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Peroxidase cytochemistry and ultrastructure of resident macrophages in fetal rat liver
DEVELOPMENTAL
Peroxidase
BIOLOGY
66,43-56
Cytochemistry
(1978)
and Ultrastructure of Resident Macrophages in Fetal Rat Liver’ A Developmental
Study2
WINFRIED DEIMANN AND H. DARIUSH FAHIMI~ Department of Anatomy, Division II, University of Heidelberg, 6900 Heidelberg, Federal Republic of Germany Received March 15, 1978; accepted in revised form April 20, 1978 The peroxidase cytochemistry and the ultrastructural characteristics of resident macrophages in fetal rat liver have been investigated. Livers of lo-, ll-, 14-, 17-, and 20-day-old fetuses were fixed by immersion or perfusion, incubated for peroxidase, and processed for transmission electron microscopy. Some 17- and 20-day-old fetuses were injected prior to sacrifice with carbon or 0% pm latex particles through the umbilical vein. Some livers were additionally processed for scanning electron microscopy @EM). The endogenous peroxidase was present in the nuclear envelope (NE) and endoplasmic reticulum (ER) of fetal macrophages with a negative reaction in the Golgi apparatus, a distribution pattern identical to that in Kupffer cells of adult rat liver. Such peroxidase-positive cells avidly took up the injected latex and carbon particles and were the only cell type in fetal liver involved in erythrophagocytosis. Furthermore, they were associated with erythropoietic elements, forming close contacts with such cells, especially normoblasts. The peroxidase pattern in leukopoietic cells differed at all stages of maturation from that in macrophages. By SEM the macrophages exhibited ruffles and lamellopodia on their surfaces and protruded often into the lumen of fetal sinusoids. Macrophages in fetal liver underwent mitotic divisions. The macrophages were first seen on gestation day 11, whereas the first mature monocytes were found on gestation day 17. These observations suggest that resident macrophages in fetal rat liver form a self-replicating cell line independent of the monocytopoietic series, although they may both arise from a common precursor cell.
of monocytes in this tissue. The existence of macrophages in fetal mammalian liver has been established by their ultrastructural characteristics and evidence of erythrophagocytosis (Sorenson, 1960; Reade and Casley-Smith, 1964; Zamboni, 1964; Rifkind, 1969; Chui and Russel, 1974; Fukuda, 1974). The endogenous peroxidase has proven to be a useful cytochemical marker for mononuclear phagocytes (Kupffer cells) in adult rat liver (Fahimi, 1970; Widmann et al., 1972; Ogawa et al., 1973; Wisse, 1974). Furthermore, since this enzyme is a sensitive indicator of monocytopoietic maturation (Nichols et al., 1971; Morris et al., 1975; Bentfeld et al., 1977), it may provide insight into the interrelationships of monocytes and their precursors with resident macrophages.
INTRODUCTION
Fetal liver as a hemopoietic organ provides a unique model for the in uivo study of the interrelationships of various blood cells. The mature blood monocytes are generally considered to be the precursors of tissue macrophages (van Furth et al., 1972). Accordingly, the macrophages in fetal liver should also arise from the transformation ’ This study is dedicated to Dr. E. H. Kass on the occasion of his sixtieth birthday. ’ Supported by National Institutes of Health Grant NS 08533 and by Sonderforschungsbereich 90 (CARVAS) from Deutsche Forschungsgemeinschaft, Bad Godesberg, Federal Republic of Germany. 3 Address alI correspondence to: Professor H. D. Fahimi, Anatomisches Institut II der Universitlit, 6900 Heidelberg, Im Neuenheimer Feld 307, Federal Republic of Germany. 43
0012-1606/78/0661-0043$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
44
DEVELOPMENTALBIOLOGY
In the present study we have applied the peroxidase technique to the fetal rat liver and have studied the development of resident macrophages and their relationships with fetal hemopoietic cells from day 11 to day 20 of gestation. In addition, the phagocytic properties of fetal macrophages were assessed by injection of tracer particles and their surfaae characteristics and position in sinusoids by scanning electron microscopic techniques. MATERIALS
AND
METHODS
Animals. Adult Sprague/Dawley rats (Zentrale Versuchstieranlage, Heidelberg University) were kept on a regular laboratory diet and water ad libitum and were used for mating in all our experiments. On the morning after the overnight housing of males and females, vaginal smears were examined; females positive for sperm were caged separately, and the day was designated as day 0 of gestation. On gestation days 10, 11, 14, 17, and 20, the abdomen was opened under light ether anesthesia and the uterine vessels were clamped. Immediately thereafter, one to two fetuses were removed from the uterus and submitted to fixation. Fixation. All material was fixed for 5 min at room temperature with 1.5% purified glutaraldehyde (Serva, Heidelberg) in 0.15 MNa-cacodylate buffer (pH 7.2) containing 0.05% CaC12. Two separate fixation procedures were used: (a) Immersion fixation for lo-, ll-, and 14-day-old fetuses. As the liver primordium of lo- and 11-day-old fetuses could not be visualized with certainty under the dissecting microscope, the entire fetus, including its intact membranes, was immersed in the fixative. It was important not to detach the fetal membranes from the fetus, because the liver primordium was easily removed with these membranes. In 14-day-old fetuses, only the abdominal cavity was immersed in the same fixative. (b) Perfusion fixation for 17- and 20-day-old fetuses. Under a dissecting microscope, the liver was perfused either through the um-
VOLUME 66,1978
bilical vein or by transparenchymal sion (Sandstrom, 1970).
perfu-
Peroxidase cytochemistry and processing for transmission electron microscopy (TEM). After fixation, tissues were rinsed several times with 0.15 M Na-cacodylate buffer. The 50+m sections were prepared using a Smith-Farquhar tissue sectioner (TC-2-Sorvall, Norwalk, Conn.) and incubated at room temperature. The incubation medium contained 0.1% 3,3’-diaminobenzidine (DAB) in 0.1 M ‘Iris-HCl buffer, pH 7.2, and 0.01% H202. The sections were first preincubated in the absence of Hz02 for 60 min, followed by incubation in the complete medium for another 60 min. Subsequently, the specimens were postfixed with 2% aqueous osmium tetroxide for 90 min, dehydrated in graded ethanol solutions, and embedded in Epon 812. Two-micrometer sections, unstained or stained with toluidine blue, were examined by light microscopy. In the cases of the lo- and 11-day-old fetuses, only those blocks containing identifiable liver tissue were selected and trimmed for ultramicrotomy. Ultrathin sections were contrasted with lead citrate, with or without uranyl acetate, and examined in a Philips EM 301 transmission electron microscope. Cytochemical controls were performed by the incubation of sections in the absence of HzOz or, in some cases, of DAB. Injected compounds. (a) Ten minutes prior to fixation, some 17- and 20-day-old fetuses were injected with 0.02 ml of 0.8~pm latex particles (Serva, Heidelberg) or colloidal carbon (Giinter Wagner, Hannover) into the umbilical vein. (b) Twelve hours prior to sacrifice, some adult rats pregnant for 16.5 days received an intraperitoneal injection of 1 mg/lOO g vinblastine (Velban, Eli Lilly Corp., Indianapolis) to arrest the dividing cells in mitosis. Cell counts. Cells with a peroxidase-positive nuclear envelope (NE) and endoplasmic reticulum (ER) were counted in a transmission electron microscope. The sections were mounted on 300-mesh copper
DEIMANN
AND FAHIMI
grids and counterstained with lead citrate only, to avoid masking the peroxidase reaction by uranyl acetate. Only cells with sectioned nuclei entirely within the grid square were counted. The counts are expressed as cells per square centimeter. Processing for scanning electron microscopy (SEM). Small cubes of liver tissue of 17- and 20-day-old fetuses were sampled for SEM. After perfusion fixation, they were immersed for an additional 2 hr in the same fixative, postfixed for 3 hr with OsO1, and dehydrated in graded ethanol solutions. Intracellular fractures were obtained according to Haggis (1972) and extracellular fractures by breaking the tissue with two forceps after critical-point drying. The latter was achieved by exchanging 100% ethanol with CO,. All specimens were sputtered with gold and examined in a SEM 500 Philips scanning electron microscope at 25 kV. RESULTS
Our observations deal mainly with the resident macrophages of fetal rat liver, which exhibited phagocytosis and the characteristic peroxidase distribution pattern. The hepatic organogenesis is not treated here, since it has already been described extensively [for a review, see Du Bois (1963)]. Light microscopy. The staining for peroxidase appeared as a diffuse brownish precipitate distributed throughout the entire cytoplasm of the macrophages, but sparing the nucleus (Figs. 1 and 2). Most of the macrophages contained phagosomes with peroxidase-positive material of probable red blood cell origin. An additional feature was their close association with erythropoietic elements, which exhibited a strong reaction due to their hemoglobin content. Furthermore, there were leukopoietic elements in fetal rat liver with strong peroxidase-positive granules (Fig. 10).
