Alpha-1 antitrypsin (AT) is a major anti-elastase and protects the lung from uncontrolled elastolysis. AT is highly susceptible to oxidation in vivo. We investigated the role of Ox-AT in the inflammatory response.
Lung epithelial (A549 and NHBE) cells were exposed to 25% CSE. Ox-AT, TNF-α, IL-6, IL-8, MCP-1, NF-kB and AP-1 were assessed by ELISA or RT-PCR. Anti-Ox-AT mAb (3F4, 10µg/ml) and N-acetylcysteine (NAC, 10–3M) were used to probe the effect of Ox-AT.
CSE (compared to PBS) significantly induced TNF-α (440.4±76.8pg/ml vs. 17.5±2.6, p<0.001) at 24h and induced IL-6 at 72h (354.7±38.4pg/ml vs. 17.6±2, p<0.001). CSE induced TNF-α mRNA at 0.5h (p<0.001) and IL-6 mRNA at 3h (p<0.001). CSE (compared to PBS) activated NF-κB (OD at 405nm, 1.325 vs. 0.315, p<0.001) and AP-1 (OD at 405nm, 0.988 vs. 0.296, p<0.001) at 0.5h. At 24h CSE (compared to PBS) resulted in significant level of Ox-AT (mean±SEM, 1372.8±162.8ng/ml vs. undetectable, p<0.001) and induced IL-8 (1415.7±92.5pg/ml vs. 57.2±10, p<0.001) and MCP-1 (15683±713pg/ml vs. 2208±157, p<0.001). At 24h Ox-AT (compared to PBS) significantly induced IL-8 (1168±9pg/ml vs. 110±8, p=0.008) and MCP-1 (14500±424pg/ml vs. 4225±470, p=0.005) in A549 cells. NAC inhibited Ox-AT, TNF-α, IL-6, IL-8, MCP-1, NF-κB and AP-1 (p<0.001 for all). 3F4 selectively inhibited Ox-AT, IL-8, MCP-1, NF-κB and AP-1 (p<0.001 for all). These findings were confirmed with NHBE cells.
In conclusion, Ox-AT generated in the airway interacts directly with epithelial cells to release MCP-1 and IL-8, so enhancing lung inflammation. This mechanism could potentially contribute to the abnormal inflammatory response seen in COPD independent of CSE. Anti-oxidant treatment inhibited both CSE and Ox-AT induced inflammatory response further supporting a role for these agents in COPD.
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