Induction of antibody-dependent and nonspecific tumor killing in human monocytic leukemia cells by nonlymphocyte factors and phorbol ester

CELLULAR

IMMUNOLOGY

71,2 15-223 (1982)

Induction of Antibody-Dependent and Nonspecific Tumor Killing in Human Monocytic Leukemia Cells by Nonlymphocyte Factors and Phorbol Ester P. RALPH,

N. WILLIAMS,

M. A. S. MOORE, AND P. B. LITCOFSKY

Department of Developmental Hematopoiesis, Sloan-Kettering Institute for Cancer Research, Walker Laboratory, Rye, New York 10580 Received February 4. 1982; accepted June 6, 1982 Induction of human cell lines to express properties characteristic of differentiated mononuclear phagocytes was investigated. Phagocytosis and receptors for immunoglobulin Fc and complement were induced or increased to variable extents in histiocytic lymphoma line U937, myeloblast line KG-l, and myelomonocytic line RC-2A. The active agents used were tumor promoter phorbol myristic acetate (PMA), supematants of human bladder carcinoma line 5637, and lymphokine preparations. The strong induction of these properties concomitant with growth inhibition of the U937 line was suggestive of a terminal differentiation process. Incubation of U937 with these agents for several days induced antibody-dependent cellular cytotoxicity (ADCC) to tumor targets in the monocyte-related line. Factors inducing U937 maturation appear to be distinct from myeloid colony-stimulating activity (CSA) since the former is present in 2-day LK, whereas more than 2 days is required for the production of most CSA. In addition to ADCC, PMA also induced nonspecific tumoricidal activity in U937. These experiments show that (i) the same monocyte cell type can mediate both nonspecific and antibodydependent lysis of tumor targets; (ii) exogenous agents and endogenous factors other than lymphokine preparations can induce ADCC, (iii) the U937 line can be used as a model for detecting nonlymphoid as well as lymphoid factors stimulating differentiation or maturation of human monocytic cells.

INTRODUCTION Macrophages are considered to play an important role in defense against cancer and can kill tumor cells in vitro by nonspecific and antibody-dependent mechanisms. Lipopolysaccharide (LPS)’ (l), lymphokines (LK) (2, 3), and interferon-containing preparations (4) stimulate nonspecific killing of tumor cells by human mononuclear phagocytes in vitro. In a model system a human immature monocyte-like cell line U937 was found to mediate antibody-dependent cellular cytotoxicity (ADCC) to RBC targets after several days stimulation with phorbol myristic acetate (PMA) (5), and to tumor and RBC targets after stimulation with LK (6). However, it is not known if ADCC and nonspecific effector mechanisms are mediated by the same cell ’ Abbreviations used: LPS, lipopolysacchatide; LK, lymphokine; ADCC, antibody-dependent cellular cytotoxicity; PMA, phorbol myristic acetate; EA, IgG antibody-erythrocytes; EAC, complement-IgM antibody-erythrocytes; CSA, myeloid colony-stimulating activity; CM, conditioned medium; MAF, macrophage activating factors. 215 0008-8749/82/ 1202 15-09$02.00/O Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.

