The anemia of chronic renal failure and chronic diseases: in vitro studies of erythropoiesis.

The presence of a serum factor in chronic renal failure (CRF) which inhibits erythropoietin-stimulated erythropoiesis was studied, using a technique in which dog marrow cells were stimulated to produce heme in the presence of human serum. In the total series comparing 27 normal sera with 52 CRF sera, less heme was synthesized when the system contained CRF sera (total series, p = 0.0001). There was no evidence of inhibition of heme synthesis by serum from 12 patients with the anemia of chronic diseases (CD). Mixing experiments with normal and CRF sera suggested that this defect in CRF serum was not due to lack of a factor necessary for heme synthesis. Addition of urea, creatinine, and guanidinosuccinic acid to normal serum did not impair its ability to support erythropoiesis in this system. These data demonstrated that serum from patients with CRF contains a material inhibiting erythropoiesis in vitro, We propose that the material is responsible, in part, for the clinically severe anemia seen in these patients.     

A Complement independent erythropoietic inhibitor acting on the progenitor cell in refractory anemia

An erythropoietic inhibitor was detected in the serum of a patient with refractory anemia. Using an in vitro heme synthesis method, the patient's serum produced tenfold inhibition of erythropoietin-stimulated radioactive iron (Fe⁵⁹) incorporation into heme of normal human marrow at 72 hours, as compared with AB serum. In a separate experiment the patient's serum produced threefold inhibition, whereas immunoglobulin G (IgG) prepared from the same serum sample produced 12-fold inhibition. To identify the site of action of the inhibitor, serum was tested in a cell culture system whereby human marrow cells, grown in a plasma clot, respond to exogenous erythropoietin with the appearance of nucleated erythroid colonies. Each colony arises from a committed erythroid progenitor. The patient's serum produced a two- to tenfold reduction in the number of colonies from normal human marrow. The effect was also demonstrated on autologous marrow obtained when the patient was in “partial clinical remission.” Serum samples obtained at various times during the course of the patient's illness all demonstrated a suppressive effect on colony growth. All serums were heat-inactivated, and total hemolytic complement could not be detected in either culture system. It is concluded that the anemia is due to an inhibitor, probably of IgG class, that acts on the erythroid progenitor cell. The absence of heat-labile complement components in the culture systems suggests that the mechanism is not due to immune cytolysis.     

Heme metabolism and in vitro erythropoiesis in anemia associated with hypochromic microcytosis

Heme metabolism and in vitro erythropoietic growth (CFU-E, BFU-E) were examined in bone marrow cells taken from two siblings with apparent familial hypochromic microcytic anemia. Bone marrow cells from both patients grew adequate numbers of CFU-E and BFU-E colonies in culture in the presence of erythropoietin. In addition, small numbers of endogenous CFU-E were seen in 7-day cultures. Assays on bone marrow cells taken from both patients revealed that baseline delta-aminolevulinic synthase activity was considerably reduced, but increased six to seven fold (to normal levels) when patients' cells were exposed to pyridoxal phosphate (PLP). In both cases, ferrochelatase and delta-aminolevulinic acid dehydratase activities were normal. Bone marrow heme oxygenase showed no significant differences in activities between normals and patients values in the absence or presence of PLP. In contrast, heme synthesis by patients' bone marrow was less than that of normals. This study demonstrates that bone marrow cells from patients with this rare disorder have some disturbances in heme metabolism, whereas erythropoiesis appeared to be normal when cultured with adequate nutrients in vitro.     

The effect of serum from uremic patients on erythropoietin

Serum from patients with chronic renal failure (CRF serum) contains a substance inhibitory to erythropoiesis in vitro. This paper explores the mechanism of the inhibition. Four experiments were performed to evaluate the effects of CRF serum on erythropoietin (EP). In the first 2 experiments, the effect of exposure of EP solutions to CRF serum was evaluated using the plethoric mouse EP assay system and a tissue culture system containing normal dog marrow cells. In the third study, dog marrow cells were preincubated with CRF serum before being stimulated with EP. Finally, EP-dose response curves were constructed in the dog marrow tissue culture system and analyzed using an enzyme kinetic model. The results show no evidence of inhibition or inactivation of EP by CRF serum, although in vitro heme synthesis is clearly depressed in the presence of CRF serum. We conclude that CRF serum inhibits erythropoiesis by directly, although reversibly, impairing the ability of erythroblasts to synthesize heme.     

