Administration of interleukin-6 stimulates multilineage hematopoiesis and accelerates recovery from radiation-induced hematopoietic depression

Hematopoietic depression and subsequent susceptibility to potentially lethal opportunistic infections are well-documented phenomena following radiotherapy. Methods to therapeutically mitigate radiation-induced myelosuppression could offer great clinical value. In vivo studies in our laboratory have demonstrated that interleukin-6 (IL-6) stimulates pluripotent hematopoietic stem cell (CFU-s), granulocyte-macrophage progenitor cell (GM-CFC), and erythroid progenitor cell (CFU-e) proliferation in normal mice. Based on these results, the ability of IL- 6 to stimulate hematopoietic regeneration following radiation-induced hematopoietic injury was also evaluated. C3H/HeN female mice were exposed to 6.5 Gy 60Co radiation and subcutaneously administered either saline or IL-6 (1,000 micrograms/kg) on days 1 through 3 or 1 through 6 postexposure. On days 7, 10, 14, 17, and 22, femoral and splenic CFU-s, GM-CFC, and CFU-e contents and peripheral blood white cell, red cell, and platelet counts were determined. Compared with saline treatment, both 3-day and 6-day IL-6 treatments accelerated hematopoietic recovery; 6-day treatment produced the greater effects. For example, compared with normal control values (N), femoral and splenic CFU-s numbers in IL-6-treated mice 17 days postirradiation were 27% N and 136% N versus 2% N and 10% N in saline-treated mice. At the same time, bone marrow and splenic GM-CFC values were 58% N and 473% N versus 6% N and 196% N in saline-treated mice; bone marrow and splenic CFU-e numbers were 91% N and 250% N versus 31% N and 130% N in saline-treated mice; and peripheral blood white cell, red cell, and platelet values were 210% N, 60% N, and 24% N versus 18% N, 39% N, and 7% N in saline- treated mice. These studies demonstrate that therapeutically administered IL-6 can effectively accelerate multilineage hematopoietic recovery following radiation-induced hematopoietic injury.     

Effects of an aplastic anemia urinary extract on mouse erythroid progenitor cells in vivo

We investigated the in vivo effects of a crude extract from the urine of aplastic anemia patients (AA urinary extract) on erythroid precursor cells in the femoral bone marrow and spleens of normal adult mice. A single intraperitoneal injection of AA urinary extract induced a significant increase in the number of splenic erythroid burst-forming units (BFU-e) and erythroid colony-forming units (CFU-e) within 24 h after injection. We then injected pure recombinant erythropoietin (Epo) equivalent to the amount present in the urinary extract. This addition increased the number of splenic CFU-e by almost the same degree as the amount induced by the AA urinary extract 24 h after injection, but failed to elicit any change in the number of splenic BFU-e. In other studies, mice were injected with the same amount of lipopolysaccharide (LPS) and/or pure Epo as that present in the AA urinary extract. Experiments with Limulus amebocyte lysate-adsorbed (endotoxin-depleted) or nonadsorbed (endotoxin-containing) AA urinary extracts showed that endotoxin contamination interfered with the increase in numbers of marrow CFU-e and enhanced the increase in splenic CFU-e numbers induced by pure Epo or Epo activity in the AA urinary extract. The number of splenic BFU-e, however, was not affected by administration of LPS and/or Epo or by adsorbed endotoxin. These data suggest that AA urinary extract contains a stimulating activity for mouse splenic BFU-e, and that this activity is not attributable to the Epo activity or endotoxin contamination within the urinary extract.     

Survival enhancement and hemopoietic regeneration following radiation exposure: therapeutic approach using glucan and granulocyte colony-stimulating factor.

