Functional heterogeneity among peritoneal macrophages

CELLULAR

38, 94-104 (19%)

IMMUNOLOGY

Functional

Heterogeneity

Among

Peritoneal

Macrophages

I. Effector Cell Activity of Macrophages Against Syngeneic and Xenogeneic Tumor Cells 1.2 D. S. WEINBERG, Department Division

of Pathology, of Immunology,

M. FISHMAN,

AND B. C. VEIT

Peter Bent Brigham Hospital, Boston, Massachzlsetts 02115 St. Jude Children’s Research Hospital, Memphis, Tenlzessee 38101 Received

December

and

21, 1977

Peritoneal exudate cells from immunized and nonimmunized animals were separated into subpopulations by centrifugation on discontinuous bovine serum albumin (BSA) density gradients. Cells in the several subpopulations were then tested for their cytostatic or cytotoxic activity against syngeneic and xenogeneic tumor cells. Nonimmune macrophages isolated at the 8 to 11% BSA interface were highly inhibitory to the growth of syngeneic and xenogeneic tumor cells during coculture for 24 to 48 hr. A second macrophage subpopulation of heavier density was not as effective in preventing tumor growth and frequently augmented it. Cytotoxic activity against (C58NT) D tumor cells could not be detected with macrophages or subpopulations of macrophages from immune as well as nonimmune animals, as determined by a 4-hr chromium release assay. The cytotoxic activity of the immune peritoneal exudate cells observed by this assay could be accounted for by the small percentage of lymphocytes present.

INTRODUCTION Populations of macrophages, like lymphocyte populations, are functionally heterogeneous. Subpopulations of macrophages separated by buoyant density (l-3) differ in their ability to (i) phagocytize inert particles and bacteria, (ii) bind antigens, (iii) form rosettes with antibody-coated erythrocytes, (iv) produce immunogenic RNA upon exposure to antigen in vitro, and (v) take up and incorporate tritiated thymidine (4). In studies of tumor immunity, macrophages have been shown to be cytotoxic or cytostatic for tumor cells and these activities were either specific (5-S) or nonspecific (9-11) for a given tumor. Although activated macrophages can destroy tumor cells in a nonspecific manner, they have been reported to be selectively cytotoxic and will not affect the growth of non-neoplastic cells (12, 13). In view of the functional heterogeneity of macrophages already cited, a study was made to determine the role of subpopulations of macrophages in specific and nonspecific immunity to syngeneic tumor cells in vitro. The results reported here emphasize two major 1 Supported by NIH Grant CA-18672, NC1 Grant CA-08480, and by ALSAC. *Part of this research was submitted by D. S. W. in partial fulfillment of the requirements for the Ph.D. degree in the Department of Pathology, New York University Medical School. 94 0008-8749/78/0381-0094$02.00/O Copyright All rights

1978 by Academic Press, Inc. o? reproduction in any form reserved

MACROPHAGE-TUMOR

CELL

INTERACTIONS

95

observations : (i) Macrophages separated from peritoneal exudate lymphocytes of immune rats by density gradient centrifugation were not active in cell-mediated cytotoxicity as measured by a 4-hr 51chromium release assay, and (ii) peritoneal macrophages from nonimmune rats were highly cytostatic against syngeneic tumor cells. Nonspecific cytostatic activity was expressed primarily by light-density macrophage populations, whereas heavy-density macrophages inhibited tumor growth only weakly and frequently augmented it. A recent report by Nathan et al. (14) described two classes of adherent peritoneal exudate cells from BCG-treated mice, one that prevented and one that augmented tumor growth. MATERIALS

