Isolation and characterization embryonic muscle culture Hitoshi
Honda,
Department
Abstract: bution
of Neurology,
We have resident
of
Hiromitsu
Kimura,
University
previously macrophages
of macrophages and
Abdolmohammad
of Pennsylvania,
described the in normal
wide rat
distriskeletal
Immunoreagents
antibodies ED1, cells with denFurthermore, we of macrophages The isolated cells, Ia
mostly ED1, expressed class I and CD4 antigens and bore complement (C3) receptors on their surfaces. The fact that cell of monocyte-macrophage lineage occur in the embryonic muscle suggests that during embryogenesis these cells may enter the developing muscle and give rise to a population of tissue-associated macrophages. J. Lcukoc. Biol. 52: 537-544; 1992. Key .
Words: macrophages immunohistochemistry . FAGS
muscle
.
culture
suiface
marker
analysis
Murine monoclonal and MRC OX43 to identify recognizes
INTRODUCTION that
tissue-associated sor
cells
with stasis
mononuclear
macrophages, in
the
bone
phagocytes, monocytes,
marrow,
form
and
a dynamic
their
precursystem
the potential to exert a modulatory role in tissue as well as local defense and tissue remodeling
Tissue
macrophages
being
present
spleen,
lymph
in node,
are
several bone
widely
organs marrow,
distributed
(e.g., and
connective tissue, and in serous cavities have demonstrated that tissue-associated present in large numbers in normal skeletal phenotypically heterogeneous populations differentiation in the muscle
and biologic as well as the
in
liver, nervous
homeo[1, 2]. the
kidney,
body,
lung,
system),
in
[3]. Recently, macrophages muscle and [4]. However,
we are form the
function of tissue other nonlymphoid
(mAbs) ED1, Indianapolis, of
ED2, IN)
ED3 [5], were used
rat macrophages. in monocytes
ED1 most
and
OX18 rat
MAbs
against
(MHC) [7]; rat Ia
class antigens
antigen,
W3/25
CD4
rat
major
histocompatibility
I antigens (monomorphic), (monomorphic), MRC [9];
and
MRC OX6 [8]; receptor,
C3
complement
MRC OX42 [10], were obtained from Bioproducts Science Inc. (Indianapolis, IN). Fluorescein isothiocyanate (FITC)-and peroxidase-labeled affinity-purified goat antimouse immunoglobulin G’s (IgG’s), F(ab’)2 fragment (Cappel, West Chester, PA), were used for indirect immunofluorescence and immunoperoxidase staining, respectively. Normal mouse IgG (Bioproducts) was used in control experiments. Anti-sheep erythrocyte antibody (Cappel) was for
Fc
receptor
detection.
sections
Hind
including cellular
antibodies (Serotec,
the subpopulations a cytoplasmic antigen
subpopulations.
complex
Muscle
is evidence
[6]
macrophage subpopultions. ED2 recognizes a membrane antigen on tissue macrophages of lymphoid and nonlymphoid organs. ED3 recognizes a membrane antigen on macrophages predominantly confined to lymphoid organs. MRC 0X43 recognizes a membrane antigen on some macrophage
used
There
Rostami
Philadelphia
muscle. In this study, we investigated the characteristics of the macrophages that occur in rat embryo muscle cultures. We showed that cells of monocyte-macrophage lineage are present in primary muscle cultures of rat embryos (18 days in gestation) and that these cells form morphologically and phenotypically heterogeneous populations, based on their reaction with monoclonal ED2, ED3, and 0X43. Constitutively dritic appearance were also observed. established the procedure for isolation from the primary muscle cultures.
from rat
macrophages organs have
limbs of 18-day-old Lewis rat embryos were removed and snap frozen in liquid nitrogen-cooled 2-methylbutane. Cryostat sections (6 jzm) of thigh muscle were cut in the transverse plane and picked up on glass slides.
Preparation macrophages Primary
of primary muscle
cultures
culture
were
and
isolation
prepared
as
of
previously
described [ii]. Briefly, hind limb muscles were removed aseptically from 18-day-old Lewis rat embryos. Muscles were dissected free of bone and skin under a dissecting microscope, washed well with Wylie’s medium, minced into small pieces, and digested with Freehold, NJ) 2.4
been demonstrated. In this paper, we show that cells of monocyte-macrophage lineage occur in primary muscle cultures and that these cells form morphologically and phenotypically heterogeneous populations. Furthermore, we have established procedures for isolation of macrophages from primary muscle cultures and analyzed their cytologic and phenotypic characteristics.
muscle
collagenase mg/ml and
(CLS, DNase
grade II, Worthington, (Sigma Chemical
Co.,
not
Abbreviations: erythrocyte
calf
DMEM, antibody;
serum;
FITC,
munodeficiency body;
IgG,
major
modified
Eagle’s
fluorescence-activated
fluorescein
virus;
MHC,
Dulbeccos
FACS,
cell
isothiocyanate;
immunoglobulin
HIV, G;
histocompatibility
medium; sorter;
complex;
mAb, PBS,
EA,
FCS,
fetal
human
im-
monoclonal
anti-
phosphate-buffered
saline.
MATERIALS
AND
Reprint
METHODS
the H.
Rats Lewis rats (Wilmington,
sity,
were
obtained MA).
from
Charles
River
ogy,
AM.
Kimura’s
Department
of Neurology,
Philadelphia,
address:
PA
Department
of
Hospital
of
19104.
Neurology,
Nagoya
Univer-
Japan.
present
address:
Children’s
March
of Leukocyte
Rostami,
Pennsylvania,
present 466,
National
Received
Journal
of
Honda’s Nagoya
H.
Laboratories
requests:
University
Department
Hospital,
10,
1992;
Biology
Tokyo
accepted
Volume
of Transplantation 157,
July
Immunol-
Japan.
1, 1992.
52,
November
1992
537
St.
Louis,
MO)
37#{176}C.After nylon
0.01
mesh,
10%
5 g/ml were plated a
times
calf
CO2
medium
passed
with
Wylie’s
medium Hazelton,
(5
1 h
Fc receptor
at
a 20-tm
medium,
(FCS,
Cells culture
humidified
for
through
Eagle’s
serum
gentamicin. in 25-cm2
5%
Wylie’s
were
modified
fetal
KS) and
37#{176}C in
cells
three
in Dulbecco’s
containing
in
the
washed
suspended
5 ml
mg/ml
digestion,
Erythrocyte to detect (dilution
and
(DMEM) Lenexa,
x 106) in a volume flasks and incubated
of
Several
atmosphere.
from
days after plating, irregular phase-bright cells apon the top layer of the cultures (Fig. iA). In the fol3-5 days, these cells became confluent over the entire and clusters of rounded, phase-bright, loosely ad-
above.
The
(13 mm diameter) were maintained plemented with
Identification
cells
cultivated
on
glass
0.1
cacodylate,
sodium
Phagocytosis
performed glutaraldehyde pH 7.4, for 30
of latex
beads
and
coverslips
living cells or buffered with mm at 4#{176}C.
cells
were
mounted
Fig.
The
limb
of
gestation)
Lewis
as
Methods. plating.
(C)
(5 days
phase-bright
Acid
vesicles
appeared
(D,
10 days
of
abundant
x200;
538
(E)
on (E,
culture).
and
of
of primary cul-
of
cytoplasculture
later
vesicles
con-
black B)
after
E) A cluster
of the
Sudan
(B) in the
These (A,
methanol
and
8 days
numerous
top
incubation). lipid
formalin-fixed
and
(D, with
in
hind days
Materials
staining
of incubaton). cells
18
views
(A)
phosphatase
large
from (at
in
4 days
mic tamed
embryos
B) Phase-contrast
cultures,
acid
xlOO;
B stain
on
(C,
D)
x500.
Journal
of Leukocyte
Biology
Volume
52,
November
to cells
nonspecific
method some Sudan
isolated esterase
for peroxicoverslips black B.
staining
with peroxidase-conjugated fragment (dilution 1:200), peroxidase was developed
cell sorter
for with
goat an2 h at 0.05%
and 0.01% hydrogen 7.6, for 10 mm at room with methyl green, deFor controls, mAb was mouse IgG (2 mg/ml).
