Int. J . Cancer: 48, 473-475 (1991) 0 1991 Wiley-Liss, Inc .
Publication of the International Union Against Cancer Publication de I’Union Internationale Contre le Cancer
LETTER TO THE EDITOR Dear Sir,
Interferon (Y induces expression of the CD69 activation antigen in human resting NK cells, while interferon y and tumor necrosis factor (Y are ineffective Natural killer (NK) cells constitute a population of lymphocytes which lyse a broad spectrum of tumor cells and virus-infected cells. The lytic activity of NK cells is potentiated both in vitro and in vivo by IL-2 and by other soluble cytokines such as inte~erons(INFs)and TNF (Trinchieri et al., 1984; Ostensen et al., 1987). Activation of the lytic function of NK cells induced by IL-2 is accompanied by a succession of events that lead to production of cytokines, expression of activation markers such as HLA-DR and, finally, cell proliferation (Trinchieri et al., 1984; London et al., 1985). In contrast, the events associated with INF-induced activation of NK cells are at present not completely understood. Cebrian et al. (1988) have described an antigen, defined as CD69, that is acquired early after activation of T lymphocytes by PMA or by mitogenic stimuli. CD69 is rapidly induced also on NK cells after stimulation with IL-2 (Lanier et al., 1988). In the present report we analyzed whether different cytokines, i.e. rINF-a, rINF-y and rTNF-a, can induce the expression of the CD69 early activation antigen in resting NK cells. To this purpose, CD3-, CD16+ NK cells were purified from PBL by a twice-repeated treatment with a mixture of anti-CD3, anti-CD4, anti-CD8 and anti-HLA DR MAb and complement. Purified NK cells were incubated f o r 18 hr in medium only or in a medium containing rINF-a, rINF-y, rTNF-a or rIL-2. Expression of the CD69 activation antigen was evaluated by indirect immunojluorescence using the recently characterized CI .I 8 MAb (Gerosa et al., 1991). Figure 1 shows that incubation of NK cells for 18 hr with rINF-a (1,000 Ulml) induced CD69 antigen expression in the large majority of NK cells to an extent comparable with the expression of CD69 antigen induced by rlL-2 (500 Ulml). In contrast, the expression of CD69 antigen after incubation with rINF-y (2,000 Ulml) or with rTNF-a (1,000 UIml), was comparable to CD69 antigen expression of NK cells incubated with medium alone. The expression of CD69 antigen induced by incubation with rINF-a was dose-dependent: maximal values of CD69+ NK cells (more than 50% CD69+ NK cells above background levels) were obtained at 1.000 Ulml rINF-a; approximately half maximal values were obtained at 100 Ulml rINF-a and low numbers of CD69+ cells were obtained at 20 Ulml of the cytokine. Maximal expression of CD69 antigen was observed after 18 hr of incubation with rINF-a (1,000 Ulml); however, high levels of expression (approximately 70% of the maximal expression) were already observed after 7 hr of incubation with the cytokine, while only minimal increases were detectable after 3 hr of incubation with rlNF-a. In contrast, incubation with rlNF-y or with rTNF-a did not result in the induction of CD69 antigen expression, independently of the dose employed or of the time of incubation (up to 48 hr). It is known that rlNF-y induces expression of HLA class-I1 antigens in diperent human tumor cell lines (Carrel et al., 1986). Therefore, to assess the biological activity of the preparation of rINF-y, the human Hu126 glioma cell line was incubated with difSerent concentrations of rlNF-y ; HLA-DR expression was evaluated after 48 hr. Incubation with rlNF-y strongly increased HLA-DR antigen expression at the various concentrations used (data not shown). In contrast to the activatory effect observed on NK cells, rINF-a did not induce surface expression of CD69 antigen on T cells enriched by separation with neuraminidase-treated sheep red blood cells (Table I ) . Phosphorylation of CD69 antigen occurs following activation with rIL-2 (Lanier et al., 