Activity of the nucleolar organizers in cultured cells from the bone marrow erythroblastic islets

The erythroblast islands of the bone marrow are the morphofunctional units of erythropoiesis. In this work, the functional state of erythroblast islands' cells of the bone marrow for the first time was defined by estimation of the activity of the nucleolar organizers of erythroid cells in the erythroblast islands cultivated during 24 and 48 hours in presence of various doses of erythropoietin. The findings indicated that an increase in doses of erythropoietin was accompanied by a corresponding increase of the activity of nucleolar organizers in erythrokaryocytes of erythroblast islands. The nucleolar organizers of erythroid cells in cultures of erythroblast islands responded with activation to very small doses of erythropoietin; besides, a proliferative response of erythrokaryocytes was observed after activation of the nucleolar organizers.     

the effect of colonystimulating macrophage factor on activity of the nuclear organisers in central macrophages of the cultures of the bone marrow erythroblastic islands

Erythroblastic islands of the bone marrow are morpho-functional units of erythropoiesis. The functional state of erythroblastic islands' cells of the bone marrow was for the first time defined by estimation of activity of the nuclear organisers of the central macrophages in the erythroblastic islands cultivated for 24 hrs in presence of various doses of the colony-stimulating macrophage factor. The findings indicate that increased doses of the colonystimulating macrophage factor was accompanied by a respective enhancement of the activity of nucleolar organisers in central macrophages of erythroblastic islands.     

Effects of various concentrations of erythropoietin on cultured erythroblast islands

Proliferative effects of increasing concentrations of erythropoietin were studied in vitro. The maximal amount of erythroblastic islands consisting of residual macrophages and erythroid cells was observed with the doses 0.5 to 1.5 ME/ml of erythropoietin. Smaller doses stimulated the reconstruction of erythroblastic islands.     

The content of ribonucleoproteids in the bone marrow erythroblastic islands in various functional states of erythropoiesis

Erythroblastic islands of the bone marrow are morpho-fuctional units of erythropoiesis. The functional state of the erythroblastic islands' erythrokariocytes and central macrophages of the bone marrow was first studied by estimation of the content of ribonucleoproteids in its cells (Brashe reaction). Acute blood loss and posttransfusion polycytemia lead to enhancement of the content of ribonucleoproteids in erythrokariocytes and central macrophages of the bone marrow of the rates.     

Dynamic changes of erythropoietin and tumor necrosis factor-alpha levels in peripheral blood and their correlation with erythropoiesis changes in posttransfusion polycythemia in animals

Posttransfusion polyglobulia reveals elevated level of the tumor necrosis factor-alpha in peripheral blood, suppresses erythropoiesis in bone marrow erythroblastic islands by diminishing the newly forming erythroblastic islands, and suppresses erythropoietin production in rats.     

Microfluorimetric research on the erythroblastic islands and macrophages of the bone marrow]

A microfluorimetric system was used to study rat bone marrow erythroblastic islands (EI) and macrophages. Measurement of fluorescence from acridine-orange-treated cells was performed on the microscope LUMAM-E3 (LOMO, Leningrad) at 530-550 and 630-650 nm. The intensity of fluorescence in 530-550 nm range depended on the intensity of proliferative processes in EI, that at 630-650 nm was associated with activity of lysosomal apparatus of EI macrophages. Erythroblast amplification in EI is parallel both to enhancement of fluorescence intensity at 530-550 nm and 630-650 nm. Intensity of fluorescence of different bone marrow macrophages was evaluated. It is suggested that a part of bone marrow macrophages has high affinity to erythroid tissue.     

