cytolytic and cytostatic activity on tumor cells of circulating human monocytes

f n t . J . Cancer: 23, 18-27 (1979)

CYTOLYTIC AND CYTOSTATIC ACTIVITY ON TUMOR CELLS OF CIRCULATING HUMAN MONOCYTES

Alberto MANTOVANI *, Thomas R. JERRELLS z, Jack H. DEAN * and Ronald B. HERBERMAN Istituto Di Ricerrhe Farmacologiche Mario Negri, Milan, Italy; Department of Immunology, Litton

Bionetics, Inc., Kensington, Md. 20795; Laboratorji of Immunodiagnosis, National Cancer Institute, Bethesda, Md. 20014, USA

Monocytes f rom the peripheral blood of normal adult human donors were found t o have appreciable levels of cytotoxic activity against murine and human tumor-cell lines. Adherent cells (>W% monocytes) were obtained from the peripheral blood of 29 normal healthy volunteers either by adherence on plastic and scraping with a rubber policeman or by adherence on microexudate-coated plastic and exposure t o ethylene diamine tetra- acetic acid. Cytolytic capacity was tested by incubating effector cells for 72 h with murine and human tumor cell lines prelabelled with tr i t iated thymidine. Cytostasis was evaluated by inhibition of [1251]iododeoxyuridine (lzSldUrd) uptake. Tumor target cells employed were: a murine SV40-transformed kidney line (TU5), a murine chemically- induced sarcoma (1023), a human breast-cancer- derived cell line (G11) and a human iung-cancer- derived cell line (CaLu). Monocyte preparations at attacker t o target cell ratios of 1:l t o 40:1, showed significant cytolytic and cytostatic activity against tumor target cells. Tumor cells showed different susceptibility t o cytolytic activity, whereas comparable levels of cytostasis were observed with the various targets: TUS and G11 tumor cells were more susceptible than 1023 and CaLu target cells t o the cytolytic capacity of human monocytes and TU5 was used In most subsequent experiments. Peak isotope release from prelabelled target cells was observed after 72 h of incubation, whereas peak inhibition of [1z51]dUrd uptake occurred after 24 h of culture. The cytotoxic capacity of monocytes isolated by either of the two methods mentioned above was similar. The monocytes had higher cytotoxic activlty than unseparated mononuclear cells, and non-adherent cells showed minimal cytotoxic effects. Cytotoxicity by natural ki l ler cells did not appear t o have a major role in these assays, since adherent cells did not lyre K562 cells in a 4-h 51Cr release assay. Treatment wi th anti-human T-cell serum and complement did not inhibit the cytotoxic capacity of the monocyte preparations, whereas exposure t o silica particles significantly inhibited the cytotoxic activity. Monocyte-mediated cytotoxicity on tumor cells was expressed in the presence of both fetal bovine serum and human AB serum.

Rodent mononuclear phagocytes can express cytotoxic activity (cytostasis and/or cytolysis) in vitro on tumor cells, either spontaneously (Meltzer, 1976; Miller and Feldman, 1976; Keller, 1978) or as a consequence of exposure to chemical or biological stimuli (reviewed by Evans and Alexander, 1976; Hibbs, 1976; Keller, 1976~). Macrophages have been shown to infiltrate experimental and human tumors (Evans, 1972; Pross and Kerbel, 1976; Gauci and Alexander, 1975; Wood and Gollahon, 1 977) and mononuclear phagocytes

obtained from lymphoid organs (Kirchner et al., 1975; Mantovani et al., 19776) or primary neo- plasms (Evans, 1973; Holden et al., 1976; Russell and McIntosh, 1977) of rodents bearing chemically- or virally-induced tumors manifested increased levels of cytotoxic activity against neoplastic cells.

Although the in vivo significance of cytotoxicity by macrophages in resistance against tumor growth is far from being firmly established (Evans, 1976), evidence has accumulated in experimental systems to support the role of mononuclear phagocytes in the regulation of tumor growth and metastasis (Birbeck and Carter, 1972; Eccles and Alexander, 1974; Pross and Kerbel, 1976; Wood and Gillespie, 1975; Zarling and Tevethia, 1973; Russell and McTntosh, 1977).

In spite of extensive studies on cytotoxicity by rodent macrophages and the possible significance of macrophages as a regulatory mechanism in neoplasia, little is known about the in vitro effects on tumor cells of human mononuclear phagocytes (Holtermann et al., 1974). The present investigation was designed to characterize the cytolytic and cytostatic activity on tumor cells of human peripheral blood monocytes.

MATERIAL AND METHODS

Mononuclear cells Heparinized peripheral venous blood (50 to

300 ml) was obtained from 29 normal healthy adult volunteers. Blood was diluted 1:4 with phosphate-buffered saline (PBS) and mononuclear cells were separated by centrifugation at 400 g for 30 min on Ficoll-Hypaque (LSM, Litton Bio- netics, Kensington, Md., USA). Mononuclear cells were washed twice with PBS and resuspended at a concentration of approximately 1 x lO6/ml in RPMI 1640 medium supplemented with 207; heat-inactivated (30 min at 56" C ) fetal bovine serum (FBS, HEM, Rockville, Md.) or aseptically obtained FBS, Microbiological Associates, Bethesda, Md.).

Monocytes Two methods were employed to separate adherent

cells from mononuclear cell suspensions. According to the first, 10-15 ml of the cell suspension were seeded in 10-cm plastic Petri dishes (3003 Falcon, Oxnard, Calif., USA) and incubated for 45 min at 37" C. At the end of the incubation, non-adherent

Received: September 12, 1978.

