The transmural migration and release of blood cells in acute myelogenous leukemia.

The transmural passage of malignant blood cells from the extravascular parenchyma into sinusoidal lumen has been studied in the bone marrow of rats with myelogenous leukemia. The Shay myelogenous leukemia was chosen as a model system because an increased bone marrow cellularity is, in this leukemia, usually accompanied by an increase in circulating myeloid cells. By means of light microscopy, transmission electron microscopy (TEM) and scanning electron microscopy (SEM) it was found that the sinusoidal endothelial lining of the bone marrow remains intact and continuous even in advanced stages of the disease. SEM shows that the malignant myeloblast-like cell enters the sinusoidal lumen by means of a temporary migration pore, which appears only during the transmural passage of the cell. Certain nondegenerative changes in the sinusoidal blood vessels are associated with the myelogenous leukemia. The normal radial alignment of sinusoids about the central sinusoid is changed into a tortuous pattern, and intraluminal cytoplasmic bridges which impede the blood flow are formed by the endothelial cells.     

Embolization by sinusoidal lining cells obstructs the microcirculation in rat sinusoidal obstruction syndrome

Mechanisms leading to the obstruction of the microcirculation in sinusoidal obstruction syndrome (SOS) have been unclear. Because this occurs at the onset of disease, this is a potential key target for therapeutic intervention. Rats were treated with monocrotaline with or without continuous intraportal infusion of glutathione and were studied at 0.5, 1, 2, 4, 6, and 10 days after monocrotaline treatment with the use of in vivo microscopy and transmission electron microscopy. Sinusoidal perfusion decreased from days 1 through 10 with a nadir on day 4. At 12 h, numerous swollen sinusoidal endothelial cells (SECs) were observed. Subsequently, red blood cells penetrated into the space of Disse through gaps between and through swollen SEC and dissected the sinusoidal lining away from the parenchymal cells. Sinusoidal blood flow was obstructed by an embolism of aggregates of sinusoidal lining cells, red blood cells, and adherent monocytes. All changes were prevented by glutathione infusion, notably the initial swelling of SEC. SOS is initiated by changes in SEC. Microcirculatory obstruction is due to dissection of the sinusoidal lining, followed by embolization of the sinusoid by sinusoidal lining cells, compounded by aggregates of monocytes adherent in the sinusoids. Glutathione prevents SOS by preserving an intact sinusoidal barrier.     

In situ fluorescence labeling of sheep lung microvascular endothelium

Summary Endothelial cells are intimately involved in a variety of biological processes such as inflammatory disorders, wound healing, and tumor invasion. The finding of endothelial heterogeneity in various tissues has led to major efforts to isolate and culture microvascular endothelial cells in human and animal tissue. In this report we have used phosphatidyl ethanolamine (PE)-labeled liposomes to fluorescently label the sheep lung microvasculature in situ. Using normotensive perfusion pressure, the PE-labeled liposomes did not extravasate into extravascular lung tissue. Mechanical and enzymatic digestion of the lung tissue demonstrated that the PE-labeled liposomes provided a stable label of the vascular lining cells during ex vivo processing. After digestion, the overwhelming majority of the fluorescent label appeared in cellular aggregates. Approximately 80% of these cells demonstrated an in vitro phenotype consistent with microvascular endothelium. A novel monoclonal antibody selective for sheep endothelial cells was developed to confirm the presence of lung endothelium in the fluorescently labeled cellular aggregates. We conclude that in situ fluorescence labeling of vascular lining cells provides an anatomic marker for relevant vascular lining cells and an opportunity to study these cells in vitro.     

Morphology and vascular anatomy of the accessory respiratory organs of the air-breathing climbing perch, Anabas testudineus (Bloch)

The vascular organization and endothelial cell specialization of the air-breathing organs of Anabas testudineus were examined by light and scanning electron microscopy of fixed tissue and vascular corrosion replicas. The vessels supplying blood to the lining of paired suprabranchial chambers and the plicated labyrinthine organs within the chambers are tripartite, having a median artery and paired, lateral veins. Hundreds of respiratory islets, the functional units of gas exchange, cover the surfaces of both the chamber and labyrinthine organ. A median islet artery supplies the central aspect of each islet and gives rise to numerous short arterioles from which the transverse channels are formed. Transverse channels are parallel capillary-sized vessels that extend in two rows away from the medial arterioles and drain laterally into one of two lateral islet veins. Basally situated single rows of endothelial cells lining the transverse channels form thick, evaginated, tongue-like cytoplasmic processes that project freely into the lumen from the tissue side of the channel. Other thin, septate, cytoplasmic extensions of the same cells form valve-like septa that extend across the channel. Both the septa and tongue-like processes appear to direct the red blood cells to the epithelial side of the channel and thus decrease the diffusion distance between the air and red cell. A large sinusoidal space lies under the transverse channels and may support the channels and even elevate them during increased oxygen demand. The epithelium covering the transverse channels is smooth, which enhances air convection and minimizes unstirred layer effects. The epithelium between the channels contains microvilli that may serve to trap bacteria or particulates and to humidify the air chambers.     