(FACS)
analysis
Flow cytometric analysis was performed to detect the cell surface expression of MHC class I, Ia, and CD4 antigens and complement C3 receptor on the cells isolated from primary muscle cultures. Immediately after isolation, 1 x 10 living cells were incubated on ice for 30 mm with mAb OX18, OX6, W3/25, or OX42 dissolved in 0.15 M PBS containing 2% FCS, 0.01% sodium azide, and 0.04% ethy-
in rat muscle
prepared
described
(A,
muscle
with
ofmacrophages were
acetic glycerol.
5%
slides
cultures
muscles
with
glass
1. Occurrence
cultures.
ture
fixed
on
applied
cells
Fluorescence-activated
ink
The cells isolated from primary muscle culture were incubated with either latex beads (1.1 jan, Sigma Chemical; final dilution 1:500) or carbon ink (no. 518, Perikan AG, Hannover, Germany; prefiltered with a 0.2-nm filter, final dilution 1:1000) at 37#{176}Cfor 30 mm. After rinsing in DMEM, the
were including
diaminobenzidine tetrahydrochloride peroxide in 0.5 M Tris buffer, pH temperature, rinsed, counterstained hydrated, and mounted in Permount. replaced with either PBS or normal
on
carbon
stains culture,
acid phosphatase [14], and Kaplow’s [iS]. For cytoplasmic lipid staining, in 10% formalin were stained with
30 mm; and incubated timouse IgG, F(ab’)2 4#{176}C.Subsequently,
was
2%
muscle
immunoperoxidase staining was carried out to idenof monocyte-macrophage lineage in the muscle cultures. Both the embryo muscle sections and cultures were fixed in absolute methanol for 10 mm and air-dried overnight at 4#{176}C.The specimens were rinsed in phosphate-buffered saline (PBS) and treated at 4#{176}Covernight with mAb ED1, ED2, ED3, or 0X43 at a dilution of 1:500; rinsed in PBS for tify
microscopy microscopy fixed in
examined antibody
Indirect
of macrophages
Phase-contrast on specimens
(EA) rosette formation was using anti-sheep erythrocyte sheep erythrocytes [i2].
staining histochemical
Immunocytochemical
at a density of 1 x i04/coverslip. Cultures and refed every 5 days with DMEM sup10% FCS and antibiotics.
Phase-contrast
M
were
primary
[13], dase fixed
herent cells were also observed (Fig. iB). Cultures were then shaken on an orbital shaker at 150 rpm for 30 mm at room temperature. Supernatant media containing detached cells were passed through a 20-sm nylon mesh and centrifuged, and the cells were suspended in the culture medium described
antibody Fc receptors 1:128)-coated
Histochemical
at
At 4-5 peared lowing surface,
detection
1992
lene diaminetetraacetic acid (all mAbs at a final dilution of 1:100); washed three times; and treated with FITC-labeled goat antimouse IgG, F(ab’)2 fragment (1:50 dilution), dissolved in the same PBS solution, on ice for 30 mm. After washing, the cells were suspended in 1.0 ml of cold 0.15 M PBS and examined with a FACS Analyzer I (BectonDickinson, Mountain View, CA).
tion
period,
OX43
striking
The proliferation activity of isolated macrophages was determined by [3H]thymidine incorporation. Immediately after isolation, cells were suspended in DMEM supplemented with 10% FCS and transferred into 96-well plates at density of 2.0 x i04/well. Cells were preincubated for 48 h with or without rat interferon-y (Genzyme, 1 or iO U/ml). After washing with DMEM, the cells were cultured for 2 days and pulsed with 0.25 tCi [3H]thymidine/well i2 h prior to the harvest. Gamma-irradiated (1500 rads) cells were used as negative controls.
Localization
of macrophages
in situ
Immunoperoxidase staining with mAbs ED1, ED2, and ED3, which recognize the distinct subpopulation of rat monocyte-macrophages [5], was carried out on cryostat sections of embryonic rat thigh muscles. Cells of monocytemacrophage lineage were localized in situ with these mAbs, which gave discrete labeling of a cell population with an ameboid appearance (Fig. 2.) EDi cells scattered in both the subcutaneous connective tissue and the interstitium of muscle (Fig. 2A), whereas ED2 cells occurred mainly in the subcutaneous and epimysial connective tissues and were rarely observed in the embryonic muscle parenchyma (Fig. 2B).
No
ED3
cells
Macrophages
were
in the
detected
primary
in the
muscle
sections.
culture
In preliminary experiments, we prepared primary muscle cultures from Lewis rats of three different ages-i8-day-old embryos, neonates, and 8-week-old adults and observed the occurrence of monocyte-macrophages in each culture for 2 weeks. Cell suspensions for primary cultures were obtained using the method mentioned in Materials and Methods. Cultures prepared from thigh muscles of 8-week-old Lewis rats
displayed a scattering of ED2 cells and clusters of OX43 cells, which were morphologically and cytochemically quite similar to those observed in the primary cultures of embryos and neonates, but irregularly shaped ED1 cells were rarely observed. In neonates, during the 2-week incuba-
Fig. 2. Immunohistochemical macrophages in the developing sections
of
the
18
thigh
muscle
of
a 1:500 dilution of ED1 (A) or ED2 and then with peroxidase-conjugated antimouse IgG antibody as described Methods.
counterstained the EDP cells muscle
of
methyl in the
(A,
x 250),
whereas
present
at
and
subcutaneous
ED2 ED1
and cells.
cells
These
in
the
observation
narrow
inter-
suggested
that
which
In primary muscle monocyte-macrophage
with
mAbs
ing
the
ED1,
ED2,
of rat embryos, lineage, based on
ED3,
and
OX43,
were
four their
cell types reaction
observed
dur-
1). Morphologically, at day 4-5 displayed a large number of irregular cells on its top layer (Fig. 1A). In the following these cells became confluent over the entire surface
8-week
the cultures phase-bright 3-5 days, the
cultures
culture
culture
(Fig.
period
1B).
At
(Table
8-10
days
of
incubation
three
kinds of cells could be distinguished under a phase-contrast microscope: a small, rounded, phase-bright, and loosely adherent cell; a “process-bearing” cell in a variety of cell shapes ranging from bipolar elongate to elaborate dendritic; and a large oval cell exhibiting several cytoplasmic vesicles (Fig. 1D). Sudan black B stain on formalin-fixed cultures showed that these vesicles contained abundant lipid (Fig. 1E). All of these cells were positive for both acid phosphatase and nonspecific esterase (Fig. 1C). Immunocytochemical staining with mAbs showed that the first type of cell was intensely positive for ED1 and the second also stained with the same antibody (Fig. 3A), whereas the third type was labeled only with
OX43
(Fig.
3B).
In
the
first
1-2
weeks
of
incubation
the
consisted almost solely of ED1 cells and clusters of OX43 cells. Later, at 3-5 weeks after plating, the cultures displayed not only ED1 and OX43 cells but also a scattering of ED2 cells and a small cluster of ED3 cells. ED2 cells had a similar morphology to ED1 cells (Fig. 3C), and ED3 cells showed several elongated spinous processes with poor primary
cultures
cytoplasm
tion
(Fig.
(5-8
cells, with
weeks)
which
were
a dendritic
Isolation
3D).
we
Furthermore,
could
at
observe
morphologically appearance
a late
some similar
(Fig.
stage
of
incuba-
constitutively to
the
Ia
EDP
cells
3E).
of macrophages
Macrophages were isolated from primary muscle cultures 10 days of incubation. The cell yield by our method was x i05/flask. The isolated cells easily adhered to glass and
at 1-3 had
irregular
cell
shapes
with
ruffled
cell
surfaces
(Fig.
4A).
The
em-
were
(B) in
lightly
green. Note that interstitium of the most
all,
macrophages,
of
number of EDi cells occurmuscle was much less than in the embryo (18 days of study demonstrated a
ED1
tissue.
only
incubated
Sections
when
epimysial (B,
with occur
were
rat
with mAb goat
cells,
gestation)
Lewis
(at
and
of
of
muscle
not
numbers
localization of muscle. Frozen
bryo
Materials
days
displayed
significant
were the major cell type in the primary cultures, seemed to be the most immature ones in the muscle. Thus, in the present study we chose the 18-dayold embryonic muscle cultures as the source of cells of monocyte-macrophage lineage in the muscle.
of
RESULTS
also
predominance
of
ED1
incorporation
cultures
but
However, a roughly estimated ring in the cultures from neonatal those from embryos. Furthermore, gestation) our immunohistochemical stitium
[3H]Thymidine
primary
cells
are
of
found
connective
the
ED2
in
the
tissues
x125).