Erythropoietin controls heme metabolic enzymes in normal human bone marrow culture

Erythropoietin (Epo) was found to act as a concentration-dependent inducer of aminolevulinic acid (ALA) synthase and porphobilinogen (PBG) deaminase in normal human bone marrow in culture. Epo increased enzymatic activities in individual plated nucleated cells. At a low concentration of Epo, heme oxygenase activity did not change in human bone marrow erythroid progenitor cells. However, Epo at a concentration of 2 U/ml increased heme oxygenase as demonstrated by an increase in both the enzyme protein and its mRNA. In experiments with an inhibitor of heme synthesis, succinylacetone (SA), Epo failed to stimulate erythroid colony-forming unit (CFU-E) growth, but this CFU-E inhibition by SA was completely overcome by the addition of hemin. Epo nevertheless potentiated induction of ALA synthase in the presence of SA. Hemin exerted its regulatory role by negative feedback on ALA synthase in the presence of SA and Epo. Heme potentiated Epo action and resulted in the increase of human marrow erythroid progenitor cell proliferation and differentiation and a concomitant stimulation of ALA synthase and PBG deaminase. The potentiating effects of hemin on CFU-E growth were observed in human bone marrow cells cultured in media supplemented with fetal calf serum or serum-free media with interleukin 3 (IL-3). These results indicate that Epo is a potent inducer of ALA synthase and PBG deaminase in normal human bone marrow. In addition, our results may explain the mechanisms by which heme potentiates Epo or IL-3 enhancement of erythropoiesis. 1) Heme may stimulate the translation of several globin and nonglobin mRNAs, including those of ALA synthase and PBG deaminase; 2) as endogenous cellular heme synthesis reaches optimal levels, heme exerts its regulatory role on ALA synthase by negative feedback inhibition. Additionally, an increase in cellular heme may lead to an increase in its own degradation by induction of heme oxygenase.     

Primary refractory anemia: clinical and laboratory study of erythropoiesis in 16 patients

Erythropoiesis was studied in vitro in 16 selected patients with primary refractory anemia without excess of blasts who have been followed for an average of 4.8 years. The number of erythroid colonies and bursts grown in vitro from the patients' marrows did not correlate with any parameter of their disease or their prognosis. The response of marrow erythroid precursor cells to erythropoietin was found to be normal. In no case was a serum or IgG inhibitor of erythropoiesis detected either by quantitation of heme synthesized by marrow cells or by the erythroblast cytotoxicity assay. A clinically significant response of the anemia to corticosteroids was noted in three out of 14 patients. Ten patients died during the followup period, eight of them as a consequence of their hematologic disorder. Bone marrow aplasia with pancytopenia developed in six cases, increased number of marrow blasts in two cases, myelofibrosis with myeloid metaplasia in one case and a spontaneous remission in another case. Refractory anemia without excess of blasts is a heterogeneous disorder with variable natural history including evolution into marrow aplasia.     

Erythroid colony studies on sickle cell anemia in hypoproliferative crisis

Bone marrow cells, peripheral blood lymphocytes, and sera from patients with sickle-cell anemia in hypoproliferative crisis were studied in the plasma clot culture system in the presence or absence of erythropoietin (Epo). Bone marrow cells from five patients demonstrated a marked ability to form erythroid colonies in the presence of Epo. These studies also suggested that bone marrow cells from some patients may have an increased sensitivity to Epo. The most outstanding observation in the present study was the marked erythroid colony inhibition by serum taken from one patient during crisis. Serum taken from the same patient two months after hypoproliferative crisis had no suppressive effect on erythroid colony formation. Lymphocytes taken from three patients in crisis had a stimulatory effect on erythroid colony formation when included in culture. The conclusion is that the defect of erythropoiesis in sickle-cell anemia during hypoproliferative crisis is not due to the absence of erythroid precursor cells or to the presence of suppressor lymphocytes, but may in some cases be associated with a circulating inhibitor of erythroid maturation.     

