macrophage mediated tumor cell cytotoxicity

MACROPHAGE MEDIATED TUMOR CELL CYTOTOXICITY *

Alan M. Kaplan and Page S . Morahan

Departments of Surgery and Microbiology and Division of Medical Oncology

MCV/ VCU Cancer Center Medical College of Virginia Richmond, Virginia 23298

INTRODUCTION

Recently, considerable evidence has emerged that implicates the macrophage as a major effector cell in both specific and nonspecific cell mediated cytotoxicity to tumors.’-* Biological reticuloendothelial stimulants such as Corynebacterium parvum, Toxoplasma gondii, and Mycobacterium bovis, as well as synthetic reticuloendothelial stimulants, such as polyriboinosinic-polyribocytodylic acid, polyacrylic acid-maleic anhydride, and pyran, are known to enhance macrophage function as well as to induce resistance to tumor G-n Moreover, macrophages from animals treated with these chemical or biological stimulants have been shown to be selectively cytotoxic for tumor cells in vitro while demon- strating quantitatively less cytotoxicity for normal cells.’, 3,

In addition to the in vitro evidence which indicates that macrophages play a role in regulation of tumor growth, several lines of evidence from in vivo experiments suggest that macrophages function as effector cells in resistance to tumors. Carter and Gershon 21 demonstrated that a nonmetastasizing hamster lymphoma stimulated a “massive proliferation of histiocytes” in the draining lymph nodes, whereas a metastasizing lymphoma did not. Morphological evidence strongly suggested that macrophages were involved in the destruction of second grafts of the lymphoma.?2 Moreover, cytochemical staining for acid phosphatase activity indicated large, intensely staining lysosomes suggestive of activated macrophages. Correlation of the increased number of tumor-associated macrophages with a decrease in metastases also implicated macrophages in tumor cell destruction.13 These data suggested that treatment of mice with agents that enhanced macrophage proliferation and activated macrophages within a tumor or directed additional macrophages to a tumor would be beneficial to the host. This is consistent with the data of Fidler 11 who demonstrated that syngeneic-activated macrophages injected intravenously (i.v.) 48 hours after i.v. inoculation of B 16 melanoma-cells significantly reduced the number of established pulmonary metastases. Moreover, increased numbers of macrophages or histiocytes were the predominant in vivo histopathologic finding at the site of intratumor BCG injection,15 and ultrastructural studies have shown histiocyte-tumor cell interactions at the sites of tumor regression in guinea pigs treated with intralesional BCG.ls Similarly, an increased number of histiocytes present in the connective tissue

lo

* Supported in part by National Institutes of Health Grant CAI0537 and Contract CB 43877. PSM is a recipient of United States Public Health Service Research Career Development Award.

134

Kaplan & Morahan: Macrophage Mediated Tumor Cell Cytotoxicity 135

surrounding the Lewis lung tumor in mice could be demonstrated after systemic treatment with pyran cop01ymer.~7 Although the functional status of such tumor-associated macrophages has not been well delineated, several investigators recently demonstrated that the predominant reactive host cell within some rat sarcomas is a macrophage-like cell 'that nonspecifically inhibited colony formation of tumor cells in vitro.lfi-?n

The common denominator of the antitumor activity of immunopotentiators (BCG, C. parvum, pyran, complete Freund's adjuvant, or chronic infection of mice with Besnoitia jellisoni or Toxoplasina gondii) appears to be activation of macrophages to selectively and/or specifically destroy tumor cells.l-l.* The present communication demonstrates the importance of activated macrophages in C. parvum and pyran-induced resistance to the Lewis lung carcinoma and a methylcholanthrene-induced fibrosarcoma (MCA 21 82).

MATERIALS AND METHODS

Mice

Male C57B1/6 mice were obtained from Jackson Memorial Laboratories (Bar Harbor, Me.) and Laboratory Supply Co. (Indianapolis, Ind.). The mice were kept under standard laboratory conditions and used for experiments after an acclimation period of at least one week. The mice have been shown to be free of lactate dehydrogenase (LDH) virus (Microbiological Associates, Be- thesda, Md.).

