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Development of macrophages in the lungs of fetal rabbits rats and hamsters.

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THE ANATOMICAL RECORD 208:103-121(1984)
Development of Macrophages in the Lungs of Fetal
Rabbits, Rats, and Hamsters
SERGE1 P. SOROKIN, RICHARD F. HOYT, JR.,AND
MARGARET M. GRANT
Devartment of Anatomy and Houseman Research Center (S.PS., R.RH.I,
a i d Department of Biochemistr-y (M.M. G.), Boston University School of
Medicine, Boston, MA 02118
ABSTRACT
Fetal rabbits (days 13-32), rats (days 14-22), and hamsters
(days 11-15) and selected postnatal animals were examined for pulmonary
macrophages or their precursors in 2-,um sections stained by PAS-lead hematoxylin (all species), electron micrographs (rabbit and rat), and cytochemical
incubations for acid phosphatase (rabbit and rat), aliesterase, and N-acetyl
glucosaminidase (rabbits). All methods revealed macrophages in perinatal
specimens. The appearance and distribution of these cells were compared in
the different preparations to establish the reliability of PAS-lead hematoxylin
for identifying them in less developed fetal lungs, where they are less active
for lysosomal enzymes the earlier the stage examined. In the sections, macrophages are seen to possess a round or indented nucleus, a n irregular contour,
and a deep purplish-gray cytoplasm containing a variety of pink PAS-stained
granules, equated with heterolysosomes by ultrastructural cytochemistry. In
less developed lungs, macrophages occur along with putative precursors having a more rounded outline and fewer PAS-stained granules. In pseudoglandular lungs these precursors predominate over rather vacuolated macrophages
resembling Hofbauer cells. In all three species both cell types first appear in
the stroma during the bronchial bud stage and are frequently seen to divide
from that time on. The earliest precursors have a relatively sparse cytoplasm
which later increases in daughter cells. Hofbauer-like cells disappear during
the canalicular stage of development, replaced by macrophages and transitional forms from the more rounded precursors. In day 21 rabbit lungs, scattered stromal cells are reactive for aliesterase, and, some days later, for acid
phosphatase and glucosaminidase. Free mononuclear cells are rare in airways
of pseudoglandular lungs but become common later. A day or two before birth
in rats, free cells range between rather undifferentiated leukocytes to typical
macrophages, but cells with the macrophage’s complete repertory of inclusions
are seen only after birth. In the fetus, typical monocytes were not identified in
either the pulmonary stroma or the airways. A replicating population of
macrophage-like cells therefore resides in fetal lungs. It is established before
bone marrow is formed and, in rats, before monocytes have appeared in the
circulation.
It has proven difficult to give a simple answer to the question, “Where do pulmonary
alveolar macrophages come from?” because
the topic is complex. Without doubt these
cells resemble the amoeboid cells called macrophages that generally are derived from
embryonic or extraembryonic mesoderm,
scavenge the tissues of nearly all Metozoans,
0 1984 ALAN R. LISS,INC.
and usually function in association with the
hematopoietic system, when phylogenetically this is present (Metchnikoff, 1893). In
the course of evolution this ancient cell type
has become adapted to work in concert with
developing serum-borne immunological deReceived March 24,1983; accepted June 6, 1983.
104
S.P. SOROKIN, R.F. HOYT, JR.,AND M.M. GRANT
fense mechanisms, but it retains its character as a relatively unspecialized cell with
some capacity for further transformation (Sorokin, 1977). In the lungs of healthy mammals, the macrophage population is distinctly
heterogeneous in the appearance and functional activity of individual cells. If all these
cells still comfortably fit within a broad definition of “macrophage” it is possible that at
any moment important components of the
population may have had different past histories, some for example having become
transformed from newly arrived monocytes
(Bluss6 and van Furth, 1979)and others having been resident for some time in the pulmonary interstitium before migrating onto
the alveolar surface (Bowden and Adamson,
1972). Furthermore, lavaged alveolar cells
are able to form colonies when cultured in
semisolid, conditioned medium, and this capacity is enhanced if the cells are obtained
from lungs subjected to mild ozone injury
(Boorman et al., 1979a,b). Granting that
these rather undifferentiated cells are able
to divide and yet are often undisposed to do
so, one might well understand why a comprehensive answer to the question of pulmonary
macrophage origin has eluded investigators
despite considerable effort to address it (Brain
et al., 1977).
This study was undertaken to provide information on the development of macrophages in fetal lungs of three mammalian
species. Certain morphological and cytochemical observations are presented in a n
attempt 1)to identify macrophage precursors
and macrophages in fetal lungs; 2) to note
the time of their first appearance in the lungs
and the location(s) in which they are first
found 3) to trace the subsequent appearance
of these cells in other pulmonary compartments, such as the airway and respiratory
surfaces; 4) to look for evidence of macrophage replication andor maturation in situ
and to place these events in the lungs a t
various gestational ages. Finally, using our
own data and those from the literature, we
comment briefly on the timing of some of the
preceding events in comparison with timings
of the first appearance and subsequent maturation of macrophages or their allies in yolk
sac, liver, and bone marrow of the same
species.
