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Macrophage developmentIII. Transformation of pulmonary macrophages from precursors in fetal lungs and their later maturation in organ culture

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THE ANATOMICAL RECORD 232551-571 11992)
Macrophage Development: 111. Transformation of Pulmonary
Macrophages From Precursors in Fetal Lungs and Their Later
Maturation in Organ Culture
SERGE1 P. SOROKIN, NANCY A. McNELLY, DANA G. BLUNT, AND
RICHARD F. HOYT, JR.
Laboratory of Pulmonary Cell Biology, Department of Anatomy and Neurobiology, Boston
University School of Medicine, Boston, Massachusetts
ABSTRACT
The fate of macrophage precursors residing in 14-day prenatal rat
lungs was followed in organ cultures to obtain a detailed, ultrastructurally resolved picture of the sequence and timing of events accompanying their transformation into typical pulmonary macrophages. Cultures were examined at close
intervals during the first day (1, 2, 3, 4, 6, 9, 12, 15, 18, and 24 hr) and at wider
intervals thereafter (2, 4,5, 7, 9, and 13 days) to yield a developmental series of
cells identified as in the macrophage line based on binding of peroxidase-coupled
isolectin B, of Griffonia simplicifolia (GSA I-B4) to cell membranes and on negligible content of peroxidase-positive granules in the cytoplasm. Organ culturing
stimulated virtually all precursors to develop into macrophages. GSA-positive cells
in explants occurred outside vessels in pulmonary connective tissue, and a t the
outset none were typical macrophages: 71% were angular cells, resembling unlabeled mesenchymal cells around them, 16% were undifferentiated leukocytes, and
the remainder were irregularly shaped cells with few vacuoles intermediate between the preceding and the macrophages. During the first 12 hr in culture the
proportion of angular cells and leukocytes fell to zero, and that of intermediate
cells first rose, then receded. In the same interval the proportion of macrophages
rose to 87.5%, and by 24 hr all GSA-positive cells were typical macrophages generally engorged with phagocytosed material; about 8 hr appear necessary for converting half the population. Notable ultrastructural changes during this period of
transformation involved the centrioles and cytoskeleton, reflecting enhanced cell
mobility and phagocytosis. A period of maturation followed, marked by disappearance of cellular debris from phagosomes and an increased prevalence of cells with
elaborate lamellipodia. This accords with earlier work showing that macrophage
Fc receptor density increases sharply during the first 24 hr, but elevated levels of
histochemically demonstrable acid phosphatase appear only later. Mitotic activity
was conspicuous in GSA-positive cells throughout both periods. 3H-thymidine labeling indices for precursors and macrophages, determined at six intervals between 1hr and 24 hr, remained steady at -34%, whereas indices of other categories of lung cells (GSA-negative stromal cells, pleural cells, and airway epithelium)
began at this level but rapidly declined, indicating that the GSA-positive cells
constitute a single population distinct from others in the lungs. Macrophages found
outside the lung cultures after 4-5 days qualify as a mature population, but having migrated away from direct contact with the lung stroma, they survive only a
week or two and no longer divide.
It is now known that precursors of pulmonary mac- nary tissues and from mitotic activity, prominent
rophages exist in mammalian lungs from earliest times among cells already recognizable as macrophages.
and that cells with morphological characteristics of Judged by many criteria, these cells are indistinguishmacrophages gradually accumulate in the organ dur- able from typical alveolar macrophages of adult lungs
ing much of fetal life, to build up more rapidly just
prior and subsequent to birth (Sorokin et al., 1984;
Received June 19, 1991; Accepted October 2, 1991.
Honda et al., 1989). Such cells appear on an accelerated
Address reprint requests to Professor Sergei P. Sorokin, Pulmonary
schedule after lungs from late embryonic rats or ham- Cell
Biology, Department of Anatomy and Neurobiology, Boston Unisters are explanted to organ culture. This results from versity School of Medicine, 80 East Concord Street, Boston, MA
rapid transformation of precursors present in pulmo- 02118.
0 1992 WILEY-LISS, INC
552
S.P. SOROKIN ET AL.
(Sorokin and Hoyt, 1987; Sorokin et al., 1989). The
origin of some alveolar macrophages is therefore attributable to early resident precursors, although the
relative importance of this source has not yet been determined, as against seeding of the lungs by circulating
stem cells or more specialized monocytes at later times
in life.
This paper reconstructs the process of transformation from precursors into typical pulmonary macrophages as it occurs in organ cultured embryonic lungs.
It is based on electron microscopy, which we consider
essential for accurate staging of events. Two major objectives of the study were to determine the time scale of
transformation and to depict morphological stages
along the pathway of transformation in some detail. In
addition, an effort was made to select out the cell type
or types of origin for this population, considered from
candidates present in the lungs at explantation. As
shown here, an outstanding difference between the developmental pathways of these “premedullary” pulmonary macrophages and typical bone marrow-derived
macrophages is that none of the candidate precursors is
a typical monocyte. This also seems true for kindred
“primitive” or “fetal” macrophages located outside the
lungs. In mice and rats this line of cells appears to
originate directly from precursor cells in blood islands
of the yolk sac (Takahashi et al., 1989, 1990) and we
have seen that a considerable buildup of the population
occurs in the embryo itself prior to the time macrophages first appear in the organs (Sorokin et al.,
1992a). Such early macrophages are later supplemented by monocyte-derived macrophages, but as in
the lungs, the extent to which their direct descendents
persist in adults is still uncertain.
