Augmentation of tumoricidal activity of human monocytes and macrophages by lymphokines

Int. J . Cancer: 25, 691-699 (1980)

AUGMENTATION OF TUMORICIDAL ACTIVITY OF HUMAN MONOCYTES AND MACROPHAGES BY LYMPHOKINES

Alberto MANTOVANI’, Jack H. DEAN2, Thomas R. JERRELLS’ and Ronald B. HERBERMAN3 Istituto di Ricerche Farmacologiche “Mario Negri”, Via Eritrea 62, Milan, Italy; Department of Immuno-

logy, Litton Bionetics, Inc., Kensington, Md. 20795; and Laboratory of Immunodiagnosis, National Cancer Institute, National Institutes of Health, Bethesda, Md . 20205, USA.

Monocytes were separated from peripheral blood mononuclear cells of normal human donors by adherence on plastic conditioned by cell lines (microexudate- coated plastic) and harvested by exposure to ethylene diamine tetra-acetic acid. Cytolytic activity was tested by incubating effector cells for 48 h with the murine SV4O-transformed TU5 kidney line or the human lung cancer-derived C a b line prelabelled with tritiated thymidine. Lymphokine-containing supernatants were obtained from in vitrocultures of lymphoid cells with phytohemagglutinin (PHA) I, purified protein derivative (PPD), or with Corynebacteriurn parvum strains CN6134 or CN5888. The monocytes had significant levels of spontaneous cytotoxicity and exposure to lymphokine supernatants markedly enhanced their tumoricidal activity. Augmentation of monocyte-mediated cytotoxicity required a minimal exposure to lymphokine supernatants for 4 h and was maximal after 24 h of preincubation. Treatment of the effector cells with anti-human T-cell serum and complement did not affect either their spontaneous or their lymphokine-stimulated cytotoxicity, whereas silica impaired both reac- tivities. Supernatants of cultures with PHA, PPD and C. parvurnCN6I 34 had significant levels of interferon (IF). Since partially purified human fibroblast or leukocyte IF was able to stimulate monocyte-mediated cytotoxicity, the IF in these supernatants could play some role in the stimulation of the monocytes. However, C. parvum CN5888 supernatants, which had little IF, enhanced monocyte cytotoxicity as effectively as the C. parvum CN6 I34 supernatants, strongly suggesting that lymphocyte mediators other than IF can augment the tumoricidal activity of these effector cells. Mature macrophages obtained by in vitro cultivation of monocytes for 4-7 days retained natural cytolytic activity and showed enhanced cytotoxicity in the presence of lymphokines. However, more prolonged in vitro cultivation (> 10 days) resulted in cultures of epithelioid and multinucleated cells which had little natural cytotoxicity and were not responsive to lymphokines.

Rodent monocytes and macrophages can express significant levels of tumoricidal activity in vitro either spontaneously (Keller, 1978; Tagliabue et al., 1979) or as a consequence of exposure to chemical or biological stimuli (reviewed by Hibbs, 1976; Keller, 1976; Evans and Alexander, 1976). Cells of the monocyte-macrophage series are a major compo- nent of the lymphoreticular infiltrate of experimental and human tumors (Evans, 1972; Pross and Ker- bel, 1976; Wood and Gollahon, 1977; Carr, 1977; Mantovani et al., 1979b) and several observations are consistent with the hypothesis that monocytes and macrophages play a role in regulation of tumor growth and metastasis (Birbeck and Carter, 1972; Russell and Mclntosh, 1977; Eccles and Alexander, 1974; Wood and Gillespie, 1975; Zarling and Tevethia, 1973; Mantovani, 1978).

In contrast to the extensive studies on macrophages in experimental animals, there is only limited information on the interaction of human rnonocytes and macrophages with tumor cells. We have recently observed that human peripheral blood monocytes have appreciable levels of natural cytolytic and cytostatic activity on tumor cell lines and that this cytotoxicity is mainly directed against transformed target cells (Mantovani et al., 1979a,b,c; 1980). Natural cytotoxicity on tumor cells is not restricted to circulating monocytes, as human macrophages obtained by in vitro cultivation of monocytes, from peritoneal exudates or from early post- partum milk samples all had significant tumoricidal activity (Mantovani et al., 1980). Lymphokines have been shown to regulate a number of functions of cells of the monocyte-macrophage lineage and, in rodents, to activate their cytotoxic capacity against tumor cells (David, 1975; Piessens et al., 1975; Chur- chill et al., 1975; Fidler, 1975; Fidler et al., 1976; Christie and Bomford, 1975; Ruco and Meltzer, 1977). The present investigation was designed to elucidate whether products of stimulated human lymphoid cells could augment the tumoricidal capacity of human monocytes and macrophages and to obtain evidence on the nature of the lymphokines capable of enhancing human monocyte cytotoxicity.

MATERIAL AND METHODS

Mononuclear cells Heparinized venous blood (100-200 ml) was ob-

tained from normal healthy adult volunteers. Blood was diluted 1:4 with Hanks’ balanced salt solution (HBSS) and mononuclear cells were separated by centrifugation at 400 g at room temperature for 30 min on Ficoll-Hypaque (LSM, Litton Bionetics, Kensington, Md.). Mononuclear cells were washed twice with HBSS and resus ended at a concentration

plemented with 20 % aseptically collected fetal bovine serum (FBS, Microbiological Associates, Bethesda, Md.). In a few experiments mononuclear cells were separated by the same procedure from leukocyte preparations obtained from healthy adult donors undergoing plateletphoresis.

of approximately 2 X 10 P . /ml in RPMI 1640 sup-

‘Abbreviations: HBSS, Hanks’ balanced salt solution; FBS, fetal bovine serum; EDTA, ethylene diamine tetraacetic acid; PHA, phytohemagglutinin; PPD, purified protein derivative; C. parvum, Corynebacterium parvum; IF, interferon; C , complement; A:T, attacker to target cell; SDS, sodium dodecyl sulfate; NK, natural killer.

Received: March 28, 1980.

