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Monocyte- and natural killer cell-mediated spontaneous cytotoxicity against human noncultured solid tumor cells
CELLULAR
IMMUNOLOGY
108,495-500
(1987)
SHORT COMMUNICATION Monocyte- and Natural Killer Cell-Mediated Spontaneous Cytotoxicity against Human Noncultured Solid Tumor Cells’ KYOGO IToH,**?
CHRIS D. l%ATSOUCAS,t
AND CHARLES
M. BALCH*
*Division ofsurgery and Department of General Surgery and TDepartment of Immunology, The University of Texas System Cancer Center, M. D. Anderson Hospital and Tumor Institute, Houston, Texas 77030 Received February 23,1987; acceptedApril 13. I987 Unstimulated human peripheral blood mononuclear cells from healthy donors exhibited spontaneous cytotoxicity against noncultured solid tumor targets in a 12- to 24-hr “Cr release or “‘In release assay. Both purified monocytes (199% monocytes) and natural killer (NK)enriched lymphocytes exhibited comparable levels of spontaneous cytotoxicity against fresh melanoma tumor targets. This cytotoxicity was observed under endotoxin-free conditions. NKdepleted lymphocytes did not lyse the melanoma targets. Culture supematants of monocytes incubated with the melanoma tumor cells did not exhibit cytotoxic activity against these targets. Purified monocytes lacked NK activity against the K562 targets in a 4-hr 5’Cr release assay. Treatment of the monocytes with anti-Leul 1b and antiLeu7 monoclonal antibodies plus complement did not reduce monocyte-mediated lysis of the melanoma targets, demonstrating that contaminating NK cells, if any, were not responsible for the lysis of noncultured melanoma targets by monocytes. In contrast, Leul I b+ NK cells were responsible for the lysis of the melanoma targets by NK-enriched lymphocytes. The addition of recombinant interferon-r (rIFNy), but not lipopolysaccharide, into the 5’Cr release assay or pretreatment of monocytes with rIF’N-y significantly increased their cytotoxicity against noncultured solid tumor cells. Monocytes cultured for 3 days with medium alone lost their cytotoxic activity. The addition of rIFNy from the beginning of these cultures prevented the loss of the cytotoxic activity of monocytes. In summary, both unstimulated monocytes and NK-enriched lymphocytes exhibit comparable levels of spontaneous cytotoxicity against fresh solid tumor targets. o 1987 Academic press, 1~.
INTRODUCTION Human monocytes and NK cells have spontaneous cytotoxic activity against several established tumor cell lines in vitro, without requiring previous immunization (I, 2). However, their ability to lyse freshly prepared solid tumor targets is usually low or undetectable when a standard 4-hr cytotoxicity assayis used (1). The present study is designed to investigate the ability of monocytes and NK cells to lyse human noncultured solid tumor targets by employing cytotoxic assaysof longer incubation periods (up to 24 hr). We report here that monocytes and NK-enriched lymphocytes ’ This work was supportedin part by GrantsCA-27197, CA-13148, CA-03013, andCA-41699 from the National Institute of Health, by Grant IM 409D from the American Cancer Society, and by a grant from the Eleanor Naylor Dana Charitable Trust. 495 0008-8749/87 $3.00 Copyright 0 1987 by Academic Press,Inc. All rights of reproduction in any form reserved.
