Erythropoiesis Inhibiting Factor(s) in Intact and Haemolyzed Red Blood Cells

A previous report have shown that both intact red blood cells (RBC) and an equal number of haemolyzed cells were able to inhibit erythropoiesis on ESF-induced erythropoiesis, suggesting a cell compound to be responsible for the inhibitory effect. Haeme and haeme compounds have been found to stimulate or inhibit both erythropoiesis and haeme synthesis. The present work presents data on the inhibitory effect of haemolyzed RBC and compounds from intact RBC on erythropoiesis. The inhibiting factor was found to be of a small molecular size and of the same range as a urinary erythropoiesis inhibiting factor (EIF). The inhibitor did not contain haeme. Both Fe²⁺ and Fe³⁺ were tested, showing no reduction of the ⁵⁹Fe incorporation into RBC in the test animals. The inhibition could not be due to dilution of the ⁵⁹Fe with unlabelled iron from the haemolyzed cells. On the contrary, Fe³⁺-ions rather stimulated erythropoiesis, probably due to increased amounts of available iron. Haemolysates were prepared from RBC with different amounts of immature cells. With increasing amounts of reticulocytes, a reduction of the inhibitory effect occurred. Also foetal cells showed less inhibition than an equal amount of adult cells. After high speed centrifugation, the inhibitory effect of haemolysates was found in the supernatant, while ghost cells exerted no inhibition. No species differences were found using both exhypoxic polycythaemic mice and rats. An inhibiting factor was liberated into the incubation medium when RBC were incubated for 20 h. No haemolysis occurred during the incubation period. Mature, adult RBC therefore contain a substance which is different from haeme, with a negative feedback on erythropoiesis.     

Erythropoiesis Inhibiting Factor (EIF) The Inhibitory Effect of Oestrogens on Erythropoiesis and the Content of Oestrogens in the Urinary EIF

An erythropoiesis inhibiting factor (EIF) with a low molecular weight has been demonstrated in urine from normal healthy individuals, indicating a dual, humoral regulatory mechanism of the erythropoietic homeostasis. Since oestrogens are known to inhibit erythropoiesis and are excreted in the urine, the question was raised whether the EIF preparations could contain eostrogens in such amounts that this could explain the inhibitory effect. Fractionated eostrogen determinations in EIF preparations revealed only micro-quantities of the different oestrogen-metabolites. An inhibitory effect on erythropoiesis was obtained with commercial oestrogens only in doses 10⁴-10⁵ times higher than the oestrogen amounts found in the EIF preparations. It is therefore most unlikely that oestrogens are causing the inhibitory effect of EIF on erythropoiesis.     

SWAP-70 regulates erythropoiesis by controlling α4 integrin

Background The regulation of normal and stress-induced erythropoiesis is incompletely understood. Integrin-dependent adhesion plays important roles in erythropoiesis, but how integrins are regulated during erythropoiesis remains largely unknown. DESIGN AND METHODS: To obtain novel insights into the regulation of erythropoiesis, we used cellular and molecular approaches to analyze the role of SWAP-70 and the control of integrins through SWAP-70. In addition, mice deficient for this protein were investigated under normal and erythropoietic stress conditions. RESULTS: We show that SWAP-70, a protein involved in cytoskeletal F-actin rearrangements and integrin regulation in mast cells, is expressed in hematopoietic stem cells and myeloid-erythroid precursors. Although Swap-70(-/-) mice are not anemic, erythroblastic differentiation is perturbed, and SWAP-70 is required for an efficient erythropoietic stress response to acute anemia and for erythropoietic recovery after bone marrow transplantation in irradiated mice. SWAP-70 deficiency impairs colony-forming unit erythroid development, while burst-forming unit erythroid development is normal, and significantly affects development of late erythroblasts in the spleen and bone marrow. The α(4) integrin is constitutively hyper-activated in Swap-70(-/-) colony-forming unit erythroid cells, which hyper-adhere to fibronectin. Blocking α(4) and β(1) integrin chains in vivo restored erythroblastic differentiation and the erythropoietic stress response in Swap-70(-/-) mice. Conclusions Our study reveals that SWAP-70 is a novel regulator of integrin-mediated red blood cell development and stress-induced erythropoiesis.     