Peroxidase cytochemistry and ultrastructure. The fetal hepatic macrophages exhibited
the same ultrastructural
and cy-
Macrophages in Fetal Liver
45
tochemical features as described previously for the adult rat liver Kupffer cells (Fahimi, 1970; Widmann et al., 1972; Ogawa et al., 1973; Wisse, 1974). The peroxidase activity was confined to the NE and ER, whereas the Golgi apparatus, including Golgi saccules and vesicles, was consistently peroxidase negative (Fig. 3, Table 1). Another prominent feature found in SO-85% of the fetal macrophages was the evidence of erythrophagocytosis. Peroxidase-positive erythropoietic cell residues at all stages of degradation were found in phagosomes and phagolysosomes, which varied widely in size, density, and shape (Fig. 3). Some macrophages occasionally contained whole normoblast nuclei (Fig. 6). The 0.8-pm latex particles injected into the umbilical veins of the 17- and 20-dayold fetuses were taken up exclusively by peroxidase-positive macrophages (Fig. 5). Discharge of peroxidase into the latex-containing phagolysosomes was not observed. Similarly, injected carbon was found only in the macrophages (Fig. 4). Peroxidatic activity in other fetal liver cells. The liver parenchymal cells and typical wall-forming endothelial cells were consistently peroxidase negative. On the other hand, evidence of peroxidase activity was observed in the erythropoietic and leukopoietic elements. The erythropoietic cell series contained reaction product diffusely throughout the cytoplasm, but sparing the cytoplasmic organelles (Fig. 3). Although this pattern of localization did not change, the intensity of the peroxidase reaction increased with the maturation of the erythropoietic cell series. The peroxidase-positive leukopoietic cells comprised the myeloid and the monocytic series (Table 1). Whereas the immature cells (promyelocytes and promonocytes) exhibited peroxidase activity in the NE, ER, Golgi apparatus, and all cytoplasmic granules (Fig. 7), the more mature cells (myelocytes and monocytes) showed no activity in their secretory apparatuses, but contained only peroxidase in some of their
46
DEVELOPMENTAL BIOLOGY VOLUME66,1978
FIGS.1-3
DEIMANN TABLE
AND FAHIMI
1
PEROXIDASE DISTRIBUTION PATTERN IN RESIDENT MACROPHAGES AND MONOCYTOPOIETIC AND GRANULOPOIETIC CELL SERIES IN FETAL RAT LIVER Cell types
Resident macrophages Promonocytes” Monocytes Promyelocytes” Myelocytes Granulocytes
Peroxidase
activity
NE and ER
Golgi
Granules
+ + -
+ -
+ -
+ -
Oh + +,--’ + + +,--’
” In spite of identical peroxidase distributions promonocytes and mature promyelocytes can be distinguished from one another, as the latter exhibit a larger number of granules and a focally dilated ER. Young promyelocytes are similar to promonocytes: Both cell types contain only a small number of peroxidase-positive granules (see also Fig. 7). ’ In macrophages of fetal rat liver, peroxidase-positive granules similar to “azurophil” granules of leukopoietic cells are absent (0). “Mature monocytes and granulocytes contain a peroxidase-positive (+) as well as a negative (-) granule population.
granules (Fig. 6). Although the various cell types of the myeloid and monocytic cell series could be easily identified on the basis of peroxidase cytochemistry, promonocytes and young promyelocytes were difficult to distinguish from each other. Both cell types showed an identical peroxidase distribution, i.e., reaction product in the NE, ER, and Golgi apparatus and in a few granules (Fig. 7). This pattern differed distinctly, however, from the peroxidase distribution
Macrophages
in Fetal Liver
47
in macrophages, since the latter lacked peroxidase in the Golgi apparatus and did not contain peroxidase-positive granules (Fig. 3). The phagolysosomes of macrophages containing peroxidase activity were heterogeneous in size, shape, and density, and thus could be easily distinguished from the rather uniform granule population in leukopoietic cells. Furthermore, there was no evidence of erythrophagocytosis in the leukopoietic series, including the mature monocytes. Surface features and position. By transmission electron microscopy, the sinusoidal surface of macrophages was irregularly outlined with pseudopods or microvilli-like processes (Fig. 3). These correspond to ruffles or lamellopodia, as seen by scanning electron microscopy (Figs. 8 and 9). Thus the macrophages could be distinguished from the endothelial cells, which exhibited a rather flat surface with occasional fenestrations. Another characteristic feature revealed clearly by the SEM technique was the position of the macrophages in the sinusoids.Whereas one part of their cell body contributed to the fetal sinusoidal lining, another part protruded into the lumen, occasionally contacting the opposite wall (Figs. 8 and 9). Contact with hemopoietic cell types. A close association of macrophages with erythropoietic elements was observed both by TEM (Figs. 1-3) and by SEM techniques (Fig. 9). Thus 75% of all macrophages showed appositions to one to five erythro-
FIGS. 1 AND 2. Light micrographs of two fetal hepatic macrophages (M) showing diffuse peroxidase reaction product in the cytoplasm, which outlines the peroxidase-negative nucleus. No additional staining. Figure 1: Note the contact between this macrophage and at least four erythropoietic cells (E). Seventeen-day-old fetus. x 6800. Figure 2: This macrophage contains a large phagolysosome (arrow) with peroxidase-positive material of probable red blood cell origin. Seventeen-day-old fetus. X 6800. (Note: All figures are from fetal rat livers fixed with 1.5% glutaraldehyde and, with the exception of Figs. 8 and 9, reacted for localization of endogenous peroxidase activity. They were counterstained with lead citrate if not otherwise indicated.) FIG. 3. This electron micrograph illustrates the peroxidase distribution in a resident macrophage and two adjacent erythropoietic elements (ERY). In the macrophage, the reaction product is localized in the NE and ER with no staining in the Golgi apparatus (GO and inset). A typical feature of fetal macrophages is erythrophagocytosis, which is evidenced here by several phagolysosomes(*) containing red cell debris in various stages of degradation. The peroxidase activity in erythropoietic elements is distributed diffusely over the entire cytoplasm, sparing the mitochondria. Seventeen-day-old fetus. ~15,500. Inset, x 44,000.
48
DEVELOPMENTAL BIOLOGY VOLUME66,1978
FIGS.4-6
DEIMANN
AND FAHIMI
Macrophages in Fetal Liver
49
FIG. 7. An immature leukopoietic cell with positive peroxidase reaction in NE and ER and in small cytoplasmic granules (GR). The granule content is homogeneous in some and flocculent in others. Two granules show diffusion of peroxidase into the cytoplasm. Typically, the Golgi apparatus (GO) also contains reaction product (see inset). Eleven-day-old fetus. x 18,500. FIGS. 4 AND 5. Electron micrographs from phagocytosis experiments which show that peroxidase-positive macrophages are capable of avidly taking up injected carbon (Fig. 4) and latex particles (Fig. 5). Figure 4: Carbon particles are on the macrophage surface and in the phagolysosomes. Twenty-day-old fetus. x 34,500. Figure 5: Some of the 0.891 latex particles are still attached to the plasma membrane of the macrophage, while others are seen in phagosomes within the cell body. Seventeen-day-old fetus. x 15,000. FIG. 6. A fetal resident macrophage and a monocyte in close contact. In the macrophage the peroxidase is localized in the NE and ER, whereas in the monocyte it is confined to the cytoplasmic granules (GR). The large phagosome contains an extruded normoblast nucleus. Furthermore, the macrophage shows contacts with erythropoietic elements (ERY). The inset shows a high-power view of the contact between the two cells. By tilting the section, it was revealed that the plasma membranes of the two cells approached each other in several regions, forming close contacts but remaining at least 200 A apart from each other (arrows). No specialized type junctions were seen in such regions. In order to improve the membrane contrast, this section was additionally stained with many1 acetate, which has caused partial masking of the peroxidase staining in the macrophage. Twenty-day-old fetus. x 13,500. Inset x 35,000.
50
DEVELOPMENTALBIOLOGY
VOLUME 66,1978
FIGS. 8 AND 9. SEM micrographs of fetal liver macrophages showing the typical membrane ruffles on their surfaces (arrows). Both cells traverse the lumen of the sinusoids. Figure 8: This macrophage (MAC) with ruffles on its surface (arrows) has engulfed a large particle, probably a red blood cell, which is totally covered by the macrophage membrane (*). Note the endothelial fenestrations (F). The inset shows such fenestrations after specimen tilting. Extracellular fracture; 17-day-old fetus. x 5500. Inset, x 16,000. Figure 9: A macrophage associated with several erythroid elements (ERY). Note the membrane ruffles (arrows). Intracellular fracture; 20-day-old fetus. x 5000.
DEIMANN
Mucrophages
AND FAHIMI
poietic cells, especially normoblasts. Contacts between macrophages and leukopoietic cells, especially monocytes, occurred only rarely (Fig. 6). In order to assess the nature of such contacts, sections were tilted on a goniometer. As shown in Fig. 6 (inset), the two membranes remain approximately 200 A apart and do not form any specialized type junctions. Development. The primordial liver in rat fetus forms between gestation days 10 and 11, and hepatic hemopoiesis begins shortly thereafter (Henneberg, 1937; Elias, 1955). As early as day 11, the macrophages were present in the liver primordium, exhibiting their two typical characteristics: peroxidase activity in the NE and ER and erythrophagocytosis. Compared to immature leukopoietic cells with positive peroxidase activity, the number of macrophages on days 11 and 14 was small, but it increased substantially on days 17 and 20 (Table 2). On the other hand, the immature leukopoietic cells decreased gradually and had disappeared shortly before birth (gestation day 20). This was accompanied by a simultaneous increase in mature promyelocytes and myelocytes. The first mature monocytes with typical peroxidase-positive granules (Fig. 6) were seen in the sinusoids of 17-day-old fetuses. Macrophages in mitosis. Mitotic activity is a frequent finding in the fetal liver and is seen both in parenchymal and in nonparenchymal cells. The dividing fetal macrophages were identified by their two characteristics: erythrophagocytosis (Figs. 10
in Fetal Liver
51
and 12) and positive-peroxidase activity (Fig. 11). Although the number of dividing macrophages was small in untreated animals, it increased after vinblastin treatment.
