Inhibition of Neutrophil Elastase by α1-Protease Inhibitor at the Surface of Human Polymorphonuclear Neutrophils

Abstract

The uncontrolled proteolytic activity in lung secretions during lung inflammatory diseases might be due to the resistance of membrane-bound proteases to inhibition. We have used a new fluorogenic neutrophil elastase substrate to measure the activity of free and membrane-bound human neutrophil elastase (HNE) in the presence of α1-protease inhibitor (α1-Pi), the main physiological inhibitor of neutrophil serine proteases in lung secretions. Fixed and unfixed neutrophils bore the same amounts of active HNE at their surface. However, the HNE bound to the surface of unfixed neutrophils was fully inhibited by stoichiometric amounts of α1-Pi, unlike that of fixed neutrophils. The rate of inhibition of HNE bound to the surface of unfixed neutrophils was the same as that of free HNE. In the presence of α1-Pi, membrane-bound elastase is almost entirely removed from the unfixed neutrophil membrane to form soluble irreversible complexes. This was confirmed by flow cytometry using an anti-HNE mAb. HNE activity rapidly reappeared at the surface of HNE-depleted cells when they were triggered with the calcium ionophore A23187, and this activity was fully inhibited by stoichiometric amounts of α1-Pi. HNE was not released from the cell surface by oxidized, inactive α1-Pi, showing that active inhibitor is required to interact with active protease from the cell surface. We conclude that HNE activity at the surface of human neutrophils is fully controlled by α1-Pi when the cells are in suspension. Pericellular proteolysis could be limited to zones of contact between neutrophils and subjacent protease substrates where natural inhibitors cannot penetrate.

EPI-hNE4, a Proteolysis-Resistant Inhibitor of Human Neutrophil Elastase and Potential Anti-Inflammatory Drug for Treating Cystic Fibrosis

“…Human neutrophils from healthy volunteers, who gave their informed consent, were purified essentially as described previously (Korkmaz et al, 2005). The neutrophils recovered at the bottom of the Lymphoprep gradient were suspended in 200 l of PBS.…”

“…EPI-hNE4 and ␣ 1 -PI (control) were treated with N-chlorosuccinimide (Korkmaz et al, 2005). The inhibitory capacities of the treated and native forms of the inhibitors were assayed after incubation with equimolar amounts (10 Ϫ8 M) of HNE for 10 min at 37°C.…”

“…The fate of mHNE-EPI-hNE4 complexes was analyzed by incubating a 50-fold excess of neutrophils (10 Ϫ7 M mHNE) with a stoichiometric amount of EPI-hNE4 (Korkmaz et al, 2005) for 5 min at room temperature. The suspension was centrifuged, and the supernatant was analyzed by SDS-polyacrylamide gel electrophoresis under nonreducing conditions with 0.02% (w/v) SDS in the sample buffer.…”

“…Western blots of cell supernatants obtained after centrifugation of the inhibitor-containing neutrophil suspension (Fig. 3B) show that EPI-hNE4 formed soluble complexes, suggesting that EPIhNE4 removes the bound protease from the cell surface, as does ␣ 1 -PI (Korkmaz et al, 2005).…”

Neutrophil Elastase, Proteinase 3, and Cathepsin G as Therapeutic Targets in Human Diseases

“…These two proteases are released from neutrophil cell surfaces by high concentrations of salt (Owen et al, 1995b(Owen et al, , 1997Korkmaz et al, 2005a) and after treatment with chondroitinase ABC and heparinase (Campbell and Owen, 2007). Membrane PR3 is not solubilized by high salt concentrations, which means that its membrane association is not charge dependant (Witko-Sarsat et al, 1999a;Korkmaz et al, 2009).…”