Exceptionally high serum erythropoietin activity in an anephric patient with severe anemia

An exceptionally high serum erythropoietin (EPO) activity was documented in an anephric patient with severe anemia who required transfusions every 4 weeks. The patient's serum EPO was comparable to normal human urinary EPO in the polycythemic mouse assay and was neutralized by an antiserum against EPO. The patient's serum inhibited EPO stimulated-heme synthesis by normal human marrow cells in vitro. This finding suggests that an inhibitor played an important role in causing the anemia of this uremic patient.     

Haem Synthesis in the Diamond-Blackfan Syndrome

The response of bone marrow to erythropoietin (EPO) from five children with the Diamond-Blackfan syndrome, also known as congenital hypoplastic anaemia (CHA), was tested in tissue culture by measurement of haem synthesis. Studies of 13 control marrows indicated that the maximum EPO effect occurred at approximately 70 h incubation using an EPO concentration of 0.2–0.3 units/ml and a nucleated cell concentration of 5 × 10⁶ per culture. Under these conditions, haem synthesis was 121% greater in EPO-stimulated than in unstimulated cultures. Patients with CHA with anaemia and diminished marrow erythroids had reduced or absent haem synthesis. In one patient, haem production became normal after a spontaneous remission, and was not inhibited by autologous plasma drawn at the time of diagnosis. Plasma from three patients did not show inhibitory activity when cultured with control marrow. In contrast, plasma from an adult with acquired pure red cell aplasia produced striking inhibition of haem synthesis when cultured with control marrow. We conclude that, in comparison to some cases of the adult acquired condition, CHA is not due to inhibitors or antibodies. When present, erythroid precursors in children with CHA are capable of responding normally to EPO with increased haem synthesis.     

Mechanism of ranitidine associated anemia

Ranitidine, an H2 receptor antagonist, has been associated with hematotoxicity. The mechanism(s) underlying the toxicity is not well understood. The authors studied the mechanism of anemia in a patient with ranitidine associated anemia and thrombocytopenia. Clinical evaluation suggested drug-induced Coombs' positive reticulocytopenic hemolysis. In vitro, with the patient off ranitidine, the authors were able to induce Coombs' positivity by incubating patient's red cells with ranitidine and his serum. This process was inhibited by prior exposure of his red cells to histamine. In vitro studies using clonal assays for hematopoietic progenitors revealed that while the patient's serum or ranitidine alone did not affect the patient's or normal bone marrow hematopoiesis, the simultaneous presence of both agents significantly suppressed both patient's and normal erythroid progenitor (BFU-E) colony development. This suppressive effect was prevented by the prior exposure of marrow to histamine and was not observed when the patient's serum was heat inactivated. These studies suggest that the anemia may have resulted from complement-dependent autoimmune destruction/inhibition of progenitor/mature erythroid cells by a process critically dependent on the presence of ranitidine and possibly acting at or near the histamine receptor.     

Two antigenically different types of colony-stimulating activities in sera of patients with aplastic anemia

Sera obtained from seven normal volunteers and 11 patients with aplastic anemia were assayed for two types of colony-stimulating activity (CSA) using monolayer agar cultures containing human unfractionated and monocyte-depleted bone marrow cells. Sera obtained from normal volunteers had low CSA for unfractionated bone marrow cells and no CSA for monocyte-depleted bone marrow cells. On the other hand, sera obtained from patients with aplastic anemia (patients' sera) had moderate CSA for both unfractionated and monocyte-depleted bone marrow cells. The patients' serum CSA titer for unfractionated bone marrow cells rose markedly with the addition of a small amount of diluted control rabbit serum (control serum) into the CSA assay system, while it did not rise with addition of the same amount of diluted rabbit antiserum against partially purified human urinary colony-stimulating factor (CSF) (antiserum). The patients' serum CSA titer for monocyte-depleted human bone marrow cells rose with the addition of control serum as well as antiserum. These findings indicate that the addition of rabbit serum enhances colony formation by human bone marrow cells in the presence of aplastic anemia sera as a source of CSA, and that sera of patients with aplastic anemia contain two antigenically different types of CSA: one that is active on human unfractionated bone marrow cells and partially neutralized by the addition of antiserum, and another that is active on human monocyte-depleted bone marrow cells and is not neutralized by the addition of antiserum.     