1988). Figure 2 shows that, after stimulation with rINF-a also, phosphorylated CD69 antigen was immunoprecipitated by CI .I8 MAb from detergent-solubilized lysates of 32P-labelledNK cells. Finally, the effect of rlNF-a and rlNF-y was evaluated on the cytolytic activity of NK cells against the K562 target cell line. It was found that rINF-y increased the cytolytic activity of effector cells, although less efficiently than rINF-a (Table I l ) . In conclusion, we found that incubation with rINF-a induced expression of the CD69 antigen, while incubation with rlNF-y or rTNF-a was ineffective. Since much experimental evidence suggests that expression of the CD69 molecule is dependent on the acquisition of an activation state (Cosulich et al., 1987; Cebrian et al., 1988; Lanier et al., 1988), our data directly demonstrate that rINF-a and rIL-2 selectively induce activation of resting NK cells. Several authors described an increased cytolytic activity of NK cells induced by rlNF-a (Trinchieri et al., 1984; Brunda et al., 1986). However, to our knowledge, the ability of rlNF-a to induce sur$ace expression of activation markers has not been reported as yet. The functional role of CD69 antigen has been described for various cell types including T and B lymphocytes and platelets (Cebrihn et al., 1988; Risso et al., 1989; Testi et a[., 1990). As regards NK cells, it has been reported that NK cell clones could not be activated through this antigen to produce TNF (Gerosa et al., 1990). In addition, MAb directed to CD69 antigen failed to induce cytolytic activity of NK cells against P815 target cells (Gerosa, data not shown). Therefore, we suggest that this antigen might not be associated with a functional activity in NK cells. 6To whom correspondenceand reprint requests should be addressed at: Istituto di Scienze Immunologiche,Policlinicodi Borgo Roma, 37100 Verona, Italy.
474
FIGURE1 - Effect of rlNF-a and rINF-y on surface expression of CD69 activation antigen in purified NK cells. NK cells were purified from normal PBL by a double treatment with anti-CD3 (CBT3-M, kindly provided by F. Malavasi, Turin), anti-CD4 and antLCD8 (OKT4 and OKT8, Ortho, Raritan, NJ), and anti-DR (D1-12, kindly provided by R.S. Accolla, Verona) and complement. Dead cells were removed by centrifugation on Ficoll-Hypaque. Purified cells were 90% CD16+, less than 3% CD3+, less than 8% leu-Ml+ and less than 2% OKB22+. Cells were incubated for 18 hr with medium only or with medium containing the following cytokines: rINF-a (1,000 U/ml, kindly obtained from Takeda, Osaka, Japan through I. Suzuki, Verona), rINF-y (2,000 U/ml, kindly obtained from Glaxo, Geneva, Switzerland through R.S. Accolla, Verona), rTNF-a (1,000 U/ml, purchased from Genetech, South San Francisco, CA) and rIL-2 (500 Ulml, kindly provided by Glaxo). CD69 antigen was detected by indirect immunofluorescence with the C1.18 MAb. The background fluorescence is given by cells stained with the fluoresceinated anti-mouse Ig reagent alone. TABLE I - rINF-a DID NOT INDUCE CD69 ANTIGEN EXPRESSION ON PURIFIED T LYMPHOCYTES
TABLE I1 - EFFECT OF rINF-a and rINF-y ON THE CYTOLYTIC ACTIVlTY OF NK CELLS AGAINST THE K562 TARGET CELL LINE
% CD69' cells (mean fluorescence)
medium rINF-a (1 ,000) rINF-y (2,000)
Medium ET
T
NK
5 (3.0) 4 (2.0) 5 (2.6)
20 (19.5) 71 (22.0) 11 (19.1)
T lymphocytes, purified from normal PBL by rosetting with neuraminidase-treated sheep red blood cells, were more than 90% CD3 . NK cells were purified as described in legend to Figure 1, Cells were incubated with the cytokines or with medium alone for 18 hr before testing with CD69 antigen expression by indirect immunofluorescence. +
Exp. 1 EXp. 2
10:1 5 :1 2: 1 10:1 5: 1 2: 1
rINF-n
rINF-y
% 'kr-release
-
45
28 19 36 28 17
71 44 39 65 54
35
56 47 29 47 38 28
NK cells, purified as described in the legend to ~i~~~~ were incubated for 18 with medium only or with medium containing rINF-a (1 ,OOO U/ml)or rINF-y (2,000 U/ml) before testing for cytolytic activity.
