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Erythrocyte production is regulated by balancing precursor cell apoptosis and survival signaling. Previously, we found that BH3-only proapoptotic factor, Nix, opposed erythroblast-survival signaling by erythropoietin-induced Bcl-xl during normal erythrocyte formation. Since erythropoietin treatment of human anemia has limitations, we explored the therapeutic potential of abrogating Nix-mediated erythroblast apoptosis to enhance erythrocyte production. Nix gene ablation blunted the phenylhydrazine-induced fall in blood count, enhanced hematocrit recovery, and reduced erythroblast apoptosis, despite lower endogenous erythropoietin levels. Similar to erythropoietin, Nix ablation increased early splenic erythroblasts and circulating reticulocytes, while maintaining a pool of mature erythroblasts as erythropoietic reserve. Erythrocytes in Nix-deficient mice showed morphological abnormalities, suggesting that apoptosis during erythropoiesis not only controls red blood cell number, but also serves a “triage” function, preferentially eliminating abnormal erythrocytes. These results support the concept of targeting erythroblast apoptosis to maximize erythrocyte production in acute anemia, which may be of value in erythropoietin resistance.
Dynamic regulation of erythropoiesis is largely determined by activation of erythroblast survival pathways by erythropoietin (EPO), secreted by the kidney in response to anemia or hypoxic stress 1. EPO and its longer acting analogue, darbopoietin are widely employed therapeutically to treat anemia in cancer, myelodysplastic syndromes, renal disease and rheumatoid arthritis 2-5. While this approach of enhancing erythroblast survival is effective, therapy with these ‘erythropoiesis stimulating agents’ has raised concerns for venous thromboembolism 6, stimulation of cancer growth 7 and induction of a rare form of red-cell aplasia 8. EPO treatment has hypertensive effects that may increase mortality in patients with renal failure 9,10, and between 10-50% of the patients are hyporesponsive to EPO treatment, depending on the underlying condition 11. A better understanding of the molecular mechanisms involved in erythroblast development may help identify novel targets to treat anemia.
EPO secretion by the kidney is the major stimulus for increased erythrocyte production in anemia 12. Although multiple cellular mechanisms may be involved, one of the effects of EPO is to shift the balance between erythrocyte precursor (erythroblast) survival and programmed death 13. An important mechanism by which EPO stimulates survival signaling in erythroblasts is transcriptional upregulation of anti-apoptotic Bcl-xl via activation of the JAK2/Stat5 pathway 13,14. As a consequence of increased Bcl-xl during erythroblast maturation, there is increased production of circulating reticulocytes from erythroblast precursors. Accordingly, mouse models lacking EPO, its receptor, or its critical downstream effectors exhibit increased erythroblast apoptosis and anemia 1,14-16.
Given that Bcl-xl binds to and inhibits pro-apoptotic BH3-only members of the Bcl2 family of mitochondrial apoptosis factors, 17,18 there must be an erythroblast death pathway opposed by Bcl-xl. Recently, using gene targeting in the mouse, we identified Nix as the critical pro-apoptotic BH3 factor that causes apoptosis of maturing erythroblasts 19. The hallmark characteristic of Nix knockout mice was a reticulocytosis that occurred in the context of low/normal in vivo circulating EPO levels, leading us to conclude that absence of pro-apoptotic Nix dis-inhibits EPO/Bcl-xl-stimulated erythroblast survival pathways. These findings also suggest that inhibition of erythroblast apoptosis might be an effective therapeutic approach for anemia. Here, we performed proof-of-principle studies demonstrating that inhibiting erythroblast apoptosis by Nix ablation can promote rapid reconstitution of the circulating erythrocyte compartment in response to an anemic stress.
Nix knockout mice 19 were maintained in a mixed 129SVJ/C57BL/6 background and were housed and studied according to procedures approved by the University of Cincinnati Institutional Animal Care and Use Committee.
