S63 F alpha-1 antitrypsin polymerises and promotes an exaggerated inflammatory response

Abstract

Alpha-1 antitrypsin (AT) is the most important anti-elastase in the lung. Z-AT (342Glu>Lys) polymerises within the hepatocyte resulting in severe plasma deficiency and is the commonest genetic reason for the development of COPD. The F α1-antitrypsin variant (223Arg>Cys) has been associated with mild plasma deficiency, and when found in association with the Z allele linked to emphysema and liver cirrhosis. We investigated the properties of F-AT in a cell-model. Human F-AT cDNA was generated by site-directed mutagenesis and overexpressed into hepatocytes (HepG2 cells). Supernatants, lysates and inclusion bodies were assessed for total AT. F-AT cells were assessed for polymeric-AT, PERK, NF-kB, AP-1, TNF-α and IL-6 by ELISA, immunoblot or RT-PCR in comparison with normal (non-polymerising) M-AT and Z AT (polymerising control). F-AT cells had no cell cytotoxicity or apoptosis upto 72h. At 24h (unless stated) F-AT secretion was slightly lower, but comparable to M-AT secretion (1479.3 ± 142pg/ml vs. 1745.5 ± 102.3pg/ml, P = 0.413) Secreted F-AT had significantly reduced elastase inhibitory capacity compared to M-AT (0.972 ± 0.069(OD at 405nm) vs. 0.449 ± 0.085, P < 0.001). F-AT formed insoluble aggregates of polymeric-AT (375 ± 32pg/ml vs. undetectable), upregulated PERK mRNA (at 3h) and significantly increased NF-κB (at 16h), AP-1, TNF-α (34.5 ± 5.3pg/ml vs. 10.53 ± 3.2, P = 0.023) and IL-6 (at 48h) (132.3 ± 20.8pg/ml vs. 23.9 ± 16) (P = 0.006). All of which were inhibited by treatment with an inhibitor of polymerisation (P < 0.001 for all). In comparison to Z-AT, F-AT secretion was greater (P < 0.001) but had significantly reduced anti-elastase activity (P = 0.032) and lower ER accumulation of polymeric-F-AT (P < 0.001). In the F-AT cell, PERK mRNA was upregulated at 3h compared to 0.5h in Z-AT. Although elevated compared to M-AT cells, F-AT cells had lower NF-κB activity (P < 0.001), TNF-a production (P = 0.046) and IL-6 production (P = 0.012) compared to Z-AT. In conclusion, F-AT secretion was comparable to M-AT. However, secreted F-AT was defective as an anti-elastase. F-AT was found to polymerise and aggregate in inclusion bodies. ER accumulation of F-AT activated the ER overload response; PERK-dependant-NF-κB mediated inflammatory response, greater than M-AT but to a lesser degree than Z-AT. This data indicate that FZ phenotype may be at risk for liver and lung disease.