CELLULAR

38, 276-285 (1978)

IMMUiTOLOGY

Tumor III.

Cell

Killing with

Inhibition

by Cytochalasins,

SOMESH Departments

by Macrophages Activated Lymphocyte Mediators

D.

SHARMA

Colchicine,

AND

WILLY

and Vinblastine

F.

PIESSENS

of Medicine, Harvard Medical School and Peter Robert B. Brigham Hospitals, Boston, Massachusetts Received

in Vitro l

2 B. Brighmrz 02120

arid

December 29, 1977

The effect of cytochalasin A and B, colchicine and vinblastine on tumor cell killing by macrophages activated in vitro with lymphocyte mediators was examined. Both cytochalasins reversibly inhibited the killing of tumor cells by activated macrophages. Kinetic studies with cytochalasin B suggested that this drug exerts its effect on an early step of the cytotoxic process. Additional studies revealed that the drug inhibited the binding of tumor cells by activated macrophages. Colchicine inhibited both the binding and the killing of tumor cells by activated macrophages, whereas its structural analogue, lumicolchicine, had no effect on either macrophage function. Vinblastine also inhibited the binding and killing of tumor cells. However, this drug no longer inhibited tumor cell binding at low concentrations (< lo-” M) that still inhibited tumor cell killing. Further, vinblastine inhibited tumor cell killing when added late to an ongoing cytolytic reaction. These results suggest that the cytochalasins, colchicine and vinblastine inhibit macrophage mediated cytotoxicity by preventing intimate contact between the effector macrophages and their targets. In addition, vinblastine also appears to inhibit a later step of the cytolytic process, possibly the secretion of a cytotoxic macrophage product.

INTRODUCTION Following their activation in vitro with lymphocyte mediators, macrophages from nonimmune animals become capable of killing tumor cells in vitro by a nonphagocytic mechanism that requires close contact between the effector and the target cells (l-5). The molecular mechanisms by which activated macrophages recognize and kill their targets remain poorly understood. In a previous report we described the effect of inhibitors of protein, DNA and RNA synthesis on the cytotoxic capacity of guinea pig macrophages activated in vitro with the lymphocyte mediator macro1 Supported by USPHS Grant ROl CA19403, Training Grant lT32 HL07142 and USPHS Contract NOl-CB-64057 from the National Cancer Institute. 2Recipient of a Cancer Research Scholar Award from the American Cancer Society, Massachusetts Division, Inc. 276 000%8749/78/0382-0276$02.00/O Copyright All rights

@ 1978 by Academic Press, of reproduction in any form

Inc. reserved.

INHIBITION

OF

MACROPHAGE

MEDIATED

277

CYTOTOXICITY

phage activating factor (MAF) (6). In the present study we examine the effect of drugs that affect microfilament and microtubular functions on the capacity of activated macrophages to bind and to kill tumor cells. The results indicate that the cytochalasins A and B reversibly, and colchicine and vinblastine irreversibly inhibit both the binding and the killing of neoplastic cells by activated macrophages. Lumicolchicine, a structural analogue of colchicine that does not affect microtubular integrity (7), did not inhibit the interaction of activated macrophages with tumor cells. Kinetic studies further suggest that cytochalasin B may affect an early step, whereas colchicine and vinblastine appear to inhibit both early and later stages in the cytolytic process. MATERIALS

AND

METHODS

Drzlgs. Cytochalasin B and A (CB and CA), colchicine and vinblastine sulfate were obtained from Sigma Chemical Co., St. Louis, MO. The cytochalasins were dissolved in dimethyl sulfoxide (DMSO) to give a 10-Z M stock solution. CB was stored frozen at - 20°C. A fresh solution of CA was used for each experiment. Colchicine and vinblastine sulfate were dissolved in MEM. Lumicolchicine, a structural analogue of colchicine, was prepared by the method of Mizel and Wilson (8 1. L-[3H]proline (sp. act. 49 Ci/mM) was obtained from Schwarz-Mann, Orangeburg, N.Y. Akna1.s and tut$tor lines. Inbred strain 2 guinea pigs were obtained from the National Institutes of Health. Line 1 and line 10 hepatoma cells were maintained as previously described (3, 5). Cytotoxicity assay with MAF activated nzacrophages. The detailed procedures for the preparation of MAF-rich and control supernatants and the activation of guinea pig peritoneal exudate macrophages have been described (1, 3). The cytotoxicity assay with [3H]proline prelabeled line 1 hepatoma cells was described in a previous report of this series (9). Briefly, line 1 hepatoma cells were labeled overnight in culture medium containing 250 PC1 of [“H ] proline, collected by trypsinization, mixed with activated or control macrophages and plated in microtiter II plates (Falcon Plastics, Los Angeles, Calif.). After 6 hr of incubation the amount of cytotoxicity was determined from the residual adhering radioactivity by the formula : % Kill by activated

macrophages 1 _ Mean CPM (tumor + activated macrophages) Mean CPM (tumor + coctrol macropl-ages)

x li)? ’’