In recent years acceptance has grown that macrophage populations of adults are also heterogeneous,
and that diversity may even exist within the population of a single organ, with subsets sometimes differing
greatly in renewal kinetics (van Rooijen et al., 1989).
Although the idea that blood leukocytes give rise to
macrophages is well over 60 years old (Lewis, 19251,
and ultrastructural aspects of monocyte transformation have been known for about 25 years (Sutton and
Weiss, 1966), many important questions remain unanswered about the transition between precursor and
macrophage, or require reinvestigation in the light of
current concepts of the heterogeneity of these cells. Observations made in relation to specific macrophage subsets, like those that follow in relation to the primitive/
fetal macrophage line, may well discourage uncritical
application of the term, “monocyte,”to any immediate
precursor. Indeed, “exact description andlor identification of the transitional stages” has been considered essential to understanding the differentiation pathway of
macrophages (Kreipe et al., 1987).
more, with the assistance of a marker that adequately
selects developing macrophages and their precursors
from other cells inside these cultures, morphological
stages of transformation can be reconstructed simply
by harvesting the cultures at suitable intervals, noting
the form of labeled cells predominating at each interval, and arranging these forms in the correct temporal
sequence. The marker chosen for our work was the peroxidase-coupled isolectin B, of Griffonia simplicifolia
(GSA I-B4), which recognizes terminal a1pha-Dgalactose on cell coats. It is selective for cell membranes of macrophages and antecedents in the macrophage line, although not to the exclusion of all other
leukocytes, since it tags heterophils and eosinophils
equally well (Sorokin and Hoyt, 1992). Two special circumstances, however, made GSA I-B, an entirely satisfactory marker for the macrophage line in our organ
culture system: 1)Comparatively few cells intrinsic to
embryonic or fetal rat lungs bind this lectin. 2) The
growing macrophage population in the cultures is remarkably pure; virtually no granulocytes and few lymphocytes have ever been seen in Giemsa-stained mature cultures from embryonic lungs despite exhaustive
examination of serial sections (Sorokin and Hoyt, 1987;
Sorokin et al., 1989). Consequently, possible confusion
from GSA binding to other leukocytes was not of serious concern.
General Procedure
Eleven litters of 13.5 t o 14 day prenatal rats [crownrump (CR) length 8.5-10 mml furnished the main
specimens used for this study-either the right lung or
the left, but most frequently the paired lungs. Of these,
ten specimens were dissected out and preserved as
records of starting conditions; all were examined. The
remaining lungs were placed in organ culture and subsequently harvested for processing at the following
hourly or daily intervals (in parentheses, number of
specimens examined): l ( n = 31, 2(n = 61, 3(n = lo),
4(n = 9), 6(n = 12),9(n = 12), 12(n = l l ) , 15(n = 13),
18(n = lo), and 24 hours (n = 121, and at 2(n = 1),4(n
= 8),5(n = 2),7(n = 5),9(n = 51, and 13 days (n = 1).
The lungs and lung cultures were fixed in 1%glutaraldehyde in 0.1 M phosphate-buffered saline (pH
7.2), incubated in a solution containing peroxidase-coupled GSA I-B4, followed by development in a diaminobenzidine substrate, osmication, and embedding in
Epon. Details of organ culturing, fixation and staining
procedures, and controls used to ascertain the specificity of GSA I-B, binding were given previously (Sorokin
and Hoyt, 1992). Semi-thin (0.5 pm) sections of each
lung and culture were counterstained with 0.05% toluidine blue and examined a t 1-8 generally horizontal
planes spaced about 10 pm apart, to maximize surveillance of macrophages within each block and minimize
excessive encounters with the same cell. Subsequent
ultrastructural examination of thin sections at these
MATERIALS AND METHODS
levels proved that most cells of interest were identifiable by light microscopy, although electron microscopy
Rationale
was required to resolve cellular detail needed for stagGiven that embryonic rat lungs are free from mor- ing the transition to macrophages. Sufficient thin secphologically recognizable macrophages until after the tions a t each sampling plane were examined to provide
lungs are placed in organ culture, an opportunity serial views of certain cells, useful for determining
arises for determining the length of time needed for their shape and relationship to neighboring cells in the
precursors to transform into macrophages. Further- lungs.
DIFFERENTIATION O F LUNG MACROPHAGES
H- Thymidine Labeling Indices
3H-thymidine labeling indices were obtained in organ cultures of 14-day prenatal lungs at 1,4, 7,13, 19,
and 24 hr after explantation, for cells in the following
categories: macrophages and precursors (GSA I-B,
stained), stromal cells, pleural cells, airway epithelium, and fetal erythroblasts. Explants were placed on
medium containing 4 pCi/ml 3H-thymidine for 1hr immediately before fixation. After processing with peroxidase-coupled lectin and embedding in glycol methacrylate, 2 pm sections were cut, mounted on glass
slides, dipped in Kodak NTB-2 emulsion mixed 1:l
with distilled water, and exposed for 20 days at 4°C.
Slides were developed in Kodak D-19 developer, fixed
in 24% sodium thiosulfate, and stained with 0.05%
toluidine blue. Cells with five or more grains (above
background) over or touching the nucleus were considered labeled.
RESULTS
Morphological Form of GSA I-B,-Positive Cells
at Explantation
In lungs examined from the 14th day of gestation,
GSA I-B,-positive cells occurred only in the pulmonary
connective tissue, a t this time constituted as a loose
mesenchyme somewhat condensed about developing
bronchi. There were no labeled cells in the epithelium
nor among mesothelial cells of the visceral pleura.
Within the connective tissue positive cells were thinly
scattered throughout the extravascular compartment.
