CELL PROLIFERATION IN THE ERYTHROID COMPARTMENT OF GUINEA-PIG BONE MARROW: STUDIES WITH 3H-THYMIDINE

Single and multiple injections of ³H-TdR have been used for measuring the rate of proliferation in morphologically defined cell populations of guinea-pig bone marrow that are committed to erythroid differentiation. The conclusions are based on the analysis of absolute cell numbers in the maturational compartments, the labeling and mitotic indices, labeled mitotic curves, pulse and chase grain counts over dividing and interphase cells, and on the rate of labeling during multiple, repeated injections of ³H-TdR. The average duration of S and the rate of cycling is similar in all maturational compartments of the erythron. The majority of cells progress to the next maturational compartment by the time they divide for the second time. All proerythroblasts and basophilic erythroblasts are in cycle. Polychromatic erythroblasts incapable of incorporating ³H-TdR reach the orthochromatic population in the span of 5–6 hr. The orthochromatic population is renewed every 20–24 hr. The number of divisions between the proerythroblast and orthochromatic erythroblast does not exceed four and some cells may undergo only two divisions during the maturation pathway. Cell input from a progenitor cell population contributes to the maintenance of the erythron. The kinetic behavior of progenitor cells is similar to that of proerythroblasts. By the time of their second division, progenitor cells may reach either the proerythroblast or basophilic erythroblast compartments. The kinetic behavior of basophilic transitional cells corresponds to the predicted behavior of the erythroblast progenitor cell pool. Several of the conclusions are based on the assumption that grain count halving is the result of cell division. In view of the evidence discussed, this assumption in the present studies seems justified.     

Cyclic AMP phosphodiesterase activity during differentiation of rabbit erythroid bone marrow cells.

Changes in the activity of cyclic AMP phosphodiesterase during differentiation of rabbit bone marrow erythroid cells were investigated. The cells were separated by velocity sedimentation at unit gravity into six fractions corresponding to different stages of development: proerythroblasts, basophilic cells, polychromatic cells, early orthochromatic and late orthochromatic cells and reticulocytes. Cyclic AMP phosphodiesterase was found to be very active in the most immature cells, the proerythroblasts, which also have the highest content of cyclic AMP. After differentiation into basophilic erythroblasts, a 4-fold decrease in cyclic AMP phosphodiesterase activity was observed. In these cells the amount of cyclic AMP was about 80% lower than that in proerythroblasts. In polychromatic cells a further drop in phosphodiesterase activity occurred. After the final cell division the enzyme activity was very low and the levels of cyclic AMP in the early and late orthochromatic cells remained constant. Kinetic studies demonstrated a heterogeneity of erythroid cell cyclic AMP phosphodiesterase: high affinity, low-Km (5.5 X 10(-6) M) and low affinity, high-Km (0.1 X 10(-3) M) enzymes were found. The phosphodiesterase activity was dependent on the presence of Mg2+ and was activated by Ca2+ at low Mg2+ concentrations (1 mM). The changes in cyclic AMP phosphodiesterase activity during differentiation and maturation of erythroid cells suggest the possible importance of this enzyme in the physiological control of cyclic AMP concentrations in developing erythroblasts. The loss of cyclic AMP phosphodiesterase activity after cessation of cell division supports the concept of the significance of the final cell division in erythroblast differentiation.     

Cell kinetics in the erythroid compartment of guinea pig bone marrow: a model based on 3H-TdR studies

A model of steady-state erythropoiesis in the guinea pig is described. The model incorporates an unidentified progenitor compartment, as well as compartments representing proerythroblasts, basophilic, polychromatic and orthochromatic cells. A computer representation of the model permits a simulation of the labeling curves obtained in pulse and intermittent labeling regimes. It was found that a reasonable fit to the data can be achieved when the parameters for the various compartments are essentially identical. The results of a preliminary sensitivity analysis, carried out by perturbing the duration of S phase from the best fit value, are reported. The fit achieved to the data supports the hypothesis underlying the model that each compartment corresponds to one generation and that the flux within and between compartments is sequential.     

