Murine Hertwig's anemia: premature death after normal bone marrow transplantation is radiation dose-dependent

Marrow transplantation therapy in mice with heritable blood disorders usually leads to rapid blood cell normalization, but is sometimes followed by pancytopenia and premature death. This is especially true in mice with Hertwig's anemia (an/an). Unlike the +/+ recipients, 100% of whom survive for over a year, 66% of the mutant mice die by 6 months posttransplantation, and the rest die soon thereafter. It is not clear whether premature death is due to the radiation dose (10 Gy) or to the fact that the F1 mutant mice receive parental-type cells known to induce hybrid resistance. In the present report, experiments were designed to determine whether the F1-an/an host is more sensitive to radiation and/or resistant to continued expansion of the parental-type +/+ cells. The mutant mice are, indeed, more sensitive to irradiation, with an LD100/30 of 7 Gy as compared with an LD100/30 of 10 Gy for the +/+ mice. The times of anemia onset and death for mutant mice implanted with +/+ cells postirradiation is also radiation dose-dependent. Further evidence that death is due to host radiation damage rather than F1 hybrid resistance was provided by transplanting cells from three morbid 10 Gy-irradiation recipients into unirradiated, anemic, stem cell-deficient, F1-W/Wv secondary hosts. All recipients were repopulated by the original parental cells, were cured of their anemia, and survived for 52 weeks posttransplantation. The an/an mouse's heightened susceptibility to radiation damage appears to be the major factor in early death after transplantation therapy.     

The decrease in long-term marrow repopulating capacity seen after transplantation is not the result of irradiation-induced stromal injury

Marrow cells from nonirradiated F1-W/Wv mice repopulated slightly less well than cells from lethally irradiated recipients. Therefore, avoiding irradiation of recipients did not improve the relative repopulating ability of their marrow cells. In other experiments, F1-W/Wv mice were transplanted by parabiosis with marrow of WBB6F1-+/+ (F1-+/+) mice, avoiding cellular handling and irradiation. Marrow cells transplanted to F1-W/Wv mice by this procedure demonstrated slightly better repopulating ability than did marrow cells transplanted by injection. However, they performed no better than those transplanted by parabiosis to irradiated F1-+/+ recipients. Significant impairment of stromal function after irradiation was not indicated. Apparently, stem cell damage caused by transplantation may have greater importance in causing loss of stem cell replicative potential than effects of irradiation-induced stromal injury.     

Sl/Sld mouse bone marrow stroma in vitro contains an active radiation- sensitive inhibitor of normal hemopoiesis

Sl/Sld mice have a defective hemopoietic microenvironment. It has been assumed, based upon previous studies, that the primary abnormality in these mice is simply lack of a necessary supportive or inductive material within the hemopoietic stroma. We used in vitro long-term bone marrow cultures to characterize further the nature of the hemopoietic microenvironmental defect in Sl/Sld mice. Sl/Sld mouse bone marrow cells consistently produced less than 10% of the total hemopoietic cells and multipotent and unipotent hemopoietic progenitor cells produced in cultures of marrow from normal, congenic +/+ mice. If fresh Sl/Sld and +/+ marrow cells were mixed prior to establishing long-term marrow cultures, there was a direct correlation between number of Sl/Sld cells added and degree of inhibition of +/+ hemopoiesis. A pre- established, confluent Sl/Sld adherent stromal layer inhibited hemopoiesis by fresh +/+ marrow cells by nearly 70%, as compared with dishes with irradiated +/+ or no stroma. This inhibitory effect was abrogated by irradiation of the Sl/Sld stroma prior to addition of the fresh +/+ marrow cells. Similarly, unirradiated, but not 9 to 200 Gy irradiated Sl/Sld stroma inhibited proliferation of the factor- dependent FDC-P1 hemopoietic progenitor cell line. Thus, the Sl/Sld hemopoietic microenvironment actively inhibits hemopoiesis in vitro, and this inhibition can be at least partially eliminated by irradiation of the Sl/Sld stroma.     

