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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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Reduced DNA fragmentation but not membrane permeability in MPR-deficient L cells killed by alloreactive CTL. MS and MS9-II cells (H-2k) were prelabeled simultaneously with 51Cr and 125I-deoxyuridine, then incubated at 37°C for 4 h at the effector-to-target cell ratios indicated with alloreactive CTL raised in BALB/c (H-2d), C57BL/6, or C57BL/6.grB−/− (H-2b) mice (as described in Materials and methods). Specific release of 51Cr (indicating cell membrane permeability) and 125I-DNA (DNA fragmentation) were estimated as the mean of triplicate data points ±standard error. The experiment shown is representative of four similar experiments.
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fig7: Reduced DNA fragmentation but not membrane permeability in MPR-deficient L cells killed by alloreactive CTL. MS and MS9-II cells (H-2k) were prelabeled simultaneously with 51Cr and 125I-deoxyuridine, then incubated at 37°C for 4 h at the effector-to-target cell ratios indicated with alloreactive CTL raised in BALB/c (H-2d), C57BL/6, or C57BL/6.grB−/− (H-2b) mice (as described in Materials and methods). Specific release of 51Cr (indicating cell membrane permeability) and 125I-DNA (DNA fragmentation) were estimated as the mean of triplicate data points ±standard error. The experiment shown is representative of four similar experiments.

Mentions: The susceptibility of MPR-overexpressing and MPR- L cells was also examined in response to intact allogenic CTL. Effector cells raised in both BALB/c (d anti-k) or C57BL/6 (b anti-k) mice induced the equivalent release of 51Cr release from MS and MS9-II cells over a 4-h assay. However, MPR- MS cells demonstrated significantly reduced DNA fragmentation (Fig. 7). DNA damage in both populations of L cells was virtually totally dependent on granzyme B, since granzyme B–deficient C57BL/6 CTL were unable to induce any release of 125I-DNA from either cell line. Overall, our results were consistent with a role for the granzyme B-MPR pathway in facilitating DNA fragmentation rather than significantly influencing cell survival.


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)

Reduced DNA fragmentation but not membrane permeability in MPR-deficient L cells killed by alloreactive CTL. MS and MS9-II cells (H-2k) were prelabeled simultaneously with 51Cr and 125I-deoxyuridine, then incubated at 37°C for 4 h at the effector-to-target cell ratios indicated with alloreactive CTL raised in BALB/c (H-2d), C57BL/6, or C57BL/6.grB−/− (H-2b) mice (as described in Materials and methods). Specific release of 51Cr (indicating cell membrane permeability) and 125I-DNA (DNA fragmentation) were estimated as the mean of triplicate data points ±standard error. The experiment shown is representative of four similar experiments.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: Reduced DNA fragmentation but not membrane permeability in MPR-deficient L cells killed by alloreactive CTL. MS and MS9-II cells (H-2k) were prelabeled simultaneously with 51Cr and 125I-deoxyuridine, then incubated at 37°C for 4 h at the effector-to-target cell ratios indicated with alloreactive CTL raised in BALB/c (H-2d), C57BL/6, or C57BL/6.grB−/− (H-2b) mice (as described in Materials and methods). Specific release of 51Cr (indicating cell membrane permeability) and 125I-DNA (DNA fragmentation) were estimated as the mean of triplicate data points ±standard error. The experiment shown is representative of four similar experiments.
Mentions: The susceptibility of MPR-overexpressing and MPR- L cells was also examined in response to intact allogenic CTL. Effector cells raised in both BALB/c (d anti-k) or C57BL/6 (b anti-k) mice induced the equivalent release of 51Cr release from MS and MS9-II cells over a 4-h assay. However, MPR- MS cells demonstrated significantly reduced DNA fragmentation (Fig. 7). DNA damage in both populations of L cells was virtually totally dependent on granzyme B, since granzyme B–deficient C57BL/6 CTL were unable to induce any release of 125I-DNA from either cell line. Overall, our results were consistent with a role for the granzyme B-MPR pathway in facilitating DNA fragmentation rather than significantly influencing cell survival.

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