Homeostatic action of adenosine A3 and A1 receptor agonists on proliferation of hematopoietic precursor cells.

Two adenosine receptor agonists, N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA) and N6-cyclopentyladenosine (CPA), which selectively activate adenosine A3 and A1 receptors, respectively, were tested for their ability to influence proliferation of granulocytic and erythroid cells in femoral bone marrow of mice using morphological criteria. Agonists were given intraperitoneally to mice in repeated isomolar doses of 200 nmol/kg. Three variants of experiments were performed to investigate the action of the agonists under normal resting state of mice and in phases of cell depletion and subsequent regeneration after treatment with the cytotoxic drug 5-fluorouracil. In the case of granulopoiesis, IB-MECA 1) increased by a moderate but significant level proliferation of cells under normal resting state; 2) strongly increased proliferation of cells in the cell depletion phase; but 3) did not influence cell proliferation in the regeneration phase. CPA did not influence cell proliferation under normal resting state and in the cell depletion phase, but strongly suppressed the overshooting cell proliferation in the regeneration phase. The stimulatory effect of IB-MECA on cell proliferation of erythroid cells was observed only when this agonist was administered during the cell depletion phase. CPA did not modulate erythroid proliferation in any of the functional states investigated, probably due to the lower demand for cell production as compared with granulopoiesis. The results indicate opposite effects of the two adenosine receptor agonists on proliferation of hematopoietic cells and suggest the plasticity and homeostatic role of the adenosine receptor expression.

Ultrafiltered pig leukocyte extract (IMUNOR) decreases nitric oxide formation and hematopoiesis-stimulating cytokine production in lipopolysaccharide-stimulated RAW 264.7 macrophages.

A low-molecular-weight (<12 kDa) ultrafiltered pig leukocyte extract, IMUNOR, was tested in experiments in vitro on non-stimulated and lipopolysaccharide (LPS)-stimulated murine RAW 264.7 macrophages in order to assess modulation of nitric oxide (NO) production (measured indirectly as the concentration of nitrites), hematopoiesis-stimulating activity of the supernatant of the macrophage cells (ascertained by counting cell colonies growing from progenitor cells for granulocytes and macrophages (GM-CFC) in vitro), and the release of hematopoiesis-stimulating cytokines. No hematopoiesis-stimulating activity and cytokine or NO production were found in the supernatant of non-stimulated macrophages. It was found that IMUNOR does not influence this status. Supernatant of LPS-stimulated macrophages was characterized by hematopoiesis-stimulating activity, as well as by the presence of nitrites, interleukin-6 (IL-6), and granulocyte colony-stimulating factor (G-CSF). A key role in the hematopoiesis-stimulating activity of the supernatant of LPS-stimulated macrophages could be ascribed to G-CSF since the formation of the colonies could be abrogated nearly completely by monoclonal antibodies against G-CSF. IMUNOR was found to suppress all the mentioned manifestations of the LPS-activated macrophages. When considering these results together with those from our previous in vivo study revealing stimulatory effects of IMUNOR on radiation-suppressed hematopoiesis, a hypothesis may be formulated which postulates a homeostatic role of IMUNOR, consisting in stimulation of impaired immune and hematopoietic systems but also in cutting back the production of proinflammatory mediators in cases of overstimulation which threats with undesirable consequences.

The role of G-CSF and IL-6 in the granulopoiesis-stimulating activity of murine blood serum induced by perorally administered ultrafiltered pig leukocyte extract, IMUNOR.

IMUNOR, a low-molecular weight (< 12 kD) ultrafiltered pig leukocyte extract, has been previously found to have significant stimulatory effects on murine hematopoiesis supressed by ionizing radiation or cytotoxic drugs. This communication shows data on the mechanisms of these effects. Using ELISA assay, significantly increased levels of granulocyte colony-stimulating factor (G-CSF) and interleukin-6 (IL-6) were observed. On the contrary, no detectable levels of granulocyte-macrophage colony-stimulating factor (GM-CFC) and interleukin-3 (IL-3) have been found in blood serum of IMUNOR-treated mice. Incubation of the serum from IMUNOR-treated mice with antibodies against G-CSF caused abrogation of the ability of the sera to stimulate in vitro growth of colonies originating from granulocyte-macrophage progenitor cells (GM-CFC). In contrast, incubation of the serum with antibodies against IL-6 did not change its colony-stimulating activity. It may be inferred from these findings that G-CSF is probably the main cytokine responsible for the granulopoiesis-stimulating effects of IMUNOR. When the serum from IMUNOR-treated mice with G-CSF inactivated by anti-G-CSF antibodies (but with elevated IL-6) was added to cultures of bone marrow cells together with a suboptimum concentration of IL-3, a significant increase in the numbers of GM-CFC colonies was found. Moreover, conjoint inactivation of G-CSF and IL-6 significantly decreased the numbers of GM-CFC colonies in comparison with those observed when only G-CSF was inactivated. This observation strongly suggests that though IMUNOR-induced IL-6 is not able to induce the growth of GM-CFC colonies alone, it is able to potentiate the hematopoiesis-stimulating effect of IL-3. These findings represent a new knowledge concerning the hematopoiesis-stimulating action of IMUNOR, a promising immunomodulatory agent.