Association between leukemic erythroid progenitors and bone marrow macrophages

Previous ultrastructural investigations have shown that the erythroblastic island is composed of erythroblasts at different stages of maturation which are intimately associated with a central macrophage. However, it is still unclear at which stage of erythroid differentiation this interaction occurs, mainly because of the lack of purified populations of normal erythroid progenitors [erythroid colony-forming units (CFU-E) and erythroid burst-forming units (BFU-E)] and early precursor cells (proerythroblasts) and because of our limited knowledge of their ultrastructural characteristics. In the present work we analyzed the ultrastructure of CFU-E enriched from normal human bone marrow by avidin-biotin immune rosetting and leukemic blasts of erythroid origin from two patients. Normal and leukemic CFU-Es were defined as glycophorin A (GPA)-negative blasts, devoid of rhopheocytosis, containing some ferritin molecules, either free in the cytoplasm or associated with theta-granules (theta-Gr) in the Golgi zone. Peroxidase activity was detected in the endoplasmic reticulum of these blasts. A preproerythroblast stage was identified, which corresponded to an intermediate phenotype with few GPA sites and rhopheocytosis. In contrast to hemoglobin synthesis, which was absolutely dependent on the presence of erythropoietin (Epo) during culture for 24 hours, ferritin molecules accumulated in the absence of Epo. Interestingly, leukemic CFU-E-like blasts were always in contact with bone marrow macrophages and adhesion between these cell types resisted mechanical dissociation. This result suggests that erythroid progenitors may be part of the erythroblastic island. The mechanisms involved in erythroblast-macrophage binding are still unknown, but the expression by macrophages and erythroid progenitors of receptors for fibronectin and thrombospondin (TSP), as well as their respective ligands in the case of macrophages, suggests that these molecules could be involved in the formation of the erythroblastic island.     

Kupffer cells support extramedullary erythropoiesis induced by nitrogen-containing bisphosphonate in splenectomized mice

Our previous study indicated that injecting nitrogen-containing bisphosphonate (NBP) induced the site of erythropoiesis to shift from the bone marrow (BM) to the spleen. This was due to the depletion of BM-resident macrophages, which support erythropoiesis. In this study, we examined NBP treatment-induced extramedullary hematopoiesis in splenectomized mice, focusing on hepatic hematopoiesis. NBP-treated mice did not display anemia or significant change in erythropoietin production, while megakaryopoiesis and erythropoiesis were constantly observed in the liver. Erythroblastic islands were detected in the sinusoidal lumen. Kupffer cells expressed VCAM-1 following NBP treatment, which is an important factor for erythroblast differentiation. Cl₂MBP-liposome treatment depleted the erythroblastic islands, and decreased the number of hematopoietic cells in the liver, as determined by colony forming assays. Together, these results indicate that Kupffer cells support erythropoiesis, acting as stromal cells in the liver, and that they might act as a niche for hematopoietic precursor cells in an emergency.     

Origin of the central cells of erythroblastic islands in fetal mouse liver: ultrahistochemical studies of membrane-bound glycoconjugates

 To clarify the origin of the central cells in hepatic erythroblastic islands, glycoconjugates on the surface of cellular constituents in fetal mice liver were ultrahistochemically examined using lectin staining. At 11 days of gestation, the cells derived from mesenchyme in fetal liver, including sinusoidal macrophages, endothelial cells, and erythropoietic cells, bound Griffonia simplicifolia isoagglutinin I-B4 (GS-I-B4), but hepatocytes lacked binding sites for the isolectin. Scavenger macrophages in the hepatic cords at 13 days of gestation and the central cells in the erythroblastic islands at 15 days of gestation also bound GS-I-B4. Hepatocytes, however, exhibited no GS-I-B4 binding site at any gestational day. At 11 days of gestation, none of the cells in fetal liver had binding sites for soybean agglutinin (SBA), but cells derived from mesenchyme acquired these binding sites at 13 days of gestation. The central cells in the erythroblastic islands also bound SBA, but hepatocytes did not bind the lectin at all. The central cells in the erythroblastic islands can be considered to belong to a mesenchymal cell lineage, and primitive sinusoidal macrophages at 11 days of gestation are possible precursors of these central cells. Accepted: 22 January 1997     

Erythroblastic Islands in the Bone Marrow of Patients with Immune-Related Pancytopenia

Background

Immune-related pancytopenia (IRP) is characterized by pancytopenia caused by autoantibody-mediated bone marrow destruction or suppression. The bone marrows of IRP patients have remarkably increased erythroblastic islands (EIs).