Blood vessels contained peroxidase-positive erythroblasts and a leukocytic population consisting almost
entirely of immature megakaryocytes rounded out by a
very few undifferentiated cells of uncertain lineage.
None of these intravascular leukocytes stained by the
lectin marker; as exceptions, however, two GSA-positive leukocytes were found inside vessels of newly cultured lungs (see below), and so may have been there at
explantation. The plasmalemma of vascular endothelial cells occasionally appeared stained. When this occurred, however, it was usually on only one surface,
more frequently the luminal. Instances with both luminal and basal surface staining occurred too infrequently to certify pulmonary endothelial cells as GSAIB,-positive, and by this criterion to admit them for
consideration as macrophage precursors.
Cells with the form and ultrastructural appearance
of macrophages were not found in any of our lung specimens at 14 days of gestation. Those qualifying for consideration as macrophage precursors by the criterion of
GSA I-B, binding were primarily 1) rather angular,
fusiform cells sandwiched in among the mesenchymal
fibroblasts, having a moderately euchromatic nucleus,
a large nucleolus, and a cytoplasm drawn out into two
or more major processes each bearing a varying number of short secondary processes (Fig. 1). As seen in
thin sections, typical GSA-positive cells of this kind
rather resembled surrounding fibroblasts in size and
shape, with the bulk of cytoplasm in the processes
rather than centered about the nucleus. Such cells usually appeared to be about twice as long as their nuclear
diameter, but those fortuitously sectioned along their
major axis measured as much as 6 diameters long. The
presence of many GSA-positive, nucleus-free cell pro-
553
files in the connective tissue testified further to the
elongate, branching form of these cells. Their cytoplasm appeared undifferentiated, characterized by
abundant polysomes, moderate numbers of mitochondria, little development of cytoplasmic membrane systems, and rarely a small cluster of peroxidase-positive
granules, too meager in diameter (ca. 100 nm) and too
few in the aggregate to register by light microscopy.
Alongside equally undifferentiated fibroblasts, these
cells at times stood out by their greater electron density in addition to their GSA binding. They were often
observed brushing their cell membranes against those
of the fibroblasts but never appeared to form specific
junctions with them. In counts made from the electron
micrographic record of 14-day prenatal lungs, these
angular cells made up 71% of all GSA-positive cells
present.
2) The second largest population of GSA I-B,-positive
cells in the extravascular stroma of 14-day lungs consisted of mixed undifferentiated leukocytes. Rarely exceeding three to four cells per specimen, they made up
only a fraction of the combined total of 60-70 intraand extra-vascular leukocytes present in each lung
pair at 14 days (Sorokin and Hoyt, 1987). Some of these
labeled cells resembled medium sized lymphocytes
with an electron-dense cytoplasm rich in polysomes,
and few exhibited more than an occasional profile of
granular endoplasmic reticulum. Exceptionally, one
lung specimen contained three cells characterized by a
distinctly notched nucleus and coliform, peroxidasepositive granules within a dense, polysome-rich cytoplasm. The granules were 0.1-0.4 pm long, radially
arrayed about the cytocentrum, and visible by light
microscopy, causing the cells to stand out in semi-thin
sections. They resembled lysosomal granules present
in bone marrow-derived neutrophilic promyelocytes or
promonocytes and afforded some basis for affiliating
the cells with the granulocyte-macrophage line (GM
cells). A third type of leukocyte contained conspicuous,
peroxidase-positive cisternae of endoplasmic reticulum
and large, peroxidase-positive and distinctly more
rounded granules than in the progranulocyte-macrophages. These cells closely resembled eosinophilic promyelocytes of adult rat bone marrow (Bainton and Farquhar, 1970). None was found in any of the uncultured
lungs we examined; however, two were found in a culture a t 3 hr and another a t 4 hr. Consequently, they
likely had been present a t explantation. Cells with any
of the foregoing descriptions (Fig. 2) were simply
grouped together as leukocytes and totaled 16% of the
GSA-positive cells present.
3) The third category of GSA I-B,-positive cells in
14-day lungs was intermediate in appearance between
the angular cells and the more agranular of the leukocytes and so equally could be thought to derive from
them. Cell processes were less extended and more cytoplasm surrounded the nucleus than in the angular
cells; on the other hand, cell outlines were more irregular than among the leukocytes. Prior to explantation
or immediately afterwards, intermediate cells resembled those of the other two classes in possessing an
undifferentiated cytoplasm and very few vacuoles (Fig.
3). Peroxidase-positive granules were either absent or
inconspicuous, as in angular cells. The intermediate
cells made up 14% of the total.
554
S.P. SOROKIN ET AL.
All figures are electron micrographs of 14-day prenatal rat lungs or
of organ cultures of 14-day explants, incubated with peroxidase-coupled GSA I-B, to reveal precursors and maturing pulmonary macrophages by the binding of the isolectin to the cell membrane. All have
been counterstained with uranyl acetate and lead citrate. In the designation, 14 +(n), n equals the time in culture. Beginning at zerotime (Fig. 1)and in the time-resolved sequence ending at 7 days (Figs.
7-15), illustrations have been selected to portray the most characteristic cell o f macrophage lineage present in the culture at the stated
time. In addition, low-power views are included to illustrate GSApositive cells in relation to other pulmonary tissues as well as
changed conditions in the cultures between + 6 and + 12 hr (Figs. 3,
5, respectively). Additional illustrations (Figs. 2, 6, 16, 17) are not in
chronological order but document special points raised in the text.