Fine structure of the bone marrow of the chicken and pigeon

The structure of bone marrow from chickens and pigeons was studied with light and electron microscopy. Erythropoiesis occurs in the lumen of the medullary sinuses. Immature erythroid cells appear to adhere to the sinus wall and may thus be prevented from entering the peripheral circulation. The wall of the medullary sinuses is formed by elongated lining cells, lacking a basement membrane, which are continous except at sites where blood cells are passing through them. When viewed with the electron microscope, developing heterophil myelocytes, which occur only in the extravascular spaces, possess two populations of granules; one type is globular in content, the other is fibrillar in content. The globular type predominates during all stages of development and appears to be the specific granule. Specific granules originate from material which is formed in the Golgi complex, pinches off, and accumulates in expanded vesicles. The origin of the material in the fibrillar granules was not determined. Like the globular granules of heterophil leucocytes, granules of eosinophil leucocytes arise from material which is formed in the Golgi complex.     

The regenerating liver: a site of erythropoiesis in the adult Long-Evans rat

Erythropoiesis, which is primarily hepatic in the rat during fetal and early neonatal life, shifts almost entirely to the bone marrow in the neonatal-adolescent stage of development. In the adult, extramedullary erythropoiesis has been demonstrated in the liver and spleen under certain pathological conditions when bone marrow red cell production is insufficient. In the present study, erythropoietic foci have been found in young-adult rat liver regenerating 24-72 hr after subtotal hepatectomy. This erythropoiesis is both extravascular and sinusoidal, with some erythroblastic islands noted. The centrolobular hepatic area contains the highest concentration of erythroblasts. Peripheral blood reticulocytosis coincides with the appearance of these cells and this is considered as an indicator of effective erythropoiesis. Liver regenerating after partial hepatectomy produces significant quantities of erythropoietin (Ep) in response to hypoxia. Subtotal hepatectomy may confer upon the adult liver the ability to revert to a fetal-like condition both in its ability to produce Ep and to function as a hematopoietic inductive microenvironment for erythropoiesis.     

Ultrastructural localization of peanut lectin binding to extravascular white blood cells in the bone marrow of embryonic chicks

Summary Labelling by the galactose-specific lectin peanut agglutinin was studied in bone marrow of the embryonic chick at the electron-microscopic level by use of both a gold-conjugated lectin and an indirect, ferritin-conjugated, biotinylated lectin. Cell surface labelling is exclusively restricted to developing and mature heterophilic granulocytes, monocyte/macrophages, mast cells/basophils, all of which appear to develop and reside in the extravascular spaces of the bone marrow. Resident small lymphocytes, which comprise a minor portion of the cell population, are also labelled. Erythroid cells and thrombocytic cells, which develop inside venous sinusoidal vessels, display no labelling. The latter cells, like extravascular leukocytes, contain surface galactosyl residues located in subterminal positions on cell surfaces, since they are labelled by the galactose-specific Ricinus communis agglutinin-I. It is postulated that terminal galactosyl residues might be involved in interactions between the surfaces of extravascular leukocytes and extracellular matrix and/or stromal cell surfaces.     

Immunocytochemical localization of the receptor for asialoglycoprotein in rat liver cells

We used high-resolution immunocytochemistry on ultrathin frozen sections labeled with colloidal gold to study the subcellular distribution of the asialoglycoprotein receptor in rat liver. The receptor was localized along the entire hepatocyte plasma membrane, including the bile capillary membrane, but was scarce intracellularly. Sinusoidal lining (Kupffer) cells and blood cells showed no immunoreactivity. In liver cells of rats injected with 1 to 100 micrograms of asialoorosomucoid (ASOR) 2-15 min before tissue fixation, endocytotic internalization of receptors at the blood front was conspicuous. At all times in this interval, receptor was present in approximately 100-nm vesicles and larger vacuoles adjacent to the sinusoidal plasma membrane. No other significant intracellular receptor was noted during the 15-min exposure to ASOR; in particular, lysosomes and Golgi complex were not labeled. Our observations, in combination with data from the literature which demonstrate that, under these conditions, the ligand is transferred further to the Golgi complex-lysosome region, suggest that the receptor and ligand are dissociated in the vicinity of the plasma membrane, after which the receptor rapidly returns to the cell surface.     

