Inhibitors of Tryptase as Mast Cell-Stabilizing Agents in the Human Airways: Effects of Tryptase and Other Agonists of Proteinase-Activated Receptor 2 on Histamine Release Shaoheng He, Akhmed Aslam, Marianna D. A. Gac ¸ a, Yongsong He, Mark G. Buckley, Morley D. Hollenberg, and Andrew F. Walls Immunopharmacology Group, University of Southampton, Southampton General Hospital, Southampton, United Kingdom (S.H., A.A., M.D.A.G., Y.H., M.G.B., A.F.W.); Allergy and Inflammation Research Institute, Shantou University Medical College, Shantou, People’s Republic of China (S.H.); and Departments of Pharmacology and Therapeutics, and Medicine, University of Calgary Faculty of Medicine, Calgary, Canada (M.D.H.) Received October 8, 2003; accepted December 22, 2003 ABSTRACT Tryptase, the major secretory product of human mast cells, is emerging as a new target for therapeutic intervention in allergic airways disease. We have investigated the ability of tryptase and inhibitors of tryptase to modulate histamine release from human lung mast cells and have examined the potential contribution of proteinase-activated receptor 2 (PAR2). The tryptase inhibitor APC366 [ N-(1-hydroxy-2- naphthoyl)-L-arginyl-L-prolinamide hydrochloride] was highly effective at inhibiting histamine release stimulated by anti- IgE antibody or calcium ionophore from enzymatically dis- persed human lung cells. A concentration of APC366 as low as 10 M was able to inhibit anti-IgE-dependent histamine release by some 50%. Addition of leupeptin or the tryptic substrate N-benzoyl-D,L-arginine-p-nitroanilide also inhib- ited IgE-dependent histamine release. Purified tryptase in the presence of heparin stimulated a small but significant release of histamine from lung cells, suggesting that tryptase may provide an amplification signal from activated cells that may be susceptible to proteinase inhibitors. Trypsin was also able to induce histamine release apparently by a catalytic mechanism. Moreover, pretreatment of cells with metabolic inhibitors or with pertussis toxin reduced responses, indicat- ing a noncytoxic pertussis toxin-sensitive G protein- mediated signaling process. Addition to cells of the PAR2 agonists SLIGKV-NH 2 or tc-LIGRLO-NH 2 or appropriate control peptides were without effect on histamine release, and PAR2 was not detected by immunohistochemistry in tissue mast cells. The potent actions of tryptase inhibitors as mast cell-stabilizing agents could be of value in the treat- ment of allergic inflammation of the respiratory tract, possi- bly by targeting the non-PAR2-mediated actions of tryptase. Mast cell activation is prominent in allergic airways dis- ease. The mast cell has been implicated as an initial effector cell and also as a key cellular participant in later processes of acute inflammation and in tissue remodeling (Church et al., 1997). Several drugs used to treat allergic inflammation of the lower airways (such as salmeterol and salbutamol) or upper airways (terfenadine and cetirizine) possess mast cell- stabilizing activity (Naclerio et al., 1990; Butchers et al., 1991; Okayama and Church, 1992, 1994), and more recently the potential for mast cells to play a critical role in allergic inflammation has been highlighted by reports that omali- zumab, a humanized antibody specific for IgE, may be effi- cacious in the treatment of asthma and other allergic condi- tions (D’Amato, 2003). The major secretory product of human mast cells is the serine proteinase tryptase (Walls, 2000). This enzyme is emerging as a major mediator of allergic disease and as a promising target for therapeutic interven- tion. Tryptase inhibitors have been reported to be particu- larly potent as mast cell-stabilizing compounds, though their effects on mast cells of the lung have not been examined. The ability of tryptase to stimulate mast cell degranulation first became apparent in studies involving transfer of this proteinase to laboratory animals. Microvascular leakage pro- voked by injection of human tryptase into guinea pig skin was found to be blocked by antihistamine pretreatment of the Financial support from the Sir Jules Thorne Charitable Trust and Novartis, UK, is gratefully acknowledged. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. DOI: 10.1124/jpet.103.061291. ABBREVIATIONS: APC366, N-(1-hydroxy-2-naphthoyl)-L-arginyl-L-prolinamide hydrochloride; PAR2, proteinase-activated receptor 2; BAPNA, N-benzoyl-D,L-arginine-p-nitroanilide; HBSS, HEPES-balanced salt solution; NA, nitroanilide; MES, 2-(N-morpholino)ethane-sulfonic acid; MEM, minimum Eagle’s medium; FCS, fetal calf serum. 0022-3565/04/3091-119 –126$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 309, No. 1 Copyright © 2004 by The American Society for Pharmacology and Experimental Therapeutics 61291/1136359 JPET 309:119–126, 2004 Printed in U.S.A. 119