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Originally published In Press as doi:10.1074/jbc.M313108200 on February 25, 2004

J. Biol. Chem., Vol. 279, Issue 19, 20200-20210, May 7, 2004
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Granzyme-mediated Cytotoxicity Does Not Involve the Mannose 6-Phosphate Receptors on Target Cells*

Ralf Dressel{ddagger}§, Srikumar M. Raja¶, Stefan Höning||, Tim Seidler**, Christopher J. Froelich¶, Kurt von Figura||, and Eberhard Günther{ddagger}

From the {ddagger}Division of Immunogenetics, University of Göttingen, Heinrich-Düker-Weg 12, 37073 Göttingen, Germany, the Evanston Healthcare Research Institute, Feinberg School of Medicine, Northwestern University, Evanston, Illinois 60201, the ||Division of Biochemistry II, University of Göttingen, Heinrich-Düker-Weg 12, 37073 Göttingen, Germany, and the **Division of Cardiology and Pneumology, University of Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Germany

Received for publication, December 2, 2003 , and in revised form, February 19, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cytotoxic T lymphocytes (CTL) and natural killer cells secrete granzymes to kill infected or transformed cells. The mannose 6-phosphate receptor (Mpr) 300 on target cells has been reported to function as receptor for secreted granzyme B. Using lymphoblasts and mouse embryonal fibroblast lines from Mpr300 and Mpr46 knockout mice, we show here that both receptors are not essential for CTL-induced apoptosis. Similarly, cells exposed to either monomeric granzyme B or granzyme B-serglycin complexes readily internalize the granzyme and undergo apoptosis in the absence of Mpr300 and Mpr46. Further, no colocalization of granzyme B and Mpr300 could be observed in target cells after internalization. In conclusion, these results strongly argue against an Mpr300- or Mpr46-dependent pathway of granzyme-mediated killing and provide new insight in the internalization of monomeric and complexed granzyme B.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cytotoxic T lymphocytes (CTL)1 and natural killer cells use two different mechanisms of killing infected or transformed cells by direct contact: the engagement of cell surface death receptors on target cells, such as CD95 that activates the cellular death caspases (1), and the granule-exocytosis pathway that leads to the secretion of preformed toxins stored in specialized secretory lysosomes of cytotoxic lymphocytes (2–4). Essential components of cytotoxic granules are granzyme B (GrB) and perforin (PFN). PFN-formed pores in the plasma membrane have long been considered as the entry sites for granzymes into the cytoplasm of target cells (5). In contrast to this model, GrB has been shown to bind to target cells in a specific, saturable manner in the absence of PFN and to remain in endocytotic vesicles after internalization without exerting a cytotoxic activity (6). Therefore, it is assumed that GrB is internalized by a specific receptor, and a sublytic PFN concentration is then required for its release from the endocytic compartment into the cytosol of target cells (6). This hypothesis is strongly supported by the finding that endosomolytic agents other than PFN, such as non-replicating adenovirus (AD) and the bacterial toxins listeriolysin O and streptolysin O are able to substitute for PFN in GrB-mediated apoptosis (6, 7).

Recently, it has been shown that granzyme molecules in the cytotoxic granules are non-covalently bound to the ~250 kDa proteoglycan serglycin (SG), and are secreted in this macromolecular form and delivered by PFN to target cells through a process that does not require the formation of plasma membrane pores (8, 9). The finding that granzymes are secreted as macromolecular complexes with SG rather than as isolated molecules has important implications for the understanding of granule-mediated apoptosis, because GrB cell surface binding, internalization, intracellular trafficking, as well as targeting and processing of putative substrates have to be reconsidered (8, 9).

The mannose 6-phosphate receptor (Mpr) 300 has been reported to be the cell surface receptor for GrB and to be essential for CTL-mediated apoptosis of target cells and the rejection of allogeneic cells in vivo (10). Mpr300, besides the Mpr of 46 kDa, is known to be essential for the delivery of newly synthesized lysosomal enzymes from the trans-Golgi network to endosomes. Mpr300 also binds and endocytoses insulin-like growth factor II (Igf2) (11). Interestingly in this context, the same receptor is involved in sorting newly synthesized GrB molecules to the cytotoxic granules of lymphocytes (12). However, the role of Mpr300 in GrB-mediated killing is controversial, because an additional, but Mpr300-independent pathway for GrB-induced cell death, has been described (13).

The results presented here clearly establish that apoptosis induced by CTL and mediated by the granule-exocytosis killing pathway is not dependent on Mpr300 or Mpr46 expression in target cells. This conclusion is based on the analysis of genetically defined target cells that are derived from Mpr300 and, in addition, Mpr46 knockout mice in lieu of previously used (10, 13) selected lines lacking the Mpr300. Furthermore, internalization of monomeric as well as complexed GrB and their capacity to induce apoptosis remain unimpeded in Mpr300- and Mpr46-deficient targets.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—All chemicals, unless specified, were purchased from Sigma (Taufkirchen, Germany) or Merck (Darmstadt, Germany). Human GrB and PFN were prepared from cultures of YT cells as previously described (14, 15). Briefly, after cavitation of YT cells, cytotoxic granules were enriched by differential centrifugation and solubilized in buffers suitable for isolation of GrB and PFN, respectively, by cation exchange and hydrophobic interaction chromatography. The isolated GrB is fully glycosylated and phosphorylated as determined by the reduction in Mr from 32 to 27 following exposure to endoglycosidase H. For the labeled granzyme preparations used in this study, ~70% of the protease retain functionality as determined by cleavage of the IETD-pNA substrate. Human SG was purified as described previously (16). GrBAlexa 488 was fabricated as described by the manufacturer (Molecular Probes, Eugene, OR) and either used in monomeric form or complexed to SG (GrBAlexa 488-SG) (16).

Mice—Mice were bred in the central animal facility of the Medical Faculty, University of Göttingen. Animal experiments were approved by local authorities. As a source for Mpr300-deficient lymphoblasts, we used Mpr300-/-/Igf2-/-/Mpr46+/- mice that carried a mixed C57BL/6 (H2b) x 129/Sv (H2bc) background (17). The lethal effect of Mpr300 deficiency in these mice is avoided by an additional knockout of Igf2. Mice that are triple deficient for Mpr300, Mpr46, and Igf2 (obtained by intercrossing Mpr300-/-/Igf2-/-/Mpr46+/- mice, Ref. 17) were used for some experiments. OT-I mice, transgenic for a V{alpha}2V{beta}5 T cell receptor (TCR) that recognizes the H2-Kb-restricted ovalbumin-derived peptide SIINFEKL (18) served as donors for peptide-specific CTL. The OT-I mice used carry a mixed C57BL/6 x 129/Sv background. C3H/HeN (H2k), BALB/c (H2d), and FVB/N (H2q) mice were used to raise alloreactive CTL from spleen cells. From C57BL/6, 129/Sv, and (129/Sv x C57BL/6)F1 mouse lymphocytes were obtained to serve as target cells after stimulation with concanavalin A (Con A).