Thymectomized, irradiated, bone marrow reconstituted (TxB) mice were prepared by suction thymectomy of six-week-old mice, followed by 900 rads whole body irradiation from a W o source three to four weeks later, and imme- diate reconstitution with approximately lo7 syngeneic bone marrow cells.

Drugs

Pyran, a copolymer of maleic anhydride-divinyl ether (lot XA124-177 with average MW 33,200) and varying molecular weight fractions of pyran were obtained from Dr. D. Breslow, Hercules, Inc. (Wilmington, Del.), and was solubilized in 0.15 N NaCl at pH 7.0. Preparations of the killed vaccine of Corynebacterium parvum were obtained from Burroughs Wellcome, Research Triangle, N.C.

Preparation of trypan blue (Fisher Chemical Co.) included exhaustive dialysis against distilled water to remove the toxic impurities followed by lyo- philization, as originally described by Hibbs.26

Tumors

The Lewis lung carcinoma, which originated spontaneously in a C57B1/6 mouse in 195 1 2B was received from Dr. Woodman, Microbiological Associates, Bethesda, Md., and has been transplanted in our laboratory for four years. The methylcholanthrene-induced sarcoma MCA 2 182 was obtained through the courtesy of Dr. J. Berkelhammer, Institute for Cancer Research, Fox Chase, Pa.

136 Annals New York Academy of Sciences

These tumors have been maintained by SC implantation of 1 cu mm tumor fragments into C57B1/6J mice at 2- to 3-week intervals. The tumors were periodically checked for LDH virus; and if found to be positive, the tumor cells were grown in vitro for several passages to eliminate the virus, and then transplanted back into animals.

Cells and Tumor Cell Cytotoxicity Test

A continuous cell line of Lewis lung carcinoma was used as a target cell in vitro for activated macrophages. These cells were prepared and cultured in Eagle's minimum essential medium with Earle's balanced salt solution, 2 X essential amino acids, vitamins, 100 U penicillin/ml, 100 pg streptomycin/ml and 20% heat inactivated fetal bovine serum (EMEM), as previously described.3- 21 The tumor cells have been shown to be free of Mycoplasmn (Micro- biological Associates, Bethesda, Md.) . For cytotoxicity testing, peritoneal exudate cells from normal and drug-treated mice were removed, washed with Hank's balanced salt solution, and loe or 5 X lo5 cells in 0.5 ml EMEM added to duplicate 81 mm2 chambers of an 8-chambered slide (Lab-tek, Naperville, Ind.). The cells were allowed to adhere for 2 hours, washed free of nonadherent cells, overlaid with 5 X 10' tumor or normal cells, incubated for 60 hours, washed, and the remaining cells stained with Giemsa. Cytotoxicity was assessed morphologically, as previously des~r ibed ,~ by comparing the slides containing target cells and macrophages with control slides containing target cells alone, and the percent cytotoxicity was calculated from the four wells (two wells each at peritoneal cel1:target cell ratios of 20: 1 and 10: 1).

Tumor Testing in Vivo

Mice were inoculated in the footpad or leg muscle (i.m.) with a known number of tumor cells. C. parvum or macrophages were inoculated three days after tumor challenge. Tumor sue was measured twice a week, and the tumor volume calculated as described previously.27

For some experiments, Lewis lung cells were incubated in vitro with activated or normal peritoneal cells (20: 1, 10: 1 and 5: 1 peritoneal cel1:tumor cell ratios) before inoculation into animals. Tumor cells (2 x 1O0/ml) and peritoneal cells or macrophages (1-4 x 107/ml) in EMEM were incubated together for 30 min at 36" C. Each mouse was then inoculated i.m. with 0.2 ml containing 2 X lo5 tumor Cells and 1-4 x lo6 peritoneal cells or macrophages.