AND

METHODS

Animals. Wistar Furth (W/Fu) rats were originally obtained from Microhiological Associates and bred in our laboratory facilities. Peritoneal exudate (PE) 3 cell. Rats (4-10 weeks old) were injected intraperitoneally (ip) with 5 to 10 ml of either Klearol mineral oil (Ruger Chemical Company, Irvington, New Jersey) or beef heart infusion broth enriched with 10% proteosepeptone No. 3. Animals were killed 48 hr later by cervical dislocation and their peritoneal cavities were washed out with buffered saline. Oil and aqueous phases of the exudate were separated by allowing the washings to stand in a separatory funnel for 20 min at 4°C. Cells were washed with buffered saline, centrifuged at 250g in an International PR-2 centrifuge, suspended in buffered saline, and counted in hemocytometers. The peritoneal exudate consisted of 80 to 90% macrophages, and the yield per animal was approximately 1 X 108. Peritoneal exudates were also induced by ip injection of 50 ml of Klearol into rabbits weighing approximately 2 kg each. After 4 days the animals were killed by intravenous injection of 120 mg of Nembutal. Exudates were collected by washing out the peritoneal cavities with buffered saline and treated as described above for rat PE cells. The peritoneal exudate consisted of approximately 90 to 95% macrophages, and the yield per rabbit was approximately 1 x log. Tumor cells. (C58NT) D tumor cells syngeneic in W/Fu rats were obtained from Dr. R. C. Nowinski (Fred Hutchinson Cancer Research Center, Seattle, Washington) and Dr. R. Herberman (NIH). The cells were maintained in RPM1 1640 medium (containing 10% fetal calf serum) at 37°C in an atmosphere of 10% CO2 in air, and the growing cultures were split 1: 3 two or three times weekly. EL-4 tumor cells passaged in tivo in C57B1/6 mice were obtained from Dr. E. A. Boyse (Sloan-Kettering Institute, New York, New York). Tumor cells from ascites fluids were suspended in RPM1 1640 medium and adapted as an in vitro cell line and maintained as described for the (C58NT) D tumor cells. Raji (human Burkitt) cells were obtained from Dr. R. Naegele at St. Jude Children’s Research Hospital and maintained in RPM1 1640 medium. Equilibrium density gradient centrifugation. Subpopulations of rat and rabbit PE cells were prepared by centrifugation on discontinuous bovine serum albumin (BSA) density graidents, as described previously (3). The gradients were prepared by carefully layering S-ml volumes of 20, 15, 11, and 8% BSA above a 2.5-ml 8 Abbreviations used : PE, peritoneal exudate ; ip, intraperitoneally ; BSA, bovine Serum albumin; SC, subcutaneously; FCS, fetal calf serum; EC : TC, ratio of effector cells to target cells ; and GI, growth inhihitinn.

96

WEINBERG,

FISHMAN,

AND

VEIT

bottom layer of 30% stock BSA. Peritoneal exudate cells were suspended in 5% BSA to a concentration of 2 x lo7 cells/ml, and 7.5 ml of the cell suspension was layered on top of each gradient. The tubes were centrifuged at 20,OOOg for 30 min in a Spinco SW 25.1 rotor at 4°C. Cells collected at the 5 to 8, 8 to 11, 11 to 15, 1.5 to 20, and 20 to 30% BSA interfaces were designated A, B, C, D, and E, respectively. Immuniu”ation. Four- to eight-week-old W/Fu rats were injected subcutaneously (SC) in each thigh with 5 X lo7 viable (C58NT)D tumor cells suspended in buffered saline. After 7 to 9 days, the rats were injected ip with an inflammatory agent and the peritoneal exudate cells were collected 48 hr later, Tumor masses observed at the site of injection had receded at the time the animals were killed. Thromium labeling of tumor cells. Tumor cells, 1 x 107, suspended in 1 ml of buffered saline containing 5% fetal calf serum (FCS) were incubated for 40 min at 37°C with 100 to 400 &i of 51Cr (supplied as sodium chromate, sp act., 200 mCi/ mg, New England Nuclear, Boston, Massachusetts). The cells were washed three times in 50 ml of buffered saline containing 1% FCS and resuspended to a concentration of 5 X 105 cells/ml in RPM1 1640 + 10% fetal calf serum. Chrowlium release assay. A 4-hr chromium release assay, as described by Ortiz de Landazuri and Herberman (IS), was used. The effector-to-target cell (EC : TC) ratio was varied by changing the number of effector cells in each plate while keeping the number of target cells constant. Labeled (C58NT)D cells, 5 x 104, in 1 ml of medium were added to the effector cells (total PE cells or subpopulations) maintained as a monolayer in 35 X lo-mm plastic tissue culture plates. The plates were incubated at 37°C in a 10% COrair atmosphere on a rocking platform operating at 6 cycIes/min. The fluids were removed after 4 hr of incubation and centrifuged at 8OOg. The radioactivity present in the supernatants was counted in a Packard gamma counter. The maximum counts that could be released, approximately 85 to 90%, were determined after freezing and thawing the labeled tumor cells three times in dry ice and alcohol. The amount of specific chromium release (percentage lysis) was calculated as follows : Percentage