Honda
et al.
Macrophages
in rat
muscle
culture
539
TABLE
1.
Characteristics
of Cells
of Monocyte-Macrophage
Lineage
in the
Rat Cells
Cell
Short (1-2
tnarkrr
Cell
Muscle
in primary
term wk)
Long (3-6
muscle
Cultures
culture
term wk)
Isolated
cells@
activity
Phagocytosis Acid phosphatase Nonspecific
MAbs
esterase
specific
for
rat
surface
antigens
Complement
C3
“Cell
was
marker
primary 5N.D.
+
+
+
+
+
-
-
-
+
+
+
-
+
-
-
+
-
+
+
-
ND.
ND.
-
+
+
+
+
+
+
+
antigens’
Class I antigens Ia antigens CD4
+
+
monocytes-macrophages
EDI ED2 ED3 0X43 Cell
ND.
N.D.h
Peroxidase
‘
Embryonic
muscle ,
not
receptor detected
cultures
at
by cytochemical 10
days
of
or
indirect
immunoperoxidase
staining
as described
+
I
in Materials
and
Methods.
Cells
were
+
isolated
from
incubation.
done.
Determined
by
flow
cytometric
analysis.
nuclei were oval or elongated and usually occupied an eccentric position. The cytoplasms of these cells were arranged in pseudopods of varying length, width, form, and number, resulting in a variety of cell shapes (Fig. 4B). Most of the isolated cells (98-100%) showed active phagocytosis of latex beads and carbon ink (Fig. 4C and D) and formed rosettes with the antibody-coated sheep erythrocytes (Fig. 4E). Immunocytochemical staining with mAbs revealed that these cells constituted phenotypically a homogeneous population of ED1 cells (more than 98%), and, unlike the cells in the primary muscle cultures, ED2, ED3, and OX43 cells were not observed (Fig. 5A, Table 1). They were positive for both nonspecific esterase and acid phosphatase (Fig. SB), but peroxidase activity was not detected in their cytoplasm. Thus, by morphologic, cytochemical, and mAb
analyses, virtually all of the cells isolated from primary muscle cultures were identified as the EDi macrophages. In examining the isolated cells in the culture, we observed large cells that contained several round nuclei in extensive cytoplasm. These multinucleate cells were morphologically quite different from multinucleate myotubes and, in fact, they stained with ED1 mAb and contained perinuclear nonspecific esterase-positive granules (Fig. SC and D). Approximately 5-10 of such multinucleate giant cells occurred per
Cell
coverslip
surface
Cell surface mAbs and
at
5-7
days
after
plating.
antigens antigens on the isolated cells were analyzed by FACS flow cytometry. As shown in Fig. 6, more
Fig. 3. Immunocytochemical macrophage phenotypes cle cultures. Indirect staining that
was react
carried with
mAbs
analysis in the
primary
of mus-
immunoperoxidase
out
to EDI
identify (A),
the 0X43
cells (B),
ED2 (C), ED3 (D), and OX6 (E). Methanolfixed primary muscle cultures (A, B, 7 days; C, D, 21 days; E, 35 days in vitro) were incubated with a 1:500 dilution of mAb and then with the peroxidase-conjugated goat antimouse IgG antibody as described in Materials OX43
period ED3 in
540
Journal
of Leukocyte
Biology
Volume
52,
November
1992
the
and (B)
of (D), culture.
Methods.
cells were incubation, and OX6” (A-D)
Both
ED1”
observed whereas (E) cells xl25;
(A)
at an ED2 occurred (E)
x400.
and
early (C), late
Fig. and
4. Macrophages the field contains
dant
vesicular
with
either
in
the
than
structures latex
cells.
30 mm.
EA
0X42
(C) rosette
of cells
were
were
(anti-complement
of
muscle cultures. (A, and round immature (B,
ink
(D)
stained
fixed at
Cells
positive
(anti-CD4). weak, a low most
cytoplasm
formation.
cells
on
primary cells
or carbon
rinsing,
I) and W3/25 Ia) was very expressed
in their
beads
(E)
After
98%
isolated from irregular-shaped
were
with
with
37#{176}Cfor
EDI
mAb.
for 0X18
C3
cells.
After
with
anti-sheep
xlOO;
(anti-MHC
was detected
rinsing
(B-D)
(antito be
binding
D)
Phagocytic
of
activity
in DMEM, erythrocyte
class
of OX6 appeared
views of isolated cells. culture). The cells had
(C,
mm.
(A)
Furthermore,
receptor)
of the isolated cells. Control replaced by either PBS or normal the second antibody step, showed
glutaraldehyde).
30
incubated
Although binding level of Ia antigens
these
2%
B) Phase-contrast ones (A, unfixed
x500;
they antibody
(E)
For
cells were elongated
of isolated fixed
(1:128
highly adherent processes and
macrophages.
with
cold
The
acid-alcohol.
dilution)-coated
to glass contained
cells
were
incubated
Inclusions
sheep
erythrocytes
muscle
culture
surface abun-
can at
be
seen
37#{176}Cfor
x250.
of macrophages
Proliferation
large
were
These several
the
first
10 days,
number
of
were
the
primary
irregular
confluent
phase-bright,
on most
cells
that
stains, in which mAb was mouse IgG with or without no significant fluorescence.
(Fig. this case
1B). These cells seemed early period of cultivation. in the isolated macrophage
over
Fig.
the entire
of the
Almost mAb (A) B)
tive
surface
to replicate However, culture.
5. Cytochemical
staining
granules
and
their
of the culture during not the the iso-
immunocytochemical
macrophage
all the isolated and contained
a
adherent
extensively this was Although
isolated
in
displayed
loosely
cultures.
cells stained with acid phosphatase-posi-
cytoplasm
(B)
1 day
(A,
ED1 after
isolation. (C, D) Multinucleate giant cells occurred in the isolated cell cultures, 7 days in vitro. The giant cells were ED1” (C) and contamed perinuclear nonspecific esterase-positive granules (D). (A) x 125; (B) x 500, (C, D) x 400.
Honda
et al.
Macrophages
in rat
muscle
culture
541
Fig.
6.
Flow
antigens
cytometric
on
the
analysis
cells
isolated
of cell from
surface
primary
250 rat
muscle
cultures. The cell surface expression of class I (A), Ia (B), and CD4 (C) antigens and complement C3 receptors (D) was determined by flow cytometric analysis with mAbs OX18, OX6, W3/25,
and
0X42,
second-step timouse
IgG
cells
respectively,
reagent
were
using
as
Flit-labeled
antibody.
Control
stained
with
the
goat
an-
(dotted
second-step
line); antibody
only. 0
I-
z
0
LI a0
io
io2
250
U
-a ...a ‘U
U
LOG
The contamination mated by counting muscle culture was ED1 mAb staining
lated cells could be maintained in vitro and survived for up to 4 weeks, the cell density decreased gradually after plating and we could not find proliferation of the isolated cells under our culture conditions (Table 2).
of monocytemuscle culture phase-contrast with mAbs salient features
of the cells are a large eccentric nucleus and elongated cytoplasmic processes that can produce a variety of cell shapes. In the living state in vitro, these cells readily adhere to glass or plastic surfaces and show morphological features ofactive endocytosis, i.e., membrane ruffling, abundant lysosomes, and phagocytosed objects. Most of these cells formed rosettes with EA, indicating the presence of Fc receptor on their surface. Observations thus indicate that these cells in primary muscle cultures have identical properties to cells of monocyte-macrophage
TABLE
lineage
2.
[3,
Proliferation
Treatment
Exp.
incorporation
(cpm)
1
Exp.
181
Untreated
265+77
222 + 47 248 +90
+
56
IFN--y
1 (U/mI)
267+50
277
IFN--y
10 (U/mI)
190+39
189+45
Proliferation interferon--y 2 days prior tive
542
of isolated Isolated
(IFN--y,
in
96-well
to the controls.
