The liver hemopoietic environment: I. Developing hepatocytes and their role in fetal hemopoiesis

Mouse fetal liver was studied ultrastructurally to identify and characterize the developing hepatic parenchyma or prehepatocyte which may be responsible for producing the liver hemopoietic environment. It was observed that as the liver develops, there is close association of endodermal and mesenchymal cells in the region of the septum transversum. Numerous intercellular adhesions were observed between endodermal cells and mesenchymal cells. Twenty-four after endodermal and mesenchymal cells first intermingle, the liver extravascular space consisted of spherical hemopoietic cells dispersed among a heterogenous population of dark and light cells. The reticulum of prehepatocytes formed a three-dimensional cellular network which structurally supported the hemopoietic cells residing in the liver. By 12 days of gestation, prehepatocytes were a homogenous population of dark, stellate cells joined together by numerous intercellular adhesions. Broad areas of intercellular association were noted between processes and prehepatocytes and hemopoietic cells; however, no intercellular junctions between these two disparate cell populations were observed at this or any stage in development. Characteristics reflecting a cell population capable of synthesis and secretion of proteinaceous substances, namely, dilated Golgi apparati, increased numbers of polyribosomes and profiles of rough endoplasmic reticulum (RER), two types of vacuoles and/or vesicles, and intercellular microvillus-lined spaces, were observed in the prehepatocytes between 12 and 17 days gestation. By day 17 of gestation, glycogen accumulation, biliary channel development, appearance of a subendothelial microvillus surface, nuclear shape and chromatin pattern, and arrangement of cytoplasmic organelles reflected the maturation of prehepatocytes into hepatocytes, the adult liver parenchyma.     

Ultrastructural localization of peroxidase in leukocytes of rat bone marrow and blood

The differentiation of leukocytes in the bone marrow and blood of normal adult male rats was studied by electron microscopy and peroxidase cytochemistry. Tissue samples were fixed in glutaraldehyde, or paraformaldehyde-glutaraldehyde, and incubated in a peroxidase medium containing 3,3′-diaminobenzidine and H₂O₂ at pH 7.6. Mature cells of blood were identified, and then the earlier stages of maturation in bone marrow were analyzed. In immature cells of four cell lines, neutrophils, monocytes, basophils, and eosinophils, peroxidase is synthesized and could be demonstrated in the rough endoplasmic reticulum (RER), Golgi complex, and in cytoplasmic granules. Later in maturation, reaction product for peroxidase could not be found in RER or Golgi complex, indicating that peroxidase synthesis had ceased. In two cell lines, neutrophils and monocytes, peroxidase-negative granules were formed, and the mature cells contained two populations of cytochemically distinct granules. All granules of mature eosinophils were peroxidasepositive. In mature basophils, some granules were clearly peroxidase-positive; others displayed variable density, making interpretation uncertain. Mast cells were never seen in blood, but were abundant in bone marrow; peroxidase was never found in their granules by either electron microscopic cytochemistry or a variety of light microscopic methods. Hence, these cells differ from basophils, not only in morphology but also in the enzyme content of their granules.     

Immunohistochemical assessment of human bone marrow macrophages in hematologic disorders

Changes in bone marrow macrophages may be associated with abnormal hematopoiesis in various hematologic disorders. We immunohistochemically evaluated the density of macrophages in bone marrow trephine biopsies. In reactive erythroid hyperplasia (hemolytic anemia and megaloblastic anemia), the macrophages slightly increased in density, extending their cytoplasmic processes between hematopoietic cells. In erythroid hypoplasia (pure red cell aplasia), they became rounded and frequently had hemosiderin granules. There was no significant difference in the macrophage density in the hematopoietic area between erythroid hyperplasia and hypoplasia. The macrophages increased in density in myeloproliferative disorders (polycythemia vera, chronic myelogenous leukemia and primary thrombocythemia). In myelofibrosis, some macrophages became extremely elongated along the line of the fibroblastic cells. In contrast, in conditions in which myelopoietic activity is considerably impaired (aplastic anemia, acute leukemia and multiple myeloma), they significantly decreased in density. These results suggest that the morphologic change in bone marrow macrophages is associated with erythropoietic activity and that there is a correlation between macrophage density and myelopoietic activity.     