RESULTS

We have previously demonstrated that adherent peritoneal exudate cells were required for cytotoxicity and cytostasis against tumor cells in vitr0.~9 lo

Trypan blue has been reported to be an inhibitor of macrophage-mediated tumor cell cytotoxicity. In order to verify that the active adherent peritoneal exudate effector cells were macrophages, peritoneal cells from pyran and C. parvum- treated animals were incubated with 4.2 X 10-4 M trypan blue for 24 hours prior to the addition of Lewis lung cells. This treatment reduced the cytotoxicity


of the pyran-adherent peritoneal cells (PEC) by 44% and that of C. parvum adherent PEC's by 58% (TABLE 1). The growth of Lewis lung cells in vitro in the absence of macrophages or in the presence of normal macrophages was unaffected. In a similar experiment, inoculation of trypan blue i.p. 24 hours prior to harvesting pyran-activated peritoneal exudate cells reduced the cytotoxicity of the activated macrophages to 9% of the positive controls. These data substantiated earlier work,'-' suggesting that activated macrophages played a role in regulation of tumor cell growth in vitro.

To extend these observations into an in vivo situation, pyran or C. parvum- activated macrophages or normal macrophages were incubated with Lewis lung cells in vitro for 30 minutes and then the macrophage-tumor cell mixtures inoculated into syngeneic recipients. It has been demonstrated previously that

TABLE 1

REDUCTION OF MACROPHAGE MEDIATED TUMOR-CELL CYTOTOXICIIY BY TRYPAN BLUE

Treatment with Trypan Blue

- +

Percent Cytotoxicity with Macrophages Obtained from Mice inoculated

with- Pyran C. parvum

7 8 94 44 40

It1 vivo t - + 69

6 ND ND

* Mice were inoculated i.p. with 2.5 mg/kg pyran, or 70 mg/kg C. parvum; peritoneal cells harvested 6 days later, were allowed to adhere to glass for 2 hr, incubated for 24 hr with medium or medium containing 4 . 2 ~ lo4 M trypan blue, washed 3 times, Lewis lung tumor cells added, and cytotoxicity was measured 60 hr. later.

t As above, except trypan blue 200 mg/kg was inoculated i.p. 24 hr. before harvesting peritoneal cells and assay was carried out in medium without additional trypan blue.

tumor cell destruction requires a 24-hour period of interaction between tumor cells and macrophages.28 Moreover, macrophage-mediated tumor cell cytotoxicity has been shown to be a delayed event, occurring 24-48 hours after the initiation of macrophage-tumor cell cultures.'" Therefore, for the activated peritoneal exudate cells to inhibit tumor growth in the recipient mice, the macrophages in the macrophage-tumor cell inoculum would have to mediate their cytotoxic effects in vivo. The results of two such experiments are shown in TABLE 2 and in each case demonstrate a clear reduction in the incidence of tumors in the presence of activated peritoneal exudate cells, while normal or glycogen stimulated peritoneal exudate cells were unable to reduce the incidence of tumors. Preliminary experiments have suggested that similar effects can be demonstrated with adherence-purified macrophages (Kaplan-Morahan, unpub- lished observations).


These results indicated that macrophages activated by immunopotentiators in vivo could function both in viiro and in vivo to inhibit tumor cell growth. Moreover, we had previously demonstrated that pyran could activate macrophages in thymectomized irradiated bone marrow reconstituted mice (TxB) , suggesting that activation was independent of thymus derived lymphocytes.3 Therefore, in order to further define the site of immunopotentiation in vivo, we tested whether or not immunopotentiator induced tumor regression depended on the presence of thymus derived lymphocytes or only on the functional status of macrophages.

Normal and TxB mice were inoculated in the footpad with MCA 21 82; three

TABLE 2 INHIBITION OF LEWIS LUNG CARCINOMA GROWTH In Vivo BY PYRAN

AND C. Parvum ACTIVATED PERITONEAL EXUDATE CELLS *

Pretreatment of Lewis Lung cells

Incidence No. Tumors

Total Percent

EXPERIMENT Alone Pyran PEC t (20:l) Glycogen PEC (20: 1 ) Normal PEC (20: 1) Pyran PEC (5: 1) Glycogen PEC ( 5 : 1 ) Normal PEC (5 : 1 )

1 6/9 0/10 6/10

10/10 2/ 10 7/10 7/9

67 0

60 100 20 70 78

EXPERIMENT 2 Alone 20/20 100 C. Parvum PEC (1O:l) 0/20 0 Pyran PEC ( 1 O : l ) 0/10 0 Glycogen PEC (10: 1) 20/20 100