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type, and the possibility that cells other than lymphocytes may produce macrophagestimulating factors has not been fully explored. The present study investigated the effect of exogenous agents and lymphoid and nonlymphoid sources of endogenous factors on ADCC and nonspecific killing of tumor targets mediated by U937 and two other human monocytic/myeloid cell lines. MATERIALS AND METHODS Cell line cultures. Cell lines U937 from a patient with diffuse histiocytic lymphoma (7, 8), KG-1 from acute myeloid leukemia (9), and RC-2A from myelomonocytic leukemia ( 10) were grown in RPM1 1640 medium with 10% fetal calf serum. The doubling times were about 24 hr. Assays. Cell receptors for immunoglobulin Fc and for complement were assayed by rosetting with IgG antibody-coated erythrocytes (EA) and complement-IgM antibody-erythrocytes (EAC), respectively (11). Latex bead phagocytosis was performed as described (12). Cells with three or more erythrocytes or beads were considered positive. Tumoricidal assays used [‘2SI]IUdR-prelabeled mouse myeloid leukemia Ml in duplicate 20-hr incubations at 25: 1 effector-to-target ratios in the absence and presence of 10e3 rabbit anti-mouse spleen serum ( 13, 14). Background radiolabel release of 5-9s by targets alone + antiserum was subtracted. Similar assays used mouse pre-B lymphocyte leukemia 18-8 as a target, which had background radiolabel release values of 6- 15% ( 14). Myeloid colony-stimulating activity (CSA) was measured by incubating 10’ normal human bone marrow cells with 0.1 ml sample in 1 ml agar cultures and scoring colonies at Day 7 as described previously ( 15). Reagents and factors. Lipopolysaccharide (S. typhosu WO90 1, LPS, Difco Laboratories, Detroit, Mich.), tumor promoter phorbol myristic acetate (PMA, Consolidated Midland, Brewster, N.Y.), and conditioned medium from GCT cell line (GIBCO, Grand Island, N.Y.) were purchased. Lymphokine (LK) was the 2-day supematant (2- or 5-day supematant in Table 2) of human peripheral blood mononuclear cells incubated at 2 X 106/ml RPM1 1640 + 5% fetal calf serum with 1% PHA-M (Difco). Conditioned medium from human urinary bladder carcinoma line 5637 (16) was used as a nonlymphoid source of factors including granulocyte, macrophage-CSA (17). It induced 50-120 colonies of macrophages, granulocytes, and megakaryocytes per lo5 human bone marrow cells at 10% v/v. RESULTS Induction of Phagocytosis, Fc Receptors, and C Receptors Cell lines U937, KG- 1, and RC-2A normally show few differentiated markers of the macrophage or granulocyte series. The leukemic cells were incubated 5 days FIG. 1. Induction of mature markers and growth inhibition of leukemic cell lines. Results typical of four experiments for each cell line. U937 (a), RC-2A (b), and KG-l (c) cells at 4 X lO’/ml were incubated in duplicate with 0.1 or 1 pg./ml PMA, 20 &ml LPS, 20% LK, or 20% supernatant of human urinary bladder carcinoma line 5637. Aber 5 days, viable cells were counted, washed, and assayed for immunoglobulin Fc region and complement (C) by EA and EAC rosettes, respectively, and for latex bead phagocytosis. Tenfold lower concentrations of each agent had smaller effects than shown or no effect. Values are means f SE. K = ~20% viable cells; in the other conditions viability was >90’%, except for >80% with 0.1 &ml PMA treatment of U937 and KG- 1.

E 4

z

F

................... :: . . . . . . . . . . . . . . . . . . . .

::::::::::::::::::::::

0

Cells

E3

Phagocytosis

0

I G

% Growth

Inhibition

m

... 58......:..:..::. E:::::::::::::::::::::: 4 :::::::::::::::::::: F

E

0 i

% Positive

Fc and C Receptors

I

Fc and C Receptors

% Growth

Cells

Phagocytosis

I 0

% Positive

Inhibition

I Es!

0

m

1 s

% Positive

Fc and C Receptors

% Growth

1 0

Cells

Phagocytosis

lnhibitior

I 8

0

m

I s

218

RALPH

ET AL.

with several types of stimulating agents, washed, and tested for differentiation markers. In U937, the expression of Fc and C receptors and phagocytosis of latex beads was greatly stimulated or induced by exposure to PMA (Fig. 1a). LK and conditioned medium (CM) from a human urinary bladder carcinoma line 5637 also caused marked expression of Fc receptors and phagocytic capacity, and caused moderate levels of surface C receptors. Growth of U937 cells was almost completely inhibited by PMA, LK, or 5637~CM. Incubation ofU937 with LPS showed moderate increases in the macrophage markers with very little growth inhibition. The cell lines RC-2A and KG- 1 were less responsive to the differentiation signals. Phagocytosis and Fc receptors in RC-2A were not significantly altered by the agents, and C receptors were induced only by PMA and LK (Fig. 1b). Growth was inhibited more than 50% by PMA but not by the other agents tested. In KG-l, phagocytosis and Fc receptors expression were moderately stimulated by all agents, and low but significant levels of C receptors were induced by PMA and LPS (Fig. 1c). Only PMA blocked growth of KG-l by more than 50%. Induction

of Direct and Antibody-Dependent

Cytotoxicity

to Tumor

Targets

Preincubation of U937 cells with PMA greatly stimulated their direct killing of two tumor targets tested (Table 1). LK and 5637~CM had little or no effect on the TABLE Direct

