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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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Residual uptake of granzyme B by K44A mutant dynamin–overexpressing HeLa cells. (a) Confocal micrograph of HeLa cells overexpressing K44A mutant dynamin (−tet) or in which expression of mutant dynamin is suppressed (+tet) after exposure to FITC-transferrin for 120 min. (b) Confocal micrograph of K44A HeLa cells after exposure to FITC–granzyme B (50 nM) for 60 or 120 min. (c) Uptake of FITC–granzyme B (grB) or unglycosylated GFP by HeLa cells overexpressing wild-type (WT) or K44A-mutated dynamin for the times indicated. Uptake of either fluoresceinated molecule into the cell cytoplasm was quantitated by confocal laser scanning microscopy and image analysis as described (Jans, 1995; Jans et al., 1996). The results are expressed as a ratio between cytoplasmic fluorescence (Fc) 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 substruction of autofluorescence (as described in Materials and methods).
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fig1: Residual uptake of granzyme B by K44A mutant dynamin–overexpressing HeLa cells. (a) Confocal micrograph of HeLa cells overexpressing K44A mutant dynamin (−tet) or in which expression of mutant dynamin is suppressed (+tet) after exposure to FITC-transferrin for 120 min. (b) Confocal micrograph of K44A HeLa cells after exposure to FITC–granzyme B (50 nM) for 60 or 120 min. (c) Uptake of FITC–granzyme B (grB) or unglycosylated GFP by HeLa cells overexpressing wild-type (WT) or K44A-mutated dynamin for the times indicated. Uptake of either fluoresceinated molecule into the cell cytoplasm was quantitated by confocal laser scanning microscopy and image analysis as described (Jans, 1995; Jans et al., 1996). The results are expressed as a ratio between cytoplasmic fluorescence (Fc) 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 substruction of autofluorescence (as described in Materials and methods).

Mentions: Expression of K44A-dynamin has been shown to result in retention of some ligands, including transferrin at the plasma membrane, whereas other ligands fail to accumulate on the cell surface (Damke et al., 1994). To demonstrate the induction of K44A-dynamin expression, cells grown in the presence of tet or in the absence of tet for 48 h were incubated at 37°C with FITC-labeled transferrin (FITC-transferrin) and then viewed by confocal laser scanning microscopy. As expected, HeLa cells in which K44A-dynamin expression was repressed by tet demonstrated strong uptake of FITC-transferrin into cytoplasmic vesicles. In addition, most of the cells demonstrated punctate fluorescence at the plasma membrane, indicating multifocal binding to the transferrin receptor (Fig. 1a). In cells grown in tet-deficient medium, the fluorescence was largely restricted to the plasma membrane and few vesicles were internalized, consistent with defective clathrin-dependent uptake of FITC-transferrin. Fewer than 10% of cells demonstrated clear vesicular uptake under these conditions after 120 min, although some residual fluorescence was seen in the cytoplasm, consistent with constitutive uptake through fluid phase micropinocytosis, which is known to remain active in these cells (see below). Despite the demonstrated potent inhibition of receptor-dependent endocytosis in these cells (Damke et al., 1994), >90% of K44A-dynamin–expressing cells incubated with FITC–granzyme B showed cytoplasmic fluorescence, which increased with time (Fig. 1 b, −tet) and was far above levels of autofluorescence (see below). No cell surface binding of FITC–granzyme B was seen at either 4°C (unpublished data) or 37°C, and the cytoplasmic staining pattern was less obviously punctate than seen when only wild-type dynamin was expressed. These findings were again consistent with fluid phase uptake of granzyme B. Control cells expressing only wild-type dynamin showed consistent cell surface and punctate vesicular staining with FITC–granzyme B similar to that seen with FITC-transferrin (Fig. 1 b, +tet) and reminiscent of granzyme B trafficking observed previously in Jurkat or FDC-P1 cells (Jans et al., 1996; Browne et al., 1999; Motyka et al., 2000). Similar HeLa cell transfectants that overexpressed wild-type dynamin upon withdrawal of tet showed normal vesicular uptake of both FITC–granzyme B and FITC-transferrin (Damke et al., 1994; data not shown).


