Tohoku

J. exp.

Med.,

1977,

123, 371-380

Inhibition

of Cellular

Peritoneal

Macrophages

DNA

Synthesis

KENTARO TOH, NORIYUKI SATO and

by

Rat

KOKICHI KIKUCHI

Department of Pathology, Sapporo Medical College,Sapporo 060

TOH, K., SATO, N. and KIKUCHI, K. Inhibition of Cellular DNA Synthesis by Rat Peritoneal Macrophages. Tohoku J. exp. Med., 1977, 123 (4), 371-380 The DNA synthesis in syngeneie, allogeneic and xenogeneic cells was inhibited by peritoneal macrophages of normal WKA rats. This inhibitory effect of macro phages was found to be mediated by a soluble factor (MfID) released into a culture medium from macrophages which was heat-stable and non-dialyzable. The MfID was also responsible for the inhibition of lymphocyte DNA synthesis by MIX reaction. The action of MfID was indicated to suppress rather than to kill cell growth. It was interpreted that the inhibitory effect of macrophages on cellular DNA synthesis is their inherent property and macrophages may play the role of regulating the proliferation of lymphocytes and other cells. rat peritoneal macrophage; macrophage soluble factor; DNA synthesis

It is well known that macrophages are essential cells at the site of inflamma tion, wound repair, graft rejection, etc. However, the role of macrophages in these areas has not been well defined. Recently, macrophages have been shown to inhibit DNA synthesis in lymphocytes (Harris 1965; Parkhouse and Dutton 1966; Yoshinaga et al. 1972; Waldman and Gottlieb 1973; Miller and Mishell 1975; Baird and Kaplan 1977) and other cells (Keller 1973; Krahenbuhl and Remington 1974; Reed and Lucas 1975; Lejeune 1976), irrespective of whether they are derived from normal (Waldman and Gottlieb 1973; Miller and Mishell 1975; Reed and Lucas 1975; Lejeune 1976; Baird and Kaplan 1977) or activated (Keller 1973; Krahenbuhl and Remington 1974; Baird and Kaplan 1977) animals. These findings suggest that one vital function of macrophages is to regulate cellular proliferation and differentiation. Previously, we described that the peritoneal macrophages of the normal rat inhibited DNA synthesis in syngeneic tumor cells induced by 3-methylcholanthrene (Toh and Kikuchi 1977). In this report, additional studies were undertaken to elucidate the regulatory function of macrophages on cellular DNA synthesis. The results are mainly concerned with the release of a soluble factor from macrophages which is suggested to mediate the inhibition of cellular DNA synthesis by macrophages. Received

for publication,

June

6, 1977. 371

372

K.

Toh et al.

MATERIALS AND METHODS Cells. been

3-methylcholanthrene

previously

routinely

treated

by

Peritoneal by

of

incubation

at

aspirating cells

hr

order

in

the

remaining 48

dishes

37•Ž,

of

additional

to

remove

cultured dish

synthesis of

number assay

the

cover

Radiochemical from

the

The

residual

being

bases,

of

the

of

and

adult

medium ml.

centrifuged

seeded

cells

with

5

ml

one were

in

60

hr

of

removed

PBS.

was at

medium.

After

supernatant

rats After

culture

were

per

dishes

WKA PBS.

a

non-adherent

The

soluble

fluid

of

was

used

cover

slips

were

Adherent

harvested

500 •~

slip

g for

after 10

as

control.

After

for in

cold

adhering

on

was

carried

45

min

in

5% the

48

hr

of

TCA

to

be

slips

target

macro by

previously

mixed was

the 20

seeded

target

slips

for

cells

Ci/mmol; were

The removed

non-incorporated

counted

the

alone=100%.

incubation,

of

by

mixture

cover

free

were

cells

the

DNA

reflected

(methyl-3H;

Subsequently,

cover

to

was

of

the

of cellular

were

ml

each

from

adhered

suspensions

(Toh

were

inhibition

as

0.4

published

interposed

macrophages

3H-TdR

min.

be

previously

and

with

to

the

exhibited cell

been (5•~104/Ml)

as

assay

of

volume,

pulse-labeled

so

was

was

has

medium

dish

number

target

equal

ml

To

inhibition factor,

methods

0.4

a Petri

cover

an

Amersham) dipped

in

The

The

of

in

glasses.

number.

initially.

cells

set

each

cell

a

detail cells

slide

macrophages,

MEM

test.