692 MANTOVANI ET AL

Monocytes Adherent cells were obtained from mononuclear

cells by adherence on microexudate-coated plastic and exposure to ethylene diamine tetraacetic acid (EDTA) by minor modifications of the previously described procedure (Mantovani et al., 1979~). Chang or mKSA-TU5 (TU5) cells were grown to confluence in 75 cm2 tissue culture flasks (3024, Fal- con, Oxnard, Calif.) and detached by incubation with 1 mM EDTA for 10-20 min at 37°C. These microexudate-coated flasks were thoroughly washed with HBSS and stored at 4°C. Mononuclear cell suspensions (10-20 ml) were incubated in these conditioned flasks for 45 min at 37°C. Non-adherent and loosely adherent cells were thoroughly washed off and the adherent cells were recovered by incubating the flasks with 10 ml of 1 mM EDTA (Mi- crobiological Associates) for 5-10 min followed by vigorous shaking. The adherent cells were washed with 50 ml HBSS and resuspended in RPMI 1640 medium supplemented with 10% FBS. In this series of experiments, the yield of recovered adherent cells was 7.6 t 2.9% (mean k SD) of the original cell suspensions and viability exceeded 80 5%. More than 90% of the adherent cells belonged to the monocyte-macrophage lineage, as assessed by morphology, non-specific esterase staining, latex phagocytosis, and binding and phagocytosis of antibody-coated sheep erythrocytes.

In vitro matured macrophages Mature macrophages were obtained by in vitro

cultivation of monocytes essentially as previously described (Mantovani et al., 1977; Anderson and Re- mington, 1974; Anderson et al., 1976). Monocytes (3x1O5 in 0.2 ml of RPMI 1640 or Neuman and Tytell medium supplemented with 50 % autologous or pooled AB serum) were cultivated for 5-20 days in flat-bottomed 6.4 mm culture wells (3596, Costar, Cambridge, Mass.). The wells were then thoroughly washed with HBSS and the number of adherent cells was estimated with the aid of an eyegrid. More than 95 % of the adherent cells were macrophages as assessed by morphology, avid uptake of neutral red, phagocytosis of latex particles, and binding and phagocytosis of antibody-coated erythrocytes (Man- tovani et al., 1977).

Lymphokine supernatants Firmly adherent monocytes were removed from

mononuclear cell suspensions as described above. Cells not adhering to plastic were centrifuged at 200 g for 10 min and resuspended in RPMI 1640 with 10 % FBS and 50 pg/ml gentamicin. These cell preparations were only partially depleted of monocytes since 2-4 % of the cells were esterase-positive, as compared to 9-20% in the original cell suspensions (Mantovani et al., 1979~).

These lymphoid cells (2-5 X 106/ml) were cultured in plastic tubes (2070, Falcon) in 7-10 ml RPMI 1640 medium with 10% FBS in the presence of 10 pg/ml phytohemagglutinin (PHA, HA17, Wellcome Research Laboratories, Beckenham, Kent, England) or 20 p d m l purified protein derivative (PPD) (Connaught Medical Research Labo-

ratories, Toronto, Canada). In experiments in which PPD was used as a stimulus, mononuclear cells were obtained from PPD-reactive donors, as assessed by skin testing. After 20 h at 37°C in air with 5 % CO,, cells were washed three times with 50 ml RPMI 1640 medium with 10% FBS and, after resuspension in the original volume, the lymphoid cells were further cultivated for 24 h (PHA) or 48 h (PPD). Control cultures consisted either of lymphoid cells cultivated alone or of cultures to which PHA and PPD were added immediately prior to washing after the first 20 h of incubation. Since no differences were ever detected between these two types of con- trols, only results with supernatants of lymphoid cells cultivated in medium alone will be presented. At the end of the incubation the supernatants were filtered through a 0.45 pm filter (Millipore Corpora- tion, Bedford, Mass.) and assayed immediately or stored at -20°C until used.

In a series of experiments, supernatants were generated by culturing lymphoid cells with either of two strains of anaerobic coryneforms, C. parvum strain CN6134 (lot CA732) or strain CN5888 (lot E2223, Wellcome Research Laboratories). Before use, 0.5 ml of the killed bacterial cell suspensions (7 mg/ml) were washed three times with 5 ml culture medium. Lymphoid cells (2-5 X 10h/ml) were cultivated in 10 mi RPMI 1640 medium supplemented with 10% FBS (or 10% human AB serum) and 50 pg/ml gentamicin in the presence of 140 pg/ml of these two anaerobic coryneforms. After 6 days of culture at 37°C in air with 5 % COz, the cells were centrifuged at 400 g for 10 min. The supernatants were filtered through a 0.45 pm filter (Millipore Corporation). Control supernatants were obtained from cultures of lymphoid cells or bacteria alone.

Treatment with silica and anti-T-cell serum Monocytes ( 2 X lo5 cells in 0.1 ml of RPMI 1640

medium with 10% FBS) were incubated for 1 h at 37°C in 96-well tissue-culture plates (3596, Costar) with 25 pg/well silica (Santocel58, average particle size 3 pm, Monsanto Co., St. Louis, Mo.), which had been sonicated immediately before use. Lym- phokine supernatant (0.1 ml) was then added to the wells and the monocytes were cultivated for 20 h at 37°C. The medium was removed and cytolytic activity was assessed by adding labelled target cells to the wells.

To deplete any contaminating T cells, monocyte preparations were treated with a rabbit anti-human T-cell serum, as previously described (Mantovani el al., 1979~). Briefly, the serum was prepared by in. jecting rabbits with cultured T cells from a norma donor and was absorbed with a B-cell line from tht same donor (Bonnard et al., 1979; Maca et al., sub mitted for publication). This reagent, in the pre sence of complement, has been shown to lyse virtu ally all cells forming rosettes with sheep erythrc cytes, to eliminate lympho-proliferative responses t mitogens, to abrogate natural killer cell activity, bi not to affect B cells or monocytes. Monocytes (8 lo6 in 1 ml RPMI 1640 medium) were incubated plastic tubes (2074, Falcon) with a 1:12 dilution anti-T-cell serum and a 1:12 dilution of rabbit COI

HUMAN MONOCYTE CYTOTOXICITY 693

plement (C) at 37°C for 45 min. At the end of the incubation the cells were washed with 50 ml of HBSS before use in the cytotoxicity assay.

