The effect of low dose rate on recovery of hemopoietic and stromal progenitor cells in gamma-irradiated mouse bone marrow

Long-term recovery of mouse hemopoietic stem cells (CFU-S and CFU-S per colony), granulocyte-macrophage precursor cells (GM-CFC), and stromal colony-forming units (CFU-F) after doses up to 12.5 Gy was almost complete by 1 year when the dose rate was reduced to 0.0005 Gy/min compared to incomplete recovery after doses up to only 6.5 Gy given at greater than 0.7 Gy/min. This sparing effect of dose rate on long-term hemopoietic recovery is in contrast to the generally reported lack of dependence on dose rate for acute survival of hemopoietic progenitors after doses up to 5 Gy. The present results are compatible with the hypothesis that good recovery of the stroma should be reflected in the long-term recovery of hemopoiesis.     

The effects of graded doses of 1 MeV fission neutrons or X rays on the murine hematopoietic stroma.

The acute radiosensitivity in vivo of the murine hematopoietic stroma for 1 MeV fission neutrons or 300 kVp X rays was determined. Two different assays were used: (1) an in vitro clonogenic assay for fibroblast precursor cells (CFU-F) and (2) subcutaneous grafting of femora or spleens. The number of stem cells (CFU-S) or precursor cells (CFU-C), which repopulated the subcutaneous implants, was used to measure the ability of the stroma to support hemopoiesis. The CFU-F were the most radiosensitive, and the survival curves after neutron and X irradiation were characterized by D0 values of 0.75 and 2.45 Gy, respectively. For regeneration of CFU-S and CFU-C in subcutaneously implanted femora, D0 values of 0.92 and 0.84 Gy after neutron irradiation and 2.78 and 2.61 Gy after X irradiation were found. The regeneration of CFU-S and CFU-C in subcutaneously implanted spleens was highly radioresistant as evidenced by D0 values of 2.29 and 1.49 Gy for survival curves obtained after neutron irradiation, and D0 values of 6.34 and 4.85 Gy after X irradiation. The fission-neutron RBE for all the cell populations was close to 3 and varied from 2.77 to 3.28. The higher RBE values observed for stromal cells, compared to the RBE of 2.1 reported previously for hemopoietic stem cells, indicate that stromal cells are relatively more sensitive than hemopoietic cells to neutron irradiation.     

骨髓基质细胞的辐射效应及其临床意义

小鼠骨髓基质细胞团在γ线照射后的Do值为2.40Gy,但其成灶能力损伤后持续时间较久。正常骨髓基质细胞能促进骨髓GM-CFU-C的生长;照射10-80Gy后的骨髓基质细胞失去这种促进作用。文中讨论了骨髓基质细胞的辐射效应及其临床意义,提出了谨慎选择放射治疗剂量的必要性。     

Effect of Myleran On Murine Hemopoiesis

Time- and dose-dependent patterns of depletion and regeneration of hemopoietic progenitor cells in mouse femora and spleens following treatment with the antileukemic agent Myleran (Busulphan, MY) were studied using the murine spleen colony system and the agar gel in vitro colony system. MY was found to depress granulopoiesis selectively, as manifested by the development of marked prolonged neutropenia, hypoplasia of the bone marrow and (to a lesser degree) of the spleen, reduction of the incidence of multipotential hemopoietic progenitor cells (CFU-S) and of granulocytic progenitor cells (CFU-C) in both femora and spleens, and impairment of the capacity of CFU-S from either tissue to generate granulocytic colonies in the spleens of irradiated hosts. the severity and duration was greatest at high dose levels of MY (800 μ). the action of MY on CFU-S was more pronounced than that on CFU-C, suggesting that MY is a cycle-independent agent. Repopulation of the CFU-C pool preceded that of the CFU-S pool. Development of neutropenia and maximal marrow hypoplasia followed the onset of depression of CFU-S and CFU-C incidence, while recovery of normal nucleated cellularity in the blood, femur and spleen preceded repopulation of the CFU-S and CFU-C pools. MY treatment resulted in transitory stimulation of colony stimulating factor (CSF) generation by the femur but had no effect on serum CSF levels. the peak of femoral CSF generation coincided with the nadir of CFU-C depression. These findings indicated that the prolonged neutropenia following MY treatment was secondary to depletion of the progenitor cell pools, that during recovery granulopoietic repopulation took precedence over self-maintenance of the hemopoietic progenitor cell pools, and that increased generation of CSF may play a role in the early phase of granulopoietic recovery.     

