title: A clathrin/dynamin- and mannose-6-phosphate receptor-independent pathway for granzyme B-induced cell death.
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-null 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.
authors.affiliation: Cancer Immunology Laboratory, Peter MacCallum Cancer Institute, Melbourne 8006, Australia. j.trapani@pmci.unimelb.edu.au
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Trapani
authors.forename: Vivien R
authors.initials: VR
authors.lastname: Sutton
authors.forename: Kevin Y T
authors.initials: KY
authors.lastname: Thia
authors.forename: Yu Qin
authors.initials: YQ
authors.lastname: Li
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Froelich
authors.forename: David A
authors.initials: DA
authors.lastname: Jans
authors.forename: Mauro S
authors.initials: MS
authors.lastname: Sandrin
authors.forename: Kylie A
authors.initials: KA
authors.lastname: Browne
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000276:immunology*; D002462:Cell Membrane / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism; D002966:Clathrin / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D034281:Dynamins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects / Q000276:immunology*; D005260:Female; D006084:Graft Rejection / Q000235:genetics / Q000276:immunology; D053804:Granzymes; D006367:HeLa Cells; D006801:Humans; D007694:Killer Cells, Natural / Q000201:enzymology* / Q000276:immunology; D008297:Male; D008562:Membrane Glycoproteins / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009154:Mutation / Q000235:genetics; D009369:Neoplasms / Q000276:immunology / Q000378:metabolism; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins; D017527:Receptor, IGF Type 2 / Q000172:deficiency* / Q000187:drug effects / Q000235:genetics; D012697:Serine Endopeptidases / Q000172:deficiency / Q000276:immunology* / Q000494:pharmacology; D013602:T-Lymphocytes, Cytotoxic / Q000201:enzymology* / Q000276:immunology
chemical_list: D002966:Clathrin; D008562:Membrane Glycoproteins; D052899:Pore Forming Cytotoxic Proteins; D017527:Receptor, IGF Type 2; D054353:Perforin; C509213:GZMB protein, human; D053804:Granzymes; C509214:Gzmb protein, mouse; D012697:Serine Endopeptidases; D034281:Dynamins