Interferon Interferon (IF) in lymphokine supernatants was

measured in a virus plaque assay by Dr. R. Rafako (Biofluids, Inc., Rockville, Md.). Partially purified human fibroblast IF (lot 45-10-9) was obtained from HEM, Rockville, Md. Partially purified human leukocyte IF, induced by Sendai virus, was obtained from the NCI Interferon Working Group. Interferon from a human lymphoblastoid cell line (batch 4645/ 6) was a kind gift of Dr. A.J. Beale (Wellcome Re- search Laboratories).

Target cells The simian virus 40-transformed TU5 line (Kit et

af., 1969) was employed in the present study. Tumor cells were grown either in minimal essential medium or in RPMI 1640 supplemented with 10% FBS. Cells were prelabelled with [3H] thymidine by incubating non-confluent cultures in 75 cm2 tissue culture flasks (Falcon) for 24 h with 25 ml medium containing 0.5 pCi/ml of [3H] thymidine (6 Ci/mmole, Schward Mann, Orangeburg. N.Y.) . Tumor cells were exposed for 5 min to 5 ml trypsin-EDTA (GIBCO, Grand Island, N.Y.) , washed twice with 50 ml of medium, and finally resuspended in RPMI 1640 medium with 10% FBS at a concentration of lo5 cells/ml.

Cytolysis assay Target cells (104/sample) were incubated with

monocytes at 37°C in air with 5% C 0 2 for 48 h

(unless otherwise specified) in a final volume of 0.3 ml RPMI 1640 medium supplemented with 10% FBS and 50 pg/ml gentamicin in flat-bottomed 6.4 mm culture wells (3596, Costar). Unless otherwise stated, the attacker to target cell (A:T) ratio was 20:l. Tumor cell growth was checked daily under an inverted microscope. At the end of the incubation, 0.1 ml of the supernatant was mixed with 10 ml of Aquasol (New England Nuclear, Bos- ton, Mass.) and counted in a Packard 3375 liquid scintillation spectrometer. Total incorporated radioactivity was estimated from tumor cells incubated with 1 % sodium dodecyl sulfate (SDS) in wa- ter and averaged 7,830 cpm in this series of experiments. Percentage of isotope release was calculated as 100XA/B, where A was the isotope released in test samples and B was the SDS-releasable radioactivity. Spontaneous release of tumor cells incubated in the absence of effectors never exceeded 25 % over a 72-h incubation period and was usually below 20%. Specific lysis was determined by subtracting the spontaneous release.

To evaluate the augmentation of cytolysis, monocytes were preincubated with lymphokine supernatants for various times in 0.3 ml culture medium and the wells were then gently washed with 0.3 ml of medium prior to addition of the target cells. Alterna- tively, and more frequently, lymphokines were added to the wells with monocytes in a volume of 0.2 ml; target cells were then prepared (a procedure that took 30-60 min) and seeded in the wells in a volume of 0.1 ml culture medium. Lymphokine supernatants present throughout the 72-h assay did not affect the spontaneous release or the proliferative capacity (assessed by daily checking the cultures

TABLE I

EFFECT OF LYMPHOKINE SUPERNATANTS ON MONOCYTE-MEDIATED CYTOTOXICITY

Release of [?H]thymidine ' Percentage Percentage specific Final 1ys1s dilution cpm (+ SD) lysis Supernatant Monocytes

(A:T ratio)

None None - 820 (110) 13.3 -

Conditioned 1/ 3 845 ( 27) 13.8 - Medium I / 9 794 ( 31) 12.9 -

1 /27 841 (140) 13.8 - 1/ 3 864 35) 14.1 -

PHA supernatant

1/ 9 PPD-supernatant 1 /27

I/ 3

857 ( 3 i j 13.9 - 828 ( 78) 13.5 -

785 f1211 12.8 -

20: 1 I / 9 812 ( 38j 13.2 - 1 /27 778 ( 12) 12.7 - None

Conditioned - 1,954 ( 88)* 31.8 18.5 Medium I / 3 2.389 f 51)' 38.9 25.6

1/ 9 I ,818 (i7oj 29.6 16.3

1/ 3 5,094 (257)4 82.9 69.6 PHA supernatant 1 /27 1,641 (149) 26.7 13.4

1/ 9 4,610 (i98j4 75.0 61.7

1/ 3 3,840 ( 54)4 62.5 49.2 1/ 9 2,982 (178)4 48.5 35.2

PPD-supernatant 1 /27 3,111 (341)4 50.6 37.3

1 /27 2,175 (157) 35.4 22. I

' Total incorporated, SDS-releasable, radioactivity was 6,144 cpm. Supernatants were present throughout the cytotoxicity assay. - * p<O.OI versus spontaneous release. - p<O.O1 versus cytolytic activity of monocytes alone. - ' p<O.O1 versus conditioned medium.

694 MANTOVANI ET AL

TABLE 11

EFFECT OF HUMAN AND FETAL BOVINE SERUM ON MONOCYTE ACTIVATION BY LYMPHOKINE SUPERNATANTS

Supernatant Monocyres Serum' (A:T ratio)

- - FBS 20: 1 - 20: 1 Conditioned medium 20: 1 Lymphokine supernatant

20: 1 20: 1 Conditioned medium 20: 1 Lymphokine supernatant

Human serum - - -

Relenae ot ['H]thymidine cpm (+ CD)

364 ( 16) 958 ( IS)?

2,435 (121)2,' 1,205 ( 99)'.'

351 ( 34) 1,294 ( 57)* I ,480( 15)'.' 2,628( 46)2.4

Percentage lysis

8.8 23.5 29.4 59.6 8.6

31.7 36.2 64.3

Percentage specific

lysis

14.7 20.6 50.8

23. I 27.6 55.5

~ ~~~

' Production of supernatants and cytotoxicity tests were performed in RPMl 1640 medium containing 10% FBS o r human AB serum. Total incorporated, SDS-releasable, radioactivity was 4.097 cpm. Control or PHA-diluted supernatants were prcsent throughout the cytotoxicity assay at a 1/Y final dilution. - p<O.Ol VCTSUS spontaneous release of tumor cells alone. - ' p<O.OS versus cytolytic activity of monocytes in the absencc o f supernatants. - ' p<O.01 versus conditioned medium.

under an inverted microscope and by a postlabelling cytostasis assay) of tumor target cells in the absence of effector cells.