Splenocytes were separated from parenchyma by filtration through a 70μm nylon cell strainer (BD Biosciences, CA). As described 19, freshly isolated splenocytes were suspended at a density of 106 cells per 100μl of phosphate-buffered saline (PBS), stained with Ter 119-PE (1:200) and CD71-FITC (1:100) (BD Pharmingen, CA), Annexin V-APC (1:20), and 7AAD (1:20), and 5X104 cells analyzed by flow cytometry using a FACSCalibur instrument from BD Pharmingen, CA. Data analysis was performed using CellQuest™Pro software from BD Biosciences, CA.
Mice were injected intraperitonealy on each of days 0, 1, and 3 with 40mg/kg of phenylhydrazine hydrochloride (Sigma, MO) solution in PBS 20. Blood was obtained by sub-mandibular bleed on days 0, 3, 4, 6, and 9 for hematocrit and reticulocyte count measurements. Reticulocytes were enumerated by staining blood smears with Reticulocyte Stain (Sigma, MO) and counted using oil immersion microscopy. Flow cytometry of splenocytes was performed on the day after the final phenylhydrazine injection.
Mice were injected subcutaneously with 300IU/kg of Epogen (Amgen, Thousand Oaks, CA) solution in PBS for five consecutive days (days 0-4) 21. Blood was obtained by sub-mandibular bleed on days 0, 2, 5, 7, and 9 for spun hematocrit and manual reticulocyte count measurements. Reticulocytes were enumerated by staining blood smears with Reticulocyte Stain (Sigma, MO) and counted using a 100x oil objective (Olympus SPlan 100PL) on a Nikon Diaphot 300 microscope. FACS analysis was performed the day after the final erythropoietin dose.
Serum erythropoietin was assayed with a murine erythropoietin ELISA kit from R & D Systems (Minneapolis, MN), according to the manufacturer's instructions.
To study red cell enucleation, we used a modification of an ex vivo erythroid differentiation culture protocol, previously 22. Briefly, low density bone marrow cells were cultured in serum-free medium StemPro-34TM (Invitrogen, CA) supplemented with StemPro-34 medium supplement, 1% BSA, 10 μg/ml insulin, 200 μg/ml transferrin (BIT-9500, StemCell Technologies), 900 ng/ml ferrous sulfate, 90 ng/ml ferric nitrate, in three steps. On days 0-7, the medium was supplemented with 10-6 M hydrocortisone, 100ng/ml SCF, 5ng/ml IL-3, and 2 IU/ml EPO. On days 8-10, 2 X 105 cells/ml were resuspended in 6-well plates coated with fibronectin (50 μg/ml) in fresh medium supplemented with EPO. Finally, on days 10-12, the cells were cultured on fibronectin in fresh medium without cytokines. The percentage of enucleated cells in ex vivo erythropoiesis cultures were evaluated by flow cytometry on day 12. The cells from each well were collected and stained with anti-Ter119-PECy7 (BD Biosciences, CA) in 100μl PBS + 0.5% BSA + 5% mouse serum, for 20 minutes on ice. After gentle washing to remove unbound antibody, the cells were resuspended in 10mM HEPES buffer pH 7.4 containing 140mM NaCl and 5mM MgCl2 with 0.25μM SYTO16 (Molecular Probes, Invitrogen, CA), a green-fluorescent cell-permeable nucleic acid-staining dye. After gating on Ter119-positive cells, the SYTO16-low and negative population represent the enucleated red blood cells.
Peripheral blood smears were stained with MTR CMXRos (Molecular Probes, CA) and imaged using a confocal laser-scanning microscope (LSM 510; Carl Zeiss, Thornwood, NY) equipped with a 100x Plan-Apo oil-immersion objective (N.A. 1.4), with Zeiss LSM 510 software, version 3.2.
Freshly obtained red blood cells were washed and suspended in PBS to obtain a hematocrit of 20%. 10μl of resuspended RBCs were incubated with a gradient of concentrations of hypotonic to isotonic NaCl solution for 15 minutes. Hemolysis was quantitated as absorbance of released hemoglobin at 550nm as described 23.