HUMAN MONOCYTE CYTOTOXICITY 19

and loosely adherent cells were resuspended by agitation and the dishes were thoroughly washed at least five times with 10-20 ml of RPMI 1640 madium supplemented with 20% FBS. Cells washed off the dishes were collected and are hereafter referred to as '' non-adherent and loosely adherent cells." Adherent cells were recovered from the dishes by scraping with a rubber policeman and, after washing with 50 ml PBS, the cells were resuspended in RPMI 1640 medium with 10% FBS. Viability assessed by trypan blue exclusion was 70.6&3.2 ( m e a n k s ~ ) and adherent cells recovered from the dishes were 5.3*2.2% of the seeded mononuclear cells. More than 90% of the adherent cells were cells of the monocyte-macrophage series as assessed by morphology, non-specific esterase staining (Koski et al., 1976), latex phagocytosis, spreading capacity and binding and phagocytosis of ox erythrocytes coated with rabbit IgG antibody. Adherent cells will hereafter be referred to as monocytes.

In part of the experiments adherent cells were recovered from microexudate-coated plastic following a modification of a recently described method (Ackerman and Douglas, 1978). Chang liver cells were grown to confluence in 75 cm2 tissue culture flasks (3024, Falcon, Oxnard, Calif.). Cells were detached by exposure to 10 I-~M ethylene diamine tetra-acetic acid (EDTA) in PBS for 20 min at 37" C. These microexudate-coated flasks were thoroughly washed with PBS and stored at 4°C until used. Mononuclear cell suspensions (10- 20 ml) were incubated in conditioned flasks for 45 min at 37" C and non-adherent or loosely adherent cells were washed off as described above. Adherent cells were recovered by incubating the flasks for 10 rnin with 10 ml of 10 mM EDTA in PBS supplemented with 5 % FBS followed by vigorous shaking. (When the same procedure was applied to cells attached to untreated plastic, no detachment occurred). After washing with 50 ml PBS, adherent cells were resuspended in RPMI 1640 medium containing 10% FBS. Adherent cells recovered following this procedure represented 5.9*3.2% of the original mononuclear cell suspen- sicn and viability always exceeded 90%. According to the above-mentioned criteria, more than 90 % of the adherent cells were mononuclear phagocytes.

As shown in the '' Results " section, monocytes obtained by these two methods showed similar in vitro cytotoxicity on tumor cells.

Non-adherent cells Adherent cells were selectively removed from

mononuclear cell preparations by passage on Sephadex G10 columns, as recently described (Jerrells et al., 1978). Briefly, 1-2 x lo7 mononuclear cells in 1-2 ml RPMI 1640 medium supplemented with 20% FBS were run into columns consisting of 6 ml Sephadex G10 (Pharmacia Fine Chemicals, Uppsala, Sweden) in a 10-ml syringe. After incubation for 30 min at 37" C, non-adherent cells were eluted from the column with 15 ml of warm RPMI 1640 medium with 20% FBS. The non- adherent population contained < 1 % monocytes,

as assessed by morphology and non-specific esterase staining.

Anti- T-cell serum The preparation and characteristics of a potent

and specific rabbit anti-human T-cell serum have recently been described (Maca et al., in preparation). Briefly, T cells from the peripheral blood of a normal donor were cultivated in vitro (Ruscetti et al., 1977; Alvarez et al., 1978; Bonnard et a]., 1978). Rabbits were immunized against cultured T cells and the serum was absorbed with a B-cell line established from the same donor. Monocytes (4 x lo8 in 0.6 ml RPMI 1640 medium) were incubated in plastic tubes (2074 Falcon, Oxnard, Calif.) with a 1:3 dilution of anti-human T-cell serum and a 1 :12 dilution of rabbit complement at 37" C for 45 min, with agitation every 3-5 min. Under these conditions, lymphoproliferative responsiveness to concanavalin A was completely eliminated and there was a 80-90% reduction in the response to phytohemagglutinin. Moreover, treatment with anti- human T-cell serum and complement inhibited the capacity of T cells to act as helper cells for in vitro antibody production against sheep erythrocytes, without affecting antibody-producing cells (Maca et al., in preparation). Finally, the viability and phagocytic activity of human monocytes were not significantly affected under these experimental conditions.

NK activity Cytolytic activity of natural killer (NK) cells

was evaluated on 51Cr-labelled K562 myeloid leukemia cells in a 4-h assay as previously described (West et al., 1977).

Silica Silica particles (Santocel 58, average particle

size 3 ,urn, Monsanto Co., S. Louis, Mo.) were suspended and sonicated immediately before use. Monocytes (2 x lo5 cells in 0.2 ml of RPMI 1640 medium with 10% FBS) were incubated for 1 h at 37" C in 96-we11 tissue-culture plates (3596, Costar, Cambridge, Mass.) with 25 ,ug/well silica. Labelled cells were then added to the wells and the cytolysis assay performed as described below.

Target cells Target cells used in the present investigation

included: mKSA-TU5 (TU5), a BALB/c mouse kidney line transformed by SV40 (Kit et al., 1969); 1023 sarcoma, chemically induced in C3H/HeNicr mice (Meltzer and Bartlett, 1972); G11, derived from a patient with breast carcinoma (Bassin et a[., 1973); and CaLu derived from a human lung carcinoma (McCoy et al., 1977). The TU5 and 1023 cell lines were maintained in Minimal Essential Medium supplemented with 10% FBS and the G11 and CaLu lines in RPMI 1640 with 20% FBS. In a few experiments, a subline of G11 (Gll/AB) was maintained in RPMI 1640 medium with 10% human AB serum. Cells were prelabelled with [3H]methyl-thymidine by incubating non- confluent cultures in 75 cm2 tissue-culture flasks (Falcon, Oxnard, Calif.) for 24 h with 25 ml of

