Differential Regulatory and Compensatory Responses in Hematopoiesis/Erythropoiesis in α- and β-Globin Hemizygous Mice
2004; Elsevier BV; Volume: 279; Issue: 19 Linguagem: Inglês
10.1074/jbc.m309989200
ISSN1083-351X
AutoresHugues Beauchemin, Marie‐José Blouin, Marie Trudel,
Tópico(s)Blood groups and transfusion
ResumoCharacterization of hematopoiesis/erythropoiesis in thalassemias from multipotent primitive cells to mature erythrocytes is of fundamental importance and clinical relevance. We investigated this process in α- and β-globin hemizygous mice, lacking the two adult tandemly organized genes from either the α- or β-globin locus. Although both mice backcrossed on a homogeneous background exhibited similar reduced red blood cell (RBC) survival, β-globin hemizygous mice had less severe reticulocyte loss and globin chain imbalance, suggesting an apparently milder thalassemia than for α-globin hemizygous mice. In contrast, however, β-globin hemizygous mice displayed a more marked perturbation of hematologic parameters. Quantification of erythroid precursor subpopulations in marrow and spleen of β-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 α-chain than β-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 β-globin hemizygous mice. Furthermore, in response to the ineffective erythropoiesis in β-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 ∼100-fold) as well as of multipotent primitive cells (day 12 spleen colony-forming units ∼7-fold). This early compensatory mechanism was less pronounced in α-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 α- and β-thalassemia. 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 α- and β-globin hemizygous mice, lacking the two adult tandemly organized genes from either the α- or β-globin locus. Although both mice backcrossed on a homogeneous background exhibited similar reduced red blood cell (RBC) survival, β-globin hemizygous mice had less severe reticulocyte loss and globin chain imbalance, suggesting an apparently milder thalassemia than for α-globin hemizygous mice. In contrast, however, β-globin hemizygous mice displayed a more marked perturbation of hematologic parameters. Quantification of erythroid precursor subpopulations in marrow and spleen of β-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 α-chain than β-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 β-globin hemizygous mice. Furthermore, in response to the ineffective erythropoiesis in β-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 ∼100-fold) as well as of multipotent primitive cells (day 12 spleen colony-forming units ∼7-fold). This early compensatory mechanism was less pronounced in α-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 α- and β-thalassemia. Thalassemia, among the most frequent of inherited diseases, constitutes a heterogeneous disorder based on clinical severity, pathophysiology, and molecular changes. This hemoglobinopathy has been classified into two major groups, α- and β-thalassemia, reflecting impairment or absence of either α or β chain synthesis. The level of globin chain imbalance, resulting from a change in the relative ratio of α- and β-globin chains, appears directly related to the severity of thalassemia in humans. A variety of mutations including deletions, frameshifts, nonsense, and abnormal splicing lead to a thalassemic phenotype (1Coleman M.B. Lu Z.H. Smith II, C.M. Adams III, J.G. Harrell A. Plonczynski M. Steinberg M.H. J. Clin. Invest. 