Hemopoiesis recovery of irradiated rats conjugated with normo- and polycythemic animal by aortic anastomoses

The experiment was designed to observe the possible relation between myelopoietic and erythropoietic activities of circulating nucleated cells. Wistar rats were lethally irradiated with 60Co, 100 r once. Two days after irradiation the bone marrow cells had faded completely. At this stage animals were conjugated with normocythemic or polycythemic rats by aortic anastomoses. After conjugation the aplastic bone marrow of the irradiated animal rapidly regained its hemopoietic activity in cases having normocythemic and polycythemic partners. Active erythropoiesis and myelopoiesis were found 96 h after parabiosis in those having normocythemic partners. In animals having polycythemic partners, however, erythropoiesis was successfully suppressed. An increase in lymphoid cell numbers was found in place of decreased erythroid cells, but there was no change in the myeloid cell proliferation rate. No hemopoietic precursor cells or immature cells were found in circulating blood all through the experimental period before and after parabiosis. The data suggest that circulating nucleated cells have marked erythropoietic activity. Erythropoietic cells may be somehow related to lymphoid cells independent of myelopoietic activity.     

Resident sinusoidal macrophages in the liver of the brown bullhead (Ictalurus nebulosus): an ultrastructural, functional and cytochemical study

Ultrastructural, functional, and cytochemical characteristics of resident sinusoidal macrophages (RSM) in brown bullhead (Ictalurus nebulosus) liver were examined. Following perfusion fixation of the hepatic vascular bed, light micrographs revealed RSM that possessed multiple elongate cytoplasmic processes and frequently contained erythrocytes in various stages of degradation. Following brief perfusion fixation, light microscope examination of vibratome sections of bullhead liver reacted for peroxidase revealed intensely positive RSM. By transmission electron microscopy, peroxidase activity was localized to the nuclear envelope and cytoplasmic granules of RSM and in endothelial and perisinusoidal fat-storing cells. In cryostat sections of fresh-frozen liver, glucose-6-phosphate dehydrogenase (G-6-PDH) was uniformly distributed over hepatocytes, whereas intensely positive punctate staining for G-6-PDH was localized over RSM. To test for phagocytosis by RSM, latex beads (0.81 micron) were injected into a tributary of the hepatic portal vein 2 min prior to perfusion fixation. Latex beads appeared either singly or in dense aggregates within RSM. Ultrastructurally, RSM were characterized by an irregularly shaped, eccentrically located nucleus, electron-dense vacuoles, small patches of granular endoplasmic reticulum, a well-developed Golgi apparatus, elongated mitochondria, desmosomes or desmosome-like densities that served as a source of attachment to endothelial cells, and a centriole with radiating microtubules. Invaginations of the plasma membrane (vermiform processes) characteristic of mammalian Kupffer cells were not observed in bullhead RSM. The results indicated a resident cell population of sinusoidal macrophages in the bullhead liver with properties that partially resembled mammalian Kupffer cells. These results are important for the identification of the normal resident cells in the bullhead liver.     

Technique of preparation of hemopoietic cells of human fetal liver for transplantation

The preparation of single cell suspension from human fetal liver is described. The technique is based on pressing the liver through wire mesh followed by repeated aspiration into a syringe through needles of decreasing internal diameter. Subsequently, the cell suspension is depleted of cell debris by a "triple medium sedimentation procedure" without net loss of hemopoietic cells. Following centrifugation and resuspension the preparation is ready for either transplantation or storage. About 2 and 11 X 10(9) cells were obtained per liver depending on the age of fetus in the range of 16 and 24 weeks of gestation in three representative preparations. The cell suspension contained comparable numbers of hemopoietic progenitors to the adult bone marrow suspension as assayed using plasma clot diffusion chamber technique.     

Immunocytochemical localization of parathyroid hormone in bovine parathyroid glands and human parathyroid adenomas.

