Hematopoietic differentiation cell surface antigen switching in the bone marrow of different aged chickens

Abstract. Immune cytolysis and immunofluorescence were used to examine chicken fetal antigen CFA) and chicken adult antigen (CAA) expression on the differentiation/maturation series of definitive erythroid cells obtained from the bone marrow of different aged chickens. We found that erythroid cells undergo changes in CFA/CAA antigenic expression dependent on their differentiation/maturation stage as well as the developmental age of the chicken. All differentiation/maturation stages of erythroid cells in the bone marrow of 12 and 18-day-old embryos express CFA only. Erythroblasts obtained from 7-day post-hatched chickens express either CFA or CAA. All three CFA/CAA phenotypes (i.e., CFA, CAA, and CFA + CAA) are observed in subsequent maturation stages, but only the CFA + CAA phenotype is observed in mature erythroid cells in the bone marrow of 7day post-hatched chickens. Erythroblasts from 62 day post-hatched chickens exhibit all three CFA/CAA phenotypes. Cells in the subsequent maturation stages express various CFA, CAA, or CFA + CAA phenotypes resulting in a majority of the mature erythrocytes expressing both CFA and CAA, and a small population of mature erythrocytes expressing CAA only. Erythroblasts from adult chickens express both CFA and CAA; however, CFA is lost during erythroid maturation resulting in mature erythrocytes which express CAA only. These studies indicate that both the erythroid differentiation/maturation stage and the developmental age of the chicken influence CFA and CAA antigenic expression on erythroid cells undergoing cellular differentiation/maturation in the bone marrow.     

Avian developmental antigens: Expression on erythrocytes of chickens,Japanese quail,and quail-chicken hybrids

Monoclonal and polyclonal antibodies were used to examine the expression of three erythroid developmental antigen systems in the chicken, Japanese quail, and quail-chicken hybrid. Chicken fetal antigen (CFA), quail fetal antigen (QFA), and chicken adult antigen (CAA) each represent a series of cell-surface glycorproteins associated with the development of avian hematopoietic cells. Monoclonal anti-CFA antibodies from clones 190-4 and 288-1.1.1.2 supernatants were shown to react against epitopes associated with CFA determinants 8 and 2, respectively. Using complement-mediated microcytotoxicity, these reagents permitted the identification of different erythroid subpopulations in the neonatal chicken and hybrid; therefore, heterogeneity in cell surface CFA determinants among mature peripheral erythrocytes should serve as a useful tool for analyzing erythroid development. In the case of CAA, erythrocytes from adult hybrids were found to express the same complement of CAA determinants identified in the chicken, and CAA appeared much earlier in the hybrid than in either of the parental species. Similarly, two species-restricted fetal antigens associated with similar glycoproteins, CFA8 and QFA, had similar developmental profiles in their respective species, the chicken and quail. In contrast, these antigens were dominantly expressed but exhibited different developmental profiles on erythrocytes from the hybrids. While quail-chicken hybrids exhibited apparent genomic interactions in the expression of these developmental antigens, no evidence for the existence of hybrid-specific fetal antigens was obtained.     

Chemical and immunological characterization of developmentally expressed chicken erythroid surface membrane antigens

The expression of two hematopoietic-lymphoid membrane antigens, referred to as chicken fetal antigen (CFA) and chicken adult antigen (CAA) were investigated on primitive and definitive peripheral red blood cells (RBC) from different-aged chickens using chemical and immunological techniques. Differential adsorptions of antisera specific for adult RBC membrane antigens permitted the serological dissection of CAA into eight antigenic determinants. CFA and CAA were assayed by hemagglutination, hemolysis, and immune precipitation of radioiodinated surface antigens of RBC from different-aged chickens. Primitive RBC express CFA, while definitive RBC express three distinct phenotypes: CFA, both CFA and CAA, or CAA, depending on the developmental age of the chicken from which the RBC were obtained. When solubilized membrane extracts of radioiodinated peripheral RBC from chickens at 5 and 18 days embryonic development (E5 and E18, respectively), 13 days posthatch development (H13), and adult chickens were immunoprecipitated and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the major antigen detected by anti-CFA sera was associated with proteins having apparent molecular weights (M_r) of 50,000 daltons (50 kd). The antigens detected by anti-CAA sera were associated with proteins having apparent M_r of 102, 81, 48, and 43 kd.     

