CELLULAR IMMUNOLOGY 87, 494-503 (1984)

Monoclonal Antibody-Defined Surface Markers of Effector Cells Involved in Human Monocyte Cytotoxicity’

ANTONELLO VILLA,* GIUSEPPE Pm,-/ VINCENZO Rossr,t DoMENICO DELIA,+ AND ALBERTO MmTovAbnt*2

*Center for the Study of Peripheral Neuropathy and Neuromuscular Diseases, Department of Pharmacology, University of Milan; flstituto di Ricerche Farmacologiche “Mario Negri, ”

Via Eritrea 62, 20157 Milan; and #Istituto Nazionale per lo Studio e la Cura dei Tumori, Via Venezian, 1. 20133 Milan, Italy

Received February IO, 1984; accepted March 29, 1984

Human adherent peripheral blood mononuclear cells were cytotoxic in vitro against the murine TU5 line in a 48-hr [3H]thymidine-release assay. Monocyteenriched adherent cell preparations contain a small and variable (usually less than 5%) contamination with large granular lymphocytes as assessed by morphology and staining with monoclonal antibody markers B73.1 and HNKl. To assess whether killing was in fact mediated by monocytes, mononuclear cells or monocyte- enriched preparations were separated using monoclonal antibodies directed against mononuclear phagocytes (Mo2, UCHMl, B44.1) or natural killer (NK) cells (B73.1 and HNKl), and a flu- orescence-activated cell sorter. Cells positive for monocyte markers were highly cytotoxic against TU5, whereas negative cells were not. B73.1+ or HNKl+ cells had little or no activity. Cytotoxicity of cells positive for moncqte markers (Mo2, UCHM 1, B44.1) was augmented by in vitro exposure to lymphokines or less frequently to interferon (IFN). However, cells negative for these monocytes markers were also stimulated to kill TU5 by lymphokine or IFN to an extent similar or greater than that of positive ones. IFN or lymphokines induced killing of TU5 by monocyte-depleted, B73. l-positive, lymphoid cells. These observations demonstrate that human monocytes do kill tumor cells, either in the absence of deliberate stimulation or after exposure to agents such as lymphokines. However, the possible contribution to “monocyte” cytotoxicity of minor NK cell contaminants must be taken into account particularly when agents such as IFN and lymphokines are applied, even when a relatively NK-cell-resistant target such as TU5 is used.

INTRODUCTION

Human cell preparations enriched in monocytes or macrophages are cytotoxic in vitro to susceptible tumor cell lines of human or murine origin (l-lo). Several lines of evidence suggest that effector cells belong to the monocyte-macrophage lineage; these include their capacity to adhere, their partial susceptibility to silica, their tissue distribution, and the spectrum of target cell susceptibility (reviewed in Ref. (11)). Monocyte preparations contain a small, but appreciable contamination of lymphoid cells. Two recent studies suggested that contaminating lymphoid cells, in particular

’ This work was supported by finalized project “Oncology” from CNR, Italy, and Grant ROl CA 26824 from NCI. The generous contribution of the Fondazione Angelo e Angela Valenti, Milan, Italy, is gratefully acknowledged.

2 To whom correspondence should be addressed.

494

0008-8749184 $3.00 CopyrigJtt 8 1984 by Academic Pm.% Inc. All rights of reproduction in any form merved

MONOCYTE CYTOTOXICITY 495

large granular lymphocytes (LGL) with natural killer (NK) activity, account for most, if not all, of the cytotoxicity of “monocytes” against human lines (12, 13). These studies prompted us to reexamine the nature of effector cells involved in killing of the murine TU5 line (2, 4, 11) using monoclonal antibodies (MoAb) and the fluo- rescence-activated cell sorter (FACS).