20 MANTOVANI ET AL.

TABLE I

CYTOLYTIC ACTIVITY OF HUMAN PERIPHERAL BLOOD MONOCYTES AGAINST VARIOUS TUMOUR-CELL LINES

% specific lysis (*sD) Target cells Donor

40:l 2O:l 1 O : l 5:l 2:l 1:l

TU5 TA FV ME LP BD sc JM RW MM SD FD DS DL MA BBD GF CF

1023 BD AT DK DL FV DS

G1 I RW KN MM BD

CaLu AT AM KN MM

33.1 ( 5 ) * 48.8 (1.3) * 28.4 (2.7) * 34.6 (2.7) * 16.3 (0.9) * 17.9 (2.8) * 26.6 (0.3) * 23.4 (1.6) *

13.6 (1.8) * 14.7 (0.5) * 41.6 (4.3) 38.6 (4.2) *

-

- -

36.4 (3.5) *

4.7 (2.4) * 10.2 (1.5) *

-

- -

6.9 (0.8) * 5.5 (0.5) *

26.1 (0.7) * 20.4 (0.1) *

11.5 (4.6) * 7.3 (0.9) * 8.9 (0.5) * 7.3 (1.1) *

-

-

25.3 (1.4) * 34.5 (1.2) * 24.2 (2.2) * 29.1 (1.5) * 14.7 (0.5) * 12.4 (1) * 21.2 (0.6) * 10.6 (0.5) * 12.7 (0.3) * 12.8 (1.6) * 20.1 (1.4) * 20.8 (3.3) * 13.7 (2) * 31.9 (2.3) * 52.8 (0.6) * 7.3 (1.1) *

38.8 (0.3) * 17.5 (3.6) * 7.1 (1.5) *

19.5 (3.5) * 24.6 (0.7) * 18.1 (0.5) * 34.1 (2.4) * 7.2 (1.4) * 9.9 (3.6) * 6.2 (1.9) * 6.9 (2.5) *

-

28 (4.9) *

-

-

2 - 25 (3.8) * 14.3 (1.1) * 12.8 (1.6) * 9.8 (1.2) * 8.3 (0.1) * 7.7 (1.6) *

-

- - - - -

14.2 (3.9) * 6.9 (2)

39.9 ( I ) * 63 (2.2) * 6 (3.5) *

30 (2.3) * 20.9 (0.6) *

-

- -

11.9 (1.2) * 7.5 (0.6) *

16.6 (1.4) *

4.2 (0.4) * 5.9 (1.5) * 3.5 (1) 4.1 (0.3) *

-

- - -

6.9 (0.7) * - - - - - - - - - - - -

28.7 (5.3) *

8 (1.4)* 5.3 (0.9) * 2.1 (1.6)

-

- -

6.8 (0.2) * - - -

1.8 (0.9) 6.0 (0.9) * -

-

A:T ratio. - * Not test, - * Lysis simificantly above baseline spontaneous release, p<O.O5.

medium containing 0.5 pCi/ml of [SH]thymidine (6 Ci/mmole, Schwarz/Mann, Orangeburg, N.Y., USA). Target cells were harvested by exposure for 5 min at 37" C to 5 ml trypsin-EDTA (GIBCO, Grand Island, N.Y.) washed twice with 50 ml of RPMT 1640 medium containing 20% FBS and finally resuspended at a concentration of 1 O6 cells/ ml in RPMT 1640 medium with 10% FBS.

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

37" C in air with 5 % CO, for 72 h (unless otherwise stated) in a final volume of 0.3 ml RPMI 1640 medium containing 10% FBS and 50 pg/ml genta- mycin (Schering Corporation, Kenilworth, N.J.) in flat-bottomed 6.4-mm culture wells (3596, Costar, Cambridge, Mass.). A range of attacker to target (A:T) ratios (from 40:l to 1 :1) were usually tested. 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 Aquasol (New England Nuclear, Boston, 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 water. Percentage isotope release was calculated as 100 A x B, where A is the isotope released in test samples and B is the SDS-releasable radioactivity. Specific lysis was determined by subtracting the spontaneous release of tumor cells incubated in the absence of effectors which under these experimental conditions never exceeded 20% over a 72-h incubation period .

In a few experiments in which the cytocidal capacity of different mononuclear cell fractions was compared, a semi-log plot of the specific cytotoxicity values versus the number of effector cells per sample was obtained and the number of cells required to give 10% specific lysis was arbitrarily defined as one lytic unit (LU10).

Cytostasis assay At the end of the incubation the supernatant

was removed and the wells were gently washed twice with 0.3 ml of RPMI 1640 medium. Tumor cells were then incubated with 0.25 pCi of [1261]iododeoxyuridine (Amersham, Arlington, Ill.) in 0.2 ml of RPMI 1640 medium with 10% FBS at 37" C for 6-8 h. At the end of the incubation, the super-

HUMAN M w O C % E C f l O T O X I C I ~ 21 , I .

T 9 - 1 1 ~ - CYTOSTATIC ACTIVITY ON TUMOR CELL$ OF HUMAN PERIPHERAL BLOOD MONOCYTES

% inhibition ( ~ s D ) of ['gsldUrd uptake 'SEt Donor 40:l 20:I 10:l 5 : l 2:1 1:l

2 - TU5 TA 82.5 (1.3) * 78.4 (4) * - - - FV 84.1 (3.5) * 56 (2.3) * 41 (1.2) * - 0 0 DL 68.2 (0.8) * 35.8 (3.3) * - - - - DS 86.5 (1.1) * 62.3 (1.6) * LP 74.1 (2.7) * 54.8 (0.8) *

- - - - - - - -

- - 1023 DK - 69.5 (2.6) * 29.9 (5.8) * 0 DL - 41.7 (9) * 10.9 (2.4) 0 - - BD 88.3 (2.4) * 86.4 (1.7) * 66.8 (6.2) * - 30.6 (1.9) * -44 (2.1) *