TABLE
DISCUSSION
The observations reported here reveal a functioning resident macrophage population in fetal rat liver. These macrophages avidly took up injected latex and carbon particles and comprised the only cell type involved in erythrophagocytosis. The peroxidase was localized in the NE and ER of these cells with a negative reaction in the Golgi saccules, a pattern identical to that of Kupffer cells in adult rat liver (Fahimi, 1970; Widmann et al., 1972; Ogowa et al., 1973; Wisse, 1974). Except for two recent brief reports (Deimann and Fahimi, 1977; Naito and Wisse, 1977), endogenous peroxidase has not been demonstrated in fetal rat liver macrophages. The use of the peroxidase technique in this study facilitated the recognition of fetal macrophages and revealed their existence as early as day 11 of gestation, which is shortly after the formation of the hepatic diverticulum and the beginning of the hepatic hemopoiesis. As already mentioned, a typical feature of macrophages in fetal rat liver is erythrophagocytosis, which was found in approximately 80% of these cells. This is in contrast to the normal adult rat liver, where only a small fraction (less than 5%) of the Kupffer cells exhibits this activity (Fahimi, 1970). Furthermore, the macrophages of fetal rat 2
DEVELOPMENT OF THE CELL POPULATIONS WITH ENDOGENOUS
PEROXZDASE
IN NE
AND
EH OF FETAL RAT
LIVER
Cell types
Macrophages Immature promyelocytes cytes Mature promyelocytes
Age of fetuses” (days)
and promono-
11
14
17
20
3 +- zh lOOk
8-c7 132 f 36
79 f 42 32 f 31
154 f 32 0
14+ 11
15 * 13
52 + 35
88 f 40
‘I For each time interval, four fetuses were used. h Mean f SD. Cell counts are expressed as cells per square centimeter.
52
DEVELOPMENTAL BIOLOGY VOLUME66,1978
FIGS. 10-12
DEIMANN AND FAHIMI
liver were frequently associated with erythropoietic elements, forming close contacts with such cells, but with no evidence of endocytosis (Figs. 3 and 6). A similar association between “central macrophages” and erythropoietic elements has been reported in yolk sac (Sorenson, 1961; Fukuda, 1973; Tiedemann, 1977), bone marrow (Bessis, 1956; Sorenson, 1962; Ben-Ishay and Yoffey, 1974), spleen (Orlic et al., 1965; Pictet, 1969), and adult rat liver after phenylhydrazine treatment (Ploemacher and Soest, 1977). Although hemopoiesis in fetal liver has been extensively studied (Grass0 et al., 1962; Zamboni, 1965; Rifiind, 1969; Chui and Russel, 1974; Fukuda, 1974), this is the first description to our knowledge of such an association in fetal rat liver. The functional significance of this phenomenon has been the subject of much speculation by various authors. Thus, it has been proposed that this interrelationship may facilitate iron transfer from macrophages to erythropoietic cells (Bessis et al., 1959; Policard et al., 1959; Sorenson, 1961) or may play a role in the release of erythropoietic cells into the circulation (Ploemacher and Soest, 1977). An interesting speculation is that this association provides the morphological basis for macrophages to influence the differentiation of hemopoietic stem cells. This hypothesis is supported by the recent demonstration of erythropoietin production by resident macrophages in fetal and neonatal liver (Peschle et al., 1975; Gruber et al., 1977). The association of fetal macrophages with erythropoietic elements was also confirmed by the SEM technique (Figs. 8 and
Macrophages in Fetal Liver
53
9). In these preparations, fetal macrophages protruded into the sinusoidal lumen and exhibited ruffles and lamellopodia on their surfaces, the typical features of adult Kupffer cells (Motta and Porter, 1973; Motta, 1975; Nopanitaya and Grisham, 1975; Muto et al., 1977). In contrast, the endothelial cells had a flat appearance, forming fenestrations (Fig. 8 and inset), as already described by Bankston and de Bruyn (1974). Furthermore, the endothelial cells were peroxidase negative and did not show any evidence of phagocytic activity. These morphological, cytochemical, and functional differences between macrophages and endothelial cells are in agreement with our observations in adult rat liver (Widmann et al., 1972) and emphasize that they belong to two distinct cell lines from the beginning of hepatic ontogenesis. The origin of hepatic resident macrophages has been the subject of controversy. Whereas the kinetic and in vitro studies of van Furth et al. (1972, 1976) indicate that tissue macrophages are end cells derived from potent rapidly dividing monoblasts and promonocytes in bone marrow via the peripheral blood monocytes, there is also ample evidence that Kupffer cells are capable of replicating in situ after proper stimulation (North, 1969; Widmann and Fahimi, 1975; Volkmann, 1976). There are several observations reported here that are relevant to this controversy: (a) the time of the first sighting of macrophages and monocytes in fetal liver; (b) the difference in peroxidase localization between these two cell types; and (c) the occurrence of mitosis in fetal liver macrophages.
FIGS. 10-12. Dividing macrophages of fetal rat liver. Figure 10: Light micrograph of a macrophage (M) identified by erythrophagocytosis (arrows) 12 hr after vinblastin injection. Note the rounded cell body and the chromatin clumps, which are easily identified in this toluidin blue-stained section. An adjacent myeloid cell (MY) shows numerous cytoplasmic granules. Seventeen-day-old fetus. x 5000. Figure 11: This TEM micrograph shows a macrophage in prophase from an animal not treated with vinblastin. Note the large phagolysosomes containing granular material (*). Peroxidase reaction product is seen in segments of ER and NE, which appears disrupted. Seventeen-day-old fetus. X 8500. Figure 12: This figure, from a fetal liver 12 hr after vinblastin injection, shows a dividing macrophage with a rounded shape, containing phagocytized material (:). The chromatin clumps are visualized by additional staining with uranyl acetate. Seventeen-day-old fetus. x 15,500.
54
DEVELOPMENTAL BIOLOGY
(a) Macrophages were observed as early as day 11 of gestation in the fetal rat liver, whereas the first mature monocytes with typical peroxidase-positive granules were seen on day 17. This is in agreement with earlier studies, which indicate that monocytes are rare in fetal liver and that macrophages are usually found earlier than monocytes (Zamboni, 1965; Fukuda, 1974). Since at early stages of development (days 11 and 14) bone marrow has not yet assumed any hemopoietic activity, our observations argue against the concept of derivation of tissue macrophages from mature blood monocytes. (b) The peroxidase in macrophages of fetal rat liver is confined to the NE and ER with a negative reaction in the Golgi saccules, whereas in monocytes it is present only in cytoplasmic granules (Figs. 3 and 6, Table 1). This observation is essentially in agreement with numerous reports on the distribution of peroxidase in mononuclear phagocytes of adult animals (Fahimi, 1970; Nichols et al., 1971; Wisse, 1974; Bentfeld et al., 1977). On the other hand, van Furth et al. (1970) and van Furth and Fedorko cytochemical (1976), using a different method, found no evidence of peroxidase activity in the NE and ER of macrophages grown in an in vitro system. Bode1 et al. (1977), however, have reported that some blood monocytes develop peroxidatic activity similar to that of tissue macrophages after surface adherence in vitro. These authors considered this as evidence in favor of transformation of monocytes to macrophages. Recently, van der Rhee et al. (1978) criticized the significance of the in vitro observations of Bode1 et al. (1977) for in viva conditions. They found that in viva adherence of monocytes to subcutaneously implanted melenex plastic did not lead to the development of peroxidatic activity in the NE and ER. The major argument against the transformation of monocytes to macrophages has been the absence of intermediate cell types in uiuo, i.e., cells with positive per-
VOLUME 66.1978
oxidase in NE and ER as well as in cytoplasmic granules. Although such a distribution was found in immature leukopoietic cells (promonocytes and early promyelocytes) in fetal rat liver (Table l), the Golgi apparatuses of these cells were consistently peroxidase positive (Fig. 7). The Golgi apparatus in the intermediate cell type, however, should be peroxidase negative, since both the mature monocyte and the macrophage have a negative Golgi apparatus. The same argument also seems to speak against the transformation of promonocytes to macrophages by degranulation, although such a possibility cannot be completely ruled out on the basis of our cell counts (Table 2). (c) As demonstrated here, fetal hepatic macrophages can undergo mitosis and are thus capable of self-replication. These dividing macrophages were recognized by endogenous peroxidase (Fig. 11) and by evidence of erythrophagocytosis (Figs. 10 and 12). The findings are in agreement with earlier observations in adult rat liver (North, 1969; Widmann and Fahimi, 1975; Volkmann, 1976) and argue strongly against the end cell nature of resident hepatic macrophages. In summary, resident macrophages in fetal rat liver appear to form an independent self-replicating cell line distinct from monocytes, although a derivation from a common precursor cell cannot be ruled out (Cline and Moore, 1972). Mature functional macrophages have also been demonstrated in the yolk sac prior to the formation of liver primordium (Sorenson, 1961; Hoyes, 1969; Fukuda, 1973; Tiedemann, 1977), and it is quite likely that such yolk sac macrophages give rise to resident macrophages of fetal rat liver. We thank Ms. U. Rieth and Ms. H. Marggraf for technical assistance and Ms. G. Folsom for secretarial help. The advice of Dr. K. Tiedemann in embryological aspects of this study is gratefully acknowledged. REFERENCES BANKSTON, P. W., and DE BRUYN, P. P. H. (1974). The permeability to carbon of the sinusoidal lining