Dyserythropoiesis and severe anaemia associated with malaria correlate with deficient interleukin-12 production

Complex cytokine interactions occur during blood-stage malaria which offer a unique opportunity to study their influence on the pathogenesis of malarial anaemia. Plasmodium chabaudi AS susceptible A/J mice experience severe and fatal anaemia whereas resistant C57BL/6 (B6) mice survive following moderate anaemia. In this study we analysed the role of IL-12 in erythropoiesis and tested whether the levels of IL-12 produced in these mice correlated with the extent of anaemia. In vitro , IL-12 significantly enhanced the numbers of erythroid burst (BFU-E) and colony forming units (CFU-E) in bone marrow and spleen cells from normal and day 7 infected A/J and B6 mice. Despite the presence of IL-12 in vitro , the level of splenic erythropoiesis in infected A/J mice was significantly lower than in B6 mice. Moreover, sera from infected B6 mice, but not A/J mice, significantly up-regulated erythropoiesis in vitro and this enhancement correlated with several fold higher levels of IL-12 in the sera of B6 compared to A/J mice. Furthermore, the erythropoietic potentiating effect of sera from infected B6 mice was abrogated following depletion of IL-12. Taken together, these findings suggest that defective IL-12 production in A/J mice during the early course of infection may result in fatal anaemia.     

Changes in murine bone marrow macrophages and erythroid burst-forming cells following the intravenous injection of liposome-encapsulated dichloromethylene diphosphonate (Cl2MDP)

In order to explore the effect on bone marrow macrophages of liposome-encapsulated dichloromethylene diphosphonate (Cl2MDP), mice were injected intravenously with a preparation of such liposomes at a dose known to deplete spleen and liver macrophages. Two days later, the macrophages in the marrow of the femoral bones were quantified by flow cytometry using a macrophage-specific monoclonal antibody (F4/80), and their ultrastructure and phagocytic activity towards zymosan particles was assessed. To determine the effect on erythropoiesis of liposome-encapsulated Cl2MDP-induced changes in bone marrow macrophages, red blood cell parameters and the formation of erythroid burst-forming unit (BFU-E)-derived colonies in vitro were evaluated. In mice injected with liposome-encapsulated Cl2MDP, there was a 54% and 67% decrease in the total number of bone marrow macrophages as compared to uninjected controls and mice treated with empty liposomes, respectively. Moreover, residual macrophages showed an abnormal ultrastructure, with reduced numbers of crystalloid inclusions and increased numbers of large myelin figures. However, the phagocytic activity of these cells was unimpaired or slightly enhanced. In mice injected with liposome-encapsulated Cl2MDP there was an approximately 60% decrease in the percentage and total number of circulating reticulocytes and a 54% reduction in the BFU-E number, demonstrating deregulation of erythropoiesis under conditions of macrophage loss and impairment. The results suggest that mice treated with liposome-encapsulated Cl2MDP are a model for studying the role of macrophages in erythropoiesis.     

The Effect of Bleeding on the Number of in Vitro Colony-Forming Cells in the Bone Marrow

Bleeding 0.3 ml. daily for 3 days reduced the total number of in vitro colony-forming cells in the bone marrow of C₅₇Bl and BALB/c mice to less than 50 per cent of that in control mice, but caused a significant increase of erythropoietic cells in the bone marrow. Transfusion of packed red cells combined with bleeding prevented this fall in in vitro colony-forming cells. Bled mice developed spleen enlargement with some increase in the total number of in vitro colony-forming cells in the spleen but this did not compensate for the reduced number of such cells in the bone marrow. Splenectomy did not affect this response of in vitro colony-forming cells in the bone marrow. Cortisone reduced the bone marrow content of in vitro colony-forming cells. Serum levels of colony-stimulating factor were not elevated in response to bleeding. The results suggest that in vitro colony-forming cells are not erythropoietic cells but may share a common ancestor with erythropoietic cells.     

Erythropoiesis-inhibiting factor(s) (EIF): methodologic studies.

