Frederick Stohlman

REGULATION OF ERYTHROPOIESIS. XX. KINETICS OF RED CELL PRODUCTION

Since the last conference on erythropoiesis, much information has accumulated which has necessitated a revision of some of our notions as to the mechanism of red cell formation. It is still clear that the erythroid compartment is not a self-maintaining compartment, but is fed from a primitive stem cell pool. The work of Becker et a1.I and Whang et aL2 appeared to settle the long standing issue whether this is a unipotential or totipotential compartment. More recent evidence, however, raises the question as to whether there may not be two levels of stem cells, a more primitive totipotential stem cell compartment equivalent to the colony-forming unit of Till and McCulloch3 and a more differentiated precursor cell for each of the identifiable hematopoietic elements. This issue is by no means settled, but data will be presented favoring the latter explanation, i.e., two levels of precursor cells. The studies on the effect of erythropoietin in the hypertransfused rodent clearly demonstrated that erythropoietin is capable of differentiating the stem cell into an identifiable erythroid precursor. The mechanism by which this is accomplished is moot and the question of whether the action of erythropoietin is confined to the stem cell or whether it may also affect differentiated cells is at present undecided. In this paper it is our purpose to present data bearing on these points. The effect of erythropoietin has been studied in normal animals and in rats in which red cell production has been perturbed by hypertransfusion and/or vincristine. Critical to these considerations are studies on megakaryocytopoiesis using tritiated thymidine. These also will be reported briefly.

A Differential Effect of Hydroxyurea on Hemopoietic Stem Cell Colonies in Vitro and in Vivo

Summary When bone marrow stem cells are assayed simultaneously by the spleen colony technique and the in vitro agar system following treatment of donors with hydroxyurea a differential effect was observed. There was found to be approximately a 50% reduction in the number of bone marrow in vitro colony forming cells after DNA synthesis inhibition by hydroxyurea and only a slight change in the pluripotential stem cell compartment by the spleen colony technique. It appears therefore that the in vitro colony forming cell derives from a proliferating stem cell compartment, probably myeloid committed, and is distinct from the nonproliferating pluripotential transplantable stem cell.

Regulation of erythropoiesis. XXI. The effect of erythropoietin on the stem cell.

Summary Nine units of erythropoietin given in equally divided doses at 8-hour intervals for 23 days to the CAF1 mouse produced a significantly greater polycythemia than had been observed in this strain when exposed to 23,000 feet for a similar period of time. The splenic CFU began to increase within 24 hours after the beginning of treatment and reached levels of 5-fold those of controls between days 8 and 16. The extent to which migration of CFU from the marrow and or replication of CFU in situ is responsible for the increase in splenic CFU is discussed. It is concluded that the stem cell participates in the physiologic response to erythropoietin but without depletion of this compartment. The possibility that the previously reported decrease in CFU in hypoxic animals represents a secondary perhaps “toxic” effect of hypoxia unrelated to erythropoietin is considered.

Regulation of Erythropoiesis. XV. Neonatal Erythropoiesis and the Effect of Nephrectomy

The importance of erythropoietin as a regulator of erythropoiesis is undeniable, but that it is the sole regulator is controversial. Fried, Plzak, Jacobson, and Goldwasser (1) proposed a simple, unified concept for the regulation of red cell production. They suggested that the relationship of oxygen supply to demand governs the production of erythropoietin, which, in turn, through differentiation of the stem cells controls red cell production. An increased cell mass, e.g., through transfusion, in the presence of a normal Po2 would decrease erythropoietin production and erythropoiesis by increasing the oxygen supply. Conversely, anemia by decreasing the 02 supply would stimulate erythropoietin production. Changes in metabolic rate, e.g., starvation, would affect erythropoietin production and, hence, erythropoiesis by changes in 0° demand. As attractive as this hypothesis is, it does not appear to offer a satisfactory explanation for compensated hemolytic states (2), continued production of substantial numbers of red cells, although fewer than normal, in the polycythemic dog (3), or the stimulating effect of hemoglobin recently reported by Brown, Altschuler, and Cooper (4) and Sanchez-Medal, Labardini, and Loria (5). Jacobson, Goldwasser, Fried, and Plzak proposed the kidney as the site of production of erythropoietin (6). This conclusion was based on the striking decrease in erythropoiesis observed after bilateral nephrectomy but not ureteral ligation in the rodent. Similiar findings were reported by Naets in the dog (7), but Nathan, Schupak, Stohlman, and Merrill (8) observed substantially less suppression of red cell production in anephric