MATERIALS AND METHODS
New Zealand White Rabbits (Associated
Rabbit Industries, Easton, MA)
Light microscopy
Littermates of fetal and newly born rabbits
provided a series of developing lungs a t the
following gestational ages: 13, 15, 17, 19, 21,
23,25,26,27,29,31, and 32 days (birth); and
12 hr, 1,and 2 days postnatal. Prenatal stages
were obtained by caesarean section. Specimens taken a t 13 and 15 days were whole
fetuses; those obtained a t 17-21 days included both neck and thorax segments and
dissected lung pairs; and older stages were
represented by lungs only. Tissues were fixed
in 2% wlv formaldehyde (freshly prepared
from paraformaldehyde)-2% glutaraldehyde
buffered with 0.1 M cacodylate (pH 7.2) for
24 hr, after which they were rinsed in buffer,
dehydrated in graded ethanols, embedded in
glycol methacrylate, and sectioned a t 2 pm.
Staining was by PAS-lead hematoxylin (Sorokin and Hoyt, 1978) and Giemsa.
Electron microscopy
Electron micrographs were made from
blocks of fetal lungs aged 25,27, and 30 days
and from macrophages lavaged from lungs of
adult animals. Lung blocks were fixed by
immersion for 3-12 hr in the aldehyde mixture just described, then rinsed in 0.1 M cacodylate buffer, postfixed in 1%w/v osmium
tetroxide with 15 mglml potassium ferrocyanide for 2 hr a t 4°C (Karnovsky, 1971),
dehydrated, and embedded in Epon. Grids
were stained with uranyl acetate and lead
citrate. Pulmonary macrophages from adult
rabbits were obtained by multiple lung lavage (10 washes of 50 ml each using Dulbecco’s calcium- and magnesium-free balanced
salt solution prewarmed to 37“C), as described by Grant et al. (1979). Cells were
centrifuged, and pellets were prepared for
electron microscopy as described for lung
blocks.
Lysosomal enzyme histochemistry
Neck and thorax segments were obtained
from all previously listed developmental
stages older than the 19th fetal day. These
were fixed briefly in 4% wlv formaldehyde
and frozen on dry ice. Cryostat sections cut
a t 10 pm were then reacted either for aliesterase, using the method of Pearse (19721, in
which alpha-naphthyl acetate acts a s substrate and fast blue RR as the coupling agent,
or for acid phosphatase, using the method of
Burstone as modified by Barka and Anderson (1962), in which AS-TR phosphate is the
substrate and hexazotized pararosaniline is
the coupling agent. Incubations for esterase
were carried out a t room temperature (20°C)
for periods from 5-30 min, and those for acid
phosphatase at pH 5.0 and room temperature
for periods from 20-90 min.
DEVELOPMENT OF PULMONARY MACROPHAGES
Frozen sections cut from additional specimens fixed in 4% formaldehyde containing
1.5%w/v calcium chloride were reacted for Nacetyl-beta-glucosaminidase (Reed and Ben
nett, 1975)using a-naphthol AS-BI N-acetyl 0glucosaminide as substrate and fast garnet
GBC as coupling agent. Incubations were for
20 min a t room temperature. Sections were
counterstained with 0.1% aqueous methyl
green, air dried, and studied uncovered or
with immersion oil mounted coverslips. Photographs were taken within a day of incubation.
For each histochemical reaction, control incubations lacked substrate and were negative in all cases.
CD-1 Rats (Charles River Laboratories,
Wilmington, MA)
Light microscopy
Litters were delivered by caesarean section
from timed pregnant mothers a t 14, 15, 16,
17, 18, 19, 20, 21, and 22 days (term) of gestation; additional pups were killed 3 and 4
days postnatally. Fourteen-day specimens
were whole fetuses; dissected lung pairs were
obtained at other stages. Tissues were fixed
in 0.1 M phosphate buffered 2% formaldehyde-2% glutaraldehyde (pH 7.2-7.3) containing 0.01% picric acid (Ito and Karnovsky,
1968), embedded in glycol methacrylate, and
sectioned at 2 pm. Staining was by PAS-lead
hematoxylin and 1% aqueous toluidine blue.
Electron microscopy
Blocks of 21-day fetal and 3- and 4-day postnatal lungs were fixed in phosphate buffered
4% formaldehyde-1%glutaraldehyde (pH 7.2)
having a final buffer osmolarity of 176 mOsm
(McDowell and Trump, 1976) and postfixed
in osmium-ferrocyanide before being embedded in Epon. Grids were stained with uranyl
acetate and lead citrate.
Ultrastructural acid phosphatase
cytochemistry
Pieces of minced 22-day fetal lung (0.5
mm3) were fixed for 90 min a t 21°C in Karnovsky’s (1965) glutaraldehyde-formaldehyde solution (ph 7.2), washed in 0.02 M Trismaleate buffer (pH 5.0), and incubated a6
that pH for 1.5 h r in a medium containing
0.08% wlv lead nitrate and 2.5 mg/ml 0-glycerophosphate (Novikoff et al., 1971).After
washing in 0.1 M cacodylate buffer, tissues
were postfixed with osmium-ferrocyanide and
embedded in Epon. Grids were stained with
uranyl acetate and lead citrate.
105
Syrian Golden Hamsters (Charles River
Laboratories, Wilmington, MA)
Litters were delivered by caesarean section
from timed pregnant females on days 11, 12,
13, 14, and 15 of gestation (term 16-17 days).
Whole fetuses were taken on day 11, head
and thorax on day 12, and lung pairs on days
13-15. Tissues were fixed in buffered formaldehyde-glutaraldehyde as specified either by
Karnovsky (1965) or McDowell and Trump
(1976). They were embedded in glycol methacrylate, cut at 2 pm, and stained by PASlead hematoxylin.
RESULTS
Illustrations for this paper are grouped by
species and in order of increasing gestational
age, as follows: Rabbit, figures 1-11, light
microscopy; Figures 20-25, electron microscopy. Rat, Figures 12-17, light microscopy;
Figures 26-30 electron microscopy. Hamster,
Figures 18 and 19, light microscopy.