Data
Journal
(2.0
x
I or 10 U/mI) plates
harvest.
macrophages
cells and
pulsed
was 10), with
0.25 (1500
are
of Leukocyte
assayed
by
were
mean
+
Biology
sCi
[3H]thymidine
either
for 48 h or untreated,
Gamma-irradiated presented
which
with
cultured
[3Hjthymidine/well
rads)
cells
SE of triplicate
Volume
were
+68
in-
pretreated
were
decreased. The third and fourth types, cells stained with ED2 and ED3, respectively, formed small clusters and occurred late (usually 3 weeks or more after plating) in the primary cultures. Thus, it appeared that four distinct subpopulations of macrophages occurred in the primary muscle cultures. In addition, we found that some constitutively Ia cells appeared late in the cultures. These Ia cells were morphologically similar to the ED1 cells, exhibiting several elongated processes and a distinct dendritic appearance. The relationship of this Ia cell to the dendritic cell, which was first described in mouse lymphoid organs [17], is uncertain. Constitutive and intense expression of Ia antigens is the cardinal feature of dendritic cells, and they can be cultivated
2
Gamma-irradiated
corporation.
the cultures, and was also identified and characterized as the cell isolated from primary culture. The second, a cell labeled with 0X43 antibody, was characterized by large lipid-laden vesicles occupying its cytoplasm, and it remained attached in the cultures during the isolation procedure. These two cell types were recognized throughout the incubation of the primary culture, each exhibiting typical and consistent properties, although the number of these cells gradually
Macrophages
[‘HjThymidine
for 12 h
used
as nega-
samples.
52,
November
of peripheral blood monocytes estired blood cells in the initially prepared less than 0.i% (less than 20 cells/flask). on smears of initially prepared cell sus-
By morphological and phenotypic criteria, four types of mononuclear phagocytes were distinguished in the primary muscle cultures. One was an ED1 cell, a major cell type in
12].
of Isolated
INTENSITY
pensions showed that the antibody labeled 3-5 % of the total number of mononuclear cells. However, during the first 10 days of incubation primary cultures displayed a large number of phase-bright, loosely adherent cells identified as monocytes-macrophages, mostly ED1 cells, that were confluent over the entire surface of the culture. Therefore, although a partial contribution of blood monocytes in the culture cannot be ruled out, it seems likely that a number of monocytes-macrophages were generated as a consequence of proliferation in the primary muscle culture. Indeed, extensive replication of macrophages from nonhemopoietic tissues has been demonstrated in the presence of growth factors produced by tissue cells, such as embryo fibroblasts [16].
DISCUSSION The present study demonstrated that cells macrophage lineage occur in primary embryo of rat. These cells can be identified by microscopy and immunoperoxidase staining specific for rat monocytes-macrophages. The
FLUORESCENCE
1992
in vitro without changing their cytologic and surface properties [18]. Furthermore, the presence of Ia-bearing dendritic cells has been reported in nonlymphoid organs including skeletal muscle [4, 19]. Thus, it seems likely that the Ia cells occurring in the primary muscle cultures have a close relationship to the dendritic cells in the nonlymphoid tissues.
hemopoiesis and become macrophages in the tissues [23]. Therefore, generation of muscle macrophages from such fetal macrophage precursors, which are ontogenically independent of blood monocytes, may be equally possible. The occurrence of large multinucleate cells in isolated macrophage cultures is of interest. It has been reported that
We established with more than
human monocytes and mouse macrophages can form variable numbers of multinucleate cells in vitro [24, 25]. The present study confirmed these observations by showing that the multinucleate giant cells, which stained with ED1 antibody, are formed in rat macrophage cultures in vitro. We also observed that a number of ED1 multinucleate giant
The dish.
isolated They
our
culture
a technique 95% purity
for from
isolation primary
of macrophages muscle cultures.
adherent to a glass or plastic cytoplasmic granules positive for nonspecific esterase and acid phosphatase and showed active phagocytosis. Using [3H]thymidine incorporation, we could not find proliferation of the isolated cells in vealed body 4-week
that
cells were highly exhibited numerous
condition.
virtually
and they incubation
Immunocytochemical
all
of these
remained period.
cells
persistently Thus, unlike
staining
stained EDi the
with
ED1
throughout macrophages
re-
antithe in
the primary muscle cultures, which consisted of phenotypically heterogeneous populations, the isolated cells formed a homogeneous population of EDP macrophages, and hardly replicated under the isolated culture conditions. FACS analysis showed that almost all of the isolated cells expressed MHC class I and CD4 antigens on their surfaces. Indeed, in rat the CD4 molecule has been reported to be expressed not only on MHC class Il-restricted helper T cells but also on a subpopulation of macrophages [20]. Thus, the expression of this antigen on the isolated cells, together with the fact that they have complement (C3) receptors, provides further evidence that they are of monocyte-macrophage lineage. Whereas there were four distinct macrophage subpopulations (i.e., EDi, ED2, ED3, and OX43 cells) grown in the primary muscle cultures, this was not the case with the isolated cells, which consisted solely ofEDP cells. One explanation for the homogeneity of the isolated macrophages is that the non-EDi cells, particularly OX43 cells, firmly adhered to the culture layer and hardly detached during the isolation procedure, although the primary cultures displayed both EDP and OX43 cells. On the other hand, the observations that ED2 and ED3 cells occurred only at a late period in the primary cultures and that rat blood monocytes, which indude precursors of tissue macrophages, are predominantly EDP [4 5] suggest that in the ontogeny of macrophages the ED1 cell is a relatively immature type of macrophage and both ED2 and ED3 cells are differentiated ones. If so, it seems likely that in primary muscle cultures a cell differentiation-promoting factor(s) derived from the tissue cells might be required for differentiation of immature macrophages in the tissue, because the isolated cells persistently remained ED1 and neither ED2 nor ED3 cells occurred throughout the 4-week period of incubation. In this regard, monocytes have been shown to replicate and differentiate extensively in vitro in the presence of media conditioned by tissue cells, which served as the source of a growthand differentiationpromoting factor(s) [21]. Although we have been unable to determine whether EDi and ED2 cells are derived from macrophage-committed hemopoietic precursors in the muscle, the fact that cells of monocyte-macrophage lineage occur in the embryonic muscle suggests that during embryogenesis, monocytemacrophage precursors ofmesenchyrnal origin may enter the muscle tissue and give rise to a population of mononuclear phagocytes. Indeed, in our study macrophages in the primary
embryonic
muscle
cultures
seemed
to
replicate
tensively. Furthermore, there is evidence that tissue macrophages can self-renew and self-maintain without monocyte migration [22] and that, in embryonic tissues, fetal macrophage precursors occur before the initiation of bone marrow
ex-
cells were present days in gestation) suggest that the
in the bone marrow of rat embryos (17-19 (data not shown). Thus, our observations multinucleate giant cells (syncytia) occur in rat macrophage colonies in vivo and in vitro and that the EDi macrophages are the precursors of the multinucleate cells. The multinucleate giant cells are found in a variety of inflammatory disease processes [26]; however, the mechanisms
and
involved
in
inducing
the
syncytia
under
both
normal
pathologic
conditions remain to be elucidated. Macrophages are thought to play an important role in altered homeostasis, as in inflammation, tissue remodeling, and immune surveillance [1, 2]. Regarding muscle diseases, McDouall et al. [27] have demonstrated that among the mononuclear infiltrates in juvenile dermatomyositis and Duchenne muscular dystrophy there was a striking predominance of endomysial macrophages and suggested that the major mechanism of muscle damage is mediated in part via the macrophages. Also, in human immunodeficiency virus
(HIV)-associated
myopathy
domysial macrophages were HIV-infected macrophages muscle fibers and contribute
a
large
number
of
en-
infected with HIV and these could secrete factors toxic to to muscle fiber dysfunction
[28]. Although the precise function of macrophages in the developing muscle is unknown, evidence that macrophages can secrete multiple cell products ranging in biologic activity from induction of cell growth to cell death [29] suggests that they may modulate the process of myogenesis through their potential endocytic and secretory functions. Identification and isolation of macrophages from the rat embryonic muscle, reported here, may provide the opportunity to study the role of macrophages in the developing muscle.
ACKNOWLEDGMENT This work was 39489-02 from was a recipient
supported in par by grants NS 08075 and AR the National Institutes of Health. H. Honda of a postdoctoral fellowship from the Amen-
can
Dystrophy
Muscular
Association.
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type H.,
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of mice. markers,
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3. Immunology 57, 239-247. Rostami, AM. (1989) Expression of major histocompatibility complex class I antigens in rat muscle cultures: the possible developmental role in myogenesis. Proc. NatI. Acad. Sci. USA 86, 7007-7011. 12. Ross, GD. (1981) Detection of complement receptors and Fc receptors on macrophages. In Manual ofMacrophage Methodology (H.B. Herscowitz, H.T Holden, J.A. Bellanti, and A. Ghaffar, eds),
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tissue R.M.,
the
mononuclear
damage dystrophy.
j
(HIV)-associated tion of an Neurol. 28,
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and
the
mechanism
of
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dermatomyositis and Duchenne muscular NeuroL Sci. 99, 199-217. Smith, T.W., Blumenfeld, A., Fairchild, PG., U. (1990) Human immunodeficiency virus
28. Chad, D.A., DeGirolami,
29. Nathan, Invest.
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579-582. CF. (1987) 79, 319-326.
