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Decreased natural cytotoxicity in mice with high incidence of mammary adenocarcinoma
CLINICAL
lMMUNOLOGY
Decreased
AND
Natural
IMMUNOPATHOLOGY
38, 265-273 (1986)
Cytotoxicity in Mice with High Incidence Mammary Adenocarcinoma
of
IRA H. AMES,~ A. MARIANO GARCIA, PATRICIA A. JOHN, CATHY A. LITTY, MICHAEL A. FARRELL, AND RUSSELL H. TOMAR Departments of Anatomy and Cell Biology State University of New York, Upstate
and Pathology, Medical Center,
Division of Clinical Pathology, Syracuse, New York 13210
In an attempt to gather evidence relevant to the question of whether natural killer (NK) cells play a role in resisting the development of primary tumors, we compared natural cell-mediated cytotoxicity in two substrains of C,H mice. Animals of the C,H/ OuJ substrain are at high risk for the formation of mammary adenocarcinomas, while C,Heb/FeJ mice have a low incidence of such tumors. Natural cytotoxicity of splenic mononuclear cells was lower in the high-risk substrain, suggesting that a lesion in NK cell activity may be involved in murine mammary tumorigenesis. This difference was observed in animals between 5 and 37 weeks of age. There was no significant difference in the number of splenic large granular lymphocytes between the substrains. A significant difference in the ability of splenic lymphocytes from the two substrains to bind to the target cells was noted. Since the binding capacity of lymphocytes was greater in mice with reduced NK cell activity, the lesion in cytotoxicity may exist at a postbinding step in the lytic sequence. It is felt that the C,H mouse may provide a useful model for studying the role of NK cells in controlling primary tumors. 0 1986 Academic Press, Inc.
INTRODUCTION
Natural killer cells represent a heterogeneous subpopulation of normal lymphocytes that are present in substantial numbers in peripheral blood and spleen (1). They are characterized by the presence of prominent azurophilic cytoplasmic granules and a reniform nucleus. Considerable attention has been focused recently on the role of NK cells in immunosurveillance against neoplastic cells, and it has been suggested that they may be part of the first line of defense against malignant growth (2). The evidence supporting the conclusion that NK cells are responsible for rejection of implanted syngeneic tumors and inhibition of experimental tumor metastasis is extensive (3). However, the evidence for an effective role of these cells in resistance to growth and metastasis of spontaneous or carcinogen-induced primary tumors is meager (4). In a recent attempt to determine whether NK cells play a role in immunosurveillance against neoplastic cells, Strayer and his colleagues demonstrated an inverse relationship between familial incidence of cancer and NK cell activity (5). In other words, a lesion in natural cell-mediated cytotoxicity appears to preexist i To whom reprint requests should be addressed. 2 Abbreviations used: NK, natural killer; MMTV, mouse mammary tumor virus; RPMI, Roswell Park Memorial Institute; PBR, phosphate-buffered Ringer’s; FBS, fetal bovine serum; BBS, benign breast syndrome. 265 0090-1229/86 $1.50 Copyright AU rights
6 1986 by Academic Press, Inc. of reproduction in any form reserved.
266
AMES
ET
AL.
in individuals at risk for the development of cancer. We now report that this phenomenon also occurs in mice, and show that the defect in NK cell activity can be observed throughout most of the life span of animals having a high risk of mammary cancer. This finding may be significant with respect to the possibility of early identification of individuals at increased risk of cancer development. MATERIALS
AND METHODS
Animals. Since spontaneous mammary tumors of C,H mice are susceptible to lysis by NK cells (I), we chose to compare NK cell activity in two closely related substrains that differ markedly in their incidence of tumors. Mice of the C,H/OuJ substrain are characterized by the presence of mouse mammary tumor virus and are at high risk for the development of mammary adenocarcinomas. This substrain was isolated from the C,H/HeJ stock in 1952. Mice of the C,Heb/FeJ substrain do not carry MMTV and have a low incidence of mammary tumors. This substrain was developed by transplantation of C,H/HeJ ovaries into C57BL/6 female recipients subsequently mated to C,H/HeJ males (6). All animals were purchased from the Jackson Laboratory, Bar Harbor, Maine, at 4-5 weeks of age. They were maintained in the animal quarters at Upstate Medical Center for the duration of the experiments. Tumor target cells. YAC-1, a tissue culture cell line derived from a Moloney virus-induced lymphoma in A/Sn mice (7), was maintained as an in vitro suspension culture in RPM1 1640 medium supplemented with 10% heat-inactivated fetal bovine serum and 50 kg/ml gentamicin. For the evaluation of NK activity, 10 x lo6 YAC-1 cells in a total volume of 0.4 ml phosphate-buffered Ringer’s were labeled by incubation with 200 &i sodium “chromate (250-500 mCi/mg Cr: Amersham Corp.) for 40 min at 37°C. The labeled cells were washed three times with PBR and resuspended at a concentration of 0.2 x lo6 cells/ml in RPM1 1640 medium supplemented with 40% FBS. Preparation of effector cells. Spleens were obtained from ether-anesthetized animals, and single-cell suspensions were prepared by forcing the tissue through a stainless-steel wire mesh. Pooled cells from two to three animals were used in each experiment. Adherent cells were removed by incubating whole spleen cells for 45 min at 37°C on columns of 4 g of nylon wool in 30-ml plastic disposable syringes (8). The nonadherent cells were eluted in 50 ml of warm RPM1 1640 medium supplemented with 10% FBS and further purified by centrifugation through Percoll fraction 7 (Pharmacia Chemicals, Uppsala, Sweden). Cells from the interface between layers were collected, washed three times, and resuspended in RPM1 1640 medium (9). Evaluation ofcefl morphology. The morphology of all effector cell preparations was evaluated by microscopic analysis of Wright-Giemsa stained cytocentrifuge preparations. At least 200 cells were analyzed from each slide. Cytotoxicity assay. A 51Cr-release cytotoxicity assay in 96-well, round-bottomed microtiter plates (Linbro Scientific Co.) was used. Various numbers of effector cells were mixed with 50 p.1 of 0.2 x 106 “Cr-labeled target cells/ml in a total volume of 0.2 ml. The effector-to-target ratio ranged from 4O:l to 1O:l.