Effect of myleran on murine hemopoiesis. I. Granulocytic cell line specificity of action on progenitor cells.

Time- and dose-dependent patterns of depletion and regeneration of hemopoietic progenitor cells in mouse femora and spleens following treatment with the antileukemic agent Myleran (Busulphan, MY) were studied using the murine spleen colony system and the agar gel in vitro colony system. MY was found to depress granulopoiesis selectively, as manifested by the development of marked prolonged neutropenia, hypoplasia of the bone marrow and (to a lesser degree) of the spleen, reduction of the incidence of multipotential hemopoietic progenitor cells (CFU-S) and of granulocytic progenitor cells (CFU-C) in both femora and spleens, and impairment of the capacity of CFU-S from either tissue to generate granulocytic colonies in the spleens of irradiated hosts. The severity and duration was greatest at high dose levels of MY (800 microgram). The action of MY on CFU-S was more pronounced than that on CFU-C, suggesting that MY is a cycle-independent agent. Repopulation of the CFU-C pool preceded that of the CFU-S pool. Development of neutropenia and maximal marrow hypoplasia followed the onset of depression of CFU-S and CFU-C incidence, while recovery of normal nucleated cellularity in the blood, femur and spleen preceded repopulation of the CFU-S and CFU-C pools. MY treatment resulted in transitory stimulation of colony stimulating factor (CSF) generation by the femur but had no effect on serum CSF levels. The peak of femoral CSF generation coincided with the nadir of CFU-C depression. These findings indicated that the prolonged neutropenia following MY treatment was secondary to depletion of the progenitor cell pools, that during recovery granulopoietic repopulation took precedence over self-maintenance of the hemopoietic progenitor cell pools, and that increased generation of CSF may play a role in the early phase of granulopoietic recovery.     

Radioprotection of mice with interleukin-1: relationship to the number of spleen colony-forming units

Compared to saline-injected mice 9 days after 6.5 Gy irradiation, there were twofold more Day 8 spleen colony-forming units (CFU-S) per femur and per spleen from B6D2F1 mice administered a radioprotective dose of human recombinant interleukin-1-alpha (rIL-1) 20 h prior to their irradiation. Studies in the present report compared the numbers of CFU-S in nonirradiated mice 20 h after saline or rIL-1 injection. Prior to irradiation, the number of Day 8 CFU-S was not significantly different in the bone marrow or spleens from saline-injected mice and rIL-1-injected mice. Also, in the bone marrow, the number of Day 12 CFU-S was similar for both groups of mice. Similar seeding efficiencies for CFU-S and percentage of CFU-S in S phase of the cell cycle provided further evidence that rIL-1 injection did not increase the number of CFU-S prior to irradiation. In a marrow repopulation assay, cellularity as well as the number of erythroid colony-forming units, erythroid burst-forming units, and granulocyte-macrophage colony-forming cells per femur of lethally irradiated mice were not increased in recipient mice of donor cells from rIL-1-injected mice. These results demonstrated that a twofold increase in the number of CFU-S at the time of irradiation was not necessary for the earlier recovery of CFU-S observed in mice irradiated with sublethal doses of radiation 20 h after rIL-1 injection.     

Production of humoral factors that stimulate spleen colony-forming units in mice irradiated with moderate doses of X rays

The production of humoral factors that stimulate spleen colony-forming units (CFU-S) has been studied in irradiated mice using an in vivo diffusion chamber assay. The experiments show that a significant release of factors that stimulate CFU-S takes place in the first few days after irradiation with moderate doses of 1.5 or 5 Gy. In contrast, the release of significant amounts of these humoral factors was not seen in animals irradiated with either low (0.75 Gy) or high (10 Gy) doses of X rays. The correlation observed between the production of factors that stimulate the CFU-S and the hemopoietic regeneration kinetics of the irradiated mice suggests that these factors represent part of the physiological regulators controlling the proliferation of CFU-S.     

