Modulation of megakaryocyte emperipolesis by phlebotomy: megakaryocytes as a component of marrow-blood barrier

The presence of marrow cells within the cytoplasm of megakaryocytes has been documented and is attributed to the phenomenon of emperipolesis. In a previous study most patients demonstrating this phenomenon had or were suspected of having blood loss. To find out if these are causally related, we induced acute and chronic blood loss in rats and quantitatively measured the emperipolesis index (EI). EI dropped after acute blood loss but rose after chronic blood letting, indicating that blood loss can modulate this phenomenon. In the marrow, megakaryocytes are located preferentially on the abluminal side of sinus endothelium and it is postulated that in the state of heightened demand for cell delivery from marrow into the circulation, some cells take a transmegakaryocytic route to enter the circulation. This concept incorporates megakaryocytes as a component of the marrow-blood barrier.     

Megakaryocytes emperipolesis in bone marrow of the patients with non-Hodgkin's lymphoma

Emperipolesis of hematopoietic cells within the cytoplasm of the megakaryocytes was most often described in association with various pathologic conditions. The aim of the research was estimation of the incidence of emperipolesis in the bone marrow of the patients with non-Hodgkin's lymphoma (NHL). 30 patients with different histological types of NHL (chronic lymphocytic leukaemia--CLL, hairy cell leukaemia--HCL, multiple myeloma--MM) in compliance with clinical stage of the disease, patient's age and sex were analyzed. Trephine biopsies of the bone marrow were carried out in fixative solution and paraffin embedding. Hematoxylin and eosin and monoclonal antibody CD 61 were applied on thin sections. Phenomenon of megakaryocytic emperipolesis in human bone marrow was found in 6 cases: in 5 cases in CLL and in 1 case in HCL. In most of them emperipolesis was related to single megakaryocytes. We observed in the cytoplasm of the megakaryocytes single hematopietic cells-most often lymphocytes, rarely eosinophilic granulocytes. We found no correlations between histological types of NHL, clinical stage of the disease, patients' age, sex and the incidence of megakaryocytic emperipolesis.     

Emperipolesis--a peculiar feature of megakaryocytes as evaluated in chronic myeloproliferative diseases by morphometry and ultrastructure

A combined morphometric and ultrastructural study was performed on so called emperipolesis or internal wandering of myeloid cells in the cytoplasm of large mature megakaryocytes. Measurements were made on material from a total of 115 patients comprising a normal control group and 5 groups with subtypes of chronic myeloproliferative diseases, including primary (essential, idiopathic) thrombocythemia (PTH). A significant increase in this peculiar phenomenon was noted in myeloproliferative disorders and especially in PTH where the frequency of emperipolesis showed a positive correlation with the number of anuclear cytoplasmic fragments sectioned, with the circular deviation of the shapes of megakaryocytes and with the extent to which the peripheral thrombocyte count was elevated. Electron microscopy in selected cases displayed the integrity of the plasma membranes of engulfed hematopoietic cells within the dilated cavities of the megakaryocytic demarcation membrane system (DMS) and no evidence of phagocytosis. Moreover there was a close relationship between engulfed myeloid cells and the presumptive sites of platelet shedding which had their openings from the cisternal lumina of the DMS. Our results demonstrate that emperipolesis of hematopoietic cells within megakaryocytes should not be regarded as a special nosological feature, but as an indication of enforced thrombocytogenetic activity which is expressed particularly in PTH.     

The effects of colcemid on mouse bone marrow.

Following Colcemid administration, mitoses accumulate preferentially in the subendosteal region of the bone marrow of the mouse. This finding suggests that the most rapidly proliferating cells are localized to the subendosteal region, and complements previous radioautographic studies which have demonstrated a corresponding labelling gradient in the marrow. Quantitative estimates of cell cycle time by the stathmokinetic method were precluded by the presence of significant Colcemid induced interphase cell loss. Colcemid also affected cell differentiation in the marrow. Following Colcemid administration there was a fall in mature granulocytes in the marrow, and a concommitant rise in marrow megakaryocytes.     