TRANSFORMATION OF PRECURSORS
INTO MACROPHAGES
climbed to 65% of the population, when a new category
of GSA-positive nascent (or early) macrophages first
appeared. These cells were larger and more rounded
than the intermediates, having phagocytic vacuoles
with variable, electron-dense contents. Intermediate
cells continued to hold to around 65% of the GSA-positive population through the 6th hr. The remainder
consisted almost exclusively of nascent macrophages,
but by 9 h r these cells, augmented now by a comparatively small number of more mature-appearing macrophages, had risen to 67%of the total GSA-positive population. By 12 hr, the macrophage pool (87.5%)mainly
consisted of the more mature cells, and by 15 h r the
GSA-positive population comprised 90% macrophages
and only 10% intermediate cells. After 24 hr, immature
lectin-positive cells occasionally were present in the
cultures, although they never made up more than a
small fraction of the total.
Comparison of low-magnification electron micrographs of prenatal lungs after 6 h r and after 12 h r of
culturing underscores the dramatic shift that the GSApositive population undergoes over the interval, from a
predominance of intermediate cells to outright macrophages (Figs. 3, 5).
Immediately after the 14-day prenatal lungs were
placed in organ culture, GSA I-B,-labeled precursors
began to t u r n into macrophages. In a n initial phase of
this process, GSA I-B,-labeled cells shifted from a
mixed population of angular cells, leukocytes, and intermediate cells to one consisting exclusively of overtly
phagocytic cells having ultrastructural characteristics
of macrophages. A second phase continued past 24 hr
and was characterized by maturation changes affecting
cells already classifiable as macrophages. Only ultrastructurally visible aspects of maturation are addressed here, but some of these can be correlated with
functional aspects of maturation examined in earlier
studies on these cells. For present purposes, transformation will be defined as equivalent to the first of these
two phases and maturation to the second.
During the first hours of organ culturing, the proportion of intermediate cells in the total GSA-positive population grew rapidly while that of angular cells and
leukocytes declined (Table 1, Fig. 4). By 2 hr, they had
Fig. 1. 14-day prenatal lung a t explantation. GSA-positive angular
cell in the mesenchymatous connective tissue. x 11,140.
DIFFERENTIATION OF LUNG MACROPHAGES
555
Fig. 2. a: “Primitive” macrophage present in a pulmonary blood
vessel at 14 + 6 hr. The cell has an enlarged nucleolus and remains a n
undifferentiated leukocyte showing little response to the trauma of
lung explantation, compared to GSA-positive cells in the connective
tissue at this time. x 11,530.b Intermediate cell in pulmonary connective tissue at 14 + 4 hr. It is one of few found differing from typical
intermediate cells in possessing coliform peroxidase-positive granules
of appreciable size, possible grounds for association with GM lineage.
x 6,680. c: GSA-positive cell with round peroxidase-positive granules
and reaction product in the endoplasmic reticulum, present a t 14 + 3
hr. This cell type was not observed in cultures beyond + 4 hr. x 8,610.
The angular cells appeared to be mainstream precursors of the macrophages because they were the commonest of GSA-positive cells in 14-day explants and
underwent a steady, proportionate decline upon cultur-
ing, in tandem with a rise in intermediate cells initially and later with macrophages (Fig. 4).
Leukocyte-like GSA-positive cells also underwent
early, proportionate decline, but their initial small
556
S.P. SOROKIN ET AL.
Fig. 3. Low-power view of a lung culture at 14 +6 hr. Three intermediate cells (arrows) are present
outside blood vessels in the connective tissue, which has the texture of mesenchyme. The epithelium is
seen at the upper right, and the blood vessels contain dark-staining erythroblasts. Compare with Figure
5. ~2,340.
number and comparatively irregular occurrence in the
cultures disqualified them as prime candidate-precursors for the macrophages. We found no reason to deny
that labeled primitive leukocytes (Fig. 2a) were capable of changing into “intermediate cells,” although direct evidence was limited. Two GM cells observed in a
culture a t 4 hr were interpretable as intermediate
forms (Fig. 2b), exhibiting similarly to agranular GSA-
positive cells in their initial reaction to organ culturing. In contrast, all three promyelocytes with large peroxidase-positive granules observed in the stroma of 3
and 4 hr cultures appeared to be holding to an eosinophilic line of development, without extension of
pseudopods or other visible response to their surroundings (Fig. 2c). Similar cells were never found in older
cultures.
557
DIFFERENTIATION OF LUNG MACROPHAGES
TABLE 1. Changing composition of GSA I-B,+ cell types in lung cultures over time
Angular
cells (%)
70.5
44.0
32.3
20.8
14.1
1.8
1.2
0.0
0.0
11.1
0.0
0.0
1.1
0.0
0.0
0.0
6.2
N
Age
14 + 0 hr.
14 + 1 hr.
14 + 2 hr.
14 + 3 hr.
14 + 4 hr.
14 + 6 hr.
14 + 9 hr.
14 + 12 hr.
14 + 15 hr.
14 + 18 hr.
14 + 24 hr.
14 + 2 d.
14 + 4 d.
14 + 5 d.
14 + 7 d.
14 + 9 d.
14 + 13 d.
