Observations on the ultrastructure of sinusoids and reticular cells in human bone marrow

Summary The ultrastructural characteristics of sinusoids in human bone marrow were generally similar to those previously reported in the rat. However, human sinusoids differed from rat sinusoids in displaying frequent tight junctions between adjacent overlapping or interdigitating endothelial cells. In both species, large areas of the sinusoidal walls were devoid of much subendothelial connective tissue and of an outer adventitial cell layer. The marrow sinusoids of humans resembled those of rabbits and rats in that processes of macrophage cytoplasm protruded through endothelial cells into the sinusoidal lumen; thin veils derived from such processes were apposed over the inner surfaces of some endothelial cells. Features observed in pathological human marrow not noted in rodent marrow are the phagocytosis of extruded erythroblast nuclei and of abnormal erythroblasts by perisinusoidal adventitial cells (reticular cells) and the presence of large secondary lysosomes and siderosomes in sinusoidal endothelial cells. When compared with mouse bone marrow, human bone marrow contained very few non-phagocytic reticular cells that were unassociated with sinusoids and other blood vessels. There were no absolute ultrastructural differences between perisinusoidal adventitial cells, intersinusoidal non-phagocytic reticular cells and macrophages lacking phagosomes or containing only a few small phagosomes. Consequently, on some occasions, these cell types could not be reliably identified from the study of a single thin section. Extracellular reticulin fibres were found adjacent both to non-phagocytic reticular cells and to macrophages. Mitosis was observed in macrophages, albeit very rarely.     

Observations on the ultrastructure of sinusoids and reticular cells in human bone marrow.

The ultrastructural characteristics of sinusoids in human bone marrow were generally similar to those previously reported in the rat. However, human sinusoids differed from rat sinusoids in displaying frequent tight junctions between adjacent overlapping or interdigitating endothelial cells. In both species, large areas of the sinusoidal walls were devoid of much subendothelial connective tissue and of an outer adventitial cell layer. The marrow sinusoids of humans resembled those of rabbits and rats in that processes of macrophage cytoplasm protruded through endothelial cells into the sinusoidal lumen; thin veils derived from such processes were apposed over the inner surfaces of some endothelial cells. Features observed in pathological human marrow not noted in rodent marrow are the phagocytosis of extruded erythroblast nuclei and of abnormal erythroblasts by perisinusoidal adventitial cells (reticular cells) and the presence of large secondary lysosomes and siderosomes in sinusoidal endothelial cells. When compared with mouse bone marrow, human bone marrow contained very few non-phagocytic reticular cells that were unassociated with sinusoids and other blood vessels. There were no absolute ultrastructural differences between perisinusoidal adventitial cells, intersinusoidal non-phagocytic reticular cells and macrophages lacking phagosomes or containing only a few small phagosomes. Consequently, on some occasions, these cell types could not be reliably identified from the study of a single thin section. Extracellular reticulin fibres were found adjacent both to non-phagocytic reticular cells and to macrophages. Mitosis was observed in macrophages, albeit very rarely.     

Erythroblast iron metabolism in sideroblastic marrows

S ummary . The uptake of iron by bone marrow erythroblasts and its intracellular distribution have been studied in 23 patients with primary sideroblastic anaemia (SA), five patients with secondary SA and one patient with only non-ringed sideroblasts. EM of erythroblasts from 18 cases showed both mitochondrial iron deposits and cytoplasmic ferritin aggregates in all cases except the patient with only non-ringed sideroblasts.
Iron uptake by erythroblasts in whole bone marrow was normal but there was a decreased incorporation into haem and an increased incorporation into cell stroma. Age matching of erythroblasts using Percoll density gradient centrifugation indicated that stromal iron incorporation was high at all stages of erythroblast development even before haem synthesis had become a major metabolic activity and in intermediate and late erythroblasts a real decrease in haem synthesis appeared less certain. These observations, together with the inability to correct the abnormality in vitro with either pyridoxal phosphate or amino-laevulinic acid suggest that the primary defect in SA may be an abnormality of mitochondrial iron metabolism rather than an abnormality of haem synthesis.     