Secretory
immunocytochemical 4!) in muscle
products
identificamacrophages.
ofmacrophages.j
Ann. Clin.
Honda,
Department
Abstract: bution
of Neurology,
We have resident
of
Hiromitsu
Kimura,
University
previously macrophages
of macrophages and
Abdolmohammad
of Pennsylvania,
described the in normal
wide rat
distriskeletal
Immunoreagents
antibodies ED1, cells with denFurthermore, we of macrophages The isolated cells, Ia
mostly ED1, expressed class I and CD4 antigens and bore complement (C3) receptors on their surfaces. The fact that cell of monocyte-macrophage lineage occur in the embryonic muscle suggests that during embryogenesis these cells may enter the developing muscle and give rise to a population of tissue-associated macrophages. J. Lcukoc. Biol. 52: 537-544; 1992. Key .
Words: macrophages immunohistochemistry . FAGS
muscle
.
culture
suiface
marker
analysis
Murine monoclonal and MRC OX43 to identify recognizes
INTRODUCTION that
tissue-associated sor
cells
with stasis
mononuclear
macrophages, in
the
bone
phagocytes, monocytes,
marrow,
form
and
a dynamic
their
precursystem
the potential to exert a modulatory role in tissue as well as local defense and tissue remodeling
Tissue
macrophages
being
present
spleen,
lymph
in node,
are
several bone
widely
organs marrow,
distributed
(e.g., and
connective tissue, and in serous cavities have demonstrated that tissue-associated present in large numbers in normal skeletal phenotypically heterogeneous populations differentiation in the muscle
and biologic as well as the
in
liver, nervous
homeo[1, 2]. the
kidney,
body,
lung,
system),
in
[3]. Recently, macrophages muscle and [4]. However,
we are form the
function of tissue other nonlymphoid
(mAbs) ED1, Indianapolis, of
ED2, IN)
ED3 [5], were used
rat macrophages. in monocytes
ED1 most
and
OX18 rat
MAbs
against
(MHC) [7]; rat Ia
class antigens
antigen,
W3/25
CD4
rat
major
histocompatibility
I antigens (monomorphic), (monomorphic), MRC [9];
and
MRC OX6 [8]; receptor,
C3
complement
MRC OX42 [10], were obtained from Bioproducts Science Inc. (Indianapolis, IN). Fluorescein isothiocyanate (FITC)-and peroxidase-labeled affinity-purified goat antimouse immunoglobulin G’s (IgG’s), F(ab’)2 fragment (Cappel, West Chester, PA), were used for indirect immunofluorescence and immunoperoxidase staining, respectively. Normal mouse IgG (Bioproducts) was used in control experiments. Anti-sheep erythrocyte antibody (Cappel) was for
Fc
receptor
detection.
sections
Hind
including cellular
antibodies (Serotec,
the subpopulations a cytoplasmic antigen
subpopulations.
complex
Muscle
is evidence
[6]
macrophage subpopultions. ED2 recognizes a membrane antigen on tissue macrophages of lymphoid and nonlymphoid organs. ED3 recognizes a membrane antigen on macrophages predominantly confined to lymphoid organs. MRC 0X43 recognizes a membrane antigen on some macrophage
used
There
Rostami
Philadelphia
muscle. In this study, we investigated the characteristics of the macrophages that occur in rat embryo muscle cultures. We showed that cells of monocyte-macrophage lineage are present in primary muscle cultures of rat embryos (18 days in gestation) and that these cells form morphologically and phenotypically heterogeneous populations, based on their reaction with monoclonal ED2, ED3, and 0X43. Constitutively dritic appearance were also observed. established the procedure for isolation from the primary muscle cultures.
from rat
macrophages organs have
limbs of 18-day-old Lewis rat embryos were removed and snap frozen in liquid nitrogen-cooled 2-methylbutane. Cryostat sections (6 jzm) of thigh muscle were cut in the transverse plane and picked up on glass slides.
Preparation macrophages Primary
of primary muscle
cultures
culture
were
and
isolation
prepared
as
of
previously
described [ii]. Briefly, hind limb muscles were removed aseptically from 18-day-old Lewis rat embryos. Muscles were dissected free of bone and skin under a dissecting microscope, washed well with Wylie’s medium, minced into small pieces, and digested with Freehold, NJ) 2.4
been demonstrated. In this paper, we show that cells of monocyte-macrophage lineage occur in primary muscle cultures and that these cells form morphologically and phenotypically heterogeneous populations. Furthermore, we have established procedures for isolation of macrophages from primary muscle cultures and analyzed their cytologic and phenotypic characteristics.
muscle
collagenase mg/ml and
(CLS, DNase
grade II, Worthington, (Sigma Chemical
Co.,
not
Abbreviations: erythrocyte
calf
DMEM, antibody;
serum;
FITC,
munodeficiency body;
IgG,
major
modified
Eagle’s
fluorescence-activated
fluorescein
virus;
MHC,
Dulbeccos
FACS,
cell
isothiocyanate;
immunoglobulin
HIV, G;
histocompatibility
medium; sorter;
complex;
mAb, PBS,
EA,
FCS,
fetal
human
im-
monoclonal
anti-
phosphate-buffered
saline.
MATERIALS
AND
Reprint
METHODS
the H.
Rats Lewis rats (Wilmington,
sity,
were
obtained MA).
from
Charles
River
ogy,
AM.
Kimura’s
Department
of Neurology,
Philadelphia,
address:
PA
Department
of
Hospital
of
19104.
Neurology,
Nagoya
Univer-
Japan.
present
address:
Children’s
March
of Leukocyte
Rostami,
Pennsylvania,
present 466,
National
Received
Journal
of
Honda’s Nagoya
H.
Laboratories
requests:
University
Department
Hospital,
10,
1992;
Biology
Tokyo
accepted
Volume
of Transplantation 157,
July
Immunol-
Japan.
1, 1992.
52,
November
1992
537
St.
Louis,
MO)
37#{176}C.After nylon
0.01
mesh,
10%
5 g/ml were plated a
times
calf
CO2
medium
passed
with
Wylie’s
medium Hazelton,
(5
1 h
Fc receptor
at
a 20-tm
medium,
(FCS,
Cells culture
humidified
for
through
Eagle’s
serum
gentamicin. in 25-cm2
5%
Wylie’s
were
modified
fetal
KS) and
37#{176}C in
cells
three
in Dulbecco’s
containing
in
the
washed
suspended
5 ml
mg/ml
digestion,
Erythrocyte to detect (dilution
and
(DMEM) Lenexa,
x 106) in a volume flasks and incubated
of
Several
atmosphere.
from
days after plating, irregular phase-bright cells apon the top layer of the cultures (Fig. iA). In the fol3-5 days, these cells became confluent over the entire and clusters of rounded, phase-bright, loosely ad-
above.
The
(13 mm diameter) were maintained plemented with
Identification
cells
cultivated
on
glass
0.1
cacodylate,
sodium
Phagocytosis
performed glutaraldehyde pH 7.4, for 30
of latex
beads
and
coverslips
living cells or buffered with mm at 4#{176}C.
cells
were
mounted
Fig.
The
limb
of
gestation)
Lewis
as
Methods. plating.
(C)
(5 days
phase-bright
Acid
vesicles
appeared
(D,
10 days
of
abundant
x200;
538
(E)
on (E,
culture).
and
of
of primary cul-
of
cytoplasculture
later
vesicles
con-
black B)
after
E) A cluster
of the
Sudan
(B) in the
These (A,
methanol
and
8 days
numerous
top
incubation). lipid
formalin-fixed
and
(D, with
in
hind days
Materials
staining
of incubaton). cells
18
views
(A)
phosphatase
large
from (at
in
4 days
mic tamed
embryos
B) Phase-contrast
cultures,
acid
xlOO;
B stain
on
(C,
D)
x500.
