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. 2011 Mar 15;186(6):3497-504.
doi: 10.4049/jimmunol.1003409. Epub 2011 Feb 4.

Granulysin delivered by cytotoxic cells damages endoplasmic reticulum and activates caspase-7 in _target cells

Reena V Saini  1 , Christine Wilson, Michael W Finn, Tianhong Wang, Alan M Krensky, Carol Clayberger
Affiliations

Granulysin delivered by cytotoxic cells damages endoplasmic reticulum and activates caspase-7 in _target cells

Reena V Saini et al. J Immunol. .

Abstract

Granulysin is a human cytolytic molecule present in cytotoxic granules with perforin and granzymes. Recombinant 9-kDa granulysin kills a variety of microbes, including bacteria, yeast, fungi, and parasites, and induces apoptosis in tumor cells by causing intracellular calcium overload, mitochondrial damage, and activation of downstream caspases. Reasoning that granulysin delivered by cytotoxic cells may work in concert with other molecules, we crossed granulysin transgenic (GNLY(+/-)) mice onto perforin (perf)- or granzyme B (gzmb)-deficient mice to examine granulysin-mediated killing in a more physiologic whole-cell system. Splenocytes from these animals were activated in vitro with IL-15 to generate cytolytic T cells and NK cells. Cytotoxic cells expressing granulysin require perforin, but not granzyme B, to cause apoptosis of _targets. Whereas granzyme B induces mitochondrial damage and activates caspases-3 and -9 in _targets, cytotoxic cell-delivered granulysin induces endoplasmic reticulum stress and activates caspase-7 with no effect on mitochondria or caspases-3 and -9. In addition, recombinant granulysin and cell-delivered granulysin activate distinct apoptotic pathways in _target cells. These findings suggest that cytotoxic cells have evolved multiple nonredundant cell death pathways, enabling host defense to counteract escape mechanisms employed by pathogens or tumor cells.

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Conflict of interest statement