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)

Residual uptake of granzyme B by K44A mutant dynamin–overexpressing HeLa cells. (a) Confocal micrograph of HeLa cells overexpressing K44A mutant dynamin (−tet) or in which expression of mutant dynamin is suppressed (+tet) after exposure to FITC-transferrin for 120 min. (b) Confocal micrograph of K44A HeLa cells after exposure to FITC–granzyme B (50 nM) for 60 or 120 min. (c) Uptake of FITC–granzyme B (grB) or unglycosylated GFP by HeLa cells overexpressing wild-type (WT) or K44A-mutated dynamin for the times indicated. Uptake of either fluoresceinated molecule into the cell cytoplasm was quantitated by confocal laser scanning microscopy and image analysis as described (Jans, 1995; Jans et al., 1996). The results are expressed as a ratio between cytoplasmic fluorescence (Fc) 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 substruction of autofluorescence (as described in Materials and methods).
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Related In: Results  -  Collection

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fig1: Residual uptake of granzyme B by K44A mutant dynamin–overexpressing HeLa cells. (a) Confocal micrograph of HeLa cells overexpressing K44A mutant dynamin (−tet) or in which expression of mutant dynamin is suppressed (+tet) after exposure to FITC-transferrin for 120 min. (b) Confocal micrograph of K44A HeLa cells after exposure to FITC–granzyme B (50 nM) for 60 or 120 min. (c) Uptake of FITC–granzyme B (grB) or unglycosylated GFP by HeLa cells overexpressing wild-type (WT) or K44A-mutated dynamin for the times indicated. Uptake of either fluoresceinated molecule into the cell cytoplasm was quantitated by confocal laser scanning microscopy and image analysis as described (Jans, 1995; Jans et al., 1996). The results are expressed as a ratio between cytoplasmic fluorescence (Fc) 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 substruction of autofluorescence (as described in Materials and methods).
Mentions: Expression of K44A-dynamin has been shown to result in retention of some ligands, including transferrin at the plasma membrane, whereas other ligands fail to accumulate on the cell surface (Damke et al., 1994). To demonstrate the induction of K44A-dynamin expression, cells grown in the presence of tet or in the absence of tet for 48 h were incubated at 37°C with FITC-labeled transferrin (FITC-transferrin) and then viewed by confocal laser scanning microscopy. As expected, HeLa cells in which K44A-dynamin expression was repressed by tet demonstrated strong uptake of FITC-transferrin into cytoplasmic vesicles. In addition, most of the cells demonstrated punctate fluorescence at the plasma membrane, indicating multifocal binding to the transferrin receptor (Fig. 1a). In cells grown in tet-deficient medium, the fluorescence was largely restricted to the plasma membrane and few vesicles were internalized, consistent with defective clathrin-dependent uptake of FITC-transferrin. Fewer than 10% of cells demonstrated clear vesicular uptake under these conditions after 120 min, although some residual fluorescence was seen in the cytoplasm, consistent with constitutive uptake through fluid phase micropinocytosis, which is known to remain active in these cells (see below). Despite the demonstrated potent inhibition of receptor-dependent endocytosis in these cells (Damke et al., 1994), >90% of K44A-dynamin–expressing cells incubated with FITC–granzyme B showed cytoplasmic fluorescence, which increased with time (Fig. 1 b, −tet) and was far above levels of autofluorescence (see below). No cell surface binding of FITC–granzyme B was seen at either 4°C (unpublished data) or 37°C, and the cytoplasmic staining pattern was less obviously punctate than seen when only wild-type dynamin was expressed. These findings were again consistent with fluid phase uptake of granzyme B. Control cells expressing only wild-type dynamin showed consistent cell surface and punctate vesicular staining with FITC–granzyme B similar to that seen with FITC-transferrin (Fig. 1 b, +tet) and reminiscent of granzyme B trafficking observed previously in Jurkat or FDC-P1 cells (Jans et al., 1996; Browne et al., 1999; Motyka et al., 2000). Similar HeLa cell transfectants that overexpressed wild-type dynamin upon withdrawal of tet showed normal vesicular uptake of both FITC–granzyme B and FITC-transferrin (Damke et al., 1994; data not shown).

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