496
SHORT
COMMUNICATION
demonstrate comparable levels of spontaneous cytotoxicity against fresh solid tumor cells. MATERIALS AND METHODS Reagents. Recombinant interferon-y (rIFN-y) and recombinant interleukin 2 (rIG 2) were obtained from Biogen Corp. (Cambridge, MA) (1). All media and reagents used in these experiments were free of endotoxin as determined by the Limulus amebocyte lysate assay (Associates of Cape Cod, Inc., Woodshole, MA, sensitivity limit 0.125 rig/ml of endotoxin). Preparation ofefictor cells. Peripheral blood mononuclear cells (PBMC) prepared from healthy donors were resuspended in RPM1 1640 medium containing 10% FCS (RPMI/FCS), added to FCS-precoated tissue culture dishes (Corning, NY) (FCS dishes) at a concentration of 5 to 10 X lo6 cells/ml (5 ml/dish), and incubated for 60 min at 37°C (3). The dishes were gently washed three times with Hanks’ balanced salt solution (HBSS) and incubated for an additional 30 min. After being washed again, the adherent monocytes were detached using 0.2% ethylenediaminetetraacetic acid (EDTA) and 5% FCS as previously described (3). These monocyte preparations consisted of >99% monocytes as determined by morphological criteria, >98% yeastphagocytizing cells, and >90% LeuM3+ cells. They contained ~0.1% Leul la+, ~0.1% Leu7+ cells, and ~0.2% granular lymphocytes. Nonadherent cells, recovered from the FCS dishes, were washed and applied on a nylon wool column (1) and incubated for 45 min at 37°C to remove any residual monocytes and B cells. Nylon wool nonadherent cells were fractionated into N&Z-enriched and -depleted lymphocyte fractions according to density using discontinuous gradients of Percoll(1). These NK-enriched lymphocytes consisted of 50 + 10% granular lymphocytes, 27 f 6% Leu 11a+ cells, ~0.2% LeuM3+, and ~0.2% yeast-phagocytic cells. Tumor targets and cytotoxic assay. Tumor targets used here were noncultured melanoma tumor cells or noncultured breast tumor cells prepared from surgical specimens, the NK-resistant melanoma cell line MEL2 1, and the KS62 cells (1). Target cells were labeled with 200 PCi of Naz 5’Cr04 (Amersham Corp., IL) or 50 &i of “‘In (indium in oxyquinoline solution; Amersham) for 60 min (1). These cells were applied on a cushion of 66% Ficoll-Hypaque and centrifuged for 30 min at 400g. Cells were collected from the interface, had viability over 90%, and were used astarget cells for 4- to 24-hr 5’Cr or “‘In releaseassaysas described (1). The spontaneous 51Cr release of the noncultured tumor targets expressed as percentage of the maximum release was 20 f 5% at 4 hr, 31 + 5% at 12 hr, 40 + 3% at 18 hr, and 43 j, 5% at 24 hr. The spontaneous *“In release of the noncultured tumor targets was always less than 30% of the maximum. The spontaneous 51Crreleaseof K562 or MEL2 1 targets was always less than 20% of the maximum. Spontaneous releasewas determined by incubating labeled targets in medium alone or with LPS, rIFN-y or rIL-2. The addition of these reagents did not change the spontaneous release.Maximum releasewas determined by incubating targets with 2 N HCl solution. The maximum releasewas approximately 80% of the total releaseof the radiolabeled target cells. Mean percentage lysis in triplicate cultures was calculated as reported previously (1). Statistical analysis was performed by Student’s two-tailed t test and analysis of variance. Difference in percentage lysis of 10% was found to be statistically significant (at least P < 0.0 1 by Student’s two-tailed test).
497
SHORT COMMUNICATION 40 r
.jj
loo,-
Non-cultured
K562
melanoma
lysis
lysis
0 4
12 Assay
16
24
Time (HFO
FIG. 1. Kinetics of lysis by different cell populations. Lysis of melanoma (upper panel) and K562 (lower panel) targets by unstimulated PBMC from 13 healthy donors (X), purified monocytes from 2 1 donors (a), NK-enriched lymphocytes from 11 donors (0), and NK-depleted lymphocytes from 6 donors (A). Values represent the mean + SD of percentage lysis from all donors tested at an E/T ratio of 40. The levels of melanoma lysis by monocytes and NK-emiched lymphocytes were significantly higher (P = 0.001, analysis of variance) than that of NKdepleted lymphocytes. The level of KS62 lysis by NK-enriched lymphocytes was significantly higher (P = 0.00 1, analysis of variance) than by the other cell populations. The levels of monocyte-mediated cytotoxicity against noncultured melanoma targets were higher (P = 0.00 1, analysis of variance) than those against the K562 targets. In contrast, the level ofNK cell-mediated cytotoxicity against the K562 targets was higher (P = 0.00 1,analysis of variance) than that against the noncultured melanoma targets.
RESULTS Monocyte- and NK cell-mediated spontaneous cytotoxicity against solid tumor cells. Unstimulated PBMC were tested for cytotoxicity against noncultured melanoma targets in 4- to 24-hr “Cr release assays(Fig. I). Significant melanoma lysis (greater than 10%) was seen with PBMC from none of 13 donors in a 4-hr assay (mean % lysis: 2 + l%), from 8 of 10 donors tested in a 12-hr assay(mean % lysis: 12 f 2%), from 11 of 13 donors in an 18hr assay(mean % lysis: 17 & 4%), and from all the 13 donors in a 24-hr assay (mean % lysis: 25 + 4%). PBMC assayedwith either 10%autologous human serum or 5% human AB sera exhibited levels of cytotoxicity similar to these assayedwith 10%FCS (data not shown), indicating that this cytotoxicity is not due to the stimulation by FCS. Becausethe spontaneous release of 51Crlabeled noncultured melanomas after an 18-hr incubation was relatively high, the “‘In release assay was undertaken. The spontaneous release of “‘In-labeled melanoma targets was always lessthan 30% of the maximum release.The mean percentage lysis &SD of ‘“In-labeled melanoma targets by PBMC from 5 donors was 2 ? 2, 11 +- 1, 18 + 8, and 21 +- 4% in 4-, 12-, 18- and 24-hr ‘“In release assays,respectively. These levels of cytotoxicity and the kinetics of lysis were similar to those obtained with the “Cr releaseassay(Fig. 1).