Light and electron microscopic localization of parathyroid hormone (PTH) in human and bovine parathyroid tissue has been achieved using an indirect peroxidase labeled antibody method. Granular deposition of the reaction product was found throughout the chief cell cytoplasm. There was no nuclear staining. At the ultrastructural level, parathyroid hormone localized by this method appeared to be largely confined to the secretory granules in the cytoplasm of cells. Mitochondria and nuclei were free of reaction product. Aggregated sacs of granular endoplasmic reticulum were minimally reactive, and Golgi apparatuses did not show reaction product.     

Role of hemopoietic growth factors in regeneration of hemopoiesis during etoposide-induced myelosuppression

We studied structural and functional organization of the bone marrow, production of hemopoietic growth factors by hemopoietic cells, and plasma colony-stimulating and erythropoietic activities in CBA/CaLac mice treated with etoposide. The effects of etoposide on cultured hemopoietic and microenvironmental cells were also evaluated. Our results indicate that hemopoietic growth factors secreted by adherent bone marrow cells play the major role in the normalization of hemopoiesis during etoposide-induced myelosuppression.     

Nonparenchymal liver cells in a vertebrate without bile ducts

Summary The nonparenchymal portion of the liver of parasitic adult lampreys (Petromyzon marinus L.) consists of endothelial, Kupffer, fibroblast-like, fat-storing, and granulated cells. The fenestrae of endothelial cells are not organized into sieve plates but are of highly variable size and distribution. The dimension of large molccular size. Small numbers of Kupffer cells possess many features of these cells observed in other vertebrates but they do not have worm-like bodies and endogenous peroxidase activity. They are involved in erythrophagocytosis and perhaps the ingestion of other foreign material but they do not store iron. Fat-storing and fibroblast-like cells share many morphological features and may be different expressions of the same cell type. These perisinusoidal cells are rich in organelles suggesting protein synthesis but the fibroblast-like cells lack fat droplets. A cell with a large Golgi apparatus and associated cytoplasmic granules resembles the pit cell described in the liver of a few other vertebrates. The morphology of nonparenchymal cells of the liver in parasitic adult lampreys does not reflect the absence of bile ducts in this organism.     

The uterine progestational response in cats: Ultrastructural changes during chronic administration of progesterone to estradiol-primed and nonprimed animals

The development of a progestational response in the glandular epithelium of the cat uterus was studied using electron microscopy and special glycogen staining techniques. In ovariectomized and adrenalectomized animals, the epithelial cells were low cuboidal and lacked an active secretory apparatus. Estradiol treatment induced hypertrophy and the formation of secretory cells which contained numerous profiles of RER, an enlarged Golgi apparatus and apical electron-opaque secretory granules. The chronic administration of progesterone to estradiol-primed animals resulted in antagonism of estradiol-induced cytodifferentiation and the development of a progestational response in the glandular epithelium. After 1 day of estradiol plus progesterone treatment fewer secretory granules were observed, the RER was constricted, and the Golgi apparatus appeared less active; after 2 days, very few secretory granules were observed. Coincident with this antiestrogenic action, progesterone induced additional hypertrophy and the differentiation of cytoplasmic organelles associated with glycogen synthesis. Glycogen deposition occurred between days 2 and 4 of estradiol-plus-progesterone treatment. Numerous polyribosomes and circular profiles of membrane appeared to be associated with glycogen deposition. These membranes frequently had glycogen around their perimeter, and sometimes they were found embedded in large glycogen deposits. With continued progesterone treatment, glycogen processing occurred. The RER became extremely dilated, and a few halo granules were observed. Both the dilated RER and the halo granules appeared to be surrounding glycogen, and glycogen was frequently found bound by membrane as well as within the electron-lucent portion of the halo granule. As glycogen processing continued, the glycogen deposits began to disappear, and some cells appeared to die. The remainder differentiated into shorter residual cells which, after 10–14 days, contained numerous halo granules and constricted RER. The chronic administration of progesterone to nonprimed ovariectomized and adrenalectomized animals resulted in a cytodifferentiation sequence similar to that in the estradiol-primed animals, except that the response was delayed and was not as uniform between cells from the same animal. Thus, within each epithelial cell of the endometrial glands of the cat, the principal responses induced by progesterone in both estradiol-primed and nonprimed animals were the synthesis, deposition, and eventual disappearance of glycogen.     