Cell Kinetics In the Erythroid Compartment of Guinea Pig Bone Marrow: A Model Based On 3H-Tdr Studies

A model of steady-state erythropoiesis in the guinea pig is described. the model incorporates an unidentified progenitor compartment, as well as compartments representing proerythroblasts, basophilic, polychromatic and orthochromatic cells. A computer representation of the model permits a simulation of the labeling curves obtained in pulse and intermittent labeling regimes. It was found that a reasonable fit to the data can be achieved when the parameters for the various compartments are essentially identical. the results of a preliminary sensitivity analysis, carried out by perturbing the duration of S phase from the best fit value, are reported. the fit achieved to the data supports the hypothesis underlying the model that each compartment corresponds to one generation and that the flux within and between compartments is sequential.     

Half-life and rate of synthesis of globin messenger ribonucleic acid. Determination of half-life of messenger ribonucleic acid and its relative synthetic rate in erythroid cells

The specific radioactivity of mouse globin mRNA in blood reticulocytes was measured after injection of [(3)H]uridine into anaemic mice up to 60h before collection of reticulocytes. From these data, the decay of the acid-soluble nucleotide pool in the marrow and the relative marrow-cell composition it is possible to build models that allow the cell life-times and half-life of mRNA in the erythroid cells of the marrow to be calculated. Best fit of models to these data favour a model with either one or two cell divisions from the onset of mRNA synthesis. The single-cell-division model has cell times of 20, 13 and 7h respectively for the basophilic erythroblast, polychromatophilic erythroblast and reticulocyte. The two-cell-division model has cell times of 12, 12, 12 and 7h for the basophilic erythroblast 1 and 2, polychromatophilic erythroblast and reticulocyte respectively. Both models have an mRNA half-life of 17h and a constant rate of mRNA synthesis until enucleation at the reticulocyte stage, when synthesis stops. A declining rate of mRNA synthesis can be accommodated in a two-cell-division model, when synthesis halves at each cell division and cell times are essentially the same as above, but mRNA half-life is either 9h in the basophilic and polychromatophilic erythroblasts and 17h in the later cells, or 10h in the basophilic erythroblasts and polychromatophilic erythroblasts and 14.5h in later cells. In all cases it is clear that mRNA synthesis occurs over a time-period of only 30-36h and that mRNA cannot be pre-synthesized in precursor erythroid cells.     

Stimulation of adenylate cyclase activity by catecholamines and prostaglandins E during differentiation of rabbit bone marrow erythroid cells

After fractionation of rabbit bone marrow into erythroid cells at different developmental stages adenylate cyclase activity of membrane ghosts was assayed in the presence of sodium fluoride, catecholamines or prostaglandins E. Both basal and fluoride-stimulated adenylate cyclase decreased continuously during differentiation. Only catecholamines having beta 2-adrenergic activity stimulated adenylate cyclase and their effect was restricted to the most immature cells, the proerythroblasts and, to a lesser extent, the basophilic erythroblasts. Thus, uncoupling of beta-adrenergic receptors occurs early in erythroblast development and hormone responsiveness is lost before the final cell division. Prostaglandin E receptors and adenylate cyclase remain coupled throughout erythroid cell development.     

ERYTHROPOIETIC CELL POPULATION CHANGES DURING THE HEPATIC PHASE OF ERYTHROPOIESIS IN THE FOETAL MOUSE

Estimates have been made of the absolute numbers of hepatogenic erythropoietic cells from 12.5 days post fertilization onwards in the mouse. All stages of maturation up to reticulocytes are present in the earliest samples but the least mature cells (proerythroblasts and basophilic erythroblasts) predominate; more mature cells (orthochromatic erythroblasts, reticulocytes and erythrocytes) predominate later in development. The number of hepatogenic haemoglobinized cells increases exponentially with a population doubling time of about 8 hr until about 15.5 days post fertilization. There is then a sharp transition and the doubling time lengthens to about 2 days. The immature cells formed during the rapid phase of increase are poorly haemoglobinized; hence the increase in haemoglobin lags behind that of cells. Calculations of the rates of formation of hepatogenic haemoglobinized cells and haemoglobin per standard number of liver cells show maxima between 15 and 16 days; these findings are in accord with direct observations of rates of haemoglobin synthesis in cultured mouse foetal livers made previously.     

