Evaluation of the substrate specificity of human mast cell tryptase beta Iand demonstration of its importance in bacterial infections of the lung

Human pulmonary mast cells (MCs) express tryptases alpha and betaI, and bot h granule serine proteases are exocytosed during inflammatory events. Recom binant forms of these tryptases were generated for the first time to evalua te their substrate specificities at the biochemical level and then to addre ss their physiologic roles in pulmonary inflammation. Analysis of a tryptas e-specific, phage display peptide library revealed that tryptase betaI pref ers to cleave peptides with 1 or more Pro residues flanked by 2 positively charged residues. Although recombinant tryptase betaI was unable to activat e cultured cells that express different types of protease-activated recepto rs, the numbers of neutrophils increased > 100-fold when enzymatically acti ve tryptase betaI was instilled into the lungs of mice, In contrast, the nu mbers of lymphocytes and eosinophils in the airspaces did not change signif icantly. More important, the tryptase betaI-treated mice exhibited normal a irway responsiveness. Neutrophils did not extravasate into the lungs of try ptase alpha -treated mice, Thus, this is the first study to demonstrate tha t the two nearly identical human MC tryptases are functionally distinct in vivo. When MC-deficient W/W-nu mice were given enzymatically active tryptas e betaI or its inactive zymogen before pulmonary infection with Klebsiella prteumoniae, tryptase betaI-treated W/W-nu mice had fewer viable bacteria i n their lungs relative to zymogen-treated W/W-nu mice, Because neutrophils are required to combat bacterial infections, human tryptase betaI plays a c ritical role in the antibacterial host defenses of the lung by recruiting n eutrophils in a manner that does not alter airway reactivity,