Acute megakaryoblastic leukemia in an infant with a novel t(1;9)(p32;q34)

We report a case of a 14-month-old girl with acute megakaryoblastic leukemia (AMKL). May-Giemsa staining of the bone marrow cells revealed the proliferation of two distinct types of blasts. One type of blasts had cytoplasmic blebs, and the other showed a lymphoblastic morphology without blebs. Both types of blasts were negative for peroxidase and esterase reactions. Electron microscopic platelet peroxidase (PPO) reaction also revealed the presence of two types of blasts. One had irregular-shaped nuclei and positive PPO reaction in the nuclear envelope and rough endoplasmic reticulum but not in the Golgi apparatus. These types of blasts were considered to be megakaryoblasts. The other had an immature phenotype with round nuclei and positive PPO reaction in the nuclear envelope, rough endoplasmic reticulum, and the Golgi apparatus. The origin of this type of blasts could not be defined by their morphology. Surface marker analysis indicated that most of the leukemic cells expressed platelet markers, gpIIb, gpIIb/IIIa, gpIX, and gpIbalpha. Karyotypic analysis of the bone marrow cells of this unique subset of AMKL demonstrated a novel translocation, t(1;9)(p32;q34).     

Reactions of the erythroid hemopoietic stem and their mechanisms during blood loss

We studied changes in the erythroid hemopoietic stem during blood loss of different severity. Stimulation of erythropoiesis during the posthemorrhagic period was related to functional activation of erythroid precursors, which resulted from changes in feeder capacity of cells from the hemopoiesis-inducing microenvironment and erythropoietic activity of the plasma. The development of encephalopathy under conditions of massive blood loss was accompanied by a reduction of erythroid hyperplasia due to a decrease in the number of proliferating erythroid precursor cells, despite high secretory activity of adherent myelokaryocytes, rise in erythropoietic activity of the plasma, increased formation of erythroid hemopoietic islets, and accelerated maturation of hemopoietic cells.__________This revised version was published online in August 2005 with the addition of the issue titleTranslated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 139, No. 1, pp. 32–37, January, 2005     

Morphological and morphometric studies in pancreatic acinar cells of the rat at successive post-mortem intervals. The early appearance of a previously undescribed Golgi complex associated tubular vesicular structure

Structural alterations in rat pancreatic acinar cells were studied in thin section at 0.5, 1, 4, 8, 12, 24 and 48 h post-mortem (PM). Morphometric analyses were performed both by light and electron microscopy, at 0.5 and 1 h PM. The parameters evaluated were: a) nuclear, cytoplasmic and cellular volumes; b) volume density and absolute volume of the rough endoplasmic reticulum (RER), mitochondria, zymogen granules (ZG), Golgi complex and its subcompartments [cisternae, condensing vacuole (CV) and 56-nm diameter vesicles], dense bodies (lysosome-like structures, electron-dense vacuoles and unidentifiable granules) and cytoplasmic matrix; c) surface density, surface/volume ratio and total surface area of the RER, mitochondria, ZG, Golgi cisternae, 56-nm diameter vesicles lying at the rough ER-Golgi interface, CV, and apical and basolateral membranes. Between 0.5 and 48 h, the mitochondria were dilated, junctional complexes were preserved and autophagic vacuoles were rare or absent. Flocculent densities were present in the mitochondria and chromatin condensation was observed at 4 h PM. In thin sections from samples obtained between 0.5 and 12 h, we consistently observed a membrane bounded structure formed by tubules and vesicles, designated as a tubular vesicular structure (TVS). These TVS's were observed at positions corresponding to the 4th Golgi cisterna. Fibrillar aggregates and a reduction in the number of 56-nm vesicles on the cis side of the Golgi were seen. Morphometry revealed a 60-70% reduction in the numerical density of the 56-nm vesicles between zero (control) and 0.5 h PM. These analyses also showed a 70% increase in the total volume and 57% increase in the total membrane surface of the Golgi cisternae in the PM period. The current results suggest that during the early PM (0.5 h) there is transport between Golgi compartments, and the 56-nm diameter vesicles fuse with the cisternae.     

