Real-Time Assay of Tryptase Release from Human Umbilical Cord Blood-Derived Mast Cells

Mast cells are the central mediating cells of allergic reactions responding via the high-affinity IgE receptor (FcεRI) through binding of IgE and specific antigen (1). This activation leads to the release of preformed mediators stored in secretory granules (such as histamine, α and β tryptases, and chymase) as well as to the de novo synthesis of leukotrienes and prostaglandins and the production and release of various cytokines and chemokines (1). The monitoring of mediator release has been used in the past as a direct measurement of mast cell activation. In particular, assays to detect histamine have long been the standard used for studies of mast cell function (2–6). In addition, although the release of the mast cell protease tryptase has been used to monitor mast cell activation, those assays describe a multi-step technique in which tryptase levels are determined at a given endpoint after removal of cells (7,8). We report the development of a real-time assay for mast cell tryptase release from small numbers of cells (104 cells/assay point). This assay measures the release of tryptase from cells real-time and does not require manipulation of the cells other than incubation with appropriate stimuli. To propagate human cord blood-derived mast cells (CMBCs), human umbilical cord blood mononuclear progenitors (Poeitic Technologies, Gaithersburg, MD, USA) were placed into culture medium (RPMI/10% FBS; Invitrogen, Carlsbad, CA, USA) in the presence of stem cell factor and IL-6 (R&D Systems, Minneapolis, MN, USA), both at 100 ng/mL, at a density of 1 × 106 cells/mL (modification of Reference 9). One half of the culture medium was changed each week. After five weeks, IL-4 (R&D Systems) was introduced into the medium at 100 ng/mL. Cultures were continued for eight weeks and evaluated by staining with Toluidine blue dye. Cells used for functional assays were a minimum of 90% Toluidine blue positive. Eightto ten-week-old CBMCs were lysed by three rounds of freeze/ thaw, and the lysate was diluted in tryptase assay buffer (100 mM TrisHCl, pH 8.0, 100 mM NaCl) in a final volume of 50 μL and placed in the wells of a 96-well plate (Pierce Chemical, Rockford, IL, USA). An additional 50 μL of buffer alone or buffer containing protease inhibitors (all from Sigma St. Louis, MO, USA) was added to each well. A final 50 μL substrate [tosyl-glypro-lys-p-nitroanilide (10) (Sigma), 25 mM stock in 100% DMSO] in buffer was added to a final concentration of 500 μM, and the plate was immediately read in a plate reader (Molecular Devices, Sunnyvale, CA, USA) at 410 nm in the kinetic mode for 15 min with data collection every 30 s. Enzyme activity was determined as the rate of change of A due to cleavage of the substrate (velocity) and reported as mOD/min. The proteolytic activity present in varying numbers of cells against a serine protease substrate (tosyl-gly-pro-lys-p-nitro anilide) is shown in Figure 1 along with the activity of 5 nM rh-β-tryptase (Promega, Madison, WI, USA). Proteolytic activity above background could be detected from as few as 100 cells and was linear with cell number through the range tested (r2 = 0.99). This protease was inhibited by serine protease inhibitors (antipain, BABIM, benzamidine, leupeptin, and TLCK) and lactoferrin (which inhibits tryptase by competing for heparin) (data not shown) (11). Cysteine protease inhibitors (aprotinin, pepstatin, and TPCK) were not effective (data not shown). This pattern of inhibition suggests that the proteolytic activity being measured is indeed that of tryptase. To assay the real-time release of tryptase, CBMCs were sensitized for 24 h or longer in the presence of varying concentrations of human IgE (Biodesign, Saco, ME, USA). Before use, the cells were pelleted for 4 min at 1000× g and resuspended in RPMI at 2 × 105 cells/mL. Fifty microliters of cells (approximately 10 000 cells/well) were added to each well of a 96-well plate. Either 50 μL of buffer or buffer containing compounds to be tested was added to each well, and the plate was Benchmarks