Splenocytes were labeled with iron particle tagged anti-Ter119 antibody and magnetically enriched using MACSR cell separation columns (Miltenyi Biotech, CA). Enriched splenocytes were lysed in a buffer containing 10mmol/l HEPES, pH 7.2, 320mM sucrose, 3 mM MgCl2, 25 mM Na2P4O7,1mM DTT, 5mM EGTA and 20mM NaF and centrifuged at 100,000g for 1 hour. The resulting pellet (membrane fraction: M) and the supernatant (S) were subjected to SDS-PAGE, western transfer and immunoblotted for Nix (antiBnip3L antibody, Abcam, MA). Red blood cell ghosts were prepared as previously described 24.
Results are expressed as mean ± SEM. Statistical differences were assessed with ANOVA, unpaired t test, or paired t tests as indicated. A nonparametric test was applied when the data were not normally distributed. Significance was determined by a two-tailed P < 0.05 for t test.
To characterize the effects of anemia on apoptosis of maturing erythroblasts, we treated mice with phenylhydrazine (PHZ) to induce acute hemolytic anemia 25 and examined externalization of phosphatidylserine by annexin V staining on the surface of Ter119 positive splenocytes at day 4. In wild-type mice, the nadir of PHZ-induced anemia (hematocrit ~30%, see below) was associated with a striking decrease in annexin V positivity of splenic erythroblasts throughout the early and middle stages of the maturation sequence (Figure 1). This lower level of apoptosis during anemia recovery in wild-type mice is similar to the previously reported low rate of erythroblast apoptosis observed in mice lacking the gene for Nix (Figure 1 and reference 19). Induction of acute anemia in Nix knockout mice with PHZ had only a minor additional suppressive effect on erythroblast apoptosis (Figure 1), suggesting that apoptosis inhibition during erythroblast maturation is already maximal after Nix ablation.
To determine if chronically inhibiting erythroblast apoptosis would favorably affect clinical parameters in acute anemia, we injected ten pairs of young adult Nix null and wild-type mice with PHZ 25, and examined the time course of hematocrit decline and recovery over nine days. As previously observed with this protocol 26, the hematocrits of wild-type mice had declined by almost half on day four (Figures 2A, B), and the corrected reticulocyte count peaked at approximately 60% on the sixth day after the first PHZ injection, representing a 40-fold increase over baseline (Figures 2C, D). In contrast, the nadir of hematocrit for Nix null mice occurred on day three and declined by only 20%, compared to the starting hematocrit value (Figures 2A, B). Reticulocyte counts of Nix null mice, which are characteristically elevated at baseline, increased only modestly in response to PHZ (Figures 2C, D). Recovery from anemia was also more rapid in Nix null mice, with hematocrit values rebounding to above baseline on day six, at which time the hematocrits of wild-type mice were still depressed. The hematocrits of both groups of mice had fully recovered on day nine. Hematocrit recovery in Nix null mice was not the consequence of increased EPO secretion, as serum EPO levels measured four days after PHZ were less than one third those of wild-type mice (2815±384 pg/ml Nix null (n=5) vs. 10,354±3565 pg/ml wild type (n=4); P<0.05).
In rodents, the spleen is more important than bone marrow in EPO-mediated stress erythropoiesis27,28. Therefore, to assess in more detail the consequences of Nix ablation on erythroblast survival and maturation during acute anemic stress, we immunophenotyped splenocytes four days after PHZ treatment. In wild-type spleens, the overall proportion of erythroid cells (Ter119 positive) to lymphoid cells increased from 51±3% to 73±7% on day four after the first PHZ injection (Table 1). The numbers of cells from earlier stages of the erythroid maturation sequence (pro-erythroblasts, basophilic erythroblasts, and chromatophilic erythroblasts) increased dramatically (Figure 3, Table 1), and there was depletion of the more mature orthochromatic erythroblasts (Table 1, Figure 3). These changes reflect mobilization of the more mature cells into the peripheral circulation and increased splenic erythropoiesis. By contrast, Nix null mice started the experiment with a characteristic early stage erythroblastosis because intrinsic apoptosis suppression in the absence of Nix has the greatest effect in the early stages of the maturation sequence (Figure 3). While PHZ depleted mature erythroblasts in Nix null mice (Table 1, Figure 3), the pre-existing erythroblast reserve was available for mobilization and more rapid anemia recovery.
