A sensitive colorimetric assay for the release of tryptase from human lung mast cells in vitro.
Studies of human lung mast cells have usually focused on histamine release, 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 cell supernatants or authentic mast cell tryptase were mixed with benzoyl-DL-arginine-p-nitroaniline and incubated for up to 72 h at 37 degrees C. The appearance of nitroaniline was then measured at 410 nm in an ELISA plate reader. Cells were sonicated in H2O to measure total tryptase and histamine. Human lung mast cells contained the equivalent of 11.2 +/- 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 complete within 15 min whilst histamine release occurred within 5 min. In cells stimulated with 10 micrograms/ml anti-IgE we found a strong correlation between the release of tryptase and histamine, r = 0.95, P < 0.005. Finally, investigations with various pharmacological agents have supported our initial hypothesis that tryptase would mimic histamine release and provide an alternative marker for mast cell activation. In summary, we have utilised a simple enzymic assay as an indicator of human lung mast cell degranulation. In washed lung mast cells this assay appears be specific for granule tryptase and release of this activity into the supernatants of challenged cells correlates well with the presence of histamine. This assay offers several advantages over current methods of measuring mediator release from human lung mast cells in vitro and should provide an inexpensive and sensitive technique for following mast cell degranulation.
Lavens
S E
SE
Department of Physiology and Pharmacology, University of Southampton, Bassett Crescent East, UK.
Proud
D
D
Warner
J A
JA
D016227:Benzoquinones; D001586:Benzoylarginine Nitroanilide; D015550:Cell Degranulation / Q000187:drug effects; D053818:Chymases; D002934:Cinnamates / Q000494:pharmacology; D003124:Colorimetry / Q000379:methods*; D003594:Cytoplasmic Granules / Q000201:enzymology; D019833:Genistein; D006636:Histamine Release; D006801:Humans; D007158:Immunologic Techniques; D066298:In Vitro Techniques; D007529:Isoflavones / Q000494:pharmacology; D047029:Lactams, Macrocyclic; D008168:Lung / Q000166:cytology / Q000201:enzymology* / Q000502:physiology; D008407:Mast Cells / Q000187:drug effects / Q000201:enzymology* / Q000502:physiology; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D011809:Quinones / Q000494:pharmacology; D017828:Rifabutin / Q000031:analogs & derivatives; D012680:Sensitivity and Specificity; D012697:Serine Endopeptidases / Q000032:analysis* / Q000378:metabolism*; D014361:Trypsin Inhibitors / Q000494:pharmacology; D053802:Tryptases
D016227:Benzoquinones; D002934:Cinnamates; D007529:Isoflavones; D047029:Lactams, Macrocyclic; D011809:Quinones; D014361:Trypsin Inhibitors; D017828:Rifabutin; C067472:methyl 2,5-dihydroxycinnamate; C020754:herbimycin; D001586:Benzoylarginine Nitroanilide; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D012697:Serine Endopeptidases; C083294:chymase 2; D053818:Chymases; D053802:Tryptases