Statistical analysis Results are presented as mean S SD of 3-6 repli-

cates per experimental group and, unless otherwise specified, are representative of at least three experiments. Statistical significance was assessed by one- way analysis of variance.

RESULTS

Table I shows the results of a typical experiment in which supernatants from antigen (PPD)- or mitogen (PHA)-stimulated lymphoid cells were assayed for their effects on monocyte-mediated tumoricidal activity. In these experiments supernatants were present in the assay throughout the incubation period. As previously reported (Mantovani et al . , 1979b,a,c; 1980), human peripheral blood monocytes showed significant levels of spontaneous cytotoxicity. High concentrations (1/3) of control supernatants caused a moderate and significant increase in cytotoxicity against TUS tumor cells. Augmentation of cytotoxic- i ty by control supernatants was consistently observed in all experiments performed, including a limited series of experiments in which lymphoid cells were cultivated in the presence of 10 % AB or autologous human serum, but was only observed in the presence of high concentrations (%-!h) of the supernatants. Lymphokine supernatants from antigen-or mitogen-stimulated lymphocytes markedly increased the cytocidal efficacy of human monocytes, significantly above that induced by control supernatants. PHA-induced supernatants were somewhat more ef- fective than PPD-elicited lymphokines, in terms both of maximal absolute increase in cytolytic activity and of the minimal concentration of lymphokines required for a significant stimulatory effect. Only experiments with PHA-elicited lymphokines will be presented hereafter since the results obtained with PPD-supernatants were similar.

Previous studies with human and murine macrophages showed that natural cytolytic activity was also expressed in medium containing adult sera of

TABLE I11

ACTIVATION OF HUMAN MONOCYTES BY PREINCUBATION WITH LYMPHOKINE SUPERNATANTS

Percentage specific lysis ( 2 SD)

lymphokines for' Conditioned Lymphokinc medium supernatant

Monocytes exposed to

2 h 22.6(1.8)* 24.1(0.5)2 4 h 21.9(4.4)* 38.7(3.2)'

24 h 15.3(2.8)' 57.2(4.2)" 36 h 18.3( 1.6) 38.8(3.3)2

' Monocytes were incubated with 1:3 diluted control or PHA-supernatant as indicated. The supernatants were removed before adding target cells. Percent specific lysis of monocytes not exposed to supernatants was 20.1 ? 2.4%. The A:T ratio was 20:l. - p<0.01 vcrws spontaneous isotope release in the absence of cffector cells. - ' p<O.OS versus conditioned medium. - ' p<O.01 versus conditioned medium.

the homologous species rather than fetal bovine serum (Tagliabue et al., 1979; Mantovani et al., 1 9 7 9 ~ ) . It was of interest to evaluate whether production of lymphokines and augmentation of monocyte cytotoxicity occurred also in the presence of adult human serum. As shown in the representative experiment in Table 11, augmentation of cytotoxicity was similar in medium containing human serum or FBS.

Supernatants from antigen-or mitogen-stimulated lymphocytes did not affect the viability and proliferative capacity of TU5 tumor cells in the absence of effector cells, as assessed by morphological observation of the cultures, release of ["Hlthymidine and uptake of [-1251]deoxyuridine. To rule out further that the enhanced cytolysis observed in the presence of lymphokines was in fact due to some modification of target cells and to elucidate better the augmentation of cytotoxicity induced by lymphokine supernatants, monocytes were preincubated with supernatants, and the lyrnphokines were removed prior to addition of TU5 target cells. As shown in Table 111, the minimal exposure time resulting in a significant increase of cytocidal capacity was 4 h and optimal stimulation was observed after 20-30 h of preincubation with lymphokines. When monocytes were acti-


vated by exposure for 24 h to lymphokines and then cultivated in RPMI 1640 with 10% FBS or human AB serum for up to 36 h before testing, essentially the same levels of enhanced cytolytic activity per- sisted (results not shown).

In the representative experiments shown in Tables 1-111, the A:T ratio was 20:l and the incubation time was 48 h. It was of interest to evaluate whether the stimulatory effect of lymphokines could be detected at other ratios of effector to target cells and at shor- ter incubation times. Lymphokine supernatants enhanced the cytocidal efficacy of monocytes at A:T ratios ranging from 5:l to 40:l (results not presented). When the kinetics of target cell lysis was examined (Table IV), low but significant augmentation of cytotoxicity was already evident after 24 h of incubation, was observed throughout the 72 h observation period.

TABLE IV

KINETICS OF CYTOTOXICITY BY LYMPHOKINE-STIMULATED MONOCYTES

Percentage specific lysis (+ SD) Supernatant I

24 48 12

- 3.8(0.4) 12.8(1.3)5 20.7(2.3)' Conditioned

medium 3.5(1.2) 13.9(2.1)5 26.4(0.2)',s Lymphokine supernatant 8.9(1.5)3,4.5 2 1 4 l.7)'.4.5 41.2(3.6)'.4.5

~ ~~ ~~

' The assay was performed in the presence of 1/3 diluted control or PHA-supernatant. The A:T ratio was 201. - ' Incubation time (h). - ' p<0.01 versus monocytes in the absence of supernatants. - pt0.01 versus conditioned medium. - p<O.OI versus spontaneous release of tumor cells alone.

The enhanced cytotoxicity of monocytes exposed to lymphokines was evident when tumor growth was checked daily under an inverted microscope and when wells were fixed and stained with Wright's stain after termination of the experiments. Accordingly, enhanced growth inhibition was also detected when tumor cell proliferative capacity was measured by post-labelling with [-'251]deoxyuridine (results not presented) as previously described (Mantovani et al., 1979a).