Evidence of species differences in the ultrastructure of the Hepatic sinusoid

Summary Hepatic sinusoids in the calf have been studied by light and electron microscopy. The endothelial lining is shown to be continuous and to be associated with a prominent basement membrane. The perisinusoidal space (of Disse) contains collagen fibrils and is otherwise nearly filled with hepatic cell microvilli. Perisinusoidal cells resembling fibroblasts or reticular cells in morphology and distribution are present in moderate numbers. Hepatic sinusoids in the rat were examined for purposes of comparison. In the rat the sinusoidal lining is discontinuous and there is no identifiable basement membrane. The perisinusoidal space is larger than in the calf and contains more sparsely distributed collagen fibrils. Perisinusoidal cells have not been identified. It is suggested that the difference in structure is not artifactitious but represents a species variation. This species difference seems important to record since observations on the rat and other small laboratory animals provide the basis for most of the current generalizations about the organization of the mammalian liver sinusoid. The functional significance of the two types of sinusoidal arrangement is not clear.A preliminary report of this work was presented at the VII th International Congress of Anatomists (Wood 1960).Supported in part by Grant H-2698 from the National Institutes of Health, U. S. Public Health Service.     

Differentiation of hepatic sinusoid vessel cells in rats in the embryonal and postnatal periods of ontogenesis

Differentiation of the hepatic sinusoidal capillary cells has been studied in the embryonal and postnatal periods of the rat ontogenesis. In the embryonal liver cellular elements are differentiated divergently with formation of endotheliocytes, Kupffer cells and fat-accumulating cells. Differentiation of endotheliocytes is accompanied with their flattening, formation of fenestrae and pores in peripheral processes, increasing amount of micro- and macropinocytic vesicles, and also development of intercellular contacts, presented at first as simple, and then as tight junctions. Formation of intercellular clefts occurs by means of parting of simple junctions; this ensures migration of blood cells across the endothelial lining. Kupffer cells develop out of immature macrophagal elements of the perisinusoidal space. During differentiation they migrate into the endothelial lining. Simultaneously in their cytoplasm lysosomes accumulate and acid phosphatase activity increases. The fat-accumulating cells are formed out of poorly differentiating fibroblast-like elements of the perisinusoidal space. Their differentiation is accompanied with a progressive development of the granular endoplasmic reticulum and with accumulation of reticular fibers in the perisinusoidal space, and in cytoplasm--lipid inclusions.     

Anti-CD95-induced lethality requires radioresistant Fcgamma RII+ cells. A novel mechanism for fulminant hepatic failure

The Jo2 anti-mouse CD95 monoclonal antibody induces lethality in mice characterized by hepatocyte death and liver hemorrhage. Mice bearing a defect in Fas expression or in the Fas-mediated apoptotic pathway are resistant to Jo2. Here we show that FcgammaRII knockout mice or mice with monoclonal antibody-blocked FcgammaRII are also resistant to Jo2. The critical FcgammaRII(+) cells are radioresistant and could not be reconstituted with splenic cells. Death of sinusoidal lining cells and destruction of sinusoids were observed, consistent with the characteristic liver hemorrhage and the selective FcgammaRII expression in sinusoidal lining cells but not hepatocytes. Hemorrhage developed coincident with hepatocyte death and the sharp rise of serum alanine aminotransferase and alanine aminotransferase. Invariably, moribund mice showed severe liver hemorrhage and destruction of sinusoids. The data demonstrate a novel mechanism by which the destruction of liver sinusoids, induced by the Jo2-mediated co-engagement of Fas and FcgammaRII, leads to severe hemorrhage and lethal fulminant hepatitis.     

High endothelial venules of the lymph nodes express Fas ligand.

Fas (CD95, APO-1) is widely expressed on lymphatic cells, and by interacting with its natural ligand (Fas-L), Fas induces apoptosis through a complex caspase cascade. In this study we sought to survey Fas-L expression in vascular and sinusoidal structures of human reactive lymph nodes. Immunohistochemical Fas-L expression was present in all paracortical high endothelial venules (HEVs), in cells lining the marginal sinus wall, and in a few lymphocytes, but only occasionally in non-HEV vascular endothelium. In the paracortical zone over 60% of all vessels and all paracortical HEVs showed Fas-L expression, whereas in the medullary zone less than 10% of the blood vessels were stained with Fas-L. Normal vessels outside lymph nodes mostly showed no Fas-L expression. We show that in human reactive lymph nodes Fas-L expression is predominantly present in HEVs. Because the circulating lymphocytes gain entry to nodal parenchyma by transendothelial migration through HEVs, the suggested physiological importance of Fas-L expression in these vessels lies in the regulation of lymphocyte access to lymph node parenchyma by possibly inducing Fas/Fas-L mediated apoptosis of activated Fas-expressing lymphoid cells. The Fas-L expressing cells in the marginal sinus might have a similar function for cells accessing the node in afferent lymph.     

