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.

Figure 1.

Residual uptake of granzyme B by K44A mutant dynamin–overexpressing HeLa cells. (a) Confocal micrograph of HeLa cells overexpressing K44A mutant dynamin (−tet) or in which expression of mutant dynamin is suppressed (+tet) after exposure to FITC-transferrin for 120 min. (b) Confocal micrograph of K44A HeLa cells after exposure to FITC–granzyme B (50 nM) for 60 or 120 min. (c) Uptake of FITC–granzyme B (grB) or unglycosylated GFP by HeLa cells overexpressing wild-type (WT) or K44A-mutated dynamin for the times indicated. Uptake of either fluoresceinated molecule into the cell cytoplasm was quantitated by confocal laser scanning microscopy and image analysis as described (Jans, 1995; Jans et al., 1996). The results are expressed as a ratio between cytoplasmic fluorescence (Fc) and extracellular fluorescence (Fmed) ± standard error of the mean. Each data point was derived from measurements on at least 30 cells selected at random after substruction of autofluorescence (as described in Materials and methods).

Figure 1.

Residual uptake of granzyme B by K44A mutant dynamin–overexpressing HeLa cells. (a) Confocal micrograph of HeLa cells overexpressing K44A mutant dynamin (−tet) or in which expression of mutant dynamin is suppressed (+tet) after exposure to FITC-transferrin for 120 min. (b) Confocal micrograph of K44A HeLa cells after exposure to FITC–granzyme B (50 nM) for 60 or 120 min. (c) Uptake of FITC–granzyme B (grB) or unglycosylated GFP by HeLa cells overexpressing wild-type (WT) or K44A-mutated dynamin for the times indicated. Uptake of either fluoresceinated molecule into the cell cytoplasm was quantitated by confocal laser scanning microscopy and image analysis as described (Jans, 1995; Jans et al., 1996). The results are expressed as a ratio between cytoplasmic fluorescence (Fc) and extracellular fluorescence (Fmed) ± standard error of the mean. Each data point was derived from measurements on at least 30 cells selected at random after substruction of autofluorescence (as described in Materials and methods).

Figure 2.

Granzyme B uptake into K44A mutant dynamin–overexpressing HeLa cells is not inhibited by a vast molar excess of M6P. Cytofluorographic analysis of HeLa cells overexpressing K44A mutant dynamin (−tet, bottom) or expressing only wild-type dynamin (+tet, top) after incubation with FITC–granzyme B (50 nM) at 37°C for 20 min or in the absence of FITC–granzyme B (filled traces). Cells were preincubated either with 5 mM M6P (red) or G6P (blue) or without competing monosaccharide (green) for 15 min at 37°C.

Figure 3.

K44A-dynamin–overexpressing HeLa cells are not protected from granzyme B–induced cell death. (a) ⁵¹Cr-labeled HeLa cells overexpressing either K44A- or wild-type (WT) dynamin were incubated with sublytic perforin (Pfp) or pneumolysin (PLO) either alone or together with granzyme B (50 nM) for 90 min at 37°C. Specific ⁵¹Cr release is shown as the mean of triplicate data points ± standard error. The assay is representative of three similar experiments. (b) Clonogenic survival of K44A- or wild-type (WT) dynamin-overexpressing HeLa cells after incubation with sublytic perforin (Pfp) and various concentrations of granzyme B. 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 HeLa colonies became evident when the cells were untreated. The experiment shown is representative of four similar experiments (as described in Materials and methods).

Figure 4.

Residual uptake of granzyme B by MPR-null L cell fibroblasts. (a) Confocal micrograph of MS (MPR-null) and MS9-II (MPR-overexpressing) L cells after exposure to FITC–granzyme B (50 nM) for 60 min at 37°C or without addition of FITC–granzyme B (autofluorescence [AF], bottom). Association of granzyme B with the plasma membrane (m) and in the late endosomal compartment (l) are shown. (b) Cytofluorographic analysis of MS and MS9-II cells after incubation with FITC–granzyme B (50 nM) at 37°C for the times indicated or in the absence of FITC–granzyme B (filled traces).

Figure 4.

Residual uptake of granzyme B by MPR-null L cell fibroblasts. (a) Confocal micrograph of MS (MPR-null) and MS9-II (MPR-overexpressing) L cells after exposure to FITC–granzyme B (50 nM) for 60 min at 37°C or without addition of FITC–granzyme B (autofluorescence [AF], bottom). Association of granzyme B with the plasma membrane (m) and in the late endosomal compartment (l) are shown. (b) Cytofluorographic analysis of MS and MS9-II cells after incubation with FITC–granzyme B (50 nM) at 37°C for the times indicated or in the absence of FITC–granzyme B (filled traces).

Figure 5.

Quantitative kinetic analysis of the uptake of FITC–granzyme B into MS and MS9-II cells. Uptake of FITC–granzyme B (grB) or unglycosylated GFP by MS (MPR-null) and MS9-II (MPR-overexpressing) L cells. The uptake of both fluoresceinated molecules onto the plasma membrane (top) into the cell cytoplasm (middle) or into the late endosomal compartment (bottom) was quantitated by confocal laser scanning microscopy and image analysis as described previously (Jans, 1995). The results are expressed as a ratio between total cytoplasmic fluorescence (Fc), plasma membrane fluorescence (Fpm), or fluorescence of the late endosomal compartment (Flc) and extracellular fluorescence (Fmed) ± standard error of the mean. Each data point was derived from measurements on at least 30 cells selected at random after subtraction of autofluorescence (as described in Materials and methods).

Figure 6.

MPR-null 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.

Figure 6.

MPR-null 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.

Figure 7.

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.

Figure 8.

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.

Figure 8.

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.

Figure 8.

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.

Figure 8.

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.

Figure 9.

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×.