1995; 95: 503-509Crossref PubMed Google Scholar, 2Loukopoulos D. Ann. Hematol. 1991; 62: 85-94Crossref PubMed Scopus (8) Google Scholar, 3Harteveld C.L. Heister J.G. Giordano P.C. Batelaan D. von Delft P. Haak H.L. Wijermans P.W. Losekoot M. Bernini L.F. Br. J. Haematol. 1996; 95: 461-466Crossref PubMed Scopus (24) Google Scholar, 4Ayala S. Colomer D. Aymerich M. Abella E. Vives Corrons J.L. Br. J. Haematol. 1997; 98: 47-50Crossref PubMed Scopus (12) Google Scholar, 5Wong C. Antonarakis S.E. Goff S.C. Orkin S.H. Forget B.G. Nathan D.G. Giardina P.J. Kazazian H.H.J. Blood. 1989; 73: 914-918Crossref PubMed Google Scholar).Individuals with thalassemia display mild to severe anemia depending on their genotype (6Bunn H.F. Forget B.G. Dyson J. Hemoglobin: Molecular Genetic and Clinical Aspects. W.B. Saunders, Philadelphia1986: 322-371Google Scholar). In symptomatic patients, α- and β-thalassemia display similar abnormal red blood cell (RBC) features including microcytosis, hypochromia, anisocytosis, and poikilocytosis. The excess of one normal globin chain in RBC forms aggregates, leading to premature cell destruction. Thalassemic RBC membranes are rigid, showing instability in the case of β-thalassemia and hyperstability in α-thalassemia (7Schrier S.L. Annu. Rev. Med. 1994; 45: 211-218Crossref PubMed Scopus (59) Google Scholar). Bone marrow from affected individuals usually undergoes erythroid hyperplasia associated with increased production of erythroblasts and moderate to severe splenomegaly. Ineffective erythropoiesis, more prominent in β-than in α-thalassemia, is also observed (8Bothwell T.H. Charlton R.W. Cook J.D. Finch C.A. Iron Metabolism in Man. Blackwell Scientific Publications, London, UK1979: 190-221Google Scholar, 9Finch C.A. Deubelbeiss K. Cook J.D. Eschbach J.W. Harker L.A. Funk D.D. Marsaglia G. Hillman R.S. Slichter S. Adamson J.W. Ganzoni A. Giblett E.R. Medicine. 1970; 49: 17-53Crossref PubMed Scopus (303) Google Scholar, 10Pootrakul P. Sirankapracha P. Hemsoroach S. Moungsub W. Kumbunlue R. Piangitjagum A. Wasi P. Ma L. Schrier S.L. Blood. 2000; 96: 2606-2612Crossref PubMed Google Scholar). Individuals with severe thalassemia are dependent on regular transfusion. Although chronic transfusion improves survival, it leads progressively to iron accumulation and tissue damage in several organs.Allogeneic bone marrow transplantation has been successfully used as a therapy for thalassemia. However, the morbidity and mortality associated with this procedure as well as the difficulty in obtaining histocompatible donors remain problematic (11Giardini C. Galimberti M. Lucarelli G. Annu. Rev. Med. 1995; 46: 319-330Crossref PubMed Scopus (18) Google Scholar, 12La Nasa G. Giardini C. Argiolu F. Locatelli F. Arras M. De Stefano P. Ledda A. Pizzati A. Sanna M.A. Vacca A. Lucarelli G. Contu L. Blood. 2002; 99: 4350-4356Crossref PubMed Scopus (120) Google Scholar). These problems could potentially be alleviated by the use of autologous bone marrow transplantation following gene therapy correction. In both cases, a detailed characterization of the altered hematopoiesis and erythropoiesis in α- and β-thalassemia is necessary to develop an effective cure for thalassemic patients.Initial mouse models of α- and β-thalassemia were generated following radiation-induced or genetically induced mutations (13Martinell J. Whitney III, J.B. Popp R.A. Russell L.B. Anderson W.F. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 5056-5060Crossref PubMed Scopus (20) Google Scholar, 14Russell L.B. Russell W.L. Popp R.A. Vaughan C. Jacobson K.B. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2843-2846Crossref PubMed Scopus (41) Google Scholar, 15Skow L.C. Burkhart B.A. Johnson F.M. Popp R.A. Popp D.M. Cell. 1983; 34: 1043-1052Abstract Full Text PDF PubMed Scopus (175) Google Scholar). More recently, α- and β-globin hemizygous mice have been produced by targeted deletion of the adult globin genes (16Paszty C. Mohandas N. Stevens M.E. Loring J.F. Liebhaber S.A. Brion C.M. Rubin E.M. Nat. Genet. 1995; 11: 33-39Crossref PubMed Scopus (64) Google Scholar, 17Ciavatta D.J. Ryan T.M. Farmer S.C. Townes T.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9259-9263Crossref PubMed Scopus (110) Google Scholar). The α-globin hemizygous mice, with deletion of the two adult α-globin genes, genetically reproduce the human α-thalassemia 1. Similarly, β-globin hemizygous mice correspond genotypically to heterozygous β-thalassemia. A thorough characterization of hematopoiesis/erythropoiesis in these globin hemizygous mice is required to determine the fundamental cellular defects and whether these mice reproduce the human diseases, particularly as these globin hemizygous mice are becoming widely used as models of human thalassemia.Herein, we report such an investigation in these α- and β-globin hemizygous mice both bred and compared for the first time on an identical genetic background. These mice with half their adult α- or β-globin gene content demonstrated thalassemia. On this homogeneous background, the α-globin compared with β-globin hemizygous mice had greater globin chain imbalance in peripheral RBC, a surprising result considering that β-globin hemizygous mice had more severe anemia. However, we demonstrated that the β-globin hemizygous mice had a more severely hindered late erythroid precursor maturation attributed to an increase cell loss upon the onset of α-globin chain expression that occurred earlier than for the β-globin chain. Furthermore, consequent to the ineffective erythropoiesis, the β-globin relative to α-globin hemizygous mice underwent a more pronounced compensatory stimulation of the multipotent primitive cell populations and of early erythropoiesis. Finally, the inverse correlation between the compensatory erythropoietic/hematopoietic stimulation and the severe alterations of hematologic parameters suggested that the level of anemia might provide a reliable index for the potential effectiveness of gene therapy and bone marrow transplantation in thalassemias.EXPERIMENTAL PROCEDURESAnimal StudiesHemizygous α-globin globin mice (Hbatm1Paz/Hbaa) and β-globin mice (Hbbtm1Tow/Hbbs) were a generous gift from Drs. C. Paszty and E. Rubin and from Dr. T. Townes, respectively (16Paszty C. Mohandas N. Stevens M.E. Loring J.F. Liebhaber S.A. Brion C.M. Rubin E.M. Nat. Genet. 1995; 11: 33-39Crossref PubMed Scopus (64) Google Scholar, 17Ciavatta D.J. Ryan T.M. Farmer S.C. Townes T.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9259-9263Crossref PubMed Scopus (110) Google Scholar). Both of these lines have been backcrossed for multiple generations (>10) with C57BL/6J inbred mice to avoid the effects of various genetic backgrounds. The α-globin hemizygous mice (C57BL/6J hemi-αthal) were identified as previously described (16Paszty C. Mohandas N. Stevens M.E. Loring J.F. Liebhaber S.A. Brion C.M. Rubin E.M. Nat. Genet. 