(Figures 5-19 are color illustrations that
appear in the Color Figure Section elsewhere
in this issue. Figures 5-11 appear on pp. 8485; Figures 12-19 appear on pp. 86-87.)
Criteria Used to Identify Macrophages or
Macrophage Precursors in the Sections
Histochemistry
Mononuclear leukocytes can be identified
in the connective tissue stroma of pseudoglandular rabbit lungs after a rather long
incubation for aliesterase (30 min, compared
to 1-5 min in adult lungs). The esterase-positive cells are few in number and stand out
in 21-day fetal lungs because few pulmonary
tissues are then reactive (Fig. 1). They are
not identifiable by acid phosphatase or Nacetyl glucosaminidase cytochemistry at this
time, although these lysosomal enzyme
markers are useful for identifying alveolar
macrophages in late fetal, postnatal, and
adult lungs.
Acid phosphatase is first localizable to specific cells in fetal lungs at about the 26th day
of gestation in rabbits, when only a few cells
are active in the stroma and a n occasional,
more reactive cell appears in the lumen of
canalicular intrapulmonary airways (Fig. 2).
Thereafter, mononuclear phagocytes can be
identified in both the pulmonary stroma and
the air spaces with increasing frequency .using cytochemical methods for esterase, acid
phosphatase, or glucosaminidase and incubation times suitable for demonstrating alveolar macrophages in postnatal lungs. At
31 days of gestation, aliesterase positive cells
are demonstrable in the lungs after 5 min-
106
S.P. SOROKIN, R.F. HOYT, JR.,AND M.M. GRANT
Utes of incubation. These cells are particularly prone to aggregate in the loose
connective tissue adventitia surrounding
sizeable pulmonary arteries and veins (Fig.
3), but they also occur in the interstices between the terminal sacs and on the primitive
respiratory surface. At one day after birth,
such cells are found in increased number in
the interstitium and on the surfaces of the
primitive alveoli, as revealed by their reactivity either for esterase, acid phosphatase,
or glucosaminidase (Fig. 4).
able degree), a n indented nucleus, a cytoplasm replete with free ribosomes but poor
in granular endoplasmic reticulum, and a
small Golgi complex. Most of these cells contain a few dense bodies about 0.1 pm in diameter, but in some these range up to 0.6 pm
and resemble heterolysosomes (Fig. 21). Others contain recognizably ingested material as
well, but this is never as characteristic a
feature of the cytoplasm of mononuclear
phagocytes in fetal lungs as it becomes after
birth. As shown in a macrophage from a 22day fetal rat, the dense bodies are reactive
for acid phosphatase, which codirms their
identification as lysosomes (Fig. 261.l
Correspondence between acid phosphatase
reactive cells of late fetal lungs and
interstitial or free mononuclear cells
stainable by PAS-lead hematoxylin
Development of Macrophages in Fetal Rabbit
Lungs (Gestation 32 Days)
In 30-day fetal rabbit lungs, a n accumulation of reactive mononuclear cells is demonAs seen in glycol methacrylate sections of
strable in the perivascular adventitia as well 13-day fetuses, a very few PAS-lead hematoxby incubations for acid phosphatase (Fig. 11) ylin-positive mononuclear cells occur in the
a s for aliesterase (Fig. 3). In PAS-lead hematoxylin-stained glycol methacrylate sections
of lungs of the same gestational age, the position of these esterase or acid phosphatase po'Without phagosomes or a complement of heterolysosomes in
cytoplasm, putative macrophage precursors resemble other
sitive cells is occupied by mononuclear cells their
blast-like hematopoietic cells, including those functionally in a
made conspicuous by the dark purplish-gray lymphocytic line of development. Therefore, based on techniques
staining of a cytoplasm that contains a num- used in ths study, attribution of a cell like that in Figure 20
be made to the mononuclear phagocyte line with absolute
ber of medium pink, PAS-positive granules cannot
certainty. This same difficulty also hampers attempts to identify
(Fig. 10). These do not contain glycogen but pluripotential stem cells of the hematopoletic system, as evifrom a paper by Dicke et al. (1973) and the discussion
correspond to lysosomal granules seen in the denced
following, because few functional markers are expressed by such
cytochemical preparations.
protoplasts which, according to Weiss (1983),structurally are
lymphocytes. In the setting of the developing lung, however, and
Free cells in the airways of late fetal lungs amidst
a spectrum of morphologically related cells, some posare also reactive for acid phosphatase and sessing macrophage markers, it appears likely that the destiny
accentuated by PAS-lead hematoxylin (Figs. of these undifferentiated cells is correctly stated.
2, 9). In this location their identification as
macrophages is certifiable on morphological
grounds alone: possession of a n often irregular outline, a rounded to indented nucleus,
Fig. 1. Aliesterase in 21-day fetal rabbit lung, which
one or more nucleoli, and PAS-positive gran- is minimally reactive after a long incubation (30 min,
ules that are variable in number and size 20°C). Scattered mononuclear cells in the pulmonary
stroma (arrows) and differentiating myoblasts beneath
and become more so as term approaches. the
bronchus (right center) are the main sites of activity.
Once studied as they occur in the air spaces, The stroma has a distorted, netlike appearance in this
the cells become easier to find in the pulmo- frozen section. x 100.
nary interstitium (Fig. 7) or a s they emerge
Fig. 2. Acid phosphatase in 26-day fetal rabbit lung.
onto the surface (Fig. 8).
