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Effect of interleukin 2 on the inhibition of human natural killer activity by monolayer cells
CELLULAR
IMMUNOLOGY
110,209-2
17 ( 1987)
Effect of lnterleukin 2 on the Inhibition of Human Natural Killer Activity by Monolayer Cells’ M. HEISKALA AND T. TIMONEN Department of Pathology, University ofHelsinki, Haartmaninkatu 3, SF-00290 Helsinki, Finland Received January 5, 1987; acceptedAugust lo,1987 We have previously shown that human endogenous natural killer activity against K562 is inhibited by primary cultures of natural killer-resistant monolayer target cells. In this study we have analyzed the sensitivity of activated killer cells to this inhibitory effect. Interleukin-2 (IL2) when present during an 18-hr contact of peripheral blood lymphocytes with monolayers, did not affect the inhibition of natural killer cell activity. Pretreatment of effector cells with IL-2 for 24-62 hr before the contact with monolayer cells eradicated the inhibition causedby malignant cells, benign cells remaining inhibitory. The IL-2-pretreated effector cells killed preferentially malignant target cells, although significant cytotoxicity was also detectable against benign cell cultures. The results indicate that activation of killer cells in vitro by IL-2 involves the desensitization of effector cells to the inhibitory signals of target cells, and that the selectivity of IL-2activated killer cells toward malignant target cells involves weaker inhibition of activated killer Cells by malignant Cek. 0 1987 Academic Press,Inc.
INTRODUCTION Natural killer cells (NK cells) kill primarily in vitro propagated target cells of malignant origin (1, 2). The reasons for the insensitivity of freshly isolated target cells to NK reactivity are unknown. We have previously shown that primary cultures of both benign and malignant monolayer cells significantly inhibit NK activity, as measured by lysis of K562 target cells by both peripheral blood lymphocytes and purified large granular lymphocytes (LGL) (3). As there is an inverse correlation between the inhibitory capacity and the NK sensitivity, the inhibition of NK activity by target cells may be an important mechanism by which some cells are protected from NK cells. Activated killer cells are more potent effector cells than endogenous NK cells, and they kill also NK resistant target cells, including freshly isolated malignant cells (46). Killer cells activated by interleukin-2 (IL-2), the lymphokine activated killer cells (6), lyse preferentially malignant target cells, and the basis of this selectivity is unknown. In this study we have analyzed the sensitivity of IL-Zactivated killer cells to the inhibitory signals of solid tissue-derived target cells of both malignant and benign ’ This work was supported by the Ida Montin Foundation, the Finnish Cancer Society, the &rid Juselius Foundation, Orion Pharmaceuticals Ltd, and the National Council for Medical Sciences, Academy of Finland.
209 0008-8749/87 $3.00 Copyright 0 1987 by Academic Press,Inc. All rights of reproduction in any form reserved.
210
HEISKALA AND TIMONEN
origin. The results show that while endogenous NK activity is inhibited by monolayers of both benign and malignant cells, the IL-2-activated killer cells are preferentially inhibited by benign target cells. Thus, in addition to the endogenous NK cell system the target cell-mediated inactivation of cytotoxic cells appears to be also involved in the selectivity of activated killer cells. MATERIALS AND METHODS
Cell culture conditions. Cells were cultured at 37°C in humidified air with 5% CO2 in RPM1 1640 medium (GIBCO), supplemented with heat inactivated fetal calf serum (lo%, FCS, KC Biological, Lenexa, KS), 100 pg/ml gentamycin, 0.29 mg/ml glutamine, and 0.1 M Hepes. Inhibition tests, cytotoxicity assays,and IL-2 stimulations were performed in medium supplemented with either 10% human AB serum (Finnish Red Cross Blood Bank) or 10%FCS. Effector cells. Peripheral blood mononuclear cells were isolated from buffy coats of 400 ml of venous blood from healthy donors (Finnish Red Cross Blood Bank) by Ficoll-Isopaque gradient centrifugation (7). They were subsequently incubated in glass flasks and run through nylon wool columns to remove monocytes and B cells. Nonadherent mononuclear cells were used in the inhibition experiments and cytotoxicity tests. Part of the cells were incubated overnight at 4°C and then centrifugated through discontinuous Percoll (Pharmacia Fine Chemicals AB, Uppsala, Sweden) gradients as previously described (8). The low-density fractions 2 and 3 were pooled and run through Percoll gradients repeatedly to obtain highly purified LGLs. This resulted in cell populations consisting of >90% large granular lymphocytes. Primary cell cultures. Fetal and adult fibroblasts, ovarian carcinoma cells, renal parenchymal cells, and renal carcinoma cells were derived from the corresponding tissue samples by initiating the cultures from l-mm3 pieces. The cells were cultured in 1.55-cm diam plastic wells (Nunclon Multidish, Denmark), to be used for the inhibition experiments. For the cytotoxicity assaysthey were grown on 96-well flatbottomed micotiter plates (Lindbro Scientific Co., Hamden, CT). Confluent monolayers consisted of 1- 1.6 X lo5 and lo- 12 X 1O3cells/well, respectively. The continuous cell lines were routinely tested for the presence of mycoplasma contamination by the bisbenzimid staining using the modified method originally introduced by Russel et al. (9). Primary cultures were only tested when they failed to inhibit NK activity, becausemycoplasma contamination is known to induce IFN production in NK cells (10) and to enhance NK activity (11). Indeed, our earlier results show that the presence of both a-IFN and @YN eradicates the inhibition of NK cells by monolayer cultures ( 12). The present material did not include noninhibitory monolayers. Inhibition experiments.Effector cells (4 X lo6 in 2.0 ml of medium) were transferred onto the target cell monolayers or into empty plastic wells. After incubating for 18 hr in cell culture conditions the effector cells were removed by vigorous pipetting and their cytotoxicity against K-562 was tested immediately. The recovery of all lymphocytes from the cultures was controlled by cell counting and inverted microscopy. The percentage of the inhibition of the cytotoxicity was calculated by the following formula:
EFFECT OF IL-2 ON THE INHIBITION
% inhibition = x-y
