Macrophage heterogeneity in tumor resistance: Cytostatic and cytotoxic activity of corynebacterium parvum-activated and proteose peptone-elicited rat macrophages against Moloney sarcoma tumor cells

CELLULAR

IMMUNOLOGY

50,

153- 168 (1980)

Macrophage Heterogeneity in Tumor Resistance: Cytostatic and Cytotoxic Activity of Corynebacterium Parvum-Activated and Proteose Peptone-Elicited Rat Macrophages against Moloney Sarcoma Tumor Cells1 M. SHOLLEY,AND GLENN A. MILLERS

MELODYE W.CAMPBELL,MILTON Departments of Microbiology of Virginia Commonwealth

and Anatomy, University, Received

May

Medical Richmond,

College of Virginia Virginia 23298

3, 1979

Peritoneal macrophages from proteose peptone and Corynebacterium parvum (CP)-treated Lewis and Brown Norway rats were separated into subpopulations by centrifugation on discontinuous gradients of Ficoll. Four macrophage subpopulations were prepared and tested for cytostatic and cytotoxic activity against syngeneic and allogeneic Moloney sarcoma tumor cells. Macrophages were cocultured with tumor cells for 48 hr. whereupon either the inhibition of [‘251]iododeoxyuridine uptake was measured (cytostasis) or the tumor monolayers were observed for cytotoxic effects. CP-Activated macrophages from heavy-density portions of the gradient (8- 10% and IO%-pellet) were highly cytostatic and cytotoxic to both the syngeneic and allogeneic tumor cells while macrophages from the light-density portions (4-6 and 6-8% Ficoll bands) were not. Proteose peptone-stimulated macrophages from the heavy-density portions of the gradient were cytostatic but not cytotoxic to the tumor cells. The effector macrophages from the CP-activated pool were large, well-differentiated cells as determined by electron microscopic examinations and had enhanced phagocytic activity when contrasted with the noncytotoxic, less dense macrophages.

INTRODUCTION There is considerable evidence that macrophages participate in the regulation or destruction of syngeneic or allogeneic tumors (1,2). The mechanisms by which this is accomplished remain unresolved. In v&o, macrophages obtained by a variety of procedures can recognize transplantation antigens (3 -5) and tumor-specific antigens (6). Macrophages may become effective anti-tumor agents when activated as a result of interaction with sensitized lymphocytes (7,8) or with products of the interaction (9- 11). Macrophages can express specific effector functions mediated by antibody (12- 14) and can be activated nonspecifically to discriminate between normal and transformed cells (15). As effector cells against tumors, macrophages have been shown to cause lysis of tumor cells (7-9), growth inhibition (16), microcytotoxicity (16), and/or cytostasis (17, 18). In contrast are reports of I This work was supported in part by Grant IN-IOSB from the American Cancer Society. 2 To whom requests for reprints should be addressed: Department of Microbiology, P.O. Box 847, Medical College of Virginia, Richmond, Va. 23298. 153 0008-8749/80/030153-16$02.00/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.

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macrophage-activating immunostimulatory agents that enhance tumor growth in both humans and animals (1) and of macrophages resident in a tumor mass with the ability to enhance tumor growth (19). Also, macrophages have been shown to contribute to depression of lymphocyte responses in tumor-bearing animals (20). The diverse and often contradictory functions displayed by macrophages could be explained by assuming that macrophages are functionally heterogeneous. This argument is supported by recent studies indicating that macrophage subpopulations prepared either by Ficoll or BSA” gradients or by velocity sedimentation differ in function (21-24). Considering the accumulating evidence for the functional heterogeneity of macrophages, we began a study to determine the role of proteose peptone-elicited and Corynebacferium parvum (CP)-activated macrophage subpopulations in resistance to syngeneic and allogeneic tumor cells. Our major observation is that for both the syngeneic and allogeneic systems, in vitro cytostatic and cytotoxic anti-tumor activity is expressed by a subpopulation of large, well-differentiated, CP-activated macrophages. The large macrophage subpopulation from proteose peptone-treated rats demonstrated only cytostasis. MATERIALS

