Primitive haematopoietic progenitors in the blood of patients with sickle cell disease appear to be endogenously mobilized.

To investigate whether haematopoietic stem cells in patients with sickle cell (SS) disease might be altered, we examined the number and cycling status of 5-week long-term culture-initiating cells (LTC-ICs) and in vitro multilineage colony-forming cells (CFCs) present in the blood of a large and clinically diverse group of SS patients. The concentrations of both of these cell types per ml of blood varied over a wide range in individual patients, but, on average, were significantly elevated above normal values ( approximately sevenfold and 15-fold respectively) and to an even greater extent than the lineage-restricted CFCs in the same samples. Wide variations in the concentration of circulating progenitors, particularly the LTC-ICs, were also seen over time (in concert with changes in the white blood cell count) in SS patients. [3H]-Thymidine suicide assays showed most of the CFCs and LTC-ICs in SS blood to be quiescent like their counterparts in normal blood. However, by comparison with historical data, the SS progenitors could be recruited into the cycle more quickly (i.e. within 2 vs. 3 d), thus showing the same kinetics of activation exhibited by 'mobilized' progenitors from patients given chemotherapy and exogenous growth factors. Taken together, these findings implicate previously documented increases in endogenous Steel factor, interleukin 3 and granulocyte-macrophage colony-stimulating factor levels in SS patients in the establishment of a chronically mobilized progenitor phenotype.

Effects of erythropoietin, angiotensin II, and angiotensin-converting enzyme inhibitor on erythroid precursors in patients with posttransplantation erythrocytosis.

BACKGROUND: Angiotensin-converting enzyme inhibitors (ACEI) have become the treatment of choice for posttransplantation erythrocytosis (PTE). Yet the pathogenesis of PTE and the mechanisms of action of ACEI remain unclear. Therefore, we studied the dose response to erythropoietin (Ep), angiotensin II (AII), and the ACEI enalaprilat on the in vitro proliferation of erythroid progenitors in patients with PTE and in controls. We also evaluated ACE polymorphism in the two groups. METHODS: Twelve patients with PTE and 12 renal transplant patients without PTE were studied. Erythroid burst-forming units (BFU-E) were isolated from peripheral blood using standard methods. Ep sensitivity was determined for four patients with PTE and three control patients, using 0-3 U/ml Ep. AII dose response was studied in four patients with PTE and five control patients, using AII concentrations of 0-1000 nM. The effect of enalaprilat was studied in eight patients with PTE and eight control patients, using drug concentrations of 0-10 ng/ml. ACE gene insertion/deletion polymorphism was determined by polymerase chain reaction. RESULTS: PTE patients showed a significant shift of the Ep response curve to the left compared with controls, with 50% maximal growth occurring at a lower Ep concentration (0.3 U/ml vs. 0.95 U/ml, P<0.025.) However, there was no difference in the number of BFU-E colonies between PTE patients and controls. AII added to the growth medium produced only minor stimulation in both groups. PTE patients showed significant inhibition of BFU-E growth with 10 ng/ml enalaprilat, but controls showed no inhibition of BFU-E growth with ACEI. There was no difference in ACE polymorphism between PTE and controls. CONCLUSIONS: Our data suggest that PTE is associated with increased erythroid progenitor sensitivity to Ep. The effect of ACEI to decrease hematocrit in patients with PTE may be due to inhibition of red cell precursor growth.

Circulating cytokines response and the level of erythropoiesis in sickle cell anemia.