LOW NK CELL ACTIVITY
IN HIGH TUMOR
267
MICE
Plates were incubated for 4 hr at 37°C in a humidified 5% CO, atmosphere. Subsequent to centrifugation for 5 min at 45Og, the supernatant from each well was removed by the Titertek automatic harvesting system (Flow Laboratories) and counted in a Searle Model 1185 gamma counter for 10 min. Spontaneous release of isotope was estimated by incubating the labeled target cells alone. Maximal isotope release was estimated by exposure of labeled target cells to 10% Triton X-100. All determinations were performed in triplicate, and percent cytotoxicity was calculated as: % cytotoxicity
cpm of test group - spontaneous cpm x 100. maximal cpm - spontaneous cpm
=
Conjugation assay. In order to measure the binding capacity of the lymphocytes, 2 x lo5 effector cells were incubated with 2 x 10’ unlabeled YAC-1 cells in 2.0 ml of RPM1 1640 medium supplemented with 10% FBS for 10 min at 37°C. The suspension was centrifuged at 1OOg for 5 min, and the pellet was gently resuspended by five aspirations with a Pasteur pipet. The cells were cytocentrifuged onto microscope slides and stained with Wright-Giemsa. The frequency of conjugate-forming lymphocytes was determined by microscopic examination of 200 lymphocytes from each slide (10). Statistical analysis. The results were evaluated for statistical significance by factorial analysis of variance or Student’s t test. RESULTS
To determine the incidence of mammary tumor formation, 20 mice of each strain were examined at weekly intervals. As can be seen in Fig. 1, tumors first appeared in mice of the C,H/OuJ substrain at 29 weeks of age. At the conclusion of the observation period (37 weeks), 60% of these animals had mammary tumors. Histological examination of the tumors revealed them to be either type A or type B adenocarcinomas according to the scheme introduced by Dunn (11). Mice of the C,Heb/FeJ substrain developed no mammary tumors during the course of this investigation.
60 50
o C3H/OuJ (20 mice) C3Heb/FeJ (20 mice)
l
E x z 110 6 g E
30
2
20
z z 10 BP 0 28
29
30
32 31 Age of Mice
33 (Week?.)
34
35
36
37
FIG. 1. Cumulative incidence of mammary adenocarcinoma formation in two substrains of C,H mice.
268
AMES
ET AL.
Splenic lymphocytes from both substrains were tested for their ability to lyse highly NK-sensitive YAC-1 cells in a standard 4-hr S*Cr release assay, and the results are shown in Tables I and 2. In Table 1, the data are presented in terms of percentage cytotoxicity, and each data point represents the arithmetic mean plus or minus the standard error of the mean of three independent experiments. It is clear that splenic mononuclear cells from mice at high risk for the development of mammary adenocarcinomas (C,H/OuJ) had consistently lower NK cell activity than did comparable cells from the low-risk substrain (C,Heb/FeJ). This difference in cytotoxicity was observed at every age studied and averages about 20%. The data presented in Table 1 were tested for statistical significance by a factorial analysis of variance (12). This analysis confirmed that the difference in cytotoxicity between low- and high-risk substrains was significant (F{, = 7.8; P < 0.025); however, there was no significant difference between any of the age groups studied (F$ = 2.5; P > 0.05) or between young (5-15 weeks) and old (23-37 weeks) animals CFj4 = 2.3: P > 0.5). In order to further illustrate the difference in natural killer cell activity between substrains, we calculated the results of the cytotoxicity assays in terms of cytolytic units/lo7 cells. Each cytolytic unit represents the number of effector cells required to cause 30% lysis. TABLE NK
CELL
ACTIVITY
OF SPI.ENIC
LYMPHOCYTES DEVELOPMENT
1
FROM CJH OF MAMMARY
MICE A’T Low
.~NU HIGH
K~src
FOR IHE
TUMORS
% Cytotoxicity
Age (weeks)
E:T Ratios
g Reduction
C,H/OuJ (High)
C,Heb/FeJ (Low)
Mean reduction
5-7
4O:l 2O:l IO:1
39.8 2 6.5” 28.5 2 4.9 20.2 t 7.8
32.8 t 4.8” 22.6 2 4.8 16.3 t 3.6
17.6 20.7 19.3
19.2
S-10
40:1 2O:l IO:1
50.1 ? 2.9 40.4 + 1.0 23.5 k 4.9
38.1 f 25.8 t 19.0 i
1.1 1.0 I.2
24.0 36.1 19.2
‘6.4
13-15
40: I 20: 1 1O:l
40.9 k 5.8 30.3 k 3.9 19.8 i 3.3
36.2 2 2.4 ‘8.7 t 2.5 16.5 _t 2.5
1 I.5 53 16.7
11.2
23-25
40: 1 20: I 1O:l
36.1 -c 2.4 27.1 + 2.5 17.0 i 1.8
28.9 2 2.6 19.5 + 1.1 12.4 ir 1.4
19.Y 28.0 27.1
25.0
27-29
4O:l 20:1 1O:l
39.4 2 6.7 23.5 2 5.3 16.3 2 3.4
31.4 + 5.8 16.3 + 6.1 12.9 5 4.5
20.3 30.6 20.9
13.9
36-37
4O:l 20: 1 10:1
53.4 2 2.5 29.0 k 4.5 18.1 t 4.5
44.3 ? 5.0 27.1 t 2.5 18.8 _t I.2
17.0 6.6 -
7.9
B Each value represents independent experiments.
