Evaluation of the Substrate Specificity of Human Mast Cell Tryptase βI and Demonstration of Its Importance in Bacterial Infections of the Lung

Abstract

Human pulmonary mast cells (MCs) express tryptases ␣ and ␤I, and both granule serine proteases are exocytosed during inflammatory events. Recombinant forms of these tryptases were generated for the first time to evaluate their substrate specificities at the biochemical level and then to address their physiologic roles in pulmonary inflammation. Analysis of a tryptase-specific, phage display peptide library revealed that tryptase ␤I prefers to cleave peptides with 1 or more Pro residues flanked by 2 positively charged residues. Although recombinant tryptase ␤I was unable to activate cultured cells that express different types of protease-activated receptors, the numbers of neutrophils increased >100-fold when enzymatically active tryptase ␤I was instilled into the lungs of mice. In contrast, the numbers of lymphocytes and eosinophils in the airspaces did not change significantly. More important, the tryptase ␤I-treated mice exhibited normal airway responsiveness. Neutrophils did not extravasate into the lungs of tryptase ␣-treated mice. Thus, this is the first study to demonstrate that the two nearly identical human MC tryptases are functionally distinct in vivo. When MCdeficient W/W v mice were given enzymatically active tryptase ␤I or its inactive zymogen before pulmonary infection with Klebsiella pneumoniae, tryptase ␤I-treated W/W v mice had fewer viable bacteria in their lungs relative to zymogen-treated W/W v mice. Because neutrophils are required to combat bacterial infections, human tryptase ␤I plays a critical role in the antibacterial host defenses of the lung by recruiting neutrophils in a manner that does not alter airway reactivity.

Does β-Hexosaminidase Function Only as a Degranulation Indicator in Mast Cells? The Primary Role of β-Hexosaminidase in Mast Cell Granules

“…Other enzymes, such as proteases, are known to exert microbicidal effects through cytokine production (42)(43)(44). Our findings in this study imply that proteases may not be the primary antimicrobial component in mast cells.…”

“…For instance, mast cells sense the type 1 fimbriae of E. coli through surface-expressed CD48, resulting in release of the potent neutrophil chemoattractant TNF-a in a process that is pivotal for elimination of the bacteria (42). Additionally, mast cell tryptase b I reportedly plays a central role in the host defenses of the lung by recruiting neutrophils (43), and cecal ligation and puncture models demonstrated that mast cell chymase is essential for survival in cases of septic peritonitis (44). We revealed in the present study, to our knowledge for the first time, that although reconstitution of W/W v mice with BL/6-BMMCs reduced the lethality of S. epidermidis infection, grafting of hexb 2/2 -BMMCs did not reduce mortality.…”

Protease-Activated Receptor 2 Mediates Eosinophil Infiltration and Hyperreactivity in Allergic Inflammation of the Airway

“…Moreover, the hyperalgesic effects of tryptase in the skin are diminished in PAR2ko mice (33). A recent report has questioned the capacity of tryptase to activate PAR2 (44). The explanation of this discrepancy is unknown.…”

The mouse mast cell–restricted tetramer‐forming tryptases mouse mast cell protease 6 and mouse mast cell protease 7 are critical mediators in inflammatory arthritis

“…Thus, the mechanism(s) by which mMCP-6 and mMCP-7 control the severity of the arthritis in mBSA/IL-1␤-treated C57BL/6 mice at the molecular level remain to be determined. Mouse MCP-7 is able to induce the recruitment of eosinophils into the peritoneal cavity (42) and neutrophils and eosinophils in the conjunctiva by activating an IL-6-dependent pathway that eventually leads to the generation of other cytokines and chemokines (Miyazaki D, et al: unpublished observations). Mouse MCP-6 and its human homolog hTryptase-␤1 induce neutrophil recruitment when injected into the mouse lung or peritoneal cavity (41,46).…”