Enrichment of erythroblasts from human bone marrow using complement-mediated lysis: measurement of ferritin

S ummary . Sequential lysis of human bone marrow cells with a monoclonal antibody directed against myeloid cells (TG1) and a rabbit antiserum raised against peripheral blood mononuclear cells gave preparations in which 78–97% of the nucleated cells were erythroid, with a 24–77% recovery. Viability was high, morphology was good and the cells were able to divide and differentiate in culture. No metabolic experiments were carried out but the ferritin content of the erythroblasts was measured in four experiments and found to be about 200–2000 times higher than that found in normal erythrocytes. The H/S ratio was high in both erythroblasts and erythrocytes. Fractionation on the basis of density of two erythroblast preparations, one from a patient with sideroblastic anaemia and one from a patient with megaloblastic anaemia, showed that the most immature erythroblasts contained the highest content of ferritin and that this fell with maturation. The H/S ratio stayed the same or fell with maturation. It was concluded that this method would be valuable for the study of the role of erythroblast ferritin in normal and pathological situations.     

Immunocytochemical detection of ferritin in human bone marrow and peripheral blood cells using monoclonal antibodies specific for the H and L subunit

We have used the monoclonal antibodies 2A4 (specific for the H subunit of human ferritin) and LO3 (specific for the L subunit) for immunocytochemical detection of ferritin in bone marrow and peripheral blood cells from normal subjects and patients with various haematological disorders. Formalin-fixed slides were stained by the immunoalkaline phosphatase procedure (APAAP). In normal subjects, ferritin could be found only in bone marrow smears and appeared to be largely confined to erythroid precursors and reticuloendothelial cells. The more immature erythroid precursors contained higher concentrations of cellular ferritin. Although evaluation could be only semiquantitative, erythroblast ferritin appeared to be more reactive with the monoclonal 2A4 (15 +/- 7% positive erythroblasts) than with the monoclonal LO3 (6 +/- 5% positive erythroblasts), indicating that H-type ferritin was predominant, particularly in proerythroblasts and basophilic erythroblasts. By contrast, the ferritin present in reticuloendothelial cells appeared to be predominantly of L-type. Patients with iron deficiency showed low levels of positive erythroblast, whereas the reverse was true in patients with transfusional iron overload. Intense positivity for reticuloendothelial cell ferritin was found in patients with anaemia of chronic disease. In myelodysplastic syndromes and acute myeloid leukaemia (AML), ferritin positivity was generally very strong at any stage of erythroblast development, particularly with the monoclonal antibody 2A4. Perls-positive perinuclear granules of ring sideroblasts were not stained, confirming that mitochondrial iron deposition is not in the form of ferritin. In AML and myelodysplastic syndromes with excess of blasts, ferritin could be detected also in immature myeloid cells. These data indicate that: (a) in normal conditions ferritin is mainly expressed in red cell precursors and reticuloendothelial cells, and this is in keeping with the peculiar role of these cells in iron metabolism; (b) abnormal cell ferritin contents can be observed in both iron overload and malignancy.     

High Resolution Autoradiographic Studies of RNA, Protein and DNA Synthesis during Human Eosinophil Granulocytopoiesis: Evidence for the Presence of RNA on or within Eosinophil Granules

Human bone marrow cells which had been incubated with [3H]uridine or [3H]leucine for I h were studied using the technique of electron microscope-autoradiography. The autoradiographs revealed the presence of newly-synthesized RNA and protein molecules within or on a proportion of (I) the primary and secondary granules in all classes of eosinophil precursors and (2) the secondary granules in eosinophil granulocytes. It is suggested that the granule-associated RNA molecules may be concerned with the synthesis of at least some of the new protein molecules which were incorporated into the limiting membrane or substance of eosinophil granules long after the immature primary granule stage. Studies of eosinophil precursors which had been incubated with [3H]thymidine for I h showed that the eosinophil granules do not label with this DNA precursor.     