A hemoglobin F (HbF) level between eight and nine percent divides sickle cell anemia (SS) patients into two populations, according to the kinetics of circulating burst forming units-erythroid (BFU-E), long term culture-initialing cells (LTC-IC), and cytokine plasma concentrations. The SS patients with HbF levels lower than 8-9% are more anemic (LFSS patients) than those with HbF levels higher than 8-9% who have less severe anemia (HFSS patients). We report here that the level of erythropoiesis [evaluated by the levels of soluble transferin receptors (sTfR)] is not identical in these two patient populations, supporting the idea that a different set of regulatory mechanisms might be required to maintain the two levels of increased hematopoiesis. The plasma sTfR concentration was increased in all SS samples compared with controls (P < 0.002) and sTfR levels were negatively correlated with peripheral HbF%. (r = -0.574, P < 0.002). Furthermore, sTfR levels were higher in LFSS than in HFSS patients. Erythropoietin (Epo) levels were increased in the plasma of LFSS individuals (range = 34-215 ml U/ml), while the values in HFSS patients were in the normal range (3-20 ml U/ml). Furthermore, we identify here stem cell factor (SCF) and transforming growth factor-beta (TGF-beta) as regulatory factors specifically affected by the presence of SS genotype and its level of severity. The plasma concentrations of SCF and TGF-beta were increased compared with normal controls and high levels of SCF (up to 7,000 pg/ml) were detected in LFSS patients. The latter also showed increased proportion of SCF+ CD34 enriched circulating cells (49%). Low SCF in HFSS patients is associated with elevated TGF-beta, suggesting a regulatory role of the latter on either SCF release or c-kit expression in progenitor cells. Occasional elevation of granulocyte macrophage-colony stimulating factor (G-CSF), interleukin (IL)-7, and macrophage inflammatory protein (MIP)-1alpha in plasma of SS patients is not specific because no relation to HbF could be demonstrated. All plasma tested for leukemia inhibitory factor (LIF) were negative. Data presented here, complementing previously published information, supports a model in which HFSS patients achieve a balance between inhibitory (TGF-beta) and stimulatory (SCF, IL-3) factors, resulting in moderate erythropoietic response. In contrast, in LFSS patients, low levels of TGF-beta and the increased release of GM-CSF and SCF maintain the intense erythropoiesis in response to higher erythropoietic stress, in these more severe patients.

Embryonic stem cells release potentially novel hematopoietic factors.

We have observed that a severe decrease in the number of erythroid progenitor cells follows the long-term culture (LTC) of human primitive stem cells on an established mouse fibroblast feeder layer. This suggests that one, or several, factors necessary to support erythroid differentiation might be missing in these culture conditions. To improve the erythroid differentiation and stem cell output of LTCs, we have examined the hypothesis that the factors that regulate pluripotent stem cell proliferation and erythroid commitment can be found in media conditioned by embryonic stem (ES) cells. We have found that media conditioned by undifferentiated and differentiating ES cells can affect differentiation patterns in both short-term culture and LTC assays. Medium conditioned by undifferentiated ES cells increases the numbers of estimated LTC-initiating cells (est.LTC-IC) and potentiates granulocytic differentiation. In contrast, medium conditioned by ES cells undergoing differentiation increases the number of est.LTC-IC and is a powerful promoter of erythroid differentiation. In the presence of this medium, the number of erythroid progenitors generated after 5 weeks of LTC was increased up to 100-fold as compared with controls, and the number of est.LTC-IC was amplified up to 140-fold. These results offer a new approach for the identification of factors implicated in stem cell proliferation, self-renewal and commitment. In addition, the improved LTC conditions for the expansion of stem cells without reduction of their in vitro differentiation potential should be useful for a wide range of applications.

Circulating cytokines in sickle cell patients during steady state.

Plasma levels of GM-CSF, IL-3, IL-6 and IL-1 (alpha,beta) were investigated in steady-state sickle cell patients. We find that SS patients with low levels of HbF (< 9% [LFSS]) are characterized by elevated plasma GM-CSF, whereas in patients with high levels of HbF (HFSS), GM-CSF is not detectable. In contrast, HFSS patients exhibited increased plasma IL-3 (m = 84.8 +/- 57 pg/ml), whereas LFSS patients had lower or no detectable plasma IL-3, IL-1(alpha,beta) and IL-6 were also detected in plasma from some SS patients, but there was no correlation with HbF levels. Normal controls tested negative for all cytokines, except for one individual positive for IL-3. These results are compatible with a model in which the level of haemopoietic stress determines the level of participation of GM-CSF or IL-3 in the regulation of SS circulating BFU-E.

Inhomogeneity of the circulating BFU-E regulation in sickle cell anaemia: accessory cells properties and BFU-E growth factor response pattern.