The adherent effector cells employed in these studies consisted primarily of cells of the monocyte- macrophage series, as assessed by morphologic and functional criteria. However, it was still possible that lymphokine supernatants were in fact rendering cytotoxic an adherent cell population not belonging to the monocyte-macrophage series. To evaluate this possibility, the effect of silica, a selective macrophage toxin (Allison et al., 1966), and of anti-T- cell serum plus C on cytotoxicity was investigated. As shown in Table V and in agreement with previous data (Mantovani et al., 1979a), silica caused a marked inhibition (69 %) of natural monocyte-mediated cytotoxicity and, in addition, almost completely abrogated the enhancing effect of lymphokine super-

TABLE V

EFFECT OF SILICA AND ANTI-HUMAN T-CELL SERUM ON LYMPHOKINE-INDUCED ENHANCEMENT OF MONOCYTE-MEDIATED CYTOTOXICITY '

Percentage specific lysis

(+ SDI

Monocytes treated with Supernatants

j I

Conditioned - 21.9 (4.4)2 medium Silica

C Anti-T-cell serum 26.4 (2.3j2

Anti-T-cell serum + C 28.5 (3.9)* Lymphokine - 38.7 (3.2)2.4 supernatant Silica 12.3 (2.6)'.'

C 42.1 (1.1)'.' Anti-T-cell serum 45.4 (3.6)2.4

Anti-T-cell serum + C 46.9 (4.1)*,'

I Control and PHA-elicited supernatants were employed in this experiment at a 1/3 final dilution. The A:T ratio was 20:l. - p<0.01 versus spontaneous release of tumor cells alone. - ' p<O.01 versus monocyres not exposed to silica. - ' pi0 .01 versus conditioned medium.

natants. Exposure to anti-human T-cell serum and C did not affect natural or lymphokine-stimulated cytotoxicity (Table V).

In the experiments presented here, TU5 tumor cells were used as targets, but similar results (not presented) were obtained when we used a lung car- cinoma line (CaLu) relatively resistant to natural human monocyte cytotoxicity (Mantovani et al., 1979~).

Characterization of the lymphokine(s) responsible for augmenting the cytotoxicity of monocytes was beyond the scope of the present study. However, a series of experiments was conducted in an attempt to determine whether the effects could be attributed entirely to the presence of IF in the supernatants. IF has been shown to enhance macrophage-mediated cytostasis in mice (Schultz et al., 1977) and the antigen-or mitogen-elicited lymphokine supernatants employed in the present study were found to have significant levels of IF, with titers ranging from 20 to 1,000 unitsl0.1 ml. As recently found in separate studies (Jett, Mantovani and Herberman, 1980; Mantovani et al., 1980), monocytes exposed to IF, either during a 20-h preincubation period or throughout the cytotoxicity assay, showed enhanced cytolytic activity (Table VI). The data shown here were obtained with fibroblast IF, but similar results were obtained with leukocyte or lymphoblastoid IF. In an effort to elucidate the possible role of lymphokines other than IF in the activation of human monocyte cytotoxicity, we took advantage of the dif- ferent biological properties of lymphokines elicited by C. parvum strains CN6134 and CN5888 (Sugiya- ma and Epstein, 1978; Epstein et al., 1979). C. parvum CN6134 supernatants have relatively high concentrations of IF, whereas strain CN5888 is a poor inducer of IF in human lymphoid cell cultures (Sugiyama and Epstein, 1978; Epstein ef al., 1979). As shown in Table VII, lymphokine supernatants elicited by both strains of C. parvum markedly enhanced the cytolytic activity of human monocytes at %-% final dilutions. Supernatants of killed bacteria

696 MANTOVAN1 ET AL

TABLL V1

EFFECT OF IF ON HUMAN MONOCYTE-MEDIATED TUMORICIDAL ACTIVITY

IF (units/ml)

~ 100 300 IOIN 3000 IF preqent

Throughout the assay 23.7(4.7)' 34.S(1.4)2 58.6(2.Y)3 78.9(16.7)' NT' During 20 h preincubation 13..5(1.2) 14.2(2.7) 17.6(1.S)2 23.1( 0.7)3 28.9(2.3)'

~~ ~~ ~~

' Percent specific lysis (k S O ) with or without human fibroblast IF. The A:T ratio was 20:l. ~' Significantly ahovc cytolytic activity of monocytes not exposed to IF, p<0.05. - ' Significantly above cytolytic activity of monocytes not exposed to IF. p<O.Ol. - Nor tested.

cultivated alone did not significantly modify the cytolytic activity of these effector cells (results not presented). In agreement with previous data (Sugiyama and Epstein, 1978; Epstein et a l . , 1979), the C. par- wum CN5888 supernatant employed in the experiment shown in Table VI1 had no detectable IF, whereas the C. parvum CN6134 supernatant had an IF titer of 50 units/O.l ml. Four other subjects were tested for lymphokine production in response to anaerobic coryneforms: two yielded supernatants with biological activity similar to that shown in Table

VII, one yielded lymphokine supernatants which induced only a small, but statistically significant, 6% absolute increase in cytolysis over control supernatants at a 1/3 dilution and, in the case of the fourth donor, no significant biological activity was observed. It has been previously reported that human donors show great individual variability in their responsiveness to C. parvum as measured by blas- togenesis and IF production (Sugiyama and Epstein, 1978). In a limited series of experiments in which the C. parvum CN6134- and CN5888-elicited superna-

T A B L t V11

EFFECT OF LYMPHOKINES INDUCED RY ANAEROBIC COKYNEbOKMb ON MONOCYTE CYTOTOXICITY

Percentagc specific lysis (k $D)I

1/32 I /Y 1/81

Supernatants from lymphoid cells cultivated with

- C. parvurn strain CNh134 C. paruum strain CNS888

13.2(0.8) 29.6(3.1) 35.7(2.5)'

11.9(2.1) 21.8(3.0)' 24.4(0.8)]

10.3(0.1)

12.7(0.S) 12.6(0.2)

' Supcrnatants were present throughout the cytolysis assay. Specific cytolysis of monocytes alone was tl.R(f2.7) percentage. The A:T ratio was 20:l. - Final dilution of supernatants. - ' p<0.01 versus supernatant of lymphoid cells cultivated alone.

TABLE V111

EFFECT O F LYMPHOKINE SUPERNATANTS ON THE TUMORICIDAL ACTIVITY OF Ih' VITRO-MATUKED MONOCYTE-DERIVED MACROPHAGES

Effector cells Cultivated for

S days

12 days

Percentage specific lysis (+ Sn)

1/3' 1/9' 1/27 ' Supernaiant A:T ratio _.