1973).

The 5

Corp.)

for

medium, per

mixtures After

of

Mixed

After

ml

slips

equal

volume they

lymphocyte

were

six

days, by

were of

20

of mM

treated

and

cultures

(15-30

were

isotopes.

radioactivity

without

(MLC). ACI

in were was

and the

and

New

on

medium slips.

0.4

macrophage

at

ml

Nuclear with a

the

density

After

media

culture

et

medium

washed

cover

by

ml

England

medium,

the

(Bean

5

of

3 hr

which

fluids

of

were

or

MEM.

manner.

mixture

of

control

capped by

by

0.5

ml

cultures

loosely

pulse-labeled determined

in

replaced

above

The rats,

the

plated

proline the

method with

Ci/mmol,

suspended

and

Bean's

incubated

from

were

cold

of

were

removed

medium

in

incubated

lymphocytes

modification cells

suspensions

freed

were

were cells

these

a

[4-3H(N)]

cells

of

by

KMT-50

L-proline

culture WKA

strains

synthesis

of

of

Labeled

cover

from

individual

ml

trypsinized. 0.4

out

cultures

Subsequently,

incubation,

suspensions

test

per

hr.

ml.

incubation,

A monolayer

1CCi

24 and

5 X 104

DNA

bases

target

and

CO,

were

Dulbecco's in

2 •~ 108

have

removed.

containing

tube).

were

of

target

in

5%

of

BMT-2, embryos

laboratory.

normal

ml

culture

washing

The

which

Centre;

Cytotoxic al.

synthesis.

culture

the

ml of

medium

slips

activity

on

ml

target

seeded

slips.

5

5

flow

resuspended

density

macrophages.

the

by the

macrophage

remaining

in

be

Briefly,

the

10-fold of PEC

with on

through

repeated

were

a

a

from 50

or rat

cells.

cover

mediated

phages

To

eight

surface

by to

DNA

1977).

on

and

considered

of

Kikuchi

a

-114 WKA

from

with

PEC

incubator

incubation

Measurement and

obtained

in

at

a humidified

detached

were

min,

PEC

(Falcon)

medium

were

3

supernatant.

Petri

purchased

cavity

g for

and

18-day-old

were

PEC

peritoneal

150 •~

KMT-50

1977).

cells

(PEC). the

at

culture

diameter

by

cells

sarcoma,

Kikuchi

L-929

out

once

Macrophage

and

trypsin.

washing

centrifuged

mm

(Toh

exudated

means

being

induced

described

2ƒÊCi

measuring

of

108

viable

containing plastic per

ml 3H-TdR

1 tubes

spleen ml

from

(Falcon,

3H-TdR

for

cell the 2005

3

hr,

and

incorporation.

RESULTS

Influenceof normal lymphnode cellsor PEC on DNA synthesisin KMT-50 cells. KMT-50 cells were each co-cultured with lymph node cells or non-adherent and adherent PEC of normal rats. As shownin Fig. 1, no influencewas seen in the lymph node cells. A marked inhibition of DNA synthesis was observed in PEC, in which adherent cells (macrophages)were snore effective than non-adherent cells.

Inhibition

Fig.

1.

Influence

(•¢)

of

lymph

mean

normal

node

of

the

lymph

cells, two

of Cellular

(•ü)

node

cells

and

non-adherent

different

DNA

Synthesis

PEC

PEC,

on

373

DNA

synthesis

(•œ) adherent

in

PEC.

KMT-50

Bars

cells.

represent

the

experiments.

TABLE 1. Effect of culture fluid from lymph node cells or PEC on DNA synthesis in KMT-50 cells

*

Each

cell

was

a density

cultured

of

in

2 •~ 106

per

60

mm

Petri

dishes

(Falcon)

at

ml.