Yours sincerely, Franca GEROSA'.6, Maria SCARDONI', Marina T O M M A S I 'Claudia , B E N A T I ' ,Luca Massimo LIBONATI~, Giuseppe TRIDENTE'and Giuseppe CARRA~
SNELL14,
Giorgio GANDIN13,
415
KO
-32 .26
A
B
FIGURE2 - Autoradiograph of 11% SDS-PAGE of 32P-labelled C1.18-specific immunoprecipitates (lane B), or control Ig (lane A), under reducing conditions, from NK cells activated for 18 hr with rINF-a (1,000 U/ml).
‘Istituto di Scienze lmmunologiche and 21stituto di Chimica Biologica, Universita di Verona; 3Centro Trasfusionale, Policlinic0 di Borgo Roma, 37100 Verona; and 4Clinicu Medica I , Universita di Parma, Parma, Italy. February 14, 1991. ACKNOWLEDGEMENTS
This work was supported in part by CNR projects “Oncology” and “Biotechnology and Bioinstrumentations”, by the Minister0 Pubblica Istruzione and by the Regione Veneto. REFERENCES BRUNDA,M.J., TARNOWSKI, D. and DAVATELIS, V., Interaction of recombinant interferons with recombinant interleukin-2: differential effects on natural killer cell activity and interleukin-2-activated killer cells. I n f . J . Cancer, 37, 787-793 (1986). CARREL,S., MACH,J.P., FERREMI, P., GIUFFR~, L. and ACCOLLA, R.S., Expression of Class I1 antigens on nonhematopoietic tumor cells. In: B.G. Solheim, E. Moller and S. Ferrone (eds.), HLA Class I1 antigens. A comprehensive review of strucfure and function, pp. 412-428, Springer, New York (1986). CEBRIAN,M., YAGUE, E., R I N C ~ NM., , L6PEZ-BOTET, M., DE LANDAZURI,M.O. and SANCHEZ-MADRID, F., Triggering of T cell proliferation through AIM, an activation inducer molecule expressed on activated human lymphocytes. J . exp. Med., 168, 1621-1637 (1988). COSULICH,M.E., RUBARTELLI, A , , RISSO, A,, COZZOLINO,F. and BARGELLESI, A,, Functional characterization of an antigen involved in an early step of T-cell activation. Proc. naf. Acad. Sci. (Wash.), 84, 42054209 (1987). F., TOMMASI, M., SCARDONI, M., ACCOLLA, R.S., POZZAN,T., GEROSA, LIBONATI,M., TRIDENTE,G. and CARRA,G., Structural analysis of the CD69 early activation antigen by two monoclonal antibodies directed to different epitopes. Mol. Immunol.,28, 159-168 (1991).
LANIER,L.L., BUCK,D.W., RHODES,L., DING,A,, EVANS,E., BARNEY, C. and PHILLIPS,J.H., Interleukin 2 activation of natural killer cells rapidly induces the expression and phosphorylation of the Leu-23 activation antigen. J. exp. Med., 167, 1572-1585 (1988). LONDON,L., PERUSSIA, B. and TRINCHIERI, G., Induction of proliferation in vitro of resting human natural killer cells: expression of surface activation antigens. J. Immunol., 134, 718-727 (1985). @STENSEN, M.E., THIELE,D.L. and LIPSKY,P.E., Tumor necrosis factor-a enhances cytolytic activity of human natural killer cells. J. Immunol., 138, 4185-4191 (1987). RISSO, A., COSULICH,M.E., RUBARTELLI,A., MAZZA, M.R. and BARGELLESI, A., MLR3 molecule is an activation antigen shared by human B, T lymphocytes and T cell precursors. Europ. J . Immunol., 19, 323-328 (1989). TESTI,R., PULCINELLI,F., FRATI,L., GAZZANIGA, P.P. and SANTONI, A., CD69 is expressed on platelets and mediates platelet activation and aggregation. J. exp. Med., 172, 701-707 (1990). G., MATSUMOTO-KOBAYASHI, M., CLARK,S.C., SEEHRA, TRINCHIERI, J., LONDON,L. and PERUSSIA, B., Response of resting human peripheral blood natural killer cells to interleukin 2. J. exp. Med., 160, 1147-1169 (1984).
