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A clathrin/dynamin- and mannose-6-phosphate receptor-independent pathway for granzyme B-induced cell death.

Trapani JA, Sutton VR, Thia KY, Li YQ, Froelich CJ, Jans DA, Sandrin MS, Browne KA - J. Cell Biol. (2003)

Bottom Line: Exposure of K44A-dynamin-overexpressing and wild-type HeLa cells to granzyme B with sublytic perforin resulted in similar apoptosis in the two cell populations, both in short and long term assays.Granzyme B uptake into MPR-overexpressing L cells was more rapid than into MPR- L cells, but the receptor-deficient cells took up granzyme B through fluid phase micropinocytosis and remained sensitive to it.Entry of granzyme B into target cells and its intracellular trafficking to induce target cell death in the presence of perforin are therefore not critically dependent on MPR or clathrin/dynamin-dependent endocytosis.

View Article: PubMed Central - PubMed

Affiliation: Cancer Immunology Laboratory, Peter MacCallum Cancer Institute, Melbourne 8006, Australia. j.trapani@pmci.unimelb.edu.au

ABSTRACT
The 280-kD cation-independent mannose-6-phosphate receptor (MPR) has been shown to play a role in endocytic uptake of granzyme B, since target cells overexpressing MPR have an increased sensitivity to granzyme B-mediated apoptosis. On this basis, it has been proposed that cells lacking MPR are poor targets for cytotoxic lymphocytes that mediate allograft rejection or tumor immune surveillance. In the present study, we report that the uptake of granzyme B into target cells is independent of MPR. We used HeLa cells overexpressing a dominant-negative mutated (K44A) form of dynamin and mouse fibroblasts overexpressing or lacking MPR to show that the MPR/clathrin/dynamin pathway is not required for granzyme B uptake. Consistent with this observation, cells lacking the MPR/clathrin pathway remained sensitive to granzyme B. Exposure of K44A-dynamin-overexpressing and wild-type HeLa cells to granzyme B with sublytic perforin resulted in similar apoptosis in the two cell populations, both in short and long term assays. Granzyme B uptake into MPR-overexpressing L cells was more rapid than into MPR- L cells, but the receptor-deficient cells took up granzyme B through fluid phase micropinocytosis and remained sensitive to it. Contrary to previous findings, we also demonstrated that mouse tumor allografts that lack MPR expression were rejected as rapidly as tumors that overexpress MPR. Entry of granzyme B into target cells and its intracellular trafficking to induce target cell death in the presence of perforin are therefore not critically dependent on MPR or clathrin/dynamin-dependent endocytosis.

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Quantitative kinetic analysis of the uptake of FITC–granzyme B into MS and MS9-II cells. Uptake of FITC–granzyme B (grB) or unglycosylated GFP by MS (MPR-) and MS9-II (MPR-overexpressing) L cells. The uptake of both fluoresceinated molecules onto the plasma membrane (top) into the cell cytoplasm (middle) or into the late endosomal compartment (bottom) was quantitated by confocal laser scanning microscopy and image analysis as described previously (Jans, 1995). The results are expressed as a ratio between total cytoplasmic fluorescence (Fc), plasma membrane fluorescence (Fpm), or fluorescence of the late endosomal compartment (Flc) and extracellular fluorescence (Fmed) ± standard error of the mean. Each data point was derived from measurements on at least 30 cells selected at random after subtraction of autofluorescence (as described in Materials and methods).
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fig5: Quantitative kinetic analysis of the uptake of FITC–granzyme B into MS and MS9-II cells. Uptake of FITC–granzyme B (grB) or unglycosylated GFP by MS (MPR-) and MS9-II (MPR-overexpressing) L cells. The uptake of both fluoresceinated molecules onto the plasma membrane (top) into the cell cytoplasm (middle) or into the late endosomal compartment (bottom) was quantitated by confocal laser scanning microscopy and image analysis as described previously (Jans, 1995). The results are expressed as a ratio between total cytoplasmic fluorescence (Fc), plasma membrane fluorescence (Fpm), or fluorescence of the late endosomal compartment (Flc) and extracellular fluorescence (Fmed) ± standard error of the mean. Each data point was derived from measurements on at least 30 cells selected at random after subtraction of autofluorescence (as described in Materials and methods).