X
OF NK ACTIVITY
211
[II
X 100
where x = the percentage of control cytotoxicity and y = the percentage of the cytotoxicity after effector cell inactivation. The results at the e/t ratio 50/ 1 are shown. Cytotoxicity assays. 1. Target cells (2 X lo6 K 562) were labeled with sodium “chromate (Radiochemical Centre, Amersham, England) in 1 ml of medium supplemented with FBS for 1 hr, and the percentage cytotoxicity of the effector cells was measured by the standard 4-hr 5’chromium-release assayat the e/t ratios 50/l, 25/ 1, and 12/ 1. Total lytic units (LU) were calculated by the following formula: total LU = yt X LU/ 1O6cells,
PI
where n = the number of effector cells (in millions) in the experiment and LU = the number of cells required to cause 25% lysis of target cells. 2. The sensitivity of the monolayer target cells to NK activity was tested by labeling the target cells, grown to confluency in flat-bottomed microtiter plate wells, with 1 &i/well sodium “chromate for 6 hr. E/T ratios 20/ 1, IO/ 1, and 5/ 1 were used in an 18-hr cytotoxicity assay.Five wells for each e/t ratio were prepared. Znterleukin 2. Recombinant IL-2 (Biogen, Genova, Switzerland), was used at the concentration of 100 U/ml. In dose-response experiments, this concentration of IL2 consistently augmented cytotoxicity in all samples tested. The effector cells were pretreated for 24-62 hr with IL-2, and washed, or they were cultured in medium only. The inhibition experiments were carried out both in the presence and in the absenceof IL-2. Binding assay.To examine the binding capacity of effector cells to monolayer target cells, the monolayer target cells were grown on glass coverslips ( 12 mm diam) in plastic wells (15.5 mm diam). The effector cells (2 X 106)were labeled with 20 &i of sodium “chromate for 6 hr in 2 ml of medium. After washing three times, the effector cells were transferrred onto the monolayers (three wells for each cell line type) at e/t ratio l/ 1. Empty control wells were prepared in a similar fashion. After centrifuging for 10 min at 90 g the plates were incubated for 10 min at 37°C. The coverslips were then removed and washed in medium. The radioactivity of the coverslips was counted and the percentage of adherent effector cells was calculated by the following formula: binding % = k x Expt - c. x 1oo
Tot.
’
where Expt = the mean of the counts in binding lymphocytes. c. = the mean of the counts recovered from the empty control wells, Tot. = the total counts incorporated into the lymphocytes, and k = the ratio of the area of the plastic well/the area of the coverslip. Enriched LGL were used in binding experiments in order to diminish the effect of nonspecific binding. They were preincubated with IL-2 (100 U/ml) or in medium only for 48-96 hr before being used in binding assays. Statistics. The results were subjected to Student’s paired t test. Student’s t test for independent samples was used when different effector cells from different donors were compared.
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HEISKALA AND TIMONEN
. . i:.I)*,: :.I :. : 1. .t.
FIG. 1. The effect of IL-2 on the sensitivity of PBL to the cell contact mediated inhibition. Inhibition percentagesof (A) control PBL, cultured for O-24 hr in medium only; (B) PBL pretreated with IL-2 for 24 hr; (C) PBL pretreated with IL-2 for 48-120 hr, and benign monolayer cells were used for inhibition; (D) PBL pretreated with IL-2 for 48-120 hr, and malignant monolayer cells were used for inhibition; (E) control PBL, and inhibition assayswere carried out in the presence of IL-2. Adult fibroblasts, renal parenchymal cells, renal carcinoma cells, and ovarian carcinoma cells were usedas inhibitory cells. (0) Inhibitory monolayer of benign origin; (A) inhibitory monolayer of malignant origin. The significance of the inhibition: N. S. = not significant. **P -c0.01;***P < 0.001.
RESULTS Benign and malignant monolayer target cells were similar in their inhibitory efficacy towards unstimulated effector cells (Fig. l), and the presence of IL-2 during the 18-hr inhibitory cell contact had no effect on the inhibition. However, effector cells pretreated with 100 U/ml of IL-2 for at least 48 hr before the contact with monolayer cells was inhibited by benign monolayers only. In addition, the inactivating capacity of benign target cells was somewhat less efficient toward activated killer cells than endogenous NK cells. As seenin Fig. 2, the activated killer cells were effectively cytotoxic to the previously inhibitory and NK-resistant monolayer target cells. Target cells of malignant origin were significantly more sensitive to activated killing than the benign ones (P < 0.05) (Fig. 2), although some cytotoxicity against the benign target cells was also detected. It was also found that the insensitivity to the inhibitory influence of the target cells and the effective cytotoxicity against them appeared simultaneously in the effector cells (Fig. 3). Depending on the individual kinetics, 24- to 62-hr preincubation of the effector cells with IL-2 was required for the appearance of cytotoxicity and the disappearance of inhibition. As seenin Fig. 2, there was some minor cytotoxicity by unstimulated control effector cells against the monolayer target cells cultured in FCS-containing medium. Moreover, the cytotoxicity of the activated effector cells was not strictly selective against malignant target cells, also benign target cells being lysed to some degree. To rule out the nonspecific enhancing effect of FCS on cytotoxicity, we repeated the experiments in medium supplemented with 10% human AB serum. The selectivity pattern of the cytotoxicity was similar to the experiments conducted with FCS-con-
EFFECT OF IL-2 ON THE INHIBITION
I3
A
OF NK ACTIVITY
A
213
B
FIG. 2. The cytotoxicity of PBL against the inhibitory monolayer target cells. (m) Control PBL; (Cl) control PBL, cytotoxicity assayscarried out in the presence of IL-2; (@ PBL cultured in medium only for 24-72 hr; @I)PBL pretreated with IL-2 for 24-72 hr. P < 0.05 between the cytotoxicities of IL-2-pretreated PBL against benign (A) and malignant (B) target cells. Adult fibroblasts, renal parenchymal cells, renal carcinoma cells and ovarian carcinoma cells were used as target cells. Percentage cytotoxicities (means + SD of five experiments) at e/t ratio 20/l are shown. The lytic units at the level of 25% are indicated in front of the bars. Significance of the augmentation of the cytotoxicity: *P < 0.05; ** < 0.01.