Expression of differentiation and age-related antigens on chicken erythroleukemia cells transformed by avian erythroblastosis virus (AEV)

Immature circulating chicken red cells express on their surface two antigenic molecules referred to as Im 48 kD and Im 140 kD antigens. The Im 140 kD antigen is not present beyond the erythroblast stage while the expression of Im 48 kD antigenic molecule remains detectable on circulating erythrocytes of embryos and young chickens, but not on erythrocytes of adult animals. In addition to Im 48 kD and Im 140 kD antigens, the avian erythroblastosis virus (AEV)-transformed erythroid cells express two novel high molecular weight (MW) immature antigens referred to as Im 150 kD and Im 160 kD. Since the transformed erythroid cells are apparently blocked at a stage close to the colony-forming units erythrocytic (CFU-E), these molecules might be expressed on these progenitor cells. The age-related antigenic molecules referred to as E1 48 kD and A 40 kD/A 85 kD antigens are detected on erythrocytes of embryos (and young chickens) and adult animals respectively. The E1 48 kD antigen as well as an antigen related to the A 40 kD were also detected on AEV-transformed erythroid cells deriving from both young chicken bone marrow and yolk sac. The presence of an adult antigen on the embryonic cells might well be related to the transformation by AEV, since the yolk sac CFU-E progenitor cells do not bear the adult antigenicity.     

Chicken developmental antigens in 15I5-B-congenic lines

Six partially developed 15I5-B-congenic lines of chickens were used to assess the genetic influence on the developmental expression of selected epitopes of two avian developmental antigen systems: chicken fetal antigen (CFA) and chicken adult antigen (CAA). Both CFA and CAA are serologically and molecularly complex hematopoietic antigen systems, yet little is known about genetic influences on their expression. Using polyclonal rabbit anti-CFA, only slight variations in overall CFA expression on peripheral erythrocytes were observed during neonatal development; no consistent trend was evident. In contrast, analysis with monoclonal antibody 10C6 revealed that the incidence of CFA determinant 8 (CFA8) on erythrocytes of the early neonate was significantly reduced in line 15I5 compared with lines .6-2, .7-2 and .15I-5; line .C-12 also exhibited a reduced CFA8 incidence at hatching. Likewise, the CAA epitope detected by monoclonal antibody 3F12 was found to appear at a slower rate on erythrocytes from lines 15I5 and .C-12 than on those of other lines. Similar results were obtained using the anti-CAA monoclonal 4C2 where reduced expression was found in lines 15I5, .C-12, and .P-13. Results of complement-mediated cytolysis using the positive control 9F9 monoclonal antibody suggested that observed genetic differences were not due to inherent differences in erythroid cytolytic sensitivity. Neither could the results be explained by the incidence of circulating reticulocytes vs. mature erythrocytes within the lines. Rather, the results suggest that different genetic lines of chickens vary in the developmental kinetics of definitive erythrocyte subpopulations bearing specific phenotypes defined by monoclonal antibodies. These findings are discussed in light of previous observations using these B-congenic lines.     

E-cadherin is functionally involved in the maturation of the erythroid lineage

Differentiation and proliferation of hematopoietic progenitors take place in the bone marrow and is a tightly controlled process. Cell adhesion molecules of the integrin and immunoglobulin families have been shown to be involved in these processes, but almost nothing was known about the involvement of the cadherin family in the hematopoietic system. A PCR screening of RNA of human bone marrow mononuclear cells with specific primers for classical cadherins revealed that E-cadherin, which is mainly expressed by cells of epithelial origin, is also expressed by bone marrow cells. Western blot analysis and immunofluorescence staining of bone marrow sections confirmed this unexpected finding. A more detailed analysis using immunoaffinity columns and dual color flow cytometry showed that the expression of E- cadherin is restricted to defined maturation stages of the erythropoietic lineage. Erythroblasts and normoblasts express E- cadherin, mature erythrocytes do not. A functional role of E-cadherin in the differentiation process of the erythroid lineage was indicated by antibody-inhibition studies. The addition of anti-E-cadherin antibody to bone marrow mononuclear cultures containing exogeneous erythropoietin drastically diminished the formation of erythropoietic cells. These data suggest a non-anticipated expression and function of E-cadherin in one defined hematopoietic cell lineage.     