Humoral suppression of erythropoiesis in systemic lupus erythematosus (SLE) and rheumatoid arthritis

Anemia due to inadequate red cell production often accompanies systemic lupus erythematosus and rheumatoid arthritis. We investigated its pathogenesis in 17 patients with these disorders, using a plasma clot culture system. In serum from normal donors and nonanemic patients CFU-E derived colony formation was not significantly altered by normal marrow cells (mean 74±12 colonies/6 × 10⁴ cells), whereas colony formation was inhibited (mean 36 ± 6 colonies/6 × 10⁴ cells) in serum from 10 anemic patients. In serum from anemic patients proliferation of the more primitive BFU-E was also reduced in three cases. In two patients with a humoral inhibitor, colony growth was suppressed by autologous marrow cells. In another patient without an inhibitor, colony formation was not suppressed by autologous bone marrow.The physical properties of this inhibitor are compatible with those of an immunoglobulin. Moreover, its presence is related to disease activity and it can be removed by successful therapy with either corticosteroids or plasma exchange. Circulating inhibitors of erythropoiesis may play an important role in causing severe anemia in patients with these rheumatic diseases.     

Diamond-blackfan anemia: The role of immunoglobulin blocking factor in remission

Some patients with Diamond-Blackfan syndrome may have suppressor T-cells responsible for the failure of normal erythropoiesis. The capacity of Diamond-Blackfan syndrome mononuclear cells to react in mixed leukocyte culture to the stimulus of nucleated red cells was tested using bone marrow as the cell source. In all but one instance, Diamond-Blackfan mononuclear cells and cells from normal or a multiply-transfused control showed similar degrees of stimulation. Patient mononuclear cells reacted normally to phytohemagglutinin (PHA). The presence of a cytotoxic effect of Diamond-Blackfan mononuclear cells on normal nucleated red cells was examined using a ⁵⁹Fe-release assay. There was no evidence for lymphocyte-mediated erythroid cytotoxicity. Immunoglobulins (Ig) were removed from Diamond-Blackfan serum by affinity chromatography. Erythroid burst-forming units (BFU-E) failed to grow normally in Ig-depleted patient autologous serum, but growth failure was reversed when Ig eluted from the affinity column was added back to the culture. Diamond-Blackfan Ig added to autologous mononuclear cells in the absence of autologous serum normalized colony growth. T-cells of some Diamond-Blackfan syndrome patients may suppress BFU-E growth, but they do not react abnormally in mixed leukocyte culture to nucleated erythroid cells and are not cytotoxic for erythroblasts under our conditions of study. The serum-blocking factor in our patients appears to be an Ig that promotes BFU-E generation despite the presence of suppressor T-cells. Although it cannot be proved, this may be related to the remission of disease in our patients.     

Serum inhibitors of hematopoiesis in a patient with aplastic anemia and systemic lupus erythematosus. Recovery after exchange plasmapheresis.

A 36 year old black woman with serologic evidence of systemic lupus erythematosus had aplastic anemia that failed to respond to corticosteroid or androgen therapy. Bone marrow culture studies were performed to determine if the marrow aplasia was immunologically mediated. A potent serum inhibitor of in vitro myeloid and erythroid colony formation was demonstrated. Incubation of normal bone marrow cells with the patient's serum and a source of complement caused a 95 per cent reduction in myeloid colony formation. Under these conditions, bone marrow from 35 normal volunteer subjects formed a mean of 5 ± 1 colonies per 2 × 10⁵ marrow cells plated (control = 95 ± 8 colonies). The addition of the patient's serum plus complement to normal marrow cultured in methylcellulose also caused a 98 per cent reduction in erythroid colony numbers. Assay of serum immunoglobulin fractions obtained by gel filtration demonstrated that the inhibitory activity was contained in the immunoglobulin G (IgG) fraction. In association with a series of plasma exchanges, the titer of inhibitor fell, and marrow cellularity and peripheral blood counts returned to normal. Additional studies indicated that the inhibitor of allogeneic marrow was not directed against HLA or “B-cell” antigens. Incubation of the patient's recovery marrow with stored preplasmapheresis serum and complement did not produce a reduction in myeloid colony formation. It seems most likely that the inhibitor represents alloantibody directed against heretofore unrecognized antigens widely distributed on normal human hematopoietic progenitor cells.     

