Ivan N. Rich

The effect of reduced oxygen tension on colony formation of erythropoietic cells in vitro

Summary. The effect of reduced oxygen tension and the role of cellular components known to protect the cell against oxygen toxicity has been studied with respect to erythropoietic colony formation in vitro. Alphathioglycerol can be partially replaced by vitamin E and completely replaced by reduced glutathione (GSH) at physiological concentrations. Incubation of bone marrow and fetal Iiver early (BFU‐E) and late (CFU‐E) erythropoietic progenitor cells, in the presence of GSH, in an atmosphere containing 5% oxygen, 5% carbon dioxide and 90% nitrogen, as opposed to air supplemented with 5% carbon dioxide, resulted in an increase in colony numbers and response to erythropoietin (Epo). The number of colonies derived from bone marrow and fetal liver CFU‐E increased by 1.2‐2.8‐fold with a relative Epo sensitivity increase of 3.5‐4‐fold. Bursts obtained from bone marrow and fetal liver BFU‐E increased from 2.6‐ to 3.8‐fold with an increased response to Epo of 2‐3‐fold. The effects of GSH and low oxygen tension are interpreted as causing a reduction in oxygen toxicity of the cells, thereby increasing the life span in vitro and so increasing the number of cells capable of forming colonies. The heightened response of BFU‐E to Epo, analogous to the effect seen for CFU‐E, implies that BFU‐E may be responsive to physiological Epo concentrations at physiological oxygen tensions.

Serum erythropotietin and serum transferrin receptor levels in aplastic anaemia

Summary. Serum erythropoietin (EPO) and soluble transferrin receptor levels were serially measured in 74 patients with aplastic anaemia (AA). As control groups we investigated healthy controls (n = 24) and patients with iron‐deficiency (n = 23) or haemolytic anaemia (n = 16). There was a significant negative correlation of log EPO on haematocrit both in AA patients and in the anaemic control group. However, for the same degree of anaemia, log EPO levels in AA were significantly higher than in iron‐deficiency or haemolytic anaemia. EPO levels at diagnosis did not correlate with severity of aplastic anaemia, nor did they predict outcome after immunosuppression. During immunosuppressive treatment of AA with anti‐thymocyte globulin and cyclosporine A, EPO levels were significantly lower compared with pre‐treatment values without a corresponding change in haematocrit. This impaired EPO response to anaemia during immunosuppression might affect recovery of erythropoiesis. In AA patients, EPO levels declined with haemopoietic recovery. However, compared with normal controls, EPO levels in remission patients were still higher with respect to their haematocrit.

Erythroid colony formation (CFUe) in fetal liver and adult bone marrow and spleen from the mouse

SummaryThe methyl cellulose modification of the CFUe technique has been applied to 14 day fetal liver and adult bone marrow and spleen from CBA/CA mice. Optimized doses of fetal calf serum, α-thioglycerol, erythropoietin and cell suspensions have been obtained from dose response curves in order to standardize the technique. The slopes of the erythropoietin and cell dose response curves indicate a greater sensitivity by fetal liver to the hormone than bone marrow or spleen. The proportion of cells in the DNA synthesis phase of the cell cycle, as measured by the CFUe technique, has been estimated by administering hydroxyurea. Two hours after the drug was injected, 89% of fetal liver cells, 71% of bone marrow cells and 81% of spleen cells were found to be in the S-phase.ZusammenfassungMit einer modifizierten Methyl-Cellulose-Technik wurde der CFUe-Gehalt von fötaler Leber (Gestationsalter 14 Tage), von erwachsenem Knochenmark und Milz bei CBA/CA-Mäusen bestimmt. Die optimalen Kulturbedingungen wurden untersucht, indem Dosis-Wirkungskurven für fötales Kalbserum, α-Thioglycerol, Erythropoetin und Zelldosen durchgeführt werden. Fötale Leberzellen zeigten ein besseres Ansprechen auf Erythropoetin als die Knochenmark- und Milzzellen der Maus. Weiterhin wurde die Proliferationsaktivität der CFUe von verschiedenen hämopoetischen Geweben mit Hilfe des S-Phase-spezifischen Zytostatikums Hydroxyharnstoff untersucht. Der Anteil der CFUe in DNA-Synthese war verschieden für fötale Leber (89%), erwachsenes Knochenmark (71%) und Milz (81%).

Specific enhancement of mouse CFU-E by mouse transferrin.

Summary. Pure human and mouse transferrins were prepared by a chromatographic procedure and their effect on the growth of early (BFU‐E) and late (CFU‐E) erythropoietic precursors in mouse bone marrow is described. In the presence of optimal erythropoietin concentrations mouse bone marrow cells have a greater specificity for mouse transferrin (950 CFU‐E colonies/105 cells) than human transferrin (650 CFU‐E colonies/105 cells). Optimal transferrin concentrations for both human and mouse transferrins were 1.3 × 10−13 M and 1.3 × 10−10 M corresponding to between 7.8 × 107 and 7.8 × 1010 molecules/ml of culture. These concentrations are in excess of that calculated on a theoretical basis. Neither erythropoietic burst nor granulocyte/macrophage colony formation exhibited a dose dependent relationship for any of the transferrins employed, although higher colony numbers were obtained with mouse transferrin compared to human transferrin.