Effect of macrophage culture fluid on cellular DNA synthesis. To

define

synthesis, cellular

the

mechanism

macrophage DNA

by

culture

synthesis.

It

which

fluid can

was

be seen

macrophages showed a marked inhibitory while this effect declined in non-adherent culture

summarized to

storage phage

tested in

suppressed

for

Table

cellular

its

inhibitory

1 that

the

DNA

activity

culture

on

fluid

of

effect on DNA synthesis in KMT-50 cells, PEC and no effect was seen in the LNC

fluid.

As found

macrophages

be for

at

factor MfID

in

Table

non-dialyzable, least inhibiting

was

found

six

2,

a

resisted weeks

at

cellular neither

in

soluble

factor

heating

at

4•Ž.

This

DNA

synthesis).

supernatants

factor

of

in 56•Ž

macrophage for

was

macrophage

30

min,

designated

extracts

culture and as

was MfID

disrupted

fluid

was

stable

to

(macro

with

374

K. Toh et al.

TABLE 2.

Some characteristics of the soluble factor released by macrophages

TABLE 3.

Condition for release of MfID from macrophages

Macrophage

monolayers

Falcon

plastic

incubation,

policeman,

frozen

and

(C), lamp

at

or

4•Ž.

by

most

inhibition When synthesis shown labeled

of

cell

growth.

KMT-50

cells

KMT-50 in

the

MLC

The

it

In

order

macrophage-mediated

high

for

were

nor

in injury

MfID

release

and

determine

only Table

of

hr

of

the

surface

did loss

slight

from

MfID

DNA

a

reduced that

the

paralleled

inhibition 48

hr

loss

within

observed

at capable

the

of later

DNA it

of

3H-proline

24

hr

of

48

hr. of

was

incuba

suppressing

viability.

macrophage-mediated kinetics

MfID

and

also

was

macrophages.

hand,

was

it

possible

the

occur

incubated

but

by

MfID,

was

UV-

is

incubation

not

lymphocyte

cellular

37•Ž, it

other

In

15W

macrophages

synthesis

a

the of

12 On

(D).

of at

of

a and

37•Ž.

Thus, release

5 that

to

whether inhibition

first

at

a culture

presence

with (E),

a

fluid

DNA

to

bath

cultured

MfID

in

Prior dishes

with

3).

the

2).

culture and

much

to

in the

without

of

in

of

(Fig.

the

culture

shown

grown

MfID

the

106/ml)

5 min

ice-acetone

and

for

the for

irradiated

(Table

within

from of

9KC

sec,

in

was

control

from

200W, dry

(2 •~

medium.

scarped

a

30

PEC

5 ml

were

inhibition

is demonstrated

reaction

kinetics

the

cells

in

time

required

the

observed

presence

Finally,

be

that

of

five

release

may

shows

be

at

macrophages

4

84%

were sonicated

thawing

of

could by

cm

MfID

process

Table

20

from

with

monolayers

and

UV-irradiation

physiological

tion

from

freezing

The

thawed

macrophage

prepared

(6 cm)

macrophages

rubber

sonication

were

dishes

of

inhibition. MfID synthesis,

release PEC

parallels were

those seeded

of on

Inhibition

of Cellular

DNA

Synthesis

375

TABLE4. Effect of macrophage culture fluid on DNA synthesis and growth of KMT -50 cells

*

KMT-50 of

cells

medium)

2.

50

and

of the

cells

macrophage

cover one

of

slips

at of

ml.

of

DNA

no

presence

dishes

48h,

and

duplicate

or Petri

After

using

the

suspended a

(5 ml

cell

layers in

hematometer.

5 ml Each

cultures.

synthesis

in

of macrophage

cells

no

in

KMT-50

culture

presence

of slips

conditions

3H-proline (•£)

5 •~ 105 in

the

to

during

harvest in 12

of

per

cells

fluid

on

by

macrophage

3H-proline

ml.

3,

hr

after

(•ü) cells

culture

After

dish

culture

labeled

KMT-

the

freeing

overlayed

inhibition,

while

dishes

macrophages

rapidly of

the

culture.

or were

no

presence

(•œ)

in

condition

grown

of

fluid.

was

Such

onset

presence

KMT-50

macrophage

same

MfID.