Marrow transplantation in the treatment of a murine heritable hemolytic anemia

Mice with hemolytic anemia, sphha/sphha, have extremely fragile RBCs with a lifespan of approximately one day. Neither splenectomy nor simple transplantation of normal marrow after lethal irradiation cures the anemia but instead causes rapid deterioration and death of the mutant unless additional prophylactic procedures are used. In this report, we show that normal marrow transplantation preceded by sublethal irradiation increases but does not normalize RBC count. The mutant RBCs but not all the WBCs are replaced by donor cells. Splenectomy of the improved recipient causes a dramatic decrease in RBC count, indicating that the mutant spleen is a site of donor-origin erythropoiesis as well as of RBC destruction. Injections of iron dextran did not improve RBC counts. Transplantation of primary recipient marrow cells into a secondary host with a heritable stem cell deficiency (W/Wv) corrects the defect caused by residence of the normal cells in the sphha/sphha host. The original +/+ donor cells replace the RBCs of the secondary host, and the RBC count is normalized. Results indicate that the environment in the sphha/sphha host is detrimental to normal (as well as mutant) erythroid cells but the restriction is not transmitted.     

The cause of anemia in mutant mice of Sl/Slt genotype: hypoplasia in bone marrow and restricted hyperplasia in spleen

The cause of the severe anemia in Sl/Sld mice is attributed to (1) hypoproduction of erythrocytes due to a defect in the erythropoietic microenvironment and (2) bleeding from stomach ulcers. Sl/Slt mice also showed a moderate anemia, but bleeding from stomach ulcers was excluded as a cause of the anemia, because no significant amount of radioactivity was excreted in feces after the injection of 59Fe-labeled erythrocytes. The activity of erythropoiesis in the bone marrow and spleen was compared between Sl/Slt and congenic +/+ mice using three different criteria: the number of erythroblasts, 59Fe incorporation, and the number of erythropoietic precursor cells. All three parameters in the femur were lower, and those in the spleen were higher in Sl/Slt mice than in +/+ mice, suggesting that the low erythropoietic potential in the bone marrow of Sl/Slt mice is partially compensated by the spleen. In fact, splenectomy aggravated the anemia of Sl/Slt mice. The enhanced erythropoiesis in Sl/Slt spleens may explain our previous finding that numbers and sizes of spleen colonies were normal when bone marrow cells were injected into irradiated Sl/Slt mice. Sl/Slt mice may be a useful model for studying biological characteristics of the hematopoietic microenvironment.     

Anemia and mast cell depletion in mutant rats that are homozygous at "white spotting (Ws)" locus

Mice possessing two mutant alleles at the W or Sl locus are anemic and deficient in mast cells. These mouse mutants have black eyes and white hair. Because homozygous mutant rats at the newly found white spotting (Ws) locus were also black-eyed whites, the numbers of erythrocytes and mast cells were examined. Suckling Ws/Ws rats showed a severe macrocytic anemia and were deficient in mast cells. When bone marrow cells of normal (+/+) control or Ws/Ws rats were injected into C3H/He mice that had received cyclophosphamide injection and whole-body irradiation, remarkable erythropoiesis occurred in the spleen of +/+ marrow recipients but not in the spleen of Ws/Ws marrow recipients. When skin pieces of Ws/Ws embryos were grafted under the kidney capsule of nude athymic rats, mast cells did develop in the grafted skin tissues. Therefore, the anemia and mast cell deficiency of Ws/Ws rats were attributed to a defect of precursors of erythrocytes and mast cells. Because the magnitude of the anemia decreased and that of the mast cell deficiency increased in adult Ws/Ws rats, this mutant is potentially useful for investigations about differentiation and function of mast cells.     

Experimental hypoplastic marrow failure in the mouse.

In order to study the pathogenesis of aplastic anemia in man, hemopoietic stem cells were investigated in 'aplastic mice' the aplasia being induced by the immunological method. C3H/He (H-2k, Mlsc) received 600 rad whole body x-irradiation followed by the transplantation of 10(7) lymph node cells prepared from B10.BR mic e (H-2k, Mlsb). The C3H/He mice developed pancytopenia and marrow hypoplasia 21 days after these treatments. The total number of nucleated cells, CFU-S and CFU-C in the marrow and the wet weight and CFU-C of the spleen were markedly reduced. These findings are consistent with those of aplastic anemia in man and the model may provide a useful tool for the investigation of the pathogenesis of this anemia. Control mice that received irradiation only recovered from the damage 21 days later, while control mice that receive lymph node cells only showed no hematological changes.     

