The Role of Phorbol Ester-Sensitive Protein Kinase C Isoforms in Lymphokine-Activated Killer Cell-Mediated Cytotoxicity: Dissociation between Perforin-Dependent and Fas-Dependent Cytotoxicity

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.

235, 461–464 (1997)

RC976733

The Role of Phorbol Ester-Sensitive Protein Kinase C Isoforms in Lymphokine-Activated Killer Cell-Mediated Cytotoxicity: Dissociation between Perforin-Dependent and Fas-Dependent Cytotoxicity Yasushi Ohmi,*,† Akio Ohta,* Yuichi Sasakura,† Naoko Sato,* Takashi Yahata,* Kazuki Santa,* Sonoko Habu,* and Takashi Nishimura*,1 *Department of Immunology, Tokai University School of Medicine, Bohseidai, Isehara 259-11, Japan; and †1st Department of Oral and Maxillofacial Surgery, Kanagawa Dental College, 82 Inaoka, Yokosuka 238, Japan

Received April 30, 1997

Treatment of lymphokine-activated killer (LAK) cells with phorbol ester (PMA) caused the downmodulation of LAK activity concomitantly with the inhibition of serine esterase (SE) release, which has been shown as a marker for perforin-dependent cell-mediated cytotoxicity. The reduction of perforin-dependent LAK activity by PMA-treatment appeared to be due to the disappearance of PMA-sensitive protein kinase C (PKC) isoforms such as PKCa, g, e, u. In contrast, Fasmediated LAK activity was refractory against PMAinduced downregulation. Treatment of LAK cells with PMA caused a disappearance of cytotoxicity against Fas0 L5178Y tumor cells, while cytotoxicity against Fas/ transfectants was not affected by PMA treatment. Moreover, Fas-mediated LAK activity of perforinknockout mice was not inhibited by PMA treatment. These results clearly demonstrated that Fas-mediated cytotoxicity could be dissociated from perforin-mediated cytotoxicity by their different requirement of PMA-sensitive PKC isoforms. q 1997 Academic Press

It has been well demonstrated that protein kinase C (PKC) is one of the important signaling molecules involved in a variety of immune responses such as T cell activation, cytokine production and regulation of cell-mediated cytotoxicity (1-5). PKC consists of several different isoforms with distinct properties (6). PKC a,

bI, bII, and g are designated as classical PKC isoforms, which are Ca2/ dependent and activated by diacylglycerol (DAG) and phorbol 12-myristate 13-acetate (PMA). There are also Ca2/-independent isoforms, like PKC d, e, u, which can be activated by DAG or PMA. The third atypical PKC isoforms are PKC z and l, which are insensitive to Ca2/ and DAG/PMA. Recently, it has been demonstrated that PKC isoforms might play a different role in immune responses (7-9). A useful tool to investigate the role of PKC isoforms is the desensitization of PMA-sensitive isoforms by long term treatment of the cells with PMA (10-12). In previous paper, we initially demonstrated that the long-term treatment of cytotoxic T lymphocytes (CTL) or LAK cells with PMA caused the desensitization of PKC in parallel with the inhibition of cell-mediated cytotoxicity and SE release (5, 13, 14). Therefore, PMAsensitive PKC isoforms were predicted as essential signal transducing molecules in cell-mediated cytotoxicity. However, little has been clarified about the role of PKC isoforms in both perforin and Fas-mediated cytotoxicity (15-17). In this paper, we investigated the role of PMA-sensitive PKC isoforms in both perforin and Fas-dependent cytotoxicity using short-term cultured LAK cells generated from normal and perforin-knock out (PKO) mice. Our results clearly demonstrated that PMA-sensitive PKC isoforms were involved in perforin-dependent LAK-mediated cytotoxicity but not in Fas-dependent LAK cytotoxicity.