Erythropoiesis-inhibiting factors (EIF) have been demonstrated in plasma from hypertransfused animals and from polycythemic individuals during periods of hyperoxia, but there is a decided discrepancy in the data published. In the present paper methodologic variations of a bioassay for demonstrating the erythropoiesis-inhibiting factor are discussed. In these studies no inhibitor of erythropoiesis could be demonstrated in plasma from hypoxia-induced polycythemic mice (HPM) on posthypoxic day 5. Injections of RBC or an equal amount of hemolyzed RBC were capable of suppressing the stimulatory effects of ESF, indicating that a red cell constituent may be responsible for the inhibitory effect observed. Transfusion-induced polycythemic mice (TPM) were therefore considered to be less suitable for demonstrating erythropoiesis inhibitors. Our results from testing several doses of a urinary EIF in normal mice, TPM and HPM, indicated that the HPM provided the most sensitive assay system. A similar effect was obtained with hypoxia-induced polycythemic rats. The most marked effect was seen in HPM when the EIF was injected shortly before administering the ESF, while the effect was less pronounced when the EIF was injected 24 hr before or after the ESF.     

Rac1 and Rac2 GTPases are necessary for early erythropoietic expansion in the bone marrow but not in the spleen

Background

The small Rho GTPases Rac1 and Rac2 have both overlapping and distinct roles in actin organization, cell survival, and proliferation in various hematopoietic cell lineages. The role of these Rac GTPases in erythropoiesis has not yet been fully elucidated.

Design and Methods

Cre-recombinase-induced deletion of Rac1 genomic sequence was accomplished on a Rac2-null genetic background, in mouse hematopoietic cells in vivo. The erythroid progenitors and precursors in the bone marrow and spleen of these genetically engineered animals were evaluated by colony assays and flow cytometry. Apoptosis and proliferation of the different stages of erythroid progenitors and precursors were evaluated by flow cytometry.

Results

Erythropoiesis in Rac1^−/−;Rac2^−/− mice is characterized by abnormal burst-forming unit-erythroid colony morphology and decreased numbers of megakaryocyte-erythrocyte progenitors, erythroid colony-forming units, and erythroblasts in the bone marrow. In contrast, splenic erythropoiesis is increased. Combined Rac1 and Rac2 deficiency compromises proliferation of the megakaryocyte-erythrocyte progenitor population in the bone marrow, while it allows increased survival and proliferation of megakaryocyte-erythrocyte progenitors in the spleen.

Conclusions

These data suggest that Rac1 and Rac2 GTPases are essential for normal bone marrow erythropoiesis but that they are dispensable for erythropoiesis in the spleen, implying different signaling pathways for homeostatic and stress erythropoiesis.     

In vivo suppression of erythropoiesis by tumor necrosis factor-alpha (TNF-alpha): reversal with exogenous erythropoietin (EPO)

Tumor necrosis factor-alpha (TNF-alpha) selectively kills tumor cells in vitro and in vivo and is being tested as a cancer therapeutic agent. We have shown that TNF-alpha significantly suppresses late-stage erythropoiesis, leading to anemia in chronically treated mice. These erythropoietic effects could limit the clinical use of TNF-alpha. Therefore, we have examined whether erythropoietin (EPO) could be used to prevent TNF-alpha-induced erythroid suppression. Normal mice were treated with a single dose of recombinant murine TNF-alpha (10(5) U/mouse, i.p.) with and without various concentrations of recombinant human EPO. After 3 days, effects on late-stage erythropoiesis were measured by determining the number of mature erythroid colony-forming cells (CFU-E) in the spleen and bone marrow. Simultaneous treatment with EPO abrogated the suppressive effect of TNF-alpha in a dose-dependent manner. EPO treatment also prevented the decrease in peripheral blood-hematocrit that was observed with chronic (5 x 10(4) U/mouse/day for 5 days) administration of TNF-alpha. TNF-alpha-induced hemorrhagic necrosis of tumors and stimulation of macrophage (CFU-M) progenitors were unaffected by EPO treatment. These results demonstrate that simultaneous injection of EPO can abrogate the TNF-alpha-induced suppressive effects on erythropoiesis.     