Journal
of Leukocyte
Biology
Volume
52,
November
to cells
nonspecific
method some Sudan
isolated esterase
for peroxicoverslips black B.
staining
with peroxidase-conjugated fragment (dilution 1:200), peroxidase was developed
cell sorter
for with
goat an2 h at 0.05%
and 0.01% hydrogen 7.6, for 10 mm at room with methyl green, deFor controls, mAb was mouse IgG (2 mg/ml).
(FACS)
analysis
Flow cytometric analysis was performed to detect the cell surface expression of MHC class I, Ia, and CD4 antigens and complement C3 receptor on the cells isolated from primary muscle cultures. Immediately after isolation, 1 x 10 living cells were incubated on ice for 30 mm with mAb OX18, OX6, W3/25, or OX42 dissolved in 0.15 M PBS containing 2% FCS, 0.01% sodium azide, and 0.04% ethy-
in rat muscle
prepared
described
(A,
muscle
with
ofmacrophages were
acetic glycerol.
5%
slides
cultures
muscles
with
glass
1. Occurrence
cultures.
ture
fixed
on
applied
cells
Fluorescence-activated
ink
The cells isolated from primary muscle culture were incubated with either latex beads (1.1 jan, Sigma Chemical; final dilution 1:500) or carbon ink (no. 518, Perikan AG, Hannover, Germany; prefiltered with a 0.2-nm filter, final dilution 1:1000) at 37#{176}Cfor 30 mm. After rinsing in DMEM, the
were including
diaminobenzidine tetrahydrochloride peroxide in 0.5 M Tris buffer, pH temperature, rinsed, counterstained hydrated, and mounted in Permount. replaced with either PBS or normal
on
carbon
stains culture,
acid phosphatase [14], and Kaplow’s [iS]. For cytoplasmic lipid staining, in 10% formalin were stained with
30 mm; and incubated timouse IgG, F(ab’)2 4#{176}C.Subsequently,
was
2%
muscle
immunoperoxidase staining was carried out to idenof monocyte-macrophage lineage in the muscle cultures. Both the embryo muscle sections and cultures were fixed in absolute methanol for 10 mm and air-dried overnight at 4#{176}C.The specimens were rinsed in phosphate-buffered saline (PBS) and treated at 4#{176}Covernight with mAb ED1, ED2, ED3, or 0X43 at a dilution of 1:500; rinsed in PBS for tify
microscopy microscopy fixed in
examined antibody
Indirect
of macrophages
Phase-contrast on specimens
(EA) rosette formation was using anti-sheep erythrocyte sheep erythrocytes [i2].
staining histochemical
Immunocytochemical
at a density of 1 x i04/coverslip. Cultures and refed every 5 days with DMEM sup10% FCS and antibiotics.
Phase-contrast
M
were
primary
[13], dase fixed
herent cells were also observed (Fig. iB). Cultures were then shaken on an orbital shaker at 150 rpm for 30 mm at room temperature. Supernatant media containing detached cells were passed through a 20-sm nylon mesh and centrifuged, and the cells were suspended in the culture medium described
antibody Fc receptors 1:128)-coated
Histochemical
at
At 4-5 peared lowing surface,
detection
1992
lene diaminetetraacetic acid (all mAbs at a final dilution of 1:100); washed three times; and treated with FITC-labeled goat antimouse IgG, F(ab’)2 fragment (1:50 dilution), dissolved in the same PBS solution, on ice for 30 mm. After washing, the cells were suspended in 1.0 ml of cold 0.15 M PBS and examined with a FACS Analyzer I (BectonDickinson, Mountain View, CA).
tion
period,
OX43
striking
The proliferation activity of isolated macrophages was determined by [3H]thymidine incorporation. Immediately after isolation, cells were suspended in DMEM supplemented with 10% FCS and transferred into 96-well plates at density of 2.0 x i04/well. Cells were preincubated for 48 h with or without rat interferon-y (Genzyme, 1 or iO U/ml). After washing with DMEM, the cells were cultured for 2 days and pulsed with 0.25 tCi [3H]thymidine/well i2 h prior to the harvest. Gamma-irradiated (1500 rads) cells were used as negative controls.
Localization
of macrophages
in situ
Immunoperoxidase staining with mAbs ED1, ED2, and ED3, which recognize the distinct subpopulation of rat monocyte-macrophages [5], was carried out on cryostat sections of embryonic rat thigh muscles. Cells of monocytemacrophage lineage were localized in situ with these mAbs, which gave discrete labeling of a cell population with an ameboid appearance (Fig. 2.) EDi cells scattered in both the subcutaneous connective tissue and the interstitium of muscle (Fig. 2A), whereas ED2 cells occurred mainly in the subcutaneous and epimysial connective tissues and were rarely observed in the embryonic muscle parenchyma (Fig. 2B).
No
ED3
cells
Macrophages
were
in the
detected
primary
in the
muscle
sections.
culture
In preliminary experiments, we prepared primary muscle cultures from Lewis rats of three different ages-i8-day-old embryos, neonates, and 8-week-old adults and observed the occurrence of monocyte-macrophages in each culture for 2 weeks. Cell suspensions for primary cultures were obtained using the method mentioned in Materials and Methods. Cultures prepared from thigh muscles of 8-week-old Lewis rats
displayed a scattering of ED2 cells and clusters of OX43 cells, which were morphologically and cytochemically quite similar to those observed in the primary cultures of embryos and neonates, but irregularly shaped ED1 cells were rarely observed. In neonates, during the 2-week incuba-
Fig. 2. Immunohistochemical macrophages in the developing sections
of
the
18
thigh
muscle
of
a 1:500 dilution of ED1 (A) or ED2 and then with peroxidase-conjugated antimouse IgG antibody as described Methods.
counterstained the EDP cells muscle
of
methyl in the
(A,
x 250),
whereas
present
at
and
subcutaneous
ED2 ED1
and cells.
cells
These
in
the
observation
narrow
inter-
suggested
that
which
In primary muscle monocyte-macrophage
with
mAbs
ing
the
ED1,
ED2,
of rat embryos, lineage, based on
ED3,
and
OX43,
were
four their
cell types reaction
observed
dur-
1). Morphologically, at day 4-5 displayed a large number of irregular cells on its top layer (Fig. 1A). In the following these cells became confluent over the entire surface
8-week
the cultures phase-bright 3-5 days, the
cultures
culture
culture
(Fig.
period
1B).
At
(Table
8-10
days
of
incubation
three
kinds of cells could be distinguished under a phase-contrast microscope: a small, rounded, phase-bright, and loosely adherent cell; a “process-bearing” cell in a variety of cell shapes ranging from bipolar elongate to elaborate dendritic; and a large oval cell exhibiting several cytoplasmic vesicles (Fig. 1D). Sudan black B stain on formalin-fixed cultures showed that these vesicles contained abundant lipid (Fig. 1E). All of these cells were positive for both acid phosphatase and nonspecific esterase (Fig. 1C). Immunocytochemical staining with mAbs showed that the first type of cell was intensely positive for ED1 and the second also stained with the same antibody (Fig. 3A), whereas the third type was labeled only with
OX43
(Fig.
3B).
In
the
first
1-2
weeks
of
incubation
the
consisted almost solely of ED1 cells and clusters of OX43 cells. Later, at 3-5 weeks after plating, the cultures displayed not only ED1 and OX43 cells but also a scattering of ED2 cells and a small cluster of ED3 cells. ED2 cells had a similar morphology to ED1 cells (Fig. 3C), and ED3 cells showed several elongated spinous processes with poor primary
cultures
cytoplasm
tion
(Fig.
(5-8
cells, with
weeks)
which
were
a dendritic
Isolation
3D).
we
Furthermore,
could
at
observe
morphologically appearance
a late
some similar
(Fig.
stage
of
incuba-
constitutively to
the
Ia
EDP
cells
3E).
of macrophages
Macrophages were isolated from primary muscle cultures 10 days of incubation. The cell yield by our method was x i05/flask. The isolated cells easily adhered to glass and
at 1-3 had
irregular
cell
shapes
with
ruffled
cell
surfaces
(Fig.
4A).