Publication of the International Union Against Cancer Publication de I’Union Internationale Contre le Cancer
LETTER TO THE EDITOR Dear Sir,
Interferon (Y induces expression of the CD69 activation antigen in human resting NK cells, while interferon y and tumor necrosis factor (Y are ineffective Natural killer (NK) cells constitute a population of lymphocytes which lyse a broad spectrum of tumor cells and virus-infected cells. The lytic activity of NK cells is potentiated both in vitro and in vivo by IL-2 and by other soluble cytokines such as inte~erons(INFs)and TNF (Trinchieri et al., 1984; Ostensen et al., 1987). Activation of the lytic function of NK cells induced by IL-2 is accompanied by a succession of events that lead to production of cytokines, expression of activation markers such as HLA-DR and, finally, cell proliferation (Trinchieri et al., 1984; London et al., 1985). In contrast, the events associated with INF-induced activation of NK cells are at present not completely understood. Cebrian et al. (1988) have described an antigen, defined as CD69, that is acquired early after activation of T lymphocytes by PMA or by mitogenic stimuli. CD69 is rapidly induced also on NK cells after stimulation with IL-2 (Lanier et al., 1988). In the present report we analyzed whether different cytokines, i.e. rINF-a, rINF-y and rTNF-a, can induce the expression of the CD69 early activation antigen in resting NK cells. To this purpose, CD3-, CD16+ NK cells were purified from PBL by a twice-repeated treatment with a mixture of anti-CD3, anti-CD4, anti-CD8 and anti-HLA DR MAb and complement. Purified NK cells were incubated f o r 18 hr in medium only or in a medium containing rINF-a, rINF-y, rTNF-a or rIL-2. Expression of the CD69 activation antigen was evaluated by indirect immunojluorescence using the recently characterized CI .I 8 MAb (Gerosa et al., 1991). Figure 1 shows that incubation of NK cells for 18 hr with rINF-a (1,000 Ulml) induced CD69 antigen expression in the large majority of NK cells to an extent comparable with the expression of CD69 antigen induced by rlL-2 (500 Ulml). In contrast, the expression of CD69 antigen after incubation with rINF-y (2,000 Ulml) or with rTNF-a (1,000 UIml), was comparable to CD69 antigen expression of NK cells incubated with medium alone. The expression of CD69 antigen induced by incubation with rINF-a was dose-dependent: maximal values of CD69+ NK cells (more than 50% CD69+ NK cells above background levels) were obtained at 1.000 Ulml rINF-a; approximately half maximal values were obtained at 100 Ulml rINF-a and low numbers of CD69+ cells were obtained at 20 Ulml of the cytokine. Maximal expression of CD69 antigen was observed after 18 hr of incubation with rINF-a (1,000 Ulml); however, high levels of expression (approximately 70% of the maximal expression) were already observed after 7 hr of incubation with the cytokine, while only minimal increases were detectable after 3 hr of incubation with rlNF-a. In contrast, incubation with rlNF-y or with rTNF-a did not result in the induction of CD69 antigen expression, independently of the dose employed or of the time of incubation (up to 48 hr). It is known that rlNF-y induces expression of HLA class-I1 antigens in diperent human tumor cell lines (Carrel et al., 1986). Therefore, to assess the biological activity of the preparation of rINF-y, the human Hu126 glioma cell line was incubated with difSerent concentrations of rlNF-y ; HLA-DR expression was evaluated after 48 hr. Incubation with rlNF-y strongly increased HLA-DR antigen expression at the various concentrations used (data not shown). In contrast to the activatory effect observed on NK cells, rINF-a did not induce surface expression of CD69 antigen on T cells enriched by separation with neuraminidase-treated sheep red blood cells (Table I ) . Phosphorylation of CD69 antigen occurs following activation with rIL-2 (Lanier et al., 1988). Figure 2 shows that, after