1

To whom correspondence should be addressed. Fax: /81 463 94 2976. E-mail: [email protected]. Abbreviations used: PKC, protein kinase C; LAK, lymphokine-acvtivated killer; DAG, diacylglycerol; PMA, phorbol 12-myristate 13acetate; SE, serine esterase; CTL, cytotoxic T lymphocytes; PKO, perforin-knock out; PKO-LAK, LAK cells generated from PKO mouse spleen cells.

MATERIALS AND METHODS Mice. C57BL/6 mice were purchased from Charles River Japan Inc. (Yokohama, Japan). C57BL/6 perforin-knock out (PKO) mice 0006-291X/97 $25.00

461

Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.

AID

BBRC 6733

/

6930$$$$21

06-11-97 08:03:28

bbrcg

AP: BBRC

Vol. 235, No. 3, 1997

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

FIG. 1. The inhibition of LAK cell-mediated cytotoxicity by PMA and PKC inhibitor. (A) LAK cells generated from C57BL/6 mice were incubated with (closed bars) or without (hatched bars) PMA (20 ng/ml) for 24 hr. Then, their cytotoxicity against various target cells was measured by 4 hr-51Cr-release assay. (B) SE release of LAK cells, which were pretreated with none or PMA for 24 hrs, was induced by stimulation with (closed bar) or without (hatched bar) PMA (20 ng/ml) plus A23187 (500 ng/ml) for 2 hrs. SE-release (%) was calculated as described under Materials and Methods. (C) LAK cells were incubated with PMA (24 hrs) or PKC-inhibitor, GF 109203X (5 mM; 30 min) and their cytotoxicity and SE-release were measured. As control, LAK cells were incubated alone (None). The bars represent mean { SE of triplicate samples.

were purchased from Taconic (NY12526) (18). All animals were female and used at 5-8 weeks of age. The generation of LAK cells. LAK cells were generated from spleen cells obtained from C57BL/6 or C57BL/6 PKO mice by culturing with IL-2 (2000 U/ml, kind gift of Shionogi Pharmaceutical Institute Co., Ltd., Osaka, Japan) for 4 days at 377C using 12 well culture plates at the cell density of 5 1 106/well (13). Cytotoxicity assay. The cytotoxicity of killer cells was measured by 4 hr 51Cr-release assay as described previously (5, 13). MBL-2 T lymphoma cells (H-2b), P815 mastocytoma cells (H-2d), YAC-1 T lymphoma cells (H-2a), WEHI 164 fibrosarcoma cells (H-2d), and L5178Y T lymphoma cells (H-2d) were used as target cells for LAK

cells. For investigating Fas-mediated cytotoxicity, L5178Y cells transfected with mouse Fas gene (A1 cells) or mutated Fas gene (F10 cells) were kindly donated from Dr. Shin Yonehara (Kyoto University, Kyoto, Japan) (19). Serine esterase (SE) release assay. SE release was determined by the method described in the previous paper (5, 13). Briefly, cells (2 1 105/ml) were cultured with PMA (20 ng/ml) plus A23187 (500 ng/ ml) for 1-2 hrs in a plastic tube. An 100 ml of culture supernatant was trasferred to 96 well flat-bottomed plate and the SE activity was measured using N-a-benzyloxycarbonyl-L-lysine thiobenzyl ester (Sigma, St. Louis, MO) and Ellman’s reagent (5,5*-dithio-bis(2-nitrobenzoic acid)) to quantitate the released sulfhydryl product reading the OD at 405 nm in a plate reader. Spontaneous SE release was determined by culturing LAK cells alone. Total cellular content of SE activity was determined by lysing of the cells (105) with 0.1% Triton X-100 (500 ml). Specific SE release (%) was equal to: (SE release for LAK cells cultured with PMA plus A23187 - spontaneous SE release) / Total contents of SE activity in LAK cells 1 100. Detection of PKC isoforms by immunoblotting. LAK cells (106) were cultured with or without PMA (20 ng/ml) for 24 hrs, lysed with 5 ml of TNE buffer {150 mM NaCl, 1 mM EDTA, 1% NP-40, 1 mM PMSF, 1 mM aprotinin in 10 mM Tris-HCl buffer (pH 7.5)} and separated on 10% SDS-PAGE. The proteins were transferred to polyvinylidene difluoride (PVDF), and immunoblot analyses were performed using mAbs specific to PKC-a, g, e, u, l, z, which were purchased from Transduction Laboratories Inc. (Lexington, KY). In some experiments, GF 109203X (Sigma, St Louis, MO) (20) was used as a specific inhibitor of PKC at the concentration of 5 mM.