Fig.

of

labeled

macrophage-mediated

indicated cells

or

cover

KMT-50 As

grown fluid.

a density

the

measure

KMT-50

were

(•¢)

to

time.

of

per

presence

diameter

performed

mean

effect

culture

presence

part

served

inhibition

cytotoxic

of

mm

trypsinized,

was

the

60

5 •~ 104

medium, count

conditions

in

cells.

KMT-50

of

cell

represents

Kinetics

fluid

of

the A

in

fluid

a density

removed

medium. value

cultured

culture at

were

Fig.

were

macrophage

were

of

non-adherent

with the

other

prepared

inhibited

cells,

KMT-50

DNA

Subsequently,

cells

and

remained

free

for

test

each synthesis the

gradual

in

376

K. Toh

TABLE 5.

Equal added * •õ

Each

to

Effect of macrophage culture fluid on MLC reaction

volumes the value

The

viability

with

each

et al.

of

macrophage

lymphocyte represents of

the

prior

of was

to

fluid

and

MEM

as

control

were

mixture.

mean

lymphocytes

viability

culture

reaction

duplicate

cultures.

determined

by

trypan-blue

exclusion

incubation=100%.

Fig. 3. Kinetics of MfID release and of macrophage-mediated inhibition of DNA synthesis in KMT-50 cells. The open and closed circles show the inhibition of DNA synthesis in KMT-50 cells by MfID and macrophages, respectively. Bars represent the mean of the two different experiments.

inhibition release and

was followed in Fig.

paralleled

until

3, in which

48 hr. MfID

macrophage-mediated

There

was

was rapidly inhibition.

a similar released

kinetic

during

pattern

12 hr

of MfID

of incubation

Inhibition

Fig.

4.

Comparison

effect

on

WKA

rats

After

three

PEC

were

was

The

random

in

open

in the

0.4

normal columns

respectively.

ml

freeing

of

number fields

of

at

400 •~

Bars

density

the represent

the

other

one

of mean

part

were DNA of

the

of

the

the

cover was

the

inhibitory

two

were cover

of

different

(Bacto).

slips

(B)

seeded slips

target

equal. by

peptone injected

confirmed

practically

synthesis

10%

ml

free

it

of peptone

per

remained to

ml

and

5 •~ 105

and

macrophages

15

(A)

adhering

inhibition the

with

of

cells,

and

concerning

productivity.

control

magnification,

peptone-induced indicate

a

non-adherent cells

MfID

cavity

from

macrophages

of

and

peritoneal

medium

377

macrophages

cells

obtained

KMT-50

1000 and

the

were

DNA Synthesis

peptone-induced

KMT-50

into

PEC

overlayed

MfID.

and in

injected

days,

After

dish

normal

synthesis

contained

slips.

of

of

DNA

of Cellular

in

cells

to

cover

the

same

harvest

were

counted

that

the

The

and

at

number

closed

macrophages

rats.

on

and MfID,

experiments.

Comparisonof normal and peptone-inducedmacrophagesconcernedwith MfID prod uctivity or macrophage-mediated inhibition. Experiments

were designed

or macrophage-mediated

in an attempt

inhibition

between

to compare normal

and

the

MfID

productivity

peptone-induced

macro

phages. It is evident from Fig. 4 that the peptone-induced macrophages diminished the inhibitory effect on KMT-50 cells corresponding with a decrease in the amount of MfID release.

The susceptibility of different cell types to the inhibitory effect of macrophagesor MfID, Four tissue culture cell lines and one strain were tested for their DNA synthesis on macrophages or MfID. The results are displayed in Table 6. A high degree of macrophage-mediated inhibition was consistently observed through four transformed cell lines, irrespective of whether they were of syngeneic (KMT-50 and -114), allogeneic (BMT-2), or xenogeneic (L-929) origin. Comparing with these cell lines, syngeneic embryo cells were inhibited to a relatively low degree by macrophages. Also, Table 6 indicates that the susceptibility of tested cell types to MfID was comparable to macrophage-mediated inhibition.