Chicken developmental antigens: analysis of erythroid populations with monoclonal antibodies

Fusions were performed between the mouse PAI myeloma cell line and spleen cells from Balb/c mice immunized with intact erythrocytes from 1-day Cornell K-strain White Leghorn chickens. Following single cell cloning, four hybridoma clones were found to secrete erythroid specific monoclonal antibodies. Based on its pattern of reactivity, the antibody (IgG2a, kappa) secreted by clone 10C6 detects a specific avian oncodevelopmental antigen associated with the hematopoietic system: chicken fetal antigen (CFA). Two other clones, designated as 3F12 and 4C2, produced antibodies (IgM, kappa) that recognize another avian developmental antigen: chicken adult antigen (CAA). A fourth clone, 9F9, produced an antibody (IgM, kappa) that reacts with all peripheral erythrocytes from both Japanese quail and chicken regardless of age. Clone 10C6 antibody apparently detects an erythrocyte specific (ES) determinant of CFA associated with determinant #8 while antibodies of clones 3F12 and 4C2 recognize a chicken specific determinant of CAA. Analysis by complement mediated microcytotoxicity indicated that the epitopes detected by 10C6 vs 3F12 and 4C2 antibodies were expressed on erythrocytes in a reciprocal fashion during development. Furthermore, strain variations in the incidence of erythrocytes carrying these epitopes were observed. The usefulness of these monoclonal antibodies for the study of erythroid populations is discussed.     

Reactivation of the nuclei of reticulocytes and erythrocytes in heterokaryons]

The reactivation of the nuclei of erythrocytes, reticulocytes and bone marrow cells has been studied by means of hybridization of the pigeon erythroid cells with the human embryonic cells A1. The process of reactivation of the erythroid nucleus was shown to depend on the stage of erythroid cell differentiation. The nuclei of cells at the earlier stages of differentiation give a higher percentage of heterocaryons (40%, 70%) than those of more mature cells (9%). The activated nuclei of immatur cells formed nucleoli already 24 hrs, whereas those of mature erythrocytes only 3-5 days after the cell fusion.     

A novel way to induce erythroid progenitor self renewal: cooperation of c-Kit with the erythropoietin receptor

Red blood cells are of vital importance for oxygen transport in vertebrates. Thus, their formation during development and homeostasis requires tight control of both progenitor proliferation and terminal red cell differentiation. Self renewal (i.e. long-term proliferation without differentiation) of committed erythroid progenitors has recently been shown to contribute to this regulation. Avian erythroid progenitors expressing the EGF receptor/c-ErbB (SCF/TGFalpha progenitors) can be induced to long-term proliferation by the c-ErbB ligand transforming growth factor alpha and the steroids estradiol and dexamethasone. These progenitors have not yet been described in mammals and their factor requirements are untypical for adult erythroid progenitors. Here we describe a second, distinct type of erythroid progenitor (EpoR progenitors) which can be established from freshly isolated bone marrow and is induced to self renew by ligands relevant for erythropoiesis, i.e. erythropoietin, stem cell factor, the ligand for c-Kit and the glucocorticoid receptor ligand dexamethasone. Limiting dilution cloning indicates that these EpoR progenitors are derived from normal BFU-E/CFU-E. For a detailed study, mEpoR progenitors were generated by retroviral expression of the murine Epo receptor in bone marrow erythroblasts. These progenitors carry out the normal erythroid differentiation program in recombinant differentiation factors only. We show that mEpoR progenitors are more mature than SCF/TGFalpha progenitors and also do no longer respond to transforming growth factor alpha and estradiol. In contrast they are now highly sensitive to low levels of thyroid hormone, facilitating their terminal maturation into erythrocytes.     