AND METHODS

Animals. Lewis (LE) and Brown Norway (BN) rats of both sexes were obtained from Microbiological Associates, Bethesda, Maryland, and were maintained in the Animal Care Facilities of the Medical College of Virginia. Rats ranged in weight from 150 to 200 g. Tumor cells. The LE Moloney sarcoma tumor cell line (LM2) was derived from LE rat fibroblasts that had been exposed to Moloney sarcoma virus (MSV) extracted from BALB/c mice. LM2 carried LE histocompatibility (AgB 1) antigens on cell surfaces. The BN Moloney sarcoma cell line (MST) was derived from a tumor that appeared in a BN rat inoculated with MSV and was adapted to in vitro culture. MST produced mouse C-type virus and carried BN histocompatibility antigens AgB3 on cell surfaces (25). LM2 cells were prepared from cultures of the 15th to 40th in vitro passages and MST from the 35th to 60th in vitro passages. Macrophages. Peritoneal exudate cells (PEC) were harvested from the peritoneal cavities of rats treated either 7 days earlier with 7 mg C. parvumlkg body wt (Burroughs Wellcome, Research Triangle Park, N.C.) or 3 days earlier with 7 ml of 10% proteose peptone (Difco, Detroit, Mich.). PEC from C. parvum-treated rats are termed activated and PEC from proteose peptone-treated rats are termed elicited. Peritoneal exudate macrophages (PEM) were separated from nonadherent cells by adherence to collagen-coated surfaces (16). Three milliliters of rat tail collagen was poured into petri dishes 10 cm in diameter and gelled by exposure to 28% ammonium hydroxide for 1 hr. Gels were dialyzed against distilled water overnight and saturated with complete minimum essential medium (CMEM) (14) on the day of use. Fifty million PEC were incubated in 15 ml of medium for 2 hr at 37°C in 95% air and 5% COZ. After nonadhering cells had been rinsed off, the adherent cells were 3 Abbreviations used: PEC, peritoneal exudate cells; PEM, peritoneal exudate macrophage; C. parvum, Corynebacferium parvum; LE, Lewis rat; BN, Brown Norway rat; LM2, LE Moloney sarcoma; MST, BN Moloney sarcoma; MEM, minimum essential media; CMEM, complete media containing MEM plus additives and 10% calf serum; MI#J, macrophage; BSA, bovine serum albumin.

MACROPHAGE

HETEROGENEITY

1.55

released by a 15min incubation with 0.1% collagenase (Type IV, Worthington Biochemical Corp., Freehold, N.J.). The cells were then washed three times with CMEM and counted. Preparation of subpopulations. PEM were separated into four subpopulations using a discontinuous Ficoll gradient according to the procedure of Walker (2 1,22). In general, Ficoll was prepared by dissolving Ficoll 400 (Pharmacia Fine Chemicals, Uppsula, Sweden) in deionized water and dialyzing it against several changes of deionized water for 2 days. The Ficoll was lyophilized and redissolved in CMEM without fetal bovine serum to a concentration of 20%. The stock solution was stored at -20°C. Ficoll solutions (4, 6, 8, and 10%) were prepared in CMEM from the 20% stock solution. This discontinuous gradient was then formed in a 15ml conical centrifuge tube (Falcon, Oxnard, Calif.) by overlaying 2.0 ml of 10% Ficoll with 3.0 ml of 8,6, and 4% Ficoll. The PEM were included in the 4% Ficoll. Gradients were centrifuged at 700 rpm for 7 min at 4°C in a Beckman J-6 centrifuge fitted with a JS-4.2 rotor. The cells sedimenting at the density interfaces were collected, washed three times, and resuspended to the appropriate concentration in CMEM. Cytostatic assay. A cytostatic assay was used to assess macrophage effects on target cell proliferation in vitro. Measurement of the incorporation of a pulse of [lY]iododeoxyuridine ([rz51]UdR) was used and has proved to be a sensitive assay. This assay was also feasible because, under the conditions employed, macrophages manifest a very low proliferative rate that does not exceed background level of [12sI]UdR incorporation. Inhibition of uptake of [‘251]UdR seems to reflect macrophage-mediated inhibition of DNA synthesis in target cells since greater than 90% of the label incorporated by a monolayer of tumor cells in the absence of macrophages could be precipitated with trichloroacetic acid. Tumor target cells (5 x lo4 in 0.2 ml) were allowed to adhere in the wells of flat-bottom 96-well microtiter plates (Costar) for 4-6 hr at 37°C (95% air and 5% CO,). The plates were then washed and macrophages were added in volumes of 0.2 ml to give macrophage:tumor cell ratios of 1: 1 to 10: 1. The plates were incubated for 48 hr and washed twice with MEM to remove any thymidine released from the macrophages which could compete with the [‘251]UdR. Each well was then pulsed with 0.1 PCi [1251]UdR (New England Nuclear, Boston, Mass.) in a volume of 0.1 ml CMEM containing lop6 M fluorodeoxyuridine (Sigma Chemical Co., St. Louis, MO.). After an additional 8-hr incubation, the wells were washed twice with MEM, the cells fixed with 70% methanol and sprayed with clear laquer (Illinois Bronze Paint Co., Lake Zurich, Ill.). The individual wells were separated using a band saw and the radioactivity counted in a Beckman gamma spectrometer. Four to six replicates of each macrophage:tumor cell ratio were prepared. Data are presented as the mean counts per minute of the replicates *SE and as percentage inhibition of the control counts per minute (tumor cells incubated without macrophages). This was calculated as: 100 x 1 -

cpm tumor cells + macrophages cpm tumor cells

I.