1995; 11: 33-39Crossref PubMed Scopus (64) Google Scholar). The β-globin hemizygous mice (C57BL/6J hemi-βthal) were also identified following PCR amplification using three primers; two were localized in the wild type β-globin gene, forward (5′-GAGCAATGTGGACAGAGAAGGAG-3′) and reverse (5′-TGATGTCTGTTTCTGGGGTTGTG-3′), producing a 450-bp amplicon and a third neomycin-specific reverse primer (5′-TGAAGAGCTTGGCGGCGAATGGG-3′) that will generate a 650-bp amplicon. The DNA was amplified in a PCR buffer (10 mm Tris, pH 8.9, 50 mm KCl, 1.0 mm MgCl2) containing 0.2 mm each dNTP, 0.4 μm concentration of the forward and 0.2 μm concentration of each reverse primer, and 3.0 units of Taq, in a 20-μl reaction volume. Amplification was performed for 30 cycles of 92 °C (20 s), 65 °C (35 s), and 68 °C (105 s).Globin Chain AnalysisAnalysis of globin chain synthesis was carried out on a population of reticulocyte-enriched cells (11–33.6%) including RBC that can no longer synthesize globin chains. Cells were collected in heparinized Micro-Hematocrit capillary tubes from the tail vein. C57BL/6J hemi-αthal, C57BL/6J hemi-βthal, and C57BL/6J controls were analyzed in duplicate for globin chain synthesis levels. Packed erythroid cells composed of reticulocytes and RBC 1The abbreviations used are: RBC, red blood cell; hemi-αthal, α-globin hemizygous; hemi-βthal, β-globin hemizygous; PBS, phosphate-buffered saline; CFU-E, erythroid colony-forming units; BFU-E, erythroid burst-forming units; CFU-GM, granulocyte/macrophage colony-forming units; CFU-M, macrophage colony-forming units; CFU-GEMM, granulocyte-erythroid-macrophage-megakaryocyte colony-forming units; CFU-S12, day 12 spleen colony-forming units. (>99.4%) were washed three times in 1× Dulbecco's PBS and incubated in 1 ml of [3H]leucine labeling mix (18Fabry M.E. Suzuka S.M. Weinberg R.S. Lawrence C. Factor S.M. Gilman J.G. Costantini F. Nagel R.L. Blood. 2001; 97: 410-418Crossref PubMed Scopus (48) Google Scholar) supplemented with 50 μg of transferrin for 1, 2, and 2.5 h with periodic shaking under 5% CO2 in air (15Skow L.C. Burkhart B.A. Johnson F.M. Popp R.A. Popp D.M. Cell. 1983; 34: 1043-1052Abstract Full Text PDF PubMed Scopus (175) Google Scholar). The RBC were then lysed, and the hemoglobin solution was quantified as previously described (19Masala B. Manca L. Methods Enzymol. 1994; 231: 21-44Crossref PubMed Scopus (52) Google Scholar). The hemoglobin solution (100 μg) was used to determine the globin chain content by high pressure liquid chromatography on a Vydac large pore C4 column (4.6 × 250 mm; Grace Vydac, Hesperia, CA). Globin chains were resolved with a helium-degassed modified trifluoroacetic acid/acetonitrile gradient: phase A (0.18% trifluoroacetic acid (w/v) in 36% acetonitrile) and phase B (0.18% trifluoroacetic acid (w/v) in 46% acetonitrile) (20Fabry M.E. Sengupta A. Suzuka S.M. Costantini F. Rubin E.M. Hofrichter J. Christoph G. Manci E. Culberson D. Factor S.M. Nagel R.L. Blood. 1995; 86: 2419-2428Crossref PubMed Google Scholar, 21Shelton J.B. Shelton J.R. Schroeder W.A. J. Liquid Chromatogr. 1984; 7: 1969-1977Crossref Scopus (409) Google Scholar). Globin chains were eluted by increasing mobile phase B from 33 to 37% over 15 min and, subsequently, to 70% over 35 min at a flow rate of 1 ml/min, and the elution profiles were followed by UV detection at 220 nm. One-ml sample fractions were collected, counted, and corrected for background levels to evaluate α- or β-globin chain synthesis and to determine the ratio of α- or β-globin on total globin chains synthesized.To prepare RBC ghosts, 1 ml of blood obtained by cardiac puncture was washed three times in 0.9% cold saline. The packed RBCs were lysed as described previously (22Rouyer-Fessard P. Leroy-Viard K. Domenget C. Mrad A. Beuzard Y. J. Biol. Chem. 1990; 265: 20247-20251Abstract Full Text PDF PubMed Google Scholar) in 8.5 ml of cold lysis buffer (7.5 mm sodium phosphate, 1 mm disodium EDTA, pH 7.5) containing 0.12 mm phenylmethylsulfonyl fluoride and 2.9 μm pepstatin A (23Bennett V. Methods Enzymol. 