Reduced severity of anemia and early recovery after PHZ in Nix null mice in the context of lower circulating levels of erythropoietin could be because: 1. Erythropoiesis in Nix null mice is essentially independent of EPO, in which case there would be insensitivity to exogenous EPO administration. 2. Or, Nix ablation may greatly increase sensitivity to EPO, in which case we would observe a robust erythroblastosis and reticulocytosis in response to exogenous EPO, and resistance to anemia.
To assess the integrity of in vivo EPO responsiveness in Nix null mice, we profiled circulating red blood cells and splenic erythroblasts in ten pairs of wild-type and Nix null mice that received EPO, 10U daily for five days. As previously observed with this EPO treatment protocol 21, the hematocrits of wild-type mice peaked on day seven (i.e. two days after the last EPO dose; Figures 4A, B). In contrast, hematocrit peaked two days earlier in Nix null mice (Figures 4A, B), and in proportion to the individual respective starting hematocrit values, tended to increase more rapidly in response to EPO (Figure 4B). The time course for reticulocyte count to increase with EPO treatment was similar in wild-type and Nix null mice, peaking on day five, i.e. the day following the last EPO dose (Figures 4C, D).
Spleens of EPO-treated wild-type and Nix null mice showed similar increases in the proportion of total Ter119 positive cells (Table 1). In wild-type mice this was largely attributable to a ~25-fold increase in the basophilic erythroblast population compared to baseline, with qualitatively similar, but quantitatively more modest changes in the EPO-treated Nix null mice due to the pre-existing large numbers of erythroblasts at this maturational stage (blue colored cells in Figure 3, Table 1).
Along with characteristic reticulocytosis, Nix null mice are reported to exhibit abnormal erythrocyte morphology in the form of polychromasia and poikilocytosis 19,29 (Figure 5A). Given the high rate of erythroblast development in these mice, we considered that rapid red blood cell production might be associated with an escape from normal mechanisms of quality control during erythroblast maturation. Indeed, recent studies have identified an additional role for Nix in removal of mitochondria during terminal erythroid maturation 29,30. Alternately, Nix ablation might create an abnormality of late erythroblast development, such as terminal enucleation, or it could even have a protective effect in mature erythrocytes. Accordingly, we examined these possibilities. We compared enucleation in cultured bone marrow cells from Nix null and wild-type mice. As shown in Figure 5B, at day 12, the percent of enucleated erythroblasts was similar in Nix null and wild type mice, which does not support a role for Nix in removal of erythroblast nuclei. Analogous to the recent studies29,30, we also found persistence of mitochondria in the circulating red blood cell population, in Nix null mice (Figure 5C). Additionally, Nix null erythrocytes demonstrated increased osmotic fragility (Figure 5D), suggesting an additional mechanism for the decreased survival of Nix null erythrocytes reported by Sandoval et al30, in addition to increased phagocytosis of erythrocytes.
Since increased fragility could be due to a role of Nix in stabilizing circulating erythrocyte membranes, as postulated for Bcl-xl 31, we first determined whether Nix was present in circulating erythrocytes. Immunoreactive Nix was detected in splenic Ter119 positive erythroblasts and Ter119 negative splenocytes, but not in circulating erythrocytes (Figure 5E). As previously reported 31, Bcl-xl was present in erythrocytes (Figure 5E). These studies suggest a dual role for Nix during erythroblast maturation, namely, regulation of erythroblast apoptosis as a mechanism to regulate ‘quantity’ or cell number and clearance of mitochondria to regulate ‘cell quality’ resulting in mature circulating erythrocytes.