Cells, Cell Culture, and Generation of CTL—The mouse embryonal fibroblast (MEF) lines (Table I) derived from Mpr knockout or control mice have been described earlier (19, 20). Their genetic background is of mixed C57BL/6 and 129/Sv origin. RMA cells (H2b) served as highly CTL susceptible controls. All cell lines were maintained in NaHCO3-buffered Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS) (Biochrom, Berlin, Germany) in Petri dishes for tissue culture (Sarstedt, Nümbrecht, Germany) at 37 °C in a 10% CO2 atmosphere. Lymphocytes were obtained from spleens using a Tenbroeck homogenizer. Lymphoblasts to be used as target cells in cytotoxicity experiments were generated from spleen cells by 4 days of mitogenic stimulation with 5 µg/ml Con A and were then separated by density gradient centrifugation on Ficoll-Hypaque (Biochrom). To generate alloreactive CTL, mice of the inbred strains C3H/HeN (H2k), BALB/c (H2d), and FVB/N (H2q) were injected intraperitoneally with 30 x 106 spleen cells from C57BL/6 mice (H2b). Ten days after immunization spleen cells were restimulated in round-bottomed microtiter plates (Nunc, Wiesbaden, Germany) with C57BL/6 spleen cells. For this purpose 0.75 x 106 responder cells were cocultured with 0.75 x 106-irradiated (30 Gray) stimulator cells in 200 µl of NaHCO3-buffered DMEM, supplemented with 10% FCS, 10-5 M 2-mercaptoethanol, and 50 µl of supernatant from Con A-stimulated lymphocytes. After 4 days of mixed lymphocyte culture, vital mononuclear cells were separated by density gradient centrifugation on Ficoll-Hypaque and used as alloreactive CTL for cytotoxicity assays. To obtain peptide-specific CTL, spleen cells from naive OT-I mice were stimulated in vitro with irradiated C57BL/6 spleen cells in the presence of 1 nM SIINFEKL (Ovalbumin 257–264; Bachem Biochemica, Heidelberg, Germany) under conditions described above for alloreactive CTL.


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  		TABLE I
Mpr status of the MEF lines The MEF lines have been described previously (19, 20). At several time points throughout experimentation for this study, Mpr300 and Mpr46 expression has been controlled and confirmed by flow cytometry or immunofluorescence using specific antibodies (see "Experimental Procedures").

 
51Cr Release Assay—Target cells were labeled by incubating 1 x 106 cells in 350 µl of HEPES-buffered DMEM containing 50 µl of FCS and 50 µCi of Na251CrO4 (ICN Biomedicals, Eschwege, Germany) for 1 h at 37 °C and washed three times with HEPES-buffered DMEM. Effector cells were added to 104 51Cr-labeled target cells in triplicate at ratios of 100:1 to 1.5:1 for alloreactive CTL and 10:1 to 0.6:1 for OT-I-derived peptide-specific CTL in 200 µl of HEPES-buffered DMEM, 10% FCS per well of round-bottomed microtiter plates. Spontaneous release was determined by incubation of target cells in the absence of CTL. When OT-I effector cells were used, 0.5 µg/ml SIINFEKL peptide was added. To determine the calcium dependence of killing, 2 mM EGTA and 4 mM MgCl2 were added (21). The microtiter plates were centrifuged for 5 min at 40 x g and incubated at 37 °C for 4 h before being centrifuged again, and supernatant and sediment were separately taken to determine radioactivity in each well using a Wallac MicroBeta Trilux counter (PerkinElmer Life Sciences, Köln, Germany). Percentage of specific lysis was calculated by subtracting percent spontaneous 51Cr release.

[3H]Thymidine Release Assay—Target cells were labeled by adding 5 µCi/ml [methyl-3H]thymidine (Amersham Biosciences, Freiburg, Germany) for 20 h to cell cultures in the logarithmic growth phase. Afterward, the cells were washed three times with HEPES-buffered DMEM, and effector cells were added to target cells in a final volume of 100 µl of medium as described above for 51Cr release assays. The pan-caspase inhibitor Z-VAD-FMK (at a final concentration of 40 µM) and the caspase-3-specific inhibitor Ac-AAVALLPAVLLALLAPDEVD-CHO (at a final concentration of 20 µM) were purchased from Alexis Biochemicals (Grünberg, Germany) and were added to certain tests in order to determine caspase dependence of apoptosis. The solvent dimethyl sulfoxide (Me2SO) alone was added to the appropriate controls. After incubation at 37 °C for 4 h, the samples were manipulated as described (10, 22). Briefly, the cells were lysed with an equal volume (100 µl) of 1% Triton X-100 in 100 mM Tris HCl, 50 mM EDTA, transferred to 1.5-ml Eppendorf tubes, vortexed, and centrifuged for 10 min at 4 °C at 15,000 x g to pellet intact nuclei. For analysis of total DNA labeling, the respective samples (designated as "totals") were lysed with an equal volume of 2% SDS in 0.1 N NaOH and vigorously vortexed. Afterward 50 µl of supernatant or 50 µl of the total were removed to scintillation plates, mixed with 200 µl of scintillation mixture (PerkinElmer Life Sciences), and counted on a Wallac MicroBeta Trilux counter. The specific 3H release was calculated as described (10).

Chromogenic GrB Assay—To determine GrB release from CTL 105 target cells were incubated in HEPES-buffered phenol red-free DMEM with OT-I-derived CTL as described above for 51Cr release assays in triplicate at ratios of 1:1, 0.5:1, and 0:1. After incubation for 4 h at 37 °C, the supernatant was harvested. The GrB activity in these samples was determined as described (23) by hydrolysis of the paranitroanilide substrate Ac-IEPD-pNA (Alexis Biochemicals). Briefly, 50 µl of supernatant were incubated at 37 °C with 200 µM substrate in a final volume of 100 µl of reaction buffer (50 mM HEPES pH 7.5, 10% (w/v) sucrose, 0.05% (w/v) CHAPS, and 5 mM dithiothreitol) in flat-bottomed 96-well plates. Released chromogenic paranitroanilide was measured as absorbance at 405 nm on a MR580 Microelisa Auto Reader (Dynatech, Denkendorf, Germany) after 4 h.