Mechanisms of lymphocytic choriomeningitis virus-induced hemopoietic dysfunction.

Results of this study showed that lymphocytic choriomeningitis virus infection causes a marked activation of natural killer (NK) cells not only in the spleen but also in the bone marrow. This activity reached its peak at about day 3 of infection and declined after days 6 to 7. Enhanced NK cell activity was found to correlate with decreased receptivity for syngeneic stem cells in bone marrow and spleen, with the notable exception that decreased receptivity persisted longer in bone marrow. Treatment of infected recipients with anti-asialo GM1 (ganglio-N-tetraosylceramide) significantly increased the receptivity for syngeneic hemopoietic cells. These findings are consistent with the hypothesis that NK cell activation causes rejection of syngeneic stem cells, thus resulting in hemopoietic depression. To understand the mechanisms behind the prolonged decrease in bone marrow receptivity (and bone marrow function in the intact mouse) mentioned above, we followed the changes in the number of pluripotential stem cells (CFU-S) circulating in the peripheral blood and in endogenous spleen colonies in irradiated mice, the limbs of which were partially shielded. It was found that following a marked early decline, both parameters increased to normal or supranormal levels at about day 9 after infection. Because the bone marrow pool of CFU-S is only about 20% of normal at this time after infection, a marked tendency for CFU-S at this stage in the infection to migrate from the bone marrow to the spleen is suggested. It seems, therefore, that as NK cell activity declines, the spleen regains the ability to support growth of hemopoietic cells and the bone marrow resumes an elevated export of stem cells to the spleen. This diversion of hemopoiesis could explain both the long-standing deficiencies of the bone marrow compartment and the prolonged decrease in the receptivity of this organ.     

Alkylating Damage by Dipin of Hematopoietic and Stromal Cells of the Bone Marrow

Effect of alkylating agent dipin was studied on hematopoietic (CFU-S) and stromal (CFU-F) progenitor cells. Single administration of dipin (0.06 mg/g) to adult (CBA × C57Bl/6) F1 hybrid mice induced a long-term (2 years) oscillations in the numbers of day 7 CFU-S and day 11 CFU-S in the bone marrow and spleen. Dipin also damaged the hematopoietic stroma as indicated by decreased numbers of CFU-F which remained low for at least a year. The capacity of stromal cells to form ectopic hematopoietic foci was considerably decreased and also remained low for 10 months. The obtained data suggest high dipin sensitivity of the earliest hematopoietic and stromal cells. The dynamics of CFU-S numbers in the hematopoietic organs supports their functioning on the basis of clonal succession (Kay, 1965).__________Translated from Izvestiya Akademii Nauk, Seriya Biologicheskaya, No. 3, 2005, pp. 267–272.Original Russian Text Copyright © 2005 by Domaratskaya, Bueverova, Payushina, Starostin.     

Early and late effects in the bone marrow of mice following 2 Gy (6 MeV) neutron irradiation

Summary Following 5 Gy gamma irradiation, residual damage in bone marrow persisted up to one year and was ascribed to genetic defects in hemopoietic stem cells (von Wangenheim et al. 1986). To see whether high LET radiation is more efficient in inducing late effects, mice were whole-body irradiated with a single dose of 2 Gy neutrons ( = 6 MeV) and femoral cellularity, CFU-S number, proliferation ability of bone marrow cells (PF) and the compartment ratio (CR), i.e. the splenic 125-iodo-deoxyuridine incorporation per transfused CFU-S were measured up to one year after the radiation insult. Within 12 weeks, femoral cellularity, PF and CR recovered to control or near-control level, whereas CFU-S numbers remained significantly below control. No further recovery was observed. On the contrary, PF and CR deteriorated again after 12 and 26 weeks, respectively. CFU-S per femur tended to decrease as well. Thus it is demonstrated that a single dose of 2 Gy 6 MeV neutrons causes significant injury in function (PF) and structure (CFU-S numbers, CR) of bone marrow which persisted up to one year. While this residual injury can be attributed to genetic defects in hemopoietic stem cells, its increasing expression is probably due to late evolving damage in microenvironmental cells. The RBE of 6 MeV neutrons for the introduction of late effects in the bone marrow is in the range of 3.     