DEIMANN
AND FAHIMI
cells of the embryonic rat liver and rat bone marrow. Amer. J. Anat. 141,281-290. BEN-&HAY, Z., and YOFFEY, J. M. (1974). Ultrastructural studies of erythroblastic islands of rat bone marrow. III. Effects of sublethal irradiation. Lab. Invest. 30,320-332. BENTFELD, M. E., NICHOLS, B. A., and BAINTON, D. F. (1977). Ultrastructural localization of peroxidase in leukocytes of rat bone marrow and blood. Anat. Rec. l&7,219-240. BESSIS, M. C., and BRETON-GORIUS, J. (1956). Granules ferrugineux observes dans les cellules de la moelle osseuse et dans les siderocytes. C. R. Acad. Sci. (Paris) 243, 1235-1237. BESSIS, M. C., and BRETON-GORIUS, J. (1959). Ferritin and ferruginous micelles in normal erythroblasts and hypochromic hypersideremic anemias. Blood 14,423-432. BODEL, P. T., NICHOLS, B. A., and BAINTON, D. F. (1977). Appearance of peroxidase reactivity within the rough endoplasmic reticulum of blood monocytes after surface adherence. J. Exp. Med. 145, 264-274. CHUI, D. H. K., and RUSSEL, E. S. (1974). Fetal erythropoiesis in steel mutant mice. I. A morphological study of erythroid cell development in fetal liver. Develop. Biol. 40, 256-269. CLINE, M. J., and MOORE, M. A. S. (1972). Embryonic origin of the mouse macrophage. Blood 39,842~849. DEIMANN, W., and FAHIMI, H. D. (1977). The ontogeny of mononuclear phagocytes in fetal rat liver using endogenous peroxidase as a marker. In “Kupffer Cells and Other Liver Sinusoidal Cells” (E. Wisse and D. L. Knook, eds.). Elsevier/North Holland Biomedical Press, Amsterdam, pp. 487-495. Du BOIS. A. M. (1963). The embryonic liver. In “The Liver, Vol. I” (C. Rouiller, ed.), pp. l-32. Academic Press, New York. ELIAS, H. (1955). Origin and early development of the liver in various vertebrates. Acta Hepatol. 3, l-56. FAHIMI, H. D. (1970). The fine structural localization of endogenous and exogenous peroxidase activity in Kupffer cells of rat liver. J. Cell Biol. 47, 247-262. FUKUDA, T. (1973). Fetal hemopoiesis. I. Electron microscopic studies on human hepatic hemopoiesis. Virchows Arch. B. 16,249-270. GOUD, T. J. L. M., SCHOTTE, C., and VAN FURTH, R. (1975). Identification and characterization of the monoblast in mononuclear phagocyte colonies grown in vitro. J. Exp. Med. 142, 1180-1199. GRASSO, J. A., SWIFT, H., and ACKERMAN, G. A. (1962). Observations on the development of erthrocytes in mammalian fetal liver. J. Cell Biol. 14, 235-254. GRUBER, D. F., ZUCALI, J. R., and MIRAND, E. A. (1977). Identification of erythropoietin producing cells in fetal mouse liver cultures. Exp. Hematol. 5, 392-398. HAGGIS, G. H. (1972). Freeze-fracture for scanning electron microscopy. “Proceedings of the Fifth Eu-
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in Fetal Liver
55
ropean Congress on Electron Microscopy,” pp. 250-255. Institute of Physics, London and Bristol. HENNEBERG, B. (1937). Normentafel zur Entwicklungsgeschichte der Wanderratte. In “Normentafeln ZUIEntwicklungsgeschichte der Wirbeltiere” (F. Keibel, ed.), 15 Erganzungsheft. Verlag von Gustav Fischer, Jena. HOYES, A. D. (1969). The human foetal yolk sac. An ultrastructural study of four specimens. Z. Zellforsch. 99, 469-490. MORRIS, R. R., NICHOLS, B. A., and BAINTON, D. F. (1975). Ultrastructure and peroxidase cytochemistry of normal human leukocytes at birth. Develop. Biol. 44,223-238. MOTTA, P., and PORTER, K. R. (1974). Structure of rat liver sinusoids and associated tissue species as revealed by scanning electron microscopy. Cell Tissue Res. 148, 111-125. MOTTA, P. (1975). A scanning electron microscopic study of the rat liver sinusoid. Endothelial and Kupffer cells. Cell Tissue Res. 164, 371-385. MUTO, M., NISHI, M., and FUJITA, T. (1977). Scanning electron microscopy of human liver sinusoids. Arch. Histol. Japan. 40, 137-151. NAITO, M., and WISSE, E. (1977). Observations on the fine structure and cytochemistry of sinusoidal cells in neonatal and fetal rat liver. In “Kupffer Cells and Other Liver Sinusoidal Cells” (E. Wisse and D. L. Knook, eds.). Elsevier/North Holland Biomedical Press, Amsterdam, pp. 497-505. NICHOLS, B. A., BAINTON, D. F., and FARQUHAR, M. G. (1971). Differentiation of monocytes. Origin, nature, and fate of their azurophil granules. J. Cell Biol. 50,498-515. NOPANITAYA, W., and GRISHAM, J. W. (1975). Scanning electron microscopy of mouse intrahepatic structures. Exp. Mol. Pathol. 23,441-458. NORTH, R. J. (1969). The mitotic potential of fixed phagocytes in the liver as revealed during the development of cellular immunity. J. Exp. Med. 130, 315-326. OGAWA, K., MINASE, T., YOKOYAMA, S., and ONO& T. (1973). An ultrastructural study of peroxidatic and phagocytic activities of two types of sinusoidal lining cells in rat liver. Z’ohohu J. Exp. Med. 111,253-269. ORLIC, D., GORDON, A. S., and RHODIN, J. A. G. (1965). An ultrastructural study of erythropoietin-induced red cell formation in mouse spleen. J. Ultrastruct. Res. 13,516-542. PESCHLE, C., MARONE, G., GENOVESE, A., CILLO, L., MAGLI, C., and CONDORELLI, M. (1975). Erythropoietin production by the liver in fetal-neonatal life. Life&i. 1'7,1325-1330. PICTET, R., ORCI, L., FORSSMANN, W. G., and GIRARDIER, L. (1968). An electron microscope study of the perfusion-fixed spleen. Z. Zellforsch. 96, 400-417. PLOEMACHER, R. E., and VAN SOEST, P. L. (1977). Morphological investigation on phenylhydrazine-induced erythropoiesis in the adult mouse liver. Cell
56
DEVELOPMENTAL BIOLO~3Y
i’lksue Res. 178,435-461. POLICARD, A., and BESSIS, M. (1959). La penetration des substances dans la cellule et ses mecanismes (dialyse, phagocytose, pinocytose, ropheocytose). Rev. Franc. Etud. Clin. Biol. 4,839~345. READE, P. C., and CASLEY-SMITH, J. R. (1965). The functional development of the reticula-endothelial system. II. The histology of blood clearance by the fixed macrophages of foetal rats. Immunology 9, 61-66. RIFKIND, R. A., CHUI, D., and EPLER, H. (1969). An ultrastructural study of early morphogenetic events during the establishment of fetal hepatic erythropoiesis. J. Cell Biol. 40, 343-365. SANDSTRBM, B. (1970). Liver fixation for electron microscopy by means of transparenchymal perfusion with glutaraldehyde. Lab. Invest. 23, 71-73. SORENSON, G. D. (1960). An electron microscopic study of hematopoiesis in the liver of the fetal rabbit. Amer. J. Anat. 106, 27-40. SORENSON, G. D. (1961). An electron microscopic study of hematopoiesis in the yolk sac. Lab. Invest. 10,178-193. SORENSON, G. D. (1962). Electron microscopic observations of bone marrow from patients with sideroblastic anemia. Amer. J. Pathol. 40, 297-304. TIEDEMANN, K. (1977). On the yolk sac of the cat. II. Erythropoietic phases, ultrastructure of aging primitive erythroblasts, and blood vessels. Cell Tissue Res. 183, 71-89. VAN DER RHEE, H. J., BREDEROO, P., and DAEMS, W. Th. (1978). In vivo adherence of monocytes does not lead to the appearance of peroxidatic activity in the
VOLUME 66,1978
rough endoplasmic reticulum. Cell Biol. Int. Rep. 2, 99-102. VAN FURTH, R., COHN, Z. A., HIRSCH, J. G., HUMPHREY, J. H., SPECTOR, W. G., and LANGVOORT, H. L. (1972). The mononuclear phagocyte system: A new classification of macrophages, monocytes, and their precursor cells. Bull. WHO 46, 845-852. VAN FLJRTH, R. (1976). Origin and kinetics of mononuclear phagocytes. Ann. N.Y. Acad. Sci. 278, 161-175. VAN FURTH, R., and FEDORKO, M. E. (1976). Ultrastructure of mouse mononuclear phagocytes in bone marrow colonies grown in vitro. Lab. Invest. 34, 440-450. VOLKMANN, A. (1976). Disparity in origin of mononuclear phagocyte populations. J. Reticuloendothel. Sot. 19, 249-268. WIDMANN, J. J., COTRAN, R. S., and FAHIMI, H. D. (1972). Mononuclear phagocytes (Kupffer cells) and endothelial cells. Identification of two functional cell types in rat liver sinusoids by endogenous peroxidase activity. J. Cell Biol. 52, 159-170. WIDMANN, J. J., and FAHIMI, H. D. (1975). Proliferation of mononuclear phagocytes (Kupffer cells) and endothelial cells in regenerating rat liver. Amer. J. Pathol. 80.349-366. WISSE, E. (1974). Observations on the tine structure and peroxidase cytochemistry of normal rat liver Kupffer cells. J. Ultrastruct. Res. 46, 393-426. ZAMBONI, L. (1965). Electron microscopic studies of blood embryogenesis in humans. I. The ultrastructure of the fetal liver. J. Ultrastruct. Res. 12, 509-524.