Ultrastructural abnormalities and arrest of protein biosynthesis in some erythroblasts from homozygotes for haemoglobin C and double heterozygotes for haemoglobin C and beta-thalassaemia

Various ultrastructural abnormalities were found in the erythroblasts of three homozygotes for haemoglobin C (HbC), one patient with HbC/beta(+)-thalassaemia and one patient with HbC/beta (0) thalassaemia. These included a coarsely granular or reticular appearance and altered electron-density of the heterochromatin, loss of parts of the nuclear membrane, and oozing of nuclear material into the cytoplasm. In addition, the two patients with HbC/beta-thalassaemia, but not the others, showed precipitated intracytoplasmic alpha-chains in a few profiles of polychromatic erythroblasts and marrow reticulocytes. Electron microscope autoradiographic studies of bone marrow cells from two of the patients with HbC disease and the patient with HbC/beta (0)-thalassaemia showed a marked depression or failure of incorporation of 3H-leucine into protein in some of the ultrastructurally abnormal erythroblasts. This impairment of protein synthesis may lead to alterations in the erythroblast membrane that are involved in the recognition and phagocytosis of the abnormal erythroblasts by macrophages.     

Observations on the Ultrastructure of Erythropoietic Cells and Reticulum Cells in the Bone Marrow of Patients with Homozygous β-Thalassaemia

An electron microscopic study of marrow fragments from patients with homozygous beta-thalassaemia has shown that 3% of early polychromatic erythroblast profiles and 20% of late polychromatic erythroblast profiles contain intracytoplasmic alpha-chain precipitates. Various nuclear abnormalities were found including the loss of parts of the nuclear membrane and the presence of intranuclear alpha-chain precipitates, and these abnormalities were virtually confined to the non-dividing, late polychromatic erythroblasts. As most profiles of the proliferating early polychromatic erythroblasts did not contain intracytoplasmic or intranuclear alpha-chain precipitates, it is suggested that the arrest of many of these cells in the G1 phase of the cell cycle may be related to the presence of an excess of free alpha-chains rather than to the presence of alpha-chain precipitates within them. The cytoplasm of the bone marrow reticulum cells contained early and late polychromatic erythroblasts at various stages of degradation, providing direct evidence of ineffective erythropoiesis.     

Storage of iron in bone marrow plasma cells. Ultrastructural characterization, mobilization, and diagnostic significance

The physiological site of iron storage in the human bone marrow is the macrophage (orthotopic iron store). Iron-storing plasma cells, as bone marrow indicators of iron overload, and/or chronic alcoholism represent a pathological (heterotopic) iron store. They were demonstrated by light and electron microscopy in 23 iron-overloaded patients: transfusional siderosis (n = 8), genetic haemochromatosis (GH; n = 11), iron-loading anaemias (n = 4) and in patients with bone marrow impairment due to chronic alcoholism (n = 6). The pattern of iron storage in bone marrow plasma cells was monitored during iron loading in patients receiving continuous transfusional therapy (n = 7) and also upon reversal of iron overload by repeated phlebotomies in GH (n = 9) and in iron-loading anaemias (n = 4). The first ultrastructural evidence of iron storage in plasma cells was the appearance of free ferritin molecules in the cytosol, thus indicating endogenous ferritin synthesis. Accumulation of iron proceeded with the additional formation of ferritin- and haemosiderin-containing lysosomes (siderosomes) in these cells. Lysosomal ferritin partially showed a paracrystalline array. The siderosomes originated from autophagocytosis of cytoplasmic areas containing free ferritin molecules. In addition, the formation of ferritin-containing vesicles in the proximity of the Golgi apparatus was observed. The heterotopic iron store of bone marrow plasma cells was less readily mobilizable by blood-letting than the orthotopic store of macrophages.     

Dyserythropoiesis in homozygous haemoglobin C disease

Summary. Electron microscope studies of the bone marrow of three patients with homozygous haemoglobin C (HbC) disease have shown marked ultrastructural abnormalities in several of the polychromatic erythroblasts and marrow reticulocytes and the presence of phagocytosed erythroblasts within the macrophages. Such abnormalities were not found in the bone marrow of three patients with sickle cell anaemia indicating that the abnormalities represented a feature of HbC disease rather than a disturbance secondary to peripheral haemolysis. The characteristic ultrastructural finding in the polychromatic erythroblasts in HbC disease was the presence of grossly-disorganized nuclei showing multiple intranuclear clefts, the loss of parts of the nuclear membrane, oozing of nuclear material into the cytoplasm and an alteration of the structure and stainability of the nuclear chromatin. It is proposed that both the dyserythropoiesis and ineffective erythropoiesis in HbC disease may have resulted from the formation in vivo of very small aggregates of HbC within erythropoietic cells.     