Cytotoxicity assay. Fifty thousand target cells were allowed to adhere to the surface of wells of flat-bottom 96-well microtiter plates (Costar) for 4-6 hr in a

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volume of 0.2 ml at 37°C (95% air and 5% CO*). The plates were then washed and macrophages were added in volumes of 0.2 ml to result in macrophage:tumor cell ratios of 1: 1 to 10: 1. The plates were incubated for 48 hr and observed for the loss of tumor cells. Percentage cytotoxicity of tumor cells was gauged according to the following scale: 0: 1 +: 2+: 3+: 4+:

no cytotoxicity, 10 to 25% cells lost, 25 to 50% cells lost, 50 to 75% cells lost, 75 to 100% cells lost.

Four to six replicates of each macrophage:tumor ratio were observed. Tumor cells that detached from the wells of microtiter plates were judged killed by trypan blue staining and also because they failed to grow when injected into appropriate rats. Microscopy. Macrophages were prepared for transmission electron microscopy by fixing cell pellets for 1 hr in cold 2.5% purified glutaraldehyde (Tousimis Research Corp.) in 0.1 M sodium cacodylate buffer (pH 7.3), containing 0.05% calcium chloride. They were then rinsed in 0.1 M cacodylate buffer, postfixed for 1 hr in cold 1% osmium tetroxide in 0.1 M cacodylate, dehydrated through graded ethanols, and embedded in Epon 812 (26). Thin sections were cut on a Porter-Blum MT2-B ultramicrotome, stained with uranyl acetate and lead citrate (27) and examined in a Hitachi HU-12 electron microscope. Thick sections for light microscopy were also prepared and stained with Azure II/methylene blue (28). Esterase. The test for monocyte nonspecific esterase activity followed the method of Tucker et al. (29), using reagents purchased premixed from Technicon Instruments, Tarrytown, New York. Fc receptor activity. Sheep erythrocytes (E) were sensitized with the appropriate amount of rabbit anti-SRBC antibody (A) and the EA were incubated with macrophages at a ratio of 50: 1, centrifuged at 1000 r-pm for 10 min in a Beckman J-6, and permitted to stand an additional 15 min. The percentage of rosette-forming cells was determined by counting at least 2000 cells and scoring those with at least three EA as a rosette. Phagocytosis. Ten million macrophages were mixed with 5 x lOa latex beads (0.81 pm diameter, Bacto-Latex, Difco, Detroit, Mich.) and rotated for 30 min. Excess beads were washed away, smears or thick Epon sections made, and the preparations examined. Cells were scored as phagocytic when they had internalized three or more beads. RESULTS Effectiveness of C. parvum-Activated and Elicited Cytostasis and Cytotoxicity of Tumor Cells

Macrophages

in Causing

Macrophages from rats treated with the immunopotentiator C. parvum very effectively induced cytostasis of both syngeneic and allogeneic tumor cells, as assessed by the inhibition of [1251]UdR uptake. The results of an experiment in which activated macrophages from eight C. parvum-treated LE rats were pooled and tested against the LM2 (syngeneic) or MST (allogeneic) tumor lines are shown in Table 1. At macrophage:tumor cell ratios of 1: 1, a clear distinction is seen

MACROPHAGE

TABLE Inhibition

157

HETEROGENEITY 1

of [iZ51]UdR Uptake in Tumor Cells by C. parvum-Activated and Proteose-Elicited Macrophages [‘*“I]UdR uptake (mean cpm t SE)O

Macrophage source

LM2

M@TC”

MST

LE

Activated Elicited Activated Elicited Activated Elicited

1:l 1:l 5:l 5:l 1O:l 10: 1

3,571 56,979 353 9,317 236 1,096

+ k k + + +

124 1,550 18 2,671 2 104

(95)e,d (26) (99) (88) (99) (99)

7,862 93,030 3,147 2,663 732 1,833

2 + f ? f k

1,532 10,426 356 199 150 268

(93) (14) (97) (98) (99) (98)