1

Unfractionated peritoneal exudates containing at least SOP macrophages were used as the source of effector cells. All experiments were done in triplicate with macrophage: tumor cell ratios of 70 : 1 previously found to be optimal (9). Efect of inhibitors on cytotoxicity by mediator activated rnacroPhages. The effect of cytochalasin, colchicine and vinblastine on macrophage mediated cytotoxicity was evaluated in two different ways. In a first series of experiments, the inhibitors were added directly to mixtures of effector and target cells and thus were present throughout the entire assay. In a second series of experiments the effector macrophages were pretreated for 1 hr with various concentrations of the inhibitors and then washed extensively before they were used in the cytotoxicity

278

SHARMA

AND

PIESSENS

assay. The first approach was used as a convenient way to determine if the inhibitor under study affected the killing of tumor cells by activated macrophages. The second approach was used to determine if the observed effect resulted from the action of the drug on the macrophage, on the target tumor or on both. In all experiments with the drugs present during the assay, tumor cells were also cultured in the absence of macrophages but in medium containing the inhibitor, to determine if the drug by itself was toxic for line 1 tumor cells. In all experiments, the degree of cell killing by activated macrophages exposed to the drugs was compared with that of cells from the same pool not exposed to the inhibitors but otherwise processed in parallel. A wide dose range of inhibitors was tested (see Results). Cytoadherence assay. The details of this assay have been described elsewhere (5). In brief, established monolayers of activated or control macrophages are overlaid with [SH]proline labeled line 10 hepatoma cells, and incubated for 1 hr while maintained on a rocking platform. The nonadhering tumor cells are then removed by vigorous washings and the adhering radioactivity is determined. Tumor cell binding is expressed as adhering radioactivity (CPM) per 100 pg macrophage protein. Our previous studies indicate that line 10 tumor cells do not spontaneously adhere to the glass slides used for these experiments, and that the adhering radioactivity is associated with tumor cells attached to macrophages (5). To study the effect of drugs on the binding of tumor cells by activated macrophages, established macrophage monolayers were preincubated for 30 min with the appropriate drug concentration. Tumor cells were then added either directly or after the monolayers had been washed extensively to remove the inhibitors. The percent inhibition by the drugs of tumor cell binding to activated macrophages was calculated as previously described (6). RESULTS Efect of Cytochalasin B and A on Tumor Cell Killing by Activated Macrophages The addition of 10e6 M cytochalasin B (CB) to mixtures of tumor cells and macrophages resulted in a marked inhibition of macrophage mediated cytotoxicity (mean 67% inhibition in five experiments) ; this effect was rapidly lost when lower drug concentrations were used (Table 1A). Cytochalasin A inhibited macrophage mediated cytotoxicity to a similar extent (Table 1B). The addition to culture medium of the same amount of DMSO as that used to dissolve the cytochalasins (0.01%) had no effect on tumor killing by activated macrophages; nor did the cytochalasins affect the survival of tumor cells plated in the absence of macrophages. Indeed, in four experiments, the mean residual adhering radioactivity of tumor cells plated in the absence of macrophages in medium containing lo-” M CA or CB was 98 fi 25% of that of tumor cells pIated in drug-free medium. Further, the drugs did not render the tumor cells more susceptible to the effect of nonactivated macrophages nor did they impair the viability of the effector macrophages, as assessed by trypan blue exclusion. When macrophages were pretreated with CA or CB, and washed extensively prior to their use in the cytotoxicity assay, the inhibitory effect of the cytochalasins was almost completely reversed. A representative experiment is illustrated in Table 2. Similar pretreatments of tumor cells with these drugs also did not inhibit macrophage mediated cytotoxicity (results not shown).

INHIBITION

OF

MACROPHAGE

MEDIATED

TABLE Effect

of Cytochalasins

1

B and A on Tumor

Killing

by Mediator

A. Cytochalasin

Activated

Macrophages

B -

Drug concentration (W

279

CYTOTOXICITY

......__~~~

y. Killa ---__ Expt.