- - - - - AT 75 (3.1) * FV 68.1 (3.6) * - - - - - DS 64.9 (1.6) * 49.2 (2.7) * - - - -

G11 BD - 64.5 (0.6) * - 0 -28.3 (0.3) * - - - - RW - 69.1 (4.5) * CaLu AT 66.1 (2.3) * 58 (0.5) * 15.4 (0.5) * 0 0 0

AM 69.5 (0.4) * 56.7 (3.4) * 0 0 0 -18 (1.3) *

A:T ratio. - Not tested. - a Stimulation of [L''I]dUrd uptake above baseline uptake by target cells alone. * Uptakeof [L'sI]dUrdsignificantly different from that oftarget cellscultured alone, p <O.O5.(Mean uptake in controlcultures was 14,850cpm

for TU5,6,740 cpm for 1023, 10,840 cpm for GI 1 and 5,920 cpm for CaLu).

natant fluid was discarded and the wells were gently washed three times with 0.3 ml of RPMI 1640 medium. The cells were lysed by expolsure to 0.3 ml of 1 % SDS in water and [129]dUrd uptake determined in a Beckman gamma spectrometer. The percentage of inhibition of [lZ6I]dUrd uptake was calculated according to the formula; (I-A/B) x 100, where A is the radioactivity in-, corporated in the presence of effector cells and B is the isotope uptake in control cultures. Isotope uptake by monocytes cultivated alone was negligible (<200 cpm) and was not taken into account in the calculations.

In a series of experiments, wells were fixed and stained with Diff Quick (Harleco, Gibbstown, N.J.) and the number of tumor cells was determined

TABLE I11

CYTOTOXIC ACTIVITY OF MONOCYTES ISOLATED BY ADHERENCE TO PLASTIC AND RUBBER POLICEMAN DETACHMENT OR BY ADHERENCE TO MICROEXUDATE-COATED

PLASTIC AND EDTA DETACHMENT

% Inhibition

uptake

Monocyte separation I Target %,Specific ( ~ s D ) of procedure cells lysls ( f s ~ ) ["S[]dUrd

Plastic and rubber policeman 1023 4.3 (1.5) * 47 (3.5) * Microexudatet EDTA 1023 4.3 (1.2) * 43 (2.8) *

TU5 29.1 (2.2) * -'

TU5 27.6 (1.4)* - Monocytes from donor AT were tested at an A:T ratio of 20:l.

Incubation time was 48 h. - * Not tested. - * No significant differ- ence compared to monocytes isolated by scraping with a rubber policeman. - * Significantly different from control cultures, p 10.05.

microscopically with the aid of an eyegrid. The percentage of growth inhibition was calculated according to the above formula, A and B in this case indicating the number of tumor cells in test and control cultures, respectively.

Statistical analysis Results are presented as mean ~ S D of three

replicates per experimental group. Statistical significance was assessed by one-way analysis of variance.

RESULTS

Table I summarizes data on the cytolytic activity against tumor cells of human peripheral blood monocytes of 21 donors tested in a 72-h test. All adherent cell preparations showed significant cytocidal effects on target cells with the levels of cytotoxicity varying with the A:T ratio. The various tumor-cell lines differed markedly in their susceptibility to cytotoxicity. TU5 and G11 were most susceptible to the cytolytic capacity of human monocytes, with specific isotope release at an A:T ratio of 20:l against TU5 ranging in different individuals from 10.6 to 52.8 %.

Although CaLu and 1023 tumor cells were clearly more resistant than TU5 and G11 to the cytocidal activity of human monocytes, these target cells were also consistently lysed to a significant extent by monocytes from all donors tested.

In a more limited series of experiments (10 donors), [1261]dUrd uptake by target cells was measured as an estimate of cytostasis. As shown in Table 11, monocyte preparations consistently inhibited [1251]dUrd uptake of tumor cells at A:T ratios of 10 to 4O:l. In contrast to the varying susceptibility of different target cells to cytolysis, monocytes

22 MANTOVANI ET AL.

TABLE IV

KINETICS OF THE CYTOLYTIC EFFECTS OF CIRCULATING HUMAN MONOCYTES ON TUMOR CELLS

Incubation time (h)

24 48 72 Target cells Donor A:T ratio

I023

GI1

TU5 TA 20: I .- 2 13.3 (3) * 25.3 (1.4) * FV 20:1 5.5 (0.1) * 12.9 (1.8) * 34.5 (1.2) *

2:l 0 10.6 (1.3) * 14.1 (1.3) * 1 :1 0 7.3 (1.3) * 12.8 (1.4) *

40: 1 9.7 (0.6) * - 13.6 (1.4) * 20:l 8.4 (0.7) * - 12.7 (0.3) * SD

LP 40:l 4.2 (0.4) * 13.6 (1.8) * 34.7 (2.7) * 20: I 4.1 (0.9) * 12.3 (0.3) * 29.1 (1.5) *

0 8.4 (0.3) * 14.3 (1.1) * 1O:l 5:1 0 4.6 ( I .2) * 6.9 (0.7) *

DK 10: I 0 12.7 (0.3) * 30 (2.3) * DL 1O:l 0 7.7 (0.5) * 20.9 (0.6) * MM 40:l - 9.5 (0.7) * 20.4 (0.1 ) *

20:l - 6 (0.7) * 18.1 (0.5) * 10:l - 4.2 (0.5) * 16.6 (1.4):

CaLu AT 40: I 0 6.2 t0.3 * 11.5rt4.5 * BD 20:l - 13.1 (1.3) * 34.1 (2.4)

' Results are percentage specific lysis (&so). - Not tested. - * Significantly above spontaneous release values, p < 0 . 0 5 % . Spontaneous release levels for TU5 were 6% at 24 h, 13.6% at 48 hand 19% st 72 h ; for 1023 7.5%, 14.3% and 15.3%: for GI1 7.9% at 48 h and 15.8% at 72 h; and for CaLo 5.8% at 24 h, 11.6% at 48 h, and 15.3:/, at 72 h.

inhibited the growth of each of the cell lines to similar degrees.