Tissues are rather unreactive overall in both the epitheCorrespondence between the cells
highlighted by PAS-lead hematoxylin
and mononuclear phagocytes identified
by electron microscopy
As seen in electron micrographs of fetal
rabbit lungs aged 25-30 days of gestation
(Figs. 20-231, the cells corresponding in location and number to those made prominent in
25-30 day fetal lungs by PAS-lead hematoxylin are mononuclear leukocytes frequently
of a n undifferentiated appearance, characterized by surface projections (present to a vari-
lium (lower left) and stroma of this canalicular lung,
except for an occasional free mononuclear phagocyte (upper right). (Incubation 45 min, 20°C). Frozen section;
x290.
Fig. 3. Aliesterase in 31-day fetal rabbit lung after a
short incubation (5 min, 20°C). A large number of reactive mononuclear phagocytes are gathered in the adventitia outside the pulmonary artery, which runs alongside
the bronchus (upper left.) Frozen section; X550.
Fig. 4. N-acetyl glucosaminidase activity in newborn
rabbit lung (20 min, 20°C). The most reactive cells are
macrophages in the alveolar interstitium and on the
surface (arrow). Esterase has a similar localization. Frozen section; ~ 4 0 0 .
DEVELOPMENT OF PULMONARY MACROPHAGES
107
108
S.P. SOROKIN, R.F. HOYT, JR.,AND M.M. GRANT
undifferentiated connective tissue surrounding the bronchial buds just a s they do in the
connective tissue of the body wall. Such cells
are more prevalent in the stroma of pseudoglandular lungs at 15 days. They are smaller
than the macrophages of late fetal lungs and
are more difficult to identify because they
usually have few PAS-positive granules. A
minority of these putative macrophage precursors are rather larger and more vacuolated than the remainder, and some of the
vacuoles are faintly stainable by PAS. The
latter cells resemble macrophage-like elements, termed Hofiauer cells, that occur in
the placenta as well as in other fetal organs.
The pulmonary stroma has a mesenchymatous appearance a t this time and for several
days thereafter. It consists largely of stellate
cells that extend throughout a matrix containing few extracellular solids and stain
more lightly purplish than the macrophage
precursors. The tissue is supplied by a loose
plexus of sinusoidal capillaries.
Examination of serial Giemsa-stained sections confirms that formation of blood cells is
under way in the livers of 13- and 15-day
specimens but that the bone marow is not
yet established. In the liver the most prominent blood cells undergoing formation are
megakaryocytes and those of the erythrocytic series. Some basiphil mononuclear cells
of a decidedly undifferentiated appearance
occur among them, but few precursors of
granulocytes are recognizable. Mitotic figures are observed in blood cells located both
in the hepatic cords and sinusoids and to a
more limited extent in the lumens of distant
blood vessels, including those of the forming
lungs. In these preparations the basiphil
mononuclear cells can also be observed
sparsely inhabiting the pulmonary stroma.
They are generally smaller than the mesodermal cells of the developing lungs and compare in size to the PAS-lead hematoxylinpositive cells, to which they evidently
correspond.
During the 17th, 19th, and 21st days of
gestation, fetal rabbit lungs contain increased numbers of PAS-lead hematoxylinstained precursors of macrophages, and mitotic figures are not infrequently observed in
them (Fig. 5). Like typical mononuclear cells
found in the lungs a t 15 days, those present
from the 17th to 21st days possess a high
nucleuslcytoplasm ratio and comparatively
few PAS-positive granules, although these
are more conspicuous than at 15 days. Most
of the cells remain well away from the developing bronchial tree within the pulmonary
stroma. Nonetheless, a n occasional mononuclear cell may occur in the lumens of both
large and small airways a t any time during
this interval. Such cells have little activity
for acid phosphatase or glucosaminidase and
share morphological characteristics with the
macrophage precursors located in the stroma
(Fig. 6).
Subsequent to 25 days, macrophage-like
cells become more frequently observable
along the internal surface of the lungs, by
then a t a canalicular stage of development.
Most of these are better provided with PASpositive granules than their counterparts in
the stroma, many of which are stainable only
about the centrosome (Figs. 7,8). This difference is in keeping with the greater lysosomal
activity surface cells tend to exhibit, compared with those in the tissues. Once gained
in later fetal life, the preeminence of free
macrophages in granule content and lysosoma1 enzyme activity is maintained thereafter.
Development of Macrophages in Lungs of
Fetal Rats (Gestation 22 Days) and Hamsters
(Gestation 16 Days)
The pattern of macrophage development in
fetal lungs of rats and hamsters is similar to
that just described in rabbits.
Rats
A very few PAS-positive mononuclear cells
are observable in the stroma of the rat lung
a t 14 days of gestation, when the main bronchus and buds for the lobar bronchi have
appeared on each side and pulmonary development is a t a late lung bud stage (Fig. 12).
These cells are seen more frequently in subsequent days. Their increase results in part
from mitotic activity in situ, as shown in
pseudoglandular lungs at 16 days (Fig. 13)as
well as during the terminal sac stage at 21
days (Fig. 27). As illustrated in the field from
16 day lungs, the fetal macrophage population includes vacuolated cells of the Hofbauer type (Fig. 13); nonetheless, the
impression gained from examination of our
whole fetal series is that the stromal macrophage population of rat lungs consists mainly
of cells with comparatively few PAS-positive
granules, still fewer than in typical stromal
macrophages of fetal rabbits. Undifferentiated mononuclear cells are first seen in very
small number on the airway surface a t 16
DEVELOPMENT OF PULMONARY MACROPHAGES
109
Ultrastructural Appearance of Maturing
Macrophages in Fetal and Postnatal Lungs
Rabbits
As seen in 25-day fetal lungs, the greater
number of mononuclear leukocytes occurring
in the pulmonary stroma outside of the blood
vessels are rounded cells with an undifferentiated appearance as already described (Fig.