Methodology and Principal Findings

We determined the immunoglobulin G (IgG) autoantibodies in some parts of EIs of IRP patients using immunofluorescence to investigate the biological function of EIs with IgG in the pathophysiology of IRP. The dominant class of autoantibodies detected in mononuclear cells was IgG (CD34 IgG, CD15 IgG, and GlycoA IgG), specifically IgG on GlycoA-positive cells (GlycoA IgG). Results show that extravascular hemolysis occurred in IRP through IgG autoantibodies in the EIs. These data included a high percentage of reticulocytes in the peripheral blood, hypererythrocytosis in the bone marrow, and high serum bilirubin. Furthermore, we examined the macrophages in the bone marrow of IRP patients. The results show that the number of activated macrophages relatively increased, and the phagocytic activity of macrophages significantly increased.

Conclusions and Significance

Increased EIs with IgG were the sites of erythroblast phagocytosis by the activated macrophages, rather than erythropoietic niches. The IgG autoantibodies in the EIs possibly functioned as adhesion molecules for a ring of erythroblasts around the macrophages, thereby forming morphologic EIs.     

Effect of acute congestive splenomegaly on erythropoiesis in rats

Spleen veins ligature (SVL) led to acute congestive splenomegaly in rats with subsequent normochromic anaemia disappearing on the 21st day after the SVL. An appreciable depression of the bone marrow erythropoiesis, particularly of the so called "proliferating erythroblastic islets" number, was evident on the 7th and 14th days of post-SVL period. The post-SVL anaemia was also associated with occurrence of the "islets" missing central macrophages and eosinophil-enriched "islets" in bone marrow.     

Isolation and short-term culture of mouse splenic erythroblastic islands

We isolated and cultured erythroblastic islands (EI) from the spleens of phlebotomized mice using a combination of collagenase digestion, unit gravity sedimentation, and Percoll density gradients separation. The isolated EI were composed of surrounding erythroid cells and central stromal macrophages (M phi), which were identified by Forssman antigen. While 60% of the erythroblasts incorporated bromodeoxyuridine, the M phi did not. EI could be maintained on a plastic dish for a short period in the presence of erythropoietin. Two hours later, the central M phi spread well and bound to erythroblasts via cytoplasmic processes. One day later, erythropoietic activity on the M phi surface continued, although their processes had retracted. Some EI showed synchronized expansion of erythroblasts and others showed differentiation to reticulocytes. Two days later, about 50% of the EI still showed erythropoietic activity and most erythroblasts differentiated to the orthochromatic stage. On the other hand, the M phi secreted colony-stimulating activity during the culture. It was infrequently observed that erythroid and myeloid populations simultaneously expanded on a central M phi. These results indicate that this EI culture system is useful for studying interactions between the stomal M phi and hematopoietic cells.     

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.     

The effect of various stage maturity of erythrocytes upon proliferatio rate of erythroid cells in "crown" of erythroblastic islands

Joint cultivation of small amounts of young and mature erythrocytes with bone marrow erythroblastic islands during 24 hours in liquid culture system results in erythropoiesis stimulation due to increase of number of cells in the state of mitosis in island's "crown". Prolonged joint cultivation and/or joint cultivation with plenty of premature and mature erythrocytes leads to inhibition of the erythropoiesis in the state of mitosis in the island's "crown". The maximum of the inhibitory effect was shown by erythrocytes from polycythemia rats.     

Erythropoietic function of the kidneys under conditions of abnormally high oxygen pressure]

Three to five hours after 5-hour exposure of rabbits to high oxygen pressure (2 ata) the erythropoietin proved to disappear from both the arterial and the venous blood plasma of the kidneys. At the same time the blood plasma from the renal vein began to suppress the mitotic activity of erythroblastic cells in the bone marrow culture. These data testify to the fact that under hyperbaric hyperoxia the kidneys secreted the inhibitor of erythropoiesis. No erythropoiesis inhibitor was revealed 24 hours after the hyperoxia.     