1

Expt.

2

3

Expt.

Expt.

4

Expt.

~-

Mean “h inhibition of kill

5

49

None 10-h 10-S 10-1 1O-8

(::,

(Z) -

-

(T:! 21

(0) 42 -

24

B. Cytochalasin Drug concentration (w None 10-h 10-b 10-G 10-7 u Numbers in parenthesis medium containing 0.017c

(T:) (47) 56 58

67 19 15 7

A

y. Kill” Expt.

1

Expt.

2

Expt.

3

Expt.

Mean 56 inhibition of kill

4

20

25

49

45

--

(4) (0) 24 -

(0)

(0) 40 44

(0) (31) 43

90 86 10 7

are statistically DMSO.

(14) different

(P < 0.05)

from

the percent

kill

in drug-free

The effect of CB ( 1O-5 M) on an ongoing cytolytic reaction varied according to the time when the drug was added. When added 30 min after the re.action had been started, CB inhibited macrophage mediated killing to the same extent as when the drug was present during the entire assay period. When the drug was added after 60 min, its inhibitory effect was less marked albeit still significant. In contrast, CB no longer inhibited the cytolytic process when added to mixtures of macrophages and tumor cells previously cultured for 2 hr in the absence of the drug. The results of two representative experiments of this type are shown in Fig. 1.

TABLE Reversibility

of the Effect

of Cytochalasins

2

A and B on Macrophage

Inhibitor

in parenthesis are significantly containing O.OlcjO DMSO.

Cytotoxicity

c$, Kill Drug present

a Numbers free medium

Mediated

tliffcrent

Ihg washed ala-a)

(1’ < 0.05)

from

the pcrccn~

kill

io ~lrrlg-

280

SHARMA

AND

PIESSENS

40=1 J P 30M 50

20 1

e

(0 i OL

L 0

I 4

I

I

2

3

TIME WHEN DRUG WAS ADDED

fhrj

FIG. 1. Effect of cytochalasin B on ongoing macrophage mediated cytotoxicity reaction. Cytochalasin B (10” M) was added to mixtures of tumor cells and macrophages previously incubated in drug-free medium for the number of hours indicated in the abscissa. Percent kill was determined after 6 hr of culture. The results of two separate experiments are illustrated. Each point is the mean of triplicate determinations. Percent kill in the absence of added drug is shown on the extreme left. Cytochalasin B added up to 1 hr after the beginning of the cultures significantly inhibited tumor killing by activated macrophages (P < 0.05).

Efect

of Colchicine

and Vinblastine

on Tumor

Killing

by Activated

Macrophages

Colchicine also inhibited tumor killing by activated macrophages in a dose dependent fashion, but in contrast to the effect of the cytochalasins, the inhibition was not reversed by extensive washing of drug pretreated effector cells. The results of six experiments are shown in Table 3. With 10e5 M colchicine macrophage mediated cytotoxicity was significantly inhibited (mean 81 s inhibition). The inhibition was less marked with lO-‘j M (63%) and no longer significant (11%) with lo-? M TABLE Effect

of Colchicine

and

Lumicolchicine

3 on Macrophage

Mediated

Cytotoxizity

A. Colchicinea Drug concentration (w

% Kill6 Expt.

1

Expt.

2

Expt.

3

Expt.

4

Expt.

5

Expt.

6

Mean y0 inhibition of kill

None

26

17

38

27

-

10-S 10-C 10-T

(2)

(3)

(5) (13) (23)

(0) (8) 32

81 63 11

(8) B. Lumicolchicinec Drug concentration wf) None 10-b 10-e

% Kill Expt. 17 22 23

1

Expt.

2

31 39 29

a In experiments 1 to 3 colchicine was present throughout the assay; in experiments macrophages were pretreated and washed before use. * Values in parenthesis are significantly different (P < 0.05) from y0 kill in drug-free c Macrophages were pretreated with lumicolchicine and washed before use.

4 to 6 medium.

INHIBITION

OF

MACROPHAGE

MEDIATED

TABLE EH’cct Drug concentration (Jo INone 10-S lo- fl 1O-7 10-g lo--”

of Vinblastine

on Tumor

Expt.

(::,

1

Expt.

(ii,

Killing

2

281

CYTOTOXICITY

4 by

Mediator

c7c KiW,

h

Expt.

3

43 (9)

Expt.

