Erythropoietin mediates tissue protection through an erythropoietin and common beta-subunit heteroreceptor

The cytokine erythropoietin (Epo) is tissue-protective in preclinical models of ischemic, traumatic, toxic, and inflammatory injuries. We have recently characterized Epo derivatives that do not bind to the Epo receptor (EpoR) yet are tissue-protective. For example, carbamylated Epo (CEpo) does not stimulate erythropoiesis, yet it prevents tissue injury in a wide variety of in vivo and in vitro models. These observations suggest that another receptor is responsible for the tissue-protective actions of Epo. Notably, prior investigation suggests that EpoR physically interacts with the common beta receptor (betacR), the signal-transducing subunit shared by the granulocyte-macrophage colony stimulating factor, and the IL-3 and IL-5 receptors. However, because betacR knockout mice exhibit normal erythrocyte maturation, betacR is not required for erythropoiesis. We hypothesized that betacR in combination with the EpoR expressed by nonhematopoietic cells constitutes a tissue-protective receptor. In support of this hypothesis, membrane proteins prepared from rat brain, heart, liver, or kidney were greatly enriched in EpoR after passage over either Epo or CEpo columns but covalently bound in a complex with betacR. Further, antibodies against EpoR coimmunoprecipitated betacR from membranes prepared from neuronal-like P-19 cells that respond to Epo-induced tissue protection. Immunocytochemical studies of spinal cord neurons and cardiomyocytes protected by Epo demonstrated cellular colocalization of Epo betacR and EpoR. Finally, as predicted by the hypothesis, neither Epo nor CEpo was active in cardiomyocyte or spinal cord injury models performed in the betacR knockout mouse. These data support the concept that EpoR and betacR comprise a tissue-protective heteroreceptor.     

Hypoxic stress tolerance of the blind subterranean mole rat: expression of erythropoietin and hypoxia-inducible factor 1 alpha

Blind subterranean mole rats (Spalax, Spalacidae) evolved adaptive strategies to cope with hypoxia that climaxes during winter floods in their burrows. By using real-time PCR, we compared gene expression of erythropoietin (Epo), a key regulator of circulating erythrocytes, and hypoxia-inducible factor 1 alpha (HIF-1 alpha), Epo expression inducer, in the kidneys of Spalax and white rats, Rattus norvegicus. Our results show significantly higher, quicker, and longer responses to different O(2) levels in Spalax compared with Rattus. (i) In normoxia, both Spalax and Rattus kidneys produce small amounts of Epo. Maximal expression of Rattus Epo is noticed after a 4-h hypoxia at 6% O(2). Under these conditions, Spalax Epo levels are 3-fold higher than in Rattus. After 24 h of 10% O(2), Spalax Epo reaches its maximal expression, remarkably 6-fold higher than the maximum in Rattus; (ii) the HIF-1 alpha level in normoxia is 2-fold higher in Spalax than in Rattus. Spalax HIF-1 alpha achieves maximal expression after 4-h hypoxia at 3% O(2), a 2-fold increase compared with normoxia, whereas no significant change was detected in Rattus HIF-1 alpha at any of the conditions studied; (iii) at 6% O(2) for 10 h, in which Rattus cannot survive, Epo and HIF-1 alpha levels in Spalax galili, living in heavily flooded soils, are higher than in Spalax judaei, residing in light aerated soil. We suggest that this pattern of Epo and HIF-1 alpha expression is a substantial contribution to the adaptive strategy of hypoxia tolerance in Spalax, evolved during 40 million years of evolution to cope with underground hypoxic stress.     

Effects of interleukin-3 and erythropoietin on in vivo erythropoiesis and F-cell formation in primates

To test the in vivo cooperativity between interleukin-3 (IL-3) and erythropoietin (Epo) in stimulating erythropoiesis and hemoglobin F (HbF) production in primates, we administered recombinant human IL-3 and recombinant human Epo to baboons and macaques. The effect of these treatments was assessed by serial bone marrow cultures and by measuring HbF production in the progeny of bone marrow progenitors and in peripheral-blood reticulocytes. Administration of IL-3 alone to hematologically normal or anemic baboons produced an early increase in erythroid colony-forming units (CFUe) and erythroid clusters (e-clusters) with an increase in reticulocyte counts and a late increment in the relative frequency of erythroid burst-forming units (BFUe). In parallel to the increase in peripheral-blood reticulocytes, IL-3 increased the frequency of F reticulocytes in the normal and anemic animals. When administration of IL-3 was followed by administration of Epo, expansion in all classes of erythroid progenitors and increase in reticulocytes occurred, beyond the levels observed when the animals were treated with Epo alone. The combination of IL-3 and Epo, however, did not increase consistently the rate of F reticulocytes beyond the level induced by Epo alone. These results suggest that IL-3 enhances the effect of Epo on erythropoiesis, but the combination of the two growth factors does not lead to a preferential and significant enhancement of HbF production.     