20). A smaller number of this population are
polymorphous (Fig. 21). These cells usually
contain a variety of cytoplasmic inclusions,
including heterolysosomes of different sizes
and a few pinosomes or phagosomes. They
also extend protoplasmic leaflets into the
surrounding matrix. In contrast, the rounded
cells rarely contain more $han a few dense
bodies in the 1000-1500 A size range. The
rounded and polymorphous cells resemble
each other in the appearance of the nucleus,
which is indented and slightly heterochromatic, and both stand out from their connective tissue neighbors by their greater overall
electron density. Neither cell type forms
junctions with any of the neighboring cells.
At 27 days of gestation, many of the presumptive macrophage precursors and macrophages are identical in appearance with
those seen at 25 days, but among this group
an increase in the amount of ingested material is seen in some, while in others the granular endoplasmic reticulum has undergone
expansion (Fig. 22).
At 30 days, apart from the preceding, additional cells appear whose relationship to
mature rabbit alveolar macrophages is unmistakable (Fig. 23). In them the Golgi apparatus is enlarged and a number of electron
dense rod- or dumbbell-shaped bodies have
Hamsters
begun to gather in the cytoplasm (Fig. 23).
Macrophages and their precursors were These appear t o be nascent lysosomes, and it
sought in fetal hamster lungs using only is evident that in adult rabbits a great numPAS-lead hematoxylin-stained preparations. ber of similar structures are combined to proIn hamsters these cells stain like their hom- duce a smaller number of large, rounded
ologues in fetal rabbit and rat lungs. They heterolysosomes such as macrophages of
are present in the mesodermal tissue sur- many species possess (Figs. 24, 25).
rounding the bronchial buds at 11 days of
gestation and become increasingly prevalent Rats
In macrophages present in the stroma of
in 12-day and 13-daypseudoglandular lungs,
when they occur throughout the stroma just prenatal rat lungs, lysosomal enzyme prooutside the pulmonary blood vessels as well duction is at a relatively modest level, and
as near the terminal epithelial buds (Fig. 18). acid phosphatase activity is mainly confined
By 15 days the rapidly forming hamster to a few large lysosomes (Fig. 26). Among
lungs have begun to construct terminal sacs, cells found undergoing mitosis, a few share
and macrophages with PAS-positive gran- enough characteristics with other macroules are then frequently seen in the air spaces phages t o make their identification fairly
certain (Fig. 27). These cells have only a mod(Fig. 19).
days. As fetal development proceeds past the
pseudoglandular stage, the mononuclear cells
increasingly gain the appearance of typical
macrophages and increasingly appear in the
air spaces of the conducting airways or terminal sacs (Figs. 14, 15).
Macrophages versus mast cells in fetal rat
lungs. In light microscopic preparations, the
macrophage precursors of older fetal lungs
have to be differentiated from emergent mast
cells, which also occur in the stroma at that
time. These are rounded cells that also contain PAS-positive granules, only the granules tend to develop more nearly together
and to reach a more uniform size (ca. 0.5-0.8
pm) than the granules of macrophages. The
two ceII types can be distinguished easily if
adjacent sections are stained by toluidine
blue, thereby revealing the metachromasia
of the PAS-positivegranules in the mast cells
(Figs. 16, 17) and its absence from the granules of the macrophages (Figs. 14,15).By this
method the mast cells of fetal rat lungs are
first discernible at 16 days as a very few
sparsely granulated cells with relatively
scanty cytoplasm located alongside the lobar
pulmonary arteries. They become a little
more abundant at 17 days, when they are
found as well in the condensed connective
tissue just beneath the endodermal epithelium and in the looser tissue farther away.
The granules accumulate in the cytoplasm
during subsequent days, and the cells achieve
an adult pattern of distribution beneath the
airways, blood vessels, and pleura by the 20th
prenatal day, although the cells do not reach
their full size until well after birth.
Fig. 20. Putative macrophage precursor in connective
tissue of 25-day fetal rabbit lung. The cell is rounded
with inconspicuous cytoplasmic projections. The chromatin pattern of the nucleus is comparable to that of the
macrophage in Figure 21. The cytoplasm has many ribosomes, a large number in polysomal configurations,
and a small Golgi complex, located above the nuclear
notch. A few small dense bodies occur along the margin
on the left side of the cell. ~ 8 , 1 0 0 .
Fig. 21. “Fetal” macrophage in a 25-day fetal rabbit
lung. The cell shares many features with the macro-
phage precursor of Figure 20, except that the cell outline
is irregular and adorned with many lamellipodia (left
side and lower margin), and the cytoplasm contains dense
bodies in a larger range of sizes as well as clear vacuoles.
The former are considered lysosomal equivalents of the
PAS-stained granules seen in light microscopic sections;
the latter are thought to be macropinosomes. The cell
therefore is a macrophage, although not typical of those
in postnatal lungs. It closely resembles Hofbauer cells of
the term human placenta (Fig. 9 in Enders and King,
1970). ~8,100.