C3H/HeN female mice were exposed to wholebody cobalt-60 radiation and administered soluble glucan (5 mg i.v. at 1 h following exposure), recombinant human granulocyte colony-stimulating factor (G-CSF; 2.5 micrograms/day s.c., days 3-12 following exposure), or both agents. Treatments were evaluated for their ability to enhance hemopoietic regeneration, and to increase survival after radiation-induced myelosuppression. Both glucan and G-CSF enhanced hemopoietic regeneration alone; however, greater effects were observed in mice receiving both agents. For example, on day 17 following a sublethal 6.5-Gy radiation exposure, mice treated with saline, G-CSF, glucan, or both agents, respectively, exhibited 36%, 65%, 50%, and 78% of normal bone marrow cellularity, and 84%, 175%, 152%, and 212% of normal splenic cellularity. At this same time, granulocyte-macrophage colony-forming cell (GM-CFC) values in saline, G-CSF, glucan, or combination-treated mice, respectively, were 9%, 46%, 26%, and 57% of normal bone marrow values, and 57%, 937%, 364%, and 1477% of normal splenic values. Endogenous spleen colony formation was also increased in all treatment groups, with combination-treated mice exhibiting the greatest effects. Likewise, although both glucan and G-CSF alone enhanced survival following an 8-Gy radiation exposure, greatest survival was observed in mice treated with both agents. These studies suggest that glucan, a macrophage activator, can synergize with G-CSF to further accelerate hemopoietic regeneration and increase survival following radiation-induced myelosuppression.     

The effects of 5-fluorouracil on hematopoiesis: studies of murine megakaryocyte-CFC, granulocyte-macrophage-CFC, and peripheral blood cell levels

the effect of 5-fluorouracil (5-FU) on megakaryocytopoiesis in mice was studied with assays of megakaryocyte colony-forming cells (Meg-CFC) in bone marrow and spleen and simultaneous determinations of peripheral blood counts, after a single intraperitoneal dose (150 mg/kg) of 5-FU. Although only moderate thrombocytopenia (platelet count 40% of control values) occurred at 7 days following administration of 5-FU, sustained rebound thrombocytosis (platelets 200-250% of control values) was observed from days 11 to 17. No rebound leukocytosis was detected despite comparable initial leukopenia. Megakaryocyte colony-forming cells (Meg-CFC) in bone marrow and spleen were decreased for 2 and 5 days, respectively, after administration of 5-FU. Subsequently, there was a prolonged rebound increase in the total number of Meg-CFC in the spleen from days 11 to 17 after 5-FU, a phenomenon which did not occur with Meg-CFC derived from the bone marrow. Granulocyte-macrophage colony-forming cells (GM-CFC) in bone marrow and spleen exhibited alterations which were similar to those of Meg-CFC, indicating similar sensitivities of GM-CFC and Meg-CFC to 5-FU. Normal feedback mechanisms which control platelet levels are perturbed for almost 3 wk after administration of 5-FU. The simultaneous occurrence of maximal thrombocytosis and increased splenic Meg-CFC suggests that increased platelet production after 5-FU is associated with concomitant stimulation of the megakaryocyte progenitor compartment in the mouse spleen. However, the concurrence of thrombocytosis and increased splenic Meg-CFC indicates that elevated levels of Meg-CFC did not initiate the period of thrombocytosis.     

Recovery patterns of rat hemopoietic stem cells between pulse doses of 7,12-dimethylbenz(a)anthracene (DMBA) applied in a leukemogenic regimen

Male Wistar rats received repeated pulse doses of 7,12-dimethylbenz(a)anthracene (DMBA), known to elicit myelodysplasia followed by acute, mostly erythroblastic, leukemia at 10-day intervals. The recovery of spleen colony forming hemopoietic stem cells (CFU-s) surviving the cytocidal action of DMBA was examined between pulses. Recovery after a pulse of 35 mg/kg body weight varied with the organ source of the CFU-s (femoral bone marrow or spleen) and the number of preceding DMBA pulses. After a single DMBA pulse bone marrow CFU-s initially recovered faster than reported for normal bone marrow CFU-s transplanted into chemically conditioned rats. But recovery was followed by regeneration arrest. Population doubling times of marrow CFU-s increased with the number of DMBA pulses. Recovery of splenic CFU-s was slower after a single DMBA pulse than reported for normal spleen CFU-s transplanted into chemically conditioned rats. The CFU-s population doubling times were not significantly different after a single or five DMBA pulses. After three pulses, however, recovery of splenic CFU-s was exceedingly slow until day 5 and subsequently accelerated, but was still slower than after one or five pulses. In the spleen CFU-s recovery was always accompanied by regeneration of total cell numbers with a preference for erythroid regeneration. In the bone marrow this was the case after three DMBA pulses only.     