i 0
4 1
4
4
2-5
0
DAYS OF EFFECTOR
CELL IL-2
4 1 PRETREATMENT
2:
FIG. 3. The effect of IL-2 on PBL. Increasing cytotoxicity against monolayer target cells (benign, 0; malignant, A) at e/t ratio 20/l and decreasing inhibition of cytotoxicity against K-562 (benign, 0, malignant, A) at e/t ratio 50/l are shown. The means k SD have been calculated on the basis of four to six experiments with effector cells pretreated with IL-2 for 1 day, and on the basis of 7- 15 experiments with control effector cells and effector cells pretreated with IL-2 for 2-5 days. Effector cells pretreated with IL-2 for 2-5 days were significantly more cytotoxic against malignant than against benign monolayer cells (P < O.Ol), and they were significantly less sensitive to inhibition by malignant than by benign monolayer cells (P < 0.05). Adult fibroblasts, renal parenchymal cells, renal carcinoma cells, and ovarian carcinoma cells were used as monolayer targets and as inhibitory cells in cocultures.
214
HEISKALA AND TIMONEN
0 IL-2 /f.,L., z 3 ;i 0
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5 E
1
s
.
=. A
.MEOIUM ,oMEDIUM
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I
*IL-Z ,DlL-2
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PIG. 4. The effect of IL-2 on the percentage cytotoxicity ofcontrol (c.) and inactivated (inh.) PBL against K-562 at e/t ratio 50/ 1. The effector cells were cultured in the presence of IL-2 or in medium only for 24 hr between the determinations of cytotoxicity. The results of two representative experiments out of five are shown.
taining media, and still some cytotoxicity was observed against the benign target cells (data not shown). We also studied whether inactivated killer cells could be restimulated by IL-2. For this purpose, the effector cells were treated with IL-2 for 24 hr after the inhibitory cell contact. Their NK activity against K562 was found to be of the same order as that of the IL-2-stimulated control cells, indicating that IL-2 can restimulate the inactivated effector cells (Table 1). In some experiments (Experiment 1, Table 1) there occurred stimulation of cytotoxicity in the control cultures, probably due to the presence of FCS. However, also in these casesthere remained a difference of the lytic potential between control and inactivated effector cells, suggestingthat FCS is lessefficient than IL-2 in the eradication of the lymphocyte sensitivity to the inhibition by monolayer cells. In order to examine further the mechanism of the activation of cytotoxicity by IL2, we studied the effect of IL-2 on binding. As seen in Table 2, the IL-2 pretreatment of effector cells enhanced the binding of LGL to the monolayer cells. There was no difference in the binding of the activated killer cells to benign and malignant target cells, indicating that the selectivity of lysis towards malignant cells is not based on differences in the recognition step. TABLE 1 Restimulation of Inactivated NK Cells by IL-2” Ohr
24 hr (-IL-2)
24 hr (+IL-2)
Expt
Control
Inactivated
Control
Inactivated
Control
Inactivated
1. 2.
2.5 4.0
0.0 0.0
3.6 0.8
2.2 0.0
6.0 4.9
6.0 4.6
L?Cytotoxicity is expressed as total lytic units. Ovarian carcinoma cells were used as inhibitory target cells.
EFFECT OF IL-2 ON THE INHIBITION
OF NK ACTIVITY
215
TABLE 2 The Effect of IL-2 on the Binding Capacity of LGL”: % LGL Binding to Monolayer Target Cellsb Expt no.
Control LGL
IL-2 pretreated LGL
Malignant target cells 1 2 3 4 5 6 Mean + SD
10.4 12.7 13.6 14.2 34.4 41.1 21.1 f 13.2
13.9 14.7 14.5 26.7 47.0 44.8 26.9 f 15.5*
Benign target cells 7 8 9 10 II Mean + SD
2.3 11.7 13.9 14.4 23.1 13.1 + 7.4
23.5 20.4 21.7 22.9 35.0 24.7 f 5.9**
n Adult fibroblasts, renal parenchymal cells, renal carcinoma cells, and ovarian carcinoma cells were used as monolayer target cells. Experiments l-6 were performed using malignant target cells and Experiments 7- 11 using benign target cells. The difference between binding of control LGL in Experiments l-6 and 7-l 1 is not statistically significant. b Enriched LGL were cultured for 48-72 hr in medium only (control LGL) or with 100 U/ml IL-2 before being used in binding assaysat 1/ 1 ratio. * P < 0.05. **P
DISCUSSION The results of this study show that IL-2 renders naturally cytotoxic peripheral blood lymphocytes insensitive to inhibitory signals of malignant monolayer target cells. As there was a close correlation in the kinetics of effector cell activation and the disappearance of the effector cell sensitivity to the inhibitory signals of the target cells, the results support the hypothesis that effector cell inactivation is an important mechanism in target cell resistance to natural cell-mediated immunity (3). The mechanisms by which IL-2 induces the insensitivity of cytotoxic cells to the inhibitory signals has not yet been clarified. NK activity involves binding of effector cells to target cells (13), activation of lytic machinery as reflected by the orientation of the Golgi apparatus towards target cell (14, 15), secretion of cytotoxins such as natural killer cytotoxic factor (NKCF) and granule cytolysin ( 16, 17), and finally, the killer cell-independent phase of lysis ( 13, 18). In another report we have shown that the inhibitory signal of the monolayer target cells is targeted at the releaseof cytotoxic factors from the effector cells ( 19). Inhibited effector cells show unaltered conjugateforming capacity to K562, the orientation of the Golgi apparatus of effector cells toward target cells is readily detectable in spite of the inhibition, but the capacity of the inhibited LGL to produce NKCF is impaired. Therefore it seemslikely that the activation of killer cells in vitro by IL-2 affects the mechanism of cytotoxic factor