Peroxidase
BIOLOGY
66,43-56
Cytochemistry
(1978)
and Ultrastructure of Resident Macrophages in Fetal Rat Liver’ A Developmental
Study2
WINFRIED DEIMANN AND H. DARIUSH FAHIMI~ Department of Anatomy, Division II, University of Heidelberg, 6900 Heidelberg, Federal Republic of Germany Received March 15, 1978; accepted in revised form April 20, 1978 The peroxidase cytochemistry and the ultrastructural characteristics of resident macrophages in fetal rat liver have been investigated. Livers of lo-, ll-, 14-, 17-, and 20-day-old fetuses were fixed by immersion or perfusion, incubated for peroxidase, and processed for transmission electron microscopy. Some 17- and 20-day-old fetuses were injected prior to sacrifice with carbon or 0% pm latex particles through the umbilical vein. Some livers were additionally processed for scanning electron microscopy @EM). The endogenous peroxidase was present in the nuclear envelope (NE) and endoplasmic reticulum (ER) of fetal macrophages with a negative reaction in the Golgi apparatus, a distribution pattern identical to that in Kupffer cells of adult rat liver. Such peroxidase-positive cells avidly took up the injected latex and carbon particles and were the only cell type in fetal liver involved in erythrophagocytosis. Furthermore, they were associated with erythropoietic elements, forming close contacts with such cells, especially normoblasts. The peroxidase pattern in leukopoietic cells differed at all stages of maturation from that in macrophages. By SEM the macrophages exhibited ruffles and lamellopodia on their surfaces and protruded often into the lumen of fetal sinusoids. Macrophages in fetal liver underwent mitotic divisions. The macrophages were first seen on gestation day 11, whereas the first mature monocytes were found on gestation day 17. These observations suggest that resident macrophages in fetal rat liver form a self-replicating cell line independent of the monocytopoietic series, although they may both arise from a common precursor cell.
of monocytes in this tissue. The existence of macrophages in fetal mammalian liver has been established by their ultrastructural characteristics and evidence of erythrophagocytosis (Sorenson, 1960; Reade and Casley-Smith, 1964; Zamboni, 1964; Rifkind, 1969; Chui and Russel, 1974; Fukuda, 1974). The endogenous peroxidase has proven to be a useful cytochemical marker for mononuclear phagocytes (Kupffer cells) in adult rat liver (Fahimi, 1970; Widmann et al., 1972; Ogawa et al., 1973; Wisse, 1974). Furthermore, since this enzyme is a sensitive indicator of monocytopoietic maturation (Nichols et al., 1971; Morris et al., 1975; Bentfeld et al., 1977), it may provide insight into the interrelationships of monocytes and their precursors with resident macrophages.
INTRODUCTION
Fetal liver as a hemopoietic organ provides a unique model for the in uivo study of the interrelationships of various blood cells. The mature blood monocytes are generally considered to be the precursors of tissue macrophages (van Furth et al., 1972). Accordingly, the macrophages in fetal liver should also arise from the transformation ’ This study is dedicated to Dr. E. H. Kass on the occasion of his sixtieth birthday. ’ Supported by National Institutes of Health Grant NS 08533 and by Sonderforschungsbereich 90 (CARVAS) from Deutsche Forschungsgemeinschaft, Bad Godesberg, Federal Republic of Germany. 3 Address alI correspondence to: Professor H. D. Fahimi, Anatomisches Institut II der Universitlit, 6900 Heidelberg, Im Neuenheimer Feld 307, Federal Republic of Germany. 43
0012-1606/78/0661-0043$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
44
DEVELOPMENTALBIOLOGY
In the present study we have applied the peroxidase technique to the fetal rat liver and have studied the development of resident macrophages and their relationships with fetal hemopoietic cells from day 11 to day 20 of gestation. In addition, the phagocytic properties of fetal macrophages were assessed by injection of tracer particles and their surfaae characteristics and position in sinusoids by scanning electron microscopic techniques. MATERIALS
AND
METHODS
Animals. Adult Sprague/Dawley rats (Zentrale Versuchstieranlage, Heidelberg University) were kept on a regular laboratory diet and water ad libitum and were used for mating in all our experiments. On the morning after the overnight housing of males and females, vaginal smears were examined; females positive for sperm were caged separately, and the day was designated as day 0 of gestation. On gestation days 10, 11, 14, 17, and 20, the abdomen was opened under light ether anesthesia and the uterine vessels were clamped. Immediately thereafter, one to two fetuses were removed from the uterus and submitted to fixation. Fixation. All material was fixed for 5 min at room temperature with 1.5% purified glutaraldehyde (Serva, Heidelberg) in 0.15 MNa-cacodylate buffer (pH 7.2) containing 0.05% CaC12. Two separate fixation procedures were used: (a) Immersion fixation for lo-, ll-, and 14-day-old fetuses. As the liver primordium of lo- and 11-day-old fetuses could not be visualized with certainty under the dissecting microscope, the entire fetus, including its intact membranes, was immersed in the fixative. It was important not to detach the fetal membranes from the fetus, because the liver primordium was easily removed with these membranes. In 14-day-old fetuses, only the abdominal cavity was immersed in the same fixative. (b) Perfusion fixation for 17- and 20-day-old fetuses. Under a dissecting microscope, the liver was perfused either through the um-
VOLUME 66,1978
bilical vein or by transparenchymal sion (Sandstrom, 1970).
perfu-
Peroxidase cytochemistry and processing for transmission electron microscopy (TEM). After fixation, tissues were rinsed several times with 0.15 M Na-cacodylate buffer. The 50+m sections were prepared using a Smith-Farquhar tissue sectioner (TC-2-Sorvall, Norwalk, Conn.) and incubated at room temperature. The incubation medium contained 0.1% 3,3’-diaminobenzidine (DAB) in 0.1 M ‘Iris-HCl buffer, pH 7.2, and 0.01% H202. The sections were first preincubated in the absence of Hz02 for 60 min, followed by incubation in the complete medium for another 60 min. Subsequently, the specimens were postfixed with 2% aqueous osmium tetroxide for 90 min, dehydrated in graded ethanol solutions, and embedded in Epon 812. Two-micrometer sections, unstained or stained with toluidine blue, were examined by light microscopy. In the cases of the lo- and 11-day-old fetuses, only those blocks containing identifiable liver tissue were selected and trimmed for ultramicrotomy. Ultrathin sections were contrasted with lead citrate, with or without uranyl acetate, and examined in a Philips EM 301 transmission electron microscope. Cytochemical controls were performed by the incubation of sections in the absence of HzOz or, in some cases, of DAB. Injected compounds. (a) Ten minutes prior to fixation, some 17- and 20-day-old fetuses were injected with 0.02 ml of 0.8~pm latex particles (Serva, Heidelberg) or colloidal carbon (Giinter Wagner, Hannover) into the umbilical vein. (b) Twelve hours prior to sacrifice, some adult rats pregnant for 16.5 days received an intraperitoneal injection of 1 mg/lOO g vinblastine (Velban, Eli Lilly Corp., Indianapolis) to arrest the dividing cells in mitosis. Cell counts. Cells with a peroxidase-positive nuclear envelope (NE) and endoplasmic reticulum (ER) were counted in a transmission electron microscope. The sections were mounted on 300-mesh copper
DEIMANN
AND FAHIMI
grids and counterstained with lead citrate only, to avoid masking the peroxidase reaction by uranyl acetate. Only cells with sectioned nuclei entirely within the grid square were counted. The counts are expressed as cells per square centimeter. Processing for scanning electron microscopy (SEM). Small cubes of liver tissue of 17- and 20-day-old fetuses were sampled for SEM. After perfusion fixation, they were immersed for an additional 2 hr in the same fixative, postfixed for 3 hr with OsO1, and dehydrated in graded ethanol solutions. Intracellular fractures were obtained according to Haggis (1972) and extracellular fractures by breaking the tissue with two forceps after critical-point drying. The latter was achieved by exchanging 100% ethanol with CO,. All specimens were sputtered with gold and examined in a SEM 500 Philips scanning electron microscope at 25 kV. RESULTS
Our observations deal mainly with the resident macrophages of fetal rat liver, which exhibited phagocytosis and the characteristic peroxidase distribution pattern. The hepatic organogenesis is not treated here, since it has already been described extensively [for a review, see Du Bois (1963)]. Light microscopy. The staining for peroxidase appeared as a diffuse brownish precipitate distributed throughout the entire cytoplasm of the macrophages, but sparing the nucleus (Figs. 1 and 2). Most of the macrophages contained phagosomes with peroxidase-positive material of probable red blood cell origin. An additional feature was their close association with erythropoietic elements, which exhibited a strong reaction due to their hemoglobin content. Furthermore, there were leukopoietic elements in fetal rat liver with strong peroxidase-positive granules (Fig. 10).