Fig. 22. Developing macrophage in a 27-day fetal rabbit lung. The cell occupies a niche in the stroma framed
by fibroblasts sporting an endoplasmic reticulum with
dilated cisternae, whose activity in the synthesis of extracellular fibers nonetheless still largely lies ahead. In
the macrophage the granular reticulum has undergone
expansion compared to cells shown at 25 days (Figs. 20,
21). A few lysosomal granules are present above the
nucleus next to the Golgi region and to each side; they
are ovoid or dumbbell shaped. X 11,200.
Fig. 23. Stromal macrophage in a 30-day fetal rabbit
lung. The cell appears stimulated and resembles alveolar macrophages from adult animals more than the macrophage-like cells seen earlier in gestation, possessing a
nucleus with numerous nuclear pores, a cytoplasm with
moderate development of granular reticulum, an expanded Golgi complex associated with small electron
dense vesicles (above the nucleus), and a number of rod-,
club-, or dumbbell-shaped dense bodies, considered to
represent nascent lysosomes in this species. X9,OOO.
Figs. 24.25. Alveolar macrophages obtained by intrapulmonary lavage from adult rabbits. The upper cell
(Fig. 24) has many small lysosomes, most of which are
elongated, as well a s some larger ones, which are ring
shaped or spheroidal. The lower cell (Fig. 25) has fewer
lysosomes, but most of them are large and spheroidal
heterolysosomes, and only a minority are slender and
rodlike, as seen in Figures 23 and 24. Macrophages with
this kind of lysosomal development usually are seen only
in postnatal lungs, especially where there is a history of
exposure to airborne contaminants (Pratt et al., 1971).
; 25, ~ 7 , 1 0 0 .
Fig. 24, ~ 6 , 5 0 0Fig.
114
S.P. SOROKIN, R.F. HOYT, JR.,AND M.M. GRANT
DEVELOPMENT OF PULMONARY MACROPHAGES
erately expanded granular endoplasmic reticulum typically without dilated cisternae,
a few small, dense lysosomal bodies, an electron dense cytoplasm containing numerous
polysomes, and peripheral processes that occasionally contact but do not make junctions
with neighboring cells (Fig. 27). These neighbors are mostly fibroblasts; at this time they
are distinguishable from macrophages by cytoplasmic texture as well as by cell shape,
because their endoplasmic reticulum is dilated by cell products of an intermediate density, and because the cells store glycogen and
droplets of triglyceride.
Free cells in terminal air sacs of late fetal
lungs range in appearance from rather undifferentiated (Fig. 28) to rather macrophagelike (Fig. 29). In spite of its scanty cytoplasm,
the undifferentiated cell can be considered a
possible precursor of macrophages, because
its endoplasmic reticulum is more extensive
than that of lymphocytes, and because a
number of elongated lysosomal bodies are
present. Other free cells are intermediate in
cytological characteristics between this cell
and typical alveolar macrophages. As judged
ultrastructurally, many of the latter contain
a greater variety of phagolysosomes than
their interstitial contemporaries and so are
considered more active in phagocytosis
(Fig. 29).
By three days after birth, a large majority
of the cells on the respiratory surface have
begun to acquire the look of experienced alveolar macrophages, given them by a richly
varied content of phagolysosomes and other
inclusions (Fig. 30), and an even stronger
impression of the same kind is made by examination of lungs a day older. In this respect the postnatal alveolar macrophage
Fig. 26. Interstitial macrophage in a 22-day fetal rat
lung. The cell is relatively immature in appearance and
lies interposed between blood vessels (above and below
the field shown) and near a great alveolar cell, one of
whose lamellar bodies is shown (lower left). Acid phosphatase activity is present in some of the dense bodies
in the macrophage cytoplasm. (Glycerophosphate, pH
5.0, 90 min); ~ 9 , 2 0 0 .
Fig. 27. Dividing macrophage precursor or macrophage in the stroma of a 21-day fetal rat lung. The cell
shares cytological features with immature rabbit and
rat macrophages (Figs. 20, 21, 26); it is unattached to
and differs in appearance from interphase and dividing
fibroblasts, myoblasts, and endothelial cells. One pair of
centrioles is shown below the metaphase figure, and
clusters of small lysosomal bodies are distributed on
either side. ~8,600.
115
population is easily distinguished from any
obtainable from normal lungs before term in
either rabbits or rats.
DISCUSSION
Using a variety of morphological and functional markers, we have shown that cells
evidently in a macrophage line of development are present in fetal lungs of rabbits,
rats, and hamsters for a considerable time
before birth, although if one starts with newborn lungs and works back to earlier stages
of development, the cells in question progressively lose their markers. They become
poorer in lysosome-likegranules stainable by
PAS, and in rabbits where this was investigated, progressively less active for lysosomal
enzymes acid phosphatase and N-acetyl glucosaminidase. In this species, the cytochemical method for aliesterase is more useful than
the preceding for identifying macrophagelike cells in early developmental stages because few other cells of the pulmonary stroma
or epithelium are active for the enzyme during that period (Sorokin and Hoyt, 1982). As
we did not intervene with the normally developing animals in any experimental way,
we had to rely on our light and electron
microscopic observations for evidence of
endocytotic activity by the pulmonary macrophages or their precursors. This could be
taken for granted in postnatal macrophages;
it also appeared evident from examination of
electron micrographs of late prenatal lungs
of both rabbits and rats that the cells are
active in phagocytosis, especially when they
are on the respiratory surface. In this respect
our findings are complementary to those of
Zeligs et al. (19771, who examined lavaged
cells from rabbits aged thirty days of gestation t o four weeks postnatal and found that
free macrophages in late fetal lungs contain
ingested cellular debris and surfactant-related phospholipids, but that ingestions of
these lipids increased dramatically shortly
after birth. In our material the macrophagelike cells found in the stroma had fewer inclusions and were less active in phagocytosis
than the surface cells, as their more sheltered position might lead one t o expect.