Sickle cell anaemia (SS) patients with low (< 9%) HbF levels (LFSS) are characterized by an increased number of circulating BFU-E in active DNA synthesis, and release of burst promoting activity (BPA) by unstimulated low density (LD) adherent cells. In contrast, circulating BFU-E from SS patients with high (> 9%) HbF levels (HFSS) are normal in number, largely in resting phase, and their LD cells do not release BPA-like activity. We report now that in LFSS patients, adherent cell depletion decreases BFU-E growth in culture and apparent BFU-E cycling. Furthermore, addition of conditioned media (CM) from LD cells of LFSS patients restored cycling BFU-E expression in culture. Neutralization analysis with anti-GM-CSF antibody demonstrated that GM-CSF is, at least, one factor responsible for BPA activity present in this CM. Thus, GM-CSF is constitutively produced by unstimulated monocytes in LFSS patients. In contrast, HFSS patients' adherent cell depletion increases cycling of BFU-E in culture. CM from HFSS patients inhibits BFU-E expression in culture. Hence, LD adherent cells from HFSS patients may release a yet unknown inhibitor factor(s). In addition, we report a distinct response pattern in SS patients' BFU-E to growth factor (GM-CSF, IL-3): (a) LFSS patients have a BFU-E population, equally responsive to GM-CSF and IL-3; (b) HFSS patients, have a subset of BFU-E exclusively dependent on IL-3 (20-40% of the circulating BFU-E). This pattern is very similar to that of normal BFU-E. In conclusion, BFU-E from LFSS patients represent an actively proliferating population, equally responsive to GM-CSF and IL-3, controlled by constitutively produced GM-CSF, suggesting a unique BFU-E behaviour in SS patients with low HbF levels and high haemopoietic stress. The heterogeneous regulation of BFU-E in SS disease seems to be the epiphenomenon of HbF levels, and not vice versa.

Early circulating erythroid progenitors (BFU-E) in sickle cell anemia.

Sickle cell anemia (SS) patients can be divided into two sub-populations according to peripheral HbF levels. Patients with low (< 9%) HbF levels (LFSS) are characterized by an increased number of circulating BFU-E in active DNA synthesis, and release of burst promoting activity (BPA) by unstimulated low density (LD) adherent cells. In contrast, circulating BFU-E from SS patients with high (> 9%) HbF levels (HFSS) are normal in number, largely in resting phase, and their LD cells do not release BPA-like activity. More recently further heterogeneity has been found among these two groups. In LFSS patients GM-CSF is constitutively produced by unstimulated monocytes. In contrast, HFSS patients' adherent cell depletion increases cycling of BFU-E in culture. CM from HFSS patients inhibits BFU-E expression in culture. Hence, LD adherent cells from HFSS patients may release an inhibitory factor(s). The nature of this factor has to be determined. In addition, there are distinct subpopulations of BFU-E responsiveness to growth factor (GM-CSF, IL-3): a) LFSS patients have a homogeneous BFU-E population, equally responsive to GM-CSF and IL-3; b) HFSS patients, in addition to this subpopulation, have a subset of BFU-E dependent exclusively on IL-3 which is 20 to 40% of the total number of circulating BFU-E. This is similar to BFU-E from normal individuals. Hence, LFSS BFU-E represent an actively proliferating population, equally responsive to GM-CSF and IL-3, controlled by at least constitutively produced GM-CSF and possibly other factors. These observations suggest a significant modification in BFU-E behavior in the subset of SS patients with low HbF levels and high hemopoietic stress. The heterogenous regulation of BFU-E in SS disease seems to be an epiphenomenon of HbF levels, and not vice-versa.

The Senegal DNA haplotype is associated with the amelioration of anemia in African-American sickle cell anemia patients.

We have previously determined that in African sickle cell anemia (SS) patients three different beta-like globin gene cluster haplotypes are associated with different percent G gamma (one of the two types of non-alpha chains comprising hemoglobin F [HbF]), mean percent HbF, and percent dense cells. We report now that in adult New York SS patients, the presence of at least one chromosome with the Senegal haplotype is associated with higher Hb levels (1.2 g/dL higher) than is found for any other non-Senegal haplotype (P less than .004). The percent reticulocytes and the serum bilirubin levels were lower in these patients. When the effect of alpha-gene number was analyzed by examining a sample of SS patients with concomitant alpha-thalassemia, the same results were obtained. Because the HbF level is significantly higher among the Senegal haplotype carriers in this sample, the inhibitory effect on sickling of this Hb variant may be one of the reasons for the haplotype effect. We conclude that the Senegal beta-like globin gene cluster haplotype is associated with an amelioration of the hemolytic anemia that characterizes sickle cell disease.

Heterogeneity in the properties of burst-forming units of erythroid lineage in sickle cell anemia: DNA synthesis and burst-promoting activity production is related to peripheral hemoglobin F levels.