Adenosine A(3) receptor agonist acts as a homeostatic regulator of bone marrow hematopoiesis.

The present study was performed to define the optimum conditions of the stimulatory action of the adenosine A(3) receptor agonist, N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA), on bone marrow hematopoiesis in mice. Effects of 2-day treatment with IB-MECA given at single doses of 200nmol/kg twice daily were investigated in normal mice and in mice whose femoral bone marrow cells were either depleted or regenerating after pretreatment with the cytotoxic drug 5-fluorouracil. Morphological criteria were used to determine the proliferation state of the granulocytic and erythroid cell systems. Significant negative correlation between the control proliferation state and the increase of cell proliferation after IB-MECA treatment irrespective of the cell lineage investigated was found. The results suggest the homeostatic character of the induced stimulatory effects and the need to respect the functional state of the target tissue when investigating effects of adenosine receptor agonists under in vivo conditions.

Effects of adenosine A(3) receptor agonist on bone marrow granulocytic system in 5-fluorouracil-treated mice.

The purpose of the experiments reported was to investigate effects of N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA), a selective adenosine A(3) receptor agonist, on the granulocytic system in femoral marrow of mice depleted by the cytotoxic drug 5-fluorouracil. In the phase of the highest cell depletion IB-MECA was injected i.p. at single doses of 200 nmol/kg given either once or twice daily in 2- and 4-day regimens starting on day 1 after 5-fluorouracil administration; the effects were evaluated on days 3 and 5, respectively. The general effect of IB-MECA in all these experiments was an enhancement of the counts of morphologically recognizable proliferative granulocytic cells, interpreted as evidence of the differentiation of committed progenitor cells. A more expressive effect was observed after IB-MECA injected twice daily. It was found that the induction of the strong differentiation pressures by IB-MECA given twice daily shortly after 5-fluorouracil treatment can be counterproductive due to the preponderance of differentiaton processes over the proliferation control. In additional experiments, it has been shown that the use of the 2-day administration of IB-MECA given twice daily in the recovery phase, i.e., on days 5 and 6 after 5-fluorouracil administration, does not induce stimulatory effects. Thus, the dosing and timing of IB-MECA treatment determines its effectivity in stimulating granulopoiesis under conditions of myelosuppression.

N6-cyclopentyladenosine inhibits proliferation of murine haematopoietic progenitor cells in vivo.

Effects of N6-cyclopentyladenosine (CPA), the selective adenosine A1 receptor agonist, on bone marrow haematopoietic progenitor cells for granulocytes and macrophages (CFC-GM) were investigated by utilizing the model of haematopoietic damage induced by 5-fluorouracil. Experiments were performed in vivo on B10CBAF1 mice. A single i.p. injection of CPA at the optimum dose of 200 nmol/kg administered 22 h before a single injection of 5-fluorouracil (100 mg/kg, i.p.) protected CFC-GM against the cytotoxic damage as determined 4 days later. Isomolar doses of the selective agonists for adenosine A2A receptors, i.e. 2-p-(2-carboxyethyl)-phenethylamino-5'-N-ethylcarboxamidoadenosine, and for adenosine A3 receptors, i.e. N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide, did not induce such effects. Because 5-fluorouracil is a cell cycle-specific drug damaging mainly cells in the S-phase, protective effects of CPA can be explained by its inhibitory action on the cell cycling. This interpretation was confirmed by experiments demonstrating that repeated administration of CPA in the hyperproliferation phase of the recovering haematopoiesis after 5-fluorouracil treatment inhibited transiently restoration of CFC-GM counts.

Basal and induced granulopoiesis in outbred, F1 hybrid and inbred mice: can inbreeding depression influence the experimental practice?