Ultrastructural changes in the rat liver during Euro-Collins storage, compared with hypothermic in vitro ischemia

The ultrastructural alterations in liver tissue induced by in vitro ischemia at 4 degrees C under conditions commonly used for transplantation (Euro-Collins perfused and stored liver tissue) have been compared with changes due to hypothermic in vitro ischemia in non-perfused liver. It was found that the process of cell deterioration in non-perfused liver occurred very slowly; signs of irreversible damage appeared in sinusoidal lining cells before hepatocytes (after 24 and 96 h, respectively). Liver perfused with, and stored in Euro-Collins solution showed acceleration of the ischemical damage in both types of cell (irreversible damage to sinusoidal lining cells after 12 h and to hepatocytes after 52 h), compared with non-perfused liver. These findings indicate that the safe period for storage of rat liver in Euro-Collins before damage to the microcirculatory system is less than 12 h. It might also be questioned whether Euro-Collins treatment is the optimal procedure for tissue preservation before liver transplantation.     

The Relationship between Fenestrations,Sieve Plates and Rafts in Liver Sinusoidal Endothelial Cells

Fenestrations are transcellular pores in endothelial cells that facilitate transfer of substrates between blood and the extravascular compartment. In order to understand the regulation and formation of fenestrations, the relationship between membrane rafts and fenestrations was investigated in liver sinusoidal endothelial cells where fenestrations are grouped into sieve plates. Three dimensional structured illumination microscopy, scanning electron microscopy, internal reflectance fluorescence microscopy and two-photon fluorescence microscopy were used to study liver sinusoidal endothelial cells isolated from mice. There was an inverse distribution between sieve plates and membrane rafts visualized by structured illumination microscopy and the fluorescent raft stain, Bodipy FL C5 ganglioside GM1. 7-ketocholesterol and/or cytochalasin D increased both fenestrations and lipid-disordered membrane, while Triton X-100 decreased both fenestrations and lipid-disordered membrane. The effects of cytochalasin D on fenestrations were abrogated by co-administration of Triton X-100, suggesting that actin disruption increases fenestrations by its effects on membrane rafts. Vascular endothelial growth factor (VEGF) depleted lipid-ordered membrane and increased fenestrations. The results are consistent with a sieve-raft interaction, where fenestrations form in non-raft lipid-disordered regions of endothelial cells once the membrane-stabilizing effects of actin cytoskeleton and membrane rafts are diminished.     

Thrombomodulin is found on endothelium of arteries, veins, capillaries, and lymphatics, and on syncytiotrophoblast of human placenta

We have used antibodies to human thrombomodulin isolated from placenta to investigate the distribution of this cofactor for protein C activation in human tissues. Thrombomodulin was found on endothelial cells of arteries, veins, capillaries, and lymphatics by immunocytochemical staining using an avidin-biotin peroxidase method. Thrombomodulin was not detected on sinusoidal lining cells of liver or on postcapillary high-endothelial venules of lymph node, although the latter contained another endothelial antigen, von Willebrand factor. Other cells noted to contain thrombomodulin antigen are those of the syncytiotrophoblast in placenta. The thrombomodulin in syncytiotrophoblast was primarily on the plasma membrane surface that forms the maternal blood sinus. Syncytiotrophoblast also stained with antibodies to von Willebrand factor, which implies that these cells have multiple endothelial functions. Thrombomodulin antigen was found in all organs studied, with the notable exception of brain.     

Stabilins are expressed in bone marrow sinusoidal endothelial cells and mediate scavenging and cell adhesive functions

Bone marrow sinusoidal endothelial cells have a specific function as a site of transmigration of hematopoietic stem and progenitor cells and mature blood cells between bone marrow and blood stream. However, the specific characteristics of bone marrow sinusoidal endothelial cells are still largely unclear. We here report that these cells express stabilin-1 and stabilin-2, which in liver sinusoidal endothelial cells have been identified as endocytic scavenger receptors for several ligands, including SPARC and hyaluronan. We show here that intravenously injected formaldehyde-treated serum albumin, advanced glycation end-products, and collagen I α-chains were taken up by bone marrow sinusoidal endothelial cells, showing that these cells have a scavenging function and thereby may modulate bone marrow vascular stem cell niches. Importantly, we show hyaluronan mediated adhesion of hematopoietic stem and progenitor cells to stabilin-2-transfected cells, suggesting that stabilin-2 contributes to adhesion and homing of circulating stem and progenitor cells to bone marrow.     