Transferrin binding capacity as a marker of differentiation and maturation of rat erythroid cells fractionated by counter current distribution in aqueous polymer two-phase systems

Rat bone marrow cell populations, containing different proportions of erythroid cells, have been fractionated by counter-current distribution in the non-charge-sensitive dextran/polyethyleneglycol two-phase systems on the basis of hydrophobic cell surface properties. Cell fractions with a low distribution coefficient, which contain non-erythroid cells and early erythoblasts, showed a low transferrin binding capacity and a low haemoglobin/cell ratio whereas cell fractions with a high distribution coefficient, which contain intermediate-late erythroblasts and mature red cells, showed an elevated transferrin binding capacity and the highest haemoglobin/cell ratio. These results support transferrin binding capacity as a good marker parameter for the erythroid bone marrow cell differentiation and maturation processes.     

CHANGES IN SUSCEPTIBILITY OF CHICK EMBRYONIC BLOOD CELLS TO NEWCASTLE DISEASE VIRUS

To analyse the age-dependent changes in susceptibility of chick embryonic cells to viral infection, observations were made on the blood cells after the inoculation of Newcastle disease virus. A lethal dose of Sato strain of Newcastle disease virus was introduced into chick embryos via injection of inoculum into the blood vessel and allantoic sac. Observations of blood cells by immunofluorescent technique revealed two types of cells, susceptible and resistant to the virus infection. Erythroblasts of both primitive and definitive lines, embryonic thromboblasts and thrombocytes were of the former type and mid- and late-polychromatic erythrocytes and mature ones were of the latter. Erythroblasts gradually decrease in their viral susceptibility as they differentiate into polychromatic erythrocytes and finally become resistant to the virus at the mid-polychromatic erythrocyte stage or later. Thromboblasts, on the other hand, retain high susceptibility to the virus throughout the course of their differentiation to mature thrombocytes. The change in the viral susceptibility of erythroid cells with differentiation is discussed in relation to the structural and functional alterations during the cell specialization.     

Developmental expression of serotonin-like immunoreactivity in the sympathoadrenal system of the chicken

Summary The avian sympathoadrenal system has been used as a model to examine the differentiation of cells expressing neuroactive substances derived from the neural crest. Previous studies have dealt with the expression of the classical neurotransmitters acetylcholine and catecholamines and of the neuropeptides somatostatin and vasoactive intestinal polypeptide. We have used immunocytochemistry to examine the developmental expression of the monoamine serotonin (5HT) in the chicken sympathoadrenal system. 5HT-like immunoreactivity (5HT-LI) was found to be transiently expressed by cells of the sympathetic ganglia very early in development (E-5 to E-8), disappearing almost entirely at more advanced embryonic stages (E-10 to E-19) and posthatched chickens where only a population of cells similar to mammalian small intensely fluorescent cells express immunoreactivity to the amine. In contrast, in the adrenal gland of embryos and post-hatched chickens, most chromaffin cells also express 5HT-LI. Double labeling experiments show that in both the adrenal gland and the sympathetic ganglia catecholaminergic properties and somatostatin immunoreactivity are co-expressed with 5-HT-LI. Moreover, the cells that transiently express 5HT-LI in sympathetic ganglia also transiently express somatostatin. The catecholaminergic cells expressing serotonin and somatostatin appear to define a biochemical phenotype common to some chromaffin cells, small intensely fluorescent cells and early sympathoblasts.     

9-O-acetylated GD3 triggers programmed cell death in mature erythrocytes

An acetylated modification of a tumor-associated ganglioside GD3 (9-O-AcGD3) is expressed in certain tumors and present during early stages of development in different tissues. However, the status and the role of 9-O-AcGD3 in the erythroid progenitor cells remain unexplored. Here, we report the level of 9-O-AcGD3 during erythropoiesis in bone marrow is down regulated during maturation. Signaling via 9-O-AcGD3 induces alteration of morphology and membrane characteristics of mature erythrocytes. This process also induces, a cell death program in these erythrocytes even in the absence of nucleus, mitochondria and other cell organelles sharing features of apoptosis in nucleated cells like membrane alterations, vesicularization, phosphatidyl serine exposure, activation of cysteine proteases like caspase-3. This is the first report of a programmed cell death pathway in mature erythrocytes, triggered by 9-O-AcGD3 contrary to their anti-apoptotic role in lymphoblasts, which suggests a cell specific role of this O-acetyl ester of GD3.     