lysis

[counts per minute (test) - counts per minute (control)] = [counts per minute (freeze-thaw) - counts per minute (control)]

’

100

or Percentage

of input

Vr

released =

[counts per minute (test) J x loo [counts per minute (input)] ’

Spontaneous release of 51Cr was usually less than 15% in a 4-hr assay, and assays showing greater than 20% spontaneous release were discarded. All assays were done in triplicate and the results were expressed as means * the range or * 1 standard deviation. Nylon fiber filtration and plastic adherence treatments. Peritoneal exudate cells were incubated in nylon fiber columns according to the procedure of Julius et al. (16). Cells, 1 x lo* per column, were incubated for 45 min at 37°C and eluted with warm medium. Approximately 30% of the incubated cells were eluted from the columns, and of the cells eluted, 34 to 36% were lymphocytes and 64 to 66% were macrophages as defined by light microscope examination.

MACROPHAGE-TUMOR

CELL

97

INTERACTIONS

To separate PE cells into plastic adherent and nonadherent populations, 5 X 10’ cells were incubated in 150 x 25-mm plastic culture dishes (Falcon No. 3025) in 15 ml of RPM1 containing 10% FCS for 30 min at 37°C. Nonadherent cells were removed and replated as described above and the resulting nonadherent population was then tested. Adherent cells from the first incubation were washed several times with warm medium and scraped off with rubber policemen. The nonadherent population contained 75 to 80% macrophages and 20 to 25% lymphocytes, and the adherent population contained 95 to 98% macrophages and 2 to 5% lymphocytes. Growth inhibition assay. A procedure similar to that described by Evans and Alexander (5) was used to measure macrophage-mediated growth inhibition. The effector cells, whole PE population, or subpopulations of PE cells were placed into 35 X IO-mm tissue culture plates and allowed to adhere at 37°C for 1 or 24 hr under tissue culture conditions (RPM1 1640 medium + 10% FCS). Nonadherent cells were removed and fresh medium was added to the monolayers. Thereafter, 2 X lo5 tumor cells were added to macrophage monolayers and incubated without rocking for various periods of time ; the viable tumor cells growing in suspension were then enumerated by the trypan blue dye exclusion test. Percentage growth inhibition (GI) was determined by the following equation : Percentage

C-E GI = 7--

x 100,

where C = the number of tumor cells in control cultures (no eff ector cells added) and E = the number of tumor cells in the experimental cultures. Tumor cells were easily distinguished from floating macrophages and lymphocytes, and all assays were done in duplicate. RESULTS Rat PE cells and PE s&populations as effector cells in the 4-hr “ICr releaseassay. Wistar Furth rats were injected with (C58NT)D tumor cells, and oil-induced peritoneal exudate cells were collected from these rats 11 days after injection were assayed for cytotoxicity against (C58NT)D cells in a 4-hr “‘Cr release assay. The results of two such experiments are shown in Table 1. At two different EC: TC ratios (100: 1 and 20: 1) the immune PE cell populations caused 37 and 26% TABLE

1

Lysis of (C58NT)D Tumor Cellsby ImmuneW/Fu Rat PeritonealExudate Cellsin the 4-Hour ChromiumRelease Assay ExperimentNo. Expt 1 Expt 2

Peritonealexudatecells”

EC:TCb

Immuned Nonimmune

100:1

Immune Nonimmune

20: 1

Percentagelysisc 36.6 f

0.3

-3.1 f 0.3

QPeritonealexudatecellsinducedwith mineraloil. bPeritonealexudatecell-to-target-cellratio. c Resultsare given as the meanf range. dRats injected 11days previouslySCwith (C58NT)D tumor cells.