The macrophage CD163 surface glycoprotein is an erythroblast adhesion receptor

Erythropoiesis occurs in erythroblastic islands, where developing erythroblasts closely interact with macrophages. The adhesion molecules that govern macrophage-erythroblast contact have only been partially defined. Our previous work has implicated the rat ED2 antigen, which is highly expressed on the surface of macrophages in erythroblastic islands, in erythroblast binding. In particular, the monoclonal antibody ED2 was found to inhibit erythroblast binding to bone marrow macrophages. Here, we identify the ED2 antigen as the rat CD163 surface glycoprotein, a member of the group B scavenger receptor cysteine-rich (SRCR) family that has previously been shown to function as a receptor for hemoglobin-haptoglobin (Hb-Hp) complexes and is believed to contribute to the clearance of free hemoglobin. CD163 transfectants and recombinant protein containing the extracellular domain of CD163 supported the adhesion of erythroblastic cells. Furthermore, we identified a 13-amino acid motif (CD163p2) corresponding to a putative interaction site within the second scavenger receptor domain of CD163 that could mediate erythroblast binding. Finally, CD163p2 promoted erythroid expansion in vitro, suggesting that it enhanced erythroid proliferation and/or survival, but did not affect differentiation. These findings identify CD163 on macrophages as an adhesion receptor for erythroblasts in erythroblastic islands, and suggest a regulatory role for CD163 during erythropoiesis.     

Enucleation of primitive erythroid cells generates a transient population of "pyrenocytes" in the mammalian fetus

Enucleation is the hallmark of erythropoiesis in mammals. Previously, we determined that yolk sac-derived primitive erythroblasts mature in the bloodstream and enucleate between embryonic day (E)14.5 and E16.5 of mouse gestation. While definitive erythroblasts enucleate by nuclear extrusion, generating reticulocytes and small, nucleated cells with a thin rim of cytoplasm ("pyrenocytes"), it is unclear by what mechanism primitive erythroblasts enucleate. Immunohistochemical examination of fetal blood revealed primitive pyrenocytes that were confirmed by multispectral imaging flow cytometry to constitute a distinct, transient cell population. The frequency of primitive erythroblasts was higher in the liver than the bloodstream, suggesting that they enucleate in the liver, a possibility supported by their proximity to liver macrophages and the isolation of erythroblast islands containing primitive erythroblasts. Furthermore, primitive erythroblasts can reconstitute erythroblast islands in vitro by attaching to fetal liver-derived macrophages, an association mediated in part by alpha4 integrin. Late-stage primitive erythroblasts fail to enucleate in vitro unless cocultured with macrophage cells. Our studies indicate that primitive erythroblasts enucleate by nuclear extrusion to generate erythrocytes and pyrenocytes and suggest this occurs in the fetal liver in association with macrophages. Continued studies comparing primitive and definitive erythropoiesis will lead to an improved understanding of terminal erythroid maturation.     

Ultrastructural abnormalities and arrest of protein biosynthesis in some erythroblasts from homozygotes for haemoglobin C and double heterozygotes for haemoglobin C and β-thalassaemia

Summary. Various ultrastructural abnormalities were found in the erythroblasts of three homozygotes for haemoglobin C (HbC), one patient with HbC/β⁺-thalassaemia and one patient with HbC/β thalassaemia. These included a coarsely granular or reticular appearance and altered electron-density of the heterochromatin, loss of parts of the nuclear membrane, and oozing of nuclear material into the cytoplasm. In addition, the two patients with HbC/β-thalassaemia, but not the others, showed precipitated intracytoplasmic α-chains in a few profiles of polychromatic erythroblasts and marrow reticulocytes. Electron microscope autoradiographic studies of bone marrow cells from two of the patients with HbC disease and the patient with HbC/β thalassaemia showed a marked depression or failure of incorporation of ³H-leucine into protein in some of the ultrastructurally abnormal erythroblasts. This impairment of protein synthesis may lead to alterations in the erythroblast membrane that are involved in the recognition and phagocytosis of the abnormal erythroblasts by macrophages.     