BN

Activated Elicited Activated Elicited

1:l 1:l 5:l 5:l

39,355 28,853 3,109 36,993

k k k k

6,909 2,097 300 3,104

(14) (37) (93) (19)

34,503 46,287 2,849 36,378

k k + f

2,662 2,951 405 6,344

(2) (0)

(92) (0)

a Macrophage-to-tumor cell ratio. * Results from six replicate cultures. e Percentage suppression of control values. In the upper study, LM2 and MST incorporated respectively 76,942 ? 2671 and 108,174 t 4174 in the absence of M4. In the lower study, LM2 and MST incorporated 45,766 ? 3964 and 35,076 + 2378 cpm, respectively, in the absence of M+. d Italicized values indicate significance at P < 0.05.

between activated and elicited macrophages. Activated macrophages caused 95% inhibition of [Y]UdR uptake by LM2 cells and 93% by MST cells, whereas elicited macrophages caused 26 and 14%, respectively. The difference between the two groups of macrophages disappeared as the ratios were increased. This experiment was also performed using the BN rat (Table 1). In this case, significant differences between activated and elicited macrophages were not present at ratios of 1: 1, but appeared at ratios of 5: 1. Table 2 shows representative data from an experiment in which C. parvumactivated or proteose peptone-elicited macrophages pooled from groups of eight LE rats were tested for cytotoxic activity against LM2 (syngeneic) and the MST (allogeneic tumor cell line. At macrophage:tumor ratios of 1: 1, the activated macrophages always caused a loss of tumor cells, whereas no visible interaction was evident with elicited cells. This was true regardless of the histocompatibility relationship of the target and effector macrophage. When macrophage:tumor ratios were increased, only a minimal amount of cytotoxicity was observed for the elicited macrophages (less than 25%). Results with the BN rat (Table 2) are similar to those of the LE rat with the exception that even at macrophage:tumor ratios of 5:1, there was no observable cytotoxic effects of elicited macrophages on either target. Preparation of Macrophage Subpopulations on Discontinuous Ficoll Gradients

Pools of macrophages from proteose peptone and C. parvum-treated rats were next subjected to separation on discontinuous Ficoll gradients and the subpopulations were tested for macrophage markers such as nonspecific esterase activity, Fc

158

CAMPBELL,

SHOLLEY, TABLE

Cytotoxic Effect of C. parvum-Activated

AND MILLER 2

and Proteose-Elicited

Macrophages on Tumor Cells Relative cytotoxicity”

Macrophage source

M+TC”

LM2

MST

LE

Activated’ Elicited” Activated Elicited Activated Elicited

1:l 1:l 5:l 5:l 10: 1 10: 1

4+ 0 4+ 1+ 4+ 1+

4+ 0 4+ 1+ 4+ 1+

BN

Activated Elicited Activated Elicited

1:l 1:l 5:l 5:l

0 0 4+ 0

0 0 4f 0

a Macrophage-to-tumor cell ratio. * Four replicate cocultures were examined: 0, no cytotoxicity; l+, lo-25% lost; 2+, 25-50% lost; 3+, 50-75% lost; 4+, 75-100% lost. c Prepared from eight rats treated 7 days earlier with 7 mg C. parvumlkg body wt. d Prepared from eight rats treated 3 days earlier with 7 ml of 10% proteose.

rosette activity, and latex bead phagocytosis. Table 3 shows a typical separation of LE cells. Greater than 90% of the cells in each subpopulation bear the various macrophage markers, characterizing all the subpopulations as macrophages. Similar results were obtained with BN macrophages. TABLE Characterization

3

of LE Peritoneal Macrophages Separated on a Discontinuous

Ficoll Gradient

Cells with activity (%) Source of macrophages

Recovered cells wo)

Esterase’

Rosette@

Phagocytosisc

Proteose elicited Entire population before separation 4-6% Band 6-8% Band 8-10% Band IO%-Pellet

100 12 20 27 48

97 98 97 97 97

97 91 92 92 93

96 91 90 93 93

CP activated Entire population before separation 4-6% Band 6-8% Band 8- 10% Band 10% Pellet

100 7 19 19 62

99 96 94 98 98

96 92 90 92 95

98 90 90 90 92

a Nonspecific esterase. b Fc rosette activity. c Latex phagocytosis.