Hematopoietic repopulation of adult mice with beta-thalassemia

Deletion of the murine beta-major globin gene on chromosome 7 causes a severe, hypochromic anemia in homozygous mice. We show that over 50% of the homozygous mice die either in utero or at birth. Mice heterozygous for the deletion have a slightly increased percentage of reticulocytes when compared with normal mice, but no clinical anemia. As a therapeutic measure, we transplanted 2 x 10(6) congenic genetically marked normal (+/+) marrow cells into adult homozygous and control heterozygous mice. Pretreatment with marrow ablative irradiation was required to obtain significant percentages of donor peripheral blood cells in the homozygous mice. Red blood cell (RBC) counts normalized after pretransplantation irradiation of thalassemic mice with nonlethal doses as low as 400 R. The thalassemic mice irradiated with 200, 400, and 600 R were erythroid-cell chimeras and remained so for at least 8 months posttransplantation, whereas those irradiated with 800 R had primarily donor erythrocytes by 8 weeks. RBC replacement preceded non- erythroid cell replacement at 200, 400, and 600 R. This selective repopulation was more noticeable in the thalassemic mice than in control mice. The fact that chimeric mice are cured, coupled with a recent observation by others that erythroid replacement occurs in unirradiated newborn thalassemic mice, suggest transplantation therapy in utero might augment survival.     

Establishment of permanent chimerism in a lactate dehydrogenase-deficient mouse mutant with hemolytic anemia

Pluripotent hemopoietic stem cell function was investigated in the homozygous muscle type lactate dehydrogenase (LDH-A) mutant mouse using bone marrow transplantation experiments. Hemopoietic tissues of LDH-A mutants showed a marked decreased in enzyme activity that was associated with severe hemolytic anemia. This condition proved to be transplantable into wild type mice (+/+) through total body irradiation (TBI) at a lethal dose of 8.0 Gy followed by engraftment of mutant bone marrow cells. Since the mutants are extremely radiosensitive (lethal dose50/30 4.4 Gy vs 7.3 Gy in +/+ mice), 8.0-Gy TBI followed by injection of even high numbers of normal bone marrow cells did not prevent death within 5-6 days. After a nonlethal dose of 4.0 Gy and grafting of normal bone marrow cells, a transient chimerism showing peripheral blood characteristics of the wild type was produced that returned to the mutant condition within 12 weeks. The transfusion of wild type red blood cells prior to and following 8.0-Gy TBI and reconstitution with wild type bone marrow cells prevented the early death of the mutants and permanent chimerism was achieved. The chimeras showed all hematological parameters of wild type mice, and radiosensitivity returned to normal. It is concluded that the mutant pluripotent stem cells are functionally comparable to normal stem cells, emphasizing the significance of this mouse model for studies of stem cell regulation.     

Treatment of autoimmune diseases in mice by a new method for allogeneic bone marrow transplantation

Remarkable advances have been made in bone marrow transplantation (BMT), which has become a powerful strategy for the treatment of leukemia, aplastic anemia, congenital immunodeficiency, and also autoimmune disease. Using various animal models, allogeneic (allo) BMT has been found to be useful in the treatment of various autoimmune diseases. In MRL/lpr mice, which are radiosensitive (<8.5 Gy) and are an animal model for autoimmune diseases, conventional BMT resulted in only transient effects; the manifestations of the autoimmune diseases recurred 3 months after BMT. Using MRL/lpr mice, we have very recently established a new strategy for allo BMT. We injected bone marrow cells (BMC) directly into the bone marrow cavity (intrabone marrow [IBM] injection) of recipients that had received fractionated irradiation. This 'IBM-BMT' was found to be effective in treating autoimmune diseases in radiation-sensitive and chimeric-resistant MRL/lpr mice. In addition, this strategy was found to be applicable for the transplantation of organs. We believe that these strategies for BMT and organ transplantation herald a new era in transplantation.     