26.0 f 3.5 -8.3 f 1.6

98

WEINBERG,

FISHMAN,

Ill-----3:1

61

AND

12:t

VEIT

25~1

50: I

100: I

EC:TC

FIG. 1. Lysis of (C58NT) D cells by W/Fu PE cell populations treated with nylon fiber or plastic adherence to separate adherent and nonadherent cell populations. A constant number of “Cr-labeled tumor cells was incubated for 4 h with various numbers of effector cells to give the EC: TC ratios indicated. Each point represents the mean of triplicate determinations i 1 standard deviation : immune PE whole population, n -m ; immune PE plasticadherent cells, 0 * * . . . . .O ; immune PE plastic-nonadherent cells, A---A ; immune PE nylon fiber-nonadherent cells, 0 - - - - - - - 0 ; normal PE whole population, n-0; normal PE plastic-adherent cells, A- - - - - -A ; normal PE plastic-nonadherent cells, A-A ; and normal PE nylon fiber nonadherent cells, l - - -0.

of the target cells. In contrast, PE cells from nonimmune rats were not cytotoxic. Next, adherent and nonadherent PE cell populations were separated by nylon fiber filtration or plastic adherence treatment and tested for cytotoxic activity. Figure 1 shows that immune nonadherent PE cells obtained by either method were significantly more cytotoxic than was the untreated population. In contrast, immune adherent PE cells were unable to effect any cytotoxic activity above control levels, even at an EC: TC ratio of 100: 1. Macrophages and lymphocytes were

lysis

60 -

20 I

40 I

60

80.1

100:1

EC:TC

FIG. 2. Lysis of (C58NT) D cells by W/Fu PE cell subpopulations. A constant number of YIr-labeled tumor cells was incubated with various numbers of PE effector cells to give the EC: TC ratios indicated. Effector cells were subpopulations of PE cells obtained by discontinuous buoyant density gradient sedimentation or the whole PE population. Each point represents the mean of triplicate determinations k 1 standard deviation : whole PE population, 0-O ; pooled gradient fractions A, B, and C, 0-O; and gradient fraction E, A-A.

MACROPHAGE-TUMOR

CELL

TABLE Growth

Inhibition

Effector cells

1-hr monolayers

24-hr monolayer

of (C58NT)D

2.5 x 106 5.0 x 106 1.0 x 10” 2.5 5.0 1 2.0

x x x x

105 106 106 10”

2

by Rat Peritoneal

Number of effector cells

99

INTERACTIONS

Exudate Monolayers

EC:TC

1.2.5:1 2.5 :1 5 :1 1.25: 1 2.5 :l 5 :1 10 :l

Percentage growth inhibition* 24 hr

48 hr

36.2 (f12.1) 89.7 (scO.0) 93.1 (dzO.0)

23.6 (f12.5) 96.5 (f0.7) 94.4 (ztO.0)

6.1 15.2 12.1 63.6

(3~0.0) (4x0.0) (f3.0) (f12.2)

12.5 - 7.4 16.9 87.5

(f6.6) (zkO.0) (f5.S) (f6.6)

a Oil-induced W/Fu cells placed in monolayer culture 1 or 24 hr before adding 2.0 X 106 (C58NT)D tumor cells. * Percentage GI f range of duplicate plates measured 24 and 48 hr after adding tumor cells to the PE cell monolayers.