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HEISKALA AND TIMONEN
releasefrom the effector cells. The activated killing may represent an alternative pathway in the triggering of the lytic machinery at the phase of releaseof cytotoxic factors, a pathway which is lesssensitive to the inhibitory signals of the target cells. IL-Zstimulated killer cells (the lymphokine-activated killer cells) are particularly interesting becauseof their selectivity towards malignant target cells (20). In this study we show that this selectivity is detectable also in allogeneic combinations, although significant cytotoxicity against benign target cells was also observed. The sensitivity of benign cells to natural cell-mediated immunity indicates that the basis for the selectivity of both NK cells and IL-2-induced killer cells toward malignant target cells is probably quantitative rather than qualitative. It has not yet been clarified at what phase of the lytic event in natural killing the selectivity toward malignant target cells emerges.In this study we show that IL-2-stimulated effector cells bind as effectively malignant as benign target cells. However, the IL-Zactivated killer cells were more sensitive to the inhibitory signals of benign cells. We have shown elsewhere that primary monolayer cultures of benign and malignant origin are equally sensitive to toxic factors secreted by NK cells (12). Therefore these results altogether suggest that at least one mechanism in the selectivity of IGZstimulated killer cells toward malignant target cells may be the more efficient killer cell inactivation by benign target cells. It could be hypothesized that the more prominent inactivating capacity of benign cells may be due to larger quantities of inactivating compounds on benign cells, or to the firmer stability of these factors on cell membrane. Perhaps becauseof these quantitative differences the inactivating capacity of malignant target cells is sufficient only for the down-regulation of relatively weakly cytotoxic endogenous NK cells. This is consistent with the results of Abrams and Brahmi (2 I), who found that contact with K-562 cells at a low e/t ratio (2/l) leads to a significant loss of NK reactivity of even IL-Zpretreated effector cells. These issuescan, however, be properly examined only when the molecular identity of the inhibitory compounds has been elucidated. To conclude, we have demonstrated that the circumvention of target cell-mediated inhibition of effector cells is involved in the augmentation of cell-mediated cytotoxicity by IL-2. As IL-2 is known to increase target cell-binding capacity and proliferation of killer cells (22-24), our results present a third mechanism by which IL-2 activates cytotoxic cells. ACKNOWLEDGMENTS The technical assistanceof Mrs. Maija-Liisa MantylP and Miss Pirkko Kalliomaki is gratefully acknowledged.The renal parenchymal specimens were kindly provided by Dr. T. Lehtonen from the II Department of Surgery, University of Helsinki. The ovarian carcinoma specimens were obtained from the I and II Departments of Gynecology and Obstetrics, University of Helsinki, through the courtesy of ProfessorsM. Sepp% and 0. Widholm.
REFERENCES 1. Pross,H. F., and Baines, M. G., Cancer Immunol. Immunother. 3,75, 1917. 2. Herberman, R. B., and Holden, H. T., In “Advances in Cancer Research“ (G. Klein, and S. Weinhouse, Eds.), p. 305. Academic Press,New York, 1978. 3. Heiskala, M. K., Ylikorkala, O., and Timonen, T., Natl. Immunol. Cell Growth Reg., 6, 1, 1987. 4. Seeley,J. K., and Golub, S. H.,J. Immunol. 120, 1415, 1978. 5. Matera, L., Francis, M. K., Herlyn, M., Tucco, M., and Santoli, D., Natl. Immunol. Cell Growth Reg. 4,90, 1985.
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6. Grimm, E. A., Mazumder, A., Zhang, H. Z., and Rosenberg, S. A., J. Exp. Med. 155, 1823, 1982. 7. Boyum, A., Stand. J. Clin. Lab. Invest. Zl(Suppl. 97), 1, 1968. 8. Timonen, T., Reynolds, C. W., Ortaldo, J. R., and Herberman, R. B., J. Zmmunol. Methods 51,269, 1982. 9. Russel, W. C., Newman, C., and Williamson, D. H., Nature (London) 253,46 1, 1975. 10. Djeu, J. Y., Timonen, T., and Herberman, R. B., In “NK Cells and Other Natural Effector Cells” (R. B. Herberman, Ed.), p. 669. Academic Press,New York, 1982. 11. Brooks., C. G., Rees, R. C., and Leach, R. H., Eur. J. Zmmunol. 9, 159, 1979. 12. Heiskala, M., Zmmunology60, 167, 1987. 13. Hiserodt, J. C., B&van, L. J., and Targan, S. R., J. Zmmunol. 129, 1782, 1982. 14. Carp&t, O., Virtanen, I., and Saksela, E., Cell. Zmmunol. 58,97, 1981. 15. Kupfer, A., Dennert, G., and Singer, S. J., J. Mol. Cell. Zmmunol. 2,37, 1985. 16. Wright, S. C., and Bonavida, B., In “NK Cells and Other Natural Effector Cells” (R. B. Herberman, Ed.), p. 96 1. Academic Press,New York, 1983. 17. Henkart, P. A., Millard, P. J., Reynolds, C. W., and Henkart, M. P., J. Exp. Med. 160,75, 1984. 18. Targan, S. C., and Newman, W. J., Immunology 131, 1149, 1983. 19. Heiskala, M., Carp&n, O., Saksela,E., and Timonen, T., J. Zmmunol. 139, 14 14, 1987. 20. Rosenberg, S., J. Natl. Cancer Inst. 75,595, 1985. 21. Abrams, S. I., and Brahmi, Z., Cell. Immunol. 101,558, 1986. 22. Timonen, T., Ortaldo, J. R., Stadler, B. M., Bonnard, G. D., Sharrow, S. O., and Herberman, R. B., Cell. Zmmunol. 72, 178, 1982. 23. Suzuki, R., Handa, K., Itoh, R., and Kumagai, K., J. Zmmunol. 130,98 1, 1983. 24. Handa, K., Suzuki, R., Matsui, H., Shimizu, Y., and Kumagai, K., J. Zmmunol. 130,988, 1983.