Hematopoietic Cells From alpha -Spectrin-Deficient Mice Are Sufficient to Induce Thrombotic Events in Hematopoietically Ablated Recipients

Thrombotic events are life-threatening complications of humanhemolytic anemias such as paroxysmal nocturnal hemoglobinuria, sicklecell disease, and thalassemia. It is not clear whether these events aresolely influenced by aberrant hematopoietic cells or also involveaberrant nonhematopoietic cells. Spherocytosis mutant(Spna1^sph/Spna1^sph; for simplicityreferred to as sph/sph) mice develop a severe hemolytic anemiapostnatally due to deficiencies in -spectrin in erythroid and otheras yet incompletely defined nonerythroid tissues. Thrombotic lesionsoccur in all adult sph/sph mice, thus providing ahematopoietically stressed model in which to assess putative causes ofthrombus formation. To determine whether hematopoietic cells fromsph/sph mice are sufficient to initiate thrombi, bone marrowfrom sph/sph or +/+ mice was transplanted into mice with nohemolytic anemia. One set of recipients was lethally irradiated; theother set was genetically stem cell deficient. All mice implanted withsph/sph marrow, but not +/+ marrow, developed severe anemia and histopathology typical of sph/sph mice. Histologicalanalyses of marrow recipients showed that thrombi were present in therecipients of sph/sph marrow, but not +/+ marrow. Theresults indicate that the -spectrin-deficient hematopoietic cellsof sph/sph mice are the primary causative agents of thethrombotic events.     

Extensive proliferation of subsequently injected marrow cells in parental-to-F1 hematopoietic chimeras that restored normal stem cell concentration after initial transplantation

We investigated the fate of donor stem cells that were injected into hosts with a normal concentration of spleen colony-forming unit (CFU-S). Radiation chimeras were used as hosts. When CFU-S concentration in the marrow and spleen recovered to preirradiation levels after the initial bone marrow transplantation, the subsequent transplantation was done without reirradiation. Giant granules of beige C57B1/6 (bg) mice were used as a marker and proliferation and differentiation of the stem cells of the subsequent donor origin were evaluated by measuring the proportion of neutrophils with giant granules. No beige-type neutrophils were detectable at week 24 after transplantation of 5 X 10(7) marrow cells from bg mice to intact (WB X C57B1/6)F1 (F1) mice, which were used as control recipients. In contrast, transplantation of 5 X 10(7) marrow cells to radiation chimeras resulted in the appearance of neutrophils of second-donor origin. The proportion of beige-type neutrophils was 12% at week 24 after transplantation of bg marrow cells to F1-to-F1 (syngenic) or C57B1/6-+/+-to-bg (B6-to-bg) (congenic) chimeras; the proportion of beige-type neutrophils was 43% when bg marrow cells were transplanted to B6-to-F1 semiallogenic chimeras; the proportion of normal-type neutrophils was 82% when F1 marrow cells were injected to bg-to-F1 semiallogenic chimeras. Thus, the interaction of the host hematopoietic microenvironment with the stem cells of the initial donor as well as with the stem cells of the second donor seems to influence the proliferation and differentiation of the latter stem cells.     

Increased longevity of hematopoiesis in continuous bone marrow cultures derived from NOS1 (nNOS, mtNOS) homozygous recombinant negative mice correlates with radioresistance of hematopoietic and marrow stromal cells

OBJECTIVE: Neuronal nitric oxide synthase (NOS1, mitochondrial NOS, neuronal NOS) homozygous deletion recombinant negative mice demonstrate ionizing irradiation resistance in vivo, attributable to the decrease in mitochondrial-localized production of peroxynitrite, a potent lipid toxic free radical species resulting from the combination of nitric oxide and superoxide. The present studies were designed to determine whether reduced mitochondrial generation of toxic radical oxygen species in NOS1-/- mice also increased the longevity of hematopoiesis in continuous bone marrow cultures and conferred radioresistance to cells in vitro. MATERIALS AND METHODS: Long-term bone marrow cultures (LTBMCs) were established from NOS1-/- and NOS1+/+ littermate mice. Radiation resistance of hematopoietic and marrow stromal cells was measured. Cell cycle analysis and measurement of glutathione and glutathione peroxidase were carried out on irradiated clonal bone marrow stromal cell lines. RESULTS: A significant increase in longevity of hematopoiesis was detected in NOS1-/- mouse LTBMCs for over 64 weeks in culture compared to 20 weeks for NOS1+/+ mouse LTBMCs (p < 0.001). Permanent bone marrow stromal cell lines derived from NOS1-/- mouse LTBMCs demonstrated increased radioresistance in vitro reflected by an increased shoulder on the survival curve with n = 32.15 +/- 1.21 compared to NOS1+/+ cells n = 10.47 +/- 3.2 (p = 0.0026), interleukin-3-dependent NOS1-/- hematopoietic progenitor cell lines also demonstrated decreased apoptosis after 10 Gy irradiation. Both pre- and postirradiation stabilization of the cellular antioxidant pool was detected in NOS1-/- cells. NOS1-/- cells showed a prolonged G1 cell cycle arrest after 10 Gy. CONCLUSIONS: Prolonged hematopoiesis in LTBMCs correlates with intrinsic radioresistance of hematopoietic and marrow stromal cells from NOS1-/- mice. The data confirm the importance to hematopoiesis of mitochondrial localized nitric oxide in both radioresistance and longevity of hematopoiesis in continuous bone marrow cultures.     