Further studies on the in vitro enhancement of erythroid stem cell (CFU-e and BFU-e) colony formation by ouabain

The cardiac glycoside ouabain has been shown to stimulate erythroid stem cell (CFU-e/BFU-e) colony formation while inhibiting both granulocyte/macrophage precursor cell (CFU-gm) and murine spleen stem cell (CFU-s) colony formation. We have examined the ability of ouabain to increase both CFU-e/BFU-e colony formation by performing time-delay and temperature-dependent studies. After pre-exposure of marrow cells to ouabain (10(-15) M) at temperatures of 27, 37, or 4 degrees C, erythropoietin (Ep) was added after time-delays ranging from 10 s to 60 min. The ouabain-induced increase in CFU-e/BFU-e colony formation was observed up to an Ep-delay of 20 s, after which time the enhancing effect of ouabain was diminished. This increase was also observed when marrow cells were first exposed to Ep prior to a similar delayed exposure to ouabain. The enhancement effect seen with ouabain was also temperature dependent, since at 37 degrees C an earlier time increase in CFU-e/BFU-e colony formation was observed when compared to identical studies performed at either room temperature or 4 degrees C. These studies suggest that ouabain-induced elevations in erythroid stem cell colony formation involve mechanisms that are both time and temperature dependent.     

Sensitivity of erythroid progenitor colonies to erythropoietin in azidothymidine treated immunodeficient mice

The anaemia induced by 3'-azido-3'dideoxythymidine (AZT) is poorly understood. We have used a murine model of AIDS, infection of female C57BL/6 mice with LP-BM5 murine leukaemia (MuLV) virus, to determine if AZT-induced anaemia is due, in part, to decreased responsiveness of erythropoietic precursors (BFU-e) to erythropoietin (EPO). Mice in the early stage of LP-BM5 MuLV disease were given AZT in their drinking water at 1.0 and 2.5 mg/ml. AZT produced anaemia in both groups, in a dose-dependent fashion. Despite the anaemia, the number of splenic and bone marrow BFU-e in AZT treated mice increased up to five-fold over levels observed in infected untreated animals after 15 d of treatment. Colony formation by splenic and bone marrow BFUe was stimulated at lower concentrations of EPO in mice receiving AZT for 15 d than for infected, untreated mice. By day 30, sensitivity of both splenic and bone marrow BFU-e of treated animals returned to that observed from cells of infected untreated animals. The mean plasma levels of EPO observed in AZT treated mice were appropriate for the degree of anaemia observed when compared with phenylhydrazine (PHZ) treated mice. The numbers of BFU-e and the percentage of bone marrow erythroblasts observed were comparable in AZT and PHZ treated mice with similar degrees of anaemia. However, reticulocytosis was inappropriate for the degree of anaemia observed in AZT treated infected mice. AZT-induced peripheral anaemia in the face of increased numbers of BFU-e and increased levels of plasma EPO suggest a lesion in terminal differentiation.     

Contamination of erythropoietin by endotoxin: in vivo and in vitro effects on murine erythropoiesis.

Endotoxin was detected in all erythropoietin preparations tested and was removed from four lots, without loss of erythropoietic activity, by adsorption with limulus amebocyte lysate. Comparison of adsorbed (endotoxin-depleted) and nonadsorbed (endotoxin-containing) erythropoietin preparations demonstrated significant inhibition of CFU-e and BFU-e in vitro by nonadsorbed erythropoietin at concentrations higher than 0.25 U/ml and 2.0 U/ml, respectively. CFU-e and BFU-e were inhibited significantly by readdition in vitro of 10(-5)-10(-3) mug of endotoxin per unit of limulus-adsorbed erythropoietin. Administration of saline or 6 U of nonadsorbed or adsorbed erythropoietin twice a day for 4 days of CF1 mice resulted in reticulocyte counts of 2.1%, 9.9%, and 15.9%, respectively. Nonadsorbed erythropoietin resulted in a 29% decrease in erythropoiesis, a 42% decrease in CFU-e, and a 16% increase in granulopoiesis in the marrow, whereas adsorbed erythropoietin caused a 28% increase in erythropoiesis, no significant change in CFU-e and a 19% decrease in granulopoiesis in the marrow. Both preparations resulted in marked increases in splenic erythropoiesis and granulopoiesis. The effects of adsorbed erythropoietin are similar to those produced following stimulation of hematopoiesis by endogenous erythropoietin. Hemopoietic changes induced by nonadsorbed erythropoietin in vivo and in vitro are affected substantially by contamination of the erythropoietin preparations with endotoxin.     