IMMUNOLOGY
110,209-2
17 ( 1987)
Effect of lnterleukin 2 on the Inhibition of Human Natural Killer Activity by Monolayer Cells’ M. HEISKALA AND T. TIMONEN Department of Pathology, University ofHelsinki, Haartmaninkatu 3, SF-00290 Helsinki, Finland Received January 5, 1987; acceptedAugust lo,1987 We have previously shown that human endogenous natural killer activity against K562 is inhibited by primary cultures of natural killer-resistant monolayer target cells. In this study we have analyzed the sensitivity of activated killer cells to this inhibitory effect. Interleukin-2 (IL2) when present during an 18-hr contact of peripheral blood lymphocytes with monolayers, did not affect the inhibition of natural killer cell activity. Pretreatment of effector cells with IL-2 for 24-62 hr before the contact with monolayer cells eradicated the inhibition causedby malignant cells, benign cells remaining inhibitory. The IL-2-pretreated effector cells killed preferentially malignant target cells, although significant cytotoxicity was also detectable against benign cell cultures. The results indicate that activation of killer cells in vitro by IL-2 involves the desensitization of effector cells to the inhibitory signals of target cells, and that the selectivity of IL-2activated killer cells toward malignant target cells involves weaker inhibition of activated killer Cells by malignant Cek. 0 1987 Academic Press,Inc.
INTRODUCTION Natural killer cells (NK cells) kill primarily in vitro propagated target cells of malignant origin (1, 2). The reasons for the insensitivity of freshly isolated target cells to NK reactivity are unknown. We have previously shown that primary cultures of both benign and malignant monolayer cells significantly inhibit NK activity, as measured by lysis of K562 target cells by both peripheral blood lymphocytes and purified large granular lymphocytes (LGL) (3). As there is an inverse correlation between the inhibitory capacity and the NK sensitivity, the inhibition of NK activity by target cells may be an important mechanism by which some cells are protected from NK cells. Activated killer cells are more potent effector cells than endogenous NK cells, and they kill also NK resistant target cells, including freshly isolated malignant cells (46). Killer cells activated by interleukin-2 (IL-2), the lymphokine activated killer cells (6), lyse preferentially malignant target cells, and the basis of this selectivity is unknown. In this study we have analyzed the sensitivity of IL-Zactivated killer cells to the inhibitory signals of solid tissue-derived target cells of both malignant and benign ’ This work was supported by the Ida Montin Foundation, the Finnish Cancer Society, the &rid Juselius Foundation, Orion Pharmaceuticals Ltd, and the National Council for Medical Sciences, Academy of Finland.
209 0008-8749/87 $3.00 Copyright 0 1987 by Academic Press,Inc. All rights of reproduction in any form reserved.
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HEISKALA AND TIMONEN
origin. The results show that while endogenous NK activity is inhibited by monolayers of both benign and malignant cells, the IL-2-activated killer cells are preferentially inhibited by benign target cells. Thus, in addition to the endogenous NK cell system the target cell-mediated inactivation of cytotoxic cells appears to be also involved in the selectivity of activated killer cells. MATERIALS AND METHODS
Cell culture conditions. Cells were cultured at 37°C in humidified air with 5% CO2 in RPM1 1640 medium (GIBCO), supplemented with heat inactivated fetal calf serum (lo%, FCS, KC Biological, Lenexa, KS), 100 pg/ml gentamycin, 0.29 mg/ml glutamine, and 0.1 M Hepes. Inhibition tests, cytotoxicity assays,and IL-2 stimulations were performed in medium supplemented with either 10% human AB serum (Finnish Red Cross Blood Bank) or 10%FCS. Effector cells. Peripheral blood mononuclear cells were isolated from buffy coats of 400 ml of venous blood from healthy donors (Finnish Red Cross Blood Bank) by Ficoll-Isopaque gradient centrifugation (7). They were subsequently incubated in glass flasks and run through nylon wool columns to remove monocytes and B cells. Nonadherent mononuclear cells were used in the inhibition experiments and cytotoxicity tests. Part of the cells were incubated overnight at 4°C and then centrifugated through discontinuous Percoll (Pharmacia Fine Chemicals AB, Uppsala, Sweden) gradients as previously described (8). The low-density fractions 2 and 3 were pooled and run through Percoll gradients repeatedly to obtain highly purified LGLs. This resulted in cell populations consisting of >90% large granular lymphocytes. Primary cell cultures. Fetal and adult fibroblasts, ovarian carcinoma cells, renal parenchymal cells, and renal carcinoma cells were derived from the corresponding tissue samples by initiating the cultures from l-mm3 pieces. The cells were cultured in 1.55-cm diam plastic wells (Nunclon Multidish, Denmark), to be used for the inhibition experiments. For the cytotoxicity assaysthey were grown on 96-well flatbottomed micotiter plates (Lindbro Scientific Co., Hamden, CT). Confluent monolayers consisted of 1- 1.6 X lo5 and lo- 12 X 1O3cells/well, respectively. The continuous cell lines were routinely tested for the presence of mycoplasma contamination by the bisbenzimid staining using the modified method originally introduced by Russel et al. (9). Primary cultures were only tested when they failed to inhibit NK activity, becausemycoplasma contamination is known to induce IFN production in NK cells (10) and to enhance NK activity (11). Indeed, our earlier results show that the presence of both a-IFN and @YN eradicates the inhibition of NK cells by monolayer cultures ( 12). The present material did not include noninhibitory monolayers. Inhibition experiments.Effector cells (4 X lo6 in 2.0 ml of medium) were transferred onto the target cell monolayers or into empty plastic wells. After incubating for 18 hr in cell culture conditions the effector cells were removed by vigorous pipetting and their cytotoxicity against K-562 was tested immediately. The recovery of all lymphocytes from the cultures was controlled by cell counting and inverted microscopy. The percentage of the inhibition of the cytotoxicity was calculated by the following formula:
EFFECT OF IL-2 ON THE INHIBITION
% inhibition = x-y
X
OF NK ACTIVITY
211
[II
X 100