46
25
31
24
71
53
86
40
51
9
8
5
94
18
21
3
16
loo
Leukocytelike cells (%)
15.9
12.0
0.0
Intermediate
cells (%)
13.6
44.0
64.5
66.7
59.2
64.2
30.2
12.5
9.8
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
2.8
0.0
1.2
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
I
Macrophages (%)
Early
Mature
0.0
0.0
0.0
0.0
3.2
0.0
12.5
0.0
23.9
0.0
34.0
0.0
52.3
15.1
22.5
65.0
29.4
60.8
22.2
66.7
0.0
100.0
0.0
100.0
14.9
84.0
0.0
100.0
0.0
100.0
33.3
66.7
0.0
93.8
1
% of cells
0
1
2
3
4
6
9
15
12
18
24
hours in culture
Fig. 4. The changing composition of GSA-positive cell types in lung cultures over time, graphic representation of data for the first 24 hr from Table 1. Half of the population of transforming cells becomes
recognizable as macrophages around + 8 hr. 0,
angular cells; (01, leukocyte-like cells; (w), intermediate
cells; (o), macrophages.
Extravascular vs. lntravascular Formation
of Macrophages
During the first 6 hours of organ culturing, all GSA
I-B,-labeled transitional forms and nascent macrophages were localized to the extravascular connective
tissue. In contrast, neither of the two intravascular
GSA-labeled cells found during this period provided
more telling morphological evidence of entry into
transformation than a large nucleolus (Fig. 2a). Nascent macrophages were first found within vessels at 9
hr. They became more numerous at 12 hr and, together
with more mature forms, common at 15 hr and thereafter, particularly after tissue breakdown had left gaps
in the endothelium for cells to pass through (Fig. 6 ) .
Inside the vessels, macrophages frequently stationed
themselves beside stagnant blood pools and by 24 hr
were engorged with fragments or entire protoplasts of
red cells and expanded to many times their original cell
volume.
Ultrastructural Changes During Transformation
and Maturation
A staged and time-resolved morphological reconstruction of the transformation process was obtained by
selecting an electron micrograph to depict the predominant GSA I-B,-positive cell in the cultures a t each
specific interval after explantation and arranging
these images in chronological order; the resulting series covers close intervals in the first 15 hr, and wider
intervals from there to 7 days (Figs. 7-15).
Ultrastructurally, cells in the “intermediate” category underwent subtle changes as they gradually took
on typical morphological characteristics of macrophages. Throughout, one or more nucleoli were con-
558
S.P. SOROKIN ET AL.
Fig. 5. Low power view of a lung culture at 14 + 12 hr. By this time the prevailing GSA-positive cell
contains phagocytosed cell debris and is recognizable as a macrophage. Several are shown above the
blood vessel in the middle and upper part of the field. Compare with Figure 3. x 2,950.
spicuous in the nucleus (Fig. 7,8). The cytoplasm only
perceptibly increased its content of granular endoplasmic reticulum and gradually developed a few clear
vacuoles (Figs. 9, lo), so that after 4 hr, few nonvacuolated cells remained. The Golgi apparatus was
rarely prominent in these cells. Early-stage macrophages graded away from this appearance as they
increased in size and phagocytic activity (Fig. 11).
The Golgi apparatus began to expand in certain young
macrophages around 9 hr, forming a small but distinct
region of cisternae and vesicles on one side of the
nucleus. This had grown into a complex of many stacks
by 15 hr, so that five or six might be included in a
section passing through the cytocentrum (Fig. 12b).
Frequently they were turned with their long axis
extending radially outward, in an “open” position
559
DIFFERENTIATION OF LUNG MACROPHAGES
TABLE 2. 3H-thymidinelabeling indices for lung explants in culture
GSA-IB,
Age
14 1 hr
+
N
14
+ 4 hr
N
+ 7 hr
14 + 13 hr
N
14 + 19 hr
N
14 + 24 hr
14
N
N
cells
+
33.63%
113
33.98%
103
32.65%
98
34.03%
238
35.61%
264
34.05%
279
Stromal
cells
Pleural
cells
Epithelial
32.20%
441
26.43%
401
26.40%
428
12.56%**
422
9.88%**
476
13.58%**
508
34.54%
414
36.43%
409
26.12%
402
8.26%**
436
6.11%**
409
9.39%**
479
cells
37.67%
438
34.70%
415
29.90%
418
18.26%**
449
15.35%**
404
30.69%
613
Fetal
erythroblasts
25.62%
441
20.33%*
861
17.68%**
1103
13.75%**
1084
0.84%**
830
1.31%**
917
Comparisons with GSA-IB,+ cells by X2 tests: *P<0.005 and **P<O.OOl.
often seen among actively phagocytic cells (Sorokin,
1983b), the stacks being separated by radial arrays of
microtubules coming from centrioles within the Golgi
ring. Most of the ribosomes of transforming cells were
arranged in free polysomal clusters. The granular endoplasmic reticulum of macrophages appeared only
moderately extended beyond that seen in older intermediate cells (Figs. 10a, 12a), not a t all like the expansion sometimes present among mature alveolar macrophages provoked into production of lysosomes (Sorokin,
1983a). Indeed, the most singular aspect of cytoplasmic
differentiation among transitional cells and macrophages was the widespread occurrence of satellites and
astral fibers about the centrioles (Figs. 11, 12b, 16a),
manifestly contrasting to stromal fibroblasts which instead sprouted a primary cilium from the diplosome
(not illustrated). Like angular cells, but unlike cells
resembling blood leukocytes, intermediate cells and all
gradations of macrophages characteristically maintained extensive surface contact with adjacent stromal
mesenchymal cells or fibroblasts, as if interacting at a
cell membrane level (Fig. 16b), and like them they
formed no specialized junctions with the fibroblasts.