Flow Cytometry—Flow cytometry was performed on a FACScan flow cytometer with CellQuest software (BD Biosciences, Heidelberg, Germany). For determining cell surface expression of major histocompatibility complex (MHC) class I molecules on MEF lines or lymphoblasts anti-H2-Kb (clone CTKb, mouse IgG2a, PE-labeled), anti-H2-Db (clone CTDb, mouse IgG2a, PE-labeled) and, as isotype control, mouse IgG2a (PE-labeled, code MG2a04) monoclonal antibody (mAb) (Caltag Laboratories, Hamburg, Germany) were used. To assess the effect of exogenous SIINFEKL peptide on H2-Kb cell surface density, cells were incubated for 4 h (the duration of a 51Cr or 3H release assay) at 37 °C in HEPES-buffered DMEM, 10% FCS with 0.5 µg/ml peptide. Then 5 x 105 cells were washed twice in 5-ml polystyrene tubes (BD Biosciences) with phosphate-buffered saline (PBS) (Biochrom) before resuspension in 100 µl of PBS containing 1 µg of the respective mAb. After incubation for 30 min at 4 °C, the cells were washed twice with PBS and analyzed. Expression of green fluorescent protein (GFP) in transfected cells (see below) was determined directly after washing the cells twice with PBS.

Cell death was determined by staining with 5 µg/ml propidium iodide (PI) in PBS at 4 °C for 15 min before flow cytometry. To measure GrB or GrB-SG-induced apoptosis, the proportion of cells appearing in the sub-G1 peak of DNA histograms was analyzed as described (24). To measure CTL-induced apoptosis by sub-G1 peak determination, the target cells had been labeled before with the fluorescent dye CM-DiI (Molecular Probes) according to the manufacturer`s instructions. Briefly, 3 x 106 cells were resuspended in 3 ml of HEPES-buffered DMEM containing 15 µl of the cell labeling solution, incubated for 15 min at 37 °C and then washed three times with HEPES-buffered DMEM. OT-I-derived CTL were added to 105 CM-Dil-labeled target cells in 5-ml polystyrene tubes at ratios of 10:1, 5:1, and 1:1 or were omitted (ratio 0:1). One of two duplicates for each ratio was supplemented with 0.5 µg/ml SIINFEKL peptide before incubation at 37 °C for 4 h in 1 ml of DMEM, 10% FCS. Afterward the cells were washed with PBS, resuspended in 0.05% trypsin, 1 mM EDTA, PBS to obtain a single cell suspension, and then fixed in 2% paraformaldehyde, 0.2% Triton-X 100, PBS for 10 min. After washing with PBS the cells were stained with 0.1 µg/ml 7-aminoactinomycin D (7AAD) and subjected to flow cytometry. The percentage of CM-Dil-labeled target cells appearing in the sub-G1 peak was determined.

To determine the internalization of GrB cells were grown in 24-well plates and exposed to the Alexa 488-labeled GrB or GrB-SG in DMEM, 1% bovine serum albumin (BSA) at the concentration of 1 µg/ml for 60 min at 37 °C. Afterward the cells were harvested with 0.05% trypsin, 1 mM EDTA, PBS and washed with PBS, 1% BSA before PI staining and flow cytometry.

To determine expression of Mpr300 and Mpr46, previously described (25) rabbit antibodies (Ab) specific for Mpr300 or the cytoplasmic tail of Mpr46 were used. The cells were fixed in 1% paraformaldehyde, PBS for 10 min at 20 °C and permeabilized with 0.25% saponin, PBS before incubation with the 1:100 diluted anti-Mpr46 or anti-Mpr300 Ab and subsequently with Cy2-conjugated goat anti-rabbit IgG secondary Ab (Jackson Laboratories, Dianova, Hamburg, Germany). In certain experiments, cells were exposed to anti-Mpr300 Ab first for 1 h at 37 °C to allow for its internalization together with Mpr300, followed by fixation, permeabilization, and incubation with Cy2-conjugated goat anti-rabbit IgG secondary Ab as described above.

Immunoblot—To determine uptake of GrB by immunoblot 1 x 105 cells were incubated with GrB or GrB-SG in DMEM, 1% BSA at the indicated concentrations for 60 min at 37 °C. Afterward the cells were washed three times with 0.05% trypsin, 1 mM EDTA, PBS. Then the cells were resuspended in 20 µl of sample buffer (0.0625 M Tris-HCl, pH 6.75, containing 2% SDS, 5% 2-mercaptoethanol, 10% glycerol, 0.001% bromphenol blue) and incubated at 100 °C for 5 min. The supernatant obtained after centrifugation (10,000 x g for 5 min at 4 °C) was separated by SDS-PAGE. Proteins were transferred to nitrocellulose (Schleicher and Schüll, Dassel, Germany) before staining with anti-GrB mAb (clone B18.1, mouse IgG1, Alexis Biochemicals) and anti-Hsc70 mAb (clone 1B5, rat IgG2a, StressGen, Biomol, Hamburg, Germany) used as loading control at dilutions of 1:2000 in PBS, 0.05% Tween 20. Subsequently, blots were incubated with goat anti-mouse IgG Ab (Jackson Laboratories) or goat anti-rat IgG + IgM Ab (Jackson Laboratories) and peroxidase-conjugated rabbit anti-goat IgG Ab (Jackson Laboratories) at a dilution of 1:10,000. Blots were analyzed using an ECL chemiluminescent Western blotting detection system (Amersham Biosciences) and a CCD camera (Fuji LAS-1000plus, Raytest, Straubenhardt, Germany) with AIDA software supplied by the manufacturer.

Immunofluorescence—MEF lines were grown on cover slips for 1 day before incubation with fluorochrome-labeled GrB or GrB-SG complexes in DMEM, 1% BSA for 30 min at 4 °C or 60 min at 37 °C. After, cells were fixed with 3% paraformaldehyde, PBS. For colocalization experiments cells were permeabilized (0.1% Triton X-100, PBS or 0.1% saponin, PBS) and incubated with 1:100 diluted rabbit anti-Mpr300 (25), anti-Mpr46 (25), anti-EEA1 Ab (BD Transduction Laboratories, Heidelberg, Germany), anti-Lamp1 (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) or anti-transferrin receptor mAb (Zymed Laboratories, Zytomed GmbH, Berlin, Germany), which were then visualized using either Cy3 or Alexa 633-conjugated secondary Ab (Dianova). Texas Red-labeled transferrin (Molecular Probes) was used for endocytosis experiments. The samples were mounted in fluorescent mounting medium (DakoCytomation, Hamburg, Germany) and analyzed with a confocal laser-scanning microscope (Leica TCS SP2 AOBS, Leica Microsystems, Mannheim, Germany).