Tables I and I1 include data obtained with monocytes separated from mononuclear cells by the two different techniques used in this study (i.e., adherence to plastic and rubber policeman versus adherence to microexudate-coated plastic and EDTA). In fact, as shown in Table 111, when the two procedures were used to prepare monocytes from the same mononuclear cell suspension, very similar levels of cytolytic and cytostatic activity were obtained.

In a number of experiments the kinetics of the cytolytic and cytostatic effect of human monocytes was investigated. As shown in Table IV, lysis of tumor target cells increased with the incubation time. Maximal isotope release occurred after 72 h

of incubation and no significant effect was measurable at 24 h except when high (20:l or 40:l) A:T ratios were employed with TU5 target cells. In contrast, peak inhibition of [lZ5I]dUrd uptake was observed early (24 h), with longer incubation times resulting in lower levels of cytostasis (Table V).

It was important to determine whether the cytotoxic activity of human rnonocytes measured by [3H]thymidine release and inhibition of [ 1261]dUrd uptake was actually a reflection of loss or inhibition of the target cells. Therefore, at the end of the incubation period, tumor cells were counted microscopically as well as by the isotopic procedures. As in Table V1, actual inhibition of TU5 tumor growth was detected, with the degree of inhibition at various times closely resembling the percentage inhibition of [le51]dUrd uptake.

TABLE V

KINETICS OF THE CYTOSTATIC EFFECT OF HUMAN MONOCYTES O N TUMOR CELLS

Incubation time (h)

24 48 72 Target cells Donor A :T ralio

TU5

1023

GI 1

TA FV

DK DL BD

20:1 ~. 89 (3.1) * 78.4 (4) * 20:. I 89.5 (3.8) * 73.9 (1.3) * 65.1 (4.6) * 2:l 21.3 (1.9) * 0 0 1 : I 14 (0.7) * 0 0

1O:l 72.5 (1.5) * 62.8 (3.8,' * 29.9 (2.1) * 1O:l 57.2 (9.1) * 26 (1.3) 10.9 (2.4) 20:l - 64.5 (5.5) * 48.5 (2.9) *

Resu!ts are percentage inhibition of [lBsIl]dUrd uptake ( ~ s D ) . - Not tested. - * Significantly different from cultures with target cells alone, p<O.05. Control uptakelevelifor TU5 were 5,120cpm at 24 h, 10,980cpm at 48 h and 13,250cpm at 72 h: for 1023 they were 2,810cpm at 24 h, 5,150 cpin at 48 h and 8.930;cpm at 72 h; and for GI 1 6,480 cpm at 48 h and 11,970 cpm at 72 h.


TABLE VI

CYTOTOXIC ACTIVITY OF HUMAN MONOCYTES AS DETECTED BY [aHJTHYMIDINE RELEASE,

INHIBITION OF [12511dUrd UPTAKE AND VISUAL COUNTING

Percentage Percentage Incubation p~~~~ inhibition ( ~ s D ) growth time 01) ' ,ysis of ['P611dUrd inhibition ( ~ s D )

uptake (visual counts)

24 5.5 (0.1) * 89.5 (3.8) * 75 (4.9) * 48 12.9 (1.8) * 79.3 (1.3) * 81.6 (5.8) * 72 34.5 (1.2) * 65.1 (4.6) * 69.2 (3.9) *

' Monocytes from donor FV were tested at an A:T ratio of 2O:l against TU5 cells. - * Significantly different from controls with target cells alone, p<O.O5.

Since all of the above studies were performed with a selected subpopulation of cells, it was important to determine how these levels of activity compared to those in unseparated mononuclear cells. The relative cytolytic capacity of different subpopulations of mononuclear cells separated by adherence was therefore investigated using TU5 cells as targets (Table VII). All cell fractions (unseparated, monocytes, non-adherent plus loosely adherent and non-adherent cells) showed significant cytolytic activity against TU5 target cells. When specific cytocidal activity of the various fractions was calculated, the fractions with higher content of monocytes had stronger cytolytic activity. The highest cytotoxicity was observed with monocytes (29.1% specific lysis at an A:T of 20:l and one LUlO = 7 . 5 ~ 1 0 ~ cells). At the other end of the spectrum, non-adherent cells ( ( 1 % non-specific esterase-positive) showed only 4.3% lysis at an A:T of 20:1, with approximately over ten times more cdls needed to produce 10% lysis. The weak cytolytic activity of non-adherent cells was consistently observed in five experiments; it never exceeded 6% lysis at the highest (4O:l) A:T radio tested and was only observed after a 72-h incubation

period. When the total number of LUlO recovered, in individual cell populations, from lo7 original mononuclear cells, was calculated by dividing the number of recovered cells by the value of 1 LUlO, it appeared that the adherent cell fraction (monocytes), despite having the highest relative specific cytolytic capacity, accounted for only a portion of the total cytotoxic activity in the unseparated population. Moreover, the LUlO recovery data indicated that cytolytic activity was not induced by the adherence techniques used to fractionate cells. Rather, if anything, some activity was lost during the fractionation procedures, with the activity in all fractions adding up to only 75% of that in the unseparated cells.

It seemed possible that some of the activity in unseparated mononuclear cells and in the non- adherent cell fraction was due to NK cells. It was also important to rule out the possibility that NK cells contributed to the activity of the adherent cell fraction. The NK activity of unseparated, adherent and non-adherent mononuclear cells was therefore investigated using the particularly sensitive K562 target cells, in a 4-h 61Cr release assay (Table VIII). In agreement with previous results (West et al., 1977), non-adherent cells were the most effective population, and no significant cytotoxicity was displayed by adherent cells under these expzrimental conditions.