where x = the percentage of control cytotoxicity and y = the percentage of the cytotoxicity after effector cell inactivation. The results at the e/t ratio 50/ 1 are shown. Cytotoxicity assays. 1. Target cells (2 X lo6 K 562) were labeled with sodium “chromate (Radiochemical Centre, Amersham, England) in 1 ml of medium supplemented with FBS for 1 hr, and the percentage cytotoxicity of the effector cells was measured by the standard 4-hr 5’chromium-release assayat the e/t ratios 50/l, 25/ 1, and 12/ 1. Total lytic units (LU) were calculated by the following formula: total LU = yt X LU/ 1O6cells,
PI
where n = the number of effector cells (in millions) in the experiment and LU = the number of cells required to cause 25% lysis of target cells. 2. The sensitivity of the monolayer target cells to NK activity was tested by labeling the target cells, grown to confluency in flat-bottomed microtiter plate wells, with 1 &i/well sodium “chromate for 6 hr. E/T ratios 20/ 1, IO/ 1, and 5/ 1 were used in an 18-hr cytotoxicity assay.Five wells for each e/t ratio were prepared. Znterleukin 2. Recombinant IL-2 (Biogen, Genova, Switzerland), was used at the concentration of 100 U/ml. In dose-response experiments, this concentration of IL2 consistently augmented cytotoxicity in all samples tested. The effector cells were pretreated for 24-62 hr with IL-2, and washed, or they were cultured in medium only. The inhibition experiments were carried out both in the presence and in the absenceof IL-2. Binding assay.To examine the binding capacity of effector cells to monolayer target cells, the monolayer target cells were grown on glass coverslips ( 12 mm diam) in plastic wells (15.5 mm diam). The effector cells (2 X 106)were labeled with 20 &i of sodium “chromate for 6 hr in 2 ml of medium. After washing three times, the effector cells were transferrred onto the monolayers (three wells for each cell line type) at e/t ratio l/ 1. Empty control wells were prepared in a similar fashion. After centrifuging for 10 min at 90 g the plates were incubated for 10 min at 37°C. The coverslips were then removed and washed in medium. The radioactivity of the coverslips was counted and the percentage of adherent effector cells was calculated by the following formula: binding % = k x Expt - c. x 1oo
Tot.
’
where Expt = the mean of the counts in binding lymphocytes. c. = the mean of the counts recovered from the empty control wells, Tot. = the total counts incorporated into the lymphocytes, and k = the ratio of the area of the plastic well/the area of the coverslip. Enriched LGL were used in binding experiments in order to diminish the effect of nonspecific binding. They were preincubated with IL-2 (100 U/ml) or in medium only for 48-96 hr before being used in binding assays. Statistics. The results were subjected to Student’s paired t test. Student’s t test for independent samples was used when different effector cells from different donors were compared.
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HEISKALA AND TIMONEN
. . i:.I)*,: :.I :. : 1. .t.
FIG. 1. The effect of IL-2 on the sensitivity of PBL to the cell contact mediated inhibition. Inhibition percentagesof (A) control PBL, cultured for O-24 hr in medium only; (B) PBL pretreated with IL-2 for 24 hr; (C) PBL pretreated with IL-2 for 48-120 hr, and benign monolayer cells were used for inhibition; (D) PBL pretreated with IL-2 for 48-120 hr, and malignant monolayer cells were used for inhibition; (E) control PBL, and inhibition assayswere carried out in the presence of IL-2. Adult fibroblasts, renal parenchymal cells, renal carcinoma cells, and ovarian carcinoma cells were usedas inhibitory cells. (0) Inhibitory monolayer of benign origin; (A) inhibitory monolayer of malignant origin. The significance of the inhibition: N. S. = not significant. **P -c0.01;***P < 0.001.
RESULTS Benign and malignant monolayer target cells were similar in their inhibitory efficacy towards unstimulated effector cells (Fig. l), and the presence of IL-2 during the 18-hr inhibitory cell contact had no effect on the inhibition. However, effector cells pretreated with 100 U/ml of IL-2 for at least 48 hr before the contact with monolayer cells was inhibited by benign monolayers only. In addition, the inactivating capacity of benign target cells was somewhat less efficient toward activated killer cells than endogenous NK cells. As seenin Fig. 2, the activated killer cells were effectively cytotoxic to the previously inhibitory and NK-resistant monolayer target cells. Target cells of malignant origin were significantly more sensitive to activated killing than the benign ones (P < 0.05) (Fig. 2), although some cytotoxicity against the benign target cells was also detected. It was also found that the insensitivity to the inhibitory influence of the target cells and the effective cytotoxicity against them appeared simultaneously in the effector cells (Fig. 3). Depending on the individual kinetics, 24- to 62-hr preincubation of the effector cells with IL-2 was required for the appearance of cytotoxicity and the disappearance of inhibition. As seenin Fig. 2, there was some minor cytotoxicity by unstimulated control effector cells against the monolayer target cells cultured in FCS-containing medium. Moreover, the cytotoxicity of the activated effector cells was not strictly selective against malignant target cells, also benign target cells being lysed to some degree. To rule out the nonspecific enhancing effect of FCS on cytotoxicity, we repeated the experiments in medium supplemented with 10% human AB serum. The selectivity pattern of the cytotoxicity was similar to the experiments conducted with FCS-con-
EFFECT OF IL-2 ON THE INHIBITION
I3
A
OF NK ACTIVITY
A
213
B
FIG. 2. The cytotoxicity of PBL against the inhibitory monolayer target cells. (m) Control PBL; (Cl) control PBL, cytotoxicity assayscarried out in the presence of IL-2; (@ PBL cultured in medium only for 24-72 hr; @I)PBL pretreated with IL-2 for 24-72 hr. P < 0.05 between the cytotoxicities of IL-2-pretreated PBL against benign (A) and malignant (B) target cells. Adult fibroblasts, renal parenchymal cells, renal carcinoma cells and ovarian carcinoma cells were used as target cells. Percentage cytotoxicities (means + SD of five experiments) at e/t ratio 20/l are shown. The lytic units at the level of 25% are indicated in front of the bars. Significance of the augmentation of the cytotoxicity: *P < 0.05; ** < 0.01.