separated further by density gradient centrifugation. The lymphocyte-free cell populations, designated as A, B, and C, were pooled and their cytotoxicity was compared with that of the subpopulation E, which contained 5 to 10% lymphocytes. Less than 10% cytotoxicity was observed with the pooled macrophage subpopulation over a wide range of EC : TC ratios (Fig. 2). High levels of cytotoxicity were produced by cells in subpopulation E, which most likely accounted for all the cytotoxic activity observed in the total PE cell population (see also Table 1). Inhibition of tumor grozwth by monolayers of rat PE cells. Our failure to observe cytotoxic activity by macrophages during a 4-hr assay period did not preclude the possibility that such activity might be expressed during a longer incubation period. The high spontaneous release of Yr during prolonged incubations made it necessary to measure tumor cell growth inhibition directly. Monolayer cultures of rat PE cells were prepared in tissue culture plates, as described under Materials and Methods. The results (given) in Table 2 show the inhibitory effects of various numbers of adherent PE cells on the growth of 2 x lo” (C58NT)D tumor cells. Tumor cell growth was inhibited at EC : TC ratios of 5 : 1 and 2.5 : 1 when 1-hr PE monolayers were employed. The 24-hr PE monolayers were not as effective as the I-hr PE monolayers in inhibiting tumor cell growth. However, substantial growth inhibition was observed at an EC : TC ratio of 10 : 1. The cytostatic effect of normal rat PE cells was greatest when the tumor cells were added to 1-hr macrophage monolayer cells. The inhibitory activity of these PE cells, which were derived from nonimmune rats, was nonspecific, since the cells also inhibited growth of xenogeneic mouse tumor (EL-4) and human tumor (Raji) cells (Table 3). Because the PE cells were obtained from stimulated (oil-injected) animals, it was important to determine if rat PE cells obtained by other means also possessed growth inhibitory activity. Peritoneal exudate cells induced with beef heart infusion broth or normal resident PE cells were compared with oil-induced cells for their ability to inhibit tumor growth. Adherent rat PE cells (1-hr monolayers) from all sources significantly inhibited tumor growth (Table 4) at one or more EC : TC ratios.

100

WEINBERG,

FISHMAN,

AND

TABLE Nonspecific

Tumor

Growth Cells

Inhibition by W/Fu

cells

VEIT

3

of (C.%NT)D, EL4, and Peritoneal Exudate Cells Percentage

growth

inhibitiona

24 hr (C58NT)D EL4 Raji

90.3 80.3 32.3

Tumor

after

48 hr

(f2.0) (f3.3) (f6.7)

0 One-hour monolayers of 2 X lo6 oil-induced W/Fu with 2 X lo6 viable tumor cells (1O:l effector-to-tumor-cell percentage growth inhibitlon f the range.

Raji

98.1 93.8 72.3 peritoneal ratio);

(i 0.3) (5 1.6) (113.6)

exudate cells were cocultured the values are expressed as

Growth inhibitory activity of PE Ynacrophage subpopulations. Subpopulations of rat PE cells isolated by density gradient centrifugation were cultured as monolayers and assayed for their ability to inhibit the growth of syngeneic (C58NT)D tumor cells. Preliminary experiments showed that when I-hr monolayer cultures of macrophages were assayed, subpopulations A, B, C, and D inhibited tumor cell growth by more than 90% at EC : TC ratios of 5 : 1 and 2.5 : 1. Since the inhibitory activity of rat PE cells was reduced by preincubating the monolayer-s for 24 hr before adding tumor cells, 24-hr macrophage subpopulations were examined to accentuate any differences in activity among the subpopulations (Table 5). At the lower EC: TC ratios, bands A and B were highly cytostatic, whereas band C had little activity and band D clearly augmented tumor growth. Rabbit PE macrophages were also capable of inhibiting the growth of the xenogeneic (C58NT)D tumor cells. To determine whether this growth-inhibitory activity observed with rabbit macrophages was present among the various density gradient-separated subpopulations, experiments similar to those described for rat PE cells were conducted. In contrast to the experiments with the rat PE cells, it was not necessary to use 24-hr monolayers of rabbit macrophage subpopulations to demonstrate functional heterogeneity. Table 6 gives the results of incubatin, u xenogeneic (CSSNT) D tumor cells with 1-hr monolayers of rabbit PE subpopulations. The light-density macrophages in band A were most active in terms of growth inhibition at both 24 and 48 hr when TABLE Inhibition

of (C58NT)D Tumor Cell Cells from Stimulated

Growth by W/Fu and Nonstimulated

Rat

Peritoneal Rats

Percentage growth inhibition various EC : TC ratios

Stimulant

1O:l Oil BHIBb Nonec

4

87.2 f 94.9 f 93.4 f

Q Growth inhibition at 48 hr f b Beef heart infusion broth. c Peritoneal cells removed from