Macrophages seen in 24 hr cultures were generally
large and replete with phagocytosed matter of considerable, though varied electron density (Fig. 13). By 48
hr these dense phagosomes had greatly receded from
the cytoplasm, being supplanted in typical stromal
macrophages by many small (ca. 0.2-0.5 pm) electronlucent (lipid-containing) vacuoles. These vacuoles remained as a characteristic of macrophages for the duration of a week or more that cultures were grown
(Figs. 14a, 14b, 15,16b). During the same period, macrophages within lung tissue appeared to increase their
complement of mitochondria, giant cells began to be
seen, and many cells, giant and smaller alike, displayed well ruffled membranes (Figs. 14, 15). These
ultrastructural changes were the most consistently
found among maturing cells and remained characteristic of macrophages as many began a centrifugal migration that resulted in a buildup of these cells at the
pleural surface and gradual clearing of the lungs (Fig.
15).
Mitotic Activity and Labeling Indices of
GSA-Positive Cells
Mitotic figures were observed among GSA I-B,-labeled cells in both intermediate and macrophage categories throughout the period the lungs were cultured.
They were first documented in electron micrographs of
intermediate cells at 4 hr, and in intermediate cells
and nascent macrophages a t 6 hr (Fig. 10b,c). They
were also seen at 9 hr (intermediate cells), 12, 15, and
18 hr (maturing macrophages), and 24 hr (maturing
and well-developed macrophages, Fig. 13).Thereafter,
all dividing GSA-positive cells observed were mature
macrophages; they were especially numerous in 4 day
cultures. In our electron microscopic survey, labeled
mitotic cells were found only in the extravascular
stroma, although dividing macrophages were sometimes observed by light microscopy in vessels of 2-day
and older cultures.
3H-thymidine labeling indices for macrophages and
precursors, stromal cells, pleural cells, airway epithelium, and fetal erythroblasts in lung cultures are given
in Table 2. At explantation ( + 1 hr), grain counts indicated a high labeling index for cells in all categories, as
expected of rapidly growing embryonic tissues. Lung
stromal and pleural cells and those in the macrophage
line (GSA I-B,-positive) labeled similarly with a third
of the cells in S-phase. This figure slightly led erythroblasts in the blood vessels (26% labeled) but trailed
epithelial cells of the terminal buds (38%labeled). During the next 18 hr, however, the labeling index of cells
in all categories except the GSA-positive pool declined,
followed by some indication of recovery in the airway
epithelium at 24 hr. The fall was steepest among the
erythroblasts but was sufficiently similar in each of the
three remaining categories as to make them appear a
single population, perhaps reflecting equal susceptibility of lung cells to an “explantation shock” that requires some hours to overcome. In marked contrast,
GSA-positive cells maintained their initial labeling index throughout the entire 24 hr period, exhibiting no
explantation shock and objectively behaving as a population distinct from other cells in the lungs, including
their immediate neighbors in the connective tissue.
Figs. 6-7.
DIFFERENTIATION O F LUNG MACROPHAGES
561
Fig. 8.14 + 2 hr. Intermediate cells are now the predominant GSA-positive cells in the lung stroma:
resembling those present a n hour earlier, they as yet contain few vacuoles. x 11,180.
Immature Cells in Older Cultures
A small number of incompletely transformed cells
may be present in lung organ cultures long past the
time when virtually all other GSA-positive cells have
become transformed into macrophages (Fig. 17).
Among the younger cultures to contain nearly 100%
macrophages (12-24 hr), the immature forms more frequently were classifiable as intermediate cells, similar
Fig. 6.Intravascular vs. extravascular origin of macrophages: 14
+ 15 hr. Macrophages (*) present in the vascular
lumen (above and
left) as well as in the pulmonary stroma (center). Intravascular cells
may have migrated into the blood vessel through a rent in the endothelial lining (arrow). x 3,320.
Fig. 7.14 + 1 hr. Compared to an angular cell, this intermediate cell
appears to have shorter processes and more cytoplasm about the nucleus. Two nucleoli are seen; the cytoplasm remains rather undifferentiated, although a few tiny granules can be seen near the centriole
below the nuclear notch. x 9,750.
to intermediate cells seen earlier except for an apparently delayed response to explantation (Fig. 17b). In
contrast, among older cultures of 4 days (Fig. 17a) or as
many as 13 days (Fig. 17~1,immature GSA-positive
cells more nearly resembled angular cells and appeared further separated from mature macrophages because transitional forms between them were not seen.
DISCUSSION
This paper presents a time-resolved, ultrastructural
reconstruction of events taking place within a distinct,
GSA I-B,-labeled population of cells in organ cultures
of 14-day prenatal rat lungs as it undergoes a shift in
composition from precursors to macrophages. There is
no doubt that macrophages are produced in large number by living cultures over a comparable interval because the process can be continuously followed under
the light microscope. In the present instance, sequential passage of cells from one stage t o another has been
inferred from study of fixed specimens which are required for electron microscopy. We are confident of its
562
S.P. SOROKIN ET AL.
Fig. 9. 14 + 3 hr. The intermediate cell has begun to exhibit a few profiles of granular endoplasmic
reticulum and to contain a few, still mostly clear vacuoles. x 11,760.
validity for a number of reasons: 1)The cultures were
collected at close, in initial phases hourly, intervals. 2)
The GSA-positive population presented a changing appearance with increasing time from explantation but
was the same for all specimens fixed at a given interval. 3) In contrast to other cell types, this population
showed no pyknosis or other visible evidence of cellular
degeneration. 4) On the contrary, it maintained a uniformly high (34%)3H-thymidine labeling index during
the transformation period. 5) Many fine gradations
were seen between the main morphological classifications.