Erythroid progenitor cell kinetics in chronic haemodialysis patients responding to treatment with recombinant human erythropoietin

The response of bone marrow and peripheral blood erythroid progenitors to human recombinant erythropoietin (rHuEPO) was studied in nine haemodialysed renal failure patients receiving this hormone for the correction of their anaemia. The haematocrit rose in all patients in response to thrice weekly injections of escalating rHuEPO doses (12-192 IU/kg). Both the numbers of CUF-e and BFU-e and their proliferative state in the bone marrow as well as BFU-e numbers in the peripheral blood were estimated before treatment and again after correction of the anaemia, at 16 h following an intravenous dose of rHuEPO. Following treatment bone marrow BFU-e numbers fell to a mean of 24.5% (P less than 0.01) of the pre-treatment values although there was no significant change in CFU-e or circulating BFU-e numbers. The mitotic rate (percentage S-phase cells) estimated by tritiated thymidine suicide rose from 45.2% to 68.4% (P less than 0.05) in the case of CFU-e and from 16.4% to 45.1% (P less than 0.05) for BFU-e following treatment with rHuEPO thus indicating in-vivo sensitivity of both the primitive as well as the mature erythroid progenitors to the hormone. The fall in BFU-e numbers in the bone marrow after several months of treatment may be due to a loss of cells from this progenitor pool by maturation that is uncompensated by replacement from the pluripotential stem cell compartment.     

Increased Haemopoietic Cell Survival in Vitro Induced by a Human Marrow Fibroblast Factor

S ummary. Human marrow fibroblasts were grown in vitro and examined for effects on human and mouse haemopoietic cells. When human marrow cells were incubated with fibroblasts or with fibroblast-conditioned medium for 1 week and then assayed for committed granulocyte/monocyte (CFU-c) and erythroid (BFU-e) progenitor cells, the numbers of CFU-c and BFU-e were considerably increased compared with controls. In contrast, human marrow-fibroblast-conditioned medium, when added directly to CFU-c or BFU-e assay cultures, had no effect on colony formation by these progenitor cells. As these results suggest that the fibroblast-derived factor may be acting on a relatively primitive progenitor cell, possibly a pluripotent haemopoietic stem cell, the effect of this factor on mouse pluripotent haemopoietic stem cells (CFU-s) was examined. Human marrow-fibroblast-conditioned medium considerably enhanced CFU-s survival after a 24 h incubation and increased the proportion of CFU-s in cell-cycle. The increase in CFU-s survival depended on the concentration of the fibroblast-conditioned medium but not on the age of the fibroblast culture. The evidence suggests therefore that human marrow fibroblasts produce a factor that acts on a human myeloid progenitor cell more primitive than BFU-e and CFU-c, possibly the pluripotent haemopoietic stem cell.     

Changes of the phenotype of erythroid progenitor cells (BFU-E, CFU-E) and their progenies during early steps of differentiation

Early differentiation processes of human erythroid progenitor cells (BFU-e, CFU-e) have been studied during in vitro proliferation using a panel of monoclonal antibodies with known reactivity on different levels of the erythroid cell line. Two antibodies recognizing structures on BFU-e (VIP-2b, BMA 021), two antibodies reactive with CFU-e and nucleated red cells (5F1, CLB-Ery-3) and one antibody directed against glycophorin A (VIE-G4) were used for this study. Normal human bone marrow cells were induced to proliferation in an erythroid progenitor cell assay and, after different periods of incubation, agar cultures were treated with these antibodies and complement. Thereafter, the remaining erythroid cells were incubated again to continue their proliferation with the same stimulators as before. The changes of the phenotype of BFU-e and CFU-e progenies during in vitro proliferation were determined by the reduction of colony formation in comparison with untreated control cultures. Our results indicate that the loss of HLA-DR antigens and the p45 structure is accompanied by the acquisition of structures recognized by the antibodies 5F1 and CLB-Ery-3. After 5-7 d of incubation BFU-e derived progenies exhibit the same antigenic structure as has been found for CFU-e. Glycophorin A expression could only be demonstrated at a late differentiation stage of the erythroid cell lineage.     