MATERIALS AND METHODS

Cell culture reagents. The following reagents were used for culture of cell lines, separation of effector cells, and cytotoxicity assays: pyrogen-free, phosphate-buffered saline (PBS) for clinical use (Bieffe, Italy); pyrogen-free distilled water (Bieffe, Italy); RPM1 1640 medium (10X concentrated; GIBCO, Glasgow, Scotland); Eagle’s min- imum essential medium (MEM) (GIBCO); glutamine (GIBCO); penicillin and strep tomycin for clinical use (Farmitalia, Milan, Italy; Squibb, Rome, Italy); gentamicin (GIBCO); aseptically collected fetal bovine serum (FBS, Lot No. 2BO27 Microbiological Associates, Walkersville, Md.; Lot No. 100222 Sterile Systems Inc., Salt Lake City, Utah); aseptically collected human cord serum (CS) prepared in this laboratory.

The routinely employed tissue culture medium was RPM1 1640 with 2 mM glu- tamine, 50 pg/ml gentamicin, 10% CS or FBS, hereafter referred to as complete medium.

Target cells. The tumor cell lines used in the present study were TUS (14) and K562 (15) lines. The lines were maintained by serial passages in RPM1 1640 medium with 20% FBS. Nonconfluent cultures of SV40-transformed mKSA TU5 kidney line (TU5) were maintained overnight at 37°C in 5 ml growth medium with 0.5 &i/ml of [methyl-3H]thymidine (5 Ci/mmol, Radiochemical Centre, Amersham, U.K.) in 25-cm* tissue culture flasks (Sterilin, Teddington, Middlesex, U.K.).

Cells were detached by a 5-min incubation with 2 ml of 0.25% trypsin-0.02% EDTA in PBS and washed twice with 50 ml medium before resuspension in growth medium.

In order to measure NK activity, the K562 line, derived from a human myeloid leukemia, was used. Tumor cells ( l-5 X 1 O6 in 1 ml growth medium) were incubated with 20-50 mCi of 5’Cr (Radiochemical Centre, Amersham, U.K.) at 37°C for 45 min. Labeled cells were washed twice with medium before the NK-lysis assay.

These cell lines were negative for mycoplasma contamination as determined by Hoechst dye staining (16).

Peripheral blood mononuclear cells (PBM). Heparinized venous blood obtained from healthy donors was diluted 1:5 with PBS, and 40 ml was then placed on 10 ml Ficoll-Hypaque (Eurobio, Paris, France) for centrifugation at 400g for 20 min at room temperature. PBM were collected at the interface, washed with PBS, and sus- pended in complete medium.

Isolation of “monocytes” by adherence. PBM were fractionated by adherence on plastic essentially as previously described in detail (2,4). Briefly, PBM were suspended in complete medium and allowed to adhere on FBS-precoated plastic flasks (Falcon Plastics, Oxnard, Calif.) for 1 hr at 37°C in 5% CO1 in air. Nonadherent cells were removed by gently washing with jets of PBS while adherent cells were detached by 5 min exposure to 0.02% ethylene diaminetetraacetic acid (EDTA; Carlo Erba, Milan, Italy). Collected adherent and nonadherent cells were washed and used as effecters in the cytotoxic assay. For the experiments in which we wanted to have nonadherent

496 VILLA ET AL.

cells profoundly depleted of monocytes, adherence on plastic was repeated twice, followed by passage through nylon wool (17) and carbonyl iron ( 18). Fractionated cells were characterized by morphology, nonspecific esterase, and reactivity with monoclonal antibodies as described below.

Fractionation of cell populations by fluorescence-activated cell sorter (FACS). MoAb Mo2 which is reactive with monocytes (19) was obtained through the courtesy of Dr. R. F. Todd (Sidney Farber Cancer Institute, Boston, Mass.) and used at a final dilution of 1:40. UCHMl MoAb, which is reactive with monocytes and tissue macrophages, was a kind gift of Dr. P. C. L. Beverley (University College, London), and it was used at a dilution of 1:5. Dr. Trinchieri (Wistar Institute, Philadelphia, Pa.) provided us with MoAb B73-1 (1:80 diluted) which is reactive with NK cells and polymor- phonuclear neutrophils (PMN) (20) and with B 44.1 (1: 100 diluted) which is reactive with monocytes (21). HNKl MoAb (22), used at a 20 &ml concentration, was a generous gift of Dr. T. Abo (University of Alabama, Birmingham). Under these conditions the MoAb did not affect cytotoxicity against K562 or TUS, measured as described below.