Hepatic hematopoiesis in phenylhydrazine-induced hemolytic anemia

Ten adult rabbits were divided into two groups: the control rabbits, which received subcutaneous injections of 0.9% NaCl in three days; the experimental animals which received 3 mg/Kg body weight of phenylhydrazine (PHZ) subcutaneously also in three days. On the 8th day from the initial treatment the control and experimental animals were sacrificed, blood was collected to determine hematological parameters and livers were cut into small pieces. Sections were prepared by pressing the pieces onto slides which were stained with the Giemsa stain. The hematocrit and the reticulocytosis of experimental animals were 25 + 3%, and 70 + 5% respectively. In the liver sections of the PHZ treated animals we found a very rich population of immature erythroblasts. In fact proerythroblasts and basophilic erythroblasts were 19%, polychromatic and orthochromatic erythroblasts were 22% and 13% respectively. On the contrary, these cells were absent in the control livers. The lymphocyte and lymphoblast population, on the other hand, was very rich in control animals with a value of 38.8% compared to 1.62% in the anemic animals. The results clearly indicate the hematopoietic function of the liver in the anemic animals although the low percentage of orthochromatic erythroblasts with respect to their precursors suggests the ineffectiveness of the process.     

Hexokinase in developing rabbit erythroid cells

The activity and isozyme distribution of hexokinase were studied in bone marrow cells from normal and anemic rabbits seperated by density centrifugation or by unit-gravity sedimentation. The specific activity of the enzyme was found to be about 150-fold higher in the basophilic erythroblasts as compared with the mature circulating erythrocytes. Mos of the falls in hexokinase activity take place whent the cell completes its final division and matures from the polychromatic stage to the orthochromatic stage. Concomitant with this strong decrease in enzyme activity, qualitative as well as quantitative changes in the hexokinase isozymic pattern become apparent. While in the basophilic and polychromatic erythroblasts the only hexokinase isozyme present is hexokinase type I, the orthochromatic cells also contain hexokinase Ib. This last isozymic form, which increases further at the reticulocyte stage, is also present in the circulating reticulocytes but not in mature red blood cells.     

PRIMITIVE ERYTHROPOIESIS IN EARLY CHICK EMBRYOGENESIS : I. Cell Cycle Kinetics and the Control of Cell Division

The primitive line of embryonic chick blood cells develop as a relatively homogeneous cohort of cells. Using an analysis based on the continuous uptake of thymidine-³H, we have established the generation time, G1, S, and G2 for progressively more mature generations of these immature erythroblasts. The data indicate that after the initiation of hemoglobin synthesis, the average cell will yield six generations of hemoglobin producing erythroblasts. The older generations of erythroblasts exhibit a longer generation time, G1, S, and G2 than the earlier generations of erythroblasts. Other methods of analysis corroborated these findings. One of these methods, an estimate of total erythrocyte productivity from the primitive stem cells (hematocytoblasts), led to the conclusion that the erythroblast cell lineage might be initiated as early as the sixth or seventh division following fertilization. In addition, primitive erythroblasts characterized by one set of cell cycle parameters, when grown in serum associated with erythroblasts of different parameters, showed no alteration in mitotic behavior. These results suggest the presence of programmed cell division not immediately cued by extracellular influence.     