5:1 2.6 1.0 4.0

range normal

43.4 f 94.4 f 92.9 f of duplicate unstimulated

at

2S:l 0.5 0.5 1.6

2.6 f 91.8 f 94.4 f

cultures. rats.

Exudate

1.3:1 0.6 1.1 0.5

4.1 l 37.2 f 44.4 f

5.1 6.6 0.5

MACROPHAGE-TUMOR

CELL

TABLE Inhibition

of (C58NT)D Tumor of W/Fu Rat Peritoneal

Band

5

Cell Growth by 24-Hour Exudate Subpopulation

Percentage

growth

inhibition

(c Growth cultures f

ND 71.4 f 7.2 75.0 i 3.6 53.6 f 17.9

inhibition the range.

was determined

Monolayers Cells

at various

EC:TC

ratiosD

1.3:1

2.5:1 A B C D

101

INTERACTIONS

at 24 hr. The

1:l.S

78.6 75.0 53.6 -35.7

f f f f

8.0 10.7 10.7 0.0

values

given

50.0 57.1 7.1 -50.0 represent

f f f f

the mean

0.0 7.1 35.8 7.1 of duplicate

tested at three different EC: TC ratios. Cells in bands B, C, and D, respectively, produced decreasing levels of growth inhibition that appeared to be coordinate with increasing cell density. Significant growth-inhibitory activity in these cell populations was observed only at high EC: TC ratios and could not be detected at a ratio of 1 : 1. In this experiment, only minimal tumor growth augmentation was observed with band D cells and this occurred only in the 24-hr cultures. DISCUSSION Macrophages function as cytotoxic effector cells against tumors, and their activity can be distinguished from that of lymphocytes following separation of these two populations. To equate the activity of adherent peritoneal exudate cells with macrophage activity can be misleading, since adherent lymphocytes have been identified as effector cells in several model systems (17-19). Our data, obtained with a 4-hr 51Cr release procedure, show that immune PE cells from rats injected with syngeneic (C58NT)D tumor cells were highly cytotoxic against these targets. After separation of macrophages from lymphocytes by adherence and especially by density gradient centrifugation, it was evident that those populations which contained relatively TABLE Inhibition

Time

6

of (C58NT)D Tumor Cell Growth by l-Hour Monolayers of Rabbit Peritoneal Exudate Subpopulation Cells

Band

Percentage

growth

inhibition

.5:1 24 hr

A B C D

75.6 34.1 4.9 -22.0

48 hr

A B C 1)

95.0 82.6 52.9 32.2

u Values

given

represent

the mean

of duplicate

at various 2.5: 1

EC:TC

ratiosa 1:l

f 0.0 f 2.5 f 2.4 f 0.0

48.8 7.3 9.8 -26.8

f z!z f f

7.3 4.9 17.1 4.8

19.5 14.6 -3.7 -4.9

f f f f

2.5 17.0 5.0 2.5

f f f f

87.6 33.1 12.4 -16.6

f zk f f

4.1 0.8 3.3 7.5

54.5 -9.9 -3.3 -3.3

f f f i

0.9 0.8 0.8 0.8

1.7 4.2 2.5 5.0 cultures

f

the range.