Transient engraftment of syngeneic bone marrow after conditioning with high-dose cyclophosphamide and thoracoabdominal irradiation in a patient with aplastic anemia

We describe the clinical course of a 16 year old girl with aplastic anemia who was treated by syngeneic bone marrow transplantation. Engraftment was not obtained by simple infusion of bone marrow without immunosuppression. The patient received a high-dose cyclophosphamide and thoracoabdominal irradiation, followed by second marrow transplantation from the same donor. Incomplete but significant hematologic recovery was observed; however, marrow failure recurred 5 months after transplantation. Since donor and recipient pairs were genotypically identical, graft failure could not be attributed to immunological reactivity of recipient cells to donor non-HLA antigens. This case report implies that graft failure in some cases of aplastic anemia might be mediated by inhibitory cells resistant to cyclophosphamide and irradiation.     

Erythropoietin stimulation decreases hepcidin expression through hematopoietic activity on bone marrow cells in mice

Erythropoiesis-stimulating agents (ESA) are now central to the treatment of renal anemia and are associated with improved clinical outcomes. It is well known that erythropoietin (EPO) is a key regulator of erythropoiesis through its promotion of red blood cell production. In order to investigate the role of ESA on iron metabolism, we analyzed the regulation of the iron regulatory hormone hepcidin by ESA treatment in a bone marrow transplant model in mouse. After treating C57BL/6 mice with continuous erythropoietin receptor activator (C.E.R.A.), recombinant human epoetin-β (rhEPO), or recombinant human carbamylated epoetin-β (rhCEPO), we investigated serum hepcidin concentrations and parameters of erythropoiesis. Serum hepcidin concentrations after rhEPO treatment were analyzed in mice subjected to total body irradiation followed by bone marrow transplantation. C.E.R.A. administration caused long-term downregulation of serum hepcidin levels. Serum hepcidin levels in rhEPO-treated mice decreased significantly, whereas there was no change in rhCEPO-treated mice. The reduction in circulating hepcidin levels after rhEPO administration was not observed in irradiated mice. Finally, bone marrow transplantation recovered the response to rhEPO administration that downregulates hepcidin concentration in irradiated mice. These results indicate that ESA treatment downregulates serum hepcidin concentrations, mainly by indirect mechanisms affecting hematopoietic activity in bone marrow cells.     

Mechanism of mast cell deficiency in mutant mice of mi/mi genotype: an analysis by co-culture of mast cells and fibroblasts

Mutant mice of mi/mi genotype are osteopetrotic and are deficient in mast cells. The osteopetrosis of mi/mi mice can be cured by bone marrow transplantation from congenic normal (+/+) mice, and therefore, the cause of the osteopetrosis is attributed to a defect of osteoclasts. Since both osteoclasts and mast cells are the progeny of multipotential hematopoietic stem cells, we examined whether mast cells were defective in mi/mi mice. In spite of the deficiency of mast cells in tissues of mi/mi mice, mast cells did develop when spleen cells of mi/mi mice were cultured with pokeweed mitogen-stimulated spleen cell conditioned medium (PWM-SCM). The proliferative response of cultured mast cells (CMC) derived from mi/mi mice to PWM-SCM was comparable with that of CMC from +/+ mice. In contrast, when CMC were co-cultured with the NIH/3T3 fibroblast cell line in culture medium lacking PWM-SCM, only +/+ CMC entered into the S phase of the cell cycle and were maintained; mi/mi CMC gradually disappeared. Moreover, fibroblasts derived from the skin of mi/mi mice normally supported the proliferation of +/+ CMC. Thus, the mast cell deficiency of mi/mi mice appears to be due to the inability of mi/mi mast cells to respond to the proliferative stimulus presented by fibroblasts.     