The cells to be sorted were labeled by indirect membrane immunofluorescence. PBM (4-6 X 107) were washed twice in PBS with 5% FBS and then were incubated at a concentration of 5 X lo7 cells in 1 ml of MoAb at the appropriate concentration for 30 min at 4°C. The cells were then washed three times in PBS, 5% FBS. The second incubation was carried out at 4°C for 30 min using fluoroscein isothiocyanate (FITC)-conjugated goat F(ab’)Z anti-mouse Ig (New England Nuclear Corp., Boston, Mass.). The sterile separations were carried out with a FACS IV cell sorter (Becton- Dickinson, Irvine, Calif.). For the experiments presented here, laser output was 1000 mW at 488 nm. Sorting of cells was carried out according to standard FACS IV specifications with a three-drop deflection criterion using a 70-pm nozzle. In some experiments, the sorted cell populations were reanalyzed by FACS, confirming the positivity or negativity (>98%) of the separated cells. Cytocentrifuge preparations of each sorted cell population were stained with May-Griinwald-Giemsa stain. In some of the experiments slides were stained for nonspecific esterase. At least 300 cells per slide were identified morphologically.

NK cytotoxicity assay. “Cr-labeled K562 cells (2 X 104) were cultured for 4 hr at 37°C with different attacker to target cell (A:T) ratios (25:1, 12:1, 6:l) in 0.2 ml growth medium in round-bottomed wells of microplates (2). Isotope release was calculated as 100 X A/B, where A is the isotope in the supematant and B is the total incorporated radioactivity released by incubation with 1% sodium dodecyl sulfate (SDS) in water. Specific lysis was calculated by subtracting spontaneous isotope release of tumor cells alone. Spontaneous “Cr release from K562 cells was 0.5-1.5% per hour of incubation.

‘Monocyte” cytotoxicity (TUS-lysis) assay. Cytolytic activity was measured as [3H]thymidine release from prelabeled TU5 cells over a period of 48 hr as previously described (2, 4). Briefly, tumor cells( 104/sample) were incubated (37°C 5% CO* in air in 0.3 ml of RPM1 1640 medium with 10% human cord serum (occasionally FBS) in 6.4-mm flat-bottomed culture wells. A range of attacker to target cell (AT) ratios (5: 1, 10: 1, 20: 1, 40: 1) was usually used. When not specified, data refer to an A:T of 1O:l. Tumor cell growth was checked daily with an inverted microscope. Isotope release percentage was calculated as 100 X A/B, where A is the isotope released in the supematant and B is the total radioactivity released by incubating target cells in

MONOCYTE CYTOTOXICITY 497

1% SDS. Specific lysis was calculated by subtracting spontaneous release of TU5 cells in the absence of mononuclear phagocytes, which did not exceed 25%. In some experiment, effector cells were exposed to interferon (IFN) or lymphokine supernatants (4, 23). IFN used was a human hybrid recombinant preparation (IFN A/D, from Hoffmann-LaRoche Inc., N.J.) and an IFN-@ (Serono, Rome, Italy). Only results with IFN A/D at the optimal concentration of 100 units/ml are presented. Lymphokine supernants, from phytohemagglutinin (PHA)-pulsed lymphocytes or from mixed lym- phocyte cultures, prepared as described previously (23) were routinely used at the optimal l/3 dilution. Control conditioned medium of unstimulated lymphocytes had no effect on cytotoxicity and this control is not presented in the tables. Three replicates per experimental group were used and significance was assessed by Student’s t test.