Delivery of GrB and GrB-SG to Induce Cell Death—PFN was used for GrB delivery as described (8). In some experiments type 5 AD subtype delta L327 containing the GFP gene under the control of the immediate early cytomegalovirus promoter was used to deliver GrB into the cytosol of target cells. The construct lacks a XbaI fragment of 1.89 kb that covers almost all of the E3 region from 28,593 to 30,471 (78.5–84.3 map units; the nucleotide positions are based on the AD5 wild-type sequence) and nucleotides 358–3,332 of the E1 region (map units 1.4–9.4). The virus was amplified in HEK293 cells (26) and purified by cesium chloride ultracentrifugation followed by dialysis against a sucrose buffer (27). Plaque purification was carried out to minimize wildtype AD contamination, which was tested by PCR with primers specific for the E1 region. Biological activity on HEK293 cells was 1011 plaque forming units/ml supernatant. To insure equivalent delivery of the GrB and GrB-SG complexes by AD, the multiplicity of infection (MOI)/cell had been adjusted in initial experiments for each fibroblast line such that 24 h later about 80% cells were found to express GFP as determined by fluorescent microscopy. Target cells were cultured in 24-well plates. For the test, culture medium was replaced by DMEM, 1% BSA, and GrB or GrB-SG complexes were added before the cells were infected with AD at a MOI of 100–500. Cell death and apoptosis were determined after incubation for 5 h at 37 °C. Transfection was assessed in simultaneously infected cells 24 h later by GFP expression using fluorescence microscopy and flow cytometry.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Lysis of Lymphoblast Target Cells by Alloreactive and Peptide-specific CTL—The Mpr300 has been reported not only to function as GrB receptor on target cells but also to be essential for the rejection of allogeneic cells (10). To analyze the role of Mpr300 for CTL killing, we used as target cells Con A-stimulated lymphoblasts derived from Mpr300 knockout mice. Allorejective CTL were raised in C3H/HeN (H2k), BALB/c (H2d), and FVB/N (H2q) mice by immunization and in vitro restimulation with spleen cells derived from C57BL/6 (H2b) mice. The 51Cr release assay was used as a standard test to determine cell lysis compared with subsequent apoptosis assays. CTL-mediated lysis of Mpr300-/- lymphoblasts was comparable to lymphoblasts derived from Mpr300+/+ C57BL/6, 129/Sv, and (C57BL/6 x 129/Sv)F1 mice (Fig. 1A). Lymphoblasts from Mpr300-/-/Mpr46-/- double knockout mice were also lysed indicating that both Mpr are dispensable on target cells for an effective CTL killing. Each of these results is representative of at least three independent experiments using alloreactive CTL derived from different mouse strains (further data not shown). Inhibition of the calcium-dependent granule-exocytosis killing pathway by EGTA reduced lysis of all target cells reproducibly although not completely (Fig. 1B), indicating that alloreactive CTL do use the granule-exocytosis pathway but also other mechanisms to kill.



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  		FIG. 1.
Lysis potential of lymphoblast target cells by alloreactive CTL does not depend on Mpr300 and Mpr46 function as determined by 51Cr release assay. A, Con A-stimulated lymphoblasts (day 4), derived from Mpr300-/- or Mpr300-/-/Mpr46-/- double knockout mice that have a mixed 129/Sv x C57BL/6 background, were used as target cells for alloreactive CTL raised in C3H/HeN mice. Wild-type control lymphoblasts were obtained from C57BL/6, 129/Sv, and (129/Sv x C57BL/6)F1 mice. Mean of triplicates and S.D. of specific lysis obtained at various effector:target ratios in one representative out of three individual experiments are shown. B, in the same experiment that is shown in A, the granule-exocytosis pathway of killing was inhibited by EGTA. Mean of triplicates and S.D. are shown. The same range of inhibition of lysis by EGTA was observed in two additional experiments.

 
To analyze killing that is almost completely dependent on the granule-exocytosis pathway, peptide-specific CTL were used. They were derived from TCR-transgenic OT-I mice, which express a TCR that recognizes the ovalbumin peptide SIINFEKL in a H2-Kb-restricted manner (18). RMA cells (Fig. 2A) were always included as positive controls to ensure that OT-I-derived CTL possessed cytotoxic activity, and EGTA treatment confirmed calcium dependence of killing (Fig. 2, A–D). Peptide-pulsed lymphoblast target cells derived from wild-type and Mpr300-/- knockout mice were lysed equally well regardless of Mpr300 status. A representative experiment is shown for Mpr300+/+ (Fig. 2B) and Mpr300-/- lymphoblasts (Fig. 2C), and the specific peptide-dependent lysis obtained in six independent experiments is summarized in Fig. 2D. Lysis of lymphoblasts from C57BL/6, 129/Sv, and (C57BL/6 x 129/Sv)F1 mice was not different (data not shown) excluding genetic background effects on the susceptibility of the target cells to CTL. MHC class I expression (H2-Kb and H2-Db) showed no difference between Mpr300-/- and Mpr300+/+ lymphoblasts (data not shown).



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  		FIG. 2.
Lysis and apoptosis of lymphoblasts by peptide-specific CTL does not depend on Mpr300 of target cells. A and E, RMA cells that served as highly CTL susceptible controls were pulsed with the SIINFEKL peptide (0.5 µg/ml) and exposed to CTL derived from TCR transgenic OT-I mice. The mean of specific lysis (51Cr release) and S.D. as well as the mean of specific apoptosis ([3H]thymidine release) and S.D. at different effector:target ratios (10:1 to 0.6:1) in the presence or absence of the SIINFEKL peptide measured by individual 4-h assays are shown. To confirm granule-exocytosis dependence of killing, EGTA was added to the test. B and F, lymphoblasts at day 4 after Con A-stimulation derived from Mpr300+/+ (C57BL/6 x 129/Sv)F1 mice were used as target cells in similar experiments as shown in A and E. C and G, Mpr300-/- lymphoblasts at day 4 after Con A-stimulation were used as target cells in parallel to the experiments shown in B and F. D and H, summary of six (D) or four (H) individual experiments presenting the mean of specific 51Cr (D) or 3H (H) release and S.D. of lymphoblasts from Mpr300-/- and Mpr300+/+ (C57BL/6 x 129/Sv)F1 mice at different effector:target ratios (10:1, 5:1, 2.5:1) in the presence or absence of the SIINFEKL peptide and EGTA.

 
CTL-induced DNA Fragmentation of Lymphoblast Target Cells—GrB-/- effector cells have been reported to show no general defects in target cell killing as determined in 51Cr release assays (28). GrB appears to be important for CTL-induced DNA fragmentation. Therefore, RMA cells as control (Fig. 2E) as well as Mpr300+/+ and Mpr300-/- lymphoblasts were labeled with [3H]thymidine before exposure to OT-I-derived CTL in order to determine 3H release, which indicates DNA fragmentation. Peptide and calcium dependence of killing was confirmed in each experiment (Fig. 2, E–H). The CTL-induced DNA fragmentation was similar in lymphoblasts of wild-type (C57BL/6 x 129/Sv)F1 and Mpr300-/- knockout mice. A representative experiment is shown for Mpr300+/+ (Fig. 2F) and Mpr300-/- lymphoblasts (Fig. 2G), and the results of four independent experiments are summarized in Fig. 2H.