In this study we examined differences in selected indices of granulopoiesis in outbred, F(1) hybrid and inbred mouse strains. Specifically, serum granulocyte colony-stimulating factor (G-CSF) levels, numbers of marrow granulocyte-macrophage progenitor cells and morphologically recognizable proliferative marrow granulocytic precursor cells were evaluated. These parameters were determined in untreated controls, and in mice exposed either to a non-specific stimulus (injection of saline) or to a granulopoiesis-enhancing stimulus (administration of a cyclooxygenase-2 inhibitor, meloxicam). Lower levels of G-CSF were detectable in the outbred ICR mice, which also demonstrated an enhanced response to both types of the stimuli. Considering the fact that outbred mice are closer to natural mammalian populations, including human ones, the possibility of using outbred mice, instead of the often used inbred strains, for experiments evaluating the effects of pharmacological interventions on hematopoiesis should be investigated.

Activation of adenosine A(3) receptors supports hematopoiesis-stimulating effects of granulocyte colony-stimulating factor in sublethally irradiated mice.

PURPOSE: Research areas of 'post-exposure treatment' and 'cytokines and growth factors' have top priority among studies aimed at radiological nuclear threat countermeasures. The experiments were aimed at testing the ability of N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA), an adenosine A(3) receptor agonist, to modulate hematopoiesis in sublethally irradiated mice, when administered alone or in a combination with granulocyte colony-stimulating factor (G-CSF) in a two-day post-irradiation treatment regimen. MATERIALS AND METHODS: A complete analysis of hematopoiesis including determination of numbers of bone marrow hematopoietic progenitor and precursor cells, as well as of numbers of peripheral blood cells, was performed. The outcomes of the treatment were assessed at days 3 to 22 after irradiation. RESULTS: IB-MECA alone has been found to induce a significant elevation of numbers of bone marrow granulocyte-macrophage progenitor cells (GM-CFC) and peripheral blood neutrophils. IB-MECA given concomitantly with G-CSF increased significantly bone marrow GM-CFC and erythroid progenitor cells (BFU-E) in comparison with the controls and with animals administered each of the drugs alone. CONCLUSIONS: The findings suggest the ability of IB-MECA to stimulate hematopoiesis and to support the hematopoiesis-stimulating effects of G-CSF in sublethally irradiated mice.

Effect of adenosine on the growth of human T-lymphocyte leukemia cell line MOLT-4.

Adenosine has been observed to suppress the growth of MOLT-4 human leukemia cells in vitro. Changes in the cell cycle, especially increased percentage of cells in S phase, prolonged generation time, and induction of apoptosis at higher adenosine concentrations have been found to be responsible for the growth suppression. Dipyridamole, a drug inhibiting the cellular uptake of adenosine, reversed partially but significantly the adenosine-induced growth suppression. It follows from these results that the action of adenosine on the MOLT-4 cells comprises its cellular uptake and intracellular operation. These findings present new data on anticancer efficacy of adenosine.

Drugs elevating extracellular adenosine promote regeneration of haematopoietic progenitor cells in severely myelosuppressed mice: their comparison and joint effects with the granulocyte colony-stimulating factor.

We tested capabilities of drugs elevating extracellular adenosine and of granulocyte colony-stimulating factor (G-CSF) given alone or in combination to modulate regeneration from severe myelosuppression resulting from combined exposure of mice to ionizing radiation and carboplatin. Elevation of extracellular adenosine was induced by joint administration of dipyridamole (DP), a drug inhibiting the cellular uptake of adenosine, and adenosine monophosphate (AMP), serving as an adenosine prodrug. DP+AMP, G-CSF or all these drugs in combination were administered in a 4-d treatment regimen starting on day 3 after induction of myelosuppression. Comparable enhancements of haematopoietic regeneration due to elevation of extracellular adenosine or to action of G-CSF were demonstrated as shown by elevated numbers of haematopoietic progenitor cells for granulocytes/macrophages (GM-CFC) and erythrocytes (BFU-E) in the bone marrow and spleen in early time intervals after termination of the drug treatment, i.e. on days 7 and 10 after induction of myelosuppression. Coadministration of all the drugs further potentiated the restoration of progenitor cell pools in the haematopoietic organs. The effects of the drug treatments on progenitor cells were reflected in the peripheral blood in later time intervals of days 15 and 20 after induction of myelosuppression, especially as significantly elevated numbers of granulocytes and less pronounced elevation of lymphocytes and erythrocytes. The results substantiate the potential of drugs elevating extracellular adenosine for clinical utilization in myelosuppressive states, e.g. those accompanying oncological radio- and chemotherapy.