Hexagonal crystals in the bone marrow in patients with myeloproliferative disease and preleukaemia

In epoxy resin-embedded bone marrow biopsies from 238 cases of myeloproliferative disease and preleukaemia, crystalline inclusions were found in the bone marrow cells of 24 (10.1%), most often in patients with acute myeloproliferative disease (23%). By light microscopy the crystals were easily recognized after Giemsa staining, lying within the cytoplasm of bone marrow macrophages. By electron microscopy the hexagonal crystals were encountered chiefly within secondary lysosomes of the macrophages, but also within immature myeloid cells in one of the two cases studied. In the latter a tight-fitting membrane around the crystals suggested that they were of lysosomal origin and represented a form of abnormal granulation. We suggest that the presence of crystals in myeloid cells represents a kind of abnormal granula formation in the neoplastic cells. In the macrophages the crystals lodge in secondary lysosomes, probably after phagocytosis of crystal-bearing non-viable myeloid cells. The accumulation of crystals in lysosomes indicates resistance to degradation.     

Abnormal haem biosynthesis in the chronic anaemia of rheumatoid arthritis.

OBJECTIVES--The chronic microcytic anaemia of rheumatoid arthritis (RA) occurs despite the presence of adequate reticulo-endothelial iron stores. The red cell microcytosis is evidence of impaired haemoglobin production. This study has examined possible abnormalities of erythroid haem biosynthesis that may contribute to the anaemia. METHODS--5-Aminolaevulinate (ALA) synthase and ferrochelatase activities were assayed in whole bone marrow and in purified erythroblasts from patients with RA and in control subjects. All patients were iron replete with demonstrable iron in the bone marrow. RESULTS--ALA synthase activity was significantly reduced in both whole bone marrow and purified erythroblasts from patients with the anaemia of RA. Erythrocyte protoporphyrin levels were raised in nine of 12 patients tested while ferrochelatase activity was normal. CONCLUSION--These abnormalities provide absolute evidence of abnormal erythroblast haem biosynthesis and iron metabolism in the anaemia of RA and most likely reflect decreased ALA synthase mRNA translation and some abnormality of erythroblast iron transport. Further studies using highly purified erythroblast populations will attempt to identify the causal factors leading to this abnormal erythroblast metabolism.     

Thiamine responsive anaemia: a study of two further cases

A brother and sister of Pakistani origin suffered from sensorineural deafness, diabetes mellitus and a macrocytic anaemia. Their bone marrows showed megaloblastic erythropoiesis and contained many ringed sideroblasts. Electron microscope studies of the bone marrow revealed (1) iron-laden mitochondria in many erythroblasts, (2) non-specific abnormalities indicative of dyserythropoiesis in some erythroblasts, and (3) evidence of ineffective erythropoiesis. The deoxyuridine suppression test indicated that the megaloblastic changes were not caused by an impairment of the methylation of deoxyuridylate. Studies of nucleic acid synthesis in the bone marrow cells showed that the rate of incorporation of [3H]thymidine into DNA was increased and that the rates of incorporation of [14C]glycine and [14C]adenine into both DNA and RNA were essentially within the normal range. The anaemia did not respond to therapy with hydroxocobalamin, folic acid or pyridoxine but responded to 25 mg thiamine, daily, by mouth. In one of the cases a post-thiamine marrow aspirate showed a considerable improvement in both the megaloblastic and sideroblastic changes.     

Aberrant iron accumulation and oxidized status of erythroid-specific delta-aminolevulinate synthase (ALAS2)-deficient definitive erythroblasts

Alas2 encodes the erythroid-specific delta-aminolevulinate synthase (ALAS2 or ALAS-E), the first enzyme in heme biosynthesis in erythroid cells. Mice with the Alas2-null phenotype showed massive cytoplasmic, but not mitochondrial, iron accumulation in their primitive erythroblasts. Because these animals died by day 11.5 in utero, studies of iron metabolism in definitive erythroblasts were not possible using the in vivo model. In this study, embryonic stem (ES) cells lacking the Alas2 gene were induced to undergo differentiation to the definitive erythroblast stage in culture, and the phenotype of Alas2-null definitive erythroblasts was examined. Alas2-null definitive erythroblasts cell pellets were entirely colorless due to a marked deficiency of heme, although their cell morphology was similar to that of the wild-type erythroblasts. The level of expression of erythroid-specific genes in Alas2-null definitive erythroblasts was also similar to that of the wild-type erythroblasts. These findings indicate that Alas2-null definitive erythroblasts developed to a stage similar to that of the wild-type erythroblasts, which were also shown to be very similar to the bone marrow erythroblasts in vivo. In contrast, Alas2-null definitive erythroblasts contained 15 times more nonheme iron than did the wild-type erythroblasts, and electron microscopy found this iron to be distributed in the cytoplasm but not in mitochondria. Consistent with the aberrant increase in iron, Alas2-null definitive erythroblasts were more peroxidized than wild-type erythroblasts. These findings suggest that ALAS2 deficiency itself does not interfere with the development of definitive erythroid cells, but it results in a profound iron accumulation and a peroxidized state in erythroblasts.     