In order to assure that the OT-I-derived CTL kill target cells in a GrB-dependent manner, the release of GrB from CTL was confirmed by assaying the enzymatic activity of GrB in the supernatant of co-cultures of CTL with peptide-pulsed Mpr300+/+ and Mpr300-/- lymphoblast target cells (Fig. 3A). Furthermore, GrB-mediated DNA fragmentation has been reported (28, 29) to be caspase-dependent, and the CTL-induced DNA fragmentation was indeed largely inhibited by a caspase-3 specific inhibitor and blocked almost completely by the pan-caspase inhibitor Z-VAD-FMK in both Mpr300+/+ and Mpr300-/- lymphoblast target cells (Fig. 3B).



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  		FIG. 3.
Peptide-specific CTL release GrB and induce apoptosis in Mpr300+/+ and Mpr300-/- lymphoblasts in a caspase-dependent manner. A, results from one of two essentially similar experiments assaying GrB release from CTL. Mpr300+/+ and Mpr300-/- lymphoblasts were exposed to CTL derived from TCR-transgenic OT-I mice at different effector:target ratios (1:1, 0.5:1, and 0:1) in the presence or absence of the SIINFEKL peptide (0.5 µg/ml). After 4 h, the supernatant was harvested and analyzed for GrB activity by hydrolysis of the substrate Ac-IEPD-pNA that was measured as absorbance at 405 nm as described under "Experimental Procedures." B, a summary of four individual experiments presenting the mean of specific 3H release and S.D. of lymphoblasts from Mpr300-/- and Mpr300+/+ (C57BL/6 x 129/Sv)F1 mice in the presence of caspase inhibitors at a effector:target ratio of 10:1 is shown. [3H]Thymidine-labeled lymphoblasts were exposed in triplicates to OT-I-derived CTL in the presence of the SIINFEKL peptide (0.5 µg/ml). The caspase-3-specific inhibitor Ac-AAVALLPAVLLALLAPDEVD-CHO (DEVD) at a final concentration of 20 µM and the pan-caspase inhibitor Z-VAD-FMK (ZVAD) at a final concentration of 40 µM were added to the tests. Control tests (co) were performed in the presence of the solvent Me2SO only.

 
CTL-mediated Lysis and DNA Fragmentation in MEF Target Cells—Immortalized MEF lines that were derived from Mpr300-/- knockout and normal control mice were analyzed to verify the results obtained with lymphoblast target cells in another type of target cells. Besides Mpr300-/- single knockout, Mpr46-/- single knockout, Mpr300-/-/Mpr46-/- double knockout as well as Mpr300-/-/Mpr46-/- double knockout MEF lines reconstituted with a human Mpr300 (Table I) were included in the experiments with OT-I-derived CTL as effector cells.

The specific peptide and calcium-dependent lysis obtained in three independent 51Cr release assays for each MEF line is summarized in Fig. 4A. The Mpr300-/- single knockout fibroblast line appeared to be the most susceptible MEF target cell line (Fig. 4A). Similar results were obtained by 3H release assays that indicate DNA fragmentation (Fig. 4B). Since both lysis and DNA fragmentation was lower for the MEF lines (Fig. 4, A and B) than for lymphoblast or RMA target cells (see Fig. 2), the MHC class I cell surface expression on the fibroblasts was analyzed. The H2-Kb cell surface expression was indeed low (Fig. 4C) compared with RMA cells (see legend to Fig. 4) and was hardly increased by the 4-h peptide pulse (Fig. 4C) that otherwise could have lead to a cell surface stabilization of H2-Kb molecules. Interestingly, the class I levels did not correlate with susceptibility to the H2-Kb-restricted CTL. This is emphasized by the most susceptible Mpr300-/- single knockout MEF line showing the lowest H2-K cell surface expression. Although differences in susceptibility to lysis and to apoptosis by CTL were apparent among the MEF lines, this variation clearly did not depend on Mpr300 or Mpr46 status.



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  		FIG. 4.
Lysis and apoptosis of MEF lines by peptide-specific CTL does not depend on Mpr300 and Mpr46 expression of target cells and is not correlated with H2-Kb cell surface expression level. A, summary of six individual experiments presenting the mean of specific lysis (51Cr release) and S.D. of MEF lines with different Mpr receptor status at different effector:target ratios (10:1, 5:1, 2.5:1) in the presence of the SIINFEKL peptide (0.5 µg/ml). The peptide specificity and calcium dependence of the killing was confirmed in each experiment. B, summary of four individual experiments presenting the mean of specific apoptosis (3H release) and S.D. of MEF lines with different Mpr receptor status at different effector:target ratios (10:1, 5:1, 2.5:1) in the presence of the SIINFEKL peptide (0.5 µg/ml). C, summary of H2-Kb expression on the MEF lines tested before and after peptide pulsing (SIINFEKL, 0.5 µg/ml, 4 h) by flow cytometry using a specific mAb. Mean and S.D. of the specific mean fluorescence intensity (MFI) (anti-H2-Kb mAb minus isotype control) are shown (n = 3). Identical instrument settings were used for the measurement of the various MEF lines. Flow cytometry was done in parallel to 51Cr release assays shown in A. The MFI of RMA cells that were tested in parallel was 649 (mean, n = 3) and increased to 730 (mean, n = 3) after a 4-h peptide pulse.

 
CTL-mediated Apoptosis as Determined by Sub-G1 Peaks in Target Cells—To further verify the result that CTL-induced DNA fragmentation is unimpaired in Mpr300-/- lymphoblast and fibroblast target cells, sub-G1 peaks were determined by flow cytometry as an independent assay for DNA fragmentation and condensation. To mark the target cells they were labeled with the fluorescent dye CM-Dil before exposure to peptide-specific CTL for 4 h. Afterward they were 7AAD-stained and sub-G1 peaks were analyzed in dye-labeled target cells by flow cytometry. The level of CTL-induced apoptotic DNA condensation and fragmentation was comparable in Con A lymphoblasts of Mpr300+/+ and Mpr300-/- mice as shown by an individual experiment (Fig. 5A) and the summary of five experiments (Fig. 5B). When the different MEF lines were tested, the specific increase of sub-G1 peak was modest but again no dependence on Mpr300 or Mpr46 status was apparent as summarized in Fig. 5C. Thus, when sub-G1 peak determination is used as readout, Mpr300 and Mpr46 turn out to be dispensable in target cells for apoptosis that is mediated by the granule-exocytosis killing pathway of CTL.