Cytotoxicity

1

and ADCC

Induced

Percentage Agent

Concentration

-Ab Ml

Medium PMA

LPS LK 5637

0.01 rg/ml 0.1 jig/ml 1.0 fig/ml

20 e/ml 200 fig/ml 20% 20%

in U937”

cytotoxicity Percentage ADCC

+Ab

Target 2*0 2+1 21 f 3 6&l 4+1 4+1 6-cl 1+0

5+1 6+1 48 + 19+ 8+1 9+1 38 + 32 2

3 1

2 2

3 4 27 13 4 5 32 31

18-8 Target Medium PMA

LPS LK 5637

0.01 ag/ml 0.1 pg/ml 1 .O pgg/ml 20 &ml 200 20% 20%

0 2+0 18 + 1 19 + 2 0 If1 6+1 3fO

0 l&O 39 * 42 + 0 2+1 32 f 32 +

3 2

2 3

0 0 21 23 0 1 26 29

D Cells were incubated 5 days with agents as in Fig. 1, washed and assayed for specific killing of ‘*‘Ilabeled Ml or 18-8 leukemia targets without or with antitarget antibody (Ab). ADCC calculated as cytotoxicity with antibody minus cytotoxicity without antibody. Results typical of two experiments using each target.

INDUCTION

IN HUMAN

MONOCYTIC

TABLE Induction of Nonspecific and Antibody-Dependent

219

LEUKEMIAS

2 Tumor Cytotoxicity in Monocyte-Related

Lines”

Inducer

PMA hsg/ml) Line KG-l

E:T

Cytotoxicity

Medium

0.01

0.1

1

5

Nonspecific ADCC Nonspecific ADCC

0 0 3&O 1 &O

0 0 4fl 1+1

2+1 0 I5 k 1* 3+1

0 0 8+ I* 2+1

0 0 2+0 0

0 0 0 0

Nonspecific ADCC Nonspecific ADCC

0 0 5*1 0

0 0 5*0 0

3fl 0 22 + 2* 2*2

0 0 751 0

0 0 5+1 2*1

0 0 4+0 0

Nonspecific ADCC Nonspecific ADCC

0 0 7+1 5+1

0 0 6*0 5+1

5+ 7f 26 k 24 f

25 RC-2A

5 25

u937

5 25

o* 1* 2* 3*

6+ 1* 2fl 15 + 1* 16 -e 2*

LK

0 9+ 1* 11 + 1 38 f 4*

5637-CM

0 3+1 7+0 27 + 2*

0 Induction of nonspecific and antibody-dependent tumor cytotoxicity in monocyte-related lines. Cells were incubated 5 days as in Table 1, washed, and assayed for killing ‘251-labeled myeloid leukemia M 1 without and with antitarget serum at E:T ratios of 5 and 25. ADCC calculated as in Table 1. * Statistically significant induction, P < 0.05.

