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1-Protease Inhibitor at the Surface of Human Polymorphonuclear Neutrophils1
* University François Rabelais, Institut National de la Santé et de la Recherche Médicale Unité 618 Protéases et Vectorisation Pulmonaires, and Institut Fédératif de Recherche 135 Imagerie Fonctionnelle;
Institut National de la Santé et de la Recherche Médicale Equipé 211 Nutrition et Cancer, Tours, France; and
Universidade Federal, Departamento de Biofísica, Escola Paulista de Medicina, São Paulo, Brazil
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 
1-protease inhibitor (1-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 1-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 1-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 1-Pi. HNE was not released from the cell surface by oxidized, inactive 1-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 1-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.
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  S. Attucci, A. Gauthier, B. Korkmaz, P. Delepine, M. F.-D. Martino, F. Saudubray, P. Diot, and F. Gauthier EPI-hNE4, a Proteolysis-Resistant Inhibitor of Human Neutrophil Elastase and Potential Anti-Inflammatory Drug for Treating Cystic Fibrosis J. Pharmacol. Exp. Ther., August 1, 2006; 318(2): 803 - 809. [Abstract] [Full Text] [PDF]
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