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  		FIG. 5.
Apoptosis mediated by peptide-specific CTL and analyzed sub-G1 peak determination is not dependent on Mpr300 or Mpr46. A, an individual experiment showing sub-G1 peak determination in Mpr300+/+ (C57BL/6 x 129/Sv)F1 and Mpr300-/- lymphoblasts (day 4 after Con A stimulation) stained with CM-Dil and incubated with CTL (ratio 5:1) for 4 h at 37 °C in the presence or absence of SIINFEKL (0.5 µg/ml). The cells were fixed and permeabilized before cellular DNA was stained with 7AAD. Sub-G1 peaks were determined in CM-Dil-labeled target cells by flow cytometry after exclusion of cell doublets, and the increase in the peptide-pulsed target cells was calculated. B, summary of five individual experiments presenting mean and S.D. for the peptide-dependent percentage of cells in the sub-G1 peak (% cells in sub-G1 peak in the presence of SIINFEKL minus % cells in sub-G1 peak in the absence of SIINFEKL) at different effector:target ratios (10:1, 5:1, 1:1, 0:1). C, summary of three individual experiments presenting mean and S.D. for the peptide-dependent percentage of RMA and MEF cells in the sub-G1 peak (% cells in sub-G1 peak in the presence of SIINFEKL minus % cells in sub-G1 peak in the absence of SIINFEKL) are given. The various fibroblast lines, or as positive control, RMA cells were stained with CM-Dil and incubated with CTL at different effector:target ratios for 4 h at 37 °C in the presence or absence of the SIINFEKL peptide (0.5 µg/ml).

 
Internalization of GrB by Target Cells—To further analyze the postulated role of Mpr300 (10) and that of Mpr46 for internalization of granzyme into target cells we used isolated GrB either monomeric or complexed with SG (GrB-SG), the complex as the form in which GrB is secreted by cytotoxic cells (8). The MEF lines were exposed to monomeric GrB or GrB-SG complexes (1 µg/ml), a concentration that induces apoptosis in target cells (8). However, immunoblot for intracellular GrB yielded only a faint band in all lines representing, as estimated by densitometry, less than 1 ng of GrB per 1 x 105 cells (not shown). After increasing the concentration of GrB and GrB-SG to 10 µg/ml, the granzyme was detectable in all MEF lines as exemplified for GrB (Fig. 6A) and GrB-SG (Fig. 6B) in the Mpr300+/+ and Mpr300-/- MEF line. Flow cytometry then was used as an alternate approach to assess internalization of the granzyme. We produced a fluorochrome-labeled GrB in the monomeric (GrBAlexa 488) as well as in the complexed form (GrBAlexa 488-SG). After incubation of wild-type as well as Mpr300-/- and Mpr46-/- fibroblasts with 1 µg/ml of either the labeled monomeric or complexed GrB for 1 h at 37 °C, assessment by flow cytometry revealed uptake in all lines tested, shown in an individual experiment (Fig. 6C) and in a summary of three experiments (Fig. 6D).



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  		FIG. 6.
Internalization of GrB by target cells does not depend on the Mpr300 and Mpr46 receptors. A, Mpr300+/+ and Mpr300-/- MEF lines were exposed (+)to10 µg/ml monomeric GrB for 1 h at 37 °C or incubated with medium alone (-). Afterward, the cells were washed in the presence of trypsin and protein lysates derived from 1 x 105 cells were separated by SDS-PAGE. The blots were probed with an anti-GrB mAb and as loading control for the cell lysates with an anti-Hsc70 mAb. A dilution series of isolated GrB (that is of course free of Hsc70) was also loaded onto the gel. Approximately 15 ng of GrB were detected in Mpr300+/+ and 22 ng in Mpr300-/- cell lysates as determined by densitometry. B, Mpr300+/+ and Mpr300-/- MEF lines were exposed (+) to 10 µg/ml GrB-SG complexes for 1 h at 37 °C or incubated with medium alone (-), and immunoblot analysis was performed as described for A. On this gel a dilution series of isolated GrB-SG was loaded. GrB is non-covalently bound to SG in GrB-SG complexes and therefore separated from SG by SDS-PAGE. C, wild-type (Mpr300+/+, Mpr46+/+) and knockout (Mpr300-/-, Mpr46-/-) MEF lines were incubated with 1 µg/ml monomeric GrBAlexa 488 or GrBAlexa 488-SG complexes for 1 h at 37 °C. Internalization of the Alexa 488-labeled reagents was analyzed by flow cytometry on PI-negative cells. The dotted lines indicate the autofluorescence of the cells, and bold lines represent cells exposed to monomeric GrBAlexa 488 or GrBAlexa 488-SG complexes. The specific MFI (fluorescence of cells exposed to the Alexa 488-labeled GrB or GrB-SG minus autofluorescence of culture medium-treated cells) is given by numbers. The data were acquired with identical instrument settings for the different MEF lines. D, a summary of three independent experiments with wild-type (Mpr300+/+, Mpr46+/+) and knockout (Mpr300-/-, Mpr46-/-) MEF lines is shown. Mean and S.D. of the specific MFI (fluorescence of cells exposed to the Alexa 488-labeled GrB or GrB-SG minus autofluorescence of culture medium-treated cells) are given. Similar results are obtained when ratios (mean fluorescence of cells exposed to the Alexa 488-labeled GrB or GrB-SG divided by mean fluorescence of culture medium-treated cells) are calculated.

 
Subcellular Localization of GrB in Target Cells—For subcellular localization of GrB in target cells confocal laser scanning microscopy (CLSM) was used. MEF lines were incubated with 1 µg/ml of GrB, either monomeric or complexed to SG, for 1 h and 37 °C. Uptake was readily apparent for all MEF lines as exemplified by the vesicular pattern observed after addition of GrBAlexa 488-SG to the Mpr300+/+ MEF line (Fig. 7A, green stain). In the experiment shown, Mpr300+/+ cells exposed to GrBAlexa 488-SG (Fig. 7A) were also incubated with an anti-Mpr300 Ab and the corresponding labeled secondary Ab (red staining). The labeled GrBAlexa 488 and the Mpr300 Ab reactive compartment remained completely separate as shown by lack of yellow staining in the overlay (Fig. 7A). Monomeric GrBAl- exa 488 also did not colocalize with Mpr300 after internalization by the Mpr300+/+ MEF line (data not shown).



