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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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Adoptive transfer of anti–MS9-II antiserum mediates graft rejection. MS9-II cells were inoculated under the kidney capsule of groups of four BALB/c.scid.scid recipients (as described in Materials and methods). The groups received nonimmune C3H serum (a) or anti–MS9-II antiserum as described in Materials and methods. Magnification, 200×.
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fig9: Adoptive transfer of anti–MS9-II antiserum mediates graft rejection. MS9-II cells were inoculated under the kidney capsule of groups of four BALB/c.scid.scid recipients (as described in Materials and methods). The groups received nonimmune C3H serum (a) or anti–MS9-II antiserum as described in Materials and methods. Magnification, 200×.

Mentions: Since rejection of both MS and MS9-II tumors by immunocompetent mice did not require either the perforin/granzyme or Fas pathways, we hypothesized that rejection in this model might be antibody mediated. Consistent with this hypothesis, when MS9-II cells were used to inoculate syngeneic C3H mice, the tumors also failed to grow and the mice developed high titer antibodies that reacted with MS9-II cells and at lower but significant levels with MS cells. Typically, the antibody titers by indirect immunofluorescence were ∼1/1,600 on MS9-II cells and 1/200 on MS cells (unpublished data). We concluded that human MPR expressed on MS9-II cells is a target of the anti–MS9-II antibody response. As a result of long term culture in vitro, it is also likely that MS and MS9-II express other immunogenic antigens responsible for perforin/granzyme B–independent rejection of MS cells. To definitively demonstrate a role for antibody in tumor rejection in vivo, we adoptively transferred serum from C3H mice that had been challenged twice with MS9-II cells into four BALB/c.scid.scid mice 1 d before, and on the same day as they were implanted with MS9-II cells under the kidney capsule. Control mice received serum from unimmunized C3H mice. 7 d later, tumor growth was clearly evident in four out of four control mice; however, three out of four mice pretreated with anti–MS9-II antiserum showed complete absence of tumor, whereas the fourth mouse showed significantly reduced tumor mass (representative data in Fig. 9). These results clearly indicated that rejection of MS9-II was antibody mediated in immunocompetent animals and related to expression of immunogenic (human) MPR by these cells. Therefore, MPR played no role in cell-mediated tumor allograft rejection in these or the previously reported studies.


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)

Adoptive transfer of anti–MS9-II antiserum mediates graft rejection. MS9-II cells were inoculated under the kidney capsule of groups of four BALB/c.scid.scid recipients (as described in Materials and methods). The groups received nonimmune C3H serum (a) or anti–MS9-II antiserum as described in Materials and methods. Magnification, 200×.
© Copyright Policy
Related In: Results  -  Collection

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

fig9: Adoptive transfer of anti–MS9-II antiserum mediates graft rejection. MS9-II cells were inoculated under the kidney capsule of groups of four BALB/c.scid.scid recipients (as described in Materials and methods). The groups received nonimmune C3H serum (a) or anti–MS9-II antiserum as described in Materials and methods. Magnification, 200×.
Mentions: Since rejection of both MS and MS9-II tumors by immunocompetent mice did not require either the perforin/granzyme or Fas pathways, we hypothesized that rejection in this model might be antibody mediated. Consistent with this hypothesis, when MS9-II cells were used to inoculate syngeneic C3H mice, the tumors also failed to grow and the mice developed high titer antibodies that reacted with MS9-II cells and at lower but significant levels with MS cells. Typically, the antibody titers by indirect immunofluorescence were ∼1/1,600 on MS9-II cells and 1/200 on MS cells (unpublished data). We concluded that human MPR expressed on MS9-II cells is a target of the anti–MS9-II antibody response. As a result of long term culture in vitro, it is also likely that MS and MS9-II express other immunogenic antigens responsible for perforin/granzyme B–independent rejection of MS cells. To definitively demonstrate a role for antibody in tumor rejection in vivo, we adoptively transferred serum from C3H mice that had been challenged twice with MS9-II cells into four BALB/c.scid.scid mice 1 d before, and on the same day as they were implanted with MS9-II cells under the kidney capsule. Control mice received serum from unimmunized C3H mice. 7 d later, tumor growth was clearly evident in four out of four control mice; however, three out of four mice pretreated with anti–MS9-II antiserum showed complete absence of tumor, whereas the fourth mouse showed significantly reduced tumor mass (representative data in Fig. 9). These results clearly indicated that rejection of MS9-II was antibody mediated in immunocompetent animals and related to expression of immunogenic (human) MPR by these cells. Therefore, MPR played no role in cell-mediated tumor allograft rejection in these or the previously reported studies.

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