THE EFFECTS OF COLCEMID ON MOUSE BONE MARROW

Following Colcemid administration, mitoses accumulate preferentially in the subendosteal region of the bone marrow of the mouse. This finding suggests that the most rapidly proliferating cells are localized to the subendosteal region, and complements previous radioautographic studies which have demonstrated a corresponding labelling gradient in the marrow. Quantitative estimates of cell cycle time by the stathmokinetic method were precluded by the presence of significant Colcemid induced interphase cell loss. Colcemid also affected cell differentiation in the marrow. Following Colcemid administration there was a fall in mature granulocytes in the marrow, and a concommitant rise in marrow megakaryocytes.     

Reactivation of a hematopoietic endocrine program of pregnancy contributes to recovery from thrombocytopenia

Regulation of blood platelet levels involves an array of cytokines, including the placental hormone PRL-like protein E (PLP-E). The PLP-E receptor is present on megakaryocytes in pregnant mice, nonpregnant female mice, and male mice. Other known megakaryocytic cytokines do not share the PLP-E receptor, and thus the presence of this receptor in nonpregnant animals suggests that PLP-E may be expressed in tissues other than the placenta. Consistent with this prediction, PLP-E is produced in thrombocytopenic mouse bone marrow, primarily in granulocytes, but not in normal mouse bone marrow. Serum from thrombocytopenic mice, purified thrombopoietin or IL-6, or pregnancy can induce bone marrow cell expression of PLP-E. The induction of PLP-E gene expression in response to thrombocytopenia is physiologically significant, as injection of PLP-E into thrombocytopenic mice restores normal platelet levels with no effect on granulocytes, erythrocytes, and total white blood cell counts. We conclude that inducible expression of PLP-E in bone marrow is part of the mechanism of recovery from thrombocytopenia. These results also suggest a more general concept: that the endocrine program of pregnancy, which in mammals has evolved to support the intrauterine growth and development of the fetus, can also be harnessed to respond to pathophysiology.     

Asialogangliosides as differentiation markers of rat erythropoietic and granulopoietic cells in rat bone marrow. Flow cytometric analysis of rat bone marrow cells using anti-asialoganglioside antibodies

Flow cytometric analysis using anti-glycolipid antiserum was used on rat bone marrow cells to determine the relation between the glycolipid species expressed on cell surfaces and cell differentiation. Four kinds of antibodies against gangliotriaosylceramide (Gg3Cer), gangliotetraosylceramide (Gg4Cer), fucogangliotetraosylceramide (IV2 alpha Fuc-Gg4Cer) and IV3 alpha Gal-fucogangliotetraosylceramide (IV3 alpha GalIV2 alpha Fuc-Gg4Cer, blood group B lipid) were used. The cells sorted out by each anti-glycolipid antiserum were stained with May-Grünwald-Giemsa reagent and identified by microscopy. In the erythropoietic group, only polychromatic erythroblasts had these four glycolipids on their cell surfaces; none appeared on differentiated erythrocytes. These glycolipids were expressed during the early stages of immature granulocytes, especially in the promyelocyte and myelocyte stages of eosinophilic and neutrophilic granulocytes. Very limited populations of lymphocytes were sorted out as asialoganglioside-expressing cells. We concluded that asialogangliosides are useful differentiation markers for the erythropoietic and granulopoietic cells of rat bone marrow, and that anti-asialoganglioside antibody-flow cytometry is a very useful technique with which to isolate immature granulocytes and erythropoietic cells from rat bone marrow cells.     

Distribution of cells bearing receptors for a colony-stimulating factor (CSF-1) in murine tissues