Peroxidase cytochemistry and ultrastructure. The fetal hepatic macrophages exhibited
the same ultrastructural
and cy-
Macrophages in Fetal Liver
45
tochemical features as described previously for the adult rat liver Kupffer cells (Fahimi, 1970; Widmann et al., 1972; Ogawa et al., 1973; Wisse, 1974). The peroxidase activity was confined to the NE and ER, whereas the Golgi apparatus, including Golgi saccules and vesicles, was consistently peroxidase negative (Fig. 3, Table 1). Another prominent feature found in SO-85% of the fetal macrophages was the evidence of erythrophagocytosis. Peroxidase-positive erythropoietic cell residues at all stages of degradation were found in phagosomes and phagolysosomes, which varied widely in size, density, and shape (Fig. 3). Some macrophages occasionally contained whole normoblast nuclei (Fig. 6). The 0.8-pm latex particles injected into the umbilical veins of the 17- and 20-dayold fetuses were taken up exclusively by peroxidase-positive macrophages (Fig. 5). Discharge of peroxidase into the latex-containing phagolysosomes was not observed. Similarly, injected carbon was found only in the macrophages (Fig. 4). Peroxidatic activity in other fetal liver cells. The liver parenchymal cells and typical wall-forming endothelial cells were consistently peroxidase negative. On the other hand, evidence of peroxidase activity was observed in the erythropoietic and leukopoietic elements. The erythropoietic cell series contained reaction product diffusely throughout the cytoplasm, but sparing the cytoplasmic organelles (Fig. 3). Although this pattern of localization did not change, the intensity of the peroxidase reaction increased with the maturation of the erythropoietic cell series. The peroxidase-positive leukopoietic cells comprised the myeloid and the monocytic series (Table 1). Whereas the immature cells (promyelocytes and promonocytes) exhibited peroxidase activity in the NE, ER, Golgi apparatus, and all cytoplasmic granules (Fig. 7), the more mature cells (myelocytes and monocytes) showed no activity in their secretory apparatuses, but contained only peroxidase in some of their
46
DEVELOPMENTAL BIOLOGY VOLUME66,1978
FIGS.1-3
DEIMANN TABLE
AND FAHIMI
1
PEROXIDASE DISTRIBUTION PATTERN IN RESIDENT MACROPHAGES AND MONOCYTOPOIETIC AND GRANULOPOIETIC CELL SERIES IN FETAL RAT LIVER Cell types
Resident macrophages Promonocytes” Monocytes Promyelocytes” Myelocytes Granulocytes
Peroxidase
activity
NE and ER
Golgi
Granules
+ + -
+ -
+ -
+ -
Oh + +,--’ + + +,--’
” In spite of identical peroxidase distributions promonocytes and mature promyelocytes can be distinguished from one another, as the latter exhibit a larger number of granules and a focally dilated ER. Young promyelocytes are similar to promonocytes: Both cell types contain only a small number of peroxidase-positive granules (see also Fig. 7). ’ In macrophages of fetal rat liver, peroxidase-positive granules similar to “azurophil” granules of leukopoietic cells are absent (0). “Mature monocytes and granulocytes contain a peroxidase-positive (+) as well as a negative (-) granule population.
granules (Fig. 6). Although the various cell types of the myeloid and monocytic cell series could be easily identified on the basis of peroxidase cytochemistry, promonocytes and young promyelocytes were difficult to distinguish from each other. Both cell types showed an identical peroxidase distribution, i.e., reaction product in the NE, ER, and Golgi apparatus and in a few granules (Fig. 7). This pattern differed distinctly, however, from the peroxidase distribution
Macrophages
in Fetal Liver
47
in macrophages, since the latter lacked peroxidase in the Golgi apparatus and did not contain peroxidase-positive granules (Fig. 3). The phagolysosomes of macrophages containing peroxidase activity were heterogeneous in size, shape, and density, and thus could be easily distinguished from the rather uniform granule population in leukopoietic cells. Furthermore, there was no evidence of erythrophagocytosis in the leukopoietic series, including the mature monocytes. Surface features and position. By transmission electron microscopy, the sinusoidal surface of macrophages was irregularly outlined with pseudopods or microvilli-like processes (Fig. 3). These correspond to ruffles or lamellopodia, as seen by scanning electron microscopy (Figs. 8 and 9). Thus the macrophages could be distinguished from the endothelial cells, which exhibited a rather flat surface with occasional fenestrations. Another characteristic feature revealed clearly by the SEM technique was the position of the macrophages in the sinusoids.Whereas one part of their cell body contributed to the fetal sinusoidal lining, another part protruded into the lumen, occasionally contacting the opposite wall (Figs. 8 and 9). Contact with hemopoietic cell types. A close association of macrophages with erythropoietic elements was observed both by TEM (Figs. 1-3) and by SEM techniques (Fig. 9). Thus 75% of all macrophages showed appositions to one to five erythro-
FIGS. 1 AND 2. Light micrographs of two fetal hepatic macrophages (M) showing diffuse peroxidase reaction product in the cytoplasm, which outlines the peroxidase-negative nucleus. No additional staining. Figure 1: Note the contact between this macrophage and at least four erythropoietic cells (E). Seventeen-day-old fetus. x 6800. Figure 2: This macrophage contains a large phagolysosome (arrow) with peroxidase-positive material of probable red blood cell origin. Seventeen-day-old fetus. X 6800. (Note: All figures are from fetal rat livers fixed with 1.5% glutaraldehyde and, with the exception of Figs. 8 and 9, reacted for localization of endogenous peroxidase activity. They were counterstained with lead citrate if not otherwise indicated.) FIG. 3. This electron micrograph illustrates the peroxidase distribution in a resident macrophage and two adjacent erythropoietic elements (ERY). In the macrophage, the reaction product is localized in the NE and ER with no staining in the Golgi apparatus (GO and inset). A typical feature of fetal macrophages is erythrophagocytosis, which is evidenced here by several phagolysosomes(*) containing red cell debris in various stages of degradation. The peroxidase activity in erythropoietic elements is distributed diffusely over the entire cytoplasm, sparing the mitochondria. Seventeen-day-old fetus. ~15,500. Inset, x 44,000.
48
DEVELOPMENTAL BIOLOGY VOLUME66,1978
FIGS.4-6
DEIMANN
AND FAHIMI
Macrophages in Fetal Liver
49
FIG. 7. An immature leukopoietic cell with positive peroxidase reaction in NE and ER and in small cytoplasmic granules (GR). The granule content is homogeneous in some and flocculent in others. Two granules show diffusion of peroxidase into the cytoplasm. Typically, the Golgi apparatus (GO) also contains reaction product (see inset). Eleven-day-old fetus. x 18,500. FIGS. 4 AND 5. Electron micrographs from phagocytosis experiments which show that peroxidase-positive macrophages are capable of avidly taking up injected carbon (Fig. 4) and latex particles (Fig. 5). Figure 4: Carbon particles are on the macrophage surface and in the phagolysosomes. Twenty-day-old fetus. x 34,500. Figure 5: Some of the 0.891 latex particles are still attached to the plasma membrane of the macrophage, while others are seen in phagosomes within the cell body. Seventeen-day-old fetus. x 15,000. FIG. 6. A fetal resident macrophage and a monocyte in close contact. In the macrophage the peroxidase is localized in the NE and ER, whereas in the monocyte it is confined to the cytoplasmic granules (GR). The large phagosome contains an extruded normoblast nucleus. Furthermore, the macrophage shows contacts with erythropoietic elements (ERY). The inset shows a high-power view of the contact between the two cells. By tilting the section, it was revealed that the plasma membranes of the two cells approached each other in several regions, forming close contacts but remaining at least 200 A apart from each other (arrows). No specialized type junctions were seen in such regions. In order to improve the membrane contrast, this section was additionally stained with many1 acetate, which has caused partial masking of the peroxidase staining in the macrophage. Twenty-day-old fetus. x 13,500. Inset x 35,000.
50
DEVELOPMENTALBIOLOGY
VOLUME 66,1978
FIGS. 8 AND 9. SEM micrographs of fetal liver macrophages showing the typical membrane ruffles on their surfaces (arrows). Both cells traverse the lumen of the sinusoids. Figure 8: This macrophage (MAC) with ruffles on its surface (arrows) has engulfed a large particle, probably a red blood cell, which is totally covered by the macrophage membrane (*). Note the endothelial fenestrations (F). The inset shows such fenestrations after specimen tilting. Extracellular fracture; 17-day-old fetus. x 5500. Inset, x 16,000. Figure 9: A macrophage associated with several erythroid elements (ERY). Note the membrane ruffles (arrows). Intracellular fracture; 20-day-old fetus. x 5000.
DEIMANN
Mucrophages
AND FAHIMI
poietic cells, especially normoblasts. Contacts between macrophages and leukopoietic cells, especially monocytes, occurred only rarely (Fig. 6). In order to assess the nature of such contacts, sections were tilted on a goniometer. As shown in Fig. 6 (inset), the two membranes remain approximately 200 A apart and do not form any specialized type junctions. Development. The primordial liver in rat fetus forms between gestation days 10 and 11, and hepatic hemopoiesis begins shortly thereafter (Henneberg, 1937; Elias, 1955). As early as day 11, the macrophages were present in the liver primordium, exhibiting their two typical characteristics: peroxidase activity in the NE and ER and erythrophagocytosis. Compared to immature leukopoietic cells with positive peroxidase activity, the number of macrophages on days 11 and 14 was small, but it increased substantially on days 17 and 20 (Table 2). On the other hand, the immature leukopoietic cells decreased gradually and had disappeared shortly before birth (gestation day 20). This was accompanied by a simultaneous increase in mature promyelocytes and myelocytes. The first mature monocytes with typical peroxidase-positive granules (Fig. 6) were seen in the sinusoids of 17-day-old fetuses. Macrophages in mitosis. Mitotic activity is a frequent finding in the fetal liver and is seen both in parenchymal and in nonparenchymal cells. The dividing fetal macrophages were identified by their two characteristics: erythrophagocytosis (Figs. 10
in Fetal Liver
51
and 12) and positive-peroxidase activity (Fig. 11). Although the number of dividing macrophages was small in untreated animals, it increased after vinblastin treatment.