With fewer functional attributes expressed
by macrophage-likecells the earlier the stage
of development being examined, it was necessary for us t o rely on morphology to find
the earliest examples present. We chose to
examine the lungs using light microscopy so as to survey the developing organ
Figs. 28, 29. The range of mononuclear cells present
on the respiratory surface in 21-day fetal rat lungs. The
upper cell (Fig. 28) is the less mature and yet possesses
a greater expansion of the granular reticulum than is
present in “uncommitted” mononuclear leukocytes.
Slender threadlike bodies (arrows) are not unlike small
lysosomes in pulmonary macrophages. The lower cell
(Fig. 29) is a more typical alveolar macrophage, as seen
in late fetal lungs. It exhibits moderate development of
cytoplasmic organelles and includes lysosomes of various sizes including large ones (***I that would be conspicuous in PAS-lead hematoxylin stained preparations.
Fig. 28, x 11,200;Fig. 29, ~ 1 0 , 3 0 0 .
DEVELOPMENT OF PULMONARY MACROPHAGES
Fig. 30. Alveolar macrophage present in 3-day postnatal rat lung. Compared to cells in prenatal lungs,
postnatal ones are more highly charged with lysosomes
117
of greatly varied configurations: micron-sized and submicronic, electron dense and lucent, solid and lamellated. x 12,500.
118
S.P. SOROKIN, R.F. HOYT, JR.,AND M.M. GRANT
with sufficient thoroughness to find the rnacrophage precursors even if only a few were
present at a given stage, and we employed 2pm plastic-embedded sections stained by
compatible dyes in order to obtain optimum
tissue resolution to aid our search The PASlead hematoxylin-stained cells we singled out
as macrophage-like were also present in more
developed fetal lungs, where their staining
pattern was recognizably similar as seen in
cells on the respiratory surface and in the
interstitium, and where their correspondence was shown to mononuclear cells having
elevated lysosomal enzyme activity and to
macrophage-like cells identified in electron
micrographs.
Using PAS-lead hematoxylin we first found
macrophage precursors in the lungs of rabbits, rats, and hamsters very early in development at a bronchial bud stage. The cells
were located in the stroma without being
preferentially sited nearer the developing epithelial bronchial tree, the pleura, or the
blood vessels. They appeared detached from
other cells in this compartment and stained
differently from the largely undifferentiated
mesenchymal cells that occupy most of it in
early lungs. Cells intermediate between
stromal cells and macrophages were not observed in our preparations, just as they were
not seen in fetal rat lungs by Collet and Des
Biens (19751, although we obtained no better
evidence than this to rule against such
a derivation from pulmonary mesoderm.
Nevertheless, because a circulation is established in the lung from the earliest time, it
may seem likely that the stroma is seeded,
perhaps repeatedly, from the blood stream.
The cells considered to be precursors of
macrophages in fetal lungs are rounded
mononuclear cells of undifferentiated appearance. In the earlier stages of development they occur side by side with more
obvious macrophages having a n irregular
outline and a vacuolated cytoplasm (Figs. 13,
21) that we have likened to Hofbauer cells
(Enders and King, 1970). As development
proceeds, the rounded leukocytes are gradually supplanted by forms transitional between them and typical macrophages of periand postnatal lungs, whereas the vacuolated
cells tend to disjappear.
Hofbauer-like cells were also found in fetal
rat lungs by Collet and Des Biens (1975),who
saw them as transient cells of mesodermal
origin in evidence only during days 16 and
17 of gestation and apparently unrelated to
macrophages, which were not seen until days
20-21. In our interpretation, these cells are
but one manifestation of the presence of rnacrophages and related forms that is continuous from the inception of pulmonary development. As studied by Enders and King
(1970) in placentas of bats, guinea pigs, and
human beings, Hofbauer cells were found to
be more vacuolated during early pregnancy
when they occur in tissue poorly furnished
with extracellular fibers and demonstrable
ground substance. They become less vacuolated in late pregnancy when most are indistinguishable from macrophages of adult
tissues, and their recognition by light microscopy is made more difficult by their inclusion
in a stroma of increased density. These authors considered the vacuolated appearance
to reflect active engagement in macropinocytosis. More recently, mononuclear cells
from human placentas have been shown to
possess receptors for the Fc segment of immunoglobulin G and complement factor C3,
to bind antibodies cytophilically on the cell
surface, and to ingest bacteria Coke et al.,
1982);they therefore satisfy functional criteria for macrophages. In fetal lungs the early
appearance and later apparent fading away
of Hofbauer-like cells may result from changing environmental conditions that parallel
events described in the placenta.
The rounded leukocytes observed in the developing lungs evidently are precursors of
the pulmonary macrophages because no
other suitable candidates were present, but
it remains to see if they compare with any of
the cell types present in the maturation sequence from monoblast to macrophage proposed for bone marrow-derived macrophages
in adult mammals. Participants in this sequence are all mononuclear leukocytes possessing a variety of characteristics in common (esterase and peroxidase activity, receptors for Fc and C3, and capacity for both
macro- and microendocytosis) but differing in
mitotic capability, cytological features (nucleuskytoplasm ratio, nuclear chromatin
pattern, ribosome content of cytoplasm, extent of development of endoplasmic reticulum and Golgi apparatus, etc.), and the
intracellular distribution pattern of certain
enzymic activities. For practical purposes, ultrastructural morphology and the intracellular distribution of peroxidase activity have
been considered sufficient to distinguish
among monoblast, promonocyte, monocyte,
and macrophage (van der Meer et al., 1982).