* Lewis lung carcinoma cells ( 2 x 10B/ml) were incubated alone or with peritoneal exudate cells (PEC) at 20: 1, 10: 1 or 5: 1 ratios of PEC to tumor cells. After incubation for 30 min at 36" C, mice were inoculated with 0.2 ml of the mixture which con- tained 2 x 106 tumor cells.

t PEC were obtained seven days after i.p. inoculation of C. parvum (70 mg/kg) or pyran (50 mg/kg) or five days after inoculation or glycogen (2.5 mg/mouse).

days later C. parvum was inoculated intralesionally and the number of tumor regressions determined (TABLE 3). At high doses of C. parvum (>35 mg/ kg) , no difference in the percentage of regressor animals between normal or TxB mice was seen, while a trend toward fewer regressions in the TxB mice than in the normal mice was seen at lower doses of C. parvum (<17.5 mg/kg). The combined results showed 39 of 57 (68% ) tumor regressions in the normal mice and 31 of 60 (52%) tumor regressions in the TxB mice, indicating that thymus derived lymphocytes were not an absolute requirement for C. parvum induced regressions.

To determine the role of macrophages in C. parvum induced MCA 2182 tumor regression the effects of C. parvum were assayed in the presence of trypan


TABLE 3 EFFECT OF T-CELL DEPLETION ON C . Parvuni

INDUCED REGRESSION OF MCA 2182 * - ._ ~ ~~

Treatment C. Purvuni Regressors/Total

(mg/kg) Normal Mice TxB ,Mice ~~~~~ ~ ~~

Control 0/10 (0) t 0110 (0) 70.0 8/10 (80) 7/10 (70) 35.0 8/10 (80) 9/10 (90) 17.5 7/10 (70) 5/10 (50) 8.8 7/10 (70) 5/10 (50) 4.4 518 (63) 4/10 (40) 2.2 419 (44) 1/10 (10) ~ _ _ _ -~

~~~~

Normal or TxB mice were inoculated with 5 x lo' MCA 2182 cells on day 0 and on day 3 the appropriate dose of C. parvum was inoculated intralesionally.

t Numbers in parentheses are percentages.

blue, an inhibitor of macrophage lysosomal hydrolases and macrophage mediated cytotoxicity in vitro (TABLE 1 and Reference 26). Normal or trypan blue treated mice were inoculated in the footpad with MCA 2182 cells on day 0 and on day 3 C. parvum was inoculated intralesionally. A reduction in the number of regressions from 12 of 50 (24%) in the normal mice to 1 of 37 (2.7%) in the trypan blue treated mice was observed (TABLE 4) . These results suggested that the effect of C. parvum induced regression of the MCA 2182 tumor was mediated by macrophages. Therefore, we predicted that we should be able to mimic the effects of intralesional C. parvum by inoculating C. parvum activated macrophages intralesionally. Mice were inoculated with MCA 2 182 cells in the footpad on day 0, and on day 3 glycogen stimulated or C. parvum activated adherence purified macrophages were injected intralesionally. Thirty five days

TABLE 4

INDUCED REGRESSION OF MCA 2182 * EFFECT OF TRYPAN BLUE ON c. PUrVUWl

Normal Mice Regressors

Treatment MST2ZSE Total

Control 42.9k 1.6 0/10 C. Parvum i 37.42 1.9 12/50

~ ~~

(24)$

Trypan Blue Treated Mice

Regressors MST & SE Total

43.12Z2.6 0/8 4 0 . 4 2 3.5 1/37

(2.7)

* Normal or trypan blue treated (150 mg/kg on day 0 and 37.5 mg/kg 2 times weekly thereafter) mice were inoculated with 5x 10' MCA 2182 cells on day 0 and on day 3 C. parvum was inoculated intralesionally.

?The data was pooled over a dose range of C. parvum from 4.4 mg/kg to 70 mg/kg.

t Numbers in parentheses are percentages.

140

after tumor inoculation, a 75% reduction in tumor volume was seen in the group receiving C. parvum activated macrophages, as compared to those receiving glycogen stimulated macrophages (FIGURE 1). On day 39 tumor volume

Annals New York Academy of Sciences

550

600

450

400

m- E 350 E Y

J 300 0 >

250 K 0 5 200

- ._ 20 25 30 35 40

DAYS AFTER TUMOR INOCULATION

FIGURE 1. Effect on intralesional inoculation of C. parvum activated macrophages on the growth of MCA 2182. MCA 2182 cells (2.5 x lo') were inoculated into the footpad of C57B1/6 mice and on day 3 (1 ) no additional cells 0- - -0, (2) 2.5 x loG peritoneal macrophages from mice inoculated i.p. with 2.5 mg of glycogen 5 days earlier 0- - -0, or (3) 2.5 x 10'' peritoneal macrophages from mice inoculated i.p. with 70 mg/kg C . parvum 7 days earlier 0- - -0 were inoculated intralesionally into the tumor. The numbers in parentheses represent the number of survivors over the total tested at day 39. The bars represent the mean 2 I SEM.