Oral administration of K-11706 inhibits GATA binding activity, enhances hypoxia-inducible factor 1 binding activity, and restores indicators in an in vivo mouse model of anemia of chronic disease

Erythropoietin (Epo) gene expression is under the control of hypoxia-inducible factor 1 (HIF-1), and is negatively regulated by GATA. Interleukin 1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha), which increase the binding activity of GATA and inhibit Epo promoter activity, are increased in patients with anemia of chronic disease (ACD). We previously demonstrated the ability of K-7174 (a GATA-specific inhibitor), when injected intraperitoneally, to improve Epo production that had been inhibited by IL-1beta or TNF-alpha treatment. In the present study, we examined the ability of both K-11706, which inhibits GATA and enhances HIF-1 binding activity, and K-13144, which has no effect on GATA or HIF-1 binding activity, to improve Epo production following inhibition by IL-1beta or TNF-alpha in Hep3B cells in vitro and in an in vivo mouse assay. Oral administration of K-11706 reversed the decreases in hemoglobin and serum Epo concentrations, reticulocyte counts, and numbers of erythroid colony-forming units (CFU-Es) induced by IL-1beta or TNF-alpha. These results raise the possibility of using orally administered K-11706 for treating patients with ACD.     

Erythropoietin, an autocrine regulator? Serum-free production of erythropoietin by cloned erythroid cell lines

Production of lymphoid and myeloid growth regulatory factors by hematopoietic cells is well documented. On the other hand, the major site of production of erythropoietin (Epo), which regulates physiologic red blood cell development, is thought to be the kidney. Here we report the isolation of multiple erythroleukemia cell lines that produce erythropoietic factors and present extensive biological, immunologic, and biochemical evidence to document that the active agent is Epo. The erythropoietic activity was neutralized by Epo antiserum and exhibited physical properties indistinguishable from those of human and sheep Epo. Positive lines produced between 0.1 and 1.5 U/mL of Epo, which stimulated erythropoiesis in vivo and in vitro in nine biological assays. Twenty sublines derived from single cells were inducible for hemoglobin and spectrin synthesis. All the sublines produced Epo. Production of the hormone continued when the cells were seeded in the absence of serum. Our finding that multiple independent isolates produce Epo raises the possibility that Epo production by erythroid precursors may play a role in normal erythropoiesis or, alternatively, that Epo gene activation may be a relatively common occurrence that contributes to, or is associated with, certain forms of virus-induced leukemias.     

Effect of inflammatory cytokines on hypoxia-induced erythropoietin production.

The effects of the inflammatory cytokines interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-6, transforming growth factor-beta (TGF-beta), and tumor necrosis factor-alpha (TNF-alpha) on erythropoietin (Epo) production in Hep3B cells were examined. The addition of IL-1 alpha, IL-1 beta, or TNF-alpha resulted in a dose-dependent inhibition of hypoxia-induced Epo production by as much as 89%. IL-1 beta was the most effective cytokine tested, demonstrating half-maximal inhibition at 0.4 U/mL compared with 1.0 and 10.0 U/mL for IL-1 alpha and TNF-alpha, respectively. TGF-beta also inhibited hypoxia-induced Epo production, but only by as much as 56%. In contrast to IL-1 alpha, IL-1 beta, TNF-alpha, and TGF-beta, the addition of IL-6 to hypoxic Hep3B cells resulted in a dose-dependent stimulation of hypoxia-induced Epo production by as much as 81%. However, IL-6 did not stimulate Epo synthesis in the absence of hypoxia, and was thus synergistic with hypoxia in inducing Epo production. Combinations of IL-1 alpha, TNF-alpha, and IL-6 were found to be additive in their effects on hypoxia-induced Epo production. By Northern blot analysis, Epo messenger RNA levels in Hep3B cells grown in 1% O2 were decreased when concurrently exposed to either IL-1 alpha or TNF-alpha. The effects that IL-1 alpha, IL-1 beta, TGF-beta, TNF-alpha, and IL-6 have on hypoxia-induced Epo production may provide new insights into the signal transduction pathway by which hypoxia leads to changes in gene expression. In addition, the effects of these inflammatory cytokines on hypoxia-induced Epo production in vitro suggest that in various inflammatory disorders these cytokines may affect Epo production in vivo and may play a significant role in the pathogenesis of the anemia of chronic disease.     