Circulating 14-day erythroid progenitors (BFU-E) from 28 sickle cell anemia (SS) patients with hemoglobin F (HbF) levels ranging from 2% to 16% were studied to determine their sensitivity to [3H] thymidine kill and burst-promoting activity (BPA)-like factor production. We find that the proportion of BFU-E sensitive to 3H-dT kill, and hence active in DNA synthesis, was inversely correlated with the percent of peripheral HbF when light density (LD) mononuclear cells were used for plating. Regression analysis showed that the correlation between HbF level and BFU-E kill was highly significant (r = .88; P less than .00003). We confirmed the BPA-like factor(s) production by LD mononuclear cells of SS patients, and found, in addition, that this phenomenon is restricted to the population of SS patients with HbF levels lower than 9%. Circulating BFU-E of patients with high HbF levels are not sensitive to 3H-dT, and their mononuclear cells do not release BPA-like factor. In summary, SS patients exhibit differences in the capacity of their mononuclear cells to produce BPA activity according to their peripheral HbF level, as well as to the DNA synthesis-state of their circulating BFU-E. We conclude that erythroid progenitors differ among SS patients in relation to their peripheral HbF level.

Circulating BFU-E in sickle cell anemia: relationship to percent fetal hemoglobin and BPA-like activity.

Circulating 14-day erythroid progenitors (BFU-E) from sickle cell anemia (SS) patients were studied in culture to determine their frequency and their sensitivity to erythropoietin (Epo). Increased numbers of circulating BFU-E were found in half of the patients studied, whereas the remainder had a normal count. Patients with high circulating BFU-E counts had lower fetal hemoglobin (HbF) percentages (congruent to 4.5%) than patients with low circulating BFU-E counts (HbF congruent to 13%). This difference was highly significant (p less than 0.0001). In addition, SS circulating BFU-E expressed increased sensitivity to Epo due, at least partially, to an increased production of burst-promoting activity-like factor(s) generated by light-density mononuclear cells. These findings emphasize the possible role of the HbF level (and the extent of sickling) in BFU-E regulation under the continuous hemopoietic stress of SS disease.

Increased BPA production modulates Epo sensitivity of circulating BFU-E in sickle cell anemia.

We have examined the possibility that permanent "stress" hemopoiesis in sickle cell anemia (SS) patients leads to the modification of the behavior of circulating 14 day erythroid progenitor cells (BFU-E). In these patients we find that peripheral blood BFU-E are increased in number and have high sensitivity to erythropoietin (Epo). Maximal number of BFU-E are generated from peripheral blood of SS patients at 0.3-0.75 Epo/ml of culture compared to 1.5-2.0 U Epo/ml of culture in normals. Peripheral blood adherent cells depletion leads to the shift of Epo dose response curve, so that the Epo sensitivity of BFU-E significantly decreases. This result suggests that apparent Epo hypersensitivity reflects, in fact, an increased production of a burst promoting activity (BPA) by SS peripheral blood light density adherent (PB-LDA) cells. Experiments with conditioned medium by SS PB-LDA cells confirmed this interpretation. When peripheral blood light density non-adherent (PB-LDNA) cells of SS patients or normal individuals were plated in the presence of various concentrations of SS PB-LD cells conditioned medium and constant amounts of Epo, a dose dependent increase of the number of BFU-E was observed. When the same target cells were plated in the presence of PB-LD cells conditioned medium from normal individuals, such effect does not occur. We conclude that increased BPA production may play a role in the erythropoietic regulation during constant hemopoietic stress in sickle cell anemia and might partially explain the lower than expected Epo levels in these patients.

Differences among myeloproliferative disorders in the behavior of their restricted progenitor cells in culture.

We have studied the behavior in culture of circulating restricted hemopoietic progenitor cells from patients with idiopathic myelofibrosis (IMF), polycythemia vera (PV), and essential thrombocytopenia (ET). We have found differences in circulating granulocyte-macrophage, erythroid, and megakaryocytic progenitors that appear to be specific for these chronic myeloproliferative disorders. In IMF, most affected were granulocyte-macrophage progenitor cells (CFU-C), which circulated in increased numbers and were heterogeneous in their sensitivity to the regulatory factor(s) present in phytohemagglutinin (PHA) stimulated T-lymphocyte conditioned medium (CM). Most CFU-C were either highly sensitive to, or independent from, stimulatory factors, while others showed normal sensitivity. In some IMF patients, circulating megakaryocytic progenitors (CFU-M) were present that were capable of giving rise to colonies in the absence of added CM or erythropoietin (EPO). In PV, we confirmed the presence of circulating erythroid progenitor cells that give rise to colonies in culture without the addition of EPO. The number of circulating CFU-C was normal and they responded normally to CM. In ET, failure to detect 7-day circulating restricted progenitor cells was a common observation; the level of other circulating restricted progenitors was in the low normal range. Thus, despite certain common features, including a primary lesion at the level of the pluripotential hemopoietic stem cell, the myeloproliferative disorders differ with respect to the behavior in culture of their circulating restricted progenitor cells. These results have led us to postulate a second regulatory lesion in the pluripotential stem cell that differs in these disorders and is expressed at the level of the respective restricted progenitor cells.

