Induction of target cell DNA release by the cytotoxic T lymphocyte granule protease granzyme A.

The rapid breakdown of target cell DNA during CTL-mediated lysis has been difficult to explain by the granule exocytosis model of cytotoxicity. The involvement of CTL granule proteases in this process was strongly suggested by experiments in which CTL were pretreated with the serine protease inhibitor PMSF, in combination with agents that raise the pH of acidic intracellular compartments. While PMSF pretreatment alone had little effect on target lysis or DNA breakdown, the combination of PMSF and NH4Cl or monensin profoundly reduced target cell DNA release, while little effect was observed on target lysis, as measured by 51Cr release. CTL granule extracts cause release of 125I-DNA from detergent-permeabilized cells. This nuclear DNA-releasing (NDR) activity is inhibited by serine esterase inhibitors that also inhibit the granule BLT-esterase activity, and is specifically immunoabsorbed by antibodies to the CTL granule protease granzyme A. The NDR activity comigrates with BLT-esterase activity during subcellular fractionation, solubilization, gel filtration, and aprotinin-Sepharose affinity chromatography. SDS-PAGE analysis of the affinity-purified product indicates a molecular mass of 60,000 daltons under non-reducing conditions, which moves to 30,000 daltons upon reduction, consistent with previously reported behavior of granzyme A. When the purified material was reduced and alkylated, both esterase and NDR activities comigrated at 30,000 daltons upon gel filtration. Although fully lytic concentrations of purified LGL granule cytolysin alone failed to induce target cell DNA release, a combination of purified granzyme A and the cytolysin induces substantial DNA release.

Possible involvement of CTL granule proteases in target cell DNA breakdown.

We have carried out experiments to test whether the granule exocytosis model for lymphocyte cytotoxicity can account for the rapid target DNA breakdown seen during CTL-induced cytotoxicity. Dense granules isolated from cloned mouse CTL and from rat NK tumor cells cause target DNA breakdown during granule cytolysin-mediated lysis of tumor cells, while the purified granule cytolysin caused lysis without DNA breakdown. When target cells are permeabilized with detergent, granule extracts have the ability to release 125I-DNA from nuclei in the absence of detectable cytolysin activity. This activity formed the basis for a nuclear DNA release (NDR) assay; this activity was a property of dense granules of cytotoxic lymphocytes but generally not of other types of lymphoid cells. NDR activity in NK tumor granules had a pH optimum of 7 and was inhibited by micromolar levels of Zn+2, and could be purified away from the granule cytolysin by column chromatography. NDR activity in CTL dense granules could be inactivated by submillimolar concentrations of the protease inhibitors PMSF and DFP (but not soybean trypsin inhibitor or TLCK). In support of the relevance to CTL cytotoxicity of these findings with the NDR assay, pretreatment of CTL with PMSF in the presence of agents raising the intragranular pH inactivated 125I-DNA release from target cells (but not the 51Cr release). These results suggest that a CTL granule component(s), probably a protease, is required for target DNA breakdown.

Biochemical and functional properties of serine esterases in acidic cytoplasmic granules of cytotoxic T lymphocytes.

Percoll gradient fractions of homogenates of murine cloned cytotoxic T lymphocytes (CTL) were analyzed for the trypsin-like enzyme alpha-N-benzyloxy-carbonyl-L-lysinethiobenzyl ester (BLT) esterase recently described in CTL homogenates. Enzymatic activity was found in three areas of the gradient: the dense cytolysin containing granules; a light granule fraction; and a variable amount in the soluble fraction at the top of the gradient. Gel filtration columns showed a major peak of BLT esterase activity eluted at the position of a 60-kDa protein, and an additional, minor BLT esterase peak eluting at about 27 kDa. The separated enzymes were both significantly inhibited by the serine protease inhibitors diisopropylfluorophosphate and phenylmethyl sulfonyl fluoride (PMSF), indicating they are both serine proteases, but showed different patterns of inhibition by a series of inhibitors, suggesting the larger enzyme is not a simple dimer of the smaller. pH activity profiles of both CTL BLT esterases showed an optimum at about pH 8. PMSF inactivation of BLT esterase in detergent extracts of CTL diminished sharply as the pH was dropped below 7. Agents which raise the pH of acidic intracellular compartments were found to markedly enhance the PMSF inactivation of BLT esterase in intact CTL, showing that the granules have a low internal pH. Similarly, [3H]diisopropylfluorophosphate labeling of intact CTL gave four protein bands on non-reduced gels, of which two were labeled threefold more effectively in the presence of chloroquine. In parallel studies of inactivation of CTL lytic activity, PMSF pretreatment caused a 50% reduction of the lytic activity under conditions where greater than 90% of the BLT esterase activity was inactivated. Addition of agents raising the intragranular pH dramatically enhanced the BLT esterase inactivation but did not concomitantly reduce CTL lytic activity. These results indicate that inactivation of lytic function by PMSF is unlikely to be due to its reaction with protease in acidic granules, and suggest that the activity of these enzymes may not be required for cytotoxicity.