378

K. Toh et al.

DISCUSSION

The present studies show that normal macrophages inhibit cellular DNA synthesis, and release a soluble factor (MfID) responsible for the inhibition of DNA synthesis. The inhibitory effect of macrophages on cellular DNA synthesis is unrelated to their immunological relevance for eliminating foreign materials, since DNA synthesis of various cell types, irrespective of whether they are of syngeneic, allogeneic, and xenogeneic origin, was comparably inhibited by macrophages (Table 6). TABLE6. The susceptibility of different cell types to inhibitory effects of macrophages or MfID

The value of 3H-TdR incorporation the two different experiments.

(%) represents

the result of

MfID was found to be heat-stable and non-dialysable (Table 2). Some investigators have described the soluble factors suppressing cellular DNA synthesis which are released by macrophages of "normal" rats (Waldman and Gottlieb 1973; Reed and Lucas 1975) or mice (Calderon et al. 1974; Lejeune 1976). These are consistent with our factor on thermostability. But the feature of MfID against dialysis differed from the factors of mouse origin which are dialyzable. Especially, it is in confusion, still to be characterized, that there is a diametrical difference between MfID and MDF (Waldman and Gottlieb 1973), in spite of the fact that both are of rat origin. This study represents some evidence that the inhibitory effect of macrophages may be mediated by MfID : 1) The kinetic experiments revealed that the release of MfID was concurrent with macrophage-mediated inhibition (Fig. 3). 2) The decline of the inhibitory effect shown by peptone-induced macrophages was simultaneously accompanied by a decrease in MfID productivity (Fig. 4). 3) A considerable number of macrophages were required for the detection of macrophage-mediated inhibition or of MfID (Figs. 3 and 4). 4) The susceptibility of various cell types to macro phage-mediated inhibition was comparable to MfID (Table 6). The inhibition of 3H-TdR incorporation into cells by MfID paralleled the suppression of cell growth (Table 4). But the suppressing activity was not due to the cytotoxicity of MfID, since the rate of 3H-proline labeled target cells detached from culture surface was very slight as compared with the inhibition of 3H-TdR incorporation (Fig. 2). Also, lymphocyte DNA synthesis was inhibited without