Mentions: To characterize the residual uptake of granzyme B into MPR- MS cells, we next performed a quantitative, kinetic analysis of granzyme B uptake using quantitative laser-scanning microscopy of several hundred cells. Cells were preincubated with FITC–granzyme B at 4°C to allow surface binding and then rapidly transferred to 37°C to permit uptake into the cytoplasm. Consistent with the overexpression of MPR on the surface of MS9-II cells, FITC–granzyme B binding at the plasma membrane (expressed as the ratio of fluorescence at the plasma membrane to that in the extracellular medium, Fpm/Fmed) had reached maximal levels within 5 min and gradually diminished as the cells took up granzyme B into their cytoplasm (Fig. 5 a). Consistent with uptake through a cell surface receptor (MPR), preincubation with a 10-fold excess of unlabeled granzyme B virtually abolished plasma membrane fluorescence (Fig. 5 a, t = 5 min and later time points). By comparison, the level of plasma membrane staining of MS cells was much lower, did not vary over time, and was unaffected by the addition of unlabeled competitor. The finding that unlabeled granzyme B could block cell surface fluorescence of MS9-II but not MS cells indicated that although uptake of granzyme B into MS9-II cells was largely dependent on binding to MPR, the uptake into MS cells was not mediated by a cell surface receptor. Despite this, and consistent with findings presented above (Fig. 4), the cytoplasmic accumulation of FITC–granzyme B (Fc/Fmed) was not abolished in MS cells, although it was, as expected, reduced in comparison with MS9-II cells (Fig. 5 b). Significant and equivalent fluid phase uptake of unglycosylated GFP was seen into both cell lines, which did not vary appreciably over the 2 h of study. The trafficking of FITC–granzyme B to the late endosomal compartment (measured as the ratio of fluorescence of the late endosomal compartment and extracellular fluorescence, Flc/Fmed) was also much more rapid in MS9-II cells than MS cells and was markedly slowed by preaddition of unlabeled competitor, whereas trafficking to this compartment in MS cells was far less prominent and not influenced by competitor (Fig. 5 c).


A clathrin/dynamin- and mannose-6-phosphate receptor-independent pathway for granzyme B-induced cell death.

Trapani JA, Sutton VR, Thia KY, Li YQ, Froelich CJ, Jans DA, Sandrin MS, Browne KA - J. Cell Biol. (2003)

Quantitative kinetic analysis of the uptake of FITC–granzyme B into MS and MS9-II cells. Uptake of FITC–granzyme B (grB) or unglycosylated GFP by MS (MPR-) and MS9-II (MPR-overexpressing) L cells. The uptake of both fluoresceinated molecules onto the plasma membrane (top) into the cell cytoplasm (middle) or into the late endosomal compartment (bottom) was quantitated by confocal laser scanning microscopy and image analysis as described previously (Jans, 1995). The results are expressed as a ratio between total cytoplasmic fluorescence (Fc), plasma membrane fluorescence (Fpm), or fluorescence of the late endosomal compartment (Flc) and extracellular fluorescence (Fmed) ± standard error of the mean. Each data point was derived from measurements on at least 30 cells selected at random after subtraction of autofluorescence (as described in Materials and methods).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172645&req=5