TABLE
DISCUSSION
The observations reported here reveal a functioning resident macrophage population in fetal rat liver. These macrophages avidly took up injected latex and carbon particles and comprised the only cell type involved in erythrophagocytosis. The peroxidase was localized in the NE and ER of these cells with a negative reaction in the Golgi saccules, a pattern identical to that of Kupffer cells in adult rat liver (Fahimi, 1970; Widmann et al., 1972; Ogowa et al., 1973; Wisse, 1974). Except for two recent brief reports (Deimann and Fahimi, 1977; Naito and Wisse, 1977), endogenous peroxidase has not been demonstrated in fetal rat liver macrophages. The use of the peroxidase technique in this study facilitated the recognition of fetal macrophages and revealed their existence as early as day 11 of gestation, which is shortly after the formation of the hepatic diverticulum and the beginning of the hepatic hemopoiesis. As already mentioned, a typical feature of macrophages in fetal rat liver is erythrophagocytosis, which was found in approximately 80% of these cells. This is in contrast to the normal adult rat liver, where only a small fraction (less than 5%) of the Kupffer cells exhibits this activity (Fahimi, 1970). Furthermore, the macrophages of fetal rat 2
DEVELOPMENT OF THE CELL POPULATIONS WITH ENDOGENOUS
PEROXZDASE
IN NE
AND
EH OF FETAL RAT
LIVER
Cell types
Macrophages Immature promyelocytes cytes Mature promyelocytes
Age of fetuses” (days)
and promono-
11
14
17
20
3 +- zh lOOk
8-c7 132 f 36
79 f 42 32 f 31
154 f 32 0
14+ 11
15 * 13
52 + 35
88 f 40
‘I For each time interval, four fetuses were used. h Mean f SD. Cell counts are expressed as cells per square centimeter.
52
DEVELOPMENTAL BIOLOGY VOLUME66,1978
FIGS. 10-12
DEIMANN AND FAHIMI
liver were frequently associated with erythropoietic elements, forming close contacts with such cells, but with no evidence of endocytosis (Figs. 3 and 6). A similar association between “central macrophages” and erythropoietic elements has been reported in yolk sac (Sorenson, 1961; Fukuda, 1973; Tiedemann, 1977), bone marrow (Bessis, 1956; Sorenson, 1962; Ben-Ishay and Yoffey, 1974), spleen (Orlic et al., 1965; Pictet, 1969), and adult rat liver after phenylhydrazine treatment (Ploemacher and Soest, 1977). Although hemopoiesis in fetal liver has been extensively studied (Grass0 et al., 1962; Zamboni, 1965; Rifiind, 1969; Chui and Russel, 1974; Fukuda, 1974), this is the first description to our knowledge of such an association in fetal rat liver. The functional significance of this phenomenon has been the subject of much speculation by various authors. Thus, it has been proposed that this interrelationship may facilitate iron transfer from macrophages to erythropoietic cells (Bessis et al., 1959; Policard et al., 1959; Sorenson, 1961) or may play a role in the release of erythropoietic cells into the circulation (Ploemacher and Soest, 1977). An interesting speculation is that this association provides the morphological basis for macrophages to influence the differentiation of hemopoietic stem cells. This hypothesis is supported by the recent demonstration of erythropoietin production by resident macrophages in fetal and neonatal liver (Peschle et al., 1975; Gruber et al., 1977). The association of fetal macrophages with erythropoietic elements was also confirmed by the SEM technique (Figs. 8 and
Macrophages in Fetal Liver
53
9). In these preparations, fetal macrophages protruded into the sinusoidal lumen and exhibited ruffles and lamellopodia on their surfaces, the typical features of adult Kupffer cells (Motta and Porter, 1973; Motta, 1975; Nopanitaya and Grisham, 1975; Muto et al., 1977). In contrast, the endothelial cells had a flat appearance, forming fenestrations (Fig. 8 and inset), as already described by Bankston and de Bruyn (1974). Furthermore, the endothelial cells were peroxidase negative and did not show any evidence of phagocytic activity. These morphological, cytochemical, and functional differences between macrophages and endothelial cells are in agreement with our observations in adult rat liver (Widmann et al., 1972) and emphasize that they belong to two distinct cell lines from the beginning of hepatic ontogenesis. The origin of hepatic resident macrophages has been the subject of controversy. Whereas the kinetic and in vitro studies of van Furth et al. (1972, 1976) indicate that tissue macrophages are end cells derived from potent rapidly dividing monoblasts and promonocytes in bone marrow via the peripheral blood monocytes, there is also ample evidence that Kupffer cells are capable of replicating in situ after proper stimulation (North, 1969; Widmann and Fahimi, 1975; Volkmann, 1976). There are several observations reported here that are relevant to this controversy: (a) the time of the first sighting of macrophages and monocytes in fetal liver; (b) the difference in peroxidase localization between these two cell types; and (c) the occurrence of mitosis in fetal liver macrophages.
FIGS. 10-12. Dividing macrophages of fetal rat liver. Figure 10: Light micrograph of a macrophage (M) identified by erythrophagocytosis (arrows) 12 hr after vinblastin injection. Note the rounded cell body and the chromatin clumps, which are easily identified in this toluidin blue-stained section. An adjacent myeloid cell (MY) shows numerous cytoplasmic granules. Seventeen-day-old fetus. x 5000. Figure 11: This TEM micrograph shows a macrophage in prophase from an animal not treated with vinblastin. Note the large phagolysosomes containing granular material (*). Peroxidase reaction product is seen in segments of ER and NE, which appears disrupted. Seventeen-day-old fetus. X 8500. Figure 12: This figure, from a fetal liver 12 hr after vinblastin injection, shows a dividing macrophage with a rounded shape, containing phagocytized material (:). The chromatin clumps are visualized by additional staining with uranyl acetate. Seventeen-day-old fetus. x 15,500.
54
DEVELOPMENTAL BIOLOGY
(a) Macrophages were observed as early as day 11 of gestation in the fetal rat liver, whereas the first mature monocytes with typical peroxidase-positive granules were seen on day 17. This is in agreement with earlier studies, which indicate that monocytes are rare in fetal liver and that macrophages are usually found earlier than monocytes (Zamboni, 1965; Fukuda, 1974). Since at early stages of development (days 11 and 14) bone marrow has not yet assumed any hemopoietic activity, our observations argue against the concept of derivation of tissue macrophages from mature blood monocytes. (b) The peroxidase in macrophages of fetal rat liver is confined to the NE and ER with a negative reaction in the Golgi saccules, whereas in monocytes it is present only in cytoplasmic granules (Figs. 3 and 6, Table 1). This observation is essentially in agreement with numerous reports on the distribution of peroxidase in mononuclear phagocytes of adult animals (Fahimi, 1970; Nichols et al., 1971; Wisse, 1974; Bentfeld et al., 1977). On the other hand, van Furth et al. (1970) and van Furth and Fedorko cytochemical (1976), using a different method, found no evidence of peroxidase activity in the NE and ER of macrophages grown in an in vitro system. Bode1 et al. (1977), however, have reported that some blood monocytes develop peroxidatic activity similar to that of tissue macrophages after surface adherence in vitro. These authors considered this as evidence in favor of transformation of monocytes to macrophages. Recently, van der Rhee et al. (1978) criticized the significance of the in vitro observations of Bode1 et al. (1977) for in viva conditions. They found that in viva adherence of monocytes to subcutaneously implanted melenex plastic did not lead to the development of peroxidatic activity in the NE and ER. The major argument against the transformation of monocytes to macrophages has been the absence of intermediate cell types in uiuo, i.e., cells with positive per-
VOLUME 66.1978
oxidase in NE and ER as well as in cytoplasmic granules. Although such a distribution was found in immature leukopoietic cells (promonocytes and early promyelocytes) in fetal rat liver (Table l), the Golgi apparatuses of these cells were consistently peroxidase positive (Fig. 7). The Golgi apparatus in the intermediate cell type, however, should be peroxidase negative, since both the mature monocyte and the macrophage have a negative Golgi apparatus. The same argument also seems to speak against the transformation of promonocytes to macrophages by degranulation, although such a possibility cannot be completely ruled out on the basis of our cell counts (Table 2). (c) As demonstrated here, fetal hepatic macrophages can undergo mitosis and are thus capable of self-replication. These dividing macrophages were recognized by endogenous peroxidase (Fig. 11) and by evidence of erythrophagocytosis (Figs. 10 and 12). The findings are in agreement with earlier observations in adult rat liver (North, 1969; Widmann and Fahimi, 1975; Volkmann, 1976) and argue strongly against the end cell nature of resident hepatic macrophages. In summary, resident macrophages in fetal rat liver appear to form an independent self-replicating cell line distinct from monocytes, although a derivation from a common precursor cell cannot be ruled out (Cline and Moore, 1972). Mature functional macrophages have also been demonstrated in the yolk sac prior to the formation of liver primordium (Sorenson, 1961; Hoyes, 1969; Fukuda, 1973; Tiedemann, 1977), and it is quite likely that such yolk sac macrophages give rise to resident macrophages of fetal rat liver. We thank Ms. U. Rieth and Ms. H. Marggraf for technical assistance and Ms. G. Folsom for secretarial help. The advice of Dr. K. Tiedemann in embryological aspects of this study is gratefully acknowledged. REFERENCES BANKSTON, P. W., and DE BRUYN, P. P. H. (1974). The permeability to carbon of the sinusoidal lining