Role of splenic stroma in the action of bacterial lipopolysaccharides on radiation mortality: a study in mice carrying the Slj allele

Slj/+ mice display a slight macrocytic anaemia due to a defect in their haemopoietic organ stroma. They have a deficient endogenous spleen colony (CFU-end) formation following sublethal doses of gamma-radiation compared with their normal +/+ littermates, which is likely to be due to the low pre-irradiation CFU-S content of the Slj/+ spleen. CFU-S in these congenic mice do not differ in their sensitivity to gamma-irradiation or stem cell-activating factor. While injection of +/+ mice with 10 micrograms of lipopolysaccharide-W (LPS) one day prior to irradiation led to a substantial increase in their survival, the survival of Slj/+ mice was only slightly increased. Irradiation induced a similar dose-related reduction in the numbers of CFU-S in the spleen and femora of LPS-injected Slj/+ mice compared to similarly treated +/+ mice when measured directly after irradiation. At Day 9 after irradiation, injection of LPS led to a significantly higher CFU-end formation and higher numbers of CFU-S and nucleated cells in the Slj/+ spleens compared to LPS-injected +/+ mice. No such differences in the radioprotective effect of LPS were observed in the +/+ and Slj/+ mice with respect to the splenic and femoral 59Fe-incorporation and the femoral CFU-S numbers at Day 9. These data strongly suggest a contribution by immigrating CFU-S to the CFU-S numbers and endogenous colony formation in at least the Slj/+ spleen after LPS injection and subsequent sublethal irradiation. The observations also imply that the splenic organ stroma may play a mediatory role in the radioprotective action of LPS. In addition, the data represent an extreme example of a lack of correlation between animal survival and haemopoietic parameters. Caution should be taken when applying endogenous colony counts as a means of screening potential anti-radiation drugs.     

THE EFFECT OF FETUIN AND OF SPLEEN EXTRACTS ON HEMATOPOIETIC PRECURSORS AND ON DIFFERENTIATED HEMATOPOIETIC CELLS IN IRRADIATED MICE

Injection of extracts from normal mouse spleen tissue into irradiated mice enhance the rate of regeneration of colony forming units (CFU-S) in the femoral marrow. This effect was most pronounced when spleen extract was injected between 24 hr before and 24 hr after the time of irradiation, and was observed only during the first week after a single injection of extract. Another result of injecting spleen extract was an immediate and transient decrease in the marrow cellularity and particularly in the number of mature myeloid cells in the marrow. Fetuin produced comparable effects on the rate of regeneration of CFU-S and on the numbers of mature myeloid cells in the marrow. On the basis of these results it is tempting to speculate that injection of spleen extracts and of fetuin primarily cause a rapid depletion of the marrow's granulocyte reserve. This in turn releases the precursor cell compartment from the inhibitory effects of cell–cell interaction and results in an acceleration of the rate of CFU-S regeneration. It is equally plausible that factors present in spleen extract and in fetuin cause a depletion of the marrow granulocyte reserve and, by an unrelated mechanism, directly accelerate the rate of regeneration of CFU-S.     

Role of splenic stroma in the action of bacterial lipopolysaccharides on radiation mortality: a study in mice carrying the Slj allele

Abstract. Sl_j/+ mice display a slight macrocytic anaemia due to a defect in their haemopoietic organ stroma. They have a deficient endogenous spleen colony (CFU-end) formation following sublethal doses of gamma-radiation compared with their normal +/+ littermates, which is likely to be due to the low pre-irradiation CFU-S content of the Sl_j/+ spleen. CFU-