fig5: Quantitative kinetic analysis of the uptake of FITC–granzyme B into MS and MS9-II cells. Uptake of FITC–granzyme B (grB) or unglycosylated GFP by MS (MPR-) and MS9-II (MPR-overexpressing) L cells. The uptake of both fluoresceinated molecules onto the plasma membrane (top) into the cell cytoplasm (middle) or into the late endosomal compartment (bottom) was quantitated by confocal laser scanning microscopy and image analysis as described previously (Jans, 1995). The results are expressed as a ratio between total cytoplasmic fluorescence (Fc), plasma membrane fluorescence (Fpm), or fluorescence of the late endosomal compartment (Flc) and extracellular fluorescence (Fmed) ± standard error of the mean. Each data point was derived from measurements on at least 30 cells selected at random after subtraction of autofluorescence (as described in Materials and methods).
Mentions: To characterize the residual uptake of granzyme B into MPR- MS cells, we next performed a quantitative, kinetic analysis of granzyme B uptake using quantitative laser-scanning microscopy of several hundred cells. Cells were preincubated with FITC–granzyme B at 4°C to allow surface binding and then rapidly transferred to 37°C to permit uptake into the cytoplasm. Consistent with the overexpression of MPR on the surface of MS9-II cells, FITC–granzyme B binding at the plasma membrane (expressed as the ratio of fluorescence at the plasma membrane to that in the extracellular medium, Fpm/Fmed) had reached maximal levels within 5 min and gradually diminished as the cells took up granzyme B into their cytoplasm (Fig. 5 a). Consistent with uptake through a cell surface receptor (MPR), preincubation with a 10-fold excess of unlabeled granzyme B virtually abolished plasma membrane fluorescence (Fig. 5 a, t = 5 min and later time points). By comparison, the level of plasma membrane staining of MS cells was much lower, did not vary over time, and was unaffected by the addition of unlabeled competitor. The finding that unlabeled granzyme B could block cell surface fluorescence of MS9-II but not MS cells indicated that although uptake of granzyme B into MS9-II cells was largely dependent on binding to MPR, the uptake into MS cells was not mediated by a cell surface receptor. Despite this, and consistent with findings presented above (Fig. 4), the cytoplasmic accumulation of FITC–granzyme B (Fc/Fmed) was not abolished in MS cells, although it was, as expected, reduced in comparison with MS9-II cells (Fig. 5 b). Significant and equivalent fluid phase uptake of unglycosylated GFP was seen into both cell lines, which did not vary appreciably over the 2 h of study. The trafficking of FITC–granzyme B to the late endosomal compartment (measured as the ratio of fluorescence of the late endosomal compartment and extracellular fluorescence, Flc/Fmed) was also much more rapid in MS9-II cells than MS cells and was markedly slowed by preaddition of unlabeled competitor, whereas trafficking to this compartment in MS cells was far less prominent and not influenced by competitor (Fig. 5 c).

Bottom Line: Exposure of K44A-dynamin-overexpressing and wild-type HeLa cells to granzyme B with sublytic perforin resulted in similar apoptosis in the two cell populations, both in short and long term assays.Granzyme B uptake into MPR-overexpressing L cells was more rapid than into MPR- L cells, but the receptor-deficient cells took up granzyme B through fluid phase micropinocytosis and remained sensitive to it.Entry of granzyme B into target cells and its intracellular trafficking to induce target cell death in the presence of perforin are therefore not critically dependent on MPR or clathrin/dynamin-dependent endocytosis.

View Article: PubMed Central - PubMed

Affiliation: Cancer Immunology Laboratory, Peter MacCallum Cancer Institute, Melbourne 8006, Australia. j.trapani@pmci.unimelb.edu.au

ABSTRACT
The 280-kD cation-independent mannose-6-phosphate receptor (MPR) has been shown to play a role in endocytic uptake of granzyme B, since target cells overexpressing MPR have an increased sensitivity to granzyme B-mediated apoptosis. On this basis, it has been proposed that cells lacking MPR are poor targets for cytotoxic lymphocytes that mediate allograft rejection or tumor immune surveillance. In the present study, we report that the uptake of granzyme B into target cells is independent of MPR. We used HeLa cells overexpressing a dominant-negative mutated (K44A) form of dynamin and mouse fibroblasts overexpressing or lacking MPR to show that the MPR/clathrin/dynamin pathway is not required for granzyme B uptake. Consistent with this observation, cells lacking the MPR/clathrin pathway remained sensitive to granzyme B. Exposure of K44A-dynamin-overexpressing and wild-type HeLa cells to granzyme B with sublytic perforin resulted in similar apoptosis in the two cell populations, both in short and long term assays. Granzyme B uptake into MPR-overexpressing L cells was more rapid than into MPR- L cells, but the receptor-deficient cells took up granzyme B through fluid phase micropinocytosis and remained sensitive to it. Contrary to previous findings, we also demonstrated that mouse tumor allografts that lack MPR expression were rejected as rapidly as tumors that overexpress MPR. Entry of granzyme B into target cells and its intracellular trafficking to induce target cell death in the presence of perforin are therefore not critically dependent on MPR or clathrin/dynamin-dependent endocytosis.

Show MeSH
Related in: MedlinePlus