependent protein kinase A (Paper 2). The recognition of LPS is principally mediated by the membrane-bound or soluble form of the glycoprotein CD14 and CD14-associated signal transducer, toll-like receptor 4 (TLR4). We discovered that a longer term incubation (18 h) of monocytes with combined AAT/LPS results in a significant reduction in the expression of both CD14 and TLR4, and inhibition of TNF?, IL-1 and IL-8 expression as compared to LPS alone (Paper 3). Recent studies have also found that AAT is able to inhibit intracellular proteases. Matriptase, a cell surface serine protease involved in the activation of epithelial sodium channels, is one such protease. We found that AAT is a slow, tight-binding inhibitor of the catalytic domain of matriptase with a second order reaction rate constant of 0.31 x 103 M-1 s-1 (Paper 4). Because matriptase is known to be involved in the activation of epithelial sodium channels (ENaC), we performed a series of studies in Xenopus oocytes, injected with human alpha, beta, gamma ENaC cRNAs, in human lung Clara-like (H441) cells and in mice models, in order to investigate whether AAT has any effect on amiloride-sensitive Na+ transport and alveolar fluid clearance. We found that AAT can decrease amiloride-sensitive currents in oocytes and H441 cells, and reduce Na+-dependent alveolar fluid clearance in vivo. We believe that these effects were the result of AAT inactivation of membrane-bound proteases, such as matriptase and prostasin, present on the surface of oocytes and H441 cells (Paper 5). Findings that AAT inactivates the catalytic domain of matriptase in vitro and inhibits epithelial sodium transport both in vitro and in vivo (Paper 4 and 5) support the hypothesis postulate suggestion that the inhibition of matriptase by AAT may offer a pharmacological target for improving mucociliary clearance in both chronic obstructive pulmonary disease and cystic fibrosis. Several studies have shown that AAT inhibits the activity directly of caspase-3, an intracellular cysteine protease which plays an essential role in cell apoptosis. Taken together, recent findings highlight the potential involvement of AAT in several proteolytic pathways and also indicate that it has broader antiprotease activities than previously anticipated. Thus, the biological properties of AAT and their complex interplay with environmental factors appear to extend AAT functional activities well beyond those of protease inhibitor activity. AAT may exhibit anti-inflammatory activities independent of its protease inhibitor function, which suggests the existence of receptor-mediated AAT intracellular internalization, and provides further evidence of a broad protective role of AAT in vivo.