1983; 96: 313-324Crossref PubMed Scopus (157) Google Scholar). Protein biosynthesis was measured by incubating the RBCs with 3 ml of translation [3H]leucine labeling mix for 1, 1.5, and 2 h prior to lysis. Membrane proteins were separated by urea-Triton PAGE, stained with Coomassie Brilliant Blue, and quantified with ImageQuant software version 5.0 or by autoradiography on a Kodak Biomax MR film, as described previously (24Blouin M.-J. Beauchemin H. Wright A. DePaepe M. Sorette M. Bleau A.-M. Nakamoto B. Ou C.-N. Stamatoyannopoulos G. Trudel M. Nat. Med. 2000; 6: 177-182Crossref PubMed Scopus (48) Google Scholar).Hematologic ParametersBlood was analyzed using flow cytometry-based hematology with the mouse archetype of multispecies software version 2.2.06 (Bayer Advia 120, Tarrytown, NY). A Mie scatter theory was used to determine the volume and hemoglobin concentration for each cell by analysis of low and high angle light scattering as previously described (24Blouin M.-J. Beauchemin H. Wright A. DePaepe M. Sorette M. Bleau A.-M. Nakamoto B. Ou C.-N. Stamatoyannopoulos G. Trudel M. Nat. Med. 2000; 6: 177-182Crossref PubMed Scopus (48) Google Scholar). The percentage of hypochromic RBCs (mean cellular hemoglobin concentration, less than 22 g/dl) and the percentage of microcytic cells (volume less than 25 fl) were evaluated by appropriate gating of the cellular hemoglobin concentration mean and the mean cellular volume. Reticulocyte counts were obtained by specific RNA staining with the oxazine 750 dye using the reticulocyte channel of the Bayer Advia 120.RBC SurvivalRBC survival was evaluated using biotinylation of the entire RBC cohort and monitoring for RBC replacement. Biotinylation of RBCs was carried out by intravenous injection of 250 μl of 3.6 mg/ml of sulfo-N-hydroxysuccinimide-biotin (VWR; Montreal, Canada) for three consecutive days. RBCs (∼3 × 106) obtained from 1–5 μl of tail vein blood were labeled with 9.2 μg of fluorescein isothiocyanate-conjugated avidin (BIO/CAN; Toronto, Canada) in 1 ml of PBS. The number of biotinylated cells in circulating blood was determined at t = 0 and monitored at regular intervals by flow cytometry on a FACScan (Becton Dickinson, San Jose, CA).Fluorescence-activated Cell Sorting Analysis and QuantitationFlow cytometry analysis was performed on bone marrow and spleen samples from C57BL/6J, C57BL/6J hemi-αthal, and C57BL/6J hemi-βthal mice. Bone marrow cells were harvested by flushing one femur with PBS containing 2% fetal calf serum. Spleen cells were suspended in 2% fetal calf serum/PBS by subsequent passage through decreasing size needles (18-, 20-, and 23-gauge). Cells (5 × 105 or 7.5 × 105) were incubated in 2% fetal calf serum/PBS with antibodies for 30 min on ice according to standard techniques. The labeling with anti-mouse TER119-phycoerythrin (0.2 ng) and biotin-conjugated anti-mouse CD71 (transferrin receptor) (0.5 ng) was detected with streptavidin-allophycocyanin (0.2 ng) (BD Biosciences). Apoptosis as defined by phosphatidylserine exposure on erythroid precursors was detected with 1–1.5 μl of Annexin V-fluorescein isothiocyanate (BD Biosciences). Samples were analyzed on a flow cytometer FACSCalibur (Becton Dickinson) using CellQuest Pro version 4.0.2 software, and quantification was carried out with WinMDI version 2.8 software. Small cell debris was excluded by gating on a forward scatter plot, and erythroid precursors were identified as TER119+ CD71+ cells.Quantitation of cell loss at specific subpopulation stage depends on the following: 1) subpopulation between two consecutive stages: stage i ⇒ stage i + 1; 2) percentage (P) of cells at two consecutive stages for control and hemizygous (Picont⇒Pi+1cont; Pihe⇒Pi+1he; and 3) total number of cells in a subpopulation (Ns).The expected number of cells based on controls from stage i ⇒ stage i + 1 is as follows. PiheNs⇒PihePi+1contPicontNs(Eq. 1) The absolute cell loss can be defined as the difference between the expected number of cells and the observed number of cells. [PihePi+1contPicont−Pi+1he]Ns(Eq. 2) The cell loss relative to controls can be written as follows. PihePi+1contPilcont−Pi+1hePihePi+1contPicont×100(Eq. 3) The equation can be rearranged to give the following, (1−Pi+1hePicontPiheP i+1 cont)×100(Eq. 4) which equals the percentage of cell loss from the Pi subpopulation.Hematopoietic Progenitor StudiesClonogenic Assays—Clonogenic assays were performed on C57BL/6J hemi-αthal, C57BL/6J hemi-βthal, and control C57BL/6J mice. Progenitor cell analyses were carried out on three hematopoietic tissues: bone marrow, peripheral blood, and spleen. Peripheral blood cells were obtained from the buffy coat, washed twice in Iscove's modified Dulbecco's medium plus 5% fetal calf serum and once in PBS; RBC lysis was obtained following incubation in 5 ml of Gey's solution (8.3 g/liter NH4Cl, 1 g/liter KHCO3, pH 7.2) for 2 min at 37 °C, and the cells were resuspended in Iscove's modified Dulbecco's medium. Bone marrow cells, peripheral blood mononuclear cells, and spleen single-cell suspensions were plated, respectively, at a density of 105, 106, and 5 × 105 cells/ml in 1% methylcellulose/Iscove's modified Dulbecco's medium as previously described (25Trudel M. De Paepe M.E. Chretien N. Saadane N. Jacmain J. Sorette M. Hoang T. Beuzard Y. Blood. 1994; 84: 3189-3197Crossref PubMed Google Scholar). All experiments were performed in duplicate for each animal. Erythroid colony-forming units (CFU-E) were counted after 2 days in culture, whereas burst-forming units (BFU-E), granulocyte/macrophage colony-forming units (CFU-GM), and macrophage colony-forming units (CFU-M) were counted at 7 days, and granulocyte-erythroid-macrophage-megakaryocyte colony-forming units (CFU-GEMM) was counted on day 11. Results were expressed as the mean ± S.D. from all animals analyzed. For each genotype, the percentage of spleen weight per total body weight was also determined.Day 12 Spleen Colony-forming Unit (CFU-S12) Evaluation—Peripheral blood, bone marrow, and spleen cell suspensions were used to quantify multipotent CFU-S12 (26Blouin M.-J. De Paepe M. Trudel M. Blood. 