the arithmetic
mean
plus or minus
the standard
error
of the mean of three
LOW
NK
CELL
ACTIVITY
IN HIGH
TUMOR
269
MICE
This data is presented in Table 2. Once again, the high-risk mice had lower natural cell-mediated cytotoxicity than the low-risk animals at every age studied. This difference averages about 30%. The data in Table 2 were tested for statistical significance by the paired Student’s t test (12) which confirmed that the difference between the substrains was significant (P < 0.001). It is conceivable that the observed difference in in vitro cytotoxicity between splenic lymphocytes from the two substrains was due to differences in the number of NK cells in the final effector cell preparations. To examine this possibility, the morphology of all such preparations was evaluated by microscopic analysis. The cellular composition of final preparations of effector cells is shown in Table 3. One can see that the distribution of cell types was virtually identical in the preparations used for the cytotoxicity assays and that, in particular, the percentage of cells with the morphology of large granular lymphocytes did not differ between the two substrains. Since the number of large granular lymphocytes recovered from the spleen appears to be the same in the high- and low-risk animals, the percentage of mononuclear cells capable of forming conjugates with the target cells was determined. The results of the conjugation assays are presented in Table 4. These data show that there was a significant difference in the ability of lymphocytes from TABLE NK
2
CELL ACTIVITY OF SPLENIC LYMPHOCYTESFROM C,H MICE AT LOWAND HIGH RISK FORTHE DEVELOPMENT OF MAMMARY TUMORS Cytolytic
units
Expt no.
C,Heb/FeJ (Low)
CsH/OuJ (High)
1 2 3
118.8” 39.0 24.1
60.1” 21.3 21.4
49.4 45.4 11.2
1 2
93.1 66.1
45.6 41.3
51.3 37.5
13-15
1 2 3
45.3 11.4 28.8
44.6 56.7 33.7
1.5 26.7 (14.5)
4.6
23-25
1 2 3
37.5 52.4 49.6
15.0 31.0 21.9
60.0 40.8 55.8
52.2
27-29
1 2 3
64.3 31.9 23.1
50.3 29.3 10.5
21.8 22.7 54.5
33.0
36-37
1 2 3
17.1 54.8 49.8
55.6 58.9 33.0
27.9 (7.0) 33.7
18.2
Age (weeks)
5-7 8-10
0 The results are expressed as lytic cells required to cause 30% lysis.
units/lo’
cells,
with
YO Reduction
a lytic
unit being
Mean reduction
35.3 44.4
the number
of effector
270
AMES ET AL. TABLE 3 CELLULAR
COMPOSITION
OF FINAL
EFFECTOR
C,Heb/FeJ (low risk) Small-medium lymphocytes Large agranular lymphocytes Large granular lymphocytes Granulocytes Others
74.2 16.6 6.5 1.9 1.2
k r 2 f -c
CELL PREPARATIONS
C,H/OuJ (high risk)
1.2” 0.7 0.6 0.3 0.2
72.0 18.5 6.4 1.8 1.3
k 2 " 2 ?
1.0" I.0 0.6 0.3 0.2
lb
P
1.4085 I.5565 0.1179 0.2357 0.3536
‘co.20 <0.20 -co.95 co.90 <0.80
a Arithmetic mean f standard error of the mean of percentages of cell types b -o-tailed Student’s r test with 32 df
the two substrains to bind to the YAC-1 cells. It should be noted that the binding capacity of the cells from high-risk mice was greater than that of lymphocytes from low-risk animals. Because the cells had been cytocentrifuged onto slides and subsequently stained, it was possible to study the target-binding capacity of the lymphocyte subpopulations. One can see that the difference in total binding was attributable to increases in the conjugation of both small-medium and large granular lymphocytes with the target cells. DISCUSSION
At present, the bulk of our knowledge of the role of NK cells in immunosurveillance against neoplastic cells is derived from studies of in Go model systems utilizing transplanted tumors or experimental metastases (3). Little work has been reported on the role of these cells in resisting the growth and metastasis of induced or spontaneous primary tumors. The major problem in this respect has been the lack of a proper model system. As noted by Stutman (13), such a system should involve animals with the same genetic background that differ in their levels of NK cell activity. It appears that the C,H mouse may provide that system. To the best of our knowledge mice of the C,H/OuJ and C,Heb/FeJ substrains are genetically identical. They do not differ for any of the genetic markers that have been studied, including histocompatibility (6). We now have demonstrated that TABLE 4 PERCENTAGE
Small-medium lymphocytes Large agranular lymphocytes Large granular lymphocytes Total
OF TOTAL LYMPHOCYTES
BOUND
TO TARGET CELLS
C,Heb/FeJ (low risk)
C,HlOuJ thigh risk)
7.8 1.6 4.6 13.9
11.4 2.0 7.0 20.3
-t 2 2 f
0.7" 0.4 0.6 0.9
t 1.4" I? 0.5 +- 0.6 IT 2.2
tb
P
2.3000 0.6247 2.8285 2.6925
CO.05 co.60 co.01 ‘co.02
(1Each value represents the arithmetic mean + standard error of the mean of three independent experiments. b ‘No-tailed Student’s t test with 28 d$
LOW
NK
CELL
ACTIVITY
IN
HIGH
TUMOR
MICE
271
these substrains differ in terms of their levels of NK cell activity. In addition, we have confirmed that they also differ markedly with respect to their susceptibility to the development of mammary tumors. Mice of the C&I/OuJ substrain carry MMTV and have a high incidence of mammary adenocarcinomas, while C,Heb/ FeJ mice are free of MMTV and seldom develop mammary gland tumors. The mechanism of MMTV-induced tumorigenesis is not completely understood. Most of the work in this area has focused upon the transforming effect of the retrovirus on mammary epithelial cells, and the model of insertional mutagenesis is compelling (14). It is interesting that a high level of MMTV antigen is expressed in lymphocytes (15), and it is conceivable that the virus is present in NK cells. If this is the case, the ability of infected NK cells to lyse tumor cells may be impaired. Such a lesion could enhance the expression of malignant disease. Strayer er al. (5) have demonstrated a correlation between high familial incidence of cancer and low natural cell-mediated cytotoxicity in humans. Our data indicate that the same is true for C,H mice. Animals of the C,H/OuJ substrain that are at high risk for the development of mammary tumors had consistently lower NK cell activity than C,Heb/FeJ mice that have a