A case of acquired pure red cell anemia studied by cloning of erythroid progenitor cells in vitro

A 70-year-old woman developed typical clinical symptoms of pure red cell anemia (PRCA) following a history of rheumatoid arthritis (RA). The patient's bone marrow erythropoietic progenitors cells were cloned in a micro agar culture system several times over a period of 11 months, revealing a diminished frequency of bone marrow erythroblasts paralleled by a markedly reduced number of CFU-e and BFU-e in vitro. No inhibitory activity in the patient's IgG fraction could be detected either by preincubation with IgG and/or rabbit complement, or in the continuous presence of IgG. Depletion of T lymphocytes from the patient's bone marrow cells led to an improved in vitro erythroid proliferation. Cytostatic therapy with cyclophosphamide clinically induced a marked increase in the bone marrow erythroblast and reticulocyte number, correlated in vitro by normalization of CFU-e levels and increase in the number of BFU-e. Nevertheless, BFU-e values never attained normal levels, which could be attributed to a reduced stem cell pool resulting from previous therapy with cyclophosphamide and/or antirheumatic drugs. Two independent factors, a reduced pool of committed stem cells as well as an autoimmune cell-mediated suppression, may both contribute to the pathomechanism of the disease in this patient.     

Effect of human plasmalipoproteins on erythropoietic progenitor cells in serum-free cultures

Summary The purpose of the present study was to investigate the influence of human lipoproteins on CFU-e and BFU-e proliferation from human bone marrow in a serum-free system. In our previously described miniaturized agar system the main lipoprotein-density-classes from human plasma, namely very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), low density lipoproteins (LDL), high density lipoproteins₂ (HDL₂) and HDL₃ and a mixture of all the five lipoproteins were added in rising concentrations (from 1/10 to normal human plasma concentration) to serum-free medium containing delipidated and deionized bovine serum albumin (BSA), iron saturated transferrin and erythropoietin. The results demonstrate that all lipoproteins markedly increased the CFU-e and BFU-e proliferation after 7 and 14 days of incubation, respectively. Moreover, the lipoproteins induced a shift towards a lower threshold concentration of erythropoietin. Serumlike conditions were obtained if LDL and the mixture of lipoproteins were added to serum-free medium. Furthermore, in the serum-free cultures a maturation to the mature erythrocyte could be found.     

Erythropoiesis in vitro. III. The role of potassium ions in erythroid colony formation.

The role of potassium as an essential promotor of erythroid progenitor growth (BFU-e & CFU-e) from normal murine hematopoietic tissues was studied. Dialyzed fetal calf serum, over a wide range of concentrations, was shown to reduce the numbers of BFU-e and CFU-e that could be cultured from normal murine bone marrow. A dose-dependent addition of 1 M KC1 restored erythroid progenitor colony growth to the levels generally seen when normal, non-dialyzed fetal calf serum was used. Furthermore, when [K] was increased in some human urinary and sheep plasma erythropoietin preparations, the number of erythroid progenitor cells cultured also increased. This influence is crucial to the differentiation of committed stem cells into the erythroid pathway and must therefore be considered in the development of serum-free growth media.     

Separation of erythroid progenitor cells in mouse bone marrow by isokinetic-gradient sedimentation.

Isokinetic-gradient sedimentation employing a shallow linear gradient of Ficoll in tissue culture medium was used to isolate erythroid progenitor cells (CFU-e) from mouse bone marrow. Following gradient sedimentation, 34% of the total nucleated cells and 48% of the CFU-e applied to the gradient were recovered, and three distinct modal populations of CFU-e could be distinguished. The slowest-migrating population did not require exposure to exogenous erythropoietin in order to form erythroid colonies in vitro. The other two modal populations of CFU-e required exposure to exogenous erythropoietin for differentiation. One of these, constituting 64% of the hormone-dependent CFU-e recovered, migrated with the bulk of the marrow cells, whereas the other migrated ahead of the bulk of the marrow cells. This latter population, which contained 34% of the CFU-e, was recovered with 11% of the marrow cells, representing a twofold to threefold enrichment. BFU-e migrated more slowly than the erythropoietin-dependent CFU-e. Resedimentation studies suggested that the two erythropoietin-dependent CFU-e populations were distinct modal populations. When cells from the fastest-migrating population of erythropoietin-dependent CFU-e were cocultured with unseparated marrow cells, a further twofold to threefold enhancement of erythroid colony formation was obtained. Comparison of isokinetic-gradient sedimentation with discontinuous and continuous albumin density-gradient sedimentation revealed that isokinetic-gradient sedimentation was a more efficient method than the former and a more rapid method than the latter for isolating CFU-e from mouse bone marrow.     

