Heme oxygenase-1 deficiency alters erythroblastic island formation,steady-state erythropoiesis and red blood cell lifespan in mice

Heme oxygenase-1 is critical for iron recycling during red blood cell turnover, whereas its impact on steady-state erythropoiesis and red blood cell lifespan is not known. We show here that in 8- to 14-week old mice, heme oxygenase-1 deficiency adversely affects steady-state erythropoiesis in the bone marrow. This is manifested by a decrease in Ter-119⁺-erythroid cells, abnormal adhesion molecule expression on macrophages and erythroid cells, and a greatly diminished ability to form erythroblastic islands. Compared with wild-type animals, red blood cell size and hemoglobin content are decreased, while the number of circulating red blood cells is increased in heme oxygenase-1 deficient mice, overall leading to microcytic anemia. Heme oxygenase-1 deficiency increases oxidative stress in circulating red blood cells and greatly decreases the frequency of macrophages expressing the phosphatidylserine receptor Tim4 in bone marrow, spleen and liver. Heme oxygenase-1 deficiency increases spleen weight and Ter119⁺-erythroid cells in the spleen, although α4β1-integrin expression by these cells and splenic macrophages positive for vascular cell adhesion molecule 1 are both decreased. Red blood cell lifespan is prolonged in heme oxygenase-1 deficient mice compared with wild-type mice. Our findings suggest that while macrophages and relevant receptors required for red blood cell formation and removal are substantially depleted in heme oxygenase-1 deficient mice, the extent of anemia in these mice may be ameliorated by the prolonged lifespan of their oxidatively stressed erythrocytes.     

Erythrophagocytosis in the Human Bone Marrow as Disclosed by Iliacal Bone Biopsies

Erythrophagocytosis in human bone marrow has been studied in selected series of iliacal bone biopsies embedded in methacrylate or epoxy resin. Using established light microscopic criteria for recognition of the early digestive erythrophagic vacuole, intense erythrophagic activity was encountered in a series of 5 patients with haemolytic disease. A series of 32 biopsies, selected to represent ‘normal’ bone marrow, demonstrated low or modest erythrophagic activity in all cases. The findings are in accordance with previous demonstration of erythrophagocytosis in human bone marrow at autopsy and point to the bone marrow as the main site for destruction of aged red cells in the healthy human organism.     

Erythroblasts are a source of angiogenic factors

In adult bone marrow, mature erythroblasts are produced within structures called erythroblastic islands and then cross the endothelial barrier to reach circulation. Erythroblastic islands are composed of a central macrophage surrounded by maturing erythroblasts. In this study, it is shown that erythroid cells, but not the other mature hematopoietic cells, coexpress 2 angiogenic factors, vascular endothelial growth factor A (VEGF-A) and placenta growth factor (PlGF). Secretion of both VEGF-A and PlGF increases during in vitro erythroid differentiation. Erythroblast-conditioned medium can induce both migration of monocytes and endothelial cells and the permeability of endothelial cells. These effects are inhibited by anti-PlGF and/or anti-VEGF antibodies. Finally, it is shown that VEGF-A and PlGF proteins are expressed by bone marrow erythroblasts in vivo. Angiogenic factors secreted by erythroblasts may promote interactions either with macrophages in erythroblastic islands or with endothelial cells that would facilitate the passage of erythroid cells through the endothelial barrier. (Blood. 2001;97:1968-1974)     

Bone marrow infiltration by multiple myeloma causes anemia by reversible disruption of erythropoiesis

Multiple myeloma (MM) infiltrates bone marrow and causes anemia by disrupting erythropoiesis, but the effects of marrow infiltration on anemia are difficult to quantify. Marrow biopsies of newly diagnosed MM patients were analyzed before and after four 28‐day cycles of nonerythrotoxic remission induction chemotherapy. Complete blood cell counts and serum paraprotein concentrations were measured at diagnosis and before each chemotherapy cycle. At diagnosis, marrow area infiltrated by myeloma correlated negatively with hemoglobin, erythrocytes, and marrow erythroid cells. After successful chemotherapy, patients with less than 30% myeloma infiltration at diagnosis had no change in these parameters, whereas patients with more than 30% myeloma infiltration at diagnosis increased all three parameters. Clinical data were used to develop mathematical models of the effects of myeloma infiltration on the marrow niches of terminal erythropoiesis, the erythroblastic islands (EBIs). A hybrid discrete‐continuous model of erythropoiesis based on EBI structure/function was extended to sections of marrow containing multiple EBIs. In the model, myeloma cells can kill erythroid cells by physically destroying EBIs and by producing proapoptotic cytokines. Following chemotherapy, changes in serum paraproteins as measures of myeloma cells and changes in erythrocyte numbers as measures of marrow erythroid cells allowed modeling of myeloma cell death and erythroid cell recovery, respectively. Simulations of marrow infiltration by myeloma and treatment with nonerythrotoxic chemotherapy demonstrate that myeloma‐mediated destruction and subsequent reestablishment of EBIs and expansion of erythroid cell populations in EBIs following chemotherapy provide explanations for anemia development and its therapy‐mediated recovery in MM patients. Am. J. Hematol. 91:371–378, 2016. © 2016 Wiley Periodicals, Inc.     