Despite the lack of evidence for NK cells in the adherent cell fraction, it was important to rule out some role of a small number of contaminating T cells. The effect of treatment with anti-human T-cell serum and C on monocyte-mediated cytotoxicity was therefore investigated, using TU5 target cells (Table IX). Anti-T-cell serum-treated monocytes were as cytolytic as the untreated effector cells.

In a further effort to characterize the effector cell@) involved in the cytolytic activity of human adherent mononuclear cells, these cells were incubated with silica particles for 1 h before the addition of TU5 target cells. As shown in Table X,

TABLE VII

C Y r o L Y r I c AcrIvITY OF DIFFERENT MONONUCLEAR CELL FRACTIONS

LU'?

Effector cells ~~~~~ Recovery - (cells from 10 Percentage specific lysis (*sD) LU,, recovered Percentage

x lo-') mononuclear 40:l 2O:l 10:l 5 : l cells cells

Unseparated mononuclear 9 100 15.9 (0.8) * 8.9 (0.8) * 3.8k0.2 * 0 23 43.5 cells

Monocytes 94 6 34.7(2.7)* 29.1 (2.5)* 14.3+1.1 * 6.9(0.7)* 7.5 8 Non-adheren t

and looseIy adherent 4 83 11 (0.3) * 7.9 (0.9) * 0 0 35 23.7

Non-adherent < 1 74 5.9 (0.8) * 4.3 (0.4) * 0 0 80 9.2

Effector cells from donor LP were tested for 12 h at different A:T ratios against TUS target ce!ls. - Significantly above values for non- adherent, non-adherent and loosely adherent, and unseparated mononuclear cells, P ~ 0 . 0 5 . - a Significantly less than values for non-adherent and loosely adherent and unseparated mononuclear cells, p<O.OS. - * Significantly above spontaneous release, p<O.O5.

24 MANTOVANI ET AL.

TABLE VIII

NK ACTIVITY OF UNSEPARATED MONONUCLEAR CELLS, NON-ADHERENT CELLS AND MONOCYTES

ON %r-LABELLED K562 CELLS

Effector cells ’ % specific lysis ( ~ s D )

Unseparated 21.2 (1.9) * Non-adheren t 33.8 (2.4) * Monocytes 1.9 (0.8)

’ Effector cells from donor LJ were tested at an A:T ratio of 2O:l in a 4-h assay. - Significantly above spontaneous release, p i 0 . 0 5 .

exposure to silica markedly inhibited the cytolytic capacity of the monocytes, with a percentage specific lysis of 32.9 and 10.3 in the untreated and silica-treated groups, respectively.

Serum factors have been reported to modulate the expression of the cytotoxic activity of murine activated macrophages, with no cytotoxicity by mouse effector cells tested in adult sera from a variety of animal species including man (Hibbs et a!., 1977). It was therefore of interest to test the cytolytic potential of human monocytes isolated in RPMI 1640 medium supplemented with 20% human AB serum and tested in the same medium with 10% AB serum. As shown in Table XI, monocytes showed comparable cytotoxicity in the presence of FBS or human AB serum against TU5. To further rule out some requirement for FBS, G11 cells were passaged for some time (four passage generations) in RPMI 1640 with 10% AB serum rather than FBS. High levels of cytotoxicity by monocytes isolated and tested in AB serum were observed against the GlI/AB cells. Since spontaneous isotope release was significantly (p t0.05) higher with human AB serum than with FBS, no meaningful quantitative comparison can be made between cytotoxicity levels expressed by monocytes with the two different sera.

DISCUSSION

The results presented here show that adherent mononuclear cells isolated from human peripheral blood of normal healthy volunteers express significant cytolytic and cytostatic activity in vitro on human and murine tumor cells. Several pieces

TABLE 1X

EFFECT OF ANTI-HUMAN T-CELL SERUM ON MONOCYTB-MEDIATED CYTOTOXICITY

Monocytes treated with Percentage specific lysis ( ~ s D )

- 23.1 (0.4) * Rabbit C’ 19.2 (3.7) * An ti -T-cell serum 22 (2.1) * Anti-T-cell serum+C’ 20.8 (2.6) *

~~

Monocytes were obtained from donor KM and tested at 2O:l A:T ratio against TUS target cells. - Significantly above spontaneous release, p <0.05.

of evidence indicated that the effector cells were monocytes. More than 90% of the adherent cells were monocytes as assessed by morphologic and functional criteria. Moreover, adherent cells showed no cytotoxicity in vitro on K562 myeloid leukemia cells in a 4-h T r release assay, used as a sensitive measure of NK cell activity. A role of NK cells or other cells in the T-cell lineage was ruled out by the lack of inhibition following treatment with anti-human T-cell serum and C, which has been found very effective in eliminating cells with re- ceptors for sheep erythrocytes or with NK activity (R. Maca and J. Ortaldo, unpublished observations). Removal of monocytes by passage through a Sephadex G10 column, a procedure which, in agreement with previous data (West el al., 19771,

TABLE X

EFFECT OF SOLICA O N THE CYTOLYTIC ACTIVITY OF HUMAN MONOCYTES AGAINST

TUS TARGET CELLS

[*H]thymidine release Monocytes Silica cpm (+so) % lysis

- - 1,449 (15) 18.4 - + 1,755 (65) 22.4 + - 4,026 (59) * 51.3 + t 2,565 (43) * 32.7

SDS 7,854 (32)

’ Monocytes from donor CF were tested for 72 h at an A C T ratio of 2O:l. - Significantly reduced below results with untreated monocytes. - * Significantly above spontaneous release (with or without silica), pi0.05.

did not inhibit NK activity on K562 cells, significantly reduced the cytolytic activity of peripheral blood mononuclear cells on TU5 target cells in a 72-h[3H]thymidine release assay. Finally, silica, a well-known macrophage toxin (Allison et al., 1966) which can interfere with macrophage cytotoxicity in rodents (Keller, 1978), markedly inhibited the cytolytic capacity of the human adherent mononuclear cells.

