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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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Growth of MS and MS9-II L cell tumors in immune-deficient BALB/c.scid/scid mice, but rejection by immunocompetent mice and mice lacking perforin expression. MS or MS9-II cells were inoculated under the kidney capsule of groups of four recipients of the mouse strains indicated (as described in Materials and methods). Either 7 or 14 d later, the mice were killed and histological analysis of the kidneys was performed using hematoxalin and eosin staining of the formaldehyde-fixed tissues. Magnifications: (a–j) 200×; (k) 400×. BV, blood vessel; CT, connective tissue; L, mononuclear infiltrate.
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fig8: Growth of MS and MS9-II L cell tumors in immune-deficient BALB/c.scid/scid mice, but rejection by immunocompetent mice and mice lacking perforin expression. MS or MS9-II cells were inoculated under the kidney capsule of groups of four recipients of the mouse strains indicated (as described in Materials and methods). Either 7 or 14 d later, the mice were killed and histological analysis of the kidneys was performed using hematoxalin and eosin staining of the formaldehyde-fixed tissues. Magnifications: (a–j) 200×; (k) 400×. BV, blood vessel; CT, connective tissue; L, mononuclear infiltrate.

Mentions: The previous report by Motyka et al. (2000) indicated that MS cells (H-2k) implanted beneath the kidney capsule of BALB/c mice (H-2d) were not rejected and grew to form tumors, whereas MPR-overexpressing MS9-II cells were rapidly rejected. This finding suggested a pivotal role for MPR in allotransplantation, with possible significant implications for the therapy of allograft rejection. Because of their potential importance, we decided to perform similar experiments with some additional controls. Consistent with the previous study, both MS and MS9-II cells formed rapidly growing tumors when implanted beneath the kidney capsule of immune-compromised BALB/c.scid/scid mice. MS9-II tumors grew somewhat more rapidly and invaded the kidney parenchyma more readily than MS (Fig. 8, a and b). By contrast, both cell lines failed to form tumors in immunocompetent BALB/c mice. 7 d after implantation, small areas of MS9-II tumor could be identified histologically, invariably infiltrated with mononuclear cells, whereas MS tumor could not be identified (Fig. 8, c and d). By 14 d, both tumors were completely eradicated (Fig. 8, e and f). To determine the role of the granule (i.e., perforin/granzyme) pathway in this tumor rejection model, syngeneic perforin-deficient animals were inoculated with the same tumors. Surprisingly, both tumors were again rejected. Once more, transient tumor growth was seen in some animals at 7 d; however, complete rejection of both tumors occurred by day 14 (Fig. 8, g–j), leaving only dilated blood vessels, capsular thickening, and a patchy mononuclear cell infiltrate (Fig. 8 k). To assess tumor rejection in a different strain combination, we repeated the experiment in C57BL/6 (H-2b) and granzyme B–deficient animals on the C57BL/6 background. Once again, both tumors were rejected (unpublished data). Collectively, our findings clearly indicated that (a) MPR is not required for allograft rejection across a major histocompatibility mismatch and (b) rejection of both MS and MS9-II cells occurs independently of the granule pathway. In other experiments (unpublished data), we also found that Fas ligand mutant (gld) mice rejected MS and MS9-II tumors, indicating that cell death was not achieved through the Fas pathway.


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)

Growth of MS and MS9-II L cell tumors in immune-deficient BALB/c.scid/scid mice, but rejection by immunocompetent mice and mice lacking perforin expression. MS or MS9-II cells were inoculated under the kidney capsule of groups of four recipients of the mouse strains indicated (as described in Materials and methods). Either 7 or 14 d later, the mice were killed and histological analysis of the kidneys was performed using hematoxalin and eosin staining of the formaldehyde-fixed tissues. Magnifications: (a–j) 200×; (k) 400×. BV, blood vessel; CT, connective tissue; L, mononuclear infiltrate.
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Related In: Results  -  Collection

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fig8: Growth of MS and MS9-II L cell tumors in immune-deficient BALB/c.scid/scid mice, but rejection by immunocompetent mice and mice lacking perforin expression. MS or MS9-II cells were inoculated under the kidney capsule of groups of four recipients of the mouse strains indicated (as described in Materials and methods). Either 7 or 14 d later, the mice were killed and histological analysis of the kidneys was performed using hematoxalin and eosin staining of the formaldehyde-fixed tissues. Magnifications: (a–j) 200×; (k) 400×. BV, blood vessel; CT, connective tissue; L, mononuclear infiltrate.
Mentions: The previous report by Motyka et al. (2000) indicated that MS cells (H-2k) implanted beneath the kidney capsule of BALB/c mice (H-2d) were not rejected and grew to form tumors, whereas MPR-overexpressing MS9-II cells were rapidly rejected. This finding suggested a pivotal role for MPR in allotransplantation, with possible significant implications for the therapy of allograft rejection. Because of their potential importance, we decided to perform similar experiments with some additional controls. Consistent with the previous study, both MS and MS9-II cells formed rapidly growing tumors when implanted beneath the kidney capsule of immune-compromised BALB/c.scid/scid mice. MS9-II tumors grew somewhat more rapidly and invaded the kidney parenchyma more readily than MS (Fig. 8, a and b). By contrast, both cell lines failed to form tumors in immunocompetent BALB/c mice. 7 d after implantation, small areas of MS9-II tumor could be identified histologically, invariably infiltrated with mononuclear cells, whereas MS tumor could not be identified (Fig. 8, c and d). By 14 d, both tumors were completely eradicated (Fig. 8, e and f). To determine the role of the granule (i.e., perforin/granzyme) pathway in this tumor rejection model, syngeneic perforin-deficient animals were inoculated with the same tumors. Surprisingly, both tumors were again rejected. Once more, transient tumor growth was seen in some animals at 7 d; however, complete rejection of both tumors occurred by day 14 (Fig. 8, g–j), leaving only dilated blood vessels, capsular thickening, and a patchy mononuclear cell infiltrate (Fig. 8 k). To assess tumor rejection in a different strain combination, we repeated the experiment in C57BL/6 (H-2b) and granzyme B–deficient animals on the C57BL/6 background. Once again, both tumors were rejected (unpublished data). Collectively, our findings clearly indicated that (a) MPR is not required for allograft rejection across a major histocompatibility mismatch and (b) rejection of both MS and MS9-II cells occurs independently of the granule pathway. In other experiments (unpublished data), we also found that Fas ligand mutant (gld) mice rejected MS and MS9-II tumors, indicating that cell death was not achieved through the Fas pathway.

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