RESULTS

In agreement with previous studies (2,4, 1 l-l 3), PBM were significantly cytotoxic in vitro against TU5 cells in a 48-hr [3H]thymidine release assay, as illustrated by the experiment reported in Table 1. Cytotoxicity of monocytes in the absence of deliberate stimulation was best observed using human CS as a serum supplement (24). Non- adherent monocyte-depleted cells had little cytotoxic activity under these conditions, whereas adherent cells consisting mainly of monocytes (93% in the experiment shown in Table 1) had considerable (18.8% at an A:T of 10: 1) cytotoxic activity against this target. The converse was true for ‘lCr labeled K562 cells, that were essentially killed by nonadherent cells (Table 1). Adherent cells consisted mainly of monocytes (as assessed by morphology, esterase staining, and phenotypic analysis with monoclonal antibodies) as shown in Table 2 where results of this series of 30 consecutive experiments are summarized. However, adherent cell preparations contained a minor and variable, but appreciable (from 0.5 to 7% depending on the marker and the individual experiment considered) contamination of cells with LGL morphology (25,26) and with positivity with NK cell markers. It was therefore of interest to evaluate whether adherent NK or NK-like cells were in fact responsible for killing of TU5 cells. PBM or adherent cells were fractionated according to the expression of surface markers using a FACS apparatus. Figure 1 shows one representative experiment in which PBM were separated using Mo2 as a marker. Rapid killing of “Cr-labeled K562 cells provided a functional control of the separations obtained and, as illustrated in Table 3, NK activity was

TABLE 1

Cytotoxicity by Adherent and Nonadherent PBM against TU5 and K562

% Specific lysis k SD

Effector cells” % Monocytes” % LGL” TU5 b K562’

PBM 28.8 5.1 10.9 f 2.2 22.5 + 4.0 Nonadherent cells 0.8 7.1 -1.9 + 0.1 29.1 + 4.0 Adherent cells 93.0 1.2 18.8 + 2.2 4.8 + 1.0

’ Assessed by morphology. Values for monocytes were confirmed by staining for esterase. b 48-hr [3H]Thymidine-release assay. ’ 4-hr “Cr-release assay.

498 VILLA ET AL.

TABLE 2

Cellular Composition of Adherent and Nonadherent Cell Fractions

Marker Assigned specificity PBM Nonadherent cells Adherent cells

Morphology (Giemsa) Morphology (Giemsa) Esterase Mo2 B44.1 UCHM I B73. I HNKI

Monocytes LGL Monocytes Monocytes Monocytes Monocytes NK cells NK cells

3 I .7 (11.2-49) 8.9 (2.2-35.7)

27.4 (24-29.8) 17.9 (10.9-27.3) 16.2 (14.5-18) 18.2 (1 l-34) 9.8 (1.9-27.6)

15.8 (9-24)

1.7 (0.6-5) 12.6 (4.8-22)

1.1 (0.5-1.7) 0.3 (O-0.5) 0.1 (O-3) 1.5 (O-3) 9.1 (5-15.2)

12.6 (4-28)

85.8 (64-96) 1.6 (l-3.2)

86 (70-94) 71 (63-79) 76.5 (69-80.5) 80.2 (64-95) 2 (0.5-3.4) 4.8 (1.2-7)

Note. Results (mean with range in parentheses) are compounds of 3-30 experiments performed.

mediated by cells that were negative for monocyte markers (UCHM 1, Mo2, B44.1) and positive for LGL markers (B73.1, HNKl), as expected. Cytotoxicity against [3H]thymidine-labeled TU5 cells was mediated by cells distinct from NK cells, as judged on the basis of reactivity with monoclonal antibodies (Table 4). In the rep resentative experiments shown in Table 4, cells negative for UCHM 1, Mo2, or B44.1 had little cytotoxicity on TU5, with specific lysis (A:T = 20: 1) of 2.5, 8.8, and 12.0% respectively. In contrast UCHM I+, Mo2+, and B44.1+ cells had 40.2,60.7, and 58.6% specific cytotoxicity, respectively. On the other hand, cells positive for NK cell markers (B73.1, HNKl) had little or no cytotoxicity against TU5. In the experiments presented in Table 4, PBM were used as a starting population, but similar results were obtained in a more limited number of assays in which monocyte-enriched adherent cells or Percoll-enriched monocytes (27) were used for labeling and sorting.

wo2+ .-..

; ;... ; ‘:.

i : %..