The
em-
were
(B) in
lightly
green. Note that interstitium of the most
all,
macrophages,
of
number of EDi cells occurmuscle was much less than in the embryo (18 days of study demonstrated a
ED1
tissue.
only
incubated
Sections
when
epimysial (B,
with occur
were
rat
with mAb goat
cells,
gestation)
Lewis
(at
and
of
of
muscle
not
numbers
localization of muscle. Frozen
bryo
Materials
days
displayed
significant
were the major cell type in the primary cultures, seemed to be the most immature ones in the muscle. Thus, in the present study we chose the 18-dayold embryonic muscle cultures as the source of cells of monocyte-macrophage lineage in the muscle.
of
RESULTS
also
predominance
of
ED1
incorporation
cultures
but
However, a roughly estimated ring in the cultures from neonatal those from embryos. Furthermore, gestation) our immunohistochemical stitium
[3H]Thymidine
primary
cells
are
of
found
connective
the
ED2
in
the
tissues
x125).
Honda
et al.
Macrophages
in rat
muscle
culture
539
TABLE
1.
Characteristics
of Cells
of Monocyte-Macrophage
Lineage
in the
Rat Cells
Cell
Short (1-2
tnarkrr
Cell
Muscle
in primary
term wk)
Long (3-6
muscle
Cultures
culture
term wk)
Isolated
cells@
activity
Phagocytosis Acid phosphatase Nonspecific
MAbs
esterase
specific
for
rat
surface
antigens
Complement
C3
“Cell
was
marker
primary 5N.D.
+
+
+
+
+
-
-
-
+
+
+
-
+
-
-
+
-
+
+
-
ND.
ND.
-
+
+
+
+
+
+
+
antigens’
Class I antigens Ia antigens CD4
+
+
monocytes-macrophages
EDI ED2 ED3 0X43 Cell
ND.
N.D.h
Peroxidase
‘
Embryonic
muscle ,
not
receptor detected
cultures
at
by cytochemical 10
days
of
or
indirect
immunoperoxidase
staining
as described
+
I
in Materials
and
Methods.
Cells
were
+
isolated
from
incubation.
done.
Determined
by
flow
cytometric
analysis.
nuclei were oval or elongated and usually occupied an eccentric position. The cytoplasms of these cells were arranged in pseudopods of varying length, width, form, and number, resulting in a variety of cell shapes (Fig. 4B). Most of the isolated cells (98-100%) showed active phagocytosis of latex beads and carbon ink (Fig. 4C and D) and formed rosettes with the antibody-coated sheep erythrocytes (Fig. 4E). Immunocytochemical staining with mAbs revealed that these cells constituted phenotypically a homogeneous population of ED1 cells (more than 98%), and, unlike the cells in the primary muscle cultures, ED2, ED3, and OX43 cells were not observed (Fig. 5A, Table 1). They were positive for both nonspecific esterase and acid phosphatase (Fig. SB), but peroxidase activity was not detected in their cytoplasm. Thus, by morphologic, cytochemical, and mAb
analyses, virtually all of the cells isolated from primary muscle cultures were identified as the EDi macrophages. In examining the isolated cells in the culture, we observed large cells that contained several round nuclei in extensive cytoplasm. These multinucleate cells were morphologically quite different from multinucleate myotubes and, in fact, they stained with ED1 mAb and contained perinuclear nonspecific esterase-positive granules (Fig. SC and D). Approximately 5-10 of such multinucleate giant cells occurred per
Cell
coverslip
surface
Cell surface mAbs and
at
5-7
days
after
plating.
antigens antigens on the isolated cells were analyzed by FACS flow cytometry. As shown in Fig. 6, more
Fig. 3. Immunocytochemical macrophage phenotypes cle cultures. Indirect staining that
was react
carried with
mAbs
analysis in the
primary
of mus-
immunoperoxidase
out
to EDI
identify (A),
the 0X43
cells (B),
ED2 (C), ED3 (D), and OX6 (E). Methanolfixed primary muscle cultures (A, B, 7 days; C, D, 21 days; E, 35 days in vitro) were incubated with a 1:500 dilution of mAb and then with the peroxidase-conjugated goat antimouse IgG antibody as described in Materials OX43
period ED3 in
540
Journal
of Leukocyte
Biology
Volume
52,
November
1992
the
and (B)
of (D), culture.
Methods.
cells were incubation, and OX6” (A-D)
Both
ED1”
observed whereas (E) cells xl25;
(A)
at an ED2 occurred (E)
x400.
and
early (C), late
Fig. and
4. Macrophages the field contains
dant
vesicular
with
either
in
the
than
structures latex
cells.
30 mm.
EA
0X42
(C) rosette
of cells
were
were
(anti-complement
of
muscle cultures. (A, and round immature (B,
ink
(D)
stained
fixed at
Cells
positive
(anti-CD4). weak, a low most
cytoplasm
formation.
cells
on
primary cells
or carbon
rinsing,
I) and W3/25 Ia) was very expressed
in their
beads
(E)
After
98%
isolated from irregular-shaped
were
with
with
37#{176}Cfor
EDI
mAb.
for 0X18
C3
cells.
After
with
anti-sheep
xlOO;
(anti-MHC
was detected
rinsing
(B-D)
(antito be
binding
D)
Phagocytic
of
activity
in DMEM, erythrocyte
class
of OX6 appeared
views of isolated cells. culture). The cells had
(C,
mm.
(A)
Furthermore,
receptor)
of the isolated cells. Control replaced by either PBS or normal the second antibody step, showed
glutaraldehyde).
30
incubated
Although binding level of Ia antigens
these
2%
B) Phase-contrast ones (A, unfixed
x500;
they antibody
(E)
For
cells were elongated
of isolated fixed
(1:128
highly adherent processes and
macrophages.
with
cold
The
acid-alcohol.
dilution)-coated
to glass contained
cells
were
incubated
Inclusions
sheep
erythrocytes
muscle
culture
surface abun-
can at
be
seen
37#{176}Cfor
x250.
of macrophages
Proliferation
large
were
These several
the
first
10 days,
number
of
were
the
primary
irregular
confluent
phase-bright,
on most
cells
that
stains, in which mAb was mouse IgG with or without no significant fluorescence.
(Fig. this case
1B). These cells seemed early period of cultivation. in the isolated macrophage
over
Fig.
the entire
of the
Almost mAb (A) B)
tive
surface
to replicate However, culture.
5. Cytochemical
staining
granules
and
their
of the culture during not the the iso-
immunocytochemical
macrophage
all the isolated and contained
a
adherent
extensively this was Although
isolated
in
displayed
loosely
cultures.
cells stained with acid phosphatase-posi-
cytoplasm
(B)
1 day
(A,
ED1 after
isolation. (C, D) Multinucleate giant cells occurred in the isolated cell cultures, 7 days in vitro. The giant cells were ED1” (C) and contamed perinuclear nonspecific esterase-positive granules (D). (A) x 125; (B) x 500, (C, D) x 400.
Honda
et al.
Macrophages
in rat
muscle
culture
541
Fig.
6.
Flow
antigens
cytometric
on
the
analysis
cells
isolated
of cell from
surface
primary
250 rat
muscle
cultures. The cell surface expression of class I (A), Ia (B), and CD4 (C) antigens and complement C3 receptors (D) was determined by flow cytometric analysis with mAbs OX18, OX6, W3/25,
and
0X42,
second-step timouse
IgG
cells
respectively,
reagent
were
using
as
Flit-labeled
antibody.
Control
stained
with
the
goat
an-
(dotted
second-step
line); antibody
only. 0
I-
z
0
LI a0
io
io2
250
U
-a ...a ‘U
U
LOG
The contamination mated by counting muscle culture was ED1 mAb staining
lated cells could be maintained in vitro and survived for up to 4 weeks, the cell density decreased gradually after plating and we could not find proliferation of the isolated cells under our culture conditions (Table 2).
of monocytemuscle culture phase-contrast with mAbs salient features
of the cells are a large eccentric nucleus and elongated cytoplasmic processes that can produce a variety of cell shapes. In the living state in vitro, these cells readily adhere to glass or plastic surfaces and show morphological features ofactive endocytosis, i.e., membrane ruffling, abundant lysosomes, and phagocytosed objects. Most of these cells formed rosettes with EA, indicating the presence of Fc receptor on their surface. Observations thus indicate that these cells in primary muscle cultures have identical properties to cells of monocyte-macrophage
TABLE
lineage
2.
[3,
Proliferation
Treatment
Exp.
incorporation
(cpm)
1
Exp.
181
Untreated
265+77
222 + 47 248 +90
+
56
IFN--y
1 (U/mI)
267+50
277
IFN--y
10 (U/mI)
190+39
189+45
Proliferation interferon--y 2 days prior tive
542
of isolated Isolated
(IFN--y,
in
96-well
to the controls.