Extramedullary eosinophilopoiesis in the liver ofSchistosoma japonicum-infected mice,with reference to hemopoietic stem cells

Extramedullary hemopoiesis recognized as hemopoietic foci increased in the liver ofSchistosoma japonicum-infected mice in parallel with kinetic changes in periovular granuloma formation. At the peak of the response, about 65% of the hepatic hemopoietic foci were of eosinophil lineage. WhenS. japonicum-infected mice were irradiated, hepatic hemopoietic foci rapidly disappeared within 3 days, whereas inflammatory cells in the periovular granulomas slowly reduced in number. When the number of hemopoietic stem cells in the liver were examined by spleen-colony assay, kinetic changes in the number of hemopoietic stem cells in hepatic nonparenchymal cells paralleled those of hepatic hemopoietic foci. Hemopoietic stem cells were rare in the granuloma cells. These results indicate that in response to the increased demand for eosinophils and other inflammatory cells, the liver acts as an extramedullary hemopoietic organ in which inflammatory cells are generated from hemopoietic stem cells.Abbreviations GM-CSF granulocyte-macrophage colony-stimulating factor - NPC nonparenchymal cell - CFU-S spleen colony forming unit - ECF eosinophil chemotactic factor     

Immunoelectron microscopic demonstration of glycoprotein subunits in the FSH producing pituitary adenomas

Immunoelectron microscopic observations disclosed the presence of FSH alpha, beta subunits in secretory granules and rough endoplasmic reticulum (RER). Golgi saccules were consistently negative. The presence of these subunits in secretory granules which were located in the vicinity of RER or perinuclear spaces (PNS) suggested the possible formation of secretory granules in RER or PNS. The tumor cells showed heterogeneity in size and immunoreactivity of the secretory granules but non-neoplastic cells showed uniformity in these aspects.     

Development of the liver in the chicken embryo. II. Erythropoietic and granulopoietic cells

Hepatic hemopoiesis is apparent in the chicken embryo on day 7 of incubation (Hamburger and Hamilton Stage 30), and a peak in hemopoietic activity occurs on day 14 (Stage 40). During this period, the differentiation of hemopoietic cells was examined by light microscopy and by transmission and scanning electron microscopy. Glycol methacrylate sections were used in lieu of smears to study hemopoietic cells, thus minimizing the problems of cell shrinkage and rupture. The sections were superior to smears for close examination of nuclear and cytoplasmic morphologies and for precise localization of hemopoietic cells to intravascular and extravascular sites. The avian liver is involved directly with erythropoiesis and granulopoiesis only. Erythropoietic cells, occurring in intravascular and extravascular locations, appear throughout the time frame examined. Blood islands with granulopoietic cells were not observed until days 8–9 (Stage 35). Granulopoiesis in the liver produces only eosinophilic leukocytes. Individual granulopoietic cells appear first in the connective tissue sheaths of hepatic vessels, and these cells subsequently congregate into blood islands. Endothelial cells of the sinusoidal linings, through asymmetric divisions, frequently release daughter cells into the circulation, and Kupffer cells are actively engaged in phagocytosis of erythrocytes. From a comparative standpoint the elements deemed critical to hemopoiesis in the mammalian liver–prehepatocyte population, hepatic vasculature, and compartments for stem cell differentiation–may not hold the same importance in the bird, owing to an inordinate reliance on intravascular hemopoiesis in this vertebrate class. © 1993 Wiley-Liss, Inc.     