rather low risk of mammary cancer. Because of the nature of their study, it was not possible for Strayer and his colleagues (5) to determine when the lesion in natural cell-mediated cytotoxicity first manifested itself in the individuals at increased risk of cancer development. If, as they suggest, it may be possible to identify such people by determining their NK cell activity, it would be important to know how early such a determination could be made. In this context, it is interesting to note that in the C,H mouse system the difference in natural cell-mediated cytotoxicity was demonstrable at 5-7 weeks of age and continued throughout most of the life span of the animals. In contrast to the results of Strayer et al. (5), Pross et al. (16) observed that NK cell activity was elevated in women at high risk for breast cancer. It is difficult to evaluate this discrepancy because the criteria for determining risk were quite different. In the work of Strayer and his colleagues, family history was the sole criterion, while in the other case personal gynecological history and presence of benign breast syndrome were also utilized. It should be noted that the entire elevation reported by Pross et al. (16) was attributable to the BBS subgroup. Strayer et al. (5) refer to the individuals in their study as normal, and report no attempt to detect BBS in these women. It has been reported that in mice NK cell activity reaches a peak at 5-8 weeks of age and then declines to low levels when the animals are 3 months old (17). While Itoh et al. (18) showed that C,H/He mice closely followed this pattern of age distribution, our data indicate that this may not be true for all C,H mice. As can be seen in Tables 1 and 2, the level of natural cell-mediated cytotoxicity was fairly constant from 5-7 to 36-37 weeks of age for both C,HIOuJ and C,Heb/ FeJ substrains. In this connection, it is interesting to note that Bartizal ef al. (19) recently reported that no differences in NK cell activity were evident in C,H/ HeCr mice of different ages. The question of whether these findings reflect true differences between substrains or subtle differences in experimental protocol requires further study.
272
AMES ET AL.
At this point in time we have examined two of the possible explanations for the observed difference in the level of NK cell activity in high- and low-risk mice. First, we studied the morphology of the effector cell preparations and determined that the substrains did not differ with regard to the number of large granular lymphocytes present in the cytotoxicity assays. Second, we performed conjugation assays in order to learn whether there was a difference in the ability of lymphocytes from the two substrains to bind to the target cells. In this case the results were surprising, since the binding capacity of splenic mononuclear cells was greater in the animals with lesser NK cell activity. Therefore, the lesion in cell-mediated cytotoxicity in C,H/OuJ mice may involve the ability of their NK cells to complete the stages of the lytic sequence, and studies designed to precisely define it are now in progress. We also plan to compare NK cells from the two substrains with respect to their ability to release natural killer cytotoxic factors, to produce and respond to interferon, and to respond to interleukin 2. In conclusion, we feel that the C,H mouse may be a useful model for studying the in viva role of NK cells in controlling primary tumors, It has been known for quite some time that the two substrains with which we are working differ with respect to their susceptibility to the formation of mammary tumors. We have now shown that they also differ in regard to their levels of NK cell activity and are currently attempting to determine whether these two phenomena are correlated in addition to being coincidental. ACKNOWLEDGMENTS This research was supported by U.S. Public Health Service Grant 2S07RR0540223, the Immunology Research, Development and Education Fund, and the Dr. Glenn H. Leak Memorial Cancer Fellowship Program of the American Cancer Society. We are grateful to Dr. Richard P. Oates for his advice with respect to the statistical analysis of the data and to Ms. Nancy Snyder for her help with the manuscript.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
Ortaldo, J. R., and Herberman, R. B., Annu. Rev. Immunol. 2, 359, 1984. Herberman, R. B., and Ortaldo, J. R., Science (Washington, D.C.) 214, 24. 1981. Trinchieri, G., and Perussia, B.. Lab. Invest. 50, 489, 1984. Herberman, R. B.. Hosp. Pracr. 17, 93, 1982. Strayer, D. R.. Carter, W. A.. Mayberry, S. D.. Pequignot, E.. and Brodsky, I., Cancer Res. 44, 370, 1984. Heiniger. H-J., and Dorey. J. J., “Handbook on Genetically Standardized Jax Mice.” The Jackson Laboratory, Bar Harbor, 1980. Cikes, M., Friberg, S.. and Klein, G.. J. Nafl. Cancer Inst. 50, 347. 1973. Julius, M. H.. Simpson, E., and Herzenberg, L. A., Eur. J. Immunol. 3, 645, 1973. Timonen, T., Reynolds, C. W.. Ortaldo, J. R.. and Herberman, R. B.. J. fmmunol. Methods 51, 269, 1982. de Landazuri, M. O., Lopez-Botet, M.. Timonen, T., Ortaldo, J. R., and Herberman, R. B., J. Zmmunol. 127, 1380, 1981. Dunn, T. B., In “The Physiopathology of Cancer” (F. Homburger, Ed.), pp. 38-84, Harper and Brothers, New York, 1959. Steel, R. G. D., and Torrie, J. H.. “Principles and Procedures of Statistics,” McGraw-Hill, New York. 1960.
LOW NK CELL ACTIVITY 13. 14. 15. 16. 17. 18. 19.
IN HIGH TUMOR
MICE
273
Stutman, O., Excerpta Med. Int. Congr. Ser. 641, 389, 1984. Hynes, N. E., Groner, B., and Michalides, R., Adv. Cancer Res. 41, 155, 1984. Dickson, C., and Peters, G., Cur-r. Top. Microbial. Immunol. 106, 1, 1983. Pross, H. E, Stems, E., and MacGillis, D. R., In?. J. Cancer 34, 303, 1984. Herberman, R. B., and Holden, H. T., Adv. Cancer Res. 27, 305, 1978. Itoh, K., Suzuki, R., Umezu, Y., Hanaumi, K., and Kumagai, K., J. Immunol. 129, 395, 1982. Bartizal, K. E, Salkowski, C., Pleasants, J. R., and Balish, E., J. Leukocyte Rio!. 36, 739, 1984.