498
SHORT
COMMUNICATION
PBMC were fractionated into monocyte, NK-enriched lymphocyte, and NK-depleted lymphocyte fractions. The monocyte fractions used here contained >99% monocytes judged by morphology, ~98% phagocytic cells, >90% LeuM3+ cells, ~0.1% Leu 11a+, and ~0.1% Leu7+ cells. Monocytes and NK-enriched lymphocytes from all donors tested caused more than 10% lysis in a 12-hr “Cr assay.Their cytotoxic activity was increased by prolonging the incubation time of the assay(Fig. I). The mean percentage lysis by monocytes or NK-enriched lymphocytes in a 24-hr assaywas 30 + 12 or 32 + lo%, respectively. Monocytes from none of 2 1 donors and NK-enriched lymphocytes from only 2 of 9 donors caused more than 10% lysis in a 4-hr assay. Treatment of purified monocytes with anti-Leul lb and anti-Leu7 mAb plus complement (C) did not decreasethe lysis of melanoma targets (data not shown). In contrast, the treatment of NK-enriched lymphocytes with anti-Leu 11b and C abolished the lysis of both the melanoma and K562 targets (data not shown), demonstrating that Leu 1I+ NK cells were responsible for the melanoma lysis mediated by NKenriched lymphocytes, but not by purified monocyte fractions. Both monocytes and NK-enriched lymphocytes exhibited comparable levels of lysis against noncultured melanoma, noncultured breast tumor targets, and cells of the MEL2 1 tumor cell line (data not shown), indicating that this cytotoxicity is not MHC-restricted. In contrast, NK-depleted lymphocytes did not lyse the melanoma targets even when prolonged assayswere used. Because monocytes can produce cytotoxic factors in culture (2), we have investigated whether supematants from monocyte cultures contain cytotoxic activity. Supematants of monocyte cultures incubated for 18 hr with medium alone or with unlabeled melanoma tumor cells did not show any cytotoxic activity against melanoma targets in an 18hr “Cr release assay (data not shown). These results suggest that cytotoxic factors produced by monocytes may not account for monocyte-mediated cytotoxicity against the melanoma targets. The kinetics of monocyte-mediated lysis of the K562 targets was also investigated in the same experiments (Fig. 1, lower panel). Monocytes from none of the 19 donors examined exhibited significant K562 lysis (NK activity) in either a 4- or a I2-hr assay. Monocytes from 9 of 17 (53%) donors caused a modest level (10 to 20%) of K562 lysis in an 18-hr assay. These levels of lysis of the K562 targets by monocytes were significantly lower than those of the melanoma targets (P = 0.001, analysis of variance). In contrast, NK-enriched lymphocytes exhibited high levels of NK activity, which were significantly higher than the levels of melanoma or K562 lysis by the other cell fractions (P = 0.00 1, analysis of variance).
Monocyte cytotoxicity against nonculturedmelanomasis not aflectedby endotoxin, but it is augmentedby IFN-7. Monocytes prepared under endotoxin-free conditions were tested for cytotoxicity in the presence of LPS (a low concentration of 1 rig/ml and a high concentration of 1 pg/ml), rIFN-7 ( lo3 U/ml), or rIL-2 ( lo3 U/ml) (Table 1). These LPS concentrations did not affect the levels of cytotoxicity, suggesting that this monocyte-mediated cytotoxicity is not affected by endotoxin. In contrast, rIFN-7 increased the levels of monocyte-mediated lysis against solid tumor cells up to threefold when added directly to the 18-hr cytotoxic assay(Table 1; P < 0.0 1). rIL-2 did not increase the levels of lysis (Table 1). Preincubation of monocytes from three donors with rIFN-y ( 1O3U/ml) for 18 hr followed by washing also resulted in a significant augmentation of monocyte cytotoxicity against noncultured melanomas (32 f 3% lysis vs 18 -t 4% lysis of the controls at an E:T of 40: 1). In contrast, preincu-
499
SHORT COMMUNICATION TABLE I
Activation of Monocyte-Mediated Lysis of Noncultured Melanomas by rIFN-7, but not by LPS or rIL2” I Lysis of noncultured melanomas in the presence of LPS Monocytes (donor)
Medium (endotoxin free)
1 t&ml
1 a/ml
rIFN-7 ( 10’ U/ml)
rIL-2 ( 10’ U/ml)
1 2 3 4 Mean + SD
34.0 22.3 27.5 5.5 22 * 12
30.4 22.5 23.4 5.1 20+-11
29.7 25.1 23.6 9.4 22 2 9
63.0 67.6 67.5 50.8 62 k 8’
34.5 24.1 26.5 7.3 23+11
a Monocytes were purified under endotoxin-free conditions by using RPMI/FCS and FCS dishes. These monocytes were tested for cytotoxicity against noncultured melanomas in an 18-hr “Cr release assay at an ET ratio of 40: 1 in the presence of RPMI/FCS alone, LPS, rIFN-7, or rIL-2. The addition of these agents to the target cells did not affect the spontaneous release. b The levels of lysis induced by rIFN-y were significantly higher than those obtained in the presence of the other agents. (P < 0.0 1, Student’s two-tailed t test.)