Prolactin exerts hematopoietic growth-promoting effects in vivo and partially counteracts myelosuppression by azidothymidine.

Prolactin (PRL) is a neuroendocrine hormone that influences immune and hematopoietic development. The mechanism of action of this hormone in vivo remains unclear; therefore, we assessed the effects of PRL on hematopoiesis in vivo and in vitro. Normal resting mice were treated with 0, 1, 10, or 100 microg of recombinant human prolactin (rhPRL) for 4 consecutive days and euthanized on the fifth day for analysis of myeloid and erythroid progenitors in the bone marrow and spleen. Both frequencies and absolute numbers of splenic colony-forming unit granulocyte-macrophage (CFU-GM) and burst-forming unit-erythroid (BFU-e) were significantly increased in mice receiving rhPRL compared to the controls that had received saline only. Bone marrow cellularities were not significantly affected by any dose of rhPRL, but the absolute numbers and frequencies of bone marrow CFU-GM and BFU-e were augmented by rhPRL. These results suggest that rhPRL can promote hematopoiesis in vivo. Because rhPRL augments myeloid development in vivo, we examined the potential of the hormone to reverse the anemia and myelosuppression induced by azidothymidine (AZT). Mice were given rhPRL injections concurrent with 2.5 mg/mL AZT in drinking water. rhPRL partially restored hematocrits in the animals after 2 weeks of treatment and increased CFU-GM and BFU-e in both spleens and bone marrow. The experiments with AZT and rhPRL support the conclusion that the hormone increases myeloid and erythroid progenitor numbers in vivo, and they suggest that the hormone is clinically useful in reversing myelosuppression induced by AZT or other myeloablative therapies.     

Interleukin 1-induced sequential myelorestoration: dynamic relation between granulopoiesis and progenitor cell recovery in myelosuppressed mice.

The myelorestorative effect of recombinant human interleukin 1 alpha (IL-1 alpha) was studied in mice treated with anticancer drugs. The treatment of mice with 5-fluorouracil (5-FU; 250 mg/kg body weight) or cyclophosphamide (CPA; 100 mg/kg) considerably decreased bone marrow or splenic colony-forming units in culture (CFU-C) or neutrophils in blood. The daily administration of IL-1 alpha (100 ng/mouse/day) after 5-FU treatment markedly accelerated the recovery of bone marrow CFU-C. This increase was followed by the recovery of splenic CFU-C and neutrophils in blood. In the CPA-treated mice the recovery of bone marrow CFU-C over the normal level was observed regardless of the administration of IL-1 alpha 3 days after CPA treatment. The daily administration of IL-1 alpha after CPA treatment markedly increased splenic CFU-C or neutrophils over the normal level following the increase of bone marrow CFU-C. Thus, in both 5-FU- and CPA-treated mice, the increase of bone marrow CFU-C after the administration of IL-1 alpha was observed several days earlier than the increase of splenic CFU-C and neutrophils in blood. The increase of splenic CFU-C and neutrophils in blood was observed concomitantly. The experiments, in which effective timing of IL-1 alpha injection was examined, indicated that the administration of IL-1 alpha within a few days after treatment with anticancer drugs was necessary for the accelerated recovery of bone marrow progenitor cells. On the other hand, the effective recovery of splenic progenitor cells and peripheral neutrophils required the administration of IL-1 alpha after the increase of bone marrow progenitor cells. Thus, the administration of IL-1 alpha daily or every other day was the most effective for recovery from myelosuppression induced by anticancer drugs.     

