Selective regulation of apoptosis: the cytotoxic lymphocyte serpin proteinase inhibitor 9 protects against granzyme B-mediated apoptosis without perturbing the Fas cell death pathway

Abstract

Cytotoxic lymphocytes (CLs) induce caspase activation and apoptosis of target cells either through Fas activation or through release of granule cytotoxins, particularly granzyme B. CLs themselves resist granule-mediated apoptosis but are eventually cleared via Fas-mediated apoptosis. Here we show that the CL cytoplasmic serpin proteinase inhibitor 9 (PI-9) can protect transfected cells against apoptosis induced by either purified granzyme B and perforin or intact CLs. A PI-9 P1 mutant (Glu to Asp) is a 100-fold-less-efficient granzyme B inhibitor that no longer protects against granzyme B-mediated apoptosis. PI-9 is highly specific for granzyme B because it does not inhibit eight of the nine caspases tested or protect transfected cells against Fas-mediated apoptosis. In contrast, the P1(Asp) mutant is an effective caspase inhibitor that protects against Fas-mediated apoptosis. We propose that PI-9 shields CLs specifically against misdirected granzyme B to prevent autolysis or fratricide, but it does not interfere with homeostatic deletion via Fas-mediated apoptosis.

FIG. 1

Interaction of PI-9 and the P₁(Asp) mutant (PI9E340D) with granzyme B (graB). Equimolar amounts of PI-9 (8 ng) or PI9E340D (8 ng) and granzyme B (5 ng) in 20 mM Tris (pH 7.4)–0.15 M NaCl were incubated for 30 min at 37°C, followed by reduction, boiling, and electrophoresis on an SDS–10% polyacrylamide gel. Protein was transferred to a nitrocellulose membrane and immunoblotted with a rabbit antiserum against PI-9 diluted 1:2,000 (A). The membrane was then stripped and reprobed with a monoclonal antibody (2C5) in ascites fluid against granzyme B diluted 1:2,000 (B). Bound antibody was detected by chemiluminescence. Dots indicate the positions of the 38- and 25-kDa cleavage products.

FIG. 2

Intracellular PI-9 protects FDC-P1 cells from purified granzyme B and perforin. (A) Efficient inhibition of granzyme B-mediated apoptosis by PI-9 requires Glu at the P₁ position and mobility of the inhibitory loop. FDC-P1 clones expressing equivalent levels of PI-9, the P₁(Asp) mutant (E340D), or the hinge mutant (T327R) were exposed to granzyme B (GrB) and perforin (P) and compared to vector-transfected cells (FDC-neo). Panels show FACS profiles of DNA fragmentation by TUNEL analysis. Cells to the right of the cursor are positive for DNA fragmentation and are shown as a percentage of the whole population. (B) Increased levels of PI-9 correlate with increased protection against apoptosis triggered by granzyme B. Panels to the left show intracellular FACS analysis for PI-9 expression in the FDC-neo control cells, a low-PI-9-expressing clone (FDC-PI9/8), and a higher-PI-9-expressing clone (FDC-PI9/6b). Panels to the right are FACS profiles of the same cells showing DNA fragmentation by TUNEL analysis, following exposure to granzyme B and perforin. All results are representative of three independent experiments.

FIG. 3

Expression level and activity of PI-9 in MCF-7 transfectants compared to those in endogenous PI-9 producers. MCF-7 Neo cells were transfected with the marker plasmid only. Cells were lysed, and 50 μg of protein with (+) or without (−) granzyme B (5 ng) in 20 mM Tris (pH 7.4)–0.15 M NaCl was incubated for 30 min at 37°C. Granzyme B was not added to the YT cell extracts. Samples were reduced and subjected to electrophoresis on an SDS–10% polyacrylamide gel. Immunoblotting with anti-PI-9 antiserum was carried out as described in the legend to Fig. 1.