Differentiation to myeloid cells of lymphoblastoid cells established from myelomonocytic leukemia

Summary It has been previously reported that a lymphoblastoid cell line (Mono-1) established from the peripheral blood of a patient with acute myelomonocytic leukemia (AMML) consisted of reticulum cells, possessing properties that were more characteristic of monocytes and macrophages than those which are traditionally attributed to lymphocytes. These cells (Mono-1-207) exhibit myeloid cell properties when cultured in arginine-deficient medium and after treatment with DNA synthetic inhibitors. A comparison has been made with lymphoblastoid cell lines derived from other types of disease.During culture in arginine-deficient condition, decreased DNA synthesis is accompanied by the appearance, at 48 h, of perinuclear pink cytoplasmic blushes; nuclear lobulation had developed by about the fifth day. At 12–14 days, the cytoplasmic granules developed from blushes could not be distinguished from azurophilic granules. Electron microscopy indicated that these granules were due to the development of lysosomes in which acid phosphatase was strongly present but which lacked any peroxidase activity. Mono-1-207 also has phagocytic activity. It is considered that the induction of differentiation of these cells in response to DNA synthesis by arginine-deficiency is related to possession of the characteristics described: they are hemopoietic precursor cells, and differentiate to myeloid cells.This work was supported by a cancer research grant 51101 and 52004 from Kawasaki Medical School.     

Fine structural and cytochemical identification of microperoxisomes in developing human erythrocytic cells.

An alkaline diaminobenzidine (DAB) medium has been used to identify peroxidase activity in small granules (0.09 to 0.2 mu in diameter) present in all forms of maturing erythrocytic cells with the exception of erythrocytes. These granules, which were more frequent in proerythroblasts (from two to seven by thin section), were distinct from pleomorphic granules present in the close proximity to the Golgi apparatus. They were also distinct from ferritin molecules which were seen as aggregates in siderosomes of polychromatophilic erythroblasts. They often appeared in close association with the smooth membrane of the nuclear envelope. Optimal conditions for the visualization of these granules by incubation in alkaline DAB were obtained when the peroxidase activity of hemoglobin was reduced by addition of low concentrations of potassium cyanide. Lack of hydrogen peroxide in the incubation media completely inhibited the staining reaction of hemoglobin, while the positive reaction persisted in the granules. Aminotriazole in the incubation media prevented the staining of these organelles. These findings suggest that small granules seen in maturing erythroblasts contain catalase and that they correspond to microperoxisomes described in other tissues. The mechanism of their disappearance during reticulocyte maturation is unknown. The relationship between particulate catalase of erythroblasts and soluble erythrocytic catalase has not been elucidated.     

Ultrastructure of the exocrine pancreas in the snake Waglerophis merremii (Wagler).

Three types of serozymogenic cells were found in the secretory compartment of the snake exocrine pancreas. Type I cell was the most common and presented a well-developed granular endoplasmic reticulum arranged in cisternal and vesicular forms. The cisternal form was located predominantly in the basal regions of the cell and the vesicular form was found in the supranuclear regions of the cell next to a prominent Golgi complex. Mature secretory granules were seen at the cell apex. The cytoplasmic matrix of the Type II cells was electron dense but had only poorly-developed organelles. Secretory granules were rare. The cytoplasmic matrix of the Type III cells was electron lucent and the granular endoplasmic reticulum in the cisternal form was located predominantly in the supranuclear region, whereas the vesicular form was randomly distributed throughout the cytoplasm. The nucleus appeared pale due to the fine dispersion of the chromatin; the nucleolus was prominent. Centroacinar and intermediate cells were also examined.     

Morphological studies on the synthesis of secretory granules in convoluted tubules of mouse submandibular gland

The synthesis of zymogen-like secretory granules in convoluted tubules of mouse submandibular gland (SMG) was investigated by histometry, light microscopy and electron microscopy. In normal males secretory granules in the SMG increased greatly from 25 days after birth and reached a maximum level 50 days after birth. Castration of adult male mice markedly decreased the level, but it was completely restored by testosterone administration. A parallel was found between change in the granule level and the amount of rough endoplasmic reticulum (RER) in the convoluted tubular cells during development or after various treatments. Development of the Golgi apparatus was also observed in the cells when the granules increased. Both the increase in the granules and in the RER induced by testosterone were prevented by actinomycin D or puromycin. These results indicate that the granule contents are synthesized on the RER under the control of testosterone, and then condensed in the Golgi apparatus.