Data
Journal
(2.0
x
I or 10 U/mI) plates
harvest.
macrophages
cells and
pulsed
was 10), with
0.25 (1500
are
of Leukocyte
assayed
by
were
mean
+
Biology
sCi
[3H]thymidine
either
for 48 h or untreated,
Gamma-irradiated presented
which
with
cultured
[3Hjthymidine/well
rads)
cells
SE of triplicate
Volume
were
+68
in-
pretreated
were
decreased. The third and fourth types, cells stained with ED2 and ED3, respectively, formed small clusters and occurred late (usually 3 weeks or more after plating) in the primary cultures. Thus, it appeared that four distinct subpopulations of macrophages occurred in the primary muscle cultures. In addition, we found that some constitutively Ia cells appeared late in the cultures. These Ia cells were morphologically similar to the ED1 cells, exhibiting several elongated processes and a distinct dendritic appearance. The relationship of this Ia cell to the dendritic cell, which was first described in mouse lymphoid organs [17], is uncertain. Constitutive and intense expression of Ia antigens is the cardinal feature of dendritic cells, and they can be cultivated
2
Gamma-irradiated
corporation.
the cultures, and was also identified and characterized as the cell isolated from primary culture. The second, a cell labeled with 0X43 antibody, was characterized by large lipid-laden vesicles occupying its cytoplasm, and it remained attached in the cultures during the isolation procedure. These two cell types were recognized throughout the incubation of the primary culture, each exhibiting typical and consistent properties, although the number of these cells gradually
Macrophages
[‘HjThymidine
for 12 h
used
as nega-
samples.
52,
November
of peripheral blood monocytes estired blood cells in the initially prepared less than 0.i% (less than 20 cells/flask). on smears of initially prepared cell sus-
By morphological and phenotypic criteria, four types of mononuclear phagocytes were distinguished in the primary muscle cultures. One was an ED1 cell, a major cell type in
12].
of Isolated
INTENSITY
pensions showed that the antibody labeled 3-5 % of the total number of mononuclear cells. However, during the first 10 days of incubation primary cultures displayed a large number of phase-bright, loosely adherent cells identified as monocytes-macrophages, mostly ED1 cells, that were confluent over the entire surface of the culture. Therefore, although a partial contribution of blood monocytes in the culture cannot be ruled out, it seems likely that a number of monocytes-macrophages were generated as a consequence of proliferation in the primary muscle culture. Indeed, extensive replication of macrophages from nonhemopoietic tissues has been demonstrated in the presence of growth factors produced by tissue cells, such as embryo fibroblasts [16].
DISCUSSION The present study demonstrated that cells macrophage lineage occur in primary embryo of rat. These cells can be identified by microscopy and immunoperoxidase staining specific for rat monocytes-macrophages. The
FLUORESCENCE
1992
in vitro without changing their cytologic and surface properties [18]. Furthermore, the presence of Ia-bearing dendritic cells has been reported in nonlymphoid organs including skeletal muscle [4, 19]. Thus, it seems likely that the Ia cells occurring in the primary muscle cultures have a close relationship to the dendritic cells in the nonlymphoid tissues.
hemopoiesis and become macrophages in the tissues [23]. Therefore, generation of muscle macrophages from such fetal macrophage precursors, which are ontogenically independent of blood monocytes, may be equally possible. The occurrence of large multinucleate cells in isolated macrophage cultures is of interest. It has been reported that
We established with more than
human monocytes and mouse macrophages can form variable numbers of multinucleate cells in vitro [24, 25]. The present study confirmed these observations by showing that the multinucleate giant cells, which stained with ED1 antibody, are formed in rat macrophage cultures in vitro. We also observed that a number of ED1 multinucleate giant
The dish.
isolated They
our
culture
a technique 95% purity
for from
isolation primary
of macrophages muscle cultures.
adherent to a glass or plastic cytoplasmic granules positive for nonspecific esterase and acid phosphatase and showed active phagocytosis. Using [3H]thymidine incorporation, we could not find proliferation of the isolated cells in vealed body 4-week
that
cells were highly exhibited numerous
condition.
virtually
and they incubation
Immunocytochemical
all
of these
remained period.
cells
persistently Thus, unlike
staining
stained EDi the
with
ED1
throughout macrophages
re-
antithe in
the primary muscle cultures, which consisted of phenotypically heterogeneous populations, the isolated cells formed a homogeneous population of EDP macrophages, and hardly replicated under the isolated culture conditions. FACS analysis showed that almost all of the isolated cells expressed MHC class I and CD4 antigens on their surfaces. Indeed, in rat the CD4 molecule has been reported to be expressed not only on MHC class Il-restricted helper T cells but also on a subpopulation of macrophages [20]. Thus, the expression of this antigen on the isolated cells, together with the fact that they have complement (C3) receptors, provides further evidence that they are of monocyte-macrophage lineage. Whereas there were four distinct macrophage subpopulations (i.e., EDi, ED2, ED3, and OX43 cells) grown in the primary muscle cultures, this was not the case with the isolated cells, which consisted solely ofEDP cells. One explanation for the homogeneity of the isolated macrophages is that the non-EDi cells, particularly OX43 cells, firmly adhered to the culture layer and hardly detached during the isolation procedure, although the primary cultures displayed both EDP and OX43 cells. On the other hand, the observations that ED2 and ED3 cells occurred only at a late period in the primary cultures and that rat blood monocytes, which indude precursors of tissue macrophages, are predominantly EDP [4 5] suggest that in the ontogeny of macrophages the ED1 cell is a relatively immature type of macrophage and both ED2 and ED3 cells are differentiated ones. If so, it seems likely that in primary muscle cultures a cell differentiation-promoting factor(s) derived from the tissue cells might be required for differentiation of immature macrophages in the tissue, because the isolated cells persistently remained ED1 and neither ED2 nor ED3 cells occurred throughout the 4-week period of incubation. In this regard, monocytes have been shown to replicate and differentiate extensively in vitro in the presence of media conditioned by tissue cells, which served as the source of a growthand differentiationpromoting factor(s) [21]. Although we have been unable to determine whether EDi and ED2 cells are derived from macrophage-committed hemopoietic precursors in the muscle, the fact that cells of monocyte-macrophage lineage occur in the embryonic muscle suggests that during embryogenesis, monocytemacrophage precursors ofmesenchyrnal origin may enter the muscle tissue and give rise to a population of mononuclear phagocytes. Indeed, in our study macrophages in the primary
embryonic
muscle
cultures
seemed
to
replicate
tensively. Furthermore, there is evidence that tissue macrophages can self-renew and self-maintain without monocyte migration [22] and that, in embryonic tissues, fetal macrophage precursors occur before the initiation of bone marrow
ex-
cells were present days in gestation) suggest that the
in the bone marrow of rat embryos (17-19 (data not shown). Thus, our observations multinucleate giant cells (syncytia) occur in rat macrophage colonies in vivo and in vitro and that the EDi macrophages are the precursors of the multinucleate cells. The multinucleate giant cells are found in a variety of inflammatory disease processes [26]; however, the mechanisms
and
involved
in
inducing
the
syncytia
under
both
normal
pathologic
conditions remain to be elucidated. Macrophages are thought to play an important role in altered homeostasis, as in inflammation, tissue remodeling, and immune surveillance [1, 2]. Regarding muscle diseases, McDouall et al. [27] have demonstrated that among the mononuclear infiltrates in juvenile dermatomyositis and Duchenne muscular dystrophy there was a striking predominance of endomysial macrophages and suggested that the major mechanism of muscle damage is mediated in part via the macrophages. Also, in human immunodeficiency virus
(HIV)-associated
myopathy
domysial macrophages were HIV-infected macrophages muscle fibers and contribute
a
large
number
of
en-
infected with HIV and these could secrete factors toxic to to muscle fiber dysfunction
[28]. Although the precise function of macrophages in the developing muscle is unknown, evidence that macrophages can secrete multiple cell products ranging in biologic activity from induction of cell growth to cell death [29] suggests that they may modulate the process of myogenesis through their potential endocytic and secretory functions. Identification and isolation of macrophages from the rat embryonic muscle, reported here, may provide the opportunity to study the role of macrophages in the developing muscle.
ACKNOWLEDGMENT This work was 39489-02 from was a recipient
supported in par by grants NS 08075 and AR the National Institutes of Health. H. Honda of a postdoctoral fellowship from the Amen-
can
Dystrophy
Muscular
Association.
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