Replication vs. Transformation of
Macrophage Precursors
Our study indicates that in the organ cultures, macrophages initially arise in extravascular connective tissue rather than the intravascular spaces. For the most
part they appear to develop from angular cell precursors situated among stromal mesenchymal cells or
fibroblasts which to a degree they resemble morphologically; angular cells differ markedly from their
neighbors in binding GSA I-B,. Seemingly, macro-
phages can also arise from undifferentiated GSA-positive leukocytes present in prenatal rat lungs, particularly if the cells first migrate into the extravascular
connective tissue. In 14-day explants, however, such
leukocytes alone could not account for more than a
small part of the macrophages existing in 12 hr cultures because too few were present to have made up the
macrophage number by direct transformation, and little time was available for these candidate precursors to
increase through mitotic activity. From what we have
learned from study of GSA-positive cells in intact rat
embryos (Sorokin et al., 1992a), this distinction may be
of small account, since GSA-labeled angular cells and
GSA-labeled agranular leukocytes equally represent
the primitive/fetal macrophage line, the angular form
probably reflecting greater length of time spent residing in the connective tissues of the lungs or other organs, compared to the more rounded, leukocyte-like
forms.
As they occurred in 14-day prenatal rat lungs, angular and other GSA-positive cells constituted an actively
growing population with a high division rate. The 3Hthymidine labeling index (34%) remained unchanged
DIFFERENTIATION O F LUNG MACROPHAGES
Fig. 10. 14 + 6 hr. a: An intermediate cell has continued trends first
noted at + 3 hr: further expansion of the granular reticulum and
increased vacuolization. GSA-positive staining of a vacuolar profile
along the lower surface (arrow) may represent infolding of cell mem-
563
brane, hence early formation of a phagosome. x 9,800. b Mitosis in
a n intermediate cell. x 8,375. c: Mitosis in a n early macrophage.
x 5,800.
564
S.P. SOROKIN ET AL.
Fig. 11. 14 + 9 hr. Typical cells at this time are nascent (early) macrophages like this one with
numerous phagocytic inclusions but as yet little differentiation of other cytoplasmic membrane systems,
and centrioles with prominent satellites. x 9,180.
for 24 hr of organ culturing while the population transformed into one made up entirely of macrophages.
From this it appears on the one hand that macrophage
precursors can replicate rapidly, as in keeping pace
with lung growth, without being stimulated to transform into macrophages. On the other hand, transformation need not be accompanied by slowing in the rate
of DNA synthesis by affected cells, at least for the first
24 hr.
Significance of Macrophage Precursors in
Embryonic Lungs
A number of our findings indicate that the main purpose of the early appearance of macrophage-like cells
in the lungs and several other organs is to establish a
resident cell population of potential macrophages in
those places, rather than primarily to meet immediate
local challenges. Such a process differs fundamentally
from the dispatching of leukocytes to an organ in response to an infectious or immunological cell. If for
argument’s sake we put aside the question of de novo
formation of angular cells in the lungs (Sorokin et al.,
1992a) and assume that the original consignment of
macrophage precursors is entirely by seeding from the
blood stream, then the events that follow seeding are
still markedly different from what is generally understood to follow the arrival of monocytes in older lungs:
In the first case the cells extend processes out among
the mesenchymal fibroblasts and blend into the tissue
as angular cells, whereas in the second monocytes begin to transform into phagocytic macrophages as soon
as they cross out of the blood vessels, much as activated
precursors behave in lung cultures, or chicken monocytes in tissue culture (Sutton and Weiss, 1966). The
angular cells neither increase greatly in volume, round
up, engage in phagocytosis, or increase production of
lysosomes but remain phagocytically quiescent, evidently responding to quite different signals from those
initiating transformation. Given the high rate of thymidine incorporation and the close matching of labeling indices between GSA-positive and other constituent cells of intact lungs, what more appropriate
behavior could be expected from cells establishing a
permanent residency?
Transformation and Maturation of Macrophages
In developing lungs resident precursors normally
transform into macrophages irregularly and a few at a
time. Extensive trauma like that brought about by dissection and organ culturing brings about transfor-
DIFFERENTIATION OF LUNG MACROPHAGES
Fig. 12. 14 + 15 hr. a: Typical macrophages are now more matureappearing than at + 9 hr. They are larger and often exhibit greater
expansion of the granular reticulum (left side and top of cell) and
prominence of mitochondria, although development of these organelles remains moderate in comparison to the full potential of alveolar macrophages. x 5,620. b: The Golgi apparatus generally is
565
larger than in nascent macrophages and may have several stacks of
lamellae. These are sometimes arrayed with their long axes raidal to
the diplosome in a stellate or “open” pattern characteristic of phagocytizing cells. The centrioles characteristically are surrounded by
many satellites. x 17,600.
566
S.P. SOROKIN ET AL.