stimulation with rINF-a also, phosphorylated CD69 antigen was immunoprecipitated by CI .I8 MAb from detergent-solubilized lysates of 32P-labelledNK cells. Finally, the effect of rlNF-a and rlNF-y was evaluated on the cytolytic activity of NK cells against the K562 target cell line. It was found that rINF-y increased the cytolytic activity of effector cells, although less efficiently than rINF-a (Table I l ) . In conclusion, we found that incubation with rINF-a induced expression of the CD69 antigen, while incubation with rlNF-y or rTNF-a was ineffective. Since much experimental evidence suggests that expression of the CD69 molecule is dependent on the acquisition of an activation state (Cosulich et al., 1987; Cebrian et al., 1988; Lanier et al., 1988), our data directly demonstrate that rINF-a and rIL-2 selectively induce activation of resting NK cells. Several authors described an increased cytolytic activity of NK cells induced by rlNF-a (Trinchieri et al., 1984; Brunda et al., 1986). However, to our knowledge, the ability of rlNF-a to induce sur$ace expression of activation markers has not been reported as yet. The functional role of CD69 antigen has been described for various cell types including T and B lymphocytes and platelets (Cebrihn et al., 1988; Risso et al., 1989; Testi et a[., 1990). As regards NK cells, it has been reported that NK cell clones could not be activated through this antigen to produce TNF (Gerosa et al., 1990). In addition, MAb directed to CD69 antigen failed to induce cytolytic activity of NK cells against P815 target cells (Gerosa, data not shown). Therefore, we suggest that this antigen might not be associated with a functional activity in NK cells. 6To whom correspondenceand reprint requests should be addressed at: Istituto di Scienze Immunologiche,Policlinicodi Borgo Roma, 37100 Verona, Italy.
474
FIGURE1 - Effect of rlNF-a and rINF-y on surface expression of CD69 activation antigen in purified NK cells. NK cells were purified from normal PBL by a double treatment with anti-CD3 (CBT3-M, kindly provided by F. Malavasi, Turin), anti-CD4 and antLCD8 (OKT4 and OKT8, Ortho, Raritan, NJ), and anti-DR (D1-12, kindly provided by R.S. Accolla, Verona) and complement. Dead cells were removed by centrifugation on Ficoll-Hypaque. Purified cells were 90% CD16+, less than 3% CD3+, less than 8% leu-Ml+ and less than 2% OKB22+. Cells were incubated for 18 hr with medium only or with medium containing the following cytokines: rINF-a (1,000 U/ml, kindly obtained from Takeda, Osaka, Japan through I. Suzuki, Verona), rINF-y (2,000 U/ml, kindly obtained from Glaxo, Geneva, Switzerland through R.S. Accolla, Verona), rTNF-a (1,000 U/ml, purchased from Genetech, South San Francisco, CA) and rIL-2 (500 Ulml, kindly provided by Glaxo). CD69 antigen was detected by indirect immunofluorescence with the C1.18 MAb. The background fluorescence is given by cells stained with the fluoresceinated anti-mouse Ig reagent alone. TABLE I - rINF-a DID NOT INDUCE CD69 ANTIGEN EXPRESSION ON PURIFIED T LYMPHOCYTES
TABLE I1 - EFFECT OF rINF-a and rINF-y ON THE CYTOLYTIC ACTIVlTY OF NK CELLS AGAINST THE K562 TARGET CELL LINE
% CD69' cells (mean fluorescence)
medium rINF-a (1 ,000) rINF-y (2,000)
Medium ET
T
NK
5 (3.0) 4 (2.0) 5 (2.6)
20 (19.5) 71 (22.0) 11 (19.1)
T lymphocytes, purified from normal PBL by rosetting with neuraminidase-treated sheep red blood cells, were more than 90% CD3 . NK cells were purified as described in legend to Figure 1, Cells were incubated with the cytokines or with medium alone for 18 hr before testing with CD69 antigen expression by indirect immunofluorescence. +
Exp. 1 EXp. 2
10:1 5 :1 2: 1 10:1 5: 1 2: 1
rINF-n
rINF-y
% 'kr-release
-
45
28 19 36 28 17
71 44 39 65 54
35
56 47 29 47 38 28
NK cells, purified as described in the legend to ~i~~~~ were incubated for 18 with medium only or with medium containing rINF-a (1 ,OOO U/ml)or rINF-y (2,000 U/ml) before testing for cytolytic activity.