102

WEINBERG,

FISHMAN,

AND

VEIT

few or no lymphocytes could not lyse the target cells in a 4-hr 51Cr release assay. Cytotoxic activity was detected only in those cell populations which did contain lymphocytes such as band E or nonadherent cells obtained by nylon fiber or plastic adherence treatments. Others have reported that macrophages play little or no role in the lysis of cultured tumor cells (20, 21), Although a longer incubation period appears to be required to demonstrate effector cell activity by macrophages, the high spontaneous release of 51Cr label from the (C58NT) D cells after 16 to 24 hr of incubation discouraged us from extending the 51Cr release assays. A more direct method for assaying macrophage activity against tumor cells was to measure tumor growth inhibition, not by the incorporation of [‘HI TdR, which has serious pitfalls (22)) but by counting viable tumor cells. This procedure was suitable in our tumor-effector cell model, since the macrophages were maintained as monolayers and the tumor cells were grown in suspension. The inhibitory activity of the PE cells that was detected by this procedure was not associated with the mineral oil used to induce the peritoneal exudate, since exudates induced by beef heart infusion or obtained as washings from nonstimulated animals were also capable of inhibiting tumor growth. The data show that macrophages obtained from the nonstimulated rats were more growth inhibitory than the PE cells from rats pretreated with mineral oil or beef heart infusion broth. Increased growth inhibitory activity noted with nonstimulated macrophages could not be accounted for by any appreciable increase in the number of light-density cells (data not presented). It is possible, however, that oil may effect in some still-unexplained way the capacity of macrophages to inhibit tumor growth. Our results appear to differ from those reported by Evans et al. (5)) who showed that normal mouse PE cells were unable to inhibit tumor growth. Data reported by Keller (23, 24), h owever, showed that normal DA rat peritoneal exudate cells elicited with proteose-peptone were effective in destroying syngeneic tumor cells in vitro. Reed and Lucas (25) reported that nonstimulated normal peritoneal macrophages from W/Fu rats were cytotoxic to a variety of tumor cell lines in vitro. The above studies were carried out with whole peritoneal exudate populations and did not take into account functional heterogeneity of antitumor activity among peritoneal macrophages, as has been reported by Nathan et al. (14) and more recently by Lee and Berry (26). In contrast, however, to the findings of Nathan et ad. (27), who suggested that subpopulations of cells with lymphocyte characteristics were active in tumor cell killing, our findings indicate that subpopulations having the greatest tumoricidal activity were devoid of lymphocytes. Results from our studies clearly show that light-density macrophages separated by BSA density gradient centrifugation inhibited tumor growth, whereas macrophages of heavier density were weakly inhibitory and frequently augmented tumor growth. It is reasonable to postulate that the relative proportions of light- and heavy-density macrophages present in a given peritoneal exudate may determine the overall activity expressed by the whole population. Therefore, it may be that the whole population of peritoneal exudate cells may appear to be inactive and yet contain a subpopulation of macrophages that is highly active. Au,gmentation of tumor growth by the heavy-density subpopulation of macrophages was not observed as frequently as was the inhibitory activity of the lightdensity subpopulation. The former cell class showed some cytotoxic activity, but only at higher effector-target ratios ( 10 : 1 or greater). The enhancement of tumor

MACROPHAGE-TUMOR

CELL

INTERACTIONS

103

growth might be due to nutritional factors released from certain macrophages acting as feeder cells. It is also possible that these subpopulations were highly pinocytotic and ingested toxic products from the medium, thereby facilitating tumor cell growth. Another explanation may be that heavy-density macrophages were selectively inhibited by tumor factors which have been shown to inhibit macrophage migration (28). The mechanism by which macrophages express tumor growth inhibition is not fully understood. Tumor growth inhibition is seen within 24 to 48 hr after incubation with macrophages or macrophage subpopulations, yet preliminary observations have indicated that the period of contact required between the effector cells and targets may only be a few hours. The mechanism by which macrophages restrict tumor growth appears to require close contact between macrophages and target cells. This relationship was suggested by preliminary experiments in which target cells were cultured directly over the macrophage monolayer or in areas of the plate not covered by the monolayer. Microscopic examination of such plates after 24 to 48 hr of incubation indicated that tumor growth was inhibited only in areas where target cells were in direct contact with the macrophage monolayer. Our findings do not exclude the role of growth inhibitory factors released from macrophages, as described by Piper and McIvor (29). A hypothesis may be formulated which states that macrophages rather than lymphocytes are responsible for immunological surveillance against tumor cells. Macrophages which can selectively act on neoplastic cells are present in most if not all animal species and their activity, demonstrated in citro, appears to be restricted to a light-density subpopulation of macrophages. Also present among the peritoneal exudate cells from normal animals is a subpopulation of heavy-density macrophages that can augment tumor growth. The ability to express macrophage effector cell activity in vitro and possibly in viva would thus be dependent on the relative proportions of these two subpopulations. According to this hypothesis an effective immunotherapy procedure (BCG or C. puruu~z) would have to enhance the activity of the light-density macrophages or decrease the activity of the heavier subpopulation of macrophages-or do both simultaneously. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