When appreciable cytotoxic activity of the separated monocytes was seen, there was some concern that the isolation procedures caused alter- ation of the monocytes. However, unseparated mononuclear cells were also cytolytic in vitro against TU5 target cells, and in experiments with Sephadex G10 column passage, most of the activity was shown to be due to adherent cells. Moreover, the cytotoxic activity of purified monocytes was similar, irrespective of the method used for separation (adherence to plastic and rubber policeman versus adherence on microexudate-coated flasks and EDTA) and was also detectable with monocytes which were never exposed to FBS.

In the tests on the various fractions of mononuclear peripheral blood cells, it appeared that more than half of the total cytotoxic activity was associated with the fraction of loosely adherent


TABLE XI

CYTOLYTIC ACTIVITY OF MONOCYTES ISOLATED AND TESTED IN MEDIUM SUPPLEMENTED WITH HUMAN AR SERUM OR FETAL BOVINE SERUM

Monocytes % ['Hlthymidine release (&so)

and tested in - a 40:l 20:l 2:1 1:l Target cells isolated

TU5 AB serum 10.7 (0.8) 39.1 (2.7) * 34.8 (1.2) * 15.2 (1.2) * 13.6 (0.7) FBS 5.6 (0.3) 33.7 (1) * 31.1 (2) * 4.8 (0.9) 4.5 (1.5)

Gll/AB AB serum 12.1 (0.2) 28.7 (2.1) * 21.8 (0.3) * NT NT FBS 4.1 (0.5) 24.1 (1.8) * 18.9 (0.6) * NT NT

Monocytes were obtained from donors MCE and MS and tested in a 48-h assay against TUSiand GI I/AB target cells, respectively. - Spontaneous release significantly below rhat in the presence of human AB serum. Target cells alone. - A:T ratio. - Not tested. -

p<O.O5. - * Significantly above spontaneous release, p 10.05.

plus non-adherent cells. Since most of this reactivity was removed by depletion of the monocytes by passage through Sephadex G10, it seems that the cytotoxic activity was due to monocytes which were not sufficiently adherent to remain attached to plastic but which could adhere to G10. The purified, firmly adherent monocyte fraction usually included only about half of the monocytes in the unseparated mononuclear cell population, and the other, less adherent monocytes appear to account for at least as much cytotoxic activity. These results are similar to those previously obtained with macrophage-mediated cytostatic activity in mice and rats (H. Kirchner, J. R. Oehler and R. B. Her- berman, unpublished observations). After preparation of plastic-adherent macrophages, much of the cytotoxic activity remained with the unattached cells. Yet, virtually all of the reactivity could be removed by passage through a rayon column or upon removal of phagocytic cells by treatment with carbonyl iron plus magnet (Kirchner et al., 1975; Oehler et al., 1977).

The cells which were non-adherent to Sephadex G10 columns, which contained less than 1 % mononuclear phagocytes, showed significant, though weak, cytolytic activity against TU5 target cells. The nature of the non-adherent effector cells remains to be defined. It is possible that the activity was due to some weak sensitivity of the target cells to NK cell reactivity during the long incubation period. Alternatively, the few remaining monocytes or monocyte precursors might have accounted for this cytotoxicity.

The cytotoxic reactivity of human monocytes was measured both as lysis and stasis of tumor target cells. The question of the relationship between these two events remains unsettled. No consistent correlation was observed between cytolysis and cytostasis of target cells. Peak growth inhibition, as detected by an [1261]dUrd uptake assay, occurred at 24 h, lower levels being observed thereafter. On the contrary, maximal [3H]thymidine release was observed at 72 h, with little or no cytolytic effect being measurable after 24 h of culture. Even more relevant to this issue appears to be the obser- vation that there was no evident correlation between the susceptibility of different target cells to monocyte- mediated cytolysis and to cytostasis. The four

tumor-cell lines tested showed comparable levels of monocyte-mediated inhibition of [1261]dUrd uptake whereas TU5 and G11 were clearly more susceptible than 1023 and CaLu target cells to the cytolytic activity of these effector cells. Similar findings were reported by Keller (Keller, 19766, 1978) using rat peritoneal macrophages as effectors. Although different explanations are tenable, one may speculate that monocyte-mediated lysis of target cells is a multi-step phenomenon, with one of the early events being inhibition of cell proliferation, and that target cells equally susceptible to cytostasis may differ in their relative resistance to subsequent development of lethal damage.

In a limited number of experiments at low A:T ratios (1:l) significant enhancement of the pro- liferative capacity of 1023, G11 and CaLu target cells was observed. There are a number of reports that rodent mononuclear phagocytes can stimulate the proliferation of tumor target cells, particularly when the tumor cells by themselves are growing poorly in tissue culture (Namba and Hanaoka, 1972; Nathan and Terry, 1975; Hewlett et al., '977; Evans, 1976) or when macrophages are tested at low (<l:l) A:T ratios (Keller, 1973, 19766). The present study was primarily designed to characterize the cytotoxic activity of human monocytes. Therefore, the above-mentioned experimental conditions (poor growth in tissue culture cells or A:T ratios of <l: l) , which in rodents appear to allow optimal expression of the growth- enhancing capacity of mononuclear phagocytes, usually were not employed. Hence further studies are needed in order better to establish the capacity of human monocytes to stimulate target cell proliferation.

