Delayed neutrophil apoptosis after total body irradiation in mice. The role of granulocyte-macrophage colony-stimulating factor in neutrophil function.

We performed an in vitro study of the long-term effects of a sublethal dose (5 Gy) of x-irradiation on the survival and function of neutrophils in adult mice. For this purpose, we incubated control neutrophils harvested from long-term bone marrow cultures (LTBMCs) with supernatant withdrawn from cultures obtained in adult mice 6 or 9 months postirradiation. We noted a significant increase in superoxide anion production, NADPH, and protein levels in these cells after 3, 6, and 15 hours of incubation compared with the same cells incubated with supernatant from control LTBMCs. We also observed a delay in apoptosis that was correlated with maintenance of adenosine triphosphate levels and survival. Similar differences were found when control LTBMC neutrophils were incubated with granulocyte-macrophage colony-stimulating factor (GM-CSF) (1.3 nM). Indeed, enzyme-linked immunosorbent assay revealed a significant overproduction of this cytokine, together with higher interleukin (IL)-6 and IL-3 levels, in the supernatant from cultures of irradiated mice. Our results suggest that GM-CSF is one of the cytokines responsible for promoting the survival and activation of neutrophil function after total body irradiation.

Glucose metabolism in bone marrow cells and granulocytes of adult mice after X-ray (5 Gy) irradiation: relationship to cell functionality.

Our experiments focused on the metabolic implications of the residual haemopoietic damage in adult mice given 5 Gy X-rays. Bone marrow cells from irradiated mice exhibited an increase in protein synthesis and a decrease in ATP levels, which could be related to the enhancement of the proliferative activity of haemopoietic precursor cells. However, the kinetic parameters (Vmax and Km) of glucose uptake, the glycolytic flux and the hexose monophosphate (HMP) shunt activity were similar to those found in the control group. On the other hand, a reduction of glucose uptake (Vmax) was found in both resting and stimulated granulocytes from irradiated mice. This reduction was accompanied by a decrease in the glycolytic rate and ATP levels. However, HMP shunt activity was similar in resting granulocytes in both the control and the irradiated mice. The stimulation by PMA produced a significantly higher increase in the activity of the pathway in granulocytes from the irradiated mice and was in accordance with the enhancement of superoxide anion production that has been previously described in these cells.

Long-term haemopoietic injury in mouse by 1 Gy X-rays at different postconceptional stages.

Haemopoietic effects in mice produced by a single acute dose of 1 Gy X-rays given on the 4th (4R) and 13th (13R) day postconception were evaluated throughout the 9-month post-irradiation. The quality of the stroma was measured in long-term bone marrow cultures (LTBMC). LTBMC granulocyte production was always markedly lower among the irradiated offspring than among controls. IL-6 supernatant levels were significantly higher in both experimental models with respect to controls. However, supernatants from 13R mice had a greater colony-stimulating activity than 4R mice and controls. Granulocytes, from culture or peripheral blood, did not show changes in their functional activity after either treatment. With regard to the content of the femoral granulocyte-macrophage colony forming cells (GM-CFC), there was an enhancement of the GM-colony number as determined from the marrow of 13R mice. In these mice, an alteration in colony size and number due to different combinations of colony-stimulating factors was observed. In summary, our results obtained with irradiation during the foetal period suggest that this 1 Gy X-rays is sufficient to produce measurable, effects on developing murine haemopoiesis.

Mechanisms towards compensation of long-term haemopoietic injury in mice after 5 Gy irradiation: in vivo and in vitro enhancement of superoxide anion production by granulocytes.

This paper analyzes the long-term (6 and 12 months) function of mouse granulocytes after total body irradiation with a single dose (5 Gy) of X-rays. Superoxide anion production has been investigated in granulocytes from peripheral blood, and also in those harvested from long term bone marrow cultures, with the aim of correlating the environmental damage induced by radiation with the functional properties of granulocytes. An in vivo and in vitro enhancement of superoxide anion production and protein levels in granulocytes from irradiated mice is described. The presence of some colony stimulating factor in the supernatant of cultures from irradiated mice could play an important role in the priming of granulocytes.

Transferrin binding of bone marrow cells and metabolic activity of erythrocytes after 5 Gy irradiation.

The effect of total body irradiation (5 Gy) on functional mouse erythroid lineage has been studied. The transferrin binding capacity by bone marrow cells and the activity of glycolytic regulatory enzymes and intracellular levels of 2,3 bisphosphoglycerate in peripheral blood erythrocytes have been determined. Results obtained along one year post-irradiation period suggest a complete recovery in the erythroid cell lineage with respect to the biological endpoints investigated.

Kinetic studies of pyruvate kinase during in vitro differentiation of GM-CFC haemopoietic precursor and bone marrow cells in mice.

Pyruvate kinase studies in the granulocyte-macrophage lineage during in vitro differentiation have been performed using culture techniques on GM-CFC cells and a study has also been done in bone marrow cells. The enzyme exhibits biphasic behaviour with respect to both of its substrates in cells derived from in vitro cultures at 5 and 7 days of incubation period. However in bone marrow cells these kinetics are only observed for ADP. The different kinetic behaviour of pyruvate kinase toward Fru-1,6-P2, Ala, Phe and ATP in the three cellular populations allows us to conclude that the expression of pyruvate kinase is associated with the differentiation of these cells.

Role of phosphofructokinase during trout haemopoiesis: physiological regulation of glycolysis.

1. The regulatory properties of phosphofructokinase (PFK) has been investigated in two cellular population representatives of trout haemopoiesis; haemopoietic cells (capable of replication and differentiation) and erythrocytes (highly specialized cells). 2. The intracellular levels of substrates and effectors have been quantified and their effect on PFK activity determined. 3. Fructose 1,6-bisphosphate anc cyclic AMP show a higher activation of the PFK from haemopoietic cells than the enzyme from erythrocytes. 4. AMP and phosphoenolpyruvate act as activators of the haemopoietic cell PFK while for erythrocytes PFK, AMP is an inhibitor and phosphoenolpyruvate does not display any effect. 5. Citrate inhibits PFK activity from haemopoietic cells but was not assayed in erythrocytes since it was not detected in these cells. 6. The differences in PFK regulation in both cellular populations may be attributed to the intracellular levels of the effectors and/or different isoenzymatic patterns. 7. The different regulation of PFK together with the higher enzymatic activity of PFK and pyruvate kinase from haemopoietic cells are related to the higher glycolytic flux that exhibits the haemopoietic cells. 8. The results shown in this investigation allow us to conclude that PFK has a specific role depending on the energetic requirements of the cellular population in which the enzyme is present. 9. The requirements are related to the physiological function of each type of cell.

Pyruvate kinase during in vitro differentiation of GM-CFC haemopoietic precursor in mice: modulation by L-alanine and L-phenylalanine.

Pyruvate kinase modulation by L-alanine and L-phenylalanine was studied in cells obtained from in vitro culture of GM-CFC at different stages of development. L-Alanine and L-phenylalanine exhibit different inhibition behaviour at both differentiation stages and at various PEP concentration ranges. Therefore, a change in PK expression during granulocyte-macrophage development is suggested.