Endotoxin-induced suppression of erythropoiesis: the role of erythropoietin and a heme synthesis stimulating factor

The regulation of erythropoiesis is primarily controlled by erythropoietin (Ep). Recently, however, other factors that both stimulate and inhibit erythropoiesis have been reported. Using an in vitro liquid culture of bone marrow cells, a factor in normal mouse serum was demonstrated that markedly stimulated heme synthesis by marrow erythroid cells. In this study, the role of this heme synthesis stimulating factor (HSF) and Ep in the erythropoietic suppression caused by endotoxin administration to mice was examined. Although HSF levels did not alter appreciably after endotoxin injection, marrow erythroid cells from these animals became unresponsive to the factor. This could be reversed if Ep was added to the culture in vitro or if the hormone was injected into the mice 18 hr prior to harvesting the marrow. This marrow erythroid cell response is identical to that seen in animals in whom Ep levels are markedly reduced, such as that found in exhypoxic polycythemia, and suggest a decrease in the hormone following endotoxin administration. Additional studies demonstrated that when Ep was injected into mice 6 hr after endotoxin administration, an increase in femoral erythroid colony-forming units (CFU-E), proerythroblast number, and 59 Fe incorporation into femoral marrow cells could be demonstrated. These findings, together with the marrow erythroid cell response to the hormone, suggest that the mechanism for suppression of erythropoiesis after endotoxin injection is a reduction in the level of circulating Ep.     

Correction of severe anaemia using immuno-regulated gene therapy is achieved by restoring the early erythroblast compartment

Patients with chronic renal failure usually require exogenous erythropoietin (epo) to alleviate anaemia resulting from inadequate epo production by the kidneys. We have recently shown that severe anaemia in genetically manipulated epo-deficient mice (EpoTAg) can be corrected by adoptively transferred epo-producing lymphocytes. The aim of this study was to investigate the precise effects of human epo administration by this route on erythropoietic development in epo-deficient mice. The erythroblast compartments of untreated and treated EpoTAg mice were analysed in comparison with wild-type mice. The early erythroblast population was reduced in the bone marrow of epo-deficient mice, whilst the number of erythroid colony-forming units (CFU-E) was not significantly compromised. This paucity in marrow early erythroblasts was restored to normal values in treated mutant mice. In addition, the early erythroblast population was expanded in the spleens of treated animals. These findings show that the early erythroblasts are important targets of epo and that epo corrects anaemia of epo-deficient mice by restoring marrow function and splenic erythropoiesis.     

Delineation of erythropoiesis in normal and malignant bone marrow using monoclonal antibody AS-E1 directed against transferrin receptors (CD71)

Abstract: We have delineated the erythropoietic compartment in normal and malignant bone marrow (BM) by using the monoclonal antibody (mAb) AS-E1 directed against the transferrin receptor by flow cytometric (FCM) analysis. In normal BM we found a bimodal expression in antigen density with a minor subset (?3%) expressing AS-E1^high and a larger subset (?15%) expressing AS-E1^low. By fluorescence activated cell sorting, morphological examination of smears stained by immunocytochemistry and by BFU–E assays the AS-E1^high fraction was shown to contain cells of erythroid origin (proerythroblasts, basophilic erythroblasts and polychromatic erythroblasts), whereas the AS-E1^low fraction consisted mainly of promyelocytes and myelocytes. In patients with malignant hematological disorders we found a more pronounced heterogeneity in the density and the degree of AS-E1^low expression compared to normal BM, and to further characterize the AS-E1^low cells in patients and to exclude that this broad reactivity interfered with the identification of the AS-E1^high cells, we employed triple-color FCM assays with mAbs directed against the myeloid surface markers CD13 and CD66 in addition to AS-E1. In all patients we found that 80–90% of the AS-E1^low cells co-expressed CD13 and/or CD66 and thus were of myeloid origin. Finally, we evaluated 2 methods for determination of the AS-E1^high subset and found an assay involving forward light scatter and logAS-E1 density to be sufficient. We conclude that AS-E1^high is a valid FCM marker for the normal erythropoiesis.     

Postirradiation Erythropoietic Recovery in Splenectomized Mice

The influence of splenectomy on erythropoietic recovery of lethally X-iradiated mice injected with different doses of syngeneic bone marrow was studied. Splenectomized animals showed less activation than unoperated controls in the lower dose range; however, by increasing the number of injected cells the response obtained was similar in both groups. Providing an adequate number of stem cells is administered to the splenectomized recipients, an enhanced erythropoietic activity of the graft may compensate for the absence of the spleen, which is an important organ in postirradiation recovery. Suggested explanations for this observation are lack of an haematopoietic inhibitory effect of the irradiated spleen, or changes in the environment provided by the spleenless host.     