RESULTS AND DISCUSSION

FIG. 2. Disappearance of PMA-sensitive PKC isoforms from LAK cells by treatment with PMA. LAK cells were cultured with (/) or without (0) PMA (20 ng/ml) for 24 hrs. The expression of PKC isoforms were detected by immunoblotting as described under Materials and Methods. (A) PKC-a; (B) PKC-g; (C) PKC-e; (D) PKC-u; (E) PKCl; (F) PKC-z.

LAK cells, which were induced from C57BL/6 mice by culturing with 2000 U/ml IL-2 for 4 days, showed a strong cytotoxicity against a variety of tumor cells. However, the treatment of LAK cells with PMA for 24 hrs caused a great inhibition of their cytotoxicity (Fig. 1A). It was also demonstrated that the SE release from LAK cells induced by PMA plus A23187 was also greatly inhibited by 24 hr-PMA-treatment (Fig. 1B).

462

AID

BBRC 6733

/

6930$$$$21

06-11-97 08:03:28

bbrcg

AP: BBRC

Vol. 235, No. 3, 1997

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

FIG. 3. PMA-sensitive PKC isoforms were not required for Fas-dependent LAK cell-mediated cytotoxicity. (A, B, C) LAK cells generated from normal C57BL/6 mice were treated with (closed circles) or without (open circles) PMA (20 ng/ml) for 24 hrs. Then, their cytotoxicity against Fas0-parental L5178Y cells (A), Fas/-A1 transfectants (B), or mutated Fas/-F10 transfectants (C) was determined by 4 hr-51Crrelease assay. (D) LAK cells induced from PKO mice (PKO-LAK) were treated with (closed bars) or without (hatched bars) PMA for 24 hrs and their cytotoxicity against L5278Y, A1 cells or F10 cells was measured. The bars represents mean { SE of triplicate samples.

Such downmodulation of both LAK activity and SErelease was also induced by PKC inhibitor, GF 109203X (Fig. 1C) though its inhibitory effect was lower than that induced by PMA. Therefore, PMA-induced downmodulation of LAK activity was considered to be derived from the inactivation of PMA-sensitive PKC isoforms. It has been demonstrated that long-term PMA treatment of the cells resulted in the inhibition of PKC activity (desensitization) (5, 10-13). Recently, it was further clarified that the desensitization of PKC activity was due to the downmodulation of PMA-sensitive PKC isoforms (7-9, 12). As shown in Fig. 2, immunoblotting study clearly demonstrated that PMA-treatment of LAK cells caused the disappearance of PKCa, g, e, and u (Fig 2, A-D), which are defined as a PMA-sensitive PKC isoform proteins (7-9). However, PMA-insensitive PKC isoforms such as PKC l, z in LAK cells was not affected by PMA-treatment (Fig. 2E and F). Thus, judging from the evidence that PMA-treatment of LAK cells downmodulated both SE release, LAK activity and