Selective malformation of the splenic white pulp border in L1-deficient mice

Lymphocytes enter the splenic white pulp by crossing the poorly characterized boundary of the marginal sinus. In this study, we describe the importance of L1, an adhesion molecule of the Ig superfamily, for marginal sinus integrity. We find that germline insertional mutation of L1 is associated with a selective malformation of the splenic marginal sinus. Other splenic structures remain intact. Immunofluorescence analysis of the extracellular framework of the spleen, using an Ab to laminin, reveals that L1 knockout mice have an irregularly shaped, discontinuous white pulp margin. Electron microscopic analysis shows that it is associated with bizarrely shaped marginal sinus lining cells at the periphery of the white pulp. These abnormalities correlate with the localization of L1 in normal mice in that L1 is normally expressed on marginal sinus lining cells at the white pulp border. These L1-immunopositive lining cells coexpress high levels of mucosal addressin cell adhesion molecule-1 and vimentin, indicating that they are of fibroblastic lineage and express a well-characterized addressin. Our findings are the first to implicate L1 in splenic lymphoid architectural development. Moreover, these findings help define the poorly characterized sinusoidal boundary across which mononuclear cells cross to enter the splenic white pulp.     

Immunohistochemical localization of NAD glycohydrolase in human and rabbit tissues

NAD glycohydrolase (NADase) is present in many organisms from bacteria to mammals. In any given organism, this enzyme is ubiquitous in many tissues. However, its precise localization and its physiological significance have not been defined. We have determined the distribution of NADase in normal human and rabbit tissues by immunoblotting and immunohistochemistry, using a polyclonal antibody raised in goats. Immunoblot analyses revealed that NADase was highly expressed in the heart, lung, stomach, and liver tissues of the rabbit. From immunohistochemical studies of NADase, high concentrations in both human and rabbit tissues were found in hepatocytes and sinusoidal lining cells, sinus histiocytes of the lymph node, spleen and thymus, glomerular capillary endothelial cells of the kidney, cardiac muscle, endothelium of blood vessles, and erythrocytes.     

Human ferritin: effects of antigen source and fixation on leucocyte staining by immunoperoxidase technique

The localization of ferritin was studied in peripheral blood cells and variously fixed tissues with the antibodies against ferritins isolated from human heart and spleen. The unlabelled antibody enzyme method (PAP) was used to detect the binding sites of antibodies. In peripheral blood cell smears both antisera gave rise to strong staining of polymorphonuclear (PMN) cell cytoplasm, whereas the monocytes stained relatively weakly. There were no staining differences between the two antisera. In human spleen sections the spleen ferritin antiserum stained the PMN cells and sinusoidal lining cells, whereas the heart ferritin antiserum stained only PMN cells. Neither of the two antisera stained monocytes in the spleen sections. This finding was observed in specimens fixed in Bouin's fixative, Baker's fixative and neutral formalin. However, the immunoreactivity of ferritin was totally destroyed by some other fixatives (Carnoy's fixative, formol sucrose and glutaraldehyde). These results suggest that ferritin is more readily released from monocytes than from PMN cells, and that mature spleen macrophages contain antigenic determinants of ferritin that are recognized only by anti-spleen ferritin antiserum.     

Changes in sialic acid content of the plasma membrane in hepatocellular proliferation

The content of sialic acid bound to the sinusoidal region of plasma membrane during the prereplicative phase after the intravenous injection of a solution containing triiodothyronine, amino acids, glucagon and heparin (T.A.G.H. solution) has been measured. The results obtained show that an important decrease in sialic acid content is produced as it occurs in the hepatic cells of hepatectomized animals. In order to know if sialidase activity is involved in the decrease of sialic acid content during liver regeneration, the activity of sinusoidal plasma membrane sialidases during the prereplicative phase after the partial hepatectomy has been studied. No modifications of sialidase activity were detected during this period of time indicating that this decrease in sialic acid content has to be produced by other mechanisms such as diminution in the synthesis of precursor molecules. On the other hand due to the importance of Ca2+-calmodulin complexes in the activation of the hepatic cell proliferation the possible implication of this complex on the loss of sialic acid, observing the effect of trifluoperazine (inhibitor of Ca2+-calmodulin complexes) during the prereplicative phase of liver regeneration has been studied. The results show a delay in the decrease of the amount of sugar studied from 10 to 12 hours compared to the results obtained with the hepatectomized rats that have not received trifluoperazine.