Znt. J . Cancer: 18, 168-175 (1976)

GENETIC ROLE OF RAT MACROPHAGE CYTOTOXICITY AGAINST TUMOR Glenn A. MILLERand Joseph D. FELDMAN Department of Immunopathology, Scripps Clinic and Research Foundation, La Jolla, California 92037, USA Macrophages fvom the Lewis ( L e ) rat strain are significantly more cytotoxic to a Moloney sarcoma tumor, both in vivo and in vitro, than are macrophages from the Brown Norway ( B N ) strain. Activity of macrophages from (Le x B N ) F , rats that are histocompatible with the Moloney sarcoma tumor is directed toward tumor andlor virus-associated antigens and is expressed as a dominant genetic trait. Experiments with backcross rats suggest that the genetic factors are unrelated to the major histocompatibility locus (AgB) of the rats. B N macrophages, although not active against tumor andlor viral antigens, can become cytotoxic to cells displaying Le alloantigens. Macrophages contribute in several ways to host resistance against tumors. They are plentiful at the site of active regression (Corer, 1956; Evans, 1972; Gauci and Alexander, 1975; Eccles and Alexander, 1974; Russell et al., 1976), and, when harvested from tumor-bearing animals, can inhibit tumor growth or destroy the neoplasm in vivo (Bennett, 1965; Tsoi and Weiser, 1968). In vitro, macrophages are also capable of causing tumor inhibition or destruction (Keller, 1974; den Otter et a]., 1972; Lohmann-Matthes et al., 1972; Granger and Weiser, 1964). Cytotoxic tumor cell killing is achieved by macrophages sensitized with specific antibody, with soluble products of sensitized lymphocytes (Evans and Alexander, 1972; Evans et al., 1972) and by activation as a result of the action of adjuvants (Zbar et al., 1971 ; Hibbs et al., 1972a) and intracellular ingestion of bacteria (Hibbs et al., 1972b). I n this paper we emphasize the role of genetics in determining the efficacy of macrophage cytotoxicity against tumor. Macrophages from the Lewis (Le) rat appear to be significantly more cytotoxic to a Moloney sarcoma tumor than are macrophages from Brown Norway (BN) rats. Furthermore, the use of macrophages from (Le x BN)F,, hereafter referred to as LBN, and backcross animals, indicates that a genetic factor, unlinked to the major histocompatibility locus (AgB), determines macrophage cytotoxicit y. MATERIAL A N D METHODS

Animals Male BN (AgB3) and Le (AgBl) rats averaging 200 g were obtained from the animal care facility of

the Scripps Clinic and Research Foundation or from Simonsen Laboratories, Gilroy, Calif., USA. LBN and backcross rats were bred at the Scripps Clinic. Tumor cells A BN Moloney sarcoma tumor cell line (MST-I) was derived from a tumor that appeared in a BN rat inoculated with Moloney sarcoma virus from BALB/c spleen (Jones et al., 1974). Tumor cells were maintained as cell suspensions in spinner minimal essential medium (MEM) supplemented with 10% fetal -bovine serum, antibiotics and glutamine, or as monolayers in complete medium. The cell line produced sarcomas when injected subcutaneously into rats.

A Le Moloney sarcoma cell line (LM2S) was derived from a tumor that appeared in a Le neonate inoculated with Moloney sarcoma virus. This tumor line was initially a non-producer of virus but after superinfection with murine leukemia virus (MuLV) became a producer. These cells were maintained as monolayers in complete medium. Macrophages Macrophages were harvested from the peritoneal cavities of normal or tumor-bearing rats by lavage with 50ml of MEM, 3 days after injection with 7.0 ml of 10% proteose peptone (Difco, Detroit, Mich., USA) in saline. Cells were pooled from 4-10 donor rats, washed three times in complete medium and adjusted to the desired concentration. Differential cell counts showed that 80-85% of the cells were macrophages by morphologic and phagocytic criteria. Peritoneal exudate macrophages (PEM) were separated from non-adherent populations by adherence to plastic or collagen-coated surfaces. For plastic adherence 2 x lo7 peritoneal exudate cells (PEC) were incubated at 37" C for 2 h in 10 ml of complete medium in tissue culture processed Petri dishes 10 cm in diameter (Falcon, Oxnard, Calif., USA). Non-adherent cells were vigorously washed off and the dishes incubated for an additional hour. Adherent cells were chilled on ice for 1 h and Received: May 5 , 1976.