MACROPHAGE

TABLE Inhibition

of [rz51]UdR Uptake of C. parvum-Activated [‘251]UdR

LE M@LM2 LE Mr#xLM2 1:l 5:l 10: 1 BN M&MST 1:l 5:l

4

in Tumor Cells by Subpopulations Syngeneic Macrophages incorporation

(cpm

6-8%

4-6%”

159

HETEROGENEITY

+ SE)

8- 10%

79,117 78,278 89,734

+ 2,219 (8)* k 1,187 (9) + 1,131 (0)

94,262 93,783 61,778

+ 1,684 (0) t 1,911 (0) + 3,220 (28)

20,423 -r 2,185 1,221 + 109 376 5 64

3,974

t 1,769 (0)” N.D.’

36,596 31,062

r 2,612 (0) + 2,394 (11)

44,579 3,657

a Ficoll interface. b Percentage inhibition of control. Control cpm = 85,779 MST. Italicized values indicate significance at P < 0.05. e Not determined.

Tumor Cytostatic

Activity for Macrophage

IO%-Pellet

(76) (98) (99)

+ 2,848 (0) 2 1,055 (90)

+ 9786 for LM2

2,098 t 174 309 k 59 264 e 41

33,678 3.611

and 35,076

(98) (99) (99)

4 2,850 (4) 2 409 (90)

+ 2378 for

Subpopulations

Macrophages from each of the four subpopulations were tested for functional heterogeneity by coculturing them with tumor cells and determining the degree of inhibition of [1251]UdR incorporation into tumor cells. In Table 4 are the results of a representative study in which subpopulations of CP-activated LE macrophages were tested for cytostasis against the syngeneic LM2 tumor line. Macrophages obtained from the 10% pellet and S-10% interfaces displayed marked cytostatic activity at all cell ratios tested. In contrast, macrophages recovered from the 4-6 or 6-S% Ficoll interfaces were not significantly cytostatic, except the macrophages of the 6-S% fraction when cocultured at ratios of 10: 1. A similar experiment was performed with C. parvum-activated BN macrophabes and MST as the target. These results are also shown in Table 4. Cytostatic activity was again displayed by macrophages of the lO%-pellet and S-10% fractions but only at macrophage:tumor ratios of 5: 1. Macrophages from the 6-S% Ficoll interface had little effect (11% inhibition of [1251]UdR uptake as contrasted with 90% for the 9- 10% Ficoll fraction). In Table 5 are results from a study in which proteose peptone-elicited LE macrophages were separated into subpopulations and tested for cytostatic activity. As with the CP-activated macrophages, cytostatic activity was localized among the heavy dense macrophage subpopulation (lo%-pellet fraction at ratios of 1: 1 and 8- 10% and IO%-pellet fractions at macrophage to tumor ratios of 5: 1. Since the preceding experiments dealt with a syngeneic macrophage to tumor cell relationship, it was of interest to determine if the pattern of activity was the same when allogeneic target cells were employed. Table 6 shows data from a representative experiment in which subpopulations of CP-activated LE macrophages were cocultured with MST cells. At ratios of 1: 1, macrophages from the 8- 10% and lO%-pellet interfaces suppressed [‘251]UdR incorporation 28 and 99%, respectively. Macrophages from the 4-6% band or the 6-S% interface failed to

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cause cytostasis. When the macrophage:tumor ratios were increased to 5: 1, cells from all four fractions were cytostatic, but macrophages from the 6-8%, 8- lo%, and lO%-pellet bands still had activity that was fourfold greater than displayed by cells from the 4-6% interface. BN macrophage subpopulations from CP-treated rats tested against the allogeneic LM2 line also induced cytostasis (Table 6). At ratios of 5: 1, the 8- 10% and lO%-pellet preparations were clearly cytostatic to LM2 (91 and 94% inhibition of [lz51]UdR uptake, respectively). The less dense subclass (6-8%) was not significantly cytostatic. Results with ratios of 1:l were not consistent and never reached levels much greater than 25%. LE macrophages from proteose-treated rats when tested against MST cells gave results almost identical to those in Table 5. The heavy dense macrophages were significantly cytostatic whereas the less dense subpopulations were not. Cytotoxic Activity