Received July 8, 1985; accepted August 16, 1985
lMMUNOLOGY
Decreased
AND
Natural
IMMUNOPATHOLOGY
38, 265-273 (1986)
Cytotoxicity in Mice with High Incidence Mammary Adenocarcinoma
of
IRA H. AMES,~ A. MARIANO GARCIA, PATRICIA A. JOHN, CATHY A. LITTY, MICHAEL A. FARRELL, AND RUSSELL H. TOMAR Departments of Anatomy and Cell Biology State University of New York, Upstate
and Pathology, Medical Center,
Division of Clinical Pathology, Syracuse, New York 13210
In an attempt to gather evidence relevant to the question of whether natural killer (NK) cells play a role in resisting the development of primary tumors, we compared natural cell-mediated cytotoxicity in two substrains of C,H mice. Animals of the C,H/ OuJ substrain are at high risk for the formation of mammary adenocarcinomas, while C,Heb/FeJ mice have a low incidence of such tumors. Natural cytotoxicity of splenic mononuclear cells was lower in the high-risk substrain, suggesting that a lesion in NK cell activity may be involved in murine mammary tumorigenesis. This difference was observed in animals between 5 and 37 weeks of age. There was no significant difference in the number of splenic large granular lymphocytes between the substrains. A significant difference in the ability of splenic lymphocytes from the two substrains to bind to the target cells was noted. Since the binding capacity of lymphocytes was greater in mice with reduced NK cell activity, the lesion in cytotoxicity may exist at a postbinding step in the lytic sequence. It is felt that the C,H mouse may provide a useful model for studying the role of NK cells in controlling primary tumors. 0 1986 Academic Press, Inc.
INTRODUCTION
Natural killer cells represent a heterogeneous subpopulation of normal lymphocytes that are present in substantial numbers in peripheral blood and spleen (1). They are characterized by the presence of prominent azurophilic cytoplasmic granules and a reniform nucleus. Considerable attention has been focused recently on the role of NK cells in immunosurveillance against neoplastic cells, and it has been suggested that they may be part of the first line of defense against malignant growth (2). The evidence supporting the conclusion that NK cells are responsible for rejection of implanted syngeneic tumors and inhibition of experimental tumor metastasis is extensive (3). However, the evidence for an effective role of these cells in resistance to growth and metastasis of spontaneous or carcinogen-induced primary tumors is meager (4). In a recent attempt to determine whether NK cells play a role in immunosurveillance against neoplastic cells, Strayer and his colleagues demonstrated an inverse relationship between familial incidence of cancer and NK cell activity (5). In other words, a lesion in natural cell-mediated cytotoxicity appears to preexist i To whom reprint requests should be addressed. 2 Abbreviations used: NK, natural killer; MMTV, mouse mammary tumor virus; RPMI, Roswell Park Memorial Institute; PBR, phosphate-buffered Ringer’s; FBS, fetal bovine serum; BBS, benign breast syndrome. 265 0090-1229/86 $1.50 Copyright AU rights
6 1986 by Academic Press, Inc. of reproduction in any form reserved.
266
AMES
ET
AL.
in individuals at risk for the development of cancer. We now report that this phenomenon also occurs in mice, and show that the defect in NK cell activity can be observed throughout most of the life span of animals having a high risk of mammary cancer. This finding may be significant with respect to the possibility of early identification of individuals at increased risk of cancer development. MATERIALS
AND METHODS
Animals. Since spontaneous mammary tumors of C,H mice are susceptible to lysis by NK cells (I), we chose to compare NK cell activity in two closely related substrains that differ markedly in their incidence of tumors. Mice of the C,H/OuJ substrain are characterized by the presence of mouse mammary tumor virus and are at high risk for the development of mammary adenocarcinomas. This substrain was isolated from the C,H/HeJ stock in 1952. Mice of the C,Heb/FeJ substrain do not carry MMTV and have a low incidence of mammary tumors. This substrain was developed by transplantation of C,H/HeJ ovaries into C57BL/6 female recipients subsequently mated to C,H/HeJ males (6). All animals were purchased from the Jackson Laboratory, Bar Harbor, Maine, at 4-5 weeks of age. They were maintained in the animal quarters at Upstate Medical Center for the duration of the experiments. Tumor target cells. YAC-1, a tissue culture cell line derived from a Moloney virus-induced lymphoma in A/Sn mice (7), was maintained as an in vitro suspension culture in RPM1 1640 medium supplemented with 10% heat-inactivated fetal bovine serum and 50 kg/ml gentamicin. For the evaluation of NK activity, 10 x lo6 YAC-1 cells in a total volume of 0.4 ml phosphate-buffered Ringer’s were labeled by incubation with 200 &i sodium “chromate (250-500 mCi/mg Cr: Amersham Corp.) for 40 min at 37°C. The labeled cells were washed three times with PBR and resuspended at a concentration of 0.2 x lo6 cells/ml in RPM1 1640 medium supplemented with 40% FBS. Preparation of effector cells. Spleens were obtained from ether-anesthetized animals, and single-cell suspensions were prepared by forcing the tissue through a stainless-steel wire mesh. Pooled cells from two to three animals were used in each experiment. Adherent cells were removed by incubating whole spleen cells for 45 min at 37°C on columns of 4 g of nylon wool in 30-ml plastic disposable syringes (8). The nonadherent cells were eluted in 50 ml of warm RPM1 1640 medium supplemented with 10% FBS and further purified by centrifugation through Percoll fraction 7 (Pharmacia Chemicals, Uppsala, Sweden). Cells from the interface between layers were collected, washed three times, and resuspended in RPM1 1640 medium (9). Evaluation ofcefl morphology. The morphology of all effector cell preparations was evaluated by microscopic analysis of Wright-Giemsa stained cytocentrifuge preparations. At least 200 cells were analyzed from each slide. Cytotoxicity assay. A 51Cr-release cytotoxicity assay in 96-well, round-bottomed microtiter plates (Linbro Scientific Co.) was used. Various numbers of effector cells were mixed with 50 p.1 of 0.2 x 106 “Cr-labeled target cells/ml in a total volume of 0.2 ml. The effector-to-target ratio ranged from 4O:l to 1O:l.