bation with LPS (1 @g/ml) for 18 hr did not increase monocyte cytotoxicity ( 18 + 3% lysis vs 18 + 4% lysis of the controls). Monocytes cultured with medium alone for 3 days lost the ability to lyse noncultured melanoma targets (mean * SD of % lysis from eight donors; 3 + 2,0, and 0% at E:T ratios of 40: 1,20: 1, and 10:1, respectively). However, the addition of r1FN-y ( IO3 U/ml) from the beginning of the culture allowed monocytes to retain their cytotoxic activity (mean +- SD of % lysis from eight donors: 18 * 3, 10 It 3, 4 + 2, at E:T ratios of 40: 1, 20: 1, and 10:1, respectively; P < 0.002). Incubation of monocytes with either LPS or rIL-2 did not prevent the cells from losing their cytotoxic activity (data not shown). DISCUSSION Unstimulated monocytes are known to exhibit spontaneous cytotoxicity against cultured tumor cell lines without activation (2,4-6). Endotoxin does not account for spontaneous monocyte cytotoxicity (5, 6), although treatment of monocytes with endotoxin enhances their cytotoxicity (2,6). In contrast, according to certain other reports unstimulated monocytes do not exhibit spontaneous cytotoxicity unless activated by endotoxin or biological response modifiers (7, 8). These investigators used 12’I-IUdR-labeled melanoma tumor cell lines astargets and incubated the monocytes with the targets for 3 days. These methods are clearly different from those that we employ here and may account for the differences in the results. We have demonstrated here that endotoxin does not affect the lysis of noncultured tumor cells by monocytes and we have excluded endotoxin as responsible for causing spontaneous monocyte cytotoxicity. Furthermore, we observed that monocytes cultured in vitro for 3 days lost their ability to spontaneously lyse solid tumors, in agreement with others (6). Therefore, it is not surprising that unstimulated monocytes cultured for 3 days with medium alone did not show spontaneous cytotoxicity against either noncultured tumor targets or melanoma tumor cell lines.
500
SHORT COMMUNICATION
Pretreatment of monocytes with rIFN-y followed by washing or the addition of rIFN-7 directly into the 18-hr 5’Cr releaseassayresulted in significant augmentation of their cytotoxicity. Furthermore, the presence of rIFN--r into 3- to 5-day cultures of monocytes maintained their cytotoxic activity. The augmentation of monocyte cytotoxicity by rIFN-7 is in agreement with the reports of others (9,lO). IFN--r exhibits macrophage-activating factor (MAF) activity and is necessaryfor macrophage activation (10). Thus, IFN-7 may be responsible for the activation of monocyte-mediated cytotoxicity against noncultured solid tumors. In conclusion, we demonstrated spontaneous cytotoxicity against noncultured solid tumors by both monocytes and NK cells. These studies may be of significance in elucidating the role of natural defense mechanisms against solid tumors. ACKNOWLEDGMENT We thank Ms. Helen Fat-r for her secretarial assistance.
