Severe deficiency of the major anti-elastase α1-antitrypsin (AT) due to the Z (Glu342Lys) variant is the commonest genetic reason for the development of COPD. Cigarette smoke (CS) accelerates decline in lung function in Z-AT homozygotes. We investigated whether Z-AT is associated with an exaggerated inflammatory response compared to normal AT (M-AT).
Lung epithelial (A549 and NHBE) cells transfected with human M-AT or Z-AT (M-AT/Z-AT cells) were exposed to 12.5% CSE generated from 1R3F cigarettes. Supernatants, lysates and inclusion bodies were assessed for total AT to confirm a successful cell-model system. Supernatant was assessed for TNF-α, IL-6, IL-8 and MCP-1, oxidised pZ-AT (Ox-pZ-AT), NF-kB and AP-1 by ELISA, immunoblot or RT-PCR. N-acetylcysteine (NAC,10–3M) was used to probe the effect of oxidants.
At 24h CSE in Z-AT (CSE-Z-AT) compared to CSE-M-AT (unless stated) significantly induced TNF-α (212±20.71pg/ml vs. 37.1±2.7), IL-6 (421.4±20.8pg/ml vs. 159.3±12.1), IL-8 (8763±497pg/ml vs. 2593±450) and MCP-1 (23564±1852pg/ml vs. 5329±706), p<0.001 for all. CSE-Z-AT had significantly induced mRNA for TNF-α, IL-6, IL-8 and MCP-1 at 0.5h (p<0.001 for all). Development of Ox-pZ-AT were exclusively detected in CSE-Z-AT inclusion (3246±433ng/ml vs. undetectable, p<0.001). CSE-Z-AT had significantly activated NF-κB (p<0.001) and AP-1 (p=0.001) at 0.5h. In CSE-Z-AT treatment with NAC significantly inhibited TNF-α, IL-6, IL-8, MCP-1, NF-κB, AP-1 and Ox-pZ-AT formation (p<0.001 for all). These findings were confirmed on NHBE cells.
In conclusion, following CS exposure Z-AT cells had significantly elevated inflammatory mediators compared to M-AT cells, which was inhibited by NAC. We propose that during CS exposed lung inflammation Z-AT monomer undergoes oxidation to form oxidised polymers thereby further reducing the level of protective monomeric AT, which predisposes to increased lung inflammation.
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