Occurrence of cytoplasmic bridge between erythroblasts--morphology and frequency in hematological disease

Peculiar cytoplasmic connection between erythroblasts was observed in the smear preparation of human bone marrow; a pair of erythroblasts in the resting stage was connected by "cytoplasmic bridge" which is very thin and sometimes long compared with the telophase bridge appeared in the usual cytokinesis. Electron microscopy revealed that small amount of microtubules were running along in the cytoplasmic bridge but mid-body was not seen. Plasma membrane about the middle of the bridge bulged to form "bulging ring". Erythroblasts in various differentiating stages were connected by the cytoplasmic bridge and the stage of each pair was almost the same. The occurrence of the cytoplasmic bridge between erythroblasts was evaluated in the smear preparations of human bone marrow with hematological diseases or normal conditions. In the normal bone marrow, the mean value was 8.1 +/- 3.6% (n = 33). In patients suffered from autoimmune hemolytic anemia, it was 4.3 +/- 2.3% (n = 12), aplastic anemia, 3.2 +/- 2.1% (n = 6) and paroxysmal nocturnal hemoglobinuria, 5.0 +/- 1.4% (n = 3). While in the erythroleukemia the occurrence was very low showing 0.2 +/- 0.1% (n = 3) of total erythroblasts. In MDS, RAEB (2.5 +/- 2.4%) and RA (2.9 +/- 2.2+%) showed statistically significant lower occurrence. In the normal bone marrow the occurrence was higher, so the formation of cytoplasmic bridge could be essential for a normal proliferation of erythroblasts.     

Ineffective Erythropoiesis in Haemoglobin Eβ -thalassaemia: an Electron Microscope Study

S ummary. Electron microscope studies have been performed on the bone marrow cells of two non-splenectomized patients and the circulating erythroblasts and reticulocytes of three splenectomized patients with HbE/β-thalassaemia. Some intracellular precipitates (probably consisting of α-chains) and mild dyserythropoietic changes were found in the early polychromatic erythroblasts within the bone marrow. Larger quantities of precipitate and more marked dyserythropoietic changes were found in the late polychromatic erythroblasts and reticulocytes both within the marrow and within the circulation. The bone marrow macrophages contained phagocytosed erythroblasts within their cytoplasm. These data indicate that the anaemia in HbE/β-thalassaemia results largely from dyserythropoiesis and ineffective erythropoiesis. The ultrastructural abnormalities encountered in the cases of HbE/β-thalassaemia were qualitatively and quantitatively similar to those seen in homozygous β-thalassaemia.     

Evidence for 1,25-dihydroxyvitamin D3 production by cultured porcine alveolar macrophages

Previous studies have demonstrated that human alveolar and bone marrow macrophages when activated in vitro can metabolize 25-hydroxyvitamin[3H]D3 to 1 alpha,25-dihydroxyvitamin[3H]D3; however, to date no animal models to study this system have been available. In the present study, cultured porcine pulmonary alveolar macrophages from two animals were assayed for their capability for metabolism of 25-hydroxyvitamin[3H]D3. The porcine alveolar macrophages constitutively produced a metabolite of 25-hydroxyvitamin[3H]D3 which was identified as 1 alpha,25-dihydroxyvitamin[3H]D3 by high performance liquid chromatography. The apparent KM was in the range of 300 nM. Unlike human macrophages, treatment of porcine alveolar macrophages with lipopolysaccharide did not stimulate 1 alpha,25-dihydroxyvitamin[3H]D3 production. Addition of 1 alpha,25-dihydroxyvitamin-D3 to macrophages cultures led to a sensitive proportional inhibition of 1 alpha,25-dihydroxyvitamin[3H]D3 synthesis.