Targeted gene deletion demonstrates that the cell adhesion molecule ICAM-4 is critical for erythroblastic island formation

Erythroid progenitors differentiate in erythroblastic islands, bone marrow niches composed of erythroblasts surrounding a central macrophage. Evidence suggests that within islands adhesive interactions regulate erythropoiesis and apoptosis. We are exploring whether erythroid intercellular adhesion molecule 4 (ICAM-4), an immunoglobulin superfamily member, participates in island formation. Earlier, we identified alpha(V) integrins as ICAM-4 counterreceptors. Because macrophages express alpha(V), ICAM-4 potentially mediates island attachments. To test this, we generated ICAM-4 knock-out mice and developed quantitative, live cell techniques for harvesting intact islands and for re-forming islands in vitro. We observed a 47% decrease in islands reconstituted from ICAM-4 null marrow compared to wild-type marrow. We also found a striking decrease in islands formed in vivo in knock-out mice. Further, peptides that block ICAM-4/alpha(V) adhesion produced a 53% to 57% decrease in reconstituted islands, strongly suggesting that ICAM-4 binding to macrophage alpha(V) functions in island integrity. Importantly, we documented that alpha(V) integrin is expressed in macrophages isolated from erythroblastic islands. Collectively, these data provide convincing evidence that ICAM-4 is critical in erythroblastic island formation via ICAM-4/alpha(V) adhesion and also demonstrate that the novel experimental strategies we developed will be valuable in exploring molecular mechanisms of erythroblastic island formation and their functional role in regulating erythropoiesis.     

Erythrokinetics in the Megaloblastic Anemia of Tropical Sprue

Blood production and destruction were measured in 10 patients with themegaloblastic anemia of tropical sprue. Methods employed included the determination of the erythroid/myeloid ratio of the marrow, plasma iron turnover, red cell utilization of Fe⁵⁹ and Cr⁵¹ red blood cell survival. Rates ofproduction and destruction were compared to normal.

Patients with the megaloblastic anemia of sprue were usually not irondeficient. Total bone marrow erythroid activity did not approach the maximal response seen in other hemolytic anemias, and there was a marked decreasein the delivery of erythrocytes to the peripheral blood. The rate of red bloodcell destruction was increased, but as the red cell volume decreased, the totalmass of erythrocytes destroyed per day varied from less than normal totwice normal. Bilirubinemia was not marked, because the amount of hemoglobin destroyed daily was usually not excessive and excretory function wasnot impaired. The severity of the anemia was largely related to the erythrocyte production defect.

     

Erythrophagocytosis in the human bone marrow as disclosed by iliacal bone biopsies.

Erythrophagocytosis in human bone marrow has been studied in selected series of iliacal bone biopsies embedded in methacrylate or epoxy resin. Using established light microscopic criteria for recognition of the early digestive erythrophagic vacuole, intense erythrophagic activity was encountered in a series of 5 patients with haemolytic disease. A series of 32 biopsies, selected to represent 'normal' bone marrow, demonstrated low or modest erythrophagic activity in all cases. The findings are in accordance with previous demonstration of erythrophagocytosis in human bone marrow at autopsy and point to the bone marrow as the main site for destruction of aged red cells in the healthy human organism.     

The anemia of the Di Guglielmo syndrome

The physiopathology of the anemia of the Di Guglielmo syndrome (erythremic myelosis) was studied in 11 patients with the acute and chronic varieties of the disease. Ferrokinetic studies were performed in three additionalpatients.

1. The anemia was normochromic and macrocytic; in contrast to the meancorpuscular volume, which was elevated, the mean corpuscular hemoglobinwas often normal. In several patients the mean corpuscular hemoglobin concentration was slightly lower than normal, suggesting slight hypochromia.

2. Reticulocytes were often increased but bore no relationship to the degree of the anemia nor to the shortening of the red cell life span. The reticulocyte count is an unreliable index of blood production in this disease.