Splenic hematopoiesis in an experimental model of hemolytic anemia

The splenic hemopoiesis of rabbits, made anemic with acetylphenylhydrazine, and of control animals was investigated. Pieces of spleen of both groups were fixed in formalin and embedded in paraffin. Paraffin sections, cut 5-7 microns in thickness, were stained with hematoxylin-eosin, Giemsa, Perls' method for tissue iron (hemosiderin), and Perls-Chayen's method for iron stored in the hemoglobin. The erythroid line in the anemic rabbits, showed a marked increase of proerythroblasts and basophilic erythroblasts, while the poli and orthochromatic erythroblasts were less than their precursors. In contrast these cells were more than their precursors in control animals. There was no notable quantitative difference in the mature elements of this line in the anemic animals and in the controls. Megaloblasts and macroblasts were frequently observed in anemic spleens but they were practically absent in the controls. Regarding to other cell lineages, we noted in the anemic spleens many macrophages containing Perls and Perls-Chayen positive material and some megakaryocytes. Our results indicate that the APH-induced anemia stimulate the erythropoietic activity of the spleen in the rabbit, but the reversion of the amplification phase of the differentiation steps reveals that the erythropoietic process is ineffective. The presence of megalo- and macroblasts provide morphological evidence of dyserythropoiesis and the megakaryocytes suggest that under the anemia condition also the platelet regenerating process is stimulated.     

Shiga toxin of enterohaemorrhagic Escherichia coli directly injures developing human erythrocytes

Haemolytic anaemia is one of the characteristics of life‐threatening extraintestinal complications in humans during infection with enterohaemorrhagic Escherichia coli (EHEC). Shiga toxins (Stxs) of EHEC preferentially damage microvascular endothelial cells of the kidney and the brain, whereby occluded small blood vessels may elicit anaemia through mechanical erythrocyte disruption. Here we show for the first time that Stx2a, the major virulence factor of EHEC, is also capable of direct targeting developing human erythrocytes. We employed an ex vivo erythropoiesis model using mobilized CD34⁺ haematopoietic stem/progenitor cells from human blood and monitored expression of Stx receptors and Stx2a‐mediated cellular injury of developing erythrocytes. CD34⁺ haematopoietic stem/progenitor cells were negative for Stx2a receptors and resistant towards the toxin. Expression of Stx2a‐binding glycosphingolipids and toxin sensitivity was apparent immediately after initiation of erythropoietic differentiation, peaked for basophilic and polychromatic erythroblast stages and declined during maturation into orthochromatic erythroblasts and reticulocytes, which became highly refractory to Stx2a. The observed Stx‐mediated toxicity towards erythroblasts during the course of erythropoiesis might contribute, although speculative at this stage of research, to the anaemia caused by Stx‐producing pathogens.     

Nucleolar changes in maturing avian erythroblasts and erythrocytes

Maturing erythroblasts and erythrocytes were studied in chickens and adult hens to provide more information on the presence and frequency of various nucleolar types in these cells. Nucleoli were present at all stages of erythroblastic and erythrocytic development except in the case of a few reticulocytes and the mature erythrocytes. The number of nucleoli per cell (expressed as the nucleolar coefficient) reached a maximum at the stage of the polychromatic erythroblast. Early erythroblasts were characterized by the presence of compact nucleoli or nucleoli with nucleolonemata. Rings shaped nucleoli and micronucleoli increased in number with further maturation. Cells of the final erythroblast stage (orthochromatic erythroblasts) contained mostly micronucleoli, and micronucleoli alone were present in reticulocytes and mature erythrocytes.     

Suppression and regeneration of rat bone marrow under the influence of methotrexate. An EM study.

Adult male rats were given daily injections of methotrexate for 12 days, during which time specimens of femoral bone marrow were taken for correlative light- and electron-microscopic study. After 12 days methotrexate was discontinued and specimens were taken the marrow recovered from the effect of methotrexate. Normal rat bone marrow was used as a basis of comparison. As methotrexate was administered, cell division ceased and the immature erythroblasts and myelocytes proceeded in the maturational process and a rapid hypocellularity of the marrow developed. Eosinophilic leukocytes, lymphocytes, large fat cells, erythrocytes, and phagocytic reticuloendothelial cells were the only formed elements remaining after 12 days of methotrexate treatment. Two days after methotrexate was discontinued, myeloblasts, myelocytes and proerythroblasts were plentiful, and on the third day the marrow exhibited an erythroid hyperplasia. By the eighth day all cell types were present though the marrow still was hypocellular. 14 days post methotrexate the marrow returned to normal.     

