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.