cells’ capacity for this nonspecific killing. However, ADCC mediated by U937 was strongly enhanced by pretreatment with PMA, LK, and 5637~CM. LPS did not induce any cytotoxic capacity in U937. Direct cytotoxicity mediated by RC-2A was greatly enhanced by incubation with PMA, and that by KG- 1 was moderately stimulated (Table 2). The two preparations of factors from human sources occasionally caused a small stimulation of ADCC mediated by RC-2A. However, in most experiments, ADCC in RC-2A and KG-1 was not significantly altered by conditions which induced ADCC in U937 (Table 2). Maximum ADCC and direct cytotoxicity was achieved in U937 at about 5 days of incubation with LK, 5637-CM, or 0.1 pg/ml PMA (Fig. 2). Slightly lower cytotoxic activity occurred using 1 &ml PMA, which peaked at Day 2-3. In six experiments of this type no significant tumoricidal activity was observed after 1 day of culture with stimulating agents, even using 10 &ml PMA; longer incubations with this high dose of PMA could not be evaluated, due to toxicity. As controls for these experiments, human T (CEM, Peer and JURKAT) and B (DAUDI, BM) cell lines similarly treated with these agents did not develop antitumor killing with or without antibody, although PMA did block their growth (not shown).

Relation of U937 Stimulating Activity to Other Lymphokine Factors Lymphocytes are usually considered the source of host factors activating macrophage cytotoxicity (3, 6). Mitogen-induced human LK preparations are also rich in colony-stimulating activity (CSA) required for bone marrow progenitors to form colonies of granulocytes and macrophages in culture (18). However, maximum CSA

220

RALPH ET AL.

0123456 DAY OF PRETREATMENT

FIG. 2. Time for stimulation of tumor&al activity in U937. Cells were incubated with 0.003 (0), 0.01 (a), 0.1 (A), or 1 &ml PMA (A), or with 5% (0) or 20% 5637 supematant (m) as in Fig. 1. At various times cells were washed and assayed for tumor hilling. The range of control values at each time point for U937 incubated without stimulating agents is shown at Day 0. Optimal tumoricidal activity occurred at 5 days incubation with 0.1 &ml PMA or 20% 5637 supematant; 20% lymphokine gave similar results.

levels are usually found in &lay LK preparations with much less CSA in 2&y preparations (18). To determine the relation between CSA and U937-stimulating activity, three human sources of CSA, 5637, GCT, and LK were compared for effects on U937 cytotoxicity capacity. Results in Table 3 show that 5&y LK and the cytokine preparations from GCT and 5637 cells contain both CSF and U937stimulating factors. In contrast, 2-day LK is almost optimal in stimulating expression of U937 Fc receptors and tumoricidal activity, but contains very little CSA. U937stimulating activity was always found in 2-day LK, whereas CSA levels were low in two of three experiments, less than 20 colony units/O.1 ml at 20% v/v dilution. Thus, CSA did not correlate with maturation factors in LK. Both CSA and ADCC stimulating factors were heat-sensitive, suggesting that they are proteins. The activity of LK was not due to carryover of PHA used to stimulate the blood cells since control supernatants prepared by adding PHA 1 hr before harvesting was inactive. Supernatants of a human T-cell line, JURKAT, that produces interleukin-2 stimulating T-lymphocyte growth (21), had no detectable CSA and did not stimulate ADCC (Table 2). DISCUSSION These experiments show that the same type of human mononuclear phagocyte can mediate both nonspecific killing and ADCC to tumor targets. Nonspecific toxicity was induced by PMA whereas antibody-dependent killing was induced not only by LK but also by other agents including nonlymphoid factors endogenous to

INDUCTION

IN HUMAN

MONOCYTIC

221

LEUKEMIAS

man. Lymphocyte-dependent activities termed macrophage-activating factors (MAF) stimulate human monocytes to kill tumor targets nonspecifically (2, 3). We may be detecting MAF activity as evidenced by low levels of nonspecific lolling induced in U937 by LK but not by factors from 5637 cells, although the ADCC reaction predominates under these conditions (Table 2). The variety of methods for inducing ADCC in the U937 line is similar to experiments in which ADCC could be stimulated in murine peritoneal macrophages (23) and macrophage cell lines (14) in vitro by pretreatment with LPS, PMA, and TABLE Properties

of Factors

3

Stimulating

U937

ADCC” U937

Agent’ Exp.

Exp.