Erythropoietin production and erythropoiesis in compensated and anaemic states of hereditary spherocytosis

A compensated haemolytic state is defined by decreased red cell life-span without anaemia, i.e. by increased erythropoiesis in the absence of the physiological stimulus for erythropoietin (Epo) production. We evaluated s-Epo levels and the expansion of erythropoiesis (as measured by circulating transferrin receptor, s-TfR) in 32 patients with hereditary spherocytosis (HS) with the aim of verifying whether the enhanced erythropoiesis of compensated haemolysis was Epo-dependent. 20 of the patients (62.5%) had normal Hb values (> 12 g/dl in females and > 13 g/dl in males). Their compensated haemolytic state was the result of up to 8.2 times normal s-Epo and up to 3.9 times normal s-TfR levels, which were maintained by physiological regulation of erythropoiesis, as documented by the inverse dependence of Hb on s-Epo levels. Considering that patients with iron-deficiency anaemia represented the predicted physiological Epo response to anaemia, the observed/predicted ln s-Epo ratio (O/P ratio) was calculated in HS patients with anaemia and was used as an index of the adequateness of Epo production. All the anaemic HS patients had an O/P ratio > 1, documenting inappropriately high s-Epo levels. This work demonstrates that the compensated haemolytic state of HS patients is produced by an inappropriately high s-Epo level, and that the pattern of Epo overproduction is a biological characteristic of the disease.     

Inadequate erythropoietin response to anemia: definition and clinical relevance

Summary The development of a specific and sensitive immunoassay for the measurement of serum erythropoietin (s-Epo) allows to improve our understanding of the model of in vivo regulation of erythropoiesis. In most anemias, circulating hemoglobin level determines Epo production which in turn stimulates erythropoietic activity. The disruption of the expected feedback mechanism of Epo production leads to an inadequately low s-Epo. The definition of inadequate Epo response to anemia relies on the documentation of a downregulated dependence of s-Epo on Hb with respect to the same dependence in patients with a physiologically regulated erythropoiesis. Literature reports a wide range of so called adequate s-Epo response to anemia and a number of criteria for judging on the adequateness of s-Epo at a certain degree of anemia. The O/P (observed/predicted) ratio allows categorization of each individual patient. The clinical syndromes in which an inadequate Epo response has been reported are numerous and the mechanisms of defective Epo production are different. A number of evidences clearly point to a relation between responsiveness to r-Hu-Epo and inadequate Epo response. This inequivocabily confirms the role inadequate Epo response plays in the pathogenesis of anemia.     

Erythropoietin production and erythropoiesis in compensated and anaemic states of hereditary spherocytosis

A compensated haemolytic state is defined by decreased red cell life-span without anaemia, i.e. by increased erythropoiesis in the absence of the physiological stimulus for erythropoietin (Epo) production. We evaluated s-Epo levels and the expansion of erythropoiesis (as measured by circulating transferrin receptor, s-TfR) in 32 patients with hereditary spherocytosis (HS) with the aim of verifying whether the enhanced erythropoiesis of compensated haemolysis was Epo-dependent. 20 of the patients (62.5%) had normal Hb values (> 12 g/dl in females and > 13 g/dl in males). Their compensated haemolytic state was the result of up to 8.2 times normal s-Epo and up to 3.9 times normal s-TfR levels, which were maintained by physiological regulation of erythropoiesis, as documented by the inverse dependence of Hb on s-Epo levels. Considering that patients with iron-deficiency anaemia represented the predicted physiological Epo response to anaemia, the observed/predicted ln s-Epo ratio (O/P ratio) was calculated in HS patients with anaemia and was used as an index of the adequateness of Epo production. All the anaemic HS patients had an O/P ratio > 1, documenting inappropriately high s-Epo levels. This work demonstrates that the compensated haemolytic state of HS patients is produced by an inappropriately high s-Epo level, and that the pattern of Epo overproduction is a biological characteristic of the disease.     

Modulation of erythropoiesis and myelopoiesis by exogenous erythropoietin in human long-term marrow cultures