. . . .

: PBM ‘.... ‘\ : _/ - -_ . . . . .

f--’ M$J2- -i -.17.-

Light scatter Fluorcsccnce intensity

FIG. 1. FACS analysis of PBM sorted according to the expression of Mo2. The left and right panels show the light scatter and fluorescence of the sorted populations, respectively.

MONOCYTE CYTOTOXICITY 499

TABLE 3

NK Activity of Cells Separated According to Surface Markers by FACS

Effector cells

Input B73.1+ B73.1-

Input HNKl+ HNKl-

Input UCHMI+ UCHMI-

Input Mo2+ Mo2-

Input B44.1+ B44.1-

% Specific lysis + SD”

13.8 + 1.1 34.6 + 0.7 6.4 + 2.0

31.0 + 0.4 41.0 + 4.0 14.0 f 0.4

25.0 + 4.2 1.2 + 1.0

18.5 f 3.0

20.9 f 2.0 1.3 z!c 1.1

31.2 f 3.5

11.8 + 1.1 N.D.

8.8 + 0.4

’ Results represent specific lysis of K562 cells in a 4-hr “Cr-release assay at an A:T ratio of 12: 1.

TABLE 4

Cytotoxicity against TU5 of PBM Separated According to Surface Markers by FACS

A:T Ratio

Effector cells 5:l lo:1 2o:l 4o:l

Input B73.1+ B73.1-

Input HNKI+ HNKI-

Input UCHMI+ UCHMI-

Input Mo2+ Mo2-

Input B44. I + B44. I -

N.D.’ N.D. N.D.

11.7 +- 3.4 -1.9 f 1.4 21.7 k 3.1

N.D. N.D. N.D.

11.3 4 1.0 25.3 f 1.0

0.8 + 0.4

11.4 k 0.9 16.2 k 0.7 1.6 f 1.7

% Specific lysis + SD”

3.7 k 2.6 12.7 f 2.5 N.D. -2.6 k 1.9

4.8 k 0.3 6.2 f 1.9

26.5 f 1.8 40.2 f 2.9 -2.0 f 0.6 2.4 f 0.9 45.8 f 0.6 51.5 f 4.0

4.3 f 0.7 12.0 f 1.7 15.1 + 2.0 40.2 + 4.9

-0.3 f 0.4 2.5 zk 0.1

24.6 2 2.8 45.0 + 1.3 56.9 k 1.0 60.7 f 2.2 4.0 f 1.0 8.8 t 0.7

16.4 f 1.9 28.1 f 6.2 25.9 f 1.4 58.6 f 1.7 4.4 f 1.3 12.0 + 0.8

33.3 f 0.7 N.D.

14.5 f 2.3

57.0 + 2.8 N.D.

70.2 I! 0.7

29.0 f 5.0 N.D.

11.7 + 1.5

64.3 f 3.4 N.D.

19.7 f 0.6

N.D. N.D. N.D.

a Results represent specific lysis of TU5 in a 48-hr [3H]thymidine-release assay at A:T ratios ranging from 5: I to 40: 1.

’ Not determined.

500 VILLA ET AL.

In the experiments discussed so far cytotoxicity was measured in the absence of any deliberate stimulation, using cord serum since cytolysis values were higher using this supplement (24). IPN and lymphokine supematants augment the cytotoxicity of human monocyte-enriched adherent cells (4, 11, 23, 28, 29) and this finding was confirmed in the present study (Table 5). However, nonadherent cells, containing very few monocytes (< 1% in the experiment in Table 5) and with little or no “spon- taneous” killing of TU5 (0.7% in the experiment shown in Table 5), were boosted by IFN or lymphokines to an extent comparable to that monocytes. The effector cells responsible for IPN- or lymphokine-boosted killing of TU5 were characterized with monoclonal antibodies (Table 6). Lymphokine supematants significantly augmented killing of cells positive for monocyte markers (Mo2, UCHMl, or B44.1) in four of five experiments. In two experiments (Nos. 5 and 7) cells negative for Mo2 and B44.1 were also significantly stimulated by lymphokines. When IPN was used as a stimulus (recombinant hybrid A/D or, in one assay not shown, natural /3), significant stimulation of cytotoxicity was detected with cells positive for monocyte markers in only one of eight experiments performed. In all experiments, cells negative for Mo2, UCHMl, or B44.1 were significantly stimulated by IFN in terms of killing of TU5.