of Macrophage

Subpopulations

on Tumor Cells

Macrophages from CP- or proteose-treated animals were also tested for the ability to cause cytotoxicity when cocultured with tumor. When subpopulations of CP-activated LE macrophages were tested against the syngeneic LM2 tumor line, macrophages from the IO%-pellet and 8- 10% interfaces of the Ficoll gradient demonstrated significant tumor killing capacity (4+ at macrophage to tumor ratios of 1: 1, 5: 1, and 10: 1). In contrast, macrophages recovered from the 4-6 or 6-8% interfaces were not effective. Cytotoxic activity of BN macrophage subpopulations against the syngeneic MST line was also found to be associated with the heavy dense macrophages (8- 10% and 10%-pellet), but was not observed until macrophage:tumor ratios were raised to 5: 1. In Tables 1 and 2 data were presented showing that while proteose-activated macrophages were cytostatic to tumor cells they were not cytotoxic. In Table 5 we presented data showing that the cytostatic activity was restricted to the heavy dense portions of the Ficoll gradient. Since proteose-elicited macrophages are not cytotoxic to tumor cells, it would be predicted that the 8-10% and IO%-pellet subpopulations of proteose-elicited macrophages would not be cytotoxic. This was tested by presenting elicited LE macrophages to LM2 monolayers. When tested at macrophage:tumor ratios of 1: 1 and 5: 1, none of the subpopulations demonstrated TABLE Inhibition

of [iz51]UdR Uptake in Tumor Cells by Subpopulations of Proteose Peptone-Elicited LE Macrophages [‘*“I]UdR

LE M@LMZ” 1:1 5:l

4-6%” 78,962 77,697

5

6-8%

t 2,439 (0)c 2 1,974 (1)

a Macrophage-to-tumor b Ficoll interface. c Percentage suppression values indicate significance

incorporation

77,431 76,432

(cpm

? SE)

8- 10%

+ 2,651 (2) + 1,551 (3)

62,571 25,131

t 2,771 (20) t 657 (68)

IO%-Pellet 59,728 9,723

k 2,501 (24) 2 725 (88)

cell ratio. of control values. at P < 0.05.

LM2

cells incorporated

78,263

+ 1974 cpm.

Italicized

MACROPHAGE

TABLE Inhibition

of [*251]UdR Uptake of C. purvurn-Activated

LE M&MST” 1:1 5:l BN

6

in Tumor Cells by Subpopulations Allogeneic Macrophages

[12jI]UdR 4-6W

161

HETEROGENEITY

incorporation

(cpm

6-S%

+ SE) IO%-Pellet

8- 10%

112,985 79,364

f 4,105 (0)r 2 3,393 (26)

109,042 4,212

t 8,392 (0) 2 1,517 (96)

77,157

35,870

k 2,338 (21) N.D.”

33,062 34,216

+ 1,557 (27) + 1,372 (3)

42,527 4,254

1,473

2 5,048

(28)

i

(98)

1,026 4/l

+ 299 (7) lr 1,279 (91)

36,561 2,590

450

-+ 283 t 134

(99) (99)

M@LM2 I:1 5:l

IL Macrophage:tumor ratio. * Ficoll interface. c Percentage inhibition of control. Control LM2. Italicized values indicated significance ” Not determined.

cpm = 107,527 at P < 0.05.

i

1497 for MST

and 45,766

t 2,787 (20) -+ 695 (94)

+- 3964 for

any cytotoxicity (index of 0). At ratios of 10: 1 the index was 1 + against the LM2 target. Cytotoxic activity of CP-activated and proteose-elicited macrophage subpopulations was also assessed employing an allogeneic relationship between macrophage and tumor. With CP-activated LE macrophages and MST cells as target, cytotoxic activity was again found to be associated with the heavy dense macrophages. The IO%-pellet fractions had a cytotoxic index of 4+ at macrophage:tumor ratios of I: 1 and 5: 1 and the 8- 10% fraction had an index of 3+ at 1: 1 and 4+ at 5: 1. With CP-activated BN macrophages and the LM2 as target, cytotoxic activity was observed only at the .5:1 ratio, but, nonetheless, was complete (4+ for both the 8- 10% and IO%-pellet macrophages). Proteose-elicited LE macrophages when tested for cytotoxic activity against MST demonstrated no detectable activity (index of 0) at any ratio employed except for an index of + 1 for the IO%-pellet subset when incubated with MST at a ratio of 5: 1. Morphologic

Evaluution

To determine whether morphological differences existed among cells of the various macrophage subsets, an electron microscopic evaluation was performed. The predominant cell found in the 8- 10% and IO%-pellet fractions from C. pauvltm-treated rats (Fig. 1) had a small eccentric nucleus and a large amount of cytoplasm, containing numerous mitochondria, scattered short segments of rough endoplasmic reticulum, a large central Golgi complex, clear peripheral vacuoles (probably representing invaginations of the ruffled plasma membrane), and one or more large heterophagic vacuoles. Such cells were much less abundant in the 4-6 and 6-S% subpopulations, which included many small macrophages, having a higher nuclear-cytoplasmic ratio and infrequently containing large heterophagic vacuoles. In all the subpopulations, there were a few small cells having the ultrastructural characteristics of lymphocytes.