REFERENCES 1. Itoh, K., Tilden, A. B., and Balch, C. M., J. Zmmunol. 136,3910, 1986. 2. Chen, A. R., McKinnon, K. P., and Koren, H. S., J. Immunol. 135,3978, 1985. 3. Kumagai, K., Itoh, K., Hinuma, S., and Tada, T., J. Immunol. Methods 29, 17, 1979. 4. Mantovani, A., Jarrells, T. R., Dean, J. H., and Herberman, R. B., Znt.J. Cancer 23, 18, 1979. 5. Sone, S., Mutsura, S., Ogawara, M., and Tsubura, E., J. Immunol. 132,2105,1984. 6. Onozaki, K., Matsushima, K., Kleinetman, E. S., Saito, T., and Oppenheim, J. J., J. Zmmunof. 135, 314,1985. 7. Fidler, I. J., Fogler, W. E., Kleinerman, E. S., and Saiki, I., J. Zmmunol. 135,4289,1985. 8. Kleinerman, E. A., and Herberman, R. B., J. Immunol. 133,4, 1984. 9. Le, J., Prensky, W., Yip, Y. K., Chang, Z., Hoffman, T., Stevenson, H. C., Balazs, I., Sadlik, J. R., and Vilcek, J., J. Immunol. 131,2821, 1983. 10. Nathan, C. F., Prendegast, T. J., Wiebe, M. E., Stanley, E. R., Platzer, E., Remold, H. G., Welte, K., Rubin, B. Y., and Murray, H. W., J. Exp. Med. 160,600, 1984.
IMMUNOLOGY
108,495-500
(1987)
SHORT COMMUNICATION Monocyte- and Natural Killer Cell-Mediated Spontaneous Cytotoxicity against Human Noncultured Solid Tumor Cells’ KYOGO IToH,**?
CHRIS D. l%ATSOUCAS,t
AND CHARLES
M. BALCH*
*Division ofsurgery and Department of General Surgery and TDepartment of Immunology, The University of Texas System Cancer Center, M. D. Anderson Hospital and Tumor Institute, Houston, Texas 77030 Received February 23,1987; acceptedApril 13. I987 Unstimulated human peripheral blood mononuclear cells from healthy donors exhibited spontaneous cytotoxicity against noncultured solid tumor targets in a 12- to 24-hr “Cr release or “‘In release assay. Both purified monocytes (199% monocytes) and natural killer (NK)enriched lymphocytes exhibited comparable levels of spontaneous cytotoxicity against fresh melanoma tumor targets. This cytotoxicity was observed under endotoxin-free conditions. NKdepleted lymphocytes did not lyse the melanoma targets. Culture supematants of monocytes incubated with the melanoma tumor cells did not exhibit cytotoxic activity against these targets. Purified monocytes lacked NK activity against the K562 targets in a 4-hr 5’Cr release assay. Treatment of the monocytes with anti-Leul 1b and antiLeu7 monoclonal antibodies plus complement did not reduce monocyte-mediated lysis of the melanoma targets, demonstrating that contaminating NK cells, if any, were not responsible for the lysis of noncultured melanoma targets by monocytes. In contrast, Leul I b+ NK cells were responsible for the lysis of the melanoma targets by NK-enriched lymphocytes. The addition of recombinant interferon-r (rIFNy), but not lipopolysaccharide, into the 5’Cr release assay or pretreatment of monocytes with rIF’N-y significantly increased their cytotoxicity against noncultured solid tumor cells. Monocytes cultured for 3 days with medium alone lost their cytotoxic activity. The addition of rIFNy from the beginning of these cultures prevented the loss of the cytotoxic activity of monocytes. In summary, both unstimulated monocytes and NK-enriched lymphocytes exhibit comparable levels of spontaneous cytotoxicity against fresh solid tumor targets. o 1987 Academic press, 1~.
INTRODUCTION Human monocytes and NK cells have spontaneous cytotoxic activity against several established tumor cell lines in vitro, without requiring previous immunization (I, 2). However, their ability to lyse freshly prepared solid tumor targets is usually low or undetectable when a standard 4-hr cytotoxicity assayis used (1). The present study is designed to investigate the ability of monocytes and NK cells to lyse human noncultured solid tumor targets by employing cytotoxic assaysof longer incubation periods (up to 24 hr). We report here that monocytes and NK-enriched lymphocytes ’ This work was supportedin part by GrantsCA-27197, CA-13148, CA-03013, andCA-41699 from the National Institute of Health, by Grant IM 409D from the American Cancer Society, and by a grant from the Eleanor Naylor Dana Charitable Trust. 495 0008-8749/87 $3.00 Copyright 0 1987 by Academic Press,Inc. All rights of reproduction in any form reserved.