The cause of anemia in mutant mice of Sl/Slt genotype: hypoplasia in bone marrow and restricted hyperplasia in spleen

The cause of the severe anemia in Sl/Sld mice is attributed to (1) hypoproduction of erythrocytes due to a defect in the erythropoietic microenvironment and (2) bleeding from stomach ulcers. Sl/Slt mice also showed a moderate anemia, but bleeding from stomach ulcers was excluded as a cause of the anemia, because no significant amount of radioactivity was excreted in feces after the injection of 59Fe-labeled erythrocytes. The activity of erythropoiesis in the bone marrow and spleen was compared between Sl/Slt and congenic +/+ mice using three different criteria: the number of erythroblasts, 59Fe incorporation, and the number of erythropoietic precursor cells. All three parameters in the femur were lower, and those in the spleen were higher in Sl/Slt mice than in +/+ mice, suggesting that the low erythropoietic potential in the bone marrow of Sl/Slt mice is partially compensated by the spleen. In fact, splenectomy aggravated the anemia of Sl/Slt mice. The enhanced erythropoiesis in Sl/Slt spleens may explain our previous finding that numbers and sizes of spleen colonies were normal when bone marrow cells were injected into irradiated Sl/Slt mice. Sl/Slt mice may be a useful model for studying biological characteristics of the hematopoietic microenvironment.     

Immunological Studies of the Renal Erythropoietic Factor (Erythrogenin)

An immunological study of the renal erythropoietic factor (REF or erythrogenin) has been described. The experiments indicate that the erythropoietic activity of REF, as assayed in the polycythaemic mouse, is neutralized in vitro by addition of serum obtained from a rabbit previously immunized with a REF preparation. These anti-REF sera had no effect on the biological activity of ESF; however, a depression in erythropoiesis was observed after injection of anti-REF into normal mice. It is concluded that the injection of an antibody developed against the REF into normal mice interrupts normal erythropoiesis by reducing REF levels; the reduced REF levels lead to decreased amounts of ESF.     

Dyserythropoiesis in homozygous haemoglobin C disease

Summary. Electron microscope studies of the bone marrow of three patients with homozygous haemoglobin C (HbC) disease have shown marked ultrastructural abnormalities in several of the polychromatic erythroblasts and marrow reticulocytes and the presence of phagocytosed erythroblasts within the macrophages. Such abnormalities were not found in the bone marrow of three patients with sickle cell anaemia indicating that the abnormalities represented a feature of HbC disease rather than a disturbance secondary to peripheral haemolysis. The characteristic ultrastructural finding in the polychromatic erythroblasts in HbC disease was the presence of grossly-disorganized nuclei showing multiple intranuclear clefts, the loss of parts of the nuclear membrane, oozing of nuclear material into the cytoplasm and an alteration of the structure and stainability of the nuclear chromatin. It is proposed that both the dyserythropoiesis and ineffective erythropoiesis in HbC disease may have resulted from the formation in vivo of very small aggregates of HbC within erythropoietic cells.     

Erythropoiesis inhibiting factor(s) in intact and haemolyzed red blood cells.

A previous report have shown that both intact red blood cells (RBC) and an equal number of haemolyzed cells were able to inhibit erythropoiesis on ESF-induced erythropoiesis, suggesting a cell compound to be responsible for the inhibitiory effect. Haeme and haeme compounds have been found to stimulate or inhibit both erythropoiesis and haeme synthesis. The present work presents data on the inhibitory effect of haemolyzed RBC and compounds from intact RBC on erythropoiesis. The inhibiting factor was found to be of a small molecular size and of the same range as a urinary erythorpoiesis inhibiting factor (EIF). The inhibitor did not contain haeme. Both Fe+2 and Fe+3 were tested, showing no reduction of the 59-Fe incorporation into RBC in the test animals. The inhibition could not be due to dilution of the 59-Fe with unlabelled iron from the haemolyzed cells. On the contrary, Fe+3-ions rather stimulated erythropoiesis, probably due to increased amounts of available iron. Haemolysates were prepared from RBC with different amounts of immature cells. With increasing amounts of reticulocytes, a reduction of the inhibitory effect occurred. Also foetal cells showed less inhibition than an equal amount of adult cells. After high speed centrifugation, the inhibitory effect of haemolysates was found in the supernatant, while ghost cells exerted no inhibition. No species differences were found using both exhypoxic polycythaemic mice and rats. An inhibiting factor was liberated into the incubation medium when RBC were incubated for 20 h. No haemolysis occurred during the incubation period. Mature, adult RBC therefore contain a substance which is different from haeme, with a negative feedback on erythropoiesis.     

