Lysis by RNK-16 cytotoxic lymphocyte granules. Rate assays and conditions to study control of cytolysis.
Dense subcellular granules of cytolytic lymphocytes can mediate rapid lysis of erythrocytes or nucleated cells. The granules contain several different proteases and proteoglycans that regulate cytolysis. We describe a rate assay that we have already used to demonstrate the requirement for serine proteases in granule-mediated lysis. In this assay, 51Cr-labeled erythrocytes are lysed by limiting concentrations of granules from RNK-16 tumor cells. Cytolysis is initiated by the addition of calcium (1 mM final concentration) and stopped at 0.5-1 min intervals by acidification to pH 6.0. The effects of the granule protein concentration, temperature, the concentration of erythrocytes, pH, and the concentration of calcium on the rate of lysis are reported. A preliminary mathematical approach is described and suggested as a means to differentiate 'lag' or activation times from the initial burst of lysis. With this rate assay, we have found four classes of protease inhibitors that block granule-mediated lysis (Hudig et al. (1987) Biochem. Biophys. Res. Commun. 149, 882). The utility of the rate assays is underscored by the observation that reversible protease inhibitors only showed rates of cytolysis whereas irreversible protease inhibitors stopped cytolysis completely. Rate assays are essential for future analyses of the complex physiological regulation of granule-mediated cytotoxicity by proteases, endogenous protease inhibitors and proteoglycans.
Hudig
D
D
Cell and Molecular Biology Program, School of Medicine, University of Nevada, Reno 89557-0046.
Callewaert
D M
DM
Redelman
D
D
Allison
N J
NJ
Krump
M
M
Tardieu
B
B
D000818:Animals; D002118:Calcium; D002460:Cell Line; D003594:Cytoplasmic Granules / Q000276:immunology*; D003601:Cytotoxicity Tests, Immunologic / Q000379:methods; D004306:Dose-Response Relationship, Immunologic; D004906:Erythrocyte Count; D006863:Hydrogen-Ion Concentration; D007694:Killer Cells, Natural / Q000276:immunology*; D008433:Mathematics; D011817:Rabbits; D051381:Rats; D013696:Temperature
D002118:Calcium