DEVELOPMENT OF PULMONARY MACROPHAGES
Furthermore, in mouse and in man, the essentially undifferentiated monoblast and immature promonocytes are capable of division,
whereas the more mature monocyte and
macrophages are considered nondividing
cells (van Furth et al., 1979). Judged by light
microscopic and ultrastructural criteria, the
rounded lekocytes we observed in developing
lungs are proximal to monocytes in this developmental sequence (Figs. 5, 6, 20, 22, 27,
28).Those present inbronchialbudandpseudoglandular lungs tend to have a larger nucleuskytoplasm ratio and fewer granules
than those formed later on (Figs. 6, 20), and
so at first are nearer monoblasts and later
promonocytes. None were observed to meet
standard criteria for identification as monocytes (Weiss, 1983). Indeed, because in all
three species these macrophage precursors
were frequently observed in division (Figs. 5,
271, they would not meet van Furth’s criteria
for monocytes, either. At the same time,
transitional forms were observed between the
precursors and macrophages, which therefore must have arisen without passing
through a monocyte stage.
If the pulmonary macrophage population is
seeded one or more times from the blood
stream, then for much of the prenatal period
the seeding cells are proximal to monocytes
and likely derived from the yolk sac or liver,
hematopoietic centers proximal to the bone
marrow. By our estimates the marrow becomes active in developing hamsters during
the 13th day, two days after macrophages
were detected in fetal lungs. In rabbits, marrow production commences with erythropoiesis on the 23rd to 25th day of gestation,
whereas other blood cell types begin to appear there a few days later (King and Ackerman, 1967), some two weeks after macrophage precursors are first found in the lungs.
In rats, marrow forms on the 19th day of
gestation (Pino and Bankston, 1979), five
days after we detected macrophage precursors in the lungs. Had we somehow failed to
notice monocytes sequestered in rat lungs,
then the error could apply only to the period
after the 17th day of gestation, the earliest
date monocytes are detectable in the circulation (Deimann and Fahimi, 1978). Thus, on
embryological grounds, the first seeding of
mononuclear phagocytes in the lungs could
not have been from monocytes nor from cells
located in the bone marrow. The active replication in fetal lungs not only of monoblastor promonocyte-like cells but also of Hof-
119
bauer-like and other macrophages (Fig. 13)is
unequivocal evidence that a resident, selfreplicating population of macrophages becomes established a t the outset of lung
formation and is continued in evidence
throughout much of the fetal period. The
macrophages we found concentrated beneath
large pulmonary blood vessels in late fetal
life do not necessarily represent a new emigaration of cells from the blood stream but
equally may have become activated by serum
exudates containing antigens or factors produced by a n awakening immunological system. Exudation into periarterial tissues
would be favored by hemodynamic arrangements during fetal life that produce a blood
pressure in the pulmonary artery and its
main branches equal to that of the aorta
(Comroe, 1974). Whether the initial population subsequently becomes diluted out to a
vanishing point by later seedings of hematopoietic cells, including those by monocytes, is
a question for further investigation.
During embryonic development, macrophages are about the first leukocytes to
appear with substantially the same characteristics they possess in adult mammals.
In mouse embryos they have been traced to
extraembryonic precursors located in the
yolk sac prior to the time this organ becomes
connected to the fetal circulation (day 10 of
gestation). These precursors resemble neither promonocytes nor monocytes; less than
0.1% possess functional Fc receptors or adhere to glass, properties their descendants
acquire after some days’ growth in agar culture (Cline and Moore, 1972). Fetal mouse
liver also serves as a source of macrophage
colonies but only after it has gained access to
yolk sac cells. These authors nevertheless
obtained “rare” glass-adherent cells from 79 day whole fetuses, so that a n embryonic
origin of the macrophages cannot be excluded, however unlikely this may seem from
the other evidence presented. Furthermore,
because macrophages are the earliest differentiated blood cells to appear in developing
human liver and initially are more concentrated in the sinusoids than elsewhere in the
fetus or placenta, it is possible that they arise
from pluripotent hepatic endothelium (Kelemen and Janossa, 1980).Whether embryonic
or extraembryonic in origin, fetal rat macrophages (Kupffer cells) appear in the liver soon
after the formation of the hepatic diverticulum, engage in phagocytosis, and form a replicating population well before monocytes or
120
S.P. SOROKIN, R.F. HOYT, JR.,AND M.M. GRANT
the bone marrow are present (Deimann and
Fahimi, 1978; Pino and Bankston, 1979). As
we have shown, a similar situation exists in
fetal lungs. In both cases, during adult life
mitotic activity of the resident macrophage
population is less apparent than during fetal
life, but it is demonstrable nonetheless (Widmann and Fahimi, 1975; Sorokin, 1983). If a
centralized system based in the bone marrow
eventually takes over the task of furnishing
tissues with macrophages, and does so by
supplying them only with end-stage (nondividing) monocytes, then either the monocytes
and the macrophages they become are not
end-stage cells or the dividing alveolar macrophages observed in adult lungs are descended from another pool, such as the fetal
macrophage population we have described.
ACKNOWLEDGMENTS
The authors wish to thank Messrs. Gregory
Anthon, Victor Carabba, Peter Lauren, and
Stephen Sarikas for technical assistance contributed to this study. The work was supported by Research Grants HL-19379, ES00583, and HL-A129175 from the National
Institutes of Health, USPH, supplemented
by funding from The Gorilla Foundation
(Boston).
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