HEMOPOIETIC STEM CELL PROLIFERATION AND MIGRATION FOLLOWING BORDETELLA PERTUSSIS VACCINE

The ability of a single injection of killed, intact bacteria to effect an increase in the proliferative rate of hemopoietic stem cells was studied. The total numbers of colony forming units in bone marrow, spleen and peripheral blood as well as the proportion of CFU in cycle was assessed. Splenic CFU were observed to rise exponentially due initially to in situ proliferation and later to proliferation in bone marrow with migration via the blood to the spleen. The results are discussed in the light of current concepts of stem cell regulation.

The Effect of Hydroxyurea on Differentiated Marrow Erythroid Precursors

Summary A single i.v. dose of hydroxyurea (0.9 g/kg) produced transient marrow erythroid depopulation and reticulocytopenia in the mouse. Within minutes after drug administration there was a virtually complete erradication of marrow DNA synthesis which persisted for approximately 2 hr. Erythroid cell cycle kinetics, estimated with colchicine, revealed an entry of erythroid cells into mitosis for 80 min followed by no further accumulation of metaphase forms until the seventh hour. Thereafter erythroid cells accumulated in mitosis at a normal rate. These findings are consistent with the hypothesis that hydroxyurea kills erythroid cells in DNA synthesis; the initial 80-min accumulation of erythroid mitoses represents the progression of G2 cells into mitosis. Unaffected cells in G1 at the time of drug administration require 7-8 hr to progress through DNA synthesis, G2, and enter mitosis. From these data it would appear that hydroxyurea may prove a useful agent in the study of erythropoiesis because of its selective effect on DNA synthesis in the erythroid series.

Erythropoiesis in the Dog: The Periodic Nature of the Steady State

In at least six of 11 normal dogs serial measurement of reticulocyte counts showed oscillation with a period of approximately 14 days. The phase of oscillation could be altered by bleeding followed by retransfusion. The observations suggest that canine erythropoiesis is an example of a physiological rhythm which has its origin in homeostatic control.

Studies on the Regulation of Granulopoiesis

Tritiated thymidine autoradiography was used to study the control of granulocyte production and release using the irradiated leg shielded mouse as an experimental model. Neutropenia resulted in a shortening of 24–36 hr in the mean transit time through the non‐proliferating granulocyte compartment. There was little difference between neutropenic and control animals in labelling indices of the cells in the proliferative granulocyte compartments, which suggests that the granulocytic hyperplasia observed in the neutropenic mice was predominantly due to differentiation of morphologically unrecognizable precursor cells rather than increased proliferation of morphologically recognizable cells. The persistence of a labelling index of approximately 60% in the myeloblast‐promyelocyte compartment 24 hr after injection suggests that these precursor cells were rapidly proliferating.

A Difference in Erythropoietin Production between Anemic and Hypoxic Mice

Summary A strain difference in the response to hypoxia of the CAF1 and CF1 mice is described. The CF1 strain developed significant erythropoietin production in response to hypoxia, and after 3 weeks marked polycythemia. In the CAF1 mouse both erythropoietin production and polycythemia were minimal. Both strains produced comparable amounts of erythropoietin in response to post-hemorrhagic or hemolytic anemia. The implications of these differences are discussed.

Humoral Regulation of Erythropoiesis XII. Effect of Erythropoietin and Iron on Cell Size in Iron Deficiency Anemia.

Summary Erythropoietin did not affect the size distribution curve of iron deficient animals even when given in high doses. The type of erythroid response occurring after treatment with iron was related to the dose of iron administered, a macrocytic response being evoked after high doses and a normocytic response after low doses. The mechanism of action of erythropoietin and its relationship to available iron is discussed and a hypothesis presented to explain the above observations.