i 0
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FIG. 3. The effect of IL-2 on PBL. Increasing cytotoxicity against monolayer target cells (benign, 0; malignant, A) at e/t ratio 20/l and decreasing inhibition of cytotoxicity against K-562 (benign, 0, malignant, A) at e/t ratio 50/l are shown. The means k SD have been calculated on the basis of four to six experiments with effector cells pretreated with IL-2 for 1 day, and on the basis of 7- 15 experiments with control effector cells and effector cells pretreated with IL-2 for 2-5 days. Effector cells pretreated with IL-2 for 2-5 days were significantly more cytotoxic against malignant than against benign monolayer cells (P < O.Ol), and they were significantly less sensitive to inhibition by malignant than by benign monolayer cells (P < 0.05). Adult fibroblasts, renal parenchymal cells, renal carcinoma cells, and ovarian carcinoma cells were used as monolayer targets and as inhibitory cells in cocultures.
214
HEISKALA AND TIMONEN
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PIG. 4. The effect of IL-2 on the percentage cytotoxicity ofcontrol (c.) and inactivated (inh.) PBL against K-562 at e/t ratio 50/ 1. The effector cells were cultured in the presence of IL-2 or in medium only for 24 hr between the determinations of cytotoxicity. The results of two representative experiments out of five are shown.
taining media, and still some cytotoxicity was observed against the benign target cells (data not shown). We also studied whether inactivated killer cells could be restimulated by IL-2. For this purpose, the effector cells were treated with IL-2 for 24 hr after the inhibitory cell contact. Their NK activity against K562 was found to be of the same order as that of the IL-2-stimulated control cells, indicating that IL-2 can restimulate the inactivated effector cells (Table 1). In some experiments (Experiment 1, Table 1) there occurred stimulation of cytotoxicity in the control cultures, probably due to the presence of FCS. However, also in these casesthere remained a difference of the lytic potential between control and inactivated effector cells, suggestingthat FCS is lessefficient than IL-2 in the eradication of the lymphocyte sensitivity to the inhibition by monolayer cells. In order to examine further the mechanism of the activation of cytotoxicity by IL2, we studied the effect of IL-2 on binding. As seen in Table 2, the IL-2 pretreatment of effector cells enhanced the binding of LGL to the monolayer cells. There was no difference in the binding of the activated killer cells to benign and malignant target cells, indicating that the selectivity of lysis towards malignant cells is not based on differences in the recognition step. TABLE 1 Restimulation of Inactivated NK Cells by IL-2” Ohr
24 hr (-IL-2)
24 hr (+IL-2)
Expt
Control
Inactivated
Control
Inactivated
Control
Inactivated
1. 2.
2.5 4.0
0.0 0.0
3.6 0.8
2.2 0.0
6.0 4.9
6.0 4.6
L?Cytotoxicity is expressed as total lytic units. Ovarian carcinoma cells were used as inhibitory target cells.
EFFECT OF IL-2 ON THE INHIBITION
OF NK ACTIVITY
215
TABLE 2 The Effect of IL-2 on the Binding Capacity of LGL”: % LGL Binding to Monolayer Target Cellsb Expt no.