Walker, W. S., Nature (New Biol.) 229, 211, 1971. 26, 1025, 1974. Walker, W. S., Immunology Rice, S. G., and Fishman, M., Cell. Zmmulzol. 11, 130, 1974. Weinberg, D. S., Rice, S. G., and Fishman, M., Fed. Proc. 33, 632, 1974. Evans, R., and Alexander, P., Immunology 23,615, 1972. den Otter, W., Evans, R., and Alexander, P., Transplantation 14, 220, 1972. Evans, R., and Alexander, P., Proc. Sot. Exp. Biot. Med. 143, 256, 1973. Zeigler, F. G., Lohmann-Matthes, M., and Fischer, H., Int. Arch. Allergy Appl. Z~w~~rmol. 48, 182, 1975. Cleveland, R., Meltzer, M. S., and Zbar, B., J. Nat. Cawer Inst. 52, 1886, 1974. Gallily, R., and Eliahu, H., Cell. Immuuol. 25, 245, 1976. Hibbs, J. A., Transplawtatiolz 19, 81, 1975. Holtermann, 0. A., Klein, E., and Casale, G. P., Cell. Imnzunol. 9, 339, 1973. Piessens, W. F., Churchill, W. H., and David, J. R., J. Zmmunol. 114, 293, 1975. Nathan, C. F., Hill, W. M., and Terry, W. D., Nature (London) 260, 146, 1976. Ortiz de Landazuri, M., and Herberman, R. B., J. Nut. Cancer Inst. 49, 147, 1972. Julius, M. H., Simpson, E., and Herzenberg, L. A., Eur. J. Immunol. 3, 645, 1973.

104

WEINBERG,

FISHMAN,

AND

VEIT

17. Herberman, R. B., Advnn. Caslcer Res. 19, 207, 1974. 18. Tucker, D., Dennert, G., and Lennox, E. S., Immunology 113, 1302, 1974. 19. Djeu, J. Y., Glaser, M., Kirchner, H., Huang, K. Y., and Herberman, R. B., Cell. Zmmunol. 12, 164, 1974. 20. Pollack, S. B., Nelson, K., and Grausz, J. D., 1. Zmmamol. 116, 944, 1976. 21. Sanderson, C. J., and Taylor, G. A., Immunology 30, 117, 1976. 22. Ortiz, H. G., Niethammer, D., Lemke, H., Ffad, H. D., and Huget, R., Cell. Zmmunol. 16, 379, 1975. 23. Keller, R., I. Exp. Med. 138, 645, 1973. 24. Keller, R., Zmmmt.ology 27, 285, 1974. 25. Reed, W. P., and Lucas, 2. J., J. Zmmunol. 115,395, 1975. 26. Lee, K. C., and Berry, D., J. Zmmunol. 118, 1530, 1977. 27. Nathan, C. F., Asofsky, R., and Terry, W. D., J. Immunol. 118, 1612, 1977. 28. Pike, M. C., and Snyderman, R., J. Zmmultol. 117, 1243, 1976. 29. Piper, C. E., and McIvor, K. L., Cell. Immunol. 17,423, 1975.