496
SHORT
COMMUNICATION
demonstrate comparable levels of spontaneous cytotoxicity against fresh solid tumor cells. MATERIALS AND METHODS Reagents. Recombinant interferon-y (rIFN-y) and recombinant interleukin 2 (rIG 2) were obtained from Biogen Corp. (Cambridge, MA) (1). All media and reagents used in these experiments were free of endotoxin as determined by the Limulus amebocyte lysate assay (Associates of Cape Cod, Inc., Woodshole, MA, sensitivity limit 0.125 rig/ml of endotoxin). Preparation ofefictor cells. Peripheral blood mononuclear cells (PBMC) prepared from healthy donors were resuspended in RPM1 1640 medium containing 10% FCS (RPMI/FCS), added to FCS-precoated tissue culture dishes (Corning, NY) (FCS dishes) at a concentration of 5 to 10 X lo6 cells/ml (5 ml/dish), and incubated for 60 min at 37°C (3). The dishes were gently washed three times with Hanks’ balanced salt solution (HBSS) and incubated for an additional 30 min. After being washed again, the adherent monocytes were detached using 0.2% ethylenediaminetetraacetic acid (EDTA) and 5% FCS as previously described (3). These monocyte preparations consisted of >99% monocytes as determined by morphological criteria, >98% yeastphagocytizing cells, and >90% LeuM3+ cells. They contained ~0.1% Leul la+, ~0.1% Leu7+ cells, and ~0.2% granular lymphocytes. Nonadherent cells, recovered from the FCS dishes, were washed and applied on a nylon wool column (1) and incubated for 45 min at 37°C to remove any residual monocytes and B cells. Nylon wool nonadherent cells were fractionated into N&Z-enriched and -depleted lymphocyte fractions according to density using discontinuous gradients of Percoll(1). These NK-enriched lymphocytes consisted of 50 + 10% granular lymphocytes, 27 f 6% Leu 11a+ cells, ~0.2% LeuM3+, and ~0.2% yeast-phagocytic cells. Tumor targets and cytotoxic assay. Tumor targets used here were noncultured melanoma tumor cells or noncultured breast tumor cells prepared from surgical specimens, the NK-resistant melanoma cell line MEL2 1, and the KS62 cells (1). Target cells were labeled with 200 PCi of Naz 5’Cr04 (Amersham Corp., IL) or 50 &i of “‘In (indium in oxyquinoline solution; Amersham) for 60 min (1). These cells were applied on a cushion of 66% Ficoll-Hypaque and centrifuged for 30 min at 400g. Cells were collected from the interface, had viability over 90%, and were used astarget cells for 4- to 24-hr 5’Cr or “‘In releaseassaysas described (1). The spontaneous 51Cr release of the noncultured tumor targets expressed as percentage of the maximum release was 20 f 5% at 4 hr, 31 + 5% at 12 hr, 40 + 3% at 18 hr, and 43 j, 5% at 24 hr. The spontaneous *“In release of the noncultured tumor targets was always less than 30% of the maximum. The spontaneous 51Crreleaseof K562 or MEL2 1 targets was always less than 20% of the maximum. Spontaneous releasewas determined by incubating labeled targets in medium alone or with LPS, rIFN-y or rIL-2. The addition of these reagents did not change the spontaneous release.Maximum releasewas determined by incubating targets with 2 N HCl solution. The maximum releasewas approximately 80% of the total releaseof the radiolabeled target cells. Mean percentage lysis in triplicate cultures was calculated as reported previously (1). Statistical analysis was performed by Student’s two-tailed t test and analysis of variance. Difference in percentage lysis of 10% was found to be statistically significant (at least P < 0.0 1 by Student’s two-tailed test).
497
SHORT COMMUNICATION 40 r
.jj
loo,-
Non-cultured
K562
melanoma
lysis
lysis
0 4
12 Assay
16
24
Time (HFO
FIG. 1. Kinetics of lysis by different cell populations. Lysis of melanoma (upper panel) and K562 (lower panel) targets by unstimulated PBMC from 13 healthy donors (X), purified monocytes from 2 1 donors (a), NK-enriched lymphocytes from 11 donors (0), and NK-depleted lymphocytes from 6 donors (A). Values represent the mean + SD of percentage lysis from all donors tested at an E/T ratio of 40. The levels of melanoma lysis by monocytes and NK-emiched lymphocytes were significantly higher (P = 0.001, analysis of variance) than that of NKdepleted lymphocytes. The level of KS62 lysis by NK-enriched lymphocytes was significantly higher (P = 0.00 1, analysis of variance) than by the other cell populations. The levels of monocyte-mediated cytotoxicity against noncultured melanoma targets were higher (P = 0.00 1, analysis of variance) than those against the K562 targets. In contrast, the level ofNK cell-mediated cytotoxicity against the K562 targets was higher (P = 0.00 1,analysis of variance) than that against the noncultured melanoma targets.