Inhibitionof CellularDNASynthesis

379

the apparent cytotoxic effect (Table5). From these findings,it was clear that MfID responded to suppress rather than to kill cell growth. Furthermore, MfID was distinguishablefrom the macrophage factor "ITI" reported by Opitz et al. (1975) which was cold thymidine-like and did not affect the rate of cell proliferation. It has been widelyreported that macrophagesdevelop a cytotoxic effect, when they are activated by various agents (Alexander and Evans 1971; Hibbs et al. 1972; Basic et al. 1974; Kaplan et al. 1974). However, normal macrophages did not show a cytotoxic effect. In this respect, the inhibitory effect shown by normal macrophages should be distinguished from their cytotoxic effect. It has been indicated that normal macrophages inhibit DNA synthesis in lymphocytes (Waldman and Gottlieb 1973; Miller and Mishell 1975; Baird and Kaplan 1977). Although Keller (1974) and Krahenbuhl and Remington (1974) reported that activated macrophages showed more inhibitory effect than non-activated macrophages against other cells, some degree of "background" inhibition by the latter cells could be seen (Reference10, Fig. 1; Reference11, Table 1). Thus, the inhibitory effect of macrophages on cellular DNA synthesis seems to be an inherent property, and it is probable that macrophagesmay play the role of regulating the proliferation of lymphocytes and other cells. References 1) Alexander,P. & Evans, R. (1971)Endotoxinand doublestrandedRNA render macrophages cytotoxic. NaturenewBiology,232, 76-78. 2) Baird, L.G. & Kaplan, A.M. (1977) Macrophage regulationof mitogen-induced blastogenesis. I. Demonstrationof inhibitorycells in the spleensand peritoneal exudatesof mice. CellularImmunol.,28, 22-35. 3) Basit, I., Milas,L., Grdina,D.J. & Withers,H.R. (1974) Destructionof hamster ovariancellculturesbyperitonealmacrophages frommicetreatedwithcorynebacterium granulosum, J. nat. CancerInst., 52, 1839-1842. 4) Bean, M.A.,Pees, H., Rosen,G. & Oettgen,H.F. (1973) Prelabelingtarget cells with 3H-prolineas a methodfor studyinglymphocytecytotoxicity.Nat. Cancer Inst. monogr., 37, 41-48. 5) Calderon,J., Williams,R.T. & Unanue,E.R. (1974) An inhibitorof cellproliferation releasedby culturesof macrophages. Proc.nat. Acad.Sci.,71, 4273-4277. 6) Harris,G.(1965) Studiesof the mechanismof antigenstimulationof DNAsynthesis in rabbit spleencultures. Immunology, 9, 529-541. 7) Hibbs,J.B., Jr. Lambert,L.H., Jr. & Remington,J.S. (1972) Possiblerole of macrophagemediatednonspecificcytotoxicityin tumour resistance. Naturenew Biology,235, 48-50. 8) Kaplan,A.M.,Morahan, P.S.&Regelson, W.(1974)Inductionofmacrophage-mediated tumor-cellcytotoxicityby pyrancopolymer.J. nat. CancerInst.,52,1919-1923. 9) Keller,R. (1973)Cytostaticeliminationof syngeneicrat tumor cellsin vitro by nonspecificially activatedmacrophages.J. exp. Med.,138, 625-644. 10) Keller,R. (1974)Mechanisms bywhichactivatednormalmacrophages destroysyngeneic rat tumour cells in vitro. Cytokinetics,non-involvement of T lymphocytes,and effectof metabolicinhibitors.Immunology, 27, 285-298. 11) Krahenbuhl,J.L. & Remington,J.S. (1974) The role of activatedmacrophagesin specificand nonspecific cytostasisof tumor cells.J. Immunol.,113,507-516. 12) Lejeune,F.J. (1976) Macrophage secretionsaffectingthe growthof other cells. In: ClinicalTumorImmunology, editedby J. Wybran& M.J. Staquet,PergamonPress, Oxford,NewYork,Toront,Paris,Sydneyand Frankfurt,pp. 9-18.

380 13) 14)

15) 16)

17) 18)

19)

K. Toh et al. Miller, C.L. & Mishell. R.I. (1975) Differential regulatory effects of accessory cells on the generation of cell-mediated immune reactions. J. Immunol., 114, 692-695. Opitz, H.G., Niethammer, D., Lemke, H., Flad, H.D. & Huget, R. (1975) Inhibition of 3H-thymidine incorporation of lymphocytes by a soluble factor from macrophages. Cellular Immunol., 16, 379-388. Parkhouse, R.M.E. & Dutton, R.W. (1966) Inhibition of spleen cell DNA synthesis by autologous macrophages. J. Immunol., 97, 663-669. Reed, W.P. & Lucas, Z.J. (1975) Cytotoxic activity of lymphocytes. V. Role of soluble toxin in macrophage-inhibited cultures of tumor cells. J. Immunol., 115, 395404. Toh, K. & Kikuchi, K. (1977) A simple method of cell-mediated cytotoxic assay by postlabeling of tritiated thymidine. Tohoku J. exp. Med., 122, 259-265. Waldman, S.R. & Gottlieb, A.A. (1973) Macrophage regulation of DNA synthesis in lymphoid cells: Effect of a soluble factor from macrophages. Cellular Immunol., 9, 142-156. Yoshinaga, M., Yoshinaga, A. & Waksman, B.H. (1972) Regulation of lymphocyte responses in vitro. I. Regulatory effect of macrophages and thymus-dependent (T) cells on the response of thymus-independent (B) lymphocytes to endotoxin. J. exp. Med., 136, 956-961.

Inhibition of cellular DNA synthesis by rat peritoneal macrophages.

Tohoku J. exp. Med., 1977, 123, 371-380 Inhibition of Cellular Peritoneal Macrophages DNA Synthesis KENTARO TOH, NORIYUKI SATO and by Rat...
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