Fig. 13.14 + 24 hr. Dividing macrophage in the lung stroma. This cell is typical of those in the cultures
a t the conclusion of the transformation process, large, filled with phagocytosed but not yet well digested
material. x 6,890.
mation of a great many cells and demonstrates the
developmental potential inherent in the normally inconspicuous resident population. As our material tends
to show, initial actions of precursor cells during the
transformation period are related to becoming mobile
and actively engaged in phagocytosis, a t once mirrored
in the cytoplasm by increased prominence of microtubular arrays about the centrioles. Earlier work has
shown by rosetting assay that few cells in 14-day lung
explants have sufficient Fc receptors to bind IgG-opsonized erythrocytes effectively, but the proportion of
cells binding them increases sharply during the first 24
hr in culture (Sorokin et al., 1989).Consequently, macrophage precursors become more efficient phagocytes
during transformation. Notable expansion of the granular endoplasmic reticulum is held off for a time,
however, and so is large-scale synthesis of lysosomal
enzymes such as acid phosphatase, which is not histochemically conspicuous in macrophages before 24 hr
and attains levels of activity comparable to activated
macrophages of postnatal lungs only a day or two later
(Sorokin and Hoyt, 1987). From these observations one
can perceive an ordering of priorities that produces efficient, motile phagocytes before they are fully capable
of digesting materials ingested; hence typical transformed macrophages in the lung cultures at 24 hr are
enlarged and their phagosomes filled with recognizable
cellular debris, and digestively competent macro-
DIFFERENTIATION OF LUNG MACROPHAGES
Fig. 14. 14 + 4 days. a: Macrophage typical of those occurring well
within the maturation period. The plasmalemma has developed elaborate lamellipodia (ruffled membranes), shown here at the advancing
edge of the cell (right), and the vacuoles are homogeneous and filled
with nearly electron-lucent contents. Ingested cellular debris has
been hydrolyzed. It is uncertain whether one or two nuclei are
567
present. x 10,100. b Binucleate macrophage in the lung stroma. The
cytoplasm appears similar to that in the macrophage above, but the
cell diameter is greater. A detail of the cytoplasm is shown to the
right. X 3,280. c: Cytocentrum of the cell a t left. More than two centrioles are present, making it likely that the cell has more than one
nucleus-an early example of giant cell formation. x 32,500..
568
S.P. SOROKIN ET AL.
Fig. 15. 14 + 7 days. Macrophages on the outer surface of a lung
culture. Pleural cells form an arc sweeping down from the middle of
the left side of the figure and are themselves GSA-negative, but they
are enmeshed in lectin-positive processes from the macrophages.
These possess electron lucent vacuoles and are typical of mature cells
produced by the organ culture system. X 4,175.
569
DIFFERENTIATION O F LUNG MACROPHAGES
Fig. 16. a: Microtubules radiating from satellites about a centriole
in a pulmonary macrophage a t 14 + 24 hr. This configuration is typical of intermediate cells and young macrophages and unlike that
seen in stromal fibroblasts during the same period. x 25,780. b: Mac-
+
rophage within lung tissue a t 14 4 days. Cell processes are extensively spread over the surfaces of two neighboring fibroblasts
(arrows). x 7,700.
570
S.P. SOROKIN ET AL.
Fig. 17. a: Elongate GSA-positive cell (*) beneath the pleural lining
of a lung culture at 14 + 4 days. The cell has accommodated itself to
the layering of the tissue, does not resemble typical macrophages
present at this time, and may represent conversion to a dendritic, or
monitor cell. x 6,840. b: Late occurrence of a transforming GSA-positive cell in a culture aged 14 +15 hr. Located just beneath the
endothelium, it resembles a blast form more than most intermediate
cells, and possibly reflects delayed activation of an originally intravascular leukocyte (Fig. 2a). x 13,060. c: Undifferentiated GSA-positive cell present in pulmonary stroma at 14 + 13 days. It is at the
borderline between an angular and intermediate cell. x 8,530.
DIFFERENTIATION OF LUNG MACROPHAGES
phages seen a day or two later have clear vacuoles. In
earlier work we had also found that these vacuoles
stain with Oil Red 0 and therefore contain lipids. Such
alteration in vacuolar content should not be taken to
imply disorder in macrophage function, as it is known
that mouse peritoneal macrophages subjected to lipidloading regimens in culture exhibit no loss in Fc receptor-mediated endocytosis and continue to produce a variety of products, although the balance secreted may be
subtly altered (Montgomery and Cohn, 1989).
“Quiescence”in Older Cultures
The normal course in the organ culture system used
here is for matured macrophages with clear vacuoles
and often well-developed lamellipodia to migrate
within lung tissue a few days but eventually to be
drawn out onto the pleural surface. There they lose
contact with stromal cells and remain largely unchanged for a week or two with the population increasing from outward migration of other macrophages and
not by local mitotic activity. A few giant cells may
appear among them, and these have many mitochondria and an outwardly more metabolically active appearance than the remaining mononuclear cells; but as
a population the cells become quiescent, scavenging
activity gradually ceases, and the population eventually dies, as if from lack of stimulation. Relatively few
macrophages are left inside the lung culture, and
among those remaining, most find their way into fluid
filled airways where they become caught up in the currents set up by action of ciliated cells of the bronchi and
bronchioles and never return to the tissues. A very few
GSA-positive cells were found not conforming to the
life pattern summarized above, retaining a habit
nearer angular cells than macrophages long after most
other GSA-positive cells had matured. It is as yet unknown whether these simply required a higher threshold of stimulation to transform or, having once transformed, subsequently converted into dendritic cells.
With respect to the vast majority of GSA-positive
precursors that become macrophages, however, it is of
interest to know whether their fate can be altered experimentally by administration of hematological colony stimulating factors or other cytokines. Macrophages on the pleural surface of the culture are present
in sufficient number for certain types of quantitative
analyses, and being easily exposed to these agents,
make good experimental subjects. In particular, they
may provide information useful for resolving uncertainty about the longevity of early pulmonary phagocytes and the possibility for continuance of this macrophage line into postnatal life. These topics are further
571
explored in the next paper of the series (Sorokin et al.,
199213).
ACKNOWLEDGMENTS
This work was supported by USPH research grant
HL-33070.
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