LOW NK CELL ACTIVITY
IN HIGH TUMOR
267
MICE
Plates were incubated for 4 hr at 37°C in a humidified 5% CO, atmosphere. Subsequent to centrifugation for 5 min at 45Og, the supernatant from each well was removed by the Titertek automatic harvesting system (Flow Laboratories) and counted in a Searle Model 1185 gamma counter for 10 min. Spontaneous release of isotope was estimated by incubating the labeled target cells alone. Maximal isotope release was estimated by exposure of labeled target cells to 10% Triton X-100. All determinations were performed in triplicate, and percent cytotoxicity was calculated as: % cytotoxicity
cpm of test group - spontaneous cpm x 100. maximal cpm - spontaneous cpm
=
Conjugation assay. In order to measure the binding capacity of the lymphocytes, 2 x lo5 effector cells were incubated with 2 x 10’ unlabeled YAC-1 cells in 2.0 ml of RPM1 1640 medium supplemented with 10% FBS for 10 min at 37°C. The suspension was centrifuged at 1OOg for 5 min, and the pellet was gently resuspended by five aspirations with a Pasteur pipet. The cells were cytocentrifuged onto microscope slides and stained with Wright-Giemsa. The frequency of conjugate-forming lymphocytes was determined by microscopic examination of 200 lymphocytes from each slide (10). Statistical analysis. The results were evaluated for statistical significance by factorial analysis of variance or Student’s t test. RESULTS
To determine the incidence of mammary tumor formation, 20 mice of each strain were examined at weekly intervals. As can be seen in Fig. 1, tumors first appeared in mice of the C,H/OuJ substrain at 29 weeks of age. At the conclusion of the observation period (37 weeks), 60% of these animals had mammary tumors. Histological examination of the tumors revealed them to be either type A or type B adenocarcinomas according to the scheme introduced by Dunn (11). Mice of the C,Heb/FeJ substrain developed no mammary tumors during the course of this investigation.
60 50
o C3H/OuJ (20 mice) C3Heb/FeJ (20 mice)
l
E x z 110 6 g E
30
2
20
z z 10 BP 0 28
29
30
32 31 Age of Mice
33 (Week?.)
34
35
36
37
FIG. 1. Cumulative incidence of mammary adenocarcinoma formation in two substrains of C,H mice.
268
AMES
ET AL.
Splenic lymphocytes from both substrains were tested for their ability to lyse highly NK-sensitive YAC-1 cells in a standard 4-hr S*Cr release assay, and the results are shown in Tables I and 2. In Table 1, the data are presented in terms of percentage cytotoxicity, and each data point represents the arithmetic mean plus or minus the standard error of the mean of three independent experiments. It is clear that splenic mononuclear cells from mice at high risk for the development of mammary adenocarcinomas (C,H/OuJ) had consistently lower NK cell activity than did comparable cells from the low-risk substrain (C,Heb/FeJ). This difference in cytotoxicity was observed at every age studied and averages about 20%. The data presented in Table 1 were tested for statistical significance by a factorial analysis of variance (12). This analysis confirmed that the difference in cytotoxicity between low- and high-risk substrains was significant (F{, = 7.8; P < 0.025); however, there was no significant difference between any of the age groups studied (F$ = 2.5; P > 0.05) or between young (5-15 weeks) and old (23-37 weeks) animals CFj4 = 2.3: P > 0.5). In order to further illustrate the difference in natural killer cell activity between substrains, we calculated the results of the cytotoxicity assays in terms of cytolytic units/lo7 cells. Each cytolytic unit represents the number of effector cells required to cause 30% lysis. TABLE NK
CELL
ACTIVITY
OF SPI.ENIC
LYMPHOCYTES DEVELOPMENT
1
FROM CJH OF MAMMARY
MICE A’T Low
.~NU HIGH
K~src
FOR IHE
TUMORS
% Cytotoxicity
Age (weeks)
E:T Ratios
g Reduction
C,H/OuJ (High)
C,Heb/FeJ (Low)
Mean reduction
5-7
4O:l 2O:l IO:1
39.8 2 6.5” 28.5 2 4.9 20.2 t 7.8
32.8 t 4.8” 22.6 2 4.8 16.3 t 3.6
17.6 20.7 19.3
19.2
S-10
40:1 2O:l IO:1
50.1 ? 2.9 40.4 + 1.0 23.5 k 4.9
38.1 f 25.8 t 19.0 i
1.1 1.0 I.2
24.0 36.1 19.2
‘6.4
13-15
40: I 20: 1 1O:l
40.9 k 5.8 30.3 k 3.9 19.8 i 3.3
36.2 2 2.4 ‘8.7 t 2.5 16.5 _t 2.5
1 I.5 53 16.7
11.2
23-25
40: 1 20: I 1O:l
36.1 -c 2.4 27.1 + 2.5 17.0 i 1.8
28.9 2 2.6 19.5 + 1.1 12.4 ir 1.4
19.Y 28.0 27.1
25.0
27-29
4O:l 20:1 1O:l
39.4 2 6.7 23.5 2 5.3 16.3 2 3.4
31.4 + 5.8 16.3 + 6.1 12.9 5 4.5
20.3 30.6 20.9
13.9
36-37
4O:l 20: 1 10:1
53.4 2 2.5 29.0 k 4.5 18.1 t 4.5
44.3 ? 5.0 27.1 t 2.5 18.8 _t I.2
17.0 6.6 -
7.9
B Each value represents independent experiments.