In spite of their ability to support myelopoiesis for several months, human long-term marrow cultures (LTMC) are unable to sustain the production of mature erythroid cells for greater than 4 weeks. Because this preference correlates with the presence of myeloid growth factors and possible absence of erythroid factors in LTMC, we studied the effects of the erythroid growth and differentiation factor erythropoietin (Epo) on both erythropoiesis and myelopoiesis in human LTMC. Either natural or recombinant Epo was added weekly to LTMC for 10 weeks, and total cell number, numbers of hemopoietic progenitors (mixed lineage colony-forming units, CFU-MIX; erythroid burst-forming units, BFU-E; erythroid CFU, CFU-E; granulocyte-macrophage CFU (CFU-GM); granulocyte CFU, CFU-G; and macrophage CFU, CFU-M), erythroblasts (early and late), granulocytes, and macrophages were quantitated separately in the adherent and nonadherent layers of the cultures. In the absence of Epo, mature erythroid cells disappeared within the first 3-4 weeks, whereas in cultures supplemented with Epo, erythropoiesis was supported for up to 8 weeks. Results indicate that erythroid maturation is blocked at the BFU-E stage and that exogenous Epo may act on a mature subpopulation of BFU-E located in the nonadherent fraction of the cultures, promoting its maturation into CFU-E, which in turn develop into erythroblasts. However, despite Epo supplementation, erythropoiesis was not restored to in vivo proportions, suggesting that additional factors or conditions necessary for erythropoiesis are lacking in LTMC. Interestingly, we found that exogenous Epo reduced the numbers of presumably more mature (nonadherent) myeloid CFU (CFU-C), granulocytes, and macrophages compared to controls and did not alter the levels of any of the most primitive hemopoietic progenitors measured (CFU-MIX, adherent BFU-E, and adherent CFU-C). Thus the data show that exogenous Epo modulates hemopoiesis in human LTMC, enhancing erythropoiesis and suppressing myelopoiesis, but that its effects appear limited to modulating levels of the nonadherent (more mature) progenitors, leaving the numbers of the adherent (immature) progenitor cells unchanged.     

Blunted erythropoietin production and decreased erythropoiesis in early pregnancy

After decreasing in the first trimester of pregnancy, the total red blood cell mass increases in the second and third trimesters to peak at term at about 120% to 125% of nonpregnant values, but how this is brought about by changes in the rate of erythropoiesis is not known. We evaluated erythropoiesis by measuring serum transferrin receptor (TfR) levels in 406 women during normal pregnancy (N = 317), at delivery (N = 63), or in the early postpartum (N = 27). Despite the presence of the placenta and the frequent occurrence of iron deficiency, TfR levels remained low in the first two trimesters and increased in the third trimester and at delivery. To explain why erythropoiesic activity was relatively low in early pregnancy, we also measured serum immunoreactive erythropoietin (Epo) in relation to the degree of anemia. There was a very strong correlation between serum TfR and Epo levels in the entire group (r = .59, P less than .0001) as well as in each period of pregnancy. Epo levels remained low for the degree of anemia and did not correlate with hematocrit in the first two trimesters, but recovered afterwards. In the early postpartum, Epo production and erythropoiesis were normal. We conclude that: (1) erythropoiesis is decreased in the first part of pregnancy but increases afterwards; and (2) blunted Epo production in early pregnancy could be responsible for that observation.     

betaMinor-globin messenger RNA accumulation in reticulocytes governs improved erythropoiesis in beta thalassemic mice after erythropoietin complementary DNA electrotransfer in muscles

Mechanisms governing the induction of effective erythropoiesis in response to erythropoietin (Epo) oversecretion have been investigated in beta thalassemic C57Bl/6(Hbbth) mice. Naked DNA encoding an expression vector for mouse Epo was introduced into skeletal muscles by electrotransfer. A transient increase of serum Epo concentrations with a proportional augmentation of hematocrit values was observed. Various parameters relevant to beta thalassemia were surveyed in blood samples taken before treatment, at the peak of Epo secretion, and when the phenotype reverted to anemia. We measured globin messenger RNA (mRNA) levels in reticulocytes by real-time quantitative polymerase chain reaction, globin chain synthesis levels, and several indicators of erythrocyte membrane quality, including bound alpha chains, bound immunoglobulins, main protein components, and iron compartmentalization. Data indicated that high serum Epo levels primarily affect betaminor-globin mRNA accumulation in reticulocytes. Other changes subsequent to intense Epo stimulation, like increased betaminor/alpha-globin chain synthesis ratio, reduced levels of alpha chains and immunoglobulins bound to membranes, improved spectrin/band 3 ratio, increased red blood cell survival, and improved erythropoiesis appeared as consequences of increased betaminor-globin mRNA levels. This conclusion is consistent with models postulating that intense Epo stimulation induces the expansion and differentiation of erythroid progenitors committed to fetal erythropoiesis. Although phenotypic correction was partial in mice, and comparable achievements will probably be more difficult to obtain in humans, naked DNA electrotransfer may provide a safe and low-cost method for reassessing the potentials of Epo as an inducer of fetal erythropoiesis reactivation in patients with beta thalassemia.     

Erythropoietin in healthy elderly subjects

The effects of aging on erythropoiesis and the factors regulating it are not well known. Erythropoietin (Epo) is a specific growth factor for the erythroid line and is mainly produced by the kidney. As data on the effects of aging on Epo are discordant we studied serum Epo concentrations in a group of apparently healthy subjects divided into age classes in order to evaluate age-related modifications and correlations with hemoglobin (Hb) and red blood cells (RBC). Our results revealed that Epo values were correlated with age. Epo was higher in the over 65 years age classes than observed in control subjects. We believe that the elevated Epo values in the latter age class may be required to maintain Hb and RBC within normal range.