Spontaneous cytotoxic activity of macrophages from normal rodents has been reported only in a limited number of studies, mostly with peritoneal macrophages used as effectors (Meltzer, 1976; Miller and Feldman, 1976; Keller, 1978). Com- parisons of animal data with the results reported here appear difficult, since circulating monocytes were examined in the present study as compared to the inflammatory or resident tissue macrophages used in experimental systems. In fact, circulating monocytes have been reported to differ from mature macrophages in many respects, including

26 MANTOVANI ET AL.

morphology, phagocytic activity, adherence (Territo and Cline, 1976) and capacity to mediate antibody- dependent cellular cytotoxicity o n tumor cells (Mantovani et al., 1977~). It is of interest that, in a parallel investigation, we have demonstrated that circulating mononuclear phagocytes of normal mice have considerable cytotoxic reactivity against some of the target cells used here (Tagliabue et al., in preparation).

The mechanism(s) responsible for inducing the cytotoxicity against tumor cells of circulating human mononuclear phagocytes is a matter of speculation. Murine peritoneal macrophages obtained after stimulation with inflammatory agents have been reported to be more responsive to lym- phokine than the resident population (Ruco and Meltzer, 1978), and, more directly, we have recently observed that circulating mouse mononuclear phagocytes are more responsive to macrophage activating factor(s) than are resident peritoneal macrophages (Tagliabue ei al., in preparation). Thus it can be envisaged that lymphokines released in vivo as a consequence of exposure to environ- mental stimuli may account for the significant levels of cytotoxicity by circulating human monocytes.

The possible in vivo significance of macrophage cytotoxicity as a defense mechanism against neoplasia still needs to be documented. However,

in experimental tumor models, there is evidence that cells of the monocyte-macrophage series may play a role in the regulation of tumor growth and metastasis (Birbeck and Carter, 1972; Eccles and Alexander, 1974; Russell and Mclntosh, 1977; Wood and Gillespie, 1975; Zarling and Tevethia, 1973). Mononuclear phagocytes infiltrate human tumors (Gauci and Alexander, 1975; Wood and Gollahan, 1977) and recently macrophages isolated from human ascitic ovarian tumors have been shown to modulate tumor growth in vitro (Manto- vani et al., 1978). Thus, the cytotoxic activity of human peripheral blood monocytes demonstrated in the present study might represent one line of natural defense and the monocyte-mediated cytotoxicity assays used here may provide useful tools to better understand the immunobiology of mononuclear phagocytes in human neoplasia.

ACKNOWLEDGEMENTS

Albert0 Mantovani was supported by a Yamagiwa Yoshida study grant of the International Union Against Cancer, Geneva, Switzerland, and by CNR, Rome, Italy. Laboratory studies were supported in part by Contract N01-CB63975 from the Division of Cancer Biology and Diagnosis of the National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.

ACTIVITBS CYTOLYTIQUE ET CYTOSTATIQUE DES MONOCYTES HUMAINS CIRCULANTS SUR LES CELLULES TUMORALES

Les auteurs ont constate que les monocytes du sang pCriphCrique de donneurs adultes normaux ont une activite cytotoxique appreciable sur des IignBes de cellules tumorales murines et humaines. Les cellules adh6rentes (>90 % de monocytes) ont CtC obtenues partir du sang peripherique de 29 donneurs volontaires normaux par I’une des deux methodes suivantes: adherence au plastique et recuperation au moyen d’une spatule de caoutchouc, ou adherence B du plastique enduit de micro- exsudat et exposition A l’acide ethylhe-diamine tetra-acktique. La capacite cytolytique a 6tC test& par incubation des cellules effectrices pendant 72 h avec des lignees de cellules tumorales murines et humaines prkmarquees a la thymidine tritike. La cytostase a 6tB BvaluCe par inhibition de I’incorporation d’iododksoxyuridine marquee a 1’1 On a utilise comme celliiles cibles une lignee de cellules rha les murines transformees par le SV40 (TU5), un sarcome murin chimio-induit (1023), une lignee provenant d’un cancer mammaire humain (GI]) et un- IignCe provenant d’un cancer pulmonaire humain (CaLu). A des rapports cellules attaquantes/cellules cibles de 1 : I a 40:1, les preparations de inonocytes avaient une activite cyiolytique et cytostatique significative contre les cellules cibles tumorales. Ces dernieres etaient plus ou moins sensibles a l’activite cytolytique, alors que les niveauv d’activite cytostatique Ctaient comparables quelle que soit la cible; les cellules TU5 et GI1 etaient plus sensibles que les cellules 1023 et CaLu B I’activitk cytolytique des monocytes huniains et ce sont les TU5 que I’on a utilisees dans la plupart des expBriences suivantes. La liberation d’isotope par les cellules cibles piemarquees n culmine apres 72h d’incubation, alorsque le pic d’inhibition de I’incorporation d’ldllrd 11’ PL]apparaissait aprPs 24 h deculture.Que I’on utilise I’une ou l’autre des methodes exposees ci-dessus pour isoler les monocytes, le pouvoir cytotoxique de ceux-ci restajt le mbme. Les monocytes avaient une activitt cytotoxique plus forte que les cellules mononucleaires non sCparees, et les cellules non adhkrentes avaient des effets cytotoxiques minimaux. La cytotoxicite spontanee ne semblait pas jouer un grand rBle dans ces experiences, puisque les cellules adherentes ne lysaient pas les cellules K562 dans un test de liberation du Crsl d’une duree de 4 h. Un traitement au serum anti-cellules T humaines et au complement n’inhibait pas la capacite cytotoxique des preparations de monocytes, alors que I’exposition B des particules de d i c e inhihait significativement cette activite. La cyto- t3xicitC B mediation monocytaire contre les cellules tumorales apparaissait en presence de serum fe ta l bovin, mais aussi de serum humain AB.

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cytolytic and cytostatic activity on tumor cells of circulating human monocytes

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