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  		FIG. 7.
GrB is internalized into vesicles of Mpr300+/+ and Mpr300-/- target cells and does not colocalize with Mpr300 but partially with Lamp-1 as determined by confocal laser scanning microscopy. A, Mpr300+/+ fibroblasts were incubated with 1 µg/ml GrBAlexa 488-SG for 1 h at 37 °C, fixed and permeabilized, and stained with anti-Mpr300 (A), anti-transferrin receptor (B), or anti-Lamp-1 (C) and labeled secondary Ab. The GrBAlexa 488-SG is always shown in green and staining with Mpr300, transferrin receptor, or Lamp-1 Ab in red. The arrows point to vesicles showing a colocalization in the representative sections, and the white bar indicates 20 µm. D, Mpr300-/- fibroblasts were incubated with 1 µg/ml GrBAlexa 488-SG for 1 h at 37 °C before fixation and confocal microscopy. All confocal images were obtained under identical scan settings.

 
The vesicles of cells that had been treated with GrBAlexa 488-SG for 1 h at 37 °C were further characterized by colocalization with standard marker proteins of subcellular compartments. GrBAlexa 488 showed limited colocalization with the transferrin receptor (Fig. 7B) or with endocytosed transferrin (data not shown), both as markers of early or recycling endosomes. Hardly any colocalization was found with the most commonly used early endosomal marker EEA-1 (data not shown), suggesting that internalized GrBAlexa 488 was delivered to compartments downstream in the endocytic pathway. Consistent with this assumption, after 1 h GrB was detectable in vesicular structures colocalizing with Lamp-1 (Fig. 7C), a marker of late endosomes and lysosomes. The internalization of GrB into Mpr300-/- cells that were exposed to GrBAlexa 488-SG is shown in Fig. 7D.

GrB and GrB-SG-mediated Apoptosis—Uptake of GrB by target cells is not sufficient for apoptotic cell death. Delivery of endocytosed GrB by PFN or known endosomolytic agents such as AD to the cytosol is an essential requirement. Therefore, we compared the apoptotic response of Mpr300-/- as well as Mpr46-/- knockout and wild-type cells after GrB was delivered by adenoviral particles. Equivalent transduction of MEF lines with the AD was ensured as outlined under "Experimental Procedures" (Fig. 8, A and D). GrB, either monomeric or complexed with SG, was added to cultures with subsequent addition of AD. After an incubation period of 5 h, membrane damage was measured by PI staining (Fig. 8, B and E), and apoptosis was assayed by sub-G1 peak determination (Fig. 8, C and F). Susceptibility to apoptosis varied for fibroblast lines, but levels were independent of Mpr300 or Mpr46 expression status. The Mpr300-/- single knockout line was the most susceptible, and one of the two wild-type control MEF lines (Mpr46+/+) was most resistant. Comparison of PI staining and sub-G1 peak determination did not reveal a prominent dissociation between these markers of cell death. In additional experiments GrB, either monomeric or complexed, was delivered by sublytic concentrations of PFN to exclude the possibility that the requirement for Mpr300 depends on the agent used for releasing the granzyme to the cytosol. Again the Mpr300-/- line was the most susceptible MEF line as measured by PI staining and sub-G1 peak determination, respectively (data not shown). Next we determined whether apoptosis of the various target cells required simultaneous treatment with granzyme and AD. The cells were incubated with GrB or GrB-SG for 1 h at 37 °C, washed extensively, exposed to AD, and assayed 5 h later (Fig. 8G). Indeed the level of apoptosis was comparable to experiments in which targets were exposed to granzyme and AD simultaneously for 5 h. Thus, the amount of granzyme internalized during the 1-h preincubation (see above, Figs. 6 and 7) is sufficient to induce apoptosis.



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  		FIG. 8.
GrB- and GrB-SG-mediated apoptosis is not dependent on Mpr300 and Mpr46. A and D, adenoviral infection of MEF target cells with a replication-deficient AD type 5 containing a GFP expression construct was employed to allow intracellular delivery of GrB or GrB-SG complexes. The transduction efficiency (mean and S.D. of four experiments for GrB and three experiments for GrB-SG) was controlled by assaying the percentage of GFP-expressing cells 24 h after infection by flow cytometry in cultures that were infected in parallel at the same time. B and E, the percentage of PI-positive (dead) cells was determined by flow cytometry 5 h after infection and GrB or GrB-SG treatment (1 µg/ml) of the MEF lines. The mean and S.D. of specific PI-positive cells (% PI-positive cells treated with GrB or GrB-SG and AD minus % PI-positive cells treated with AD only) of four (GrB) and three (GrB-SG) experiments are given. C and F, percentage of cells within the sub-G1 peak was determined by flow cytometry in fibroblasts fixed 5 h after infection and GrB or GrB-SG treatment (1 µg/ml). The mean and S.D. of specific apoptotic cells (% cells in sub-G1 peak treated with GrB or GrB-SG and AD minus % cells in sub-G1 peak treated with AD only) of four (GrB) and three (GrB-SG) experiments are given. G, the GrB internalized during 1 h at 37 °C is sufficient to induce apoptosis after subsequent AD infection. Mpr300-/- MEF were incubated for 1 h at 37 °C with GrB or GrB-SG complexes (1 µg/ml) or culture medium only. Then the cells were washed three times and infected 1 h later with AD. Apoptosis was assayed after 5 h by sub-G1 peak determination. The percentage of cells in sub-G1 peaks is given. The results shown are representative for two separate experiments.

 
Different MEF lines take up approximately equal amounts of GrB (Fig. 6). Nevertheless, it appears that the degree of apoptosis seen after AD infection varies (Fig. 8, C and F), as is evident when e.g. Mpr300-/- and Mpr46+/+ MEF lines are compared. Thus, additional target cell factors might affect susceptibility to GrB and GrB-SG. However, susceptibility clearly is not dependent on expression of Mpr.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
As shown most impressively by studies with PFN knockout mice, the granule-exocytosis pathway is critical for removal of pathogen-infected and transformed cells (30). The finding that PFN forms pores in the plasma membrane (31) encouraged a model in which the granzymes entered the target through PFN pores causing apoptotic cell death. This model has become less attractive, because GrB can be taken up by target cells into endocytotic vesicles inducing apoptosis in the presence of PFN at concentrations that do not produce transmembrane pores (6). While PFN is assumed to effect the release of GrB from endocytotic vesicles, the molecular mechanism underlying granzyme delivery remains elusive.

Motyka et al. (10) suggested that the Mpr300 on target cells mediates GrB internalization and is therefore essential for CTL-mediated apoptosis and rejection of allogeneic cells. Recently, Trapani et al. (13) described a Mpr300-independent pathway for GrB-induced cell death suggesting the Mpr300 was not critical for the induction of apoptosis. However, the data reported by Motyka et al. (10) and Trapani et al. (13) were generated with cell lines selected for the absence the Mpr300 and overexpression of the protein. Therefore, characteristics beside Mpr300 deficiency, which might influence susceptibility to CTL, are undefined, and overexpression of Mpr300 could result in physiologically irrelevant functioning of the receptor. To definitively examine the role of the Mpr300 in CTL-mediated cytotoxicity, we analyzed genetically defined target cells derived from Mpr300-/- and also Mpr46-/- knockout mice using ConA-stimulated lymphoblasts and MEF lines as target cells. In contrast to the data reported (10, 13), we found absolutely no role for Mpr300 in CTL-induced apoptosis.