Clonal analysis of differentiating embryonic stem cells reveals a hematopoietic progenitor with primitive erythroid and adult lymphoid-myeloid potential.

Embryonic stem (ES) cells differentiate into multiple hematopoietic lineages during embryoid body formation in vitro, but to date, an ES-derived hematopoietic stem cell has not been identified and subjected to clonal analysis in a manner comparable with hematopoietic stem cells from adult bone marrow. As the chronic myeloid leukemia-associated BCR/ABL oncogene endows the adult hematopoietic stem cell with clonal dominance without inhibiting pluripotent lymphoid and myeloid differentiation, we have used BCR/ABL as a tool to enable engraftment and clonal analysis. We show that embryoid body-derived hematopoietic progenitors expressing BCR/ABL maintain a primitive hematopoietic blast stage of differentiation and generate only primitive erythroid cell types in vitro. These cells can be cloned, and when injected into irradiated adult mice, they differentiate into multiple myeloid cell types as well as T and B lymphocytes. While the injected cells express embryonic (beta-H1) globin, donor-derived erythroid cells in the recipient express only adult (beta-major) globin, suggesting that these cells undergo globin gene switching and developmental maturation in vivo. These data demonstrate that an embryonic hematopoietic stem cell arises in vitro during ES cell differentiation that constitutes a common progenitor for embryonic erythroid and definitive lymphoid-myeloid hematopoiesis.     

Protein synthesis in differentiating normal and leukemic erythroid cells

Erythroleukemic cells transformed by the AEV or S13 strains of avian erythroblastosis virus differentiate in vitro either spontaneously (S13) or following a temperature induction (temperature-sensitive mutants of AEV). To study differentiation in these cells at the molecular level, homogeneous fractions of maturing cells at discrete stages of differentiation were prepared by Percoll density-gradient centrifugation. This method was also used for the fractionation of differentiating normal erythroid cells separated from total bone marrow by an immunological "panning" technique. Total protein synthesis in these cells was then analyzed by two-dimensional gel electrophoresis. The expression of several proteins was altered in differentiating leukemic cells but not in their normal counterparts. However, in general, the normal and leukemic cells from comparable stages of maturity showed closely related protein synthetic patterns. Similar early and late changes in the synthesis of a number of polypeptides were detected during maturation from early erythroid precursors to terminally differentiated erythrocytes. Further, the leukemic as well as the normal cells appeared to undergo a major switch in total protein synthetic pattern during late differentiation. These results demonstrate that normal and erythroleukemic cells differentiate along similar pathways.     

Cyclin A2 regulates erythrocyte morphology and numbers

Cyclin A2 is an essential gene for development and in haematopoietic stem cells and therefore its functions in definitive erythropoiesis have not been investigated. We have ablated cyclin A2 in committed erythroid progenitors in vivo using erythropoietin receptor promoter-driven Cre, which revealed its critical role in regulating erythrocyte morphology and numbers. Erythroid-specific cyclin A2 knockout mice are viable but displayed increased mean erythrocyte volume and reduced erythrocyte counts, as well as increased frequency of erythrocytes containing Howell-Jolly bodies. Erythroblasts lacking cyclin A2 displayed defective enucleation, resulting in reduced production of enucleated erythrocytes and increased frequencies of erythrocytes containing nuclear remnants. Deletion of the Cdk inhibitor p27^Kip1 but not Cdk2, ameliorated the erythroid defects resulting from deficiency of cyclin A2, confirming the critical role of cyclin A2/Cdk activity in erythroid development. Loss of cyclin A2 in bone marrow cells in semisolid culture prevented the formation of BFU-E but not CFU-E colonies, uncovering its essential role in BFU-E function. Our data unveils the critical functions of cyclin A2 in regulating mammalian erythropoiesis.     