PKC isoforms, PMA-sensitive PKC isoforms appeared to be essential for the induction of perforin-depedent LAK cell-mediated cytotoxicity. It is now well accepted that two distinct mechanisms are involved in cell-mediated cytotoxicity (15-17, 21). One is perforin-dependent cytotoxicity and the other is Fas-dependent cytotoxicity. To determine the role of PMA-sensitive PKC isoforms in Fas-dependent cytotoxicity, we next examined the effect of PMA-treatment on both perforin-dependent and Fas-dependent LAK activity. As shown in Fig. 3, LAK cells totally lost their cytotoxicity against Fas0 L5178Y tumor cells after treatment with PMA for 24 hrs (Fig. 3A). However, the LAK activity against Fas/ A1 cells, which were L5178Y cells transfected with mouse Fas-gene, was not inhibited by PMA-treatment (Fig. 3B). In contrast, LAK activity against mutated Fas/ F10 cells, which were L5178Y cells transfected with mutated mouse Fasgene, was inhibited by PMA-treatment (Fig. 3C). Thus, it was strongly suggested that PMA-sensitive PKC isoforms appeared not to be necessary for Fas-mediated

463

AID

BBRC 6733

/

6930$$$$21

06-11-97 08:03:28

bbrcg

AP: BBRC

Vol. 235, No. 3, 1997

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

LAK cell-mediated cytotoxicity. To demonstrate this hypothesis, we finally examined the effect of PMAtreatment on the cytotoxicity mediated by PKO mousederived LAK cells (Fig.3D). The LAK cells derived from C57BL/6 PKO mice (PKO-LAK) revealed no significant cytotoxicity against LAK-sensitive Fas0-target cells (L5178Y) or mutated Fas/-target cells (F10). However, PKO-LAK cells could lyse Fas/ target cells. Moreover, it was also demonstrated that the cytotoxicity of PKOLAK cells against Fas/-target cells (A1) was not affected by PMA treatment as well as the case of LAK cells induced from normal mouse spleen cells. Taken together with these results and the finding that PMAtreatment also induced the disappearance of PMA-sensitive PKC isoforms from PKO-LAK cells (data not shown), it was strongly suggested that PMA-sensitive PKC isoforms are not required for Fas-mediated LAK cell-mediated cytotoxicity. This paper clearly indicated that perforin-dependent and Fas-dependent LAK cell-mediated cytotoxicity was dissociated by 24 hr-PMA treatment. This phenomenon appeared to be derived from the different roles of PMAsensitive PKC isoforms between perforin-dependent and Fas-dependent cytotoxicity. This result is not inconsistent with recent finding reported by Anel et al. (22) that PMA-sensitive PKC-defected CTL clone showed no significant perforin-mediated cytotoxicity but revealed Fas-mediated cytotoxicity when they were triggered with antigens or immobilized anti-CD3 mAb. Recently, Lee et al. (23) reported that TNF was also a key cytolytic molecule in LAK cell-mediated cytotoxicity in addition to perforin and Fas ligand. We also have an evidence that PKO mouse LAK cells treated with PMA revealed an enhanced cytotoxicity against Fas0 target cells by crosslinking with anti-CD3 mAb (data not shown). Therefore, there are at least three different mechanisms in LAK cell-mediated cytotoxicity; (1) PMA-sensitive PKC isoform dependent perforin-mediated cytotoxicity, (2) PMA-sensitive PKC isoform-independent Fas-FasL-dependent cytotoxicity, (3) PMA-sensitive PKC isoform-independent TNF-dependent cytotoxicty. We are now investigating whether this phenomenon can be expanded into the killing mechanisms of CTL which are freshly generated from T cell-receptor transgenic mice. ACKNOWLEDGMENTS We thank to Dr. Shin Yonehara (Kyoto University, Japan) for providing target cells for Fas-mediated cytotoxicity. This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Area and Grant-in-Aid for Scientific Research (C) from the Ministry

of Education, Science and Culture of Japan, a Research Grant from the Ministry of Health and Welfare and a Grant-in-Aid for IL-12 project from Tokai University School of Medicine.