the arithmetic
mean
plus or minus
the standard
error
of the mean of three
LOW
NK
CELL
ACTIVITY
IN HIGH
TUMOR
269
MICE
This data is presented in Table 2. Once again, the high-risk mice had lower natural cell-mediated cytotoxicity than the low-risk animals at every age studied. This difference averages about 30%. The data in Table 2 were tested for statistical significance by the paired Student’s t test (12) which confirmed that the difference between the substrains was significant (P < 0.001). It is conceivable that the observed difference in in vitro cytotoxicity between splenic lymphocytes from the two substrains was due to differences in the number of NK cells in the final effector cell preparations. To examine this possibility, the morphology of all such preparations was evaluated by microscopic analysis. The cellular composition of final preparations of effector cells is shown in Table 3. One can see that the distribution of cell types was virtually identical in the preparations used for the cytotoxicity assays and that, in particular, the percentage of cells with the morphology of large granular lymphocytes did not differ between the two substrains. Since the number of large granular lymphocytes recovered from the spleen appears to be the same in the high- and low-risk animals, the percentage of mononuclear cells capable of forming conjugates with the target cells was determined. The results of the conjugation assays are presented in Table 4. These data show that there was a significant difference in the ability of lymphocytes from TABLE NK
2
CELL ACTIVITY OF SPLENIC LYMPHOCYTESFROM C,H MICE AT LOWAND HIGH RISK FORTHE DEVELOPMENT OF MAMMARY TUMORS Cytolytic
units
Expt no.
C,Heb/FeJ (Low)
CsH/OuJ (High)
1 2 3
118.8” 39.0 24.1
60.1” 21.3 21.4
49.4 45.4 11.2
1 2
93.1 66.1
45.6 41.3
51.3 37.5
13-15
1 2 3
45.3 11.4 28.8
44.6 56.7 33.7
1.5 26.7 (14.5)
4.6
23-25
1 2 3
37.5 52.4 49.6
15.0 31.0 21.9
60.0 40.8 55.8
52.2
27-29
1 2 3
64.3 31.9 23.1
50.3 29.3 10.5
21.8 22.7 54.5
33.0
36-37
1 2 3
17.1 54.8 49.8
55.6 58.9 33.0
27.9 (7.0) 33.7
18.2
Age (weeks)
5-7 8-10
0 The results are expressed as lytic cells required to cause 30% lysis.
units/lo’
cells,
with
YO Reduction
a lytic
unit being
Mean reduction
35.3 44.4
the number
of effector
270
AMES ET AL. TABLE 3 CELLULAR
COMPOSITION
OF FINAL
EFFECTOR
C,Heb/FeJ (low risk) Small-medium lymphocytes Large agranular lymphocytes Large granular lymphocytes Granulocytes Others
74.2 16.6 6.5 1.9 1.2
k r 2 f -c
CELL PREPARATIONS
C,H/OuJ (high risk)
1.2” 0.7 0.6 0.3 0.2
72.0 18.5 6.4 1.8 1.3
k 2 " 2 ?
1.0" I.0 0.6 0.3 0.2
lb
P
1.4085 I.5565 0.1179 0.2357 0.3536
‘co.20 <0.20 -co.95 co.90 <0.80
a Arithmetic mean f standard error of the mean of percentages of cell types b -o-tailed Student’s r test with 32 df
the two substrains to bind to the YAC-1 cells. It should be noted that the binding capacity of the cells from high-risk mice was greater than that of lymphocytes from low-risk animals. Because the cells had been cytocentrifuged onto slides and subsequently stained, it was possible to study the target-binding capacity of the lymphocyte subpopulations. One can see that the difference in total binding was attributable to increases in the conjugation of both small-medium and large granular lymphocytes with the target cells. DISCUSSION
At present, the bulk of our knowledge of the role of NK cells in immunosurveillance against neoplastic cells is derived from studies of in Go model systems utilizing transplanted tumors or experimental metastases (3). Little work has been reported on the role of these cells in resisting the growth and metastasis of induced or spontaneous primary tumors. The major problem in this respect has been the lack of a proper model system. As noted by Stutman (13), such a system should involve animals with the same genetic background that differ in their levels of NK cell activity. It appears that the C,H mouse may provide that system. To the best of our knowledge mice of the C,H/OuJ and C,Heb/FeJ substrains are genetically identical. They do not differ for any of the genetic markers that have been studied, including histocompatibility (6). We now have demonstrated that TABLE 4 PERCENTAGE
Small-medium lymphocytes Large agranular lymphocytes Large granular lymphocytes Total
OF TOTAL LYMPHOCYTES
BOUND
TO TARGET CELLS
C,Heb/FeJ (low risk)
C,HlOuJ thigh risk)
7.8 1.6 4.6 13.9
11.4 2.0 7.0 20.3
-t 2 2 f
0.7" 0.4 0.6 0.9
t 1.4" I? 0.5 +- 0.6 IT 2.2
tb
P
2.3000 0.6247 2.8285 2.6925
CO.05 co.60 co.01 ‘co.02
(1Each value represents the arithmetic mean + standard error of the mean of three independent experiments. b ‘No-tailed Student’s t test with 28 d$
LOW
NK
CELL
ACTIVITY
IN
HIGH
TUMOR
MICE
271
these substrains differ in terms of their levels of NK cell activity. In addition, we have confirmed that they also differ markedly with respect to their susceptibility to the development of mammary tumors. Mice of the C&I/OuJ substrain carry MMTV and have a high incidence of mammary adenocarcinomas, while C,Heb/ FeJ mice are free of MMTV and seldom develop mammary gland tumors. The mechanism of MMTV-induced tumorigenesis is not completely understood. Most of the work in this area has focused upon the transforming effect of the retrovirus on mammary epithelial cells, and the model of insertional mutagenesis is compelling (14). It is interesting that a high level of MMTV antigen is expressed in lymphocytes (15), and it is conceivable that the virus is present in NK cells. If this is the case, the ability of infected NK cells to lyse tumor cells may be impaired. Such a lesion could enhance the expression of malignant disease. Strayer er al. (5) have demonstrated a correlation between high familial incidence of cancer and low natural cell-mediated cytotoxicity in humans. Our data indicate that the same is true for C,H mice. Animals of the C,H/OuJ substrain that are at high risk for the development of mammary tumors had consistently lower NK cell activity than C,Heb/FeJ mice that have a rather low risk of mammary cancer. Because of the nature of their study, it was not possible for Strayer and his colleagues (5) to determine when the lesion in natural cell-mediated cytotoxicity first manifested itself in the individuals at increased risk of cancer development. If, as they suggest, it may be possible to identify such people by determining their NK cell activity, it would be important to know how early such a determination could be made. In this context, it is interesting to note that in the C,H mouse system the difference in natural cell-mediated cytotoxicity was demonstrable at 5-7 weeks of age and continued throughout most of the life span of the animals. In contrast to the results of Strayer et al. (5), Pross et al. (16) observed that NK cell activity was elevated in women at high risk for breast cancer. It is difficult to evaluate this discrepancy because the criteria for determining risk were quite different. In the work of Strayer and his colleagues, family history was the sole criterion, while in the other case personal gynecological history and presence of benign breast syndrome were also utilized. It should be noted that the entire elevation reported by Pross et al. (16) was attributable to the BBS subgroup. Strayer et al. (5) refer to the individuals in their study as normal, and report no attempt to detect BBS in these women. It has been reported that in mice NK cell activity reaches a peak at 5-8 weeks of age and then declines to low levels when the animals are 3 months old (17). While Itoh et al. (18) showed that C,H/He mice closely followed this pattern of age distribution, our data indicate that this may not be true for all C,H mice. As can be seen in Tables 1 and 2, the level of natural cell-mediated cytotoxicity was fairly constant from 5-7 to 36-37 weeks of age for both C,HIOuJ and C,Heb/ FeJ substrains. In this connection, it is interesting to note that Bartizal ef al. (19) recently reported that no differences in NK cell activity were evident in C,H/ HeCr mice of different ages. The question of whether these findings reflect true differences between substrains or subtle differences in experimental protocol requires further study.
