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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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MPR- L cells are not protected from granzyme B–induced cell death. (a) Clonogenic survival of MS and MS9-II cells after incubation with sublytic pneumolysin (PLO) alone or with various concentrations of granzyme B at 37°C for 90 min. Results are shown as the percentage inhibition of colony growth (mean of three cultures ± standard error at each granzyme concentration) compared with untreated cells. Typically, 75–150 L cell colonies grew when the cells were untreated. The experiment shown is representative of four similar experiments (as described in Materials and methods). (b) 51Cr-labeled MS or MS9-II cells were incubated with sublytic pneumolysin (PLO) alone or together with granzyme B (50 nM) at 37°C for the times indicated. Specific 51Cr release is shown as the mean of triplicate data points ± standard error. The assay is representative of three similar experiments.
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fig6: MPR- L cells are not protected from granzyme B–induced cell death. (a) Clonogenic survival of MS and MS9-II cells after incubation with sublytic pneumolysin (PLO) alone or with various concentrations of granzyme B at 37°C for 90 min. Results are shown as the percentage inhibition of colony growth (mean of three cultures ± standard error at each granzyme concentration) compared with untreated cells. Typically, 75–150 L cell colonies grew when the cells were untreated. The experiment shown is representative of four similar experiments (as described in Materials and methods). (b) 51Cr-labeled MS or MS9-II cells were incubated with sublytic pneumolysin (PLO) alone or together with granzyme B (50 nM) at 37°C for the times indicated. Specific 51Cr release is shown as the mean of triplicate data points ± standard error. The assay is representative of three similar experiments.

Mentions: Our next aim was to compare the susceptibility of MS and MS9-II cells to granzyme B–mediated death. To try to maximize any difference between the two cell lines, they were exposed to limiting concentrations of granzyme B (3 and 12 nM) together with sublytic PLO for 1 h before quantitating cell death in clonogenic assays (Fig. 6 a). At these limiting granzyme B concentrations, the MPR-overexpressing cells were about twofold more susceptible to cell death, but the MPR- MS cells were also sensitive, and there was no difference in colony formation when a slightly higher but conventional granzyme B concentration (50 nM) or higher concentrations (unpublished data) were used. To put the granzyme B concentrations we used in context, concentrations in the 1–5-μM range have been used by other investigators in similar assays (Thomas et al., 2000, 2001). The release of 51Cr from MS cells after exposure to granzyme B and lytic agent was also reduced compared with MS9-II cells, but the difference between the two cell lines diminished with time (Fig. 6 b). After 1 h, the release of 51Cr by MS cells was only 35% that of MS9-II cells, but by 6 h the release by MS cells had reached 60% of MS9-II.


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)

MPR- L cells are not protected from granzyme B–induced cell death. (a) Clonogenic survival of MS and MS9-II cells after incubation with sublytic pneumolysin (PLO) alone or with various concentrations of granzyme B at 37°C for 90 min. Results are shown as the percentage inhibition of colony growth (mean of three cultures ± standard error at each granzyme concentration) compared with untreated cells. Typically, 75–150 L cell colonies grew when the cells were untreated. The experiment shown is representative of four similar experiments (as described in Materials and methods). (b) 51Cr-labeled MS or MS9-II cells were incubated with sublytic pneumolysin (PLO) alone or together with granzyme B (50 nM) at 37°C for the times indicated. Specific 51Cr release is shown as the mean of triplicate data points ± standard error. The assay is representative of three similar experiments.
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Related In: Results  -  Collection

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fig6: MPR- L cells are not protected from granzyme B–induced cell death. (a) Clonogenic survival of MS and MS9-II cells after incubation with sublytic pneumolysin (PLO) alone or with various concentrations of granzyme B at 37°C for 90 min. Results are shown as the percentage inhibition of colony growth (mean of three cultures ± standard error at each granzyme concentration) compared with untreated cells. Typically, 75–150 L cell colonies grew when the cells were untreated. The experiment shown is representative of four similar experiments (as described in Materials and methods). (b) 51Cr-labeled MS or MS9-II cells were incubated with sublytic pneumolysin (PLO) alone or together with granzyme B (50 nM) at 37°C for the times indicated. Specific 51Cr release is shown as the mean of triplicate data points ± standard error. The assay is representative of three similar experiments.
Mentions: Our next aim was to compare the susceptibility of MS and MS9-II cells to granzyme B–mediated death. To try to maximize any difference between the two cell lines, they were exposed to limiting concentrations of granzyme B (3 and 12 nM) together with sublytic PLO for 1 h before quantitating cell death in clonogenic assays (Fig. 6 a). At these limiting granzyme B concentrations, the MPR-overexpressing cells were about twofold more susceptible to cell death, but the MPR- MS cells were also sensitive, and there was no difference in colony formation when a slightly higher but conventional granzyme B concentration (50 nM) or higher concentrations (unpublished data) were used. To put the granzyme B concentrations we used in context, concentrations in the 1–5-μM range have been used by other investigators in similar assays (Thomas et al., 2000, 2001). The release of 51Cr from MS cells after exposure to granzyme B and lytic agent was also reduced compared with MS9-II cells, but the difference between the two cell lines diminished with time (Fig. 6 b). After 1 h, the release of 51Cr by MS cells was only 35% that of MS9-II cells, but by 6 h the release by MS cells had reached 60% of MS9-II.

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