Decrease of mast cells in W/Wv mice and their increase by bone marrow transplantation.

Production of tissue mast cells was evaluated in genetically anemic mice of W/Wv genotype and was found to be abnormal. In the skin of adult W/Wv mice the number of mast cells/cm was less than 1% of the number observed in the congeneic +/+ mice. No mast cells were detectable in other tissues of the W/Wv mice. After transplantation of bone marrow cells from +/+ mice the number of mast cells in the skin, stomach, caecum, and mesentery of the W/Wv mice increased to levels similar to those of the +/+ mice. These results show that the W/Wv mouse is a useful tool for the investigations concerning the physiologic roles and the origin of mast cells.     

Defective protective capacity of W/Wv mice against Strongyloides vatti infection and its reconstitution with bone marrow cells

The susceptibility of congenitally anemic, and mast cell deficient W/Wv mice to infection with Strongyloides ratti was examined. After a primary infection, W/Wv mice showed greater and more persistent peak larval counts than did normal littermates. Worm expulsion was also slower in W/Wv mice than in +/+ mice. Furthermore, difference in susceptibility was expressed as early as 24 h after infection, suggesting not only that protective mechanisms of the gut but also of the connective tissue were defective in W/Wv mice. Reconstitution with bone marrow or spleen cells from +/+ mice was effective in restoring the protective response in W/Wv mice, whereas thymocytes or mesenteric lymph nodes had no effect. Both connective tissue and mucosal mast cells were repaired in W/Wv mice after marrow reconstitution and infection. Since relatively long incubation period was required for the expression of such reconstituting activities, bone marrow cells seem to contain precursor cells of the effector and/or regulator cells.     

Hematopoietic reconstitution after lethal irradiation and bone marrow transplantation: effects of different hematopoietic cytokines on the recovery of thymus, spleen and blood cells

Lethally irradiated mice were grafted with syngeneic bone marrow cells or left ungrafted. Mice of each group were injected with different hematopoietic cytokines for 5 consecutive days starting immediately after irradiation or left uninjected. The recovery of lymphoid tissues induced by hematopoietic cytokines 7 days after irradiation and bone marrow cell transplantation was comparable to that observed at days 21-28 in irradiated, bone marrow-grafted, but cytokine-uninjected mice. IL-11 or IL-6, in combination with IL-3, was able to hasten thymus, spleen and blood cell numbers and functions. SCF also displayed a detectable effect when used with IL-3. Conversely, the IL-6 superagonist K-7/D-6 was able, when injected alone, to induce significant recovery of thymus, spleen and blood cells. Thus, K-7/D-6 appears to be a most efficient cytokine for fast reconstitution of lymphoid tissues after irradiation and bone marrow transplantation.     

In vivo selection of wild-type hematopoietic stem cells in a murine model of Fanconi anemia.

Fanconi anemia (FA) is an autosomal recessive disorder characterized by birth defects, increased incidence of malignancy, and progressive bone marrow failure. Bone marrow transplantation is therapeutic and, therefore, FA is a candidate disease for hematopoietic gene therapy. The frequent finding of somatic mosaicism in blood of FA patients has raised the question of whether wild-type bone marrow may have a selective growth advantage. To test this hypothesis, a cohort radio-ablated wild-type mice were transplanted with a 1:1 mixture of FA group C knockout (FACKO) and wild-type bone marrow. Analysis of peripheral blood at 1 month posttransplantation showed only a moderate advantage for wild-type cells, but upon serial transplantation, clear selection was observed. Next, a cohort of FACKO mice received a transplant of wild-type marrow cells without prior radio-ablation. No wild-type cells were detected in peripheral blood after transplantation, but a single injection of mitomycin C (MMC) resulted in an increase to greater than 25% of wild-type DNA. Serial transplantation showed that the selection occurred at the level of hematopoietic stem cells. No systemic side effects were observed. Our results show that in vivo selection for wild-type hematopoietic stem cells occurs in FA and that it is enhanced by MMC administration.