1999; 94: 1451-1459Crossref PubMed Google Scholar). The numbers of CFU-S12 were determined for C57BL/6J hemi-αthal, C57BL/6J hemi-βthal, and control C57BL/6J mice using 2 × 106 nucleated cells from peripheral blood and 5 × 104 nucleated cells from bone marrow. Similarly, splenic CFU-S12 was evaluated for C57BL/6J hemi-αthal, C57BL/6J hemi-βthal, and C57BL/6J mice using, respectively, 2 × 106, 5 × 105, and 106 nucleated spleen cells. Cells were injected into the tail vein of irradiated C57BL/6J mice (at 900 rads), and CFU-S colonies were counted on day 12.Statistical MethodsUnpaired two-sample Student's t test was used for statistical analysis; p < 0.05 was considered significant.RESULTSImbalanced Globin Chains in Both Hemizygous MiceThe α- and β-globin hemizygous mice (C57BL/6J hemi-αthal and C57BL/6J hemi-βthal) have been generated by targeted deletion of the two adult tandem genes at the α- or β-globin locus, respectively. To characterize the α- and β-globin hemizygous mice on the same genetic background, we backcrossed these mice to the C57BL/6J strain for more than 10 generations. We then investigated whether soluble globin chain levels were imbalanced at three short time periods of de novo synthesis in reticulocytes and at steady state in peripheral blood, which consisted of more than 99.4% reticulocytes and RBC. Short biosynthesis periods in reticulocytes are necessary to detect severe chain imbalances apart from cell destruction that may occur with time. As shown in Table I, in the β-globin hemizygous mice, the biosynthesis of the β-globin chain was significantly decreased at all time points compared with controls (11–14%). For the α-globin hemizygous mice, the α-globin synthesis displayed a stronger decrease compared with controls (17–23%). Thus, both of these globin hemizygous mice have thalassemia, but comparison of relative globin chain levels revealed a more severe imbalance for the α-globin hemizygous mice. When the ratio of soluble globin chains in peripheral blood was evaluated at steady state, it appeared improved for both hemizygous mice (Table I), suggesting loss of reticulocytes. Whereas complete chain balance was attained for the β-globin hemizygous mice, imbalance was still detected in the α-globin hemizygous mice. To assess whether the differential response between the hemi-βthal and hemi-αthal mice is due to an increased tendency of the excess α-globin chains from hemi-βthal to associate with erythroid cell membranes, we have monitored the levels of globin chains in reticulocyte membranes in these mice as a function of total protein (Table II). A significant amount of the globin chain in excess was trapped in reticulocyte membranes for both the hemizygous mice as determined by biosynthetic labeling, whereas no membrane-bound globin chain was detected in controls. However, at all time points assessed, the hemi-βthal mice had lesser amounts of membrane-trapped globin chain than the hemi-αthal mice (Table II), consistent with the soluble globin chain data (Table I). Hence, this confirmed a more severe imbalance for the hemi-αthal rather than an increased tendency of α-chain to associate with membranes in hemi-βthal. In contrast to the de novo reticulocyte synthesis data, the percentage of membrane-trapped globin chain was similar in peripheral blood at steady state for both hemizygous mice (Table II), again supporting a loss of immature erythroid cells, mainly reticulocytes. Noticeably, the membrane-trapped globin chain levels were significantly higher in both hemizygous mice than in control mice in agreement with the thalassemic phenotype.Table IAnalysis of soluble globin chains in α- and β-hemizygous miceMiceTime of biosynthesisSteady state1 h2 h2.5 h%%%%Control: C57BL/6JPercentage of α chain/total chains47.1 ± 1.048.7 ± 1.750.0 ± 0.950.0 ± 0.9Percentage of β chain/total chains52.9 ± 1.0 (n = 4)51.3 ± 1.7 (n = 4)50.0 ± 0.9 (n = 3)50.0 ± 0.9 (n = 8)C57BL/6J hemi-αthalPercentage of α chain/total chains37.1 ± 1.9ap < 0.0005.37.1 ± 1.5bp < 0.0001.41.4 ± 2.0cp < 0.005.45.9 ± 0.6dp < 10-5.Percentage of β chain/total chains62.9 ± 1.9ap < 0.0005. (n = 4)62.9 ± 1.5bp < 0.0001. (n = 4)58.6 ± 2.0cp < 0.005. (n = 4)54.1 ± 0.6dp < 10-5. (n = 8)C57BL/6J hemi-βthalPercentage of α chain/total chains54.6 ± 0.7cp < 0.005.55.6 ± 1.2ep < 0.001.55.3 ± 0.9cp < 0.005.49.3 ± 0.6Percentage of β chain/total chains45.4 ± 0.7cp < 0.005. (n = 4)44.4 ± 1.2ep < 0.001. (n = 4)44.7 ± 0.9cp < 0.005. (n = 5)50.8 ± 0.6 (n = 9)a p < 0.0005.b p < 0.0001.c p < 0.005.d p < 10-5.e p < 0.001. Open table in a new tab Table IIAnalysis of membrane-bound globin chains in α- and β-hemizygous miceMiceTime of biosynthesisSteady state1 h1.5 h2 h%%%%Control: C57BL/6JPercentage of α chain/total proteins0.0 ± 0.00.0 ± 0.00.0 ± 0.04.4 ± 3.6Percentage of β chain/total proteins0.0 ± 0.0 (n = 3)0.0 ± 0.0 (n = 2)0.0 ± 0.0 (n = 4)3.6 ± 2.9 (n = 11)C57BL/6J hemi-αthalPercentage of α chain/total proteins0.0 ± 0.00.0 ± 0.00.0 ± 0.02.2 ± 1.4Percentage of β chain/total proteins25.2 ± 10.3ap < 0.01. (n = 4)36.5 ± 28.4bp < 0.05. (n = 2)35.8 ± 20.7bp < 0.05. (n = 4)19.4 ± 5.9cp < 10-7. (n = 12)C57BL/6J hemi-βthalPercentage of α chain/total proteins8.6 ± 5.0bp < 0.05.9.4 ± 3.3bp < 0.05.5.2 ± 1.3dp < 0.0005.16.2 ± 2.8ep < 10-8.Percentage of β chain/total proteins0.0 ± 0.0 (n = 4)0.0 ± 0.0 (n = 3)0.0 ± 0.0 (n = 4)3.7 ± 1.8 (n = 13)a p < 0.01.b p < 0.05.c p < 10-7.d p < 0.0005.e p < 10-8. Open table in a new tab Hematologic ParametersMature RBCs in the globin hemizygous mice were analyzed to determine whether the hematologic parameters were compatible with those seen in human α- and β-thalassemias (Table III). Hemoglobin concentration and hematocrit were both decreased by ∼10–20% in the hemi-αthal and by ∼50% in the hemi-βthal, indicating moderate and severe anemia, respectively. The RBCs displayed a similar reduced mean cellular volume of 18 and 23% and mean cellular hemoglobin of 25 and 30% for the hemi-αthal and hemi-βthal mice, respectively. The morphology of the RBC was also altered. Notably, the red cell distribution width was significantly increased 1.8- and 2.6-fold in hemi-αthal and hemi-βthal mice relative to controls, showing heterogeneity of cell size. Consistently, the number of microcytic and of hypochromic cells was also increased in hemi-αthal and hemi-βthal mice, with the hemi-βthal being more severely affected. Furthermore, reticulocyte numbers were significantly increased in both globin hemizygous mice relative to controls, suggesting stimulation of erythropoiesis. Thus, as shown in Table III, the hematologic parameters and the anemia were exacerbated in the hemi-βthal relative to the hemi-αthal mice. This was in contrast with the more severe globin chain imbalance observed in the hemi-αthal mice (Tables I and II).Table IIIAltered hematological parameters in α- and β-globin hemizygous miceMicenHbHctMCVMCHRDWMicro (V < 25 fl)Hypo (HC < 22 g/dl)Reticg/dl%flpg%%%Control: C57BL/6J1015.4 ± 1.951.5 ± 6.249.4 ± 1.314.7 ± 1.013.7 ± 1.10.2 ± 0.10.6 ± 0.23.7 ± 0.7C57BL/6J hemi-αthal712.3 ± 0.9ap < 0.001.45.7 ± 3.2bp < 0.05.40.5 ± 0.9ap < 0.001.10.9 ± 0.2ap < 0.001.24.4 ± 2.1ap < 0.001.6.0 ± 1.9ap < 0.001.4.9 ± 1.2ap < 0.001.7.1 ± 0.8ap < 0.001.C57BL/6J hemi-βthal36.7 ± 1.1ap < 0.001.25.0 ± 3.0ap < 0.001.37.3 ± 3.4ap < 0.001.10.2 ± 0.4ap < 0.001.36.1 ± 1.3ap < 0.001.19.9 ± 4.0ap < 0.001.19.1 ± 8.2cp < 0.01.22.9 ± 6.0ap < 0.001.a p < 0.001.b p < 0.05.c p < 0.01. Open table in a new tab Since these mice
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