Indomethacin augments granulocyte-macrophage colony-stimulating factor-induced hematopoiesis following 5-FU treatment

We have previously reported that granulocyte-macrophage colony-stimulating factor (GM-CSF) given after the administration of 5-fluorouracil (5-FU) results in augmented hematopoietic recovery as evidenced by increased white blood cell and neutrophil counts. Mice receiving GM-CSF following 5-FU administration were observed to have a marked elevation in splenic granulocyte-macrophage colony-forming cells (GM-CFC) and a decrease in the femoral bone marrow GM-CFC. Because GM-CSF has been shown to increase prostaglandin synthesis and prostaglandins are thought to provide a negative feedback signal to down-regulate myelopoiesis, we sought to determine if the cyclooxygenase inhibitor, indomethacin, could prevent the reduction in the number of femoral bone marrow GM-CFC seen when GM-CSF was administered following 5-FU. Groups of mice received a single 60 mg/kg i.p. injection of 5-FU followed 24 h later by twice-daily injections of 1 micrograms GM-CSF and daily injections of 3, 5, or 6 mg/kg indomethacin; the hematopoietic assays were performed on day 7 following 5-FU. Compared to those animals that received GM-CSF alone following 5-FU, mice receiving 5 mg/kg indomethacin plus GM-CSF following 5-FU had increased numbers of GM-CFC in their bone marrow (3923 +/- 634 vs 971 +/- 138; p less than 0.001) as well as increased neutrophil counts (18,995 +/- 2872 vs 11,497 +/- 2476; p less than 0.01). Indomethacin alone was, in part, capable of facilitating hematopoietic recovery following 5-FU administration, but not to the extent seen when used in combination with GM-CSF. Prostaglandin inhibitors may have a role in combination with hematopoietic growth factors in accelerating hematopoietic recovery following cytoreductive chemotherapy.     

The effects of acute thrombocytopenia on megakaryocyte-CFC and granulocyte-macrophage-CFC in mice: studies of bone marrow and spleen

The effects of acute thrombocytopenia, produced by platelet antiserum (PAS), on both megakaryocyte colony-forming cells (Meg-CFC) and granulocyte-macrophage colony-forming cells (GM-CFC) were studied. During the 1-hr to 14-day period following acute thrombocytopenia (platelet counts < 5% of normal), bone marrow and splenic cells of C57BL/6J mice were obtained and cultured for 7 days in 0.3% agar. Numbers of GM and Meg colonies were determined. At no times were alterations in frequency of GM-CFC and Meg-CFC detected in femoral bone marrow. In contrast, GM-CFC in spleen were increased from 3 to 7 days after PAS and from 4 to 7 days after normal serum (NS). Increase in Meg- CFC in the spleen occurred from 3 to 5 days after PAS with a lesser, not significant increase after NS. Alterations in white blood cells and hematocrit values were not detected. Similar responses were observed in germ-free mice and after rechallenge of animals that had received PAS or NS 14 days previously. The delayed increase in Meg-CFC indicates that they are unlikely to be responsible for the altered megakaryopoiesis previously reported in bone marrow after acute thrombocytopenia and was not due to inhibition by PAS. The increase in GM-CFC may reflect stimulation of the reticuloendothelial system by heterologous proteins.     

T- and B-lymphocyte differentiation potentials of spleen colony-forming cells

Cells that generate splenic colonies within 8 days (day-8 colony- forming units-spleen [CFU-s]) are generally thought to differentiate only into erythroid/myeloid cells. The T and B lymphocyte differentiation potentials of day-8 CFU-s were evaluated and compared with those of day-12 and 5-fluorouracil (5-FU) CFU-s. This was achieved by analyzing, after intravenous and intrathymic injection, the lymphocyte progeny of cells contained within individual splenic colonies collected at day 8 and day 12 post-bone marrow cell transfer into irradiated congenic recipients. A large majority of day-8 spleen colonies generated T cells when transferred intrathymically. After intravenous (IV) injection of day-8 colonies, donor-type thymocytes emerged in 33% of the animals reconstituted with only 1 day-8 colony, but in 83% of those inoculated with a pool of 5 colonies. All post-5-FU and 75% of day-12 colonies gave rise to thymocytes after IV injection. B cells were generated by a high proportion of day-8 colonies, and by all day-12 and post 5-FU colonies. These results demonstrate that progenitors of T and B lymphocytes are generated within spleen colonies produced by at least some day-8 CFU-s and virtually all day-12 CFU-s. Whether these progenitors are CFU-s themselves or committed precursors remains an open question.