RESULTS Monocyte- and NK cell-mediated spontaneous cytotoxicity against solid tumor cells. Unstimulated PBMC were tested for cytotoxicity against noncultured melanoma targets in 4- to 24-hr “Cr release assays(Fig. I). Significant melanoma lysis (greater than 10%) was seen with PBMC from none of 13 donors in a 4-hr assay (mean % lysis: 2 + l%), from 8 of 10 donors tested in a 12-hr assay(mean % lysis: 12 f 2%), from 11 of 13 donors in an 18hr assay(mean % lysis: 17 & 4%), and from all the 13 donors in a 24-hr assay (mean % lysis: 25 + 4%). PBMC assayedwith either 10%autologous human serum or 5% human AB sera exhibited levels of cytotoxicity similar to these assayedwith 10%FCS (data not shown), indicating that this cytotoxicity is not due to the stimulation by FCS. Becausethe spontaneous release of 51Crlabeled noncultured melanomas after an 18-hr incubation was relatively high, the “‘In release assay was undertaken. The spontaneous release of “‘In-labeled melanoma targets was always lessthan 30% of the maximum release.The mean percentage lysis &SD of ‘“In-labeled melanoma targets by PBMC from 5 donors was 2 ? 2, 11 +- 1, 18 + 8, and 21 +- 4% in 4-, 12-, 18- and 24-hr ‘“In release assays,respectively. These levels of cytotoxicity and the kinetics of lysis were similar to those obtained with the “Cr releaseassay(Fig. 1).
498
SHORT
COMMUNICATION
PBMC were fractionated into monocyte, NK-enriched lymphocyte, and NK-depleted lymphocyte fractions. The monocyte fractions used here contained >99% monocytes judged by morphology, ~98% phagocytic cells, >90% LeuM3+ cells, ~0.1% Leu 11a+, and ~0.1% Leu7+ cells. Monocytes and NK-enriched lymphocytes from all donors tested caused more than 10% lysis in a 12-hr “Cr assay.Their cytotoxic activity was increased by prolonging the incubation time of the assay(Fig. I). The mean percentage lysis by monocytes or NK-enriched lymphocytes in a 24-hr assaywas 30 + 12 or 32 + lo%, respectively. Monocytes from none of 2 1 donors and NK-enriched lymphocytes from only 2 of 9 donors caused more than 10% lysis in a 4-hr assay. Treatment of purified monocytes with anti-Leul lb and anti-Leu7 mAb plus complement (C) did not decreasethe lysis of melanoma targets (data not shown). In contrast, the treatment of NK-enriched lymphocytes with anti-Leu 11b and C abolished the lysis of both the melanoma and K562 targets (data not shown), demonstrating that Leu 1I+ NK cells were responsible for the melanoma lysis mediated by NKenriched lymphocytes, but not by purified monocyte fractions. Both monocytes and NK-enriched lymphocytes exhibited comparable levels of lysis against noncultured melanoma, noncultured breast tumor targets, and cells of the MEL2 1 tumor cell line (data not shown), indicating that this cytotoxicity is not MHC-restricted. In contrast, NK-depleted lymphocytes did not lyse the melanoma targets even when prolonged assayswere used. Because monocytes can produce cytotoxic factors in culture (2), we have investigated whether supematants from monocyte cultures contain cytotoxic activity. Supematants of monocyte cultures incubated for 18 hr with medium alone or with unlabeled melanoma tumor cells did not show any cytotoxic activity against melanoma targets in an 18hr “Cr release assay (data not shown). These results suggest that cytotoxic factors produced by monocytes may not account for monocyte-mediated cytotoxicity against the melanoma targets. The kinetics of monocyte-mediated lysis of the K562 targets was also investigated in the same experiments (Fig. 1, lower panel). Monocytes from none of the 19 donors examined exhibited significant K562 lysis (NK activity) in either a 4- or a I2-hr assay. Monocytes from 9 of 17 (53%) donors caused a modest level (10 to 20%) of K562 lysis in an 18-hr assay. These levels of lysis of the K562 targets by monocytes were significantly lower than those of the melanoma targets (P = 0.001, analysis of variance). In contrast, NK-enriched lymphocytes exhibited high levels of NK activity, which were significantly higher than the levels of melanoma or K562 lysis by the other cell fractions (P = 0.00 1, analysis of variance).