CSF-1 is a subclass of the colony-stimulating factors that specifically stimulates the growth of mononuclear phagocytes. We used the binding of 125I-CSF-1 at 0 degrees C by single cell suspensions from various murine tissues, in conjunction with radioautography, to determine the frequency of binding cells, their identity, and the number of binding sites per binding cell. For all tissues examined, saturation of binding sites was achieved within 2 h at 2--3 x 10(-10) M 125I-CSF-1. The binding was irreversible and almost completely blocked by a 2 h preincubation with 5 x 10(-10) M CSF-1. 125I-CSF-1 binding was exhibited by 4.3% of bone marrow cells, 7.5% of blood mononuclear cells, 2.4% of spleen cells, 20.5% of peritoneal cells, 11.8% of pulmonary alveolar cells and 0.4% of lymph node cells. Four morphologically distinguishable cell types bound 125I-CSF-1: blast cells; mononuclear cells with a ratio of nuclear to cytoplasmic area (N/C) greater than 1; cells with indented nuclei; and mononuclear cells with N/C less than or equal to 1. No CSF-1 binding cells were detected among blood granulocytes or thymus cells. Bone marrow promyelocytes, myelocytes, neutrophilic granulocytes, eosinophilic granulocytes, nucleated erythroid cells, enucleated erythrocytes, and megakaryocytes also failed to bind. The frequency distribution of grain counts per cell for blood mononuclear cells was homogenous. In contrast, those for bone marrow, spleen, alveolar, and peritoneal cells were heterogeneous. The monocytes in blood or bone marrow (small cells, with either indented nuclei or with N/C greater than 1) were relatively uniformly labeled, possessing approximately 3,000 binding sites per cell. Larger binding cells (e.g., alveolar cells) may possess higher numbers of receptors. It is concluded that CSF-1 binding is restricted to mononuclear phagocytic cells and their precursors and that it can be used to identify both mature and immature cells of this series.     

A study of GRM1 monoclonal antibody that reacts with natural killer cells and granulocytes

We have produced a monoclonal antibody, GRM1, against a prolymphocytic leukemia that defines an antigen present in neutrophilic granulocytes (PMN) and a lymphocyte subset with natural killer (NK) activity, which was identified as large granular lymphocytes. This monoclonal antibody recognizes FcR2 (CD16), an antigen composed of two polypeptides of 50 and 60 kilodaltons, respectively. This GRM1 monoclonal antibody was tested against normal T and B cells, neutrophilic granulocytes, monocytes, platelets, acute and chronic leukemias, and was positive only against granulocytes (95%) and cells with NK activity. GRM1 was able to deplete NK cell activity in complement-dependent lysis. However, GRM1 did not block NK activity nor peripheral blood lymphocyte- and PMN-mediated antibody-dependent cytotoxicity in healthy individuals. GRM1 also did not block Fc receptor in an erythrocyte antibody rosette assay. The immunochemical data and cell distribution patterns lead us to conclude that GRM1 recognizes and FcR2 receptor epitope which is not involved in the receptor's function.     

Lycramine brilliant blue JL: a new stain for human megakaryocytes

Using the acrylic textile dye Lycramine brilliant blue JL, mature and immature megakaryocytes from human bone marrow specimens stained metachromatically bright lavender. This coloration was not observed in other types of bone marrow cells. After digestion with either diastase or ribonuclease, subsequent staining of marrow specimens did not reveal a significant diminution of the intensity of staining of megakaryocytes. However, after incubation with hyaluronidase followed by staining with Lycramine brilliant blue JL, staining of megakaryocyte cytoplasm was either imperceptible or very pale blue. Accordingly, at least one of the substances responsible for the staining reaction is acid mucopolysaccharide in the cytoplasm of megakaryocytes. With further experience and comparison with established immunologic and cytochemical techniques, staining of megakaryocytes with Lycramine brilliant blue JL may be a useful addition to the cytochemistry of blood and bone marrow cells.     

Effects of gangliosides on cell maturation of murine megakaryocytes in a liquid culture system

Formation of platelet-producing megakaryocytes, the cytoplasm of which showed the terminal stage of cell maturation, heavy granulation and platelet-fields delineated with demarcation membranes, was observed in a short-term culture system, using megakaryocyte-enriched bone marrow cell suspension. Approximately 6-8% of the megakaryocytes changed to the platelet-producing megakaryocytes during 12-hour incubation. In the presence of inhibitors of energy metabolism, formation of the platelet-producing megakaryocytes was inhibited, suggesting that the process is dependent on energy producing systems. Ganglioside GD1a increased both the number of total megakaryocytes and the ratio of the platelet-producing megakaryocytes to total megakaryocytes, while GM1 did not influence the number of total megakaryocytes, but increased the ratio. Gangliosides GM2, GM3 and GD1b showed little effect on either the number of total megakaryocytes or the ratio. The results suggest that ganglioside GD1a stimulates at least two steps of megakaryocyte maturation, the change of megakaryocytic progenitors to megakaryocytes and the subsequent maturation of megakaryocytes to the platelet-producing megakaryocytes, while GM1 stimulates only the latter step of the maturation.     