Activated

4

21 (5) (5) UJ) (0) 15

u In experiments 1 and 2 the drug was present throughout macrophages were pretreated and washed before use. b Values in parenthesis are significantly different (P < 0.05)

Expt.

Macrophages

5

26 (()) (0) (4) ((U (3 the from

assay; 76 kill

Mean y0 inhibition of kill 6X 88 92 too 58 -in experiments in drug-free

3 to 5 medium.

colchicine. In contrast, pretreatment of the effector macrophages with a structural analogue, lumicolchicine, did not inhibit tumor cell killing. These results are also shown in Table 3. A more pronounced and similarly nonreversible inhibition of macrophage medated cytotoxicity was observed with vinblastine (Table 4). Pretreatment of macrophagea with as little as 10m8M vinblastine still significantly inhibited the capacity of these cells to kill syngeneic tumor cells. At the doses used, neither colchicine nor vinblastine decreased the viability of effector macrophages or of tumor cel!s plated in the absence of drugs. Further, pretreatment of tumor cells did not render these cells less susceptible to killing by activated macrophages. When lo-” M vinblastine was added to an ongoing reaction, the drug inhibited the cytotoxic process even when added 3 hr after the macrophages and tumor had been mixed together. In a single experiment, macrophage mediated cytotoxicity was inhibited even when the drug was added after 4 hr. These results are illustrated in Fig. 2.

FIG. 2. Effect of vinblastine on ongoing macrophage mediated cytotoxicity reaction. blastine (lo-’ n/r) was added to mixtures of tumor cells and macrophages previously bated in drug-free medium for the number of hours indicated in the abscissa. Percent determined after 6 hr of culture. The results of 2 separate experiments are illustrated. point is the mean of triplicate determinations. Percent kill in the absence of added S~OUW on the extreme left. Vinblastine added up to 3 hr. and in one experiment up after the beginning of the cultures significantly inhibited tumor killing by activated phages (I’ < 0.05).

\‘inincukill was Each drug is to 4 hr. macrr~

282

SHARMA

AND

PIESSENS

TABLE Effect

of Cytochalasin on Tumor

Drug concentration M

YO Inhibition Cytochalasin Expt.

10-s lo-’ 5 x lo-’ lo-’ 10-a 10-g

5

A, Colchicine, Lumicolchicine and Vinblastine Cell Binding by Activated Macrophages

1 Expt.

93 79 -

A 2

100 78 38 (0)

(0) -

Colchicine

Expt. 80 61 (7)

-

of binding%b

-

a Cytochalasin A was present throughout before use. b Results in parenthesis are not significantly

3

Expt.

1 Expt.

93 43 the assay;

Lumicolchicine 2

Expt.

100 63 -

Expt.

100 40 25 (0)

(8) macrophages

different

3

values

Efect of Cytochalasin A, Colchicine, Luwkolchicine Tumor Cells by Activated Macrophages

1 Expt.

(3)

(2)

were pretreated

from control

Vinblastine

with

2

(4)

(1)

the other

Expt.

1 Expt.

85 21 (0)

81 34 (5) (0)

-

-

drugs

2

and washed

(P > 0.05).

avzd Vinblastine

on Binding

of

Cytochalasin A inhibited the binding of tumor cells by activated macrophages in a dose dependent fashion (Table 5). Almost complete inhibition was observed with 1O-6 M drug, whereas the binding was no longer inhibited in the presence of low7 M CA. Addition of DMSO to the medium in the same concentrations as those used to dissolve CA (0.01% ) had no effect on the binding of tumor cells by activated macrophages. Further, the inhibition by CA was almost completely reversible when drug treated macrophage monolayers were washed extensively prior to being overlaid with tumor cells (Table 6). Colchicine also inhibited the binding of tumor cells by activated macrophages in a dose dependent fashion. This effect persisted when drug treated macrophage monolayers were washed before tumor cells were added (Table 5). In contrast, lumicolchicine had no effect on the capacity of activated macrophages to bind tumor cells (Table 5). TABLE

6

Reversibility of the Effect of Cytochalasin Line 10 Hepatoma Cells by Activated Treatment macrophages

CPM/100 protein

of

Control macrophages None 1OW 11f cytochalasin A, continuously present None 10m3 kf cytochalasin pretreatment only

A on Binding Macrophages

of

pg macrophage adhering too

% Inhibitionbnc of binding

Activated macrophages

5481

f

381

7534

f

658

4979

f

390

5720

f

521

5235

f

410

7098

f

701

5320

f

367

7245 f

683

64 I

A,

I1 l