3. The degree of erythroblastemia was highly variable. No direct correlation existed between the degree of erythroblastemia and the acuteness of thedisease, nor was there any relationship between the degree of erythroblastemiaand either the degree of anemia or the degree of erythrocytic destruction.

4. The bone marrow showed striking erythroblastic hyperplasia. This wasusually of the megaloblastic type. Primitive erythroblasts (erythrogones) wereconspicuous. The erythroblastic hyperplasia was out of proportion to the relatively minor reticulocytosis or the relatively slight diminution in red cellsurvival.

5. The nucleated red cells of the marrow showed variable numbers ofmegaloblasts and megaloblastoid forms, suggesting the presence of a vitaminB₁₂ deficiency (pernicious anemia). However, the vitamin B₁₂ concentrationof the serum was elevated, and there was no response to the administrationof vitamin B₁₂ or folic acid.

6. Varying numbers of erythroblasts in the bone marrow and in the peripheral blood showed periodic acid-Schiff (PAS)-positive granules in the cytoplasm. No chemical abnormalities of hemoglobin could be detected either bythe method of paper electrophoresis or by the alkali denaturation test.

7. Diminished red cell survival was present in most cases, but it was of arelatively slight degree. It was due to an "intracorpuscular" defect of the redcells.

8. The often great increase in fecal urobilinogen output as compared witha relatively minor rate of red cell destruction suggests "heme pigment diversion" or increased destruction of precursor red cells, as in pernicious anemia,where the same phenomenon has been observed.

9. The great increase in the number of erythroid cells in the bone marrowand the increased rate of iron turnover as compared with the relatively minorincrease in red cell destruction and iron utilization point to an "ineffective"type of erythropoiesis. The high degree of "ineffective erythropoiesis" seenin this disease may be characteristic of the neoplastic proliferation of the redcell series.

10. In conclusion, the anemia of the Di Guglielmo syndrome is due to acombined disturbance: (1) an "ineffective" type of erythropoiesis of markeddegree, perhaps due to an acquired (neoplastic) defect in the uptake or utilization of B₁₂ by the erythroblasts and (2) increased hemolysis resulting fromthe increased destruction of defective red cells.

Submitted on June 11, 1958 Accepted on August 13, 1958     

The Mechanism of the Development of Anemia in Untreated Chronic Lymphatic Leukemia

The following conclusions can be made concerning the cause of anemia inpatients with untreated chronic lymphatic leukemia excluding the rare casewith an autoimmune hemolytic anemia:

1. Early in the natural history of the disease there is a normal amount oferythroblastic tissue which produces red cells at a normal rate. The rate ofred cell destruction is also normal.

2. With progression of the disease the erythroblastic tissue is gradually replaced by lymphatic tissue, leading to a decrease in red cell production. Therate of red cell destruction is still normal.

3. Only late in the disease may decreased rate of red cell production be aggravated by an increased rate of red cell destruction.

Submitted on October 25, 1960 Accepted on December 9, 1960     

Iron release from macrophages after erythrophagocytosis is up-regulated by ferroportin 1 overexpression and down-regulated by hepcidin

Ferroportin 1 (FPN1) is transmembrane protein involved in iron homeostasis. In the duodenum, FPN1 localizes to the basolateral surface of enterocytes where it appears to export iron out of the cell and into the portal circulation. FPN1 is also abundantly expressed in reticuloendothelial macrophages of the liver, spleen, and bone marrow, suggesting that this protein serves as an iron exporter in cells that recycle iron from senescent red blood cells. To directly test the hypothesis that FPN1 functions in the export of iron after erythrophagocytosis, FPN1 was stably expressed in J774 mouse macrophages by using retroviral transduction, and release of 59Fe after phagocytosis of 59Fe-labeled rat erythrocytes was measured. J774 cells overexpressing FPN1 released 70% more 59Fe after erythrophagocytosis than control cells, consistent with a role in the recycling of iron from senescent red cells. Treatment of cells with the peptide hormone hepcidin, a systemic regulator of iron metabolism, dramatically decreased FPN1 protein levels and significantly reduced the efflux of 59Fe after erythrophagocytosis. Subsequent fractionation of the total released 59Fe into heme and nonheme compounds revealed that hepcidin treatment reduced the release of nonheme 59Fe by 50% and 25% from control and FPN1-overexpressing cells, respectively, but did not diminish efflux of 59Fe-heme. We conclude that FPN1 is directly involved in the export of iron during erythrocyte-iron recycling by macrophages.     

Transforming growth factor beta 1 systemically modulates granuloid, erythroid, lymphoid, and thrombocytic cells in mice.