At the cellular level, alloreactive CTL killed target cells regardless of their Mpr expression status when analyzed by 51Cr release. In previous reports (10, 13), allogeneic CTL were used as effector cells without distinguishing the contribution of the secretory and death receptor pathways. Although lysis of lymphoblasts by alloreactive CTL was not completely blocked by EGTA, OT-I mice-derived peptide-specific CTL lysed target cells in an entirely calcium-dependent manner regardless of Mpr300 expression.

GrB does not contribute to the necrotic death of target cells but is crucial for the development of apoptotic phenotype (28, 29). Therefore, it is important that apoptosis, determined at the DNA level by [3H]thymidine release and sub-G1 peak analysis, was also found to be independent of Mpr300 expression. In contrast to our results, allogeneic CTL have been reported to elicit less DNA fragmentation as measured by [3H]thymidine (10) or 125I-DNA release (13) in selected L cell lines deficient in Mpr300. On the basis of the data reported here, the apparent reduction in CTL-mediated DNA fragmentation with cells lacking Mpr300 could be due to an undefined resistance of the selected L cells to apoptosis rather than reduced expression of the Mpr300 (10, 13). Importantly, our results on CTL-mediated apoptosis were obtained primarily with lymphoblast target cells directly derived from the knockout mice. Thus, artifacts attributable to clonal heterogeneity among the L cells used by other investigators (10, 13) and alterations that might arise during in vitro culture are not obscuring the results presented here. Complementing the studies of the lymphoblasts, analysis of the MEF lines produced similar results.

We have extended the analysis from CTL to the effector protease obtaining completely concordant results. In particular, the form of the granzyme secreted by cytotoxic cells (GrBSG) has not been tested previously. Because the lymphoblasts were relatively insensitive to PFN and could not be transduced with AD, experiments were restricted to the MEF lines. Wildtype and Mpr300-/- as well as Mpr46-/- MEF lines were found to readily internalize either monomeric or complexed GrB and, in Mpr300+/+ cells, uptake of the granzyme did not colocalize with Mpr300. After exposure of target cells to GrB-SG complexes for 1 h at 37 °C, a time sufficient for apoptosis after subsequent AD infection, the granzyme was identified in a late endosomal/lysosomal compartment. Furthermore, the lack of colocalization of the granzyme with Mpr300 strongly argues against an Mpr300-dependent pathway participating in GrB-mediated killing. In accordance with this concept, delivery of GrB by AD or PFN resulted in apoptosis in Mpr300-/- and Mpr46-/- MEF lines. Remarkably, the uptake of very little GrB by target cells can be sufficient for the induction of apoptosis. From the immunoblot and apoptosis assays it can be estimated that, for example, in the Mpr300-/- MEF line, less than 1 ng of GrB internalized into 1 x 105 cells is required to induce apoptosis in 50% of the cells within 5 h, when delivered by AD.

The various MEF lines manifested reproducible differences in their susceptibility to CTL, GrB, and GrB-SG. This variability, however, did not depend on Mpr status; a finding highlighted by the observation that the Mpr300-/- MEF line was most sensitive. The observed variation also was not dependent on either differences in MHC class I expression or uptake of the granzyme. Thus, conditions acting downstream appear to influence susceptibility to killing. Importantly, the lack of a similar variation for CTL-mediated apoptosis of lymphoblasts suggests clonal heterogeneity in the MEF lines was chiefly responsible. Indeed, clonal heterogeneity becomes a dominant issue when long term cell lines are studied (10, 13).

It seems unlikely that a single GrB receptor protein will be identified. To diversify the elimination of target cells, GrB should have the capacity to interact with multiple cell surface structures (32). Since GrB is released as multicomponent complex, SG or other granule proteins could contribute to the binding. CD44 has been reported to bind SG (33) and is therefore a candidate. Finally, an Hsp70-dependent pathway for GrB-mediated apoptosis has been reported recently (34).

In summary, we have studied the role of both Mpr300 and Mpr46 in target cell apoptosis mediated by GrB at the cellular and molecular level. We have obtained multifold and concordant evidence that both receptors, although known to be responsible for trafficking of granzymes in cytotoxic cells (12), are not involved in apoptosis of target cells mediated by GrB or GrB-SG complexes and the granule-exocytosis killing pathway of intact CTL. In comparison of target cells derived from knockout and wild-type mice, we have found no evidence for an Mpr300-dependent apoptotic pathway mediated by GrB described by others either exclusively (10) or in addition to a Mpr300-independent pathway (13). Furthermore, we have provided new insight in the delivery of GrB and GrB-SG complexes into target cells and their subsequent intracellular localization.


   FOOTNOTES
 
* This work was supported in part by Grant Gu105/16-1 from the Deutsche Forschungsgemeinschaft (to E. G. and R. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence should be addressed: University of Göttingen, Division of Immunogenetics, Heinrich-Düker-Weg 12, 37073 Göttingen, Germany. Tel.: 0049-551-395884; Fax: 0049-551-395852; E-mail: rdresse{at}gwdg.de.

1 The abbreviations used are: CTL, cytotoxic T lymphocyte(s); 7AAD, 7-aminoactinomycin D; Ab, antibody; AD, adenovirus; BSA, bovine serum albumin; Con A, concanavalin A; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; GFP, green fluorescent protein; GrB, granzyme B; GrB-SG, granzyme B-serglycin; Igf2, insulin-like growth factor II; mAb, monoclonal antibody; MEF, mouse embryonal fibroblast; MFI, mean fluorescence intensity; MHC, major histocompatibility complex; MOI, multiplicity of infection; Mpr, mannose 6-phosphate receptor; PBS, phosphate-buffered saline; PFN, perforin; PI, propidium iodide; SG, serglycin; TCR, T cell receptor; Z, benzyloxycarbonyl; FMK, fluoromethylketone; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. Back


   ACKNOWLEDGMENTS
 
We thank L. Elsner for expert technical assistance, and the assistance of E. Munk in animal experiments is gratefully acknowledged. We thank Prof. Dr. D. Frankenberg and G. Urban (Division of Clinical Radiation Biology and Radiation Physics, University of Göttingen, Germany) for irradiating the cells. A breeding pair of OT-I mice were generously provided by Dr. C. Kurts (RWTH Aachen, Germany).



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 ABSTRACT
 INTRODUCTION
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 RESULTS
 DISCUSSION
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