Lycramine Brilliant Blue Jl: A New Stain for Human Megakaryocytes

Using the acrylic textile dye Lycramine brilliant blue JL, mature and immature megakaryocytes from human bone marrow specimens stained metachromatically bright lavender. This coloration was not observed in other types of bone marrow cells. After digestion with either diastase or ribonuclease, subsequent staining of marrow specimens did not reveal a significant diminution of the intensity of staining of megakaryocytes. However, after incubation with hyaluronidase followed by staining with Lycramine brilliant blue JL, staining of megakaryocyte ctyoplasm was either imperceptible or very pale blue. Accordingly, at least one of the substances responsible for the staining reaction is acid mucopolysaccharide in the cytoplasm of megakaryocytes. With further experience and comparison with established immunologic and cytochemical techniques, staining of megakaryocytes with Lycramine brilliant blue JL may be a useful addition to the cytochemistry of blood and bone marrow cells.     

The action of dipyridamole on megakaryocytopoiesis in regenerating and stationary bone marrow cell populations]

The effect of dipyridamole on megakaryocytopoiesis in regenerating and stationary populations of mouse bone marrow cells has been studied by heterotopic transplantation of the bone marrow using histological, electron microscopic and biochemical techniques. It is shown that drug administration induced destruction of megakaryocytes. In megakaryocytic cytoplasm giant lipid granules were found whose growth and number increase resulted in megakaryocytes kill. Gas-liquid chromatography was used to evaluate the effect of dipyridamole on distribution of lipid fatty acids of the stationary and regenerating populations of the bone marrow cells. A marked increase of the percentage of docosahexaenoic acid was found in lipids of the stationary population. Chronic dipyridamole administration caused an increase of percentage of myristic, palmitic oleic acids, and decrease of percentage of arachidonic and eicosapentaenoic acids in lipids of regenerating bone marrow cells population.     

Reduced variance of bone-marrow transit time of granulopoiesis—a possible pathomechanism of human cyclic neutropenia

Abstract. Human cyclic neutropenia (CN) is a haematological disorder characterized by oscillations in the numbers of neutrophilic granulocytes and other blood cells with a stable period of approximately 21 days. In most cases the neutrophils oscillate well below normal values such that these patients are chronically neutropenic. A comprehensive concept of the origin of CN is proposed. It assumes an abnormally small variance of the transit time of bone marrow cells (compared to normal human granulopoiesis) for the origin of the characteristic cycles. Furthermore, a reduced responsiveness of the immature granulopoietic bone marrow cells to the mitotic feedback stimuli is assumed to account for the subnormal neutrophil peaks. Together with feedback control provided in a simulation model of normal human granulopoiesis these two abnormalities can explain experimental and clinical cell kinetic data for bone marrow and blood in CN.     

Radiation sensitivity of human and murine peripheral blood lymphocytes,stem and progenitor cells

Immunodeficiency is a severe side effect of radiation therapy, notably at high radiation doses. It may also impact healthy individuals exposed to environmental ionizing radiation. Although it is believed to result from cytotoxicity of bone marrow cells and of immunocompetent cells in the peripheral blood, the response of distinct bone marrow and blood cell subpopulations following exposure to ionizing radiation is not yet fully explored. In this review, we aim to compile the knowledge on radiation sensitivity of immunocompetent cells and to summarize data from bone marrow and peripheral blood cells derived from mouse and human origin. In addition, we address the radiation response of blood stem and progenitor cells. The data indicate that stem cells, T helper cells, cytotoxic T cells, monocytes, neutrophils and, at a high degree, B cells display a radiation sensitive phenotype while regulatory T cells, macrophages, dendritic cells and natural killer cells appear to be more radioresistant. No conclusive data are available for basophil and eosinophil granulocytes. Erythrocytes and thrombocytes, but not their precursors, seem to be highly radioresistant. Overall, the data indicate considerable differences in radiosensitivity of bone marrow and blood normal and malignant cell populations, which are discussed in the light of differential radiation responses resulting in hematotoxicity and related clinical implications.     