169

MACROPHAGE CYTOTOXICITY AGAINST TUMOR

removed with a rubber policeman. Cellular debris was removed by centrifugation through fetal bovine serum. Cell viability at this stage as determined by trypan blue exclusion was about 50%. In some studies the number of viable cells was increased to 7 5 4 5 % by centrifugation for 10min at 400xg through Ficoll-Hypaque (Boyum, 1968). Morphologically and functionally such preparations were 96-98 % macrophages. In the collagen adherence procedure 3 m l of rat tail collagen (Ehrmann and Gey, 1956) were poured into Petri dishes 10 cm in diameter and gelled by exposure to 28% ammonium hydroxide for 1 h. Gels were dialyzed against distilled water overnight and saturated with complete medium on the day of use. Fifty million PEC were incubated in 10ml of medium for 2 h at 37" C in 95% air and 5 % CO,. After non-adhering cells had been rinsed off the remaining cells were released by a 10-min incubation with 5 ml of 0.1% collagenase in MEM (Calbiochem, San Diego, Calif., USA). The cells were then washed three times with complete medium and counted. Such preparations routinely consisted of 96 to 98% macrophages and the viability was better than 93 %.

il

I

/I I

4

I

a

I

12

I

I

16 20

I

I

1

24

2a

32

Day Post Tumor Inoculation FIGURE 2

Macrophage-mediated inhibition of MST-1 growth in vivo. Le, B N and LBN donor rats were inoculated with 2 x lo7 MST-1 subcutaneously and at intervals thereafter macrophages were harvested from groups of 7-10 rats and tested for in vivo anti-tumor activity. lo7 MST-1 were admixed with either lo7 immune or nonimmune PEM and cell mixtures injected SC into each of 6 recipient rats histocompatiblewith the PEM. Percentage inhibition of tumor growth was determined 7 days later by comparing tumor growth in groups with immune PEM versus groups with non-immune PEM. Curves are plotted to show PEM activity against tumor when PEM are harvested at various times after MST-1 inoculation.

- 25

-E E

w

* 20 +I L

Collagen-prepared macrophages were used in all

c Q1

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15

in vivo studies and plastic adherence prepared cells in in vitro studies.

0 L 0

In vivo macrophage assay

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as

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5

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,

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,

,

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,

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4 8 12 16 20 24 Day Post Tumor Inoculation FIGURE 1 MST-1 growth in BN, Le and LBN rats. Rats were inoculated subcutaneously with 2 x lo' MST-1 in 0.2 ml MEM. Mean tumor diameter is plotted against time.

Five rats were used in each group. Bars indicate standard error of the mean.

Immune macrophages were obtained from 7-10 donor rats at varying intervals after subcutaneous inoculation of tumor. Rats received either 2x10' MST-1 cells o r 2x106 ML2S cells. These doses resulted in comparable tumor growth. Non-immune control macrophages were collected from unmanipulated donor rats or from rats previously treated with Freund's complete adjuvant (FCA). For assay, a mixture of lo7pooled macrophages and lo7MST-I cells or 2 x log pooled macrophages and 2x1OE LM2S cells was inoculated subcutaneously into each of six recipients histocompatible with the macrophages. Seven days later tumors were excised and weighed. Percentage inhibition of tumor growth was calculated as follows:

170

MILLER A N D FELDMAN

Mean tumor weight

Mean tumor weight in group with non-immune PEM

x 100%.

Tumor cells in this system were sensitive to the presence of la6I-IUdR. If incorporation of radioactivity did not exceed 0.02pCi per lo5 cells, the assay was operational. With such incorporation T

In vitro macrophage assay

The assay was based on release of radiolabelled target cells from the surface of a tissue culture well and was modified according to the technique of Seeger and co-workers (Seeger ef a/., 1974). Subconfluent cultures of tumor cells growing in 25 cma tissue culture flasks (10' cells/ml in a volume of 15 ml) were pulsed for 24 to 48 h with 7.5 pCi of *251-IUdR(0.1,uCi/pl, specific activity 100 pCi/pg). Labelled target cell monolayers were rinsed with medium and cells were removed by incubation with 0.25 trypsin-EDTA. Cells were washed three times with complete medium containing 10% FCS and 20mM HEPES buffer and adjusted to 10' cells/ml. Ninety-eight per cent of the radioactivity in such cells could be precipitated with 5% cold trichloroacetic acid. Next, 1.5-2.0 ml of medium were pipetted into the wells of Linbro 16-mm TC trays (FB-16-24-TC, Linbro Chemical Company, New Haven, Conn., USA) and lo5target cells in 0.1 rnl were added. Cells were allowed to adhere overnight. Five million PEM or PEC were added to a final volume of 2.1 ml. At the end of the assay target cells which had become detached were rinsed from each well. One ml of 1.5% sodium lauryl sulfate in distilled water was added and the plates were incubated for 30min at room temperature. The sodium lauryl sulfate solution was then transferred to tubes and counted in an automatic gamma scintillation spectrometer. Percentage tumor-cell kill was calculated as follows:

Percent age tumor kill

=

1-

cpm in well with immune PEM or PEC cpm in well with non-immune

2 52 015-

E

-E

10O.

- -o W i t h S i l i c a

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Without Silica

PI c P . I

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i

b 0

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25-

c

PI m

=

2015-

105-

x 100%.