272
AMES ET AL.
At this point in time we have examined two of the possible explanations for the observed difference in the level of NK cell activity in high- and low-risk mice. First, we studied the morphology of the effector cell preparations and determined that the substrains did not differ with regard to the number of large granular lymphocytes present in the cytotoxicity assays. Second, we performed conjugation assays in order to learn whether there was a difference in the ability of lymphocytes from the two substrains to bind to the target cells. In this case the results were surprising, since the binding capacity of splenic mononuclear cells was greater in the animals with lesser NK cell activity. Therefore, the lesion in cell-mediated cytotoxicity in C,H/OuJ mice may involve the ability of their NK cells to complete the stages of the lytic sequence, and studies designed to precisely define it are now in progress. We also plan to compare NK cells from the two substrains with respect to their ability to release natural killer cytotoxic factors, to produce and respond to interferon, and to respond to interleukin 2. In conclusion, we feel that the C,H mouse may be a useful model for studying the in viva role of NK cells in controlling primary tumors, It has been known for quite some time that the two substrains with which we are working differ with respect to their susceptibility to the formation of mammary tumors. We have now shown that they also differ in regard to their levels of NK cell activity and are currently attempting to determine whether these two phenomena are correlated in addition to being coincidental. ACKNOWLEDGMENTS This research was supported by U.S. Public Health Service Grant 2S07RR0540223, the Immunology Research, Development and Education Fund, and the Dr. Glenn H. Leak Memorial Cancer Fellowship Program of the American Cancer Society. We are grateful to Dr. Richard P. Oates for his advice with respect to the statistical analysis of the data and to Ms. Nancy Snyder for her help with the manuscript.
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Ortaldo, J. R., and Herberman, R. B., Annu. Rev. Immunol. 2, 359, 1984. Herberman, R. B., and Ortaldo, J. R., Science (Washington, D.C.) 214, 24. 1981. Trinchieri, G., and Perussia, B.. Lab. Invest. 50, 489, 1984. Herberman, R. B.. Hosp. Pracr. 17, 93, 1982. Strayer, D. R.. Carter, W. A.. Mayberry, S. D.. Pequignot, E.. and Brodsky, I., Cancer Res. 44, 370, 1984. Heiniger. H-J., and Dorey. J. J., “Handbook on Genetically Standardized Jax Mice.” The Jackson Laboratory, Bar Harbor, 1980. Cikes, M., Friberg, S.. and Klein, G.. J. Nafl. Cancer Inst. 50, 347. 1973. Julius, M. H.. Simpson, E., and Herzenberg, L. A., Eur. J. Immunol. 3, 645, 1973. Timonen, T., Reynolds, C. W.. Ortaldo, J. R.. and Herberman, R. B.. J. fmmunol. Methods 51, 269, 1982. de Landazuri, M. O., Lopez-Botet, M.. Timonen, T., Ortaldo, J. R., and Herberman, R. B., J. Zmmunol. 127, 1380, 1981. Dunn, T. B., In “The Physiopathology of Cancer” (F. Homburger, Ed.), pp. 38-84, Harper and Brothers, New York, 1959. Steel, R. G. D., and Torrie, J. H.. “Principles and Procedures of Statistics,” McGraw-Hill, New York. 1960.
LOW NK CELL ACTIVITY 13. 14. 15. 16. 17. 18. 19.
IN HIGH TUMOR
MICE
273
Stutman, O., Excerpta Med. Int. Congr. Ser. 641, 389, 1984. Hynes, N. E., Groner, B., and Michalides, R., Adv. Cancer Res. 41, 155, 1984. Dickson, C., and Peters, G., Cur-r. Top. Microbial. Immunol. 106, 1, 1983. Pross, H. E, Stems, E., and MacGillis, D. R., In?. J. Cancer 34, 303, 1984. Herberman, R. B., and Holden, H. T., Adv. Cancer Res. 27, 305, 1978. Itoh, K., Suzuki, R., Umezu, Y., Hanaumi, K., and Kumagai, K., J. Immunol. 129, 395, 1982. Bartizal, K. E, Salkowski, C., Pleasants, J. R., and Balish, E., J. Leukocyte Rio!. 36, 739, 1984.
Received July 8, 1985; accepted August 16, 1985