Gene controlled negative regulation of DNA synthesis in erythropoietic progenitor cells.

Congenic strains differing by a small segment of Chromosome 9 that bears the Fv-2 locus have provided valuable material for investigating genetic resistance to Friend polycythemia virus (FV). C57BL/6 (B6) (Fv-2rr) mice have been found to differ from B6.S (Fv-2ss) mice not only in their response to this virus but also in the proliferative state of their erythropoietic progenitor cells BFU-E: in B6 mice the majority of BFU-E are normally quiescent, while in B6.S mice approximately 50% are actively synthesizing DNA at any time. We have shown that B6 but not B6.S marrow contains a macromolecule that negatively regulates DNA synthesis specifically of BFU-E in vitro. Evidence is presented that this macromolecule is a physiological negative regulator active in vivo in B6 mice. A new liquid culture system is described in which FV and erythropoietin (epo) act synergistically on Ficoll-Isopaque separated bone marrow cells to give rise after 7 days to large numbers of CFU-E detectable in 2-day plasma cultures with and without epo. Inclusion of concentrated B6 but not B6.S bone marrow supernatant in liquid cultures drastically curtailed the amplification of CFU-E by FV and epo. These studies indicate that both DNA synthesis and the BFU-E stage of differentiation are necessary conditions for initiating the effects of polycythemia-inducing FV. Genetic resistance to FV appears not to reside within the target cells for the virus.

A washable macromolecule from Fv2rr marrow negatively regulates DNA synthesis in erythropoietic progenitor cells BFU-E.

The proportion of BFU-E normally engaged in DNA synthesis is low in adult B6 (C57BL/6) mice of genotype Fv2rr (resistant to Friend erythroleukemia virus), as shown by 3H-thymidine or hydroxyurea "cell suicide" experiments in vivo and vitro. When bone marrow cells from these mice were subjected to a single wash in alpha medium, the proportion of BFU-E synthesizing DNA dramatically rose to levels as high as those normally seen among the BFU-E of congenic B6.S mice of genotype Fv2ss (sensitive to Friend erythroleukemia virus). Washing Fv2rr marrow cells did not significantly affect the proportion of CFU-S, CFU-nm (CFU-C) or CFU-E engaged in DNA synthesis. An activity responsible for keeping low the proportion of BFU-E in DNA synthesis was recovered in supernatants of FV2rr (but not FV2ss) bone marrow cells; its effect could be demonstrated on the BFU-E of either Fv2rr of Fv2ss washed bone marrow cells. This activity was nontoxic to the BFU-E, rapidly reversible and effective at, but not far below, the concentrations normally found in adult Fv2rr marrow. It was stable to 56 degrees C for 30 min, was nondialyzable, appeared in the void volume on G-25 Sephadex gel filtration and could be filtered through membranes that permitted passage of particles of less than 100,000 but not less than 50,000 daltons. Thus the Fv2 locus (or a locus closely linked to it) appears to act not in the BFU-E itself, but elsewhere, to control the amount or activity of a macromolecular negative regulator to which the BFU-E population responds by a reduction in the proportion synthesizing DNA. This study reveals the existence of a negative growth control mechanism for early erythropoietic progenitor cells, which is apparently physiological in nature and under strict genetic control.

The effect of partial body irradiation on haemopoietic stem cell migration.

Data obtained after various types of partial body irradiation support the concept of a small rapidly exchangeable pool of CFUs which seems to be exhausted rapidly after irradiation. The depletion of this pool is the most plausible explanation for the decrease in stem cell migration observed 3 hr after exposure in C3H mice. After partial body irradiation the size of the rapidly mobilizable pool is reduced in proportion to the areas of bone marrow irradiated. When only one marrow area is shielded, the recovery of this pool does not occur during the first 24 hr after exposure.

Study of a CFUs stimulating factor liberated by bone marrow cells after total and partial body irradiation.

Data reported in this paper show that a long range stimulating factor is released by irradiated bone marrow 15 minutes after exposure and that the secretion of the factor precedes CFUs proliferation. The factor seems to be elaborated by liver cells as is demonstrated by dose effect experiments as well as by the fact that protected bone marrow secretes a factor, albeit with different kinetics of secretion from those of irradiated bone marrow. The liberation of the factor seems to be regulated by the size of the stem cell compartment.