20: 1 - Conditioned medium Lymphokine supernatant

Conditioned medium Lymphokine supernatant

2S:l - Conditioned medium Lymphokine supernatants

- 18: 1

-

1S.9( 1.9)'

30.4(2.2)4.5 -

22.3(1 .4)4

35.7(2.6)'.' -

S.S(l.2)~

8,6(0.4) '

-

12.0(1 . I ) *

2S.4(1.0)4i -

18.8(1.2)'

3 4 . q 4 . 9 4 -

S.9(3.2)'

Y.O(O.5)'

-

1 1 .6(1.4)'

lS.l(I.9)' -

18.8(6.0)4

24.0(3.7)4 -

NT'

N T

I Control or PHA-elicited supernatants were present throughout the cytolysis assay. ~ ' Percent specific lysia of monocytes in the absence of Significantly ahove spontaneous release of tumor cells alonc. p<O.01. - ' p<U.01 versus con^ supcmatants. -- ' Final dilution of supernatants. -

ditioned medium. - ' Not tested.


tants were studied in terms of the kinetics of activation after preincubation and responsiveness of in vitro cultivated monocytes, results were similar to those described above for mitogen- or antigen-elicited lymphokines.

In all of the above experiments we used peripheral blood monocytes as a source of effector cells, these being the only easily accessible cells of the human monocyte-macrophage series in normal donors. In an effort to determine whether augmentation of tumoricidal activity by lymphokine supernatants was restricted to circulating monocytes or would also be seen with human mature macrophages, in vitro-matured monocyte-derived macrophages were studied. Mature macrophages obtained by in vitro culture in medium with 50 % autologous serum for 4-7 days ( 5 days in the experiment presented in Table VIII) had natural cytotoxicity values against TU5 tumor cells similar or higher than those of the fresh monocytes and responded to cultivation with lymphokines with comparable augmentation of the tumoricidal capacity (Table VIII). After further in vitro cultivation for more than 10 dayas (12 days in the experiment shown in Table VIII), effector cells, consisting primarily of epithelioid and giant cells (Sutton, 1967), showed little natural cytotoxicity on tumor cells and little stimulation in the presence of lymphokines.

DISCUSSION

The results presented here confirm previous observations that human peripheral blood monocytes can express natural cytotoxicity on tumor cell lines in vitro (Mantovani et al., 19796, a, c; 1980) and show that the cytotoxic activity of monocytes can be markedly enhanced in the presence of, or after preincubation with, lymphokine supernatants generated by lymphocyte cultures with mitogen (PHA) or antigen (PPD). The effector cells involved in the lymphokine-induced augmentation of cytotoxicity ap- pear to belong to the monocyte-macropage lineage. Silica markedly inhibited the natural cytocidal capacity of adherent cell preparations, thus confirm- ing our previous data (Mantovani et al., 1979a), and completely abolished the enhancement of cytotoxicity induced by lymphokines. In contrast, treatment with anti-T-cell serum and C, which inhibited NK cell activity (Ortaldo et al., unpublished data), did not affect natural and lymphokine-stimulated tumoricidal activity. Finally, the activity of NK cells in almost completely lost upon in vitro culture for several days in medium containing human serum (Ortaldo et al., 1977), whereas monocytes cultivated in vitro for 4-7 days with autologous or pooled human serum had natural cytotoxicity and responsiveness to lymphokines similar to fresh adherent cell preparations. Thus, it seems extremely unlikely that lymphocytes contaminating adherent cell preparations (>90 % monocytes, as assessed by morphological and functional criteria) play a significant role in the expression of these cytotoxic capacities.

Lymphocyte mediators can modulate the functional status of mononuclear phagocytes (David, 1975) and, in rodents, antigen- or mitogen-elicited lymphokines can stimulate the cytotoxic capacity of

monocytes and macrophages (Piessens et al., 1975; Churchill et al., 1975; Fidler, 1975; Fidler et al., 1976; Christie and Bomford, 1975; Ruco and Melt- zer, 1977; Tagliabue et al., 1979). The factor responsible for inducing or augmenting mouse macrophage cytotoxicity has been termed macrophage activating factor and is physico-chemically indistin- guishable from macrophage migration inhibition factor (Leonard et al., 1978). It is of interest that PHA supernatants, obtained following an experimental protocol similar to that employed in the present study, have been reported to induce a monocyte- dependent suppressive activity (Larsson and Blom- gren, 1979). It therefore seemed likely that a macrophage activating factor, analogous to that described in rodents, was responsible for the augmentation of cytotoxicity by human monocytes. How- ever, a possible role for IF also has to be considered. In mice IF has been shown capable of macrophage- mediated cytostasis (Schultz et al., 1977) and, as described in more detail elsewhere (Mantovani et al., 1980; Jett et al., in preparation), human IF can stimulate the cytolytic and cytostatic activity of human monocytes. Supernatants from PPD- or PHA- stimulated lymphocyte cultures had significant levels of IF and it is therefore possible that this lymphokine as well as, or instead of, macrophage activator factor caused the augmentation of cytotoxicity by monocytes. In an attempt to obtain some initial indications for a role of lymphokines other than IF in augmenting human monocyte-mediated tumoricidal activity, we took advantage of the recent observation that supernatants of cultures of human lymphoid cells with the C. parvum strain CN5888 contained little or no IF (Sugiyama and Epstein, 1978; Epstein et al., 1979) but inhibited the migration of human monocytes (Tagliabue et al., in preparation). These supernatants were as stimulatory on monocyte cytotoxicity as C. parvum CN6134-elicited supernatants containing appreciable levels of IF. These data strongly support the hypothesis that lymphokines other than IF, presumably a human analogue of rodent macrophage activating factor, can play a role in the stimulation of the tumoricidal activity of human monocytes and macrophages. It can therefore be proposed that there are at least two possible path- ways of activation of human monocyte-mediated cytotoxicity, one involving IF and the other involving another still undefined lymphokine(s). In supernatants containing IF, it will need to be determined whether IF or another lymphokine is reponsible for augmentation of cytotoxicity. Preliminary experiments with supernatants of cultures with C. parvum CN6134 have indicated that anti-interferon can strongly reduce the boosting activity, suggesting that in these supernatants IF plays an important role.