The experiments presented in Tables 5 and 6 show that nonadherent, Mo2-, UCHMl-, B44.1- cells have appreciable cytotoxicity against TU5 cells in a 48-hr thymidine-release assay after stimulation with IPN or lymphokine. To characterize these IFN- or lymphokine-induced effecters against TU5, PBM were profoundly depleted of monocytes by adherence on plastic, nylon wool, and carbonyl iron in sequence (to avoid the confusing contribution of mononuclear phagocytes) and sep arated according to the expression of the NK marker B73.1.

As shown in Table 7, which shows results of one out of two experiments performed, IPN-induced effector cells cytotoxic to TU5 in monocyte-depleted cell preparations were B73.1+, suggesting a relationship with NK cells.

DISCUSSION

The results reported here show that human mononuclear cells spontaneously cy- totoxic against the relatively NK resistant TU5 line express the surface markers Mo2, UCHMI, and B44.1, characteristic of mononuclear phagocytes. In the absence of stimuli, cells positive for B73.1 or HNKl, which mediate NK activity against K562 cells (20, 22), had little activity against [3H]thymidine labeled TU5 cells under these

TABLE 5

Effect of IPN and Lymphokines on Cytotoxicity against TU5 of Adherent and Nonadherent PBM’

% Specific lysis + SD

Effector cells Medium

PBM 7.7 + 1.4 Nonadherent cells 0.7 + 0.2 Adherent cells 14.9 * 1.2

IFN

34.6 +- 0.2** 24.2 + l.O** 39.9 f 5.3**

Lymphokine

46.0 f 1.3** 48.8 k 3.5** 43.1 + 2.7**

’ 48-hr [3H]thymidine-release assay. ** Significantly above cytotoxicity of effector cells incubated with medium, P < 0.0 I.

MONOCYTE CYTOTOXICITY 501

TABLE 6

Effect of IFN and Lymphokines on Cytotoxicity against TU5 of FACS-Separated PBM

% Specific lysis + SD”

Expt Effector cell Medium IFN Lymphokines

Input Mo2+ Mo2-

Input Mo2+ Mo2-

Input Mo2+ Mo2-

Input Mo2+ Mo2-

Input Mo2+ Mo2-

Input B44+ B44-

Input B44+ B44-

Input UCHMl+ UCHMl-

3.5 f 1.4 5.4 * 1.1 0.2 * 0.4

6.8 f 0.6 10.6 f 0.8 2.8 f 1.5

21.1 k 0.9 19.0 f 2.5 4.8 + 0.2

5.4 + 0.5 3.4 + 0.2 5.5 + 1.2

10.5 f 0.4 15.6 f 2.6

-0.5 + 0.7

4.1 + 0.5 10.4 -t 0.5

-1.2 f 0.7

14.2 + 3.5 20.0 f 2.2

5.6 + 1.5

2.8 f 1.8 35.3 f 3.2 -1.1 f 1.1

14.9 f 0.3** 4.9 It 1.2 6.6 * 1.4*

41.0 + 3.0** 11.6 + 3.0 25.6 f 1.4**

44.2 f 9.2+ 41.0 f 3.5** 24.7 f 0.4**

22.9 f 2.6” 5.2 f 1.6

16.0 k 0.6*

22.1 + 3.1* 14.2 + 1.4 11.0 f 1.0*

21.7 f 3.4* 15.1 Z!I 4.2 7.0 f 0.7:

29.5 + 5.7* 25.5 + 3.2 16.3 f 1.6*

11.4 + 4.4* 16.0 + 4.8 7.9 + 1.0*

8.1 f 1.2; 5.0 f 0.8 1.4 +_ 0.4

- - -

- - -

9.6 f 1.0 9.3 f 1.3* 6.1 + 0.4

25.4 + 2.7* 32.1 + 4.3* 6.2 + 1.4*

- -

19.7 + 5.3: 28.2 + 2.2s 9.3 f 0.9*

11.2 f 1.1: 42.1 f O* 0.9 ic 0.8

’ Lysis of TU5 in a 48-hr [‘Hlthymidine-release assay. * P < 0.05 versus medium. ** P < 0.01 versus medium.

conditions. Two recent studies showed that lymphoid cells, and NK-like cells in particular, are present in appreciable numbers in monocytes prepared by adherence (12, 13, G. Trinchieri, personal communication), an observation confirmed in the present study: monocyte preparations contained a variable, but detectable proportion of cells with LGL morphology and positive for B73.1 or HNKl . It has been suggested that these contaminating cells may play an important role in spontaneous killing of highly NK-sensitive human lines by “monocytes” (12, 13) or “monocytes” sponta- neously detached after overnight incubation (30).

Lymphokine supernatants and IFN have been shown to augment human monocyte or macrophage cytotoxicity (4, 11, 23, 28, 29). Cytotoxicity was attributed to mono- nuclear phagocytes mainly on the basis of the resistance of TU5 target cells to NK cells, the adherence of effectors, the partial inhibition of activity in the presence of silica particles, and the elicitation of activity in macrophage preparations from an-

502 VILLA ET AL.

TABLE 7

Effect of IFN and Lymphokines on Cytotoxicity against TU5 of Monocyte-Depleted B73.1+ or B73.1- Cells”

5% Specific lysis + SDb

Effector cell Medium IFN LK

Input -0.1 * 1.1 3.8 + 1.5 2.2 + 0.6 B73+ 4.1 * 1.4 19.1 f 3.7** 18.2 + 3.4* B73- 0.7 + 0.3 1.3 * 0.6 7.9 i 1.0

a PBM were depleted of monocytes before separation by adherence on plastic (repeated twice), nylon wool, and carbonyl iron. Monocyte contamination, assessed by morphology and esterase, was <OS%.

b Lysis of TU5 in a 48-hr [‘Hlthymidine-release assay. * P < 0.05 versus medium. ** P i 0.01 versus medium.

atomical sites (peritoneal cavity, bronchoalveolar spaces) where little or no NK is found (4, 11, 23). In the present investigation, we found that cytotoxicity of cells positive for monocyte markers (Mo2, UCHMl, B44.1) is augmented by exposure to lymphokines (four of five experiments) or less frequently (one of eight experiments) to IFN. However, cells negative for these monocyte markers were also stimulated by lymphokine supematants or by IFN. Actually the latter agent appeared more con- sistently active on negative cells than on positive ones (Table 6). Along the same line, IFN or lymphokines induced killing of TU5 in a 4%hr assay (but not in a 4-hr S’Cr-release test, data not shown) by nonadherent, monocyte-depleted, B73. l-positive cells (Tables 5-7). Thus, although these data are consistent with the possibility that human monocy-tes or macrophages do kill tumor cells, either in the absence of deliberate stimulation or after exposure to IFN and lymphokines, they also caution against the possible contribution of minor NK cell contaminants, particularly when stimuli such as IFN or lymphokines are applied, even when a relatively NK-cell-resistant target such as TU5 is used. A recently developed 5’Cr-release assay for monocyte cytotoxicity (27) or the use of macrophages from anatomical sites where very few NK cells are present (peritoneal cavity, bronchoalveolar spaces, Refs. (4, 11, 31)) might provide useful tools for studying the regulation of the tumoricidal activity of human mono- nuclear phagocytes in the absence of any contribution of NK cells.

Note added in prooJ After acceptance of the present manuscript two other papers (B. Freundlich et al., J. Immunol. 132, 1255, 1984; D. A. Horwitz et al., J. Immunol. 132,2370, 1984) were published in addition to Refs. (12, 13) dealing with the characterization of human adherent cytotoxic cells.

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MONGCYTE CYTOTOXICITY 503

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