Control LGL
IL-2 pretreated LGL
Malignant target cells 1 2 3 4 5 6 Mean + SD
10.4 12.7 13.6 14.2 34.4 41.1 21.1 f 13.2
13.9 14.7 14.5 26.7 47.0 44.8 26.9 f 15.5*
Benign target cells 7 8 9 10 II Mean + SD
2.3 11.7 13.9 14.4 23.1 13.1 + 7.4
23.5 20.4 21.7 22.9 35.0 24.7 f 5.9**
n Adult fibroblasts, renal parenchymal cells, renal carcinoma cells, and ovarian carcinoma cells were used as monolayer target cells. Experiments l-6 were performed using malignant target cells and Experiments 7- 11 using benign target cells. The difference between binding of control LGL in Experiments l-6 and 7-l 1 is not statistically significant. b Enriched LGL were cultured for 48-72 hr in medium only (control LGL) or with 100 U/ml IL-2 before being used in binding assaysat 1/ 1 ratio. * P < 0.05. **P
DISCUSSION The results of this study show that IL-2 renders naturally cytotoxic peripheral blood lymphocytes insensitive to inhibitory signals of malignant monolayer target cells. As there was a close correlation in the kinetics of effector cell activation and the disappearance of the effector cell sensitivity to the inhibitory signals of the target cells, the results support the hypothesis that effector cell inactivation is an important mechanism in target cell resistance to natural cell-mediated immunity (3). The mechanisms by which IL-2 induces the insensitivity of cytotoxic cells to the inhibitory signals has not yet been clarified. NK activity involves binding of effector cells to target cells (13), activation of lytic machinery as reflected by the orientation of the Golgi apparatus towards target cell (14, 15), secretion of cytotoxins such as natural killer cytotoxic factor (NKCF) and granule cytolysin ( 16, 17), and finally, the killer cell-independent phase of lysis ( 13, 18). In another report we have shown that the inhibitory signal of the monolayer target cells is targeted at the releaseof cytotoxic factors from the effector cells ( 19). Inhibited effector cells show unaltered conjugateforming capacity to K562, the orientation of the Golgi apparatus of effector cells toward target cells is readily detectable in spite of the inhibition, but the capacity of the inhibited LGL to produce NKCF is impaired. Therefore it seemslikely that the activation of killer cells in vitro by IL-2 affects the mechanism of cytotoxic factor
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releasefrom the effector cells. The activated killing may represent an alternative pathway in the triggering of the lytic machinery at the phase of releaseof cytotoxic factors, a pathway which is lesssensitive to the inhibitory signals of the target cells. IL-Zstimulated killer cells (the lymphokine-activated killer cells) are particularly interesting becauseof their selectivity towards malignant target cells (20). In this study we show that this selectivity is detectable also in allogeneic combinations, although significant cytotoxicity against benign target cells was also observed. The sensitivity of benign cells to natural cell-mediated immunity indicates that the basis for the selectivity of both NK cells and IL-2-induced killer cells toward malignant target cells is probably quantitative rather than qualitative. It has not yet been clarified at what phase of the lytic event in natural killing the selectivity toward malignant target cells emerges.In this study we show that IL-2-stimulated effector cells bind as effectively malignant as benign target cells. However, the IL-Zactivated killer cells were more sensitive to the inhibitory signals of benign cells. We have shown elsewhere that primary monolayer cultures of benign and malignant origin are equally sensitive to toxic factors secreted by NK cells (12). Therefore these results altogether suggest that at least one mechanism in the selectivity of IGZstimulated killer cells toward malignant target cells may be the more efficient killer cell inactivation by benign target cells. It could be hypothesized that the more prominent inactivating capacity of benign cells may be due to larger quantities of inactivating compounds on benign cells, or to the firmer stability of these factors on cell membrane. Perhaps becauseof these quantitative differences the inactivating capacity of malignant target cells is sufficient only for the down-regulation of relatively weakly cytotoxic endogenous NK cells. This is consistent with the results of Abrams and Brahmi (2 I), who found that contact with K-562 cells at a low e/t ratio (2/l) leads to a significant loss of NK reactivity of even IL-Zpretreated effector cells. These issuescan, however, be properly examined only when the molecular identity of the inhibitory compounds has been elucidated. To conclude, we have demonstrated that the circumvention of target cell-mediated inhibition of effector cells is involved in the augmentation of cell-mediated cytotoxicity by IL-2. As IL-2 is known to increase target cell-binding capacity and proliferation of killer cells (22-24), our results present a third mechanism by which IL-2 activates cytotoxic cells. ACKNOWLEDGMENTS The technical assistanceof Mrs. Maija-Liisa MantylP and Miss Pirkko Kalliomaki is gratefully acknowledged.The renal parenchymal specimens were kindly provided by Dr. T. Lehtonen from the II Department of Surgery, University of Helsinki. The ovarian carcinoma specimens were obtained from the I and II Departments of Gynecology and Obstetrics, University of Helsinki, through the courtesy of ProfessorsM. Sepp% and 0. Widholm.
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6. Grimm, E. A., Mazumder, A., Zhang, H. Z., and Rosenberg, S. A., J. Exp. Med. 155, 1823, 1982. 7. Boyum, A., Stand. J. Clin. Lab. Invest. Zl(Suppl. 97), 1, 1968. 8. Timonen, T., Reynolds, C. W., Ortaldo, J. R., and Herberman, R. B., J. Zmmunol. Methods 51,269, 1982. 9. Russel, W. C., Newman, C., and Williamson, D. H., Nature (London) 253,46 1, 1975. 10. Djeu, J. Y., Timonen, T., and Herberman, R. B., In “NK Cells and Other Natural Effector Cells” (R. B. Herberman, Ed.), p. 669. Academic Press,New York, 1982. 11. Brooks., C. G., Rees, R. C., and Leach, R. H., Eur. J. Zmmunol. 9, 159, 1979. 12. Heiskala, M., Zmmunology60, 167, 1987. 13. Hiserodt, J. C., B&van, L. J., and Targan, S. R., J. Zmmunol. 129, 1782, 1982. 14. Carp&t, O., Virtanen, I., and Saksela, E., Cell. Zmmunol. 58,97, 1981. 15. Kupfer, A., Dennert, G., and Singer, S. J., J. Mol. Cell. Zmmunol. 2,37, 1985. 16. Wright, S. C., and Bonavida, B., In “NK Cells and Other Natural Effector Cells” (R. B. Herberman, Ed.), p. 96 1. Academic Press,New York, 1983. 17. Henkart, P. A., Millard, P. J., Reynolds, C. W., and Henkart, M. P., J. Exp. Med. 160,75, 1984. 18. Targan, S. C., and Newman, W. J., Immunology 131, 1149, 1983. 19. Heiskala, M., Carp&n, O., Saksela,E., and Timonen, T., J. Zmmunol. 139, 14 14, 1987. 20. Rosenberg, S., J. Natl. Cancer Inst. 75,595, 1985. 21. Abrams, S. I., and Brahmi, Z., Cell. Immunol. 101,558, 1986. 22. Timonen, T., Ortaldo, J. R., Stadler, B. M., Bonnard, G. D., Sharrow, S. O., and Herberman, R. B., Cell. Zmmunol. 72, 178, 1982. 23. Suzuki, R., Handa, K., Itoh, R., and Kumagai, K., J. Zmmunol. 130,98 1, 1983. 24. Handa, K., Suzuki, R., Matsui, H., Shimizu, Y., and Kumagai, K., J. Zmmunol. 130,988, 1983.