Cinemicrography of human erythroblasts--direct measurement of generation time and delineation of their pedigrees

To clarify the mode of erythropoiesis, the in vitro proliferation of single human erythroblasts was recorded continuously for 80 hours by time-lapse, phase-contrast cinemicrography. Progenies from single erythroblasts were followed, their pedigrees were delineated, and their generation times were measured by counting the frames of the film. In one erythroblast pedigree, a daughter cell continued to divide three times to yield eight smaller erythroblasts; another daughter cell yielded four progenies, three of which abruptly lost most of their cytoplasm and became immobile. The generation time ranged from 15.8-30.0 hours (mean +/- SD: 23.3 +/- 4.8 hours), which corresponded to generation times calculated from in vivo data, such as the mitotic index, isotope labeling, or red cell turnover. Paired daughter cells showed very similar generation times. During succeeding mitoses, erythroblast size and nucleus/cytoplasma ratio decreased, cytoplasm darkened, and cell movement became more prominent. These studies on clonal cell proliferation using cinemicrography provide considerable information on the mechanism of hemopoiesis.     

Cell kinetics, stomatal differentiation, and diurnal rhythm in Allium cepa

Cell kinetics parameters were obtained for the three mitotic divisions leading to formation of stomata in the epidermis of the cotyledon of Allium cepa seedlings. Analysis of mitotic frequencies throughout the course of development showed that the asymmetrical divisions started at about 50 hr after germination, and the symmetrical divisions were first seen a few hours later. Guard mother cell divisions started around 70 hr after germination. The maximal frequency of both symmetrical and asymmetrical division was found between 3 and 5 days after germination, and the highest frequencies of GMC divisions were observed between 6 and 8.5 days. All divisions ceased after 11 days. The three cell populations analyzed displayed diurnal fluctuations of their mitotic frequencies which were characteristic of the type of cell division measured and seemed independent of the region of the cotyledon in which they took place. The symmetrical divisions displayed two diurnal peaks—one at about 0400 and the other at 1600 hr—and the asymmetrical mitoses showed a single peak at about 2200 hr. Atypical asymmetrical divisions were observed in some guard mother cells, suggesting a different developmental sequence for some of the stomatal complexes.     

Periodontal ligament cell kinetics following orthodontic tooth movement.

The population of periodontal ligament (PDL) fibroblasts examined in this study may include osteogenic progenitor cells. PDL fibroblast and osteoblast kinetics in the periodontal ligament of the rat were measured following orthodontic stimulation of bone formation. Both single and multiple injections of tritiated thymidine (3H-TdR) were used. In single injection experiments, the peak percentage of PDL fibroblasts labeled with 3H-TdR is 15% at 22 hr post-stimulation. In multiple injection experiments, the total percentage of fibroblasts in the PDL which respond by synthesizing DNA is 50%. 3H-TdR-labeled osteoblasts appear at the same rate as, but with a time delay after, the labeled fibroblasts. Following stimulation, the most likely source of osteoblasts at the bbone forming site is not only fibroblasts which make DNA, divide, then differentiate, but also fibroblasts which either are differentiated to osteoblasts without DNA synthesis and cell division, or are released from G2 block by the orthodontic stimulation.     

Stimulation of rabbit erythroblast multiplication and maturation in vitro by blood leukocytes: cytological observations. 3. Studies on erythropoiesis

Blood leukocytes incubated in vitro with rabbit-marrow cells induced a several-fold increase in basophilic erythroblasts and smaller increases in acidophils and reticulocytes. The main effect was nearly complete in one hour at 37°. Erythropoietin augmented the leukocyte effect; anti-erythropoietin inhibited it with or without erythropoietin. The erythroblast increase came entirely from the marrow cells; the precursor cell class has not been identified, except that division of pre-existing basophils appears to be excluded. Autologous and homologous leukocytes were about equally effective. A method is described of measuring on stained smears changes in relative concentrations of different classes of cells induced experimentally. A method of preparing highly concentrated peripheral blood leukocytes is described.