An erythropoietic stimulating factor similar to erythropoietin released by macrophages after treatment with silica

ZusammenfassungAus Zellsuspensionen von foetaler Leber, erwachsenem Knochenmark und Milz kann mittels Silika, einer makrophagenspezifischen zytotoxischen Substanz, ein Erythropoese stimulierender Faktor (ESF) freigesetzt werden. Dieser Faktor wird in Abhängigkeit von der Zelldosis freigesetzt und stimuliert sowohl foetale CFU-E-Kulturen als auch, nachdem er konzentriert wurde, die Erythropoese in polyglobulen Mäusen. Es ist daher wahrscheinlich, daß dieser ESF mit dem Erythropoetin identisch ist. Die Freisetzung dieses ESF aus Makrophagen von Mäusen wird durch eine experimentelle Polyglobulie unterdrückt und durch eine Anämie erhöht. Die Rolle dieses Makrophagen-Erythropoetins bei der Regulation der Erythropoese muß weiter abgeklärt werden.SummaryAn erythropoietic stimulating factor (ESF) can be detected in the supernatant from fetal liver and adult bone marrow and spleen cells when preincubated with the macrophage-specific cytotoxic agent, silica. Stimulation is observed in 12-day fetal liver CFU-E cultures in the absence of added erythropoietin (Ep). The concentration of ESF in the supernatant added to CFU-E cultures is dependent on the preincubated cell dose and the volume added. The stimulating activity is abolished when mice are hypertransfused and increased above normal values when mice are bled. A concentrated silicatreated spleen supernatant was able to stimulate erythropoiesis in the polycythemic mouse bioassay. It is concluded that the ESF is similar, if not identical, to Ep.

Erythroid Colony Forming Cells in Aplastic Anaemia

Summary. The concentration and erythropoietin dependence of erythropoietic progenitor cells (CFU‐E) were examined in 13 patients with aplastic anaemia at different stages of their disease. The CFU‐E incidence was shown to be quantitatively diminished in aplastic anaemia but tended to recover to normal values if the disease recovered. In addition the CFU‐E showed a qualitatively different response to stimulation by erythropoietin, being resistant to low concentrations but responsive to concentrations greater than 0.2 U/ml whereas there was a linear response in the controls up to 0.5 U/ml.

An ELISA specific for murine erythropoietin

Murine recombinant erythropoietin (EPO) was purified from an EPO‐producing cell line and used for the production of polyclonal monospecific anti‐murine EPO antibodies in rabbits. The anti‐mouse EPO antibodies were purified by two affinity chromatography procedures. In order to obtain the most sensitive ELISA, different antibody combinations were tested in the ELISA sandwich assay. The best combination was achieved with an anti‐human EPO antibody as coating and the biotinylated anti‐murine EPO antibody as detecting antibody. With this sandwich‐ELISA a sensitive standard curve in the range of 0.6–30 mU/ml could be established. The assay provides a sensitive and reliable measure of murine EPO in serum and cell culture supernatants ranging from normal to highly elevated EPO levels.

Erythropoietin Production by Macrophages: Cellular Response to Physiological Oxygen Tensions and Detection of Erythropoietin Gene Expression by In Situ Hybridization

Erythropoietin can be released from bone marrow-derived macrophages grown in culture. Evidence is presented to show that, when unseparated and unstimulated mouse bone marrow cells are incubated on hydrophobic PTE foils for 14 days under reduced oxygen tensions, a 98% pure population of macrophages results. The cells develop as a result of production of low GM-CSF concentrations by the macrophages themselves. Using phenotypic and functional markers such as the Ia antigen, 5’-nucleotidase, plasminogen activator, Interleukin-1 and lactoferrin, it is possible to classify these cells as belonging to a “resident” rather than an “inflammatory” macrophage population. It is from this population that erythropoietin can be detected in the extracellular medium. The erythropoietin activity is dependent on the prevailing physiological oxygen tension under which the cells are grown, the optimal being 3.5% O2. This erythropoietin activity can be neutralized by an anti-erythropoietin antiserum. Furthermore,addition of macrophages, derived from these cultures and grown under 2%, 3.5% or 5% oxygen tension, can stimulate bone marrow CFU-E to various degrees in the absence of exogenous erythropoietin. That the macrophage can actually produce erythropoietin is shown by the combined expression of the erythropoietin gene as detected by in situ hybridization and the binding of the monoclonal antibody directed against the mouse macrophage-specific F4/80 antigen. Because not all macrophages in the culture demonstrate in situ hybridization as detected using either a 35S- or biotin-labeled erythropoietin probe, it is possible that only a subpopulation of macrophages possess the capacity of expressing the erythropoietin gene.