DEIMANN
AND FAHIMI
cells of the embryonic rat liver and rat bone marrow. Amer. J. Anat. 141,281-290. BEN-&HAY, Z., and YOFFEY, J. M. (1974). Ultrastructural studies of erythroblastic islands of rat bone marrow. III. Effects of sublethal irradiation. Lab. Invest. 30,320-332. BENTFELD, M. E., NICHOLS, B. A., and BAINTON, D. F. (1977). Ultrastructural localization of peroxidase in leukocytes of rat bone marrow and blood. Anat. Rec. l&7,219-240. BESSIS, M. C., and BRETON-GORIUS, J. (1956). Granules ferrugineux observes dans les cellules de la moelle osseuse et dans les siderocytes. C. R. Acad. Sci. (Paris) 243, 1235-1237. BESSIS, M. C., and BRETON-GORIUS, J. (1959). Ferritin and ferruginous micelles in normal erythroblasts and hypochromic hypersideremic anemias. Blood 14,423-432. BODEL, P. T., NICHOLS, B. A., and BAINTON, D. F. (1977). Appearance of peroxidase reactivity within the rough endoplasmic reticulum of blood monocytes after surface adherence. J. Exp. Med. 145, 264-274. CHUI, D. H. K., and RUSSEL, E. S. (1974). Fetal erythropoiesis in steel mutant mice. I. A morphological study of erythroid cell development in fetal liver. Develop. Biol. 40, 256-269. CLINE, M. J., and MOORE, M. A. S. (1972). Embryonic origin of the mouse macrophage. Blood 39,842~849. DEIMANN, W., and FAHIMI, H. D. (1977). The ontogeny of mononuclear phagocytes in fetal rat liver using endogenous peroxidase as a marker. In “Kupffer Cells and Other Liver Sinusoidal Cells” (E. Wisse and D. L. Knook, eds.). Elsevier/North Holland Biomedical Press, Amsterdam, pp. 487-495. Du BOIS. A. M. (1963). The embryonic liver. In “The Liver, Vol. I” (C. Rouiller, ed.), pp. l-32. Academic Press, New York. ELIAS, H. (1955). Origin and early development of the liver in various vertebrates. Acta Hepatol. 3, l-56. FAHIMI, H. D. (1970). The fine structural localization of endogenous and exogenous peroxidase activity in Kupffer cells of rat liver. J. Cell Biol. 47, 247-262. FUKUDA, T. (1973). Fetal hemopoiesis. I. Electron microscopic studies on human hepatic hemopoiesis. Virchows Arch. B. 16,249-270. GOUD, T. J. L. M., SCHOTTE, C., and VAN FURTH, R. (1975). Identification and characterization of the monoblast in mononuclear phagocyte colonies grown in vitro. J. Exp. Med. 142, 1180-1199. GRASSO, J. A., SWIFT, H., and ACKERMAN, G. A. (1962). Observations on the development of erthrocytes in mammalian fetal liver. J. Cell Biol. 14, 235-254. GRUBER, D. F., ZUCALI, J. R., and MIRAND, E. A. (1977). Identification of erythropoietin producing cells in fetal mouse liver cultures. Exp. Hematol. 5, 392-398. HAGGIS, G. H. (1972). Freeze-fracture for scanning electron microscopy. “Proceedings of the Fifth Eu-
Macrophages
in Fetal Liver
55
ropean Congress on Electron Microscopy,” pp. 250-255. Institute of Physics, London and Bristol. HENNEBERG, B. (1937). Normentafel zur Entwicklungsgeschichte der Wanderratte. In “Normentafeln ZUIEntwicklungsgeschichte der Wirbeltiere” (F. Keibel, ed.), 15 Erganzungsheft. Verlag von Gustav Fischer, Jena. HOYES, A. D. (1969). The human foetal yolk sac. An ultrastructural study of four specimens. Z. Zellforsch. 99, 469-490. MORRIS, R. R., NICHOLS, B. A., and BAINTON, D. F. (1975). Ultrastructure and peroxidase cytochemistry of normal human leukocytes at birth. Develop. Biol. 44,223-238. MOTTA, P., and PORTER, K. R. (1974). Structure of rat liver sinusoids and associated tissue species as revealed by scanning electron microscopy. Cell Tissue Res. 148, 111-125. MOTTA, P. (1975). A scanning electron microscopic study of the rat liver sinusoid. Endothelial and Kupffer cells. Cell Tissue Res. 164, 371-385. MUTO, M., NISHI, M., and FUJITA, T. (1977). Scanning electron microscopy of human liver sinusoids. Arch. Histol. Japan. 40, 137-151. NAITO, M., and WISSE, E. (1977). Observations on the fine structure and cytochemistry of sinusoidal cells in neonatal and fetal rat liver. In “Kupffer Cells and Other Liver Sinusoidal Cells” (E. Wisse and D. L. Knook, eds.). Elsevier/North Holland Biomedical Press, Amsterdam, pp. 497-505. NICHOLS, B. A., BAINTON, D. F., and FARQUHAR, M. G. (1971). Differentiation of monocytes. Origin, nature, and fate of their azurophil granules. J. Cell Biol. 50,498-515. NOPANITAYA, W., and GRISHAM, J. W. (1975). Scanning electron microscopy of mouse intrahepatic structures. Exp. Mol. Pathol. 23,441-458. NORTH, R. J. (1969). The mitotic potential of fixed phagocytes in the liver as revealed during the development of cellular immunity. J. Exp. Med. 130, 315-326. OGAWA, K., MINASE, T., YOKOYAMA, S., and ONO& T. (1973). An ultrastructural study of peroxidatic and phagocytic activities of two types of sinusoidal lining cells in rat liver. Z’ohohu J. Exp. Med. 111,253-269. ORLIC, D., GORDON, A. S., and RHODIN, J. A. G. (1965). An ultrastructural study of erythropoietin-induced red cell formation in mouse spleen. J. Ultrastruct. Res. 13,516-542. PESCHLE, C., MARONE, G., GENOVESE, A., CILLO, L., MAGLI, C., and CONDORELLI, M. (1975). Erythropoietin production by the liver in fetal-neonatal life. Life&i. 1'7,1325-1330. PICTET, R., ORCI, L., FORSSMANN, W. G., and GIRARDIER, L. (1968). An electron microscope study of the perfusion-fixed spleen. Z. Zellforsch. 96, 400-417. PLOEMACHER, R. E., and VAN SOEST, P. L. (1977). Morphological investigation on phenylhydrazine-induced erythropoiesis in the adult mouse liver. Cell
56
DEVELOPMENTAL BIOLO~3Y
i’lksue Res. 178,435-461. POLICARD, A., and BESSIS, M. (1959). La penetration des substances dans la cellule et ses mecanismes (dialyse, phagocytose, pinocytose, ropheocytose). Rev. Franc. Etud. Clin. Biol. 4,839~345. READE, P. C., and CASLEY-SMITH, J. R. (1965). The functional development of the reticula-endothelial system. II. The histology of blood clearance by the fixed macrophages of foetal rats. Immunology 9, 61-66. RIFKIND, R. A., CHUI, D., and EPLER, H. (1969). An ultrastructural study of early morphogenetic events during the establishment of fetal hepatic erythropoiesis. J. Cell Biol. 40, 343-365. SANDSTRBM, B. (1970). Liver fixation for electron microscopy by means of transparenchymal perfusion with glutaraldehyde. Lab. Invest. 23, 71-73. SORENSON, G. D. (1960). An electron microscopic study of hematopoiesis in the liver of the fetal rabbit. Amer. J. Anat. 106, 27-40. SORENSON, G. D. (1961). An electron microscopic study of hematopoiesis in the yolk sac. Lab. Invest. 10,178-193. SORENSON, G. D. (1962). Electron microscopic observations of bone marrow from patients with sideroblastic anemia. Amer. J. Pathol. 40, 297-304. TIEDEMANN, K. (1977). On the yolk sac of the cat. II. Erythropoietic phases, ultrastructure of aging primitive erythroblasts, and blood vessels. Cell Tissue Res. 183, 71-89. VAN DER RHEE, H. J., BREDEROO, P., and DAEMS, W. Th. (1978). In vivo adherence of monocytes does not lead to the appearance of peroxidatic activity in the
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rough endoplasmic reticulum. Cell Biol. Int. Rep. 2, 99-102. VAN FURTH, R., COHN, Z. A., HIRSCH, J. G., HUMPHREY, J. H., SPECTOR, W. G., and LANGVOORT, H. L. (1972). The mononuclear phagocyte system: A new classification of macrophages, monocytes, and their precursor cells. Bull. WHO 46, 845-852. VAN FLJRTH, R. (1976). Origin and kinetics of mononuclear phagocytes. Ann. N.Y. Acad. Sci. 278, 161-175. VAN FURTH, R., and FEDORKO, M. E. (1976). Ultrastructure of mouse mononuclear phagocytes in bone marrow colonies grown in vitro. Lab. Invest. 34, 440-450. VOLKMANN, A. (1976). Disparity in origin of mononuclear phagocyte populations. J. Reticuloendothel. Sot. 19, 249-268. WIDMANN, J. J., COTRAN, R. S., and FAHIMI, H. D. (1972). Mononuclear phagocytes (Kupffer cells) and endothelial cells. Identification of two functional cell types in rat liver sinusoids by endogenous peroxidase activity. J. Cell Biol. 52, 159-170. WIDMANN, J. J., and FAHIMI, H. D. (1975). Proliferation of mononuclear phagocytes (Kupffer cells) and endothelial cells in regenerating rat liver. Amer. J. Pathol. 80.349-366. WISSE, E. (1974). Observations on the tine structure and peroxidase cytochemistry of normal rat liver Kupffer cells. J. Ultrastruct. Res. 46, 393-426. ZAMBONI, L. (1965). Electron microscopic studies of blood embryogenesis in humans. I. The ultrastructure of the fetal liver. J. Ultrastruct. Res. 12, 509-524.