The current studies were undertaken to test the approach of promoting erythropoiesis by suppressing endogenous erythroblast apoptosis. We used Nix null mice as the experimental model in these studies because Nix acts in opposition to EPO/Bcl-xl in erythroblast maturation, and elimination of Nix should de-repress erythroblast survival at baseline and in response to stress. The general principle was validated, as Nix null mice exhibit a high level of reticulocyte production that blunted the severity of experimental anemia and permitted more rapid hematocrit recovery, despite lower endogenous EPO levels.
Normal homeostatic erythropoiesis occurs continuously in order to replenish senescent erythrocytes that are removed from the circulation after a lifespan that, in the mouse, has a normal reported half-life of between 12 and 16.5 days 26,32. Under these conditions, the number of earlier maturational stage erythroblasts, basophilic and chromatophilic erythroblasts, is held in check by intrinsic apoptosis pathways that are incompletely defined 33. Thus, in the normal mouse spleen, which is more important to stress-mediated erythropoiesis than is bone marrow in this species 27,28, examination of the erythroblast maturation sequence reveals a relative paucity of these early erythrocyte precursors, with an abundance of more mature orthochromatic erythroblasts that are readily available for terminal differentiation, enucleation, and release into the circulation during anemic or hypoxic stress. Although there may be multiple mechanisms for EPO-mediated erythropoiesis, EPO stimulation of Epo/Jak2/Stat5 34, with or without involvement of GATA-1 35, increases expression of the anti-apoptotic factor Bcl-xl, thereby enhancing survival of maturing erythroblasts. Nix is a developmentally regulated pro-apoptotic factor that is co-expressed in differentiating erythroblasts with, and functionally antagonized by, Bcl-xl 36,37. Accordingly, ablation of the mouse Nix gene alters the erythroblast maturational profile, causing enrichment of early- and mid-maturational sequence erythroblasts and increasing the overall proportion of erythroblasts to non-erythroblasts in spleen.
According to the above conceptual framework, Nix acts in opposition to EPO/Bcl-xl, and absence of Nix should therefore mimic EPO treatment. This was the case for in vitro CFU-E assays performed in cultured Nix null splenocytes, which revealed EPO-independent CFU-E formation and increased sensitivity to exogenous EPO 19. The current studies show that Nix ablation closely mimics EPO in its effects on in vivo erythroblast maturation. In wild-type mice, EPO dramatically increased the numbers of basophilic erythroblasts in spleen, thus increasing the overall proportion of erythroblasts to non-erythroblasts. However, more mature orthochromatic erythroblasts continued to be present after EPO treatment. We observed that the erythroblast maturation profile of Nix null mice was similar to that of wild-type mice recovering from acute anemia (endogenous EPO secretion), or who received exogenous EPO. Thus, Nix ablation largely recapitulated the effects of exogenous EPO on erythroblast maturation.
Our results (current study and reference 19), and recent observations implicating a role for Nix in mitochondrial clearance during erythrocyte maturation 29,30, suggest that Nix has a dual role in red blood cell formation depending upon the developmental stage. During erythroblast development, Nix functions as a proapoptotic factor which serves as a mechanism to regulate erythrocyte ‘quantity’. Nix mediated apoptotic cell death in erythroblasts, in opposition to EPO induced survival signaling, may also be an important ‘triage’ mechanism exerting a ‘quality’ control in erythrocyte generation. Indeed, erythroblasts vary in their sensitivity to EPO signaling38, which is likely a mechanism that acts in concert with Nix signaling to orchestrate removal of erythroblast subpopulations. The observed role for Nix in mediating mitochondrial clearance, which apparently can proceed unimpeded in a large proportion (~40-60%) of erythrocytes even in the absence of Nix 29,30, is therefore an additional mechanism for quality control in a subpopulation of maturing erythroblasts. During anemic stress, when rapid mobilization of erythroblasts is required, stimulation of EPO production by the hypoxic stimulus in kidneys 12, enhances survival signaling in all erythroblast populations, leading to formation of additional erythrocytes.