162

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FIG. 1. Ultrastructure of a macrophage from the 10% subpopulation from C. parvum-treated cell is typical of the majority of cells in the 8 and 10% subsets. x 16,500.

rats. This

Phagocytosis As indicated previously (Table 3), 90% or more of the cells in each subpopulation were phagocytic as determined on smears. This was reinforced when macrophage subsets incubated in medium containing latex beads were examined on l-pm-thick

MACROPHAGE

HETEROGENEITY

163

FIG. 2. Macrophage from a 10% subpopulation from C. parvum-treated rats after incubation with latex beads. The large cells typical of this subpopulation phagocytosed numerous beads. x 15,600.

Epon sections and by electron microscopy. it was determined that the large macrophages which predominated in the 8-10% and IO%-pellet subpopulations usually contained numerous beads (Fig. 2); the smaller cells, present to a lesser extent in the 8-10% and IO%-pellet groups and predominating in the less dense fractions, usually contained fewer beads per cell (Fig. 3). When the number of beads

164

CAMPBELL,

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AND MILLER

FIG. 3. Macrophage from a 6% subpopulation from C. parvum-treated rats after incubation with latex beads. Compare the size ofthis cell with that in Fig. 2. The majority of cells in the 4 and 6% subpopulation were of this smaller size and usually contained fewer beads. There is one bead enclosed in a vacuole. x 15,600.

in each of 200 cells was counted on thick Epon sections, it was found that the two greater-density subpopulations had an average of 10beads per cell, whereas the less dense cells averaged 5 beads per cell.

MACROPHAGE

HETEROGENEITY

165

DISCUSSION The available evidence suggests that macrophage activation is of central importance both for cytostatic potential and for target-cell killing at effector-target ratios that are physiologically attainable. C. parvum is one of the most potent activators of the mononuclear phagocytic system (30) and has potential usefulness in cancer therapy. In addition, C. parvum-activated macrophages exhibit enhanced accessory cell function (3 l), but also suppress mitogen-induced lymphocyte proliferation (32) and the growth of tumor cells (33). Cancer immunotherapy with immunopotentiators such as C. parvum may depend on a delicate balance between suppressive and stimulatory effects. Studies of the heterogeneity of macrophages will enhance understanding of this balance and may determine the mechanisms involved in tumor-cell killing, thereby permitting control of macrophage functions. For these reasons and because we consider macrophage heterogeneity as a basic immunological problem, we studied functional heterogeneity within populations of CP-activated and proteose-elicited peritoneal macrophages. We have demonstrated that a marked degree of functional heterogeneity exists within populations of CP-activated and proteose-elicited macrophages. Anti-tumor activity measured by a cytostasis assay was expressed by populations of large and dense macrophages, whereas cytotoxic activity was expressed by the large macrophages but only when recovered from CP-treated rats. Information is scarce concerning macrophage heterogeneity as it applies to effector function in tumor immunity. Nathanet al. (34) described a subpopulation of adherent peritoneal cells which are esterase-positive and responsible for tumor growth inhibitory activity in the mouse. A more recent characterization of this cell suggests it to be either an unusual macrophage or an atypical B cell (35). It has also been suggested that a second subpopulation of cells is able to stimulate the proliferation of tumor target cells (35,36). When C. parvum-activated macrophages were separated on discontinuous Ficoll, approximately 80% of the cells were found in the two heavy-density Ficoll bands (Table 3) and, as may be seen from Tables 4-6 and in cytotoxicity experiments these were the cells expressing the most cytostatic and cytotoxic activity. This result is in contrast to the results of Weinberget al. (37), who demonstrated that cytostatic activity was found in the light-density portion of a BSA gradient and that heavy-density cells frequently enhanced tumor growth. However, Lee and Berry (24), after fractionation of C. purvum activatedmacrophages according to cell size by velocity sedimentation, found that smalland medium-sized macrophages shared enhanced immunostimulatory (accessory or A cell) activity as measured by their ability to restore the immune responsiveness of nonadherent spleen cells to sheep erythrocytes and polymeric flagellin. Gorczinski (38), using velocity sedimentation, also found that different populations of macrophages were responsible for the reconstitution of the antibody response and the cytotoxic T-cell response. We have looked at the ability of our macrophage subsets to inhibit mitogen-induced blastogenesis and have found this activity associated with heavy dense cells (data not shown). Although macrophage activation seems to be centrally important for both cytostatic potential and for target-cell killing, there are a number of studies suggesting that cytostatic and cytotoxic functions of macrophages are distinct macrophage activities (39-44). Although the mechanisms of cytotoxicity and cytostasis have not been completely defined, it appears that marked differences exist. Hibbs (15) has presented evidence that cytotoxicity involves an interaction