Treatment of hypoplastic anemia in mice with placental transplants

A genetic mutation in mice (W/Wv) causes an autosomal recessive disease characterized by hypoplastic anemia which lasts throughout life. Double- dominant W/Wv anemic mice were sublethally irradiated to facilitate repopulation of marrow with transplanted cells and were injected intravenously with suspensions of 5-10 million placental cells of 15 days gestation derived from normal, isogeneic donors. Red cell counts fell promptly after irradiation and then rose progressively over a period of weeks, reaching normal levels of the nonmutant. Mean corpuscular volume and hemoglobin electrophoresis patterns of red cells in recipient W/Wv mice resembled those of normal donor animals. The therapeutic effect lasted for the duration of the observation period, in some instances over 9 mo. W/Wv mice that were administered Hanks' solution or fetal blood, instead of placental transplants, remained anemic. Late gestation placentas (18 days) were also ineffective.     

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.     

Complete recovery of hemopoiesis following bone marrow transplant in a patient with unresponsive congenital hypoplastic anemia (Blackfan- Diamond syndrome)

Allogeneic bone marrow transplantation (BMT) was carried out on a 5- year-old boy with congenital hypoplastic anemia (CHA), who did not respond to corticosteroids and who was displaying signs of progressive hemosiderosis. Pretransplant preparation had to be modified because respiratory failure and cerebral edema supervened. This preparatory regimen consisted of busulfan (2 mg/kg for four days), cyclophosphamide (50 mg/kg for one day), and total body irradiation (750 rad). Hemopoiesis was completely restored and is still maintained 650 days after transplantation. This is the second published report on the use of BMT to treat a patient with CHA, and it is the first time it has resulted in long-term survival. BMT should be considered for patients with CHA who do not respond to corticosteroids.     

Erythrokinetics in pernicious anemia

Quantitative measurements of the erythropoietic activity of the marrow, ofcirculating red cell production and destruction have been made in patients withpernicious anemia in relapse and during response to vitamin B₁₂ therapy.

Total erythropoietic marrow activity as reflected by turnover of heme components proceeds at a rate of approximately 3 times normal. The delivery ofviable red cells to the circulating blood, however, does not increase above normal. This would indicate that the greater portion of marrow activity is ineffective in terms of blood production. This marrow dysfunction coupled with anincreased rate of cell destruction of approximately 3 times normal is responsiblefor the anemia. Total erythropoiesis is somewhat less, and effective erythropoiesisconsiderably less, than that which may be expected of the normal marrow underthe sustained stimulus of anemia.

The reticulocyte count is shown to be an unreliable index of blood productionin untreated pernicious anemia due to loss of reticulum from cytoplasma ofmany red cells before their delivery into circulation.

During the response to vitamin B₁₂ the ineffective erythropoiesis is convertedto effective erythropoiesis, whereas total erythropoiesis remains unchanged.The rate of blood production during recovery is 3 to 4 times normal.

Submitted on January 23, 1956 Accepted on May 3, 1956     

Targeted deletion of the mouse Mitoferrin1 gene: from anemia to protoporphyria

Mitoferrin1 is 1 of 2 homologous mitochondrial iron transporters and is required for mitochondrial iron delivery in developing erythroid cells. We show that total deletion of Mfrn1 in embryos leads to embryonic lethality. Selective deletion of Mfrn1 in adult hematopoietic tissues leads to severe anemia because of a deficit in erythroblast formation. Deletion of Mfrn1 in hepatocytes has no phenotype or biochemical effect under normal conditions. In the presence of increased porphyrin synthesis, however, deletion of Mfrn1 in hepatocytes results in a decreased ability to convert protoporphyrin IX into heme, leading to protoporphyria, cholestasis, and bridging cirrhosis. Our results show that the activity of mitoferrin1 is required to manage an increase in heme synthesis. The data also show that alterations in heme synthesis within hepatocytes can lead to protoporphyria and hepatotoxicity.     

Circulating Inhibitors of Granulopoiesis in Patients with Aplastic Anaemia

An inhibitor of granulopoiesis has been detected in the serum of aplastic patients by its ability to reduce colony formation by normal human marrow cells in culture. The inhibitory activity in the serum was abolished when a patient was treated with azathioprine and exposure of aplastic marrow to antithymocyte globulin (ATG) in vitro released the colony forming cells from suppression. The clinical findings in three of the patients studied support the idea that the inhibitory activity detected in vitro is associated with normal marrow suppression in vivo.