Transforming growth factor beta 1 (TGF-beta 1) has been shown to inhibit the development of most early hemopoietic progenitors in vitro. The present series of in vivo experiments show that TGF-beta 1 can simultaneously augment and suppress distinct cell lineages in peripheral and central hemopoietic compartments. Mice treated daily for 7-14 days with s.c. injections of TGF-beta 1 exhibited up to a 95% reduction in circulating platelets and a 50% reduction in red cell counts, whereas a 50%-400% increase occurred in circulating white cells with the morphology of small lymphocytes. Decreased erythrocytes were also evident in the splenic red pulp and bone marrow sinusoids. A dramatic increase in granulopoiesis occurred in the spleen and bone marrow, followed by a peripheral neutrophilia 1 week after treatments ceased. All effects were completely reversible, with normal histologic and hematologic profiles evident 2 weeks after cessation of treatments. Thus, TGF-beta 1 can differentially regulate multiple hemopoietic pathways in a systemic, reversible, and dose-dependent fashion. These actions may be mediated by the direct effects of TGF-beta 1 or through modulation of secondary cytokines and receptors.     

The bone marrow in human cerebral malaria: parasite sequestration within sinusoids

Bone marrow aspirates from patients with cerebral malaria were studied with the light and electron microscopes. Various abnormalities were found including: (1) an increase in plasma cells and macrophages, sometimes to a marked degree; (2) phagocytosis of parasitized red cells by macrophages and of merozoites by neutrophil metamyelocytes, neutrophil granulocytes and macrophages; (3) an increase in the proportion of eosinophil granulocytes and their precursors; (4) the presence of giant metamyelocytes; and (5) morphological abnormalities of erythroblasts, particularly irregularly-shaped nuclei and karyorrhexis. A high percentage of the red cells within marrow sinusoids were parasitized and the parasitized cells were attached to the endothelium. Some marrow sinusoids were packed with and completely obstructed by parasitized cells. Strands of electron-dense material were sometimes found connecting the knobs of parasitized red cells to endothelial cells or to the knobs of adjacent parasitized red cells. A striking finding was a complex interdigitation between cytoplasmic processes developed by some of the parasitized red cells and those developed by the endothelial cells to which they were attached. Occasionally, cytoplasmic processes arising from marginated parasitized red cells completely penetrated the endothelial cell and emerged extravascularly. Several parasitized red cells were also found extravascularly between haemopoietic cells. Sequestration of parasitized red cells within small blood vessels may play a part in the pathogenesis not only of the encephalopathy of cerebral malaria but also of the bone marrow dysfunction in severe malaria.     

Erythroid cell adhesion molecules Lutheran and LW in health and disease

The Lutheran and LW glycoproteins are blood group-active proteins found at the surface of human red cells. The Lutheran glycoprotein (Lu gp) is a member of the immunoglobulin superfamily (IgSF) that binds the extracellular matrix protein laminin, in particular, laminin isoforms containing the alpha 5 subunit. The LW glycoprotein (LW gp), also an IgSF member, has substantial sequence homology with the family of intercellular adhesion molecules (ICAMs). LW gp binds the integrin very late antigen-4 (VLA-4, alpha 4 beta 1) and alpha V-containing integrins. Studies on the expression of LW and Lu gps during erythropoiesis utilizing in vitro cultures of haemopoietic progenitor cells have shown that LW gp expression precedes that of Lu gp. These observations have led to the suggestion that LW gp on erythroblasts may interact with VLA-4 on macrophages to stabilize erythroblastic islands in normal bone marrow and that Lu gp may facilitate trafficking of more mature erythroid cells to the sinusoidal endothelium where alpha 5-containing laminins are known to be expressed. Levels of Lu gp and LW gp expression on sickle red cells are greater than on normal red cells and sickle red cells adhere to alpha 5-containing laminins. These data suggest that the Lu and LW molecules may contribute to the vaso-occlusive events associated with episodes of acute pain in sickle cell disease.     

Plasmodium falciparum rhoptry protein RSP2 triggers destruction of the erythroid lineage

The destruction of erythrocytes and defects in erythropoiesis are among the most frequently observed causes of morbidity in severe Plasmodium falciparum malaria. The molecular mechanisms involved remain unclear, despite extensive investigation. We show here, for the first time, that tagging with the parasite rhoptry protein ring surface protein 2 (RSP2) is not restricted to the surfaces of normal erythrocytes, as previously reported, but that it extends to erythroid precursor cells in the bone marrow of anemic malaria patients. Monoclonal mouse antibodies and human sera from patients with severe anemia, reacting with RSP2-tagged erythrocytes, induced cell destruction by phagocytosis and complement activation in vitro. Our observations reveal a new parasite mechanism implicated in the destruction of normal erythrocytes and probably dyserythropoiesis in malaria patients. These data suggest that the tagging of host cells with RSP2 may trigger anemia in falciparum malaria.     