Control supernatants of lymphoid cells cultivated alone slightly, but significantly, enhanced the cytolytic activity of human monocytes. This effect was only observed with high concentrations of the supernatants and when the supernatants were present throughout the cytotoxicity assay. The mechanism(s) responsible for the significant biological activity of control supernatants (e .g . , cell death in culture or “background” production of lymphokines)

698 MANTOVANI ET A1

remains to be elucidated. However, it is tempting to speculate that the natural cytotoxic capacity of human monocytes is in part the expression of in vivo exposure to "background" release of lymphocyte products.

Monocytes differ from mature macrophages in many respects including morphology (Territo and Cline, 1976), and efficacy in mediating antibody-dependent cellular cytotoxicity (Mantovani et al., 1977). The observation that macrophages obtained by in vitro cultivation of monocytes for 4-7 days had significant natural tumoricidal activity and this could be augmented by exposure to lymphokine supernatant suggests that these properties are not restricted to circulating monocytes. In agreement with these data Cameron and Churchill (1979) recently reported that supernatants of human lymphocytes cultivated with streptokinase-streptodornase enhanced the cytolytic capacity of human peripheral blood monocytes maintained in vitro for 5 days. However, in contrast with results reported here, they briefly mentioned that fresh monocytes were not responsive to lymphokine supernatants; in the lack of information on the actual data and experi-

mental protocols followed by these investigators, the apparent discrepancy between their results and the present study remains to be clarified.

After in vitro cultivation for 4-7 days, monocyte- derived macrophages expressed both natural and lymphokine-augmented cytotoxicity, but following more prolonged in vitro culture (> 10 days), macrophages had little natural cytotoxicity and failed to respond to lymphokines. At these times, macrophage cultures consisted primarily of epithelioid and multinucleated giant cells, as previously analy- zed in detail by others (Sutton, 1967). Hence we speculate that similar macrophage-derived cells present at sites of chronic inflammation may have little natural cytotoxicity and little responsiveness, in terms of enhanced tumoricidal activity, to lymphokines. However, these points will need to be di- rectly tested.

ACKNOWLEDGEMENTS

Albert0 Mantovani was supported by Consiglio Nazionale delle Ricerche, Rome, Italy and, in part, by Grant ROI CA26824 from the National Cancer Institute, USA.

REFERENCES

ALLISON, A.C., H A R I N C ~ ~ O N , J.S., and BIRBECK, M., An ex- amination of the cytotoxic effects of silica o n marcrophages. J. exp. Med., 124, 141-154 (1966). ANDERSON, S.E., BAUTISTA, S.. and REMINOTON, J.S.. Induc- tion of resistance to Toxoplasma gondii. in human macrophages by soluble lymphocyte products. J. Immunol., 117,

ANDERSON, S.E.JR., and REMINGTON, J.S., Effect of normal and activated human macrophages on Toxoplasma gondii. J, exp. Med., 139, 1154-1174 (1974). BIRBECK, M.S.C., and CARTER, R.L., Observations on the ul- trastructure of two hamster lymphomas with particular refer- ence to infiltrating macrophages. lnt. J. Cancer, 9, 249-257 (1972). RONNARD, G.D., SCHENDEL. D.J., WEST, W.H., ALVAREZ, J.M., MACA, R.D. , YASAKA, K . , FINE, R.L., HERBERMAN, R.B., DE LANDAZURI, M.O., and MORGAN, D.A., Continued growth of normal human T lymphocytes in culture with reten- tion of imporiant functions. ln: B. Serrou (ed.). Human lymphocyte differentiation. Its application to human cancer. pp. 319-326, North-Holland, Amsterdam (1979). CAMERON, D.J., and CHURCHILL, W.H., Cytotoxicity of human macrophages for tumor cells. Enhancement by human lymphocyte mediators. J . J. elin. Invest.. 63, 977-984 (1979). CARR, I . , Macrophages in human cancer: A review. In: K. James, B. McBride and A. Stuart (ed.), The macrophage and cancer, pp. 364-374, University of Edinburgh, Edinburgh (1977). CHRISTIE, G.H., and BOMFORD, R., Mechanisms of macrophage activation by Corynebacterium parvum. I . In vitro experiments. Cell. Immunol., 17, 141-149 (1975). CHURCHILL, W.H.JR., PIESSENS, W.F., SULIS, C.A., and DAVID, J.R., Macrophages activated as suspension cultures with lymphocyte mediators devoid of antigen become cytotoxic for tumor cells. J. Immunol., 115, 781-786 (1975). DAVID. J.R., Macrophage activation by lymphocyte mediators. Fed. Proc., 34, 1730-1 736 (1 975). ECCLES, S.A. , and ALEXANDER, P., Macrophage content of tumours in relation to metastatic spread and host immune reaction. Nature (Lond.), 250, 667-669 (1974).

381-387 (1976).

EPSTEIN, L.B.. DEAN. J.H., HERBERMAN, R.B., OPPENHEIM. J.J., ROCKLIN, R.E., and SUGIYAMA, M., The specificity of mediator induction by C. parvum, PNA, and PPD: Studies on human interferon, MIF, LIF, MF and LAF. In: Proc. 2nd. lnr. Lymphokine Workshop, Wolfsberg/Ermatingen, Switzerland (Abs. no. 64) (1979). EVANS, R. , Macrophages in syngeneic animal tumours. Trans- plantation, 14, 468-473 (1972). EVANS, R., and ALEXANDER, P.. Mechanisms of extracellular killing of nucleated mammalian cells by macrophages. In: D.S. Nelson (ed.), Immunobiology of ihe macrophages, pp. 535-576, Academic Press, New York (1976).

FIDLER, I . J . , Activation in vitro of mouse macrophages by syngeneic, allogeneic, or xenogeneic lymphocyte supernatants. J . nat. Cancer Inst., 55, 1159-1163 (1975).