The effect of actinomycin D on hemopoiesis

ZusammenfassungActinomycin D hemmt in niedriger Dosierung selektiv die Differenzierung der morphologisch erkennbaren Erythropoese, ohne die Granulopoese nennenswert zu beeinflussen. Um die cytotoxische Wirkung von Actinomycin D auf die Erythropoese zu charakterisieren und zu lokalisieren, wurden die verschiedenen hämopoetischen Vorläuferzellen (BFU-E, CFU-E, GM-CFC und CFU-S) vor und während einer sechstägigen Behandlung mit niedrigen Dosen von Actinomycin D (15μg/kg, 30μg/kg, 60μg/kg) untersucht. Die CFU-E nahmen unter Actinomycin D rasch ab und waren am sechsten Tag nur noch sehr spärlich nachweisbar. Die morphologisch differenzierten Erythroblasten zeigten einen in ihrer Kinetik ähnlichen Abfall, der allerdings etwas zeitverschoben war. Dagegen wurden die übrigen hämopoetischen Vorläuferzellen (BFU-E, GM-CFC, CFU-S) durch Actinomycin D in der verwendeten Dosis nicht nachweisbar geschädigt. Daraus wurde geschlossen, daß das Actinomycin D eine selektive Schädigung auf die CFU-E und die frühen Pronormoblasten bewirkt. Diese selektive Hemmung der CFU-E- und Pronormoblasten-Differenzierung beruht wahrscheinlich auf einer Inhibition der totalen RNA-Synthese. Aus der Zellgrößenanalyse durch die „velocity“-Sedimentationstechnik und der gegenüber den normalen CFU-E unveränderten Erythropoetin-Sensitivitätnach Actino mycin D wurde geschlossen, daß Actinomycin D eine sehr homogene CFU-E-Population „at random“ hemmt.SummaryThe effect of in vivo administration of actinomycin D (Act D) on the hemopoietic precursor compartments and, in particular, the BFU-E, CFU-E, and erythroblast populations was investigated over a 6-day period. Daily injections of mice with 15μg/kg, 30μg/kg, and 60μg/kg Act D showed a dose-dependent effect. The highest dose caused an almost complete eradication of CFU-E as well as the morphologically identifiable erythroblasts. There was no appreciable reduction in BFU-E, GM-CFU, and CFU-S. These observations indicate that Act D interferes with erythropoiesis by selectively inhibiting the CFU-E compartment. The effects are not due to altered sensitivity to erythropoietin as dose response curves were similar for control and Act D-treated cells. Although a considerable reduction in CFU-E is observed and approximately 20–30% of nucleated cells are lost from the small size region, there is no displacement in the velocity sedimentation profiles either from the remaining CFU-E and nucleated cell populations or the BFU-E and GM-CFU populations.

Introduction: Developmental Biology of Erythropoiesis

In this session, the mechanisms by which erythropoiesis, and therefore hemopoiesis, is established in vertebrates are considered through examples taken from toad, chicken, quail, mouse, and human. It is an interesting fact that the first differentiated cells seen in the embryo are hemopoietic; the heart is the first functional organ, and the circulation is the first functional system of the vertebrate body. It is the first of these that is important in the present context. The hemopoietic system originates from the mesoderm, and the formation of mesoderm is a prerequisite to understanding how the first hemopoietic stem cells are produced. Unfortunately, little is known about this mechanism at the present time, but this will probably change dramatically within the next few years. In considering how hemopoiesis is initiated in the embryo, the following questions are relevant: (1) Are the growth factors required for mesoderm induction also required for hemopoietic induction, or is a separate set of growth factors required? Furthermore, do these factors act together as part of an orchestrated event, or do they act individually? (2) Is the role of these growth factors similar in all vertebrates? (3) In which cells in the embryo are these factors produced, and at what time during development are they produced? (4) What are the mechanisms by which transcriptional factors become induced, and which transcriptional factors and genes are required for the initiation of hemopoiesis? (5 ) What is the stage of “sternness” and proliferative and differentiation potential of the first hemopoietic stem cells, and how can these be characterized considering the low number that must be present? (6) Are embryonic hemopoietic stem cells maintained in the embryo so that they can migrate and “seed” in sequential hemopoietic organs, or are different hemopoietic stem cell populations produced at different times during development for this purpose? (7) What is the role of homeotic genes in hemopoietic cells, and at what stage are they expressed? Many more questions can be asked, but the four presentations included in this session address some of the points raised above. For example, it is known that there are at least four different types of substances that are involved in inducing mesoderm from ectoderm in Xenopus, namely the fibroblast growth factor (FGF) and transforming growth factor-p (TGF-p) family of cytokines, the Wnt protooncogene, and noggin. Within the TGF-p family there are the bone morphogenic proteins (BMP), of which BMP-4 induces mesodermal structures, including blood formation. A new set of factors, namely the Xenopus fork-head transcription factors, which demonstrate conserved sequences with the human hepatocyte nuclear factors, are described in this session. In birds, the dorsal aorta is a production site of stem cells that colonize the yolk sac and other hemopoietic organs. A similar area in the mouse, called the