REFERENCES 1. Nishizuka, Y. (1984) Nature 308, 693–698. 2. Weiss, A. M., Imboden, J., Hardy, K., Manger, B., Terhorst, C., and Stobo, J. (1986) Annu. Rev. Immunol. 4, 593–619. 3. Depper, J. M., Leonard, W. J., Kro¨nke, M., Noguchi, P. D., Cunningham, R. E., Waldmann, T. A., and Greene, W. C. (1984) J. Immunol. 133, 3054–3061. 4. Hirano, T., Fujimoto, K., Teranishi, T., Nishino, N., Onoue, K., Maeda, S., and Shimada, K. (1984) J. Immunol. 132, 2165–2167. 5. Nishimura, T., Burakoff, S. J., and Herrmann, S. H. (1987) J. Immunol. 139, 2888–2891. 6. Nishizuka, Y. (1992) Science 258, 607–614. 7. Terajima, J., Tsutsumi, A., Freire-Moar, J., Cherwinski, H. M., and Ransom, J. T. (1992) Cell. Immunol. 142, 197–206. 8. Tsutsumi, A., Kubo, M., Fujii, H., Freire-Moar, J., Turck, C. W., and Ransom, J. T. (1993) J. Immunol. 150, 1746–1754. 9. Teng, J. M. C., Liu, X.-R., Mills, G. B., and Dupont, B. (1996) J. Immunol. 156, 3222–3232. 10. Collins, M. K. L., and Rozengurt, E. (1982) J. Cell. Physiol. 112, 42–50. 11. Hovis, J. G., Stumpo, D. J., Halsey, D. L., and Blackshear, P. J. (1986) J. Biol. Chem. 261, 10380–10386. 12. Isakov, N., McMahon, P., and Altman, A. (1990) J. Biol. Chem. 265, 2091–2097. 13. Nishimura, T., Burakoff, S. J., and Herrmann, S. H. (1989) J. Immunol. 142, 2155–2161. 14. Nishimura, T., and Ito, T. (1987) Biochem. Biophys. Res. Commun. 146, 1262–1269. 15. Shinkai, Y., Takio, K., and Okumura, K. (1988) Nature 334, 525– 527. 16. Suda, T., Takahashi, T., Golstein, P., and Nagata, S. (1993) Cell 75, 1169–1178. 17. Ka¨gi, D., Vignaux, F., Ledermann, B., Bu¨rki, K., Depraetere, V., Nagata, S., Hengartner, H., and Golstein, P. (1994) Science 265, 528–530. 18. Walsh, C. M., Matloubian, M., Liu, C.-C., Ueda, R., Kurahara, C. G., Christensen, J. L., Huang, M. T. F., Young, J. D., Ahmed, R., and Clark, W. R. (1994) Proc. Natl. Acad. Sci. USA 91, 10854–10858. 19. Arase, H., Arase, N., and Saito, T. (1995) J. Exp. Med. 181, 1235–1238. 20. Toullec, D., Pianetti, P., Coste, H., Bellevergue, P., Grand-Perret, T., Ajakane, M., Baudet, V., Boissin, P., Boursier, E., Loriolle, F., Duhamel, L., Charon, D., and Kirilovski, J. (1991) J. Biol. Chem. 266, 15771–15781. 21. Kojima, H., Shinohara, N., Hanaoka, S., Someya-Shirota, Y., Takagaki, Y., Ohno, H., Saito, T., Katayama, T., Yagita, H., Okumura, K., and Takayama, H. (1994) Immunity 1, 357–364. 22. Anel, A., Simon, A. K., Auphan, N., Buferne, M., Boyer, C., Golstein, P., and Schmitt-Verhulst, A. M. (1995) Eur. J. Immunol. 25, 3381–3387. 23. Lee, R. K., Spielman, J., Zhao, D. Y., Olsen, K. J., and Podack, E. R. (1996) J. Immunol. 157, 1919–1925.

464

AID

BBRC 6733

/

6930$$$$21

06-11-97 08:03:28

bbrcg

AP: BBRC