was reduced by 46%. Moreover, 34% of the mice treated with glycogen stimulated macrophages were dead whereas no mice had died in the group treated with C. parvum activated macrophages.


DISCUSSION

The role of activated macrophages as effector cells in immunopotentiator induced resistance to tumor growth has become increasingly evident.'-12 The results presented here support the hypothesis that activated macrophages can inhibit tumor cell growth both in vitro and in v i v a Moreover, we have presented evidence that the macrophage is at least one functional site of action involved in the regression of tumors following intralesional C. parvurn inoculation.

In vitro, activated macrophages selectively destroy neoplastic as opposed to normal 3 We have demonstrated that the tumoricidal activity of adherent peritoneal macrophages activated by pyran or C. parvum was inhibited by pre- incubation in vitro or pretreatment in vivo with trypan blue, an inhibitor of lysosomal hydrolases, confirming a similar observation by Hibbs 2e for BCG- activated macrophages.

In vivo studies have further implicated activated macrophages as effectors of tumor resistance induced by numerous synthetic and biologic immunopotentiators. Histopathologic studies of Lewis lung tumors in mice treated with pyran systemically revealed an increase in histiocytic infiltration l7 similar to that seen in a guinea pig tumor treated intralesionally with BCG.16 Although the functional status of such tumor-associated macrophages has not been well delineated, several investigators have recently demonstrated that the predominant reactive host cell within some rat sarcomas is a macrophage-like cell that inhibited colony formation of tumor cells in vitro.1H--2" We have also demonstrated that activated peritoneal macrophages can be recovered for at least 14 days after Lewis lung implantation and pyran treatment, suggesting that activated macrophages may be present systemically in the immunopotentiator- treated tumor-bearing host." Hibbs has demonstrated that inhibition of macrophages in vivo by injection of trypan blue abrogated the increased nonspecific resistance induced by chronic infection with BCG or Toxoplusrna to Sarcoma 180 tumor gr0wth.~3 While depletion of macrophages has been reported in some instances to abrogate immunopotentiator-induced resistance to tumor growth, augmentation of the macrophage population of an animal has been reported to enhance tumor resistance. Fidler l4 reported that activated macrophages injected i.v. inhibit pulmonary metastasis of B-16 melanoma cells; however, no data on survival of animals were reported. While we have not con- sistently been able to inhibit Lewis lung tumor growth by injecting activated macrophages into an animal bearing residual metastatic tumor, we have been repeatedly successful in inhibiting tumor growth by preincubating tumor cells with activated macrophages before injection into a syngeneic recipient. Tumor occurrence and mortality could be completely abolished in animals that were injected with Lewis lung cells preincubated with activated macrophages, while control macrophages, including unstimulated and glycogen-stimulated peritoneal cells, were unable to inhibit tumor growth (TABLE 2). Our data concerning the ineffectiveness of glycogen stimulation for activating mouse macrophages to kill tumor cells are consistent with our in vitro results on tumor cell cytotoxicity; and with numerous reports which indicate that, in the mouse, macrophages can not be activated to kill tumor cells by glycogen, starch, proteose peptone, thioglycollate, and similar agentsz4 Harmel and Zbar 34 have also recently shown that pyran-activated macrophages reduced the incidence and growth of intradermally inoculated MCA 1838 sarcoma cells.


In addition to these studies with nonspecifically activated macrophages, Zarling and Tevethia have demonstrated that effective transfer of specific tumor immunity by sensitized lymphocytes required macrophage participation at the effector level.:'" Their data were consistent with the release, by specifically sensitized lymphocytes, of lymphokines which either specifically 81 or nonspecifically 32 rendered exogenous or endogenous macrophages cytotoxic for tumor cells. Specific macrophage arming factor described by Evans et al.:<l provides a mechanism for the specific activation of macrophages to the level of cytotoxic antitumor effector cells, while Piessens et al.32 have demonstrated that lymphokine-rich supernatants can render macrophages nonspecifically cytotoxic to tumor cells.

