Kevin A. Rickard

Effect of Endotoxin on Granulopoiesis and Colony-Stimulating Factor

Abstract To study the regulation of granulopoiesis, we measured the effect of endotoxin on peripheral leukocyte counts and on colony-stimulating factor (CSF) in the serum of CF1 mice. The peripheral granulocyte level fell from 612 ± 69 (mean ± S.E.M.) to 147 ± 20 per cubic millimeter within 45 minutes of intraperitoneal injection of 5 μg of Salmonella typhosa endotoxin. In control animals, the CSF in 0.1 ml of serum stimulated the growth of 0.56 ± 0.4 in vitro myeloid colonies per 105 cells. Forty-five minutes after endotoxin the CSF activity had increased to 7.7 ± 6.1 colonies per 105 cells; after two hours it was 29.6 ± 10 colonies per 105 cells. Six hours after endotoxin the marrow was depleted of polymorphonuclear cells. Thereafter, sequential increases in the myeloblast-promyelocyte and myelocyte compartments of the marrow at 24 to 48 hours, respectively, suggested a wave of differentiation from a precursor compartment with subsequent maturation. The data suggest that intermittent endotoxemia may be ...

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.

Myeloid stem cell kinetics during erythropoietic stress.

Summary. The committed myeloid stem cell compartment, monitored by the agar colony technique, has been assayed in the bone marrow, spleen and blood of mice during erythropoietic stress. When red cell production appeared maximal, the size of the committed myeloid stem cell comparment was reduced in the bone marrow and there was usually a reciprocal rise in the spleen with varying numbers of these cells in the circulation. The role of stem cell competition and migration in the development of these changes is discussed.

The granulocytic in vitro growth potential of foetal liver

&NA; The recently described method for producing granulocytic colonies in soft agar from bone marrow cell suspensions has been extended to studies on murine foetal liver cell suspensions. The colonies in agar are considered to arise from a proliferating and committed myeloid stem cell compartment. It has been shown that a linear relationship exists between the number of foetal liver cells plated and the number of colonies produced. Further, for foetal liver and bone marrow, similar numbers of colonies are produced from similar cell numbers plated. This would imply a significantly sized committed myeloid stem cell compartment in the 161/2‐day‐old foetal liver, previously considered at this stage of development to be predominantly an erythroid organ. Wide differences in content of pluripotential stem cells have been demonstrated in foetal liver when compared to adult bone marrow.

Stem Cell Stimulatory Properties in Vitro of an Agar Colony-Stimulating Factor

Summary Bone marrow was incubated over a short period in vitro in the presence of WBI and normal serum. The number of cells capable of forming colonies in soft agar was maintained over a 48-hr period in the presence of WBI but not normal serum. Furthermore the decline in transplantable CFU was slower in the presence of WBI serum. Possible mechanisms for these results are discussed although firm conclusions cannot be drawn.

Haemopoietic stem cell stimulation in short-term tissue culture

Murine serum with high levels of agar colony stimulating factor (CSF) was examined for its ability to maintain myeloid stem cell and pluripotential stem cell numbers in a short term tissue culture of normal murine bone marrow. The serum was obtained from whole body irradiated (WBI) mice and had been previously shown to have high levels of CSF. The serum was mixed with normal bone marrow suspensions, whilst mixtures of normal serum and normal bone marrow suspensions served as controls. The content of pluripotential and myeloid stem cell compartments in these short term tissue cultures were assayed at regular intervals by bone marrow transplantation or the agar colony system respectively. In the presence of WBI serum myeloid stem cell numbers were maintained for periods up to 72 hr., whilst their numbers decreased rapidly in the presence of normal serum. Similarly, but to a lesser extent, pluripotential stem cell numbers were maintained in the presence of WBI serum. These data suggest that CSF appearing in vivo has a selective stimulatory effect on committed myeloid stem cell compartments in vitro . It is postulated that WBI serum may contain a single substance which influences both proliferation and differentiation of committed myeloid stem cells, and thus may be a specific regulator of granulopoiesis at this level. The slower decline of pluripotential stem cells in the presence of a maintained myeloid stem cell compartment may be due to a lower demand for pluripotential stem cells rather than direct stimulation at this level. Such findings are in accord with the notion that CSF may be involved in the humoral regulation of granulopoiesis.