FIDLER, I.J., DARNELL, J.H., and BUDMEN, M.B., Tumoricidal properties of mouse macrophages activated with mediators from rat lymphocytes stimulated with concanavalin A. Cancer Res., 36, 3608-3615 (1976). HIRRS, J.B.JR., the macrophages as a tumoricidal effector cells: A review of in vivo and in vitro studies on the mechanism of the activated macrophage nonspecific cytotoxic reaction. In: M.A. Fink (ed.), The macrophage in neoplasia, pp. 83-111, Academic Press, New York (1976). JETT, J.. MANTOVANI, A , , and HERBERMAN, R.B., Augmenta- tion of monocyte-mediated cytolysis by interferon Cell. Im- munol. 1980, in press. KELLER, R., Cytostatic and cytocidal effects of activated macrophages. In: D.S. Nelson (ed.), lmmunobiology of the macrophages. pp. 487-508, Academic Press, New York (1976).

KIT. S. , KURIMURA, T., and DUBBS. D.R., Transplantable mouse tumor line induced by injection of SV 40-transformed mouse kidney cells. Int. J. Cancer, 4, 384-392 (1969).

LARSSON, E.-L., and BLOMGREN, H., Evidence that soluble products released by PHA-stimulated human lymphoid cells activate immuno-suppressive monocytes, Scand. J. Immunol.,

LEONARD. E.J.. RUCO, L.P.. and MELTZER, M.S.. Characteri- zation of macrophage activation factor, a lymphokine that

9, 53-60 (1979).


causes macrophages to become cytotoxic for tumor cells. Cell. Immunol., 41, 347-357 (1978). MANTOVANI, A., Effects on in virro tumor growth of murine macrophages isolated from sarcoma lines differing in im- munogenicity and metastazing capacity. Int. J. Cancer, 22,741- 746 (1978). MANTOVANI, A., BAR SHAVIT, Z. , PERI, G., POLENTARUTTI, N., BORDINGNON, C., SESSA, C., and MANGIONI, C. . Natural cytotoxicity on tumor cells of human macrophages obtained from diverse anatomical sites. Clin. exp. Immunol., in press (1980). MANTOVANI, A , , CAPRIOLI, v. , GRITTI, P., and SPREAFICO, F., Human mature macrophages mediate antibody-dependent cellular cytotoxicity on tumour cells. Transplantation, 24, 291-293 (1977). MANTOVANI, A., JERRELLS, T.R., DEAN,J.H., and HERBER~ MAN, R.B., Cytolytic and cytostatic activity on tumor cells of circulating human monocytes. Int. J . Cancer, 23, 18-27 (1979a).

MANTOVANI, A . , PERI, G. , POLENTARUTTI, N., BOLIS, G., MANGIONI, C. , and SPREAFICO, F., Effects on in uitro tumor growth of macrophages isolated from human ascitic ovarian tumors. Int. J. Cancer, 23, 157-164 (19796).

T.R., and HERBERMAN, R.B., Cytolytic activity of circulating human monocytes on transformed and untransformed human fibroblasts. Int. J . Cancer, 23, 28-31 (1979~).

ORTALDO, J .R. , BONNARD, G.D., and HERBERMAN, R.B., Cy- totoxic reactivity of human lymphocytes cultured in vitro. J . Immunol., 119, 1351-1357 (1977). PIESSENS, W.F., CHURCHILL, W.H.JR., and DAVID, J.R., Mac- rophages activated in vitro with lymphocyte mediators kill neo- plastic but not normal cells. J. Immunol., 114, 293-299 (1975).

PROSS, H.F., and KERBEL, R.S., An assessment of intratumor phagocytic and surface marker-bearing cells in a series of autochthonous and early passaged chemically induced murine sarcomas. J. nut. Cancer Inst.. 57, 1157-1167 (1976).

MANTOVANI, A,, TAGLIABUE, A, , DEAN, J .H. , JERRELLS.

RUCO, L.P., and MELTZER, M.S., Macrophage activation for tumor cytotoxicity: Induction of tumoricidal macrophages by supernatants of PPD-stimulated Bacillus Calmette-Gukrin-immune spleen cell cultures. J. Immunol., 119, 889-896 (1977). RUSSELL, S.W., and MCIINTOSH, A.T., Macrophages isolated from regressing Moloney sarcomas are more cytotoxic than those recovered from progressing sarcomas. Nature (Lond.),

SCHULTZ, R.M., PAPAMATHEAKIS, J .D. , and CHIRIGOS, M.A., Interferon: An inducer of macrophage activation by polyan- ions. Science, 197, 674-676 (1977). SUGIYAMA. M., and EPSTEIN. L.B., Effect of Corynebacterium parvum on human T-lymphocyte interferon production and T- lymphocyte proliferation in vitro. Cancer Res., 38, 4467-4473 (1978). SUTTON, J.S.. Ultrastructural aspects of in vitro development of monocytes into macrophages, epithelioid cells, and multinucleated giant cells. Nar. Cancer Inst. Monogr., 26,71-137 (1967). TAGLIABUE, A , , MANTOVANI, A , , KILGALLEN, M., HERBER- MAN, R.B.. and McCoy, J.L., Natural cytotoxicity of mouse monocytes and macrophages. J . Immunol., 122, 2363-2370 (1979). TERRITO, M., and CLINE, M.J., Macrophages and their disor- ders in man. In: D.S. Nelson (ed.), Immunobiology of the macrophuges. pp. 593-616, Academic Press, New York (1976). WOOD, G.W., and GILLESPIE, G.Y., Studies on the role of macrophages in regulation of growth and metastasis of murine chemically induced fibrosarcomas. Znt. J. Cancer, 16, 1022- 1029 (1975). WOOD, G.W., and GOLLAHON, K.A., Detections and quantita- tion of macroohage infiltration into Drimarv human tumors

268, 69-71 (1977).

with the use df c e h r f a c e markers. j . nat. ‘Cancer Inst . , 59, 1081-1087 (1 977). Z A R L i N G , J.M.. and TEVETHIA, S.S. , Transplantation immunity to simian virus 40-transformed cells in tumor-bearing mice. 11. Evidence for macrophage participation at the effector level of tumor cell injection. J . nat. Cancer Inst., 50, 149-157 (1973).

Augmentation of tumoricidal activity of human monocytes and macrophages by lymphokines

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