Monocyte cytotoxicity against nonculturedmelanomasis not aflectedby endotoxin, but it is augmentedby IFN-7. Monocytes prepared under endotoxin-free conditions were tested for cytotoxicity in the presence of LPS (a low concentration of 1 rig/ml and a high concentration of 1 pg/ml), rIFN-7 ( lo3 U/ml), or rIL-2 ( lo3 U/ml) (Table 1). These LPS concentrations did not affect the levels of cytotoxicity, suggesting that this monocyte-mediated cytotoxicity is not affected by endotoxin. In contrast, rIFN-7 increased the levels of monocyte-mediated lysis against solid tumor cells up to threefold when added directly to the 18-hr cytotoxic assay(Table 1; P < 0.0 1). rIL-2 did not increase the levels of lysis (Table 1). Preincubation of monocytes from three donors with rIFN-y ( 1O3U/ml) for 18 hr followed by washing also resulted in a significant augmentation of monocyte cytotoxicity against noncultured melanomas (32 f 3% lysis vs 18 -t 4% lysis of the controls at an E:T of 40: 1). In contrast, preincu-
499
SHORT COMMUNICATION TABLE I
Activation of Monocyte-Mediated Lysis of Noncultured Melanomas by rIFN-7, but not by LPS or rIL2” I Lysis of noncultured melanomas in the presence of LPS Monocytes (donor)
Medium (endotoxin free)
1 t&ml
1 a/ml
rIFN-7 ( 10’ U/ml)
rIL-2 ( 10’ U/ml)
1 2 3 4 Mean + SD
34.0 22.3 27.5 5.5 22 * 12
30.4 22.5 23.4 5.1 20+-11
29.7 25.1 23.6 9.4 22 2 9
63.0 67.6 67.5 50.8 62 k 8’
34.5 24.1 26.5 7.3 23+11
a Monocytes were purified under endotoxin-free conditions by using RPMI/FCS and FCS dishes. These monocytes were tested for cytotoxicity against noncultured melanomas in an 18-hr “Cr release assay at an ET ratio of 40: 1 in the presence of RPMI/FCS alone, LPS, rIFN-7, or rIL-2. The addition of these agents to the target cells did not affect the spontaneous release. b The levels of lysis induced by rIFN-y were significantly higher than those obtained in the presence of the other agents. (P < 0.0 1, Student’s two-tailed t test.)
bation with LPS (1 @g/ml) for 18 hr did not increase monocyte cytotoxicity ( 18 + 3% lysis vs 18 + 4% lysis of the controls). Monocytes cultured with medium alone for 3 days lost the ability to lyse noncultured melanoma targets (mean * SD of % lysis from eight donors; 3 + 2,0, and 0% at E:T ratios of 40: 1,20: 1, and 10:1, respectively). However, the addition of r1FN-y ( IO3 U/ml) from the beginning of the culture allowed monocytes to retain their cytotoxic activity (mean +- SD of % lysis from eight donors: 18 * 3, 10 It 3, 4 + 2, at E:T ratios of 40: 1, 20: 1, and 10:1, respectively; P < 0.002). Incubation of monocytes with either LPS or rIL-2 did not prevent the cells from losing their cytotoxic activity (data not shown). DISCUSSION Unstimulated monocytes are known to exhibit spontaneous cytotoxicity against cultured tumor cell lines without activation (2,4-6). Endotoxin does not account for spontaneous monocyte cytotoxicity (5, 6), although treatment of monocytes with endotoxin enhances their cytotoxicity (2,6). In contrast, according to certain other reports unstimulated monocytes do not exhibit spontaneous cytotoxicity unless activated by endotoxin or biological response modifiers (7, 8). These investigators used 12’I-IUdR-labeled melanoma tumor cell lines astargets and incubated the monocytes with the targets for 3 days. These methods are clearly different from those that we employ here and may account for the differences in the results. We have demonstrated here that endotoxin does not affect the lysis of noncultured tumor cells by monocytes and we have excluded endotoxin as responsible for causing spontaneous monocyte cytotoxicity. Furthermore, we observed that monocytes cultured in vitro for 3 days lost their ability to spontaneously lyse solid tumors, in agreement with others (6). Therefore, it is not surprising that unstimulated monocytes cultured for 3 days with medium alone did not show spontaneous cytotoxicity against either noncultured tumor targets or melanoma tumor cell lines.
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SHORT COMMUNICATION
Pretreatment of monocytes with rIFN-y followed by washing or the addition of rIFN-7 directly into the 18-hr 5’Cr releaseassayresulted in significant augmentation of their cytotoxicity. Furthermore, the presence of rIFN--r into 3- to 5-day cultures of monocytes maintained their cytotoxic activity. The augmentation of monocyte cytotoxicity by rIFN-7 is in agreement with the reports of others (9,lO). IFN--r exhibits macrophage-activating factor (MAF) activity and is necessaryfor macrophage activation (10). Thus, IFN-7 may be responsible for the activation of monocyte-mediated cytotoxicity against noncultured solid tumors. In conclusion, we demonstrated spontaneous cytotoxicity against noncultured solid tumors by both monocytes and NK cells. These studies may be of significance in elucidating the role of natural defense mechanisms against solid tumors. ACKNOWLEDGMENT We thank Ms. Helen Fat-r for her secretarial assistance.
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