The iron islands: Erythroblastic islands and iron metabolism

Background

A healthy human can produce over 1?×?10¹⁵ blood cells throughout their life. This remarkable amount of biomass requires a concomitantly vast amount of iron to generate functional haemoglobin and functional erythrocytes.

Effect of doxorubicin on the functional state of the mononuclear phagocyte system]

The response of the system of mononuclear phagocytes (SMP) to doxorubicin, an antitumor antibiotic, most widely used in oncological care, was studied. It was shown that a single intraperitoneal administration of doxorubicin to CBA mice in the maximum tolerance doses induced suppression of absorptive SMP capacity and increased IL-I secretion by the bone marrow and peritoneal macrophages both in the stimulated and spontaneous tests in early periods after cytostatic administration. There was a significant rise in the ability of SMP bone marrow elements to respond to the macrophage activating factor, as well as an increase in the cytotoxic activity of bone marrow and peritoneal macrophages.     

Erythropoietin and phagocyte activity of central macrophage of the erythroblastic island in rats

Administration of erythropoietin enhanced the intensity of the phagocytosis in the erythroblastic island. The effect was more evident in case of normal erythropoiesis. The phagocyte activation of the central macrophage erythroblastic island was shown to depend on the condition of erythropoiesis in the erythroblastic island crown.     

Morphology of interactions of the haematopoietic microenvironment with the haematopoietic cells in erythroid spleen colonies

The morphology of the interactions of the cells of the haematopoietic microenvironment with haematopoietic cells was studied in exogenous erythroid spleen colonies formed in mice, on the fifth and eighth day after their irradiation with a lethal dose of gamma rays and bone marrow transplantation. The characteristic type of stromal cell interacting with less mature cells of the erythroid series was a dark, branching reticular cell. The typical structural interaction of the reticular cells with erythroblasts was the formation of very long, fine cytoplasmic processes by the reticular cells. The processes were in close contact with the erythroblasts and formed a three-dimensional network stretching long distances from the nucleus of the reticular cells. Other cells of the haematopoietic microenvironment, in contact chiefly with poly- and ortochromic erythroblasts, were macrophages and the two together formed typical erythroblastic islands. In places, the macrophages and erythroblasts formed close, firm contacts by means of their cytoplasmic membranes. These morphological observations support the conception that close functional cooperation exists between the cells of the haematopoietic microenvironment and the haematopoietic cells in differentiation and proliferation processes in the haematopoietic tissue of spleen colonies.     

Absence of erythroblast macrophage protein (Emp) leads to failure of erythroblast nuclear extrusion

In mammals, the functional unit for definitive erythropoiesis is the erythroblastic island, a multicellular structure composed of a central macrophage surrounded by developing erythroblasts. Erythroblast-macrophage interactions play a central role in the terminal maturation of erythroblasts, including enucleation. One possible mediator of this cell-cell interaction is the protein Emp (erythroblast macrophage protein). We used targeted gene inactivation to define the function of Emp during hematopoiesis. Emp null embryos die perinatally and show profound alterations in the hematopoietic system. A dramatic increase in the number of nucleated, immature erythrocytes is seen in the peripheral blood of Emp null fetuses. In the fetal liver virtually no erythroblastic islands are observed, and the number of F4/80-positive macrophages is substantially reduced. Those present lack cytoplasmic projections and are unable to interact with erythroblasts. Interestingly, wild type macrophages can bind Emp-deficient erythroblasts, but these erythroblasts do not extrude their nuclei, suggesting that Emp impacts enucleation in a cell autonomous fashion. Previous studies have implicated the actin cytoskeleton and its reorganization in both erythroblast enucleation as well as in macrophage development. We demonstrate that Emp associates with F-actin and that this interaction is important in the normal distribution of F-actin in both erythroblasts and macrophages. Thus, Emp appears to be required for erythroblast enucleation and in the development of the mature macrophages. The availability of an Emp null model provides a unique experimental system to study the enucleation process and to evaluate the function of macrophages in definitive erythropoiesis.