Fetal Erythropoiesis after Allogeneic Bone Marrow Transplantation Estimated by the Peripheral Blood Erythrocytes Containing Hemoglobin F (F-cells)

During bone marrow engraftment following BMT there is a re-establishment of fetal erythropoiesis, expressed by the increase of F-cells. This seems to depend on several factors such as underlying disease, conditioning before therapy and other mechanisms concerning both the donor and the recipient bone marrow. The aim of this work was to study the factors influencing F-cell production during bone marrow engraftment following transplantation. We studied 28 patients who underwent allogeneic bone marrow transplantation, for various hematological malignancies (CML, AML, ALL, CMML and SAA). F-cells were estimated on peripheral blood smears by indirect immunofluorescence. Overall, there was an F-cell increase after BMT in comparison with values before BMT; this increase was significant on days 15–50 (p <.01). F-cell on days 18, 25, 32 and 40 following transplantation were significantly higher (p <.01) in patients who have had increased F-cell numbers post-chemotherapy before BMT, compared with the patients who did not show any increase of the F-cell number post chemotherapy. During the first month following transplantation (day 7 to day 40) patients who were transplanted from high F-cell donors failed to show any significant differences in their F-cell numbers in comparison to those transplanted from low F-cell donors. However, the F-cell increase became significantly higher in the former group between days 50 and 120. This observation implies that the stressed erythropoiesis of the initial phase does not allow revealing the varying F-cell production of the capacities donor bone marrow, while later, when the graft has settled, the high F-cell donors reveal this property of the host.     

The soluble transferrin receptor in dysplastic erythropoiesis in myelodysplastic syndrome

OBJECTIVES: In individuals without iron deficiency, the soluble transferrin receptor (sTfR) directly reflects the erythropoietic activity. This study investigated sTfR concentrations in ineffective, dysplastic erythropoiesis in myelodysplastic syndrome (MDS). METHODS: To exclude influences of other myeloid cells on sTfR, only patients with refractory anemia (RA), refractory anemia with ringed sideroblasts (RARS) and 5q(-) syndrome were included. sTfR was measured nephelometrically (normal range 0.81-1.75 mg/L). RESULTS: Thirty-four untreated MDS patients (RA = 14, RARS = 10, 5q(-) syndrome = 10) were enrolled and analysed. The mean sTfR value of all MDS patients (1.30 +/- 0.8 mg/L, range 0.2-3.8) did not differ from our control group. In 5q(-) syndrome, the mean sTfR concentration (0.80 +/- 0.5 mg/L) was significantly lower than in RA (1.32 +/- 0.4 mg/L, P = 0.02) and RARS (1.75 +/- 1.1 mg/L, P = 0.03). Subdividing MDS according to their amount of erythroid mass in bone marrow a significant difference of sTfR between patients with decreased (0.70 +/- 0.4 mg/L), normal (1.32 +/- 0.4 mg/L) and increased (2.06 +/- 0.9 mg/L) erythropoiesis was observed. MDS patients with sTfR values below the reference range of 0.81 mg/L required transfusions in 90% of cases and showed higher erythropoietin levels compared to MDS patients with sTfR levels > or =0.81 mg/L (P = 0.01). There was a good agreement between sTfR and the amount of polychromatic erythroblasts observed (r = 0.68, P < 0.001). CONCLUSION: In conclusion, the serum concentration of sTfR reflects erythropoietic activity in MDS, but it is in particular determined by the degree of erythroid maturation and the severity of ineffective erythropoiesis. Low sTfR values in MDS are associated with a reduced, poorly differentiated erythropoiesis and requirement of blood transfusions.