Yours sincerely, Franca GEROSA'.6, Maria SCARDONI', Marina T O M M A S I 'Claudia , B E N A T I ' ,Luca Massimo LIBONATI~, Giuseppe TRIDENTE'and Giuseppe CARRA~
SNELL14,
Giorgio GANDIN13,
415
KO
-32 .26
A
B
FIGURE2 - Autoradiograph of 11% SDS-PAGE of 32P-labelled C1.18-specific immunoprecipitates (lane B), or control Ig (lane A), under reducing conditions, from NK cells activated for 18 hr with rINF-a (1,000 U/ml).
‘Istituto di Scienze lmmunologiche and 21stituto di Chimica Biologica, Universita di Verona; 3Centro Trasfusionale, Policlinic0 di Borgo Roma, 37100 Verona; and 4Clinicu Medica I , Universita di Parma, Parma, Italy. February 14, 1991. ACKNOWLEDGEMENTS
This work was supported in part by CNR projects “Oncology” and “Biotechnology and Bioinstrumentations”, by the Minister0 Pubblica Istruzione and by the Regione Veneto. REFERENCES BRUNDA,M.J., TARNOWSKI, D. and DAVATELIS, V., Interaction of recombinant interferons with recombinant interleukin-2: differential effects on natural killer cell activity and interleukin-2-activated killer cells. I n f . J . Cancer, 37, 787-793 (1986). CARREL,S., MACH,J.P., FERREMI, P., GIUFFR~, L. and ACCOLLA, R.S., Expression of Class I1 antigens on nonhematopoietic tumor cells. In: B.G. Solheim, E. Moller and S. Ferrone (eds.), HLA Class I1 antigens. A comprehensive review of strucfure and function, pp. 412-428, Springer, New York (1986). CEBRIAN,M., YAGUE, E., R I N C ~ NM., , L6PEZ-BOTET, M., DE LANDAZURI,M.O. and SANCHEZ-MADRID, F., Triggering of T cell proliferation through AIM, an activation inducer molecule expressed on activated human lymphocytes. J . exp. Med., 168, 1621-1637 (1988). COSULICH,M.E., RUBARTELLI, A , , RISSO, A,, COZZOLINO,F. and BARGELLESI, A,, Functional characterization of an antigen involved in an early step of T-cell activation. Proc. naf. Acad. Sci. (Wash.), 84, 42054209 (1987). F., TOMMASI, M., SCARDONI, M., ACCOLLA, R.S., POZZAN,T., GEROSA, LIBONATI,M., TRIDENTE,G. and CARRA,G., Structural analysis of the CD69 early activation antigen by two monoclonal antibodies directed to different epitopes. Mol. Immunol.,28, 159-168 (1991).
LANIER,L.L., BUCK,D.W., RHODES,L., DING,A,, EVANS,E., BARNEY, C. and PHILLIPS,J.H., Interleukin 2 activation of natural killer cells rapidly induces the expression and phosphorylation of the Leu-23 activation antigen. J. exp. Med., 167, 1572-1585 (1988). LONDON,L., PERUSSIA, B. and TRINCHIERI, G., Induction of proliferation in vitro of resting human natural killer cells: expression of surface activation antigens. J. Immunol., 134, 718-727 (1985). @STENSEN, M.E., THIELE,D.L. and LIPSKY,P.E., Tumor necrosis factor-a enhances cytolytic activity of human natural killer cells. J. Immunol., 138, 4185-4191 (1987). RISSO, A., COSULICH,M.E., RUBARTELLI,A., MAZZA, M.R. and BARGELLESI, A., MLR3 molecule is an activation antigen shared by human B, T lymphocytes and T cell precursors. Europ. J . Immunol., 19, 323-328 (1989). TESTI,R., PULCINELLI,F., FRATI,L., GAZZANIGA, P.P. and SANTONI, A., CD69 is expressed on platelets and mediates platelet activation and aggregation. J. exp. Med., 172, 701-707 (1990). G., MATSUMOTO-KOBAYASHI, M., CLARK,S.C., SEEHRA, TRINCHIERI, J., LONDON,L. and PERUSSIA, B., Response of resting human peripheral blood natural killer cells to interleukin 2. J. exp. Med., 160, 1147-1169 (1984).
