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.
Using monoclonal antibodies to T and B lymphocytes, to natural killer cells, and to HLA-DR antigen, we characterized the lymphocyte population within the epithelial and lamina propria regions in control intestine and colon, and in grossly involved and in grossly uninvolved intestine and colon of patients with active inflammatory bowel disease. There were significantly more intraepithelial T cells in control ileum than in control colon. In comparison to control, there was a heterogeneity of alterations in intraepithelial and lamina propria T lymphocyte subsets (T11+, T8+, T4+) in inflammatory bowel disease. B lymphocytes were not detected within the lamina propria, except when found in and adjacent to lymphoid aggregates. Leu 7+ cells were uncommon in the lamina propria of control ileum and colon and in diseased tissues. The majority of intraepithelial lymphocytes did not express HLA-DR. Epithelial cells of control colon did not express HLA-DR while epithelial cells of control ileal tissues and of diseased colonic and ileal specimens expressed HLA-DR antigen. Only small numbers of lamina propria T cells expressed HLA-DR in both control and disease tissues. There was intense expression of HLA-DR by monocytes and modest expression of HLA-DR by capillary and lymphatic endothelial cells. The induction of HLA-DR expression by diseased colonic epithelium and the observation that lymphatic endothelium expresses HLA-DR are new observations, and we established that Leu 7+ cells are present in very small numbers in both normal and diseased intestine and colon.
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.
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