166

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AND

MILLER

between activated macrophages and susceptible target cells, resulting in membrane destabilization in both cells and temporary membrane fusion. It was suggested that the subsequent transfer of lysosomes into the target cell leads to cell death as a result of release of lysosomal enzymes. Another possibility for macrophagemediated tumor-killing is based on the observation that peroxidase, hydrogen peroxide, and halide ions form a potent system for the intracytoplasmic killing of microbial species (46) and also for extracellular killing of eukaryotic cells. In addition, recent studies (41, 44) indicate that aberrant cell division may also be involved in the cytotoxic event. It is possible that tumor cells may divide without synthesizing DNA when they are cultivated with activated macrophages. It is not understood how such aberrant cell division may be related to the cytotoxic event. Studies have also indicated that macrophages require close cell-to-cell contact between functional effecters and targets as essential for attaining cytostasis. However, there are situations in which cytostasis was effective despite greatly reduced cell-to-cell contact (47). It seems possible that macrophage-mediated cytostatic action might involve a soluble factor elaborated by these cells. One such factor, arginase, which is released from macrophages, has been reported to inhibit proliferation of cells located in their vicinity (48). These results and recent studies of Kaplan and Morahan (41) are consistent with a separate mechanism for macrophage-mediated tumor cell cytostasis and cytotoxicity. Also consistent with separate mechanisms are the results shown in Table 1 in which it is seen that cytostatic activity was expressed by proteose-elicited macrophages, but only at macrophage-to-tumor ratios exceeding 5: 1. Significant cytotoxicity was never observed with elicited macrophages. Background cytotoxicity may have resulted as a consequence of our animals not being specific pathogen- or germ-free, although they were free from obvious disease. It is possible that cytostasis could be mediated by one particular macrophage subset, whereas cytotoxicity could be mediated by a more vigorous macrophage subset. In the present report, we have demonstrated that tumor cytostatic and cytotoxic activity is expressed by CP-activated macrophages found in the same region of the Ficoll gradient. It would also seem that the same population of macrophages is responsible for the effects against both syngeneic and allogeneic targets. Macrophages from proteose-treated rats, however, lacked a cytotoxic population but the cytostatic subpopulation was found in the same region of the Ficoll gradient as for the CP-activated cells. It would seem, therefore, that the population inducing cytostasis is the same large dense macrophage in both cases, but that the proteose-elicited macrophages lack the cytotoxic subset. If this is true it should be possible to resolve the heavy dense CP-activated macrophages into additional subpopulations. We are in the process of using elutriation procedures (countercurrent centrifugation) to probe the existence of functional heterogeneity within a Ficoll subset. Experiments involving the uptake of latex beads indicate that the effector populations of CP-activated macrophages (the 8- lO%-pellet cells) has enhanced phagocytic activity as contrasted with the inactive population (Figs. 2 and 3). This is in contrast to another investigation (23), in which it was demonstrated that macrophages associated with low effector activity and possible augmentation of tumor growth have strong phagocytic properties (23). It is difficult at this point to explain this contrast. Previous studies (16) have demonstrated that LE macrophages are more inhibitory to growth of Moloney sarcomas in vivo and more cytotoxic in vitro than

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BN macrophages. In addition, Freund’s complete adjuvant was shown to activate LE macrophages against tumors but not BN macrophages. It was thought that BN rats are deficient in a particular, active macrophage subpopulation. When macrophages from BN rats were separated and the individual subsets tested for activity (Tables 4-6), it was found that cytostatic activity resided in the 8- IO%-pellet fractions. This was the same as seen for LE macrophages. The only difference was that BN macrophages had to be used at greater ratios with tumor for significant cytostasis to be obtained. It would appear, therefore, that BN rats do carry cytostatically active macrophage subpopulations, but these are less effective than those in LE rats. This might be the result of a problem in recognition, level of activity, or absolute numbers of active macrophages in a subset. Although we have been able to define functionally heterogeneous macrophage subpopulations, the question remains whether the functional macrophage subpopulations represent varying states in differentiation or true specialization. Experiments are in progress to define this question. ACKNOWLEDGMENTS The authors assistance.

thank

Ms.

Olivia

G. Washington

and Ms.

Mary

Gaebel

for their

excellent

secretarial

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