Splenic hemopoiesis of the platypus (Ornithorhynchus anatinus): evidence of primary hemopoiesis in the spleen of a primitive mammal

The generation of blood cells has been observed in the spleen and in the bone marrow of the platypus. Hemopoiesis was found to be far more active in the spleen than in the bone marrow judging by the number of proliferating hemopoietic elements within a unit area of tissue from each organ. Granulocytes, erythroblasts, and megakaryocytes, with the related immature forms for each cell line, were noted in the spleen. In contrast, there were very few examples of immature forms of these cell lines and a complete absence of mature megakaryocytes in the bone marrow. These findings suggest that the spleen is the primary hemopoietic organ in the platypus. Since the platypus is one of two species representing the most primitive existing mammals, it seems likely that the spleen may be the primary hemopoietic organ in mammalian evolution.     

The biological effects in animals in relation to the accident at the Chernobyl Atomic Electric Power Station. 11. The ultrastructure of the bone marrow cells in different generations of rats]

In studying the bone marrow cell ultrastructure in male rats (F0-F2 generations) aged 3 months, which were brought up within the thirty kilometer Chernobyl A.P.S. disaster area, considerable submicroscopic changes have been revealed in the cells at all stages of maturation, including undifferentiated blasts and mature forms of cells of the neutrophilic, eosinophilic, monocytic and erythroid haemopoiesis series, as well as stromal elements of the microenvironment, megakaryocytes and endothelium. The severity of these changes increases, as the number of generations grows, displaying frequently a destructive character.     

The transmural passage of blood cells into myeloid sinusoids and the entry of platelets into the sinusoidal circulation; a scanning electron microscopic investigation.

Scanning electron microscopic observations of rat bone marrow reveal that the sinusoidal wall is continuous and has no permanent patent apertures allowing free communication between the extravascular and intravascular myeloid compartments. Blood cells migrate into the sinusoids by perforating the endothelial cell body. Platelets are derived from long intrasinusoidal "proplatelet" processes which originate from the cell body of extravascularly located megakaryocytes. Proplatelet processes frequently occur in clusters, with the probability that all processes in a cluster arise from a single megakaryocyte. The release of platelets into the circulation may be initiated by local constriction along these processes, at which places either individual platelets or larger segments of proplatelet cytoplasm are pinched off. The larger segments may subsequently undergo further fragmentation into individual platelets.     

Application of sedimentation at 1 g on a Ficoll gradient to the separation of bone marrow cells

Sedimentation at unit gravity of human bone marrow, during 15 hours at 4 degrees C on a linear density gradient of Ficoll in culture medium ranging from 1.020 to 1.065 g/ml shows that it exists a differential migration of bone marrow cells subpopulation with a precise mean densities : we find successively : 1.021 +/- 1.10(-3) g/ml for the lymphocytes, 1.024 +/- 2.5.10(-3) g/ml for the non eosinophil granulocytes, 1.025 +/- 2.5.10(-3) g/ml for the metamyelocytes, 1.030 +/- 3.5.10(-3) g/ml for the immature myeloid cells (myeloblasts, promyelocytes, myelocytes), 1.040 +/- 3.10(-3) g/ml for the eosinophil granulocytes, 1.055 +/- 10.10(-3) g/l for the megakaryocytes. The highest percentages of S phase cells, G2 and M phase cells determinated with a cytofluorograph correspond to peaks of immature myeloid cells (myeloblasts, promyelocytes and myelocytes). This method of bone marrow cells separation may be used to study the cell cycle in pathological bone marrows (leukaemia in particular) and to determine the effects and the efficiency of some antimitotics.     

Effect of bone marrow suppression on granulocyte opsonin levels.

Levels of serum opsonin for neutrophilic granulocytes were measured in dogs made neutropenic by cyclophosphamide administration. Heat-labile opsonin became elevated within 24 hr following cyclophosphamide (P less than 0.005) and remained elevated over the 4-5 day period of observation (P less than 0.005). In contrast, heat stable opsonin was not significantly effected. Bone marrow suppression by X-ray and busulfan also caused serum opsonin levels to increase. Changes in the levels of IgG, C3, and total hemolytic complement during the course of bone marrow suppression did not correlate with the granulocyte opsonin levels. These findings suggest that serum granulocyte opsonin levels respond to bone marrow suppression and may provide an improved environment for the function of transfused granulocytes.