Disclosures

The authors have no financial conflicts of interest

Figures

FIGURE 1.
FIGURE 1.
Granulysin expression in murine NK cells. A, Intracellular staining and flow cytometry analysis of granulysin expression kinetics in IL-15–activated splenocytes from GNLY+/− mice over 12 d of culture. B, Expression of granulysin in NK1.1+ and CD8+ cells after 7 d of culture of GNLY+/− and WT splenocytes with IL-15. C, Confocal microscopy of granulysin expression in IL-15–activated NK cells. NK cells were isolated from IL-15–activated splenocytes by negative selection using magnetic beads (Miltenyi). Nuclei are stained with DAPI shown in blue; cells were stained with either preimmune serum (left panel) or anti–9-kDa granulysin Ab (green; center and right panels). Data are representative of three independent experiments.
FIGURE 2.
FIGURE 2.
Cytotoxic cell-delivered granulysin requires perforin, but not granzyme B, for _target cell apoptosis. A and B, IL-15–activated splenocytes were incubated with YAC-1 cells for 4 h at the indicated E:T ratios. C, Effector cells were incubated with YAC-1 cells for 2 and 4 h at 20:1 E:T ratio. 7-AAD incorporation was measured in CFSE-labeled _target cells by flow cytometry. Data are representative of at least three independent experiments ±SEM. *p < 0.05, **p < 0.005. The p values were determined by Student t test (two-tailed, paired/unequal variances) by comparing lysis of gzmb−/− versus gzmb−/−GNLY+/− effectors.
FIGURE 3.
FIGURE 3.
Granulysin delivered by cytotoxic cells does not affect mitochondrial polarization or ROS generation in _target cells. YAC-1 cells were incubated with effectors for 2 h, and ΔΨm loss (A, C) and ROS generation (B, D) were analyzed by staining cells with DiOC6(3) or 2-HE, respectively, and analyzed by flow cytometry after gating on DDAO-SE+ cells. A and B, Representative flow diagrams showing ΔΨm and ROS generation in NK _targets. _target cells without effectors are shown in gray and used as the negative control to set gates. C and D, ΔΨm and ROS generation in YAC-1 _targets after incubation with the indicated effector cells at a 10:1 E:T ratio. E, YAC-1 cells were treated with either medium or recombinant 9-kDa granulysin (25 μM) for 2 h, and ΔΨm and ROS generation was determined as described above. Staurosporin (1 μM) and 2% H2O2 served as positive control for ΔΨm and ROS generation, respectively. DiOC6(3)low or 2-HE+ cells are indicated by the horizontal bars, and the percent is shown by number. Data presented are the average of three independent experiments ±SEM.
FIGURE 4.
FIGURE 4.
Cytotoxic cell delivery of granulysin causes DNA fragmentation and activation of caspase-7. YAC-1 cells were incubated with effector cells from the indicated strains. A, DNA fragmentation was analyzed by TUNEL assay (10:1 E:T ratio, 4 h) in the absence (open bars) or presence (black bars) of 40 μM pan-caspase inhibitor (Q-VD-OPH). *p < 0.05. B and C, Activation of caspase-3 and caspase-9 detected in _target cells after a 2-h incubation at a 10:1 E:T ratio followed by staining with Abs specific for the cleaved form of each enzyme and flow cytometry. D, Activated caspase-7 in YAC-1 _targets at a 12:1 E:T ratio after a 2-h incubation with the indicated cytotoxic cells. The horizontal bars and numbers above indicate percentage of active caspase-7+ cells. E, Caspase-7 activation at various E:T ratios after incubation with cytotoxic cells for 2 h. *p < 0.05. Results are the mean of three different experiments. Error bars represent SEM.
FIGURE 5.
FIGURE 5.
Granulysin delivered by NK cells activates caspase-12 and disrupts ER. A, YAC-1 cells were incubated with effectors, and ER disruption was measured by BFA BODIPY staining. B, YAC-1 cells were incubated with either medium (gray histogram), thapsigargin (10 μM), or 9-kDa granulysin (25 μM), and ER disruption was measured by BFA BODIPY staining. C and D, Caspase-12 activation and cytochrome c release were determined after incubating YAC-1 cells with cytotoxic cells. *p < 0.05. Data presented are the mean of at least three independent experiments ± SEM.
FIGURE 6.
FIGURE 6.
Cytotoxic cell-delivered granulysin causes apoptosis and ER disruption in human _target cells. K562 (A, C, E) and THP-1 (B, D, F) _target cells were incubated with WT and GNLY+/− effectors at various E:T ratios. A and B, Flow-based killing assay for apoptotic cells. C and D, DNA fragmentation assessed by TUNEL assay in the absence or presence (#) of pan-caspase inhibitor (Q-VD-OPH). E and F, ER stress as measured by BFA BODIPY. Results are expressed as the mean ±SEM for three independent experiments. *p < 0.05, **p < 0.005. The p values were determined by Student t test (two-tailed, paired/unequal variances) by comparing values of WT versus GNLY+/− effectors.
FIGURE 7.
FIGURE 7.
Model of apoptosis induced by granulysin or granzyme B. Recombinant 9-kDa granulysin (left panel), cytotoxic cell-delivered granulysin (middle panel), or cytotoxic cell-delivered granzyme B (right panel) activates different apoptotic pathways.
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References

    1. Lieberman J 2003. The ABCs of granule-mediated cytotoxicity: new weapons in the arsenal. Nat. Rev. Immunol 3: 361–370. - PubMed
    1. Lowin B, Hahne M, Mattmann C, and Tschopp J. 1994. Cytolytic T-cell cytotoxicity is mediated through perforin and Fas lytic pathways. Nature 370: 650–652. - PubMed
    1. Kägi D, Ledermann B, Bürki K, Seiler P, Odermatt B, Olsen KJ, Podack ER, Zinkernagel RM, and Hengartner H. 1994. Cytotoxicity mediated by T cells and natural killer cells is greatly impaired in perforin-deficient mice. Nature 369: 31–37. - PubMed
    1. Walsh CM, Matloubian M, Liu CC, Ueda R, Kurahara CG, Christensen JL, Huang MT, Young JD, Ahmed R, and Clark WR. 1994. Immune function in mice lacking the perforin gene. Proc. Natl. Acad. Sci. USA 91: 10854–10858. - PMC - PubMed
    1. Kojima H, Shinohara N, Hanaoka S, Someya-Shirota Y, Takagaki Y, Ohno H, Saito T, Katayama T, Yagita H, Okumura K, et al. 1994. Two distinct pathways of specific killing revealed by perforin mutant cytotoxic T lymphocytes. Immunity 1: 357–364. - PubMed

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