FIG. 4

PI-9 protects MCF-7 cells from killing by Lp cytotoxic cells. (A) Phase-contrast (upper panel) and propidium iodide fluorescence (lower panel) microscopy of MCF-7 cells engaged by Lp cells. The cytotoxic (Lp) cells are dark and round, with very little visible cytoplasm, and have small nuclei without visible nucleoli. MCF-7 cells (M) are larger, with prominent cytoplasm and large nuclei containing nucleoli. Several stages of apoptosis of MCF-7 cells induced by attached Lp cells are shown in this field. Cells in early apoptosis show darkened cytoplasm, reduction of cell area, and retraction of pseudopodia. Cells in later stages show nuclear crenation and condensation, cytoplasmic vesicularization, and become refractile. (B) Phase-contrast microscopy of monolayers of untransfected MCF-7 cells and PI-9 transfectants exposed to Lp cells and estimation of the numbers of cells undergoing Lp-induced apoptosis. Only cells having one or two Lp killers attached were counted and assessed for apoptotic changes in a blind assay. Untransfected MCF-7 cells are indicated by solid bars, PI-9 transfectants (clone 16-11) are indicated by open bars, and corrected values to account for negative cells in the latter population are indicated by grey bars.

FIG. 4

PI-9 protects MCF-7 cells from killing by Lp cytotoxic cells. (A) Phase-contrast (upper panel) and propidium iodide fluorescence (lower panel) microscopy of MCF-7 cells engaged by Lp cells. The cytotoxic (Lp) cells are dark and round, with very little visible cytoplasm, and have small nuclei without visible nucleoli. MCF-7 cells (M) are larger, with prominent cytoplasm and large nuclei containing nucleoli. Several stages of apoptosis of MCF-7 cells induced by attached Lp cells are shown in this field. Cells in early apoptosis show darkened cytoplasm, reduction of cell area, and retraction of pseudopodia. Cells in later stages show nuclear crenation and condensation, cytoplasmic vesicularization, and become refractile. (B) Phase-contrast microscopy of monolayers of untransfected MCF-7 cells and PI-9 transfectants exposed to Lp cells and estimation of the numbers of cells undergoing Lp-induced apoptosis. Only cells having one or two Lp killers attached were counted and assessed for apoptotic changes in a blind assay. Untransfected MCF-7 cells are indicated by solid bars, PI-9 transfectants (clone 16-11) are indicated by open bars, and corrected values to account for negative cells in the latter population are indicated by grey bars.

FIG. 5

PI-9 protects MCF-7 cells from killing by LAK cells. MCF-7 clones expressing PI-9 or cells expressing the marker gene (Neo) only were exposed to LAK cells cultured for 5 or 14 days. Killing was monitored in triplicate by ⁵¹Cr release after 4 h at different effector/target ratios (E:T). The results are representative of three independent experiments performed with two independent clones. The inset panel shows granzyme B (graB) levels in the day 5 and day 14 cultured LAK cells. Cells were lysed, and 50 μg of protein was separated by SDS–10% polyacrylamide gel electrophoresis followed by immunoblotting, as described in the legend to Fig. 1. Solid bars, LAK cells cultured for 5 days; stippled bars, LAK cells cultured for 14 days.

FIG. 6

PI-9 does not protect cells from Fas-induced apoptosis. Fas-sensitive Jurkat cells were transfected with expression vectors encoding a neomycin-selectable marker and either PI-9, PI9E340D, or CrmA. G418-resistant pools were then assessed for sensitivity to apoptosis induced by cross-linking Fas with the monoclonal immunoglobulin M antibody CH-11. Cell viability was quantitated by MTT assay. The results shown represent four independent experiments performed in triplicate. The inset panel shows an immunoblot with anti-PI-9 antiserum on total protein (50 μg) extracted from cells expressing the marker plasmid (mock), PI-9, or PI9E340D. rPI-9, purified recombinant PI-9.