This method of calculation negates the contribution of non-immune cells to the tumor kill. In some experiments in which the cytotoxicity of non-immune macrophages was tested, the formuIa was : cpm in well with non-immune PEM Percentage or PEC tumor kill = x 100%. 1- cpm in well with medium alone

Lb-dk-0 4 8 12

-4

16 20 Day Post Tumor Inoculation

FIGURE 3

Influence of silica on tumor growth in rats. BN(A) and Le(B) rats were infused intravenously with 5 mg silica at the times indicated by the arrows. On day 0 silicatreated and untreated rats were inoculated SC with 2 x lo7 MST-1 and measurements of tumor size were made at intervals thereafter. Mean tumor diameters are plotted against time. Five rats were used in each group.

171

MACROPHAGE CYTOTOXICITY AGAINST TUMOR TABLE I PEC ACTIVITY AGAINST MST-I GROWTH I N VIVO: EFFECT O F FCA I

Interval between FCA and PEC harvest (days)

Tumor mass f S E (S)

Ratio of tumor mass/body mass mg/g

0 10 0 22 0 10 0 22 0 10 0 22

2.1 1 0 . 2 1.21 0 . 2 2.010.3 1.3f0.1 1 S10.3 I .5 +O.l 5.450.6 5.6k0.8 0.8+0.1 0. I g0.04 1.1 40.2 0.710.3

11.24 1.1 6.5 5 0 . 9 7.94Z1.2 6.5 A0.4 4.8 k0.72 5.1 5 0 . 3 21.84~2.5 24.7h2.3 4.3 *0.7 0.2A0.1 4 . 7 M .5 2.1 *0.9

Lewis

BN

LBN

-

Based o n ratio of tumor mass/body mass. 1 Based on tumor mass. compared weights of tumors 7 days after inoculation of MST-I.

plating efficiency ranged from 75% to 95% and remained so for at least 72 h. No more tumor cells were found in the wells after 72 h of incubation than were initially seeded, indicating that tumor cells did not proliferate under these labelling conditions, presumably because of internal irradiation. Also, all input radioactivity could be accounted for by summing the counts in the various supernatants and washed wells. Histocompatibility typing of backcross rats

Percentage inhibition of growth

02

0' 35 0 35 0 2 0 0 0 85 0 36

42

0 18 0

0 0 0 0 95 0 55

- a Compared 10 days after inoculation of

MST-I. Other data

observed when macrophages are taken from Le and LBN donors inoculated as early as 4 days previously, but peak inhibition is seen with macrophages removed from donors 8 days or more after MST-1 inoculation. Non-immune proteose peptonestimulated PEM at ratios up to 5 PEM: 1 tumor cell do not influence the growth of tumors (data not shown). In addition, non-adherent cells (lymphocytes) that comprise the 2-4 % non-macrophage contamination of PEM fail to influence tumor growth when tested at those levels.

Backcross rats were typed for their major histocompatibility antigens (AgB1 or AgB3) by means of a binding assay with 1251-labelledLe anti-BN and BN anti-Le IgG. The procedure has been detailed (Jones and Feldman, 1975).

TABLE I1 CYTOTOXIC ACTIVITY O F IMMUNE PEM AND PEC ON MST-1 I N VITRO %tumor kills +SS E f f ~ t ~ ~Incubation ~ l l (h)

PEM'

PEM'

11f2 3047 5&4 453 743 20&6

13f2 52&3 0 8&5 20f3 4555

PEC'

RESULTS

In vivo studies with MST-I

Lewis

Tumor growth. Growth of MST-1 is shown in Figure 1. The largest tumors grow in the BN; the smallest in Le, which are allogeneic and reject at 8 days. By day 24, 90% of Le rats are free of tumor whereas 100% of BN rats still have tumors. Tumor growth is intermediate in LBN which are histocompatible with the tumor cells. LBN rats also reject their tumors and are free of tumor by day 30. Data in the Figure are representative of three separate experiments.

BN

PEMactivity. The data in Figure 2 show that there is significantly greater inhibition of MST-1 growth with Le and LBN macrophages, taken 8 days after inoculation of PEM donors, than is observed with BN PEM taken at any time up to 32 days after inoculation. Significant inhibiton of MST-1 is

LBN

24 48 24 48 24 48

352 80&4 1544 6&4 16&10 93&3

cprn in well with immune PEM or PEC x 100%. cpm in well with non-immune PEM or PEC Effector and t u n o r targets were incubated at ratios of 5O:l. Immune rats had teen inoculated with 2 x 10' MST-I SC 10 days earlier. a PEC were removed from groups of 8-10 non-immune or immune rats and pooled, then PEM were prepared by adherence to plastic. Each figure represents the mean percentage tumor kill and standard error for PEM tested in triplicate. - * Cells were prepared from 8-10 nonimmune or immune rats by adherence t o plastic followed by centrifugation through Ficoll-Hypaque. Each figure represents the mean percentage tumor kill and standard error for PEM tested in tripli6 Each figure represents the mean percentage tumor kill cate. and standard error of triplicate determinations of two experiments using four non-immune and immune rats in each study. When compared after 48 h of incubation Le and LBN cytotoxic activity was greater than that of the BN at 0.005