A SENSITIVE COLORIMETRIC ASSAY FOR THE RELEASE OF TRYPTASE FROM HUMANLUNG MAST-CELLS IN-VITRO

Studies of human lung mast cells have usually focused on histamine rel ease, although the enzymes stored in the granules may also contribute to the pathophysiology of the allergic response. We have used a simple colorimetric assay for tryptase to follow the release of proteolytic enzymes from human lung mast cells in vitro. Either human lung mast ce ll supernatants or authentic mast cell tryptase were mixed with benzoy l-DL-arginine-p-nitroaniline and incubated for up to 72 h at 37 degree s C. The appearance of nitroaniline was then measured at 410 nm in an ELISA plate reader. Cells were sonicated in H2O to measure total trypt ase and histamine. Human lung mast cells contained the equivalent of 1 1.2 +/- 0.7 pg tryptase per cell and 3.2 +/- 0.3 pg of histamine. The amount of tryptase measured colorimetrically correlated with the level of tryptase measured by radioimmunoassay (Pharmacia), r = 0.92, P < 0 .01. The inhibition profile of the proteolytic enzyme measured by the cleavage of BAPNA, was found to be identical to that of authentic lung mast cell tryptase. Over 90% of the maximum tryptase release was comp lete within 15 min whilst histamine release occurred within 5 min. In cells stimulated with 10 mu g/ml anti-IgE we found a strong correlatio n between the release of tryptase and histamine, r = 0.95, P < 0.005. Finally, investigations with various pharmacological agents have suppo rted our initial hypothesis that tryptase would mimic histamine releas e and provide an alternative marker for mast cell activation. In summa ry, we have utilised a simple enzymic assay as an indicator of human l ung mast cell degranulation. In washed lung mast cells this assay appe ars be specific for granule tryptase and release of this activity into the supernatants of challenged cells correlates well with the presenc e of histamine. This assay offers several advantages over current meth ods of measuring mediator release from human lung mast cells in vitro and should provide an inexpensive and sensitive technique for followin g mast cell degranulation.