Expression of C3d receptors during human B cell differentiation: immunofluorescence analysis with the HB-5 monoclonal antibody

We have examined human B lymphocytes at different stages of differentiation for the expression of surface receptors for the C3d fragment of complement. C3d receptors (C3dR) were identified by indirect immunofluorescence using the HB-5 monoclonal antibody, which recognizes a 145,000 m.w. C3dR molecule on B lymphocytes. Pre-B and immature B cells from fetal bone marrow and liver did not express C3dR, whereas a small subpopulation (25%) of B cells in fetal spleen were C3dR+. Approximately 50% of the B cells in adult bone marrow were C3dR+, whereas the more mature B cells in the blood of newborns and adults and in peripheral lymphoid tissue of adults uniformly expressed the C3dR. Activated B cells responsive to T cell-derived differentiation factors were C3dR+, whereas plasma cells rarely expressed C3dR. T cells, NK cells, erythrocytes, and myelomonocytic cells did not express detectable surface C3dR. These results suggest that in hematopoietic and lymphoid tissues, the expression of C3dR is a specific feature of relatively mature lymphoid cells of B lineage.     

Differential regulatory and compensatory responses in hematopoiesis/erythropoiesis in alpha- and beta-globin hemizygous mice

Characterization of hematopoiesis/erythropoiesis in thalassemias from multipotent primitive cells to mature erythrocytes is of fundamental importance and clinical relevance. We investigated this process in alpha- and beta-globin hemizygous mice, lacking the two adult tandemly organized genes from either the alpha- or beta-globin locus. Although both mice backcrossed on a homogeneous background exhibited similar reduced red blood cell (RBC) survival, beta-globin hemizygous mice had less severe reticulocyte loss and globin chain imbalance, suggesting an apparently milder thalassemia than for alpha-globin hemizygous mice. In contrast, however, beta-globin hemizygous mice displayed a more marked perturbation of hematologic parameters. Quantification of erythroid precursor subpopulations in marrow and spleen of beta-globin hemizygous mice showed more severely impaired maturation from the basophilic to orthochromatophilic erythroblasts and substantial loss of these late precursors probably as a consequence of a greater susceptibility to an excess of free alpha-chain than beta-chain. Hence, only erythroid precursors exhibiting stochastically moderate chain imbalance would escape death and mature to reticulocyte/RBC stage, leading to survival and minimal loss of reticulocytes in the beta-globin hemizygous mice. Furthermore, in response to the ineffective erythropoiesis in beta-globin hemizygous mice, a dynamic compensatory hematopoiesis was observed at earlier differentiation stage as evidenced by a significant increase of erythroid progenitors (erythroid colony-forming units approximately 100-fold) as well as of multipotent primitive cells (day 12 spleen colony-forming units approximately 7-fold). This early compensatory mechanism was less pronounced in alpha-globin hemizygous mice. The expansion of multipotent primitive and potentially stem cell populations, taken together with ineffective erythropoiesis and increased reticulocyte/RBC destruction could confer major cumulative advantage for gene targeting/bone marrow transplantation. Therefore, this study not only corroborated the strong potential effectiveness of transplantation for thalassemic hematopoietic therapy but also demonstrated the existence of a differential regulatory response for alpha- and beta-thalassemia.     

Autocrine differentiation-inhibiting factor (ADIF) from chicken erythroleukemia cells acts on human and mouse early BFU-E erythroid progenitors

tsAEV-LSCC HD3 chicken erythroid cells transformed by the avian erythroblastosis virus (AEV) secrete an autocrine differentiation-inhibiting factor, ADIF, which blocks differentiation without affecting proliferation of the chicken erythroid cells that synthesize and secrete it into the culture medium. The chicken erythroleukemia cell ADIF activity is not restricted to avians. It prevents dimethylsulfoxide (DMSO) from stimulating murine Friend erythroleukemia cells to synthesize hemoglobin. ADIF also blocks erythroid differentiation in normal human and murine bone marrow where it selectively targets the early BFU-E (burst-forming) erythroid precursor cells without affecting the more advanced CFU-E erythroid precursor cells or cells of the different granulocyte-macrophage lineage.