Erythroblastic islands: niches for erythropoiesis

Erythroblastic islands, the specialized niches in which erythroid precursors proliferate, differentiate, and enucleate, were first described 50 years ago by analysis of transmission electron micrographs of bone marrow. These hematopoietic subcompartments are composed of erythroblasts surrounding a central macrophage. A hiatus of several decades followed, during which the importance of erythroblastic islands remained unrecognized as erythroid progenitors were shown to possess an autonomous differentiation program with a capacity to complete terminal differentiation in vitro in the presence of erythropoietin but without macrophages. However, as the extent of proliferation, differentiation, and enucleation efficiency documented in vivo could not be recapitulated in vitro, a resurgence of interest in erythroid niches has emerged. We now have an increased molecular understanding of processes operating within erythroid niches, including cell-cell and cell-extracellular matrix adhesion, positive and negative regulatory feedback, and central macrophage function. These features of erythroblast islands represent important contributors to normal erythroid development, as well as altered erythropoiesis found in such diverse diseases as anemia of inflammation and chronic disease, myelodysplasia, thalassemia, and malarial anemia. Coupling of historical, current, and future insights will be essential to understand the tightly regulated production of red cells both in steady state and stress erythropoiesis.     

Hydroxyurea Treatment of Myeloproliferative Disorders

ABSTRACT Blood and bone marrow changes induced by continuous low-dose hydroxyurea treatment are described. A linear increase in mean red cell volume was observed after onset of therapy. The entire normocyte population was replaced by abnormally large erythrocytes within 150 days. The bone marrow morphology changed in megaloblastic direction. Bone marrow iron stores and number of sideroblasts increased, findings compatible with ineffective erythropoiesis. Serum folate and cobalamin levels remained normal. These morphologic changes might cause confusion when examining blood or bone marrow samples from patients treated with hydroxyurea.     

The macrophage CD163 surface glycoprotein is an erythroblast adhesion receptor

Erythropoiesis occurs in erythroblastic islands, where developing erythroblasts closely interact with macrophages. The adhesion molecules that govern macrophage-erythroblast contact have only been partially defined. Our previous work has implicated the rat ED2 antigen, which is highly expressed on the surface of macrophages in erythroblastic islands, in erythroblast binding. In particular, the monoclonal antibody ED2 was found to inhibit erythroblast binding to bone marrow macrophages. Here, we identify the ED2 antigen as the rat CD163 surface glycoprotein, a member of the group B scavenger receptor cysteine-rich (SRCR) family that has previously been shown to function as a receptor for hemoglobin-haptoglobin (Hb-Hp) complexes and is believed to contribute to the clearance of free hemoglobin. CD163 transfectants and recombinant protein containing the extracellular domain of CD163 supported the adhesion of erythroblastic cells. Furthermore, we identified a 13-amino acid motif (CD163p2) corresponding to a putative interaction site within the second scavenger receptor domain of CD163 that could mediate erythroblast binding. Finally, CD163p2 promoted erythroid expansion in vitro, suggesting that it enhanced erythroid proliferation and/or survival, but did not affect differentiation. These findings identify CD163 on macrophages as an adhesion receptor for erythroblasts in erythroblastic islands, and suggest a regulatory role for CD163 during erythropoiesis.     

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     

An Electron Microscope Study of Bone Marrow in Rheumatoid Disease

The marrow of patients with various haematological complications of rheumatoid disease has been studied with the electron microscope. Although there is evidence that reticulum cells may be reluctant to turn over body iron, yet there is no evidence of iron excess in the reticulum cells of the marrow nor of a morphological defect of normoblast maturation.
Iron transfer is normal. Whereas in the early stages of red cell development iron can be seen to enter the normoblasts from the reticulum cells by the distinct process of ropheocytosis, in the late normoblast and reticulocyte aggregates of unused iron pass back to the reticulum cells. The reticulum cell is therefore important in regulating the distribution of iron within the marrow.
The reticulo-endothelial system in rheumatoid disease shows evidence of response to antigenic stimulation. In particular a case of Felty's syndrome showed reticulum cell hyperplasia, lymphoblasts and material resembling rheumatoid factor in the granulocytes. Hyperplasia of the marrow reticulum may lead to a maturation arrest constituting 'hypersplenism'.