1

2

Concentration (o/o)

ADCC (%10)

CSFd

Medium 5637 5637 5637 Boiled 5637 GCT GCT GCT Boiled GCT JURKAT JURKAT + PHA

20 2 5 20 20 2 5 20 20 20 20

0 14 36 143
9 9 14 21 8 10 16 19 10 8 9

3*1 721 12f 29 + 9+1 4+0 12 k 26 f 5+1 3*1 4+0

Medium 5637 2d-LK 2d-LK 2d-LK Boiled LK Sd-LK Sd-LK Sd-LK Boiled LK Control LK

20 20 2 5 20 20 2 5 20 20 20

0 0 2 5 18 2 19 48 191 <8 <17

5 27 3 6 22 4 5 9 30 6 6

Of0 22 + If1 7 f 21 -t If0 If0 9 f 26 + 221 221

assaysb Heat sensitivity’ inhibition (%)

If 2/ 70 0 If 90

lf lf 2f 95 lf 2/ 92

a Control JURKAT supernatants had no interleukin-2 and after stimulation supernatants contained 9 U/ml, assayed by R. Mertelsmann (22). b U937 incubated 5 days with agents, washed, and assayed for Fc receptors and ADCC as in Figs. 1 and 2. ’ Supernatants of GCT (GIBCO, Grand Island, N.Y.), 5637, and JURKAT. FHCRC (obtained from Cell Distribution Center, Salk Institute, San Diego, Calif. + 2 days stimulation with 1% PHA-M) cell lines, and that of PHA-stimulated blood mononuclear cells (LK) collected at 2 or 5 days. Control LK prepared by incubating mononuclear cells 5 days with PHA added 1 hr before harvesting. ’ Colonies per 10’ human bone marrow cells stimulated by 0.1 ml of concentration shown, calculated from results of titrations in the linear range of the assay. ‘Samples were boiled 10 mitt, assayed, and percentage inhibition due to heating of the increase in ADCC calculated. f Statistically significant, P < 0.05.

222

RALPH ET AL.

murine LK. In the murine system, ADCC stimulation was not due to interleukin2, granulocyte or macrophage CSA, or macrophage activating factors for nonspecific cytotoxicity to sarcoma (2, 3) or Leishmaniu2 targets. Incomplete differentiation of murine immature myeloid tumor lines to granulocyte morphology (24) and lysozyme synthesis (25) can occur in the presence of crude CSA preparations. In these systems the differentiation steps are believed to be caused by molecules different from any CSAs that act on normal marrow cells (25), or by minor subsets of CSAs (24). In the present experiments, stimulation of U937 macrophage properties was not correlated with LK content of CSA. It is likely that Fc receptors (6) and perhaps other properties may have to be induced in U937 cells before activation of cytotoxicity can be effected. Therefore, the U937 system may require simultaneous presence of differentiation factors plus other factors stimulating ADCC. PMA triggers immediate tumoricidal activity in human neutrophils (26) and in murine macrophages (14, 27), and augments lysis of fibroblasts by human macrophages (28). However, the effect of PMA on the immature monocyte line U937 is a differentiation-like process requiring several days for development, similar to the induction of bactericidal activity in human myeloblast line HLlO by PMA (29). Sources of CSA stimulate colony formation by the human myeloid KG-l and HL-60 cells in semisolid media (reviewed by 9). Our results show that cytokine preparations can also induce myeloid/macrophage markers in U937 and to lesser extents in KG-l and RC-2A. However, unlike the results with colony assays, these preparations inhibit growth of the myeloid or monocyte-like lines in liquid culture. Further studies are necessary to determine the role of various CSFs, interferon (4) and other host-derived factors produced as a result of an inflammatory response to tumors in the activation of macrophage tumoricidal activity. ACKNOWLEDGMENTS We thank A. Patricia C. Sheridan and Heather M. Jackson for expert assistance; R. Mertelsmann for interleukin-2 determinations; H. E. Broxmeyer for advice; R. Bradley, J. Fogh, and D. W. Golde for cell lines and information on their properties; and G. Svet-Moldavsky for preliminary data on the 5637 line. This work was supported by Grants AI- 158 11, HL-2245 1, and CA-24300 from NIH, CH-E3 from ACS, and the Gar Reichman Foundation,

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LEUKEMIAS

223

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