Our results show that targeting erythroblast apoptosis downstream of EPO signaling may be a viable strategy to prevent severe anemia in response to an acute anemic stress. Certain aspects of Nix-mediated erythroblast apoptosis are conducive to its therapeutic targeting to promote erythroblast survival: 1. It is an inducible pro-apoptotic protein, which is expressed in a highly regulated fashion during the erythroblast developmental sequence 37. 2. Nix ablation decreases apoptosis in basophilic and chromophilic erythroblasts 19, which are the predominant population of cells upregulated in wild type mice in response to an acute anemic stress in the spleen (see Figure 3 and Table 1). 3. Importantly targeting Nix does not impact the white blood cell compartment or hematopoietic stem cell population 19, which suggests minimal potential for this approach to create undesirable hematopoietic side-effects.
Rapid and complete reconstitution of the erythrocyte compartment after PHZ induced anemia in Nix null mice, suggests enhanced erythroblast survival as the primary mechanism uncovered by Nix ablation. Indeed, in mice with enhanced erythroblast apoptosis, such as Lyn null mice 39, with resultant impairment in STAT5/Bcl-xl mediated survival signaling, PHZ treatment did not result in accelerated recovery from anemia, despite stimulation of extramedullary hematopoiesis. Importantly, survival signaling through a functional EPO receptor (mediated via STAT5 activation) was necessary for recovery from PHZ induced anemic stress 40. Based on our studies, we postulate that EPO induced Bcl-xl, antagonizes the proapoptotic action of Nix to promote erythroblast survival, both in the basal state 19 and during stress. PHZ induces hemolysis by causing oxidative damage to the erythrocyte membrane 41. Therefore, if the major role for Nix was in mitochondrial clearance from maturing reticulocytes with increased oxidative stress, as postulated by Sandoval et al 30, the response to PHZ treatment would be exactly opposite to what we observed, with much poorer recovery from anemia. Indeed, in p45NF-E2 null mice, with analogously elevated levels of reactive oxygen species in erythrocytes, PHZ treatment provoked a markedly increased severity of anemia 26.
There are aspects of this work that suggest major challenges to implementing anti-apoptosis therapy for anemia. The normal hematocrit of unstressed Nix null mice does not wholly reflect increased erythroblast production indicated by a reticulocyte count that is five-fold higher than normal. This is likely secondary to increased erythrocyte turn-over supported by the observation of morphologic changes in circulating erythrocytes (Figure 5A), increased osmotic fragility (Figure 5D) and decreased life span of circulating red blood cells 30. A similar phenomenon has been observed in polycythemic mice (induced by transgenically elevated circulating levels of EPO 42); after EPO treatment, with anemia, and after Friend virus infection in mice 32,43, as well as in human polycythemia vera 44 and a subset of patients having myelofibrosis with myeloid metaplasia, i.e. “spent polycythemia” 45-47. In each of these conditions, an abbreviated erythrocyte life span is associated with increased activity of EPO or one of its downstream effectors, just as in Nix null mice the stimulus for erythropoiesis is disinhibition of EPO-mediated cell survival signaling in erythroblasts.
The preponderance of evidence supports an important role for Nix as a key regulator of splenic erythroblast maturation. Since Nix gene ablation accelerated recovery from PHZ-induced anemia in mice, it is interesting to consider how erythroblast-specific apoptosis inhibition might prove useful to enhance rapid mobilization of erythroblast reserves, in the treatment of anemia.
Supported by NHLBI HL59888, HL77101 (to G.W.D.), the American Heart Association (Scientist Development Grant to A.D.), and the U.S. Department of Veterans Affairs. The authors declare no competing financial interests. Author contributions: A.D. designed and performed research, analyzed data and wrote paper, A.G.K. performed research and analyzed data, D.C. performed research and analyzed data, H.G. performed research and analyzed data, T.A.K. performed research and analyzed data; and G.W.D. designed and performed research, analyzed data and wrote paper.