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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 MPR- L cell fibroblasts. (a) Confocal micrograph of MS (MPR-) and MS9-II (MPR-overexpressing) L cells after exposure to FITC–granzyme B (50 nM) for 60 min at 37°C or without addition of FITC–granzyme B (autofluorescence [AF], bottom). Association of granzyme B with the plasma membrane (m) and in the late endosomal compartment (l) are shown. (b) Cytofluorographic analysis of MS and MS9-II cells after incubation with FITC–granzyme B (50 nM) at 37°C for the times indicated or in the absence of FITC–granzyme B (filled traces).
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fig4: Residual uptake of granzyme B by MPR- L cell fibroblasts. (a) Confocal micrograph of MS (MPR-) and MS9-II (MPR-overexpressing) L cells after exposure to FITC–granzyme B (50 nM) for 60 min at 37°C or without addition of FITC–granzyme B (autofluorescence [AF], bottom). Association of granzyme B with the plasma membrane (m) and in the late endosomal compartment (l) are shown. (b) Cytofluorographic analysis of MS and MS9-II cells after incubation with FITC–granzyme B (50 nM) at 37°C for the times indicated or in the absence of FITC–granzyme B (filled traces).

Mentions: Our second series of experiments used mouse L cell fibroblasts either for MPR expression or overexpressing human MPR as had been studied previously (Nolan et al., 1990; Motyka et al., 2000). The L cells overexpressing MPR (designated MS9-II) became strongly fluorescent after incubation for 10 min at 37°C with FITC–granzyme B and showed a similar pattern of fluorescence when viewed by confocal microscopy as described above for HeLa cells (Fig. 4 a). The staining pattern in most cells also clearly showed a predominant concentrated area of cytoplasmic fluorescence consistent with transport of ligand to the late endosomal compartment. The MPR- MS cells (which had been transfected with vector DNA alone) were less strongly fluorescent at the same time point but still showed significant uptake of FITC–granzyme B compared with autofluorescence controls (Fig. 4 a). As was seen with the K44A-overexpressing HeLa cells, MS cells also showed minimal cell surface binding of FITC–granzyme B, whereas strong plasma membrane binding was seen in MS9-II cells. It was evident from kinetic studies in which FITC–granzyme B uptake was quantitated on a cytofluorograph that granzyme B uptake in MS cells could be partially compensated by longer incubation. Whereas FITC–granzyme B uptake into MS9-II cells had reached maximal steady-state levels within 10 min, the fluorescence of MS cells had increased somewhat but still remained below that of MS9-II cells after 40 min (Fig. 4 b).


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 MPR- L cell fibroblasts. (a) Confocal micrograph of MS (MPR-) and MS9-II (MPR-overexpressing) L cells after exposure to FITC–granzyme B (50 nM) for 60 min at 37°C or without addition of FITC–granzyme B (autofluorescence [AF], bottom). Association of granzyme B with the plasma membrane (m) and in the late endosomal compartment (l) are shown. (b) Cytofluorographic analysis of MS and MS9-II cells after incubation with FITC–granzyme B (50 nM) at 37°C for the times indicated or in the absence of FITC–granzyme B (filled traces).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172645&req=5

fig4: Residual uptake of granzyme B by MPR- L cell fibroblasts. (a) Confocal micrograph of MS (MPR-) and MS9-II (MPR-overexpressing) L cells after exposure to FITC–granzyme B (50 nM) for 60 min at 37°C or without addition of FITC–granzyme B (autofluorescence [AF], bottom). Association of granzyme B with the plasma membrane (m) and in the late endosomal compartment (l) are shown. (b) Cytofluorographic analysis of MS and MS9-II cells after incubation with FITC–granzyme B (50 nM) at 37°C for the times indicated or in the absence of FITC–granzyme B (filled traces).
Mentions: Our second series of experiments used mouse L cell fibroblasts either for MPR expression or overexpressing human MPR as had been studied previously (Nolan et al., 1990; Motyka et al., 2000). The L cells overexpressing MPR (designated MS9-II) became strongly fluorescent after incubation for 10 min at 37°C with FITC–granzyme B and showed a similar pattern of fluorescence when viewed by confocal microscopy as described above for HeLa cells (Fig. 4 a). The staining pattern in most cells also clearly showed a predominant concentrated area of cytoplasmic fluorescence consistent with transport of ligand to the late endosomal compartment. The MPR- MS cells (which had been transfected with vector DNA alone) were less strongly fluorescent at the same time point but still showed significant uptake of FITC–granzyme B compared with autofluorescence controls (Fig. 4 a). As was seen with the K44A-overexpressing HeLa cells, MS cells also showed minimal cell surface binding of FITC–granzyme B, whereas strong plasma membrane binding was seen in MS9-II cells. It was evident from kinetic studies in which FITC–granzyme B uptake was quantitated on a cytofluorograph that granzyme B uptake in MS cells could be partially compensated by longer incubation. Whereas FITC–granzyme B uptake into MS9-II cells had reached maximal steady-state levels within 10 min, the fluorescence of MS cells had increased somewhat but still remained below that of MS9-II cells after 40 min (Fig. 4 b).

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