Recent experiments of Scott *3 have indicated that the protective effect of intratumor C. parvum against the P-815 mastocytoma in BDF, mice is blocked by depletion of T cells by thymectomy, irradiation, and bone marrow reconstitution. In contrast, the results presented here indicate that depletion of T cells has no inhibitory effect on high dose (>35 mg/kg) C. parvum induced regression of MCA 2182 and only a marginal effect on low dose (c17.5 mg/kg) C. parvum induced regression of MCA 2182. However, while most of the normal animals in which regression of the MCA tumor had been induced by C. parvum were subsequently specifically immune to challenge with MCA 2182, few of the TxB mice in which similar regressions had been induced were immune to specific tumor challenge (Kaplan & Morahan, manuscript in preparation). This difference between the two tumor systems MCA 2182, a methyl- chloanthrene induced fibrosarcoma, and P-8 15, a mastocytoma, could have been due to inherent differences in the susceptibility of the two tumors to macrophage mediated cytotoxicity, to differences in the normal macrophage content of the two tumors, to differences between syngeneic or semisyngeneic systems, or to other factors. We have been unable to demonstrate tumor immunity in the regressor TxB mice, implying that these mice were indeed adequately depleted of T cells. However, since the TxB mice in both Scott's 23 and our experiments probably had residual T cells, the difference between Scott's results and ours could have been due to varying degrees of thymic dependency in the two systems. If those animals with reduced T-cell levels required more C. parvum to induce tumor regression, it would be consistent with a decrease in regressor animals observed at low doses of C. parvum in our experiment.

Although we were unable to inhibit C. parvum induced regression by TxB, trypan blue treatment of tumor bearing animals reduced the number of regressions from 12 of 50 in the normal mice to 1 of 37 in the trypan blue treated mice. Growth of tumors in non-C. parvum treated animals was comparable in normal and trypan blue treated mice. These results are consistent with the suggestion that the ability of trypan blue to enhance the growth of an allogeneic tumor is macrophage-mediated.39 Moreover, the fact that inhibition of tumor growth can be obtained by direct injection of C. parvum activated macrophages into tumors, while little effect is seen with injection of glycogen stimulated macrophages, suggests that the selectivity of macrophage-mediated tumor cell cytotoxicity is maintained in vivo. It is not surprising that intralesional-activated macrophage injection is less efficient than direct injection of C. parvum, since it is unlikely that macrophages have the host cell recruitment potential of C. parvum. However, experiments are currently in progress utilizing larger numbers of activated macrophages in an attempt to produce complete regressions.


The evaluation of such regressor animals for subsequent delayed hypersensitivity to the tumor should help in determining the mechanism: whereby, C. parvum in conjunction with tumor cells can enhance specific delayed hypersensitivity to

SUMMARY

The following evidence from our research has implicated the macrophage as an important effector cell in pyran and/or C. parvum induced host resistance to solid tumors: ( 1 ) Increased infiltration of tumors with histiocytes following systemic treatment with pyran; (2) activated peritoneal macrophages with tumoricidal activity have been recovered from the peritoneal cavity of normal or tumor bearing mice treated with pyran or C. parvum; ( 3 ) activated peritoneal macrophages mixed with tumor cells in vitro and transplanted into syngeneic recipients inhibited tumor growth; (4 ) trypan blue, an inhibitor of macrophage function, prevented C. parvum induced regression of methylcholanthrene tumors; and (5) direct intralesional injection of activated macrophages into the MCA 2182 tumor inhibited tumor growth and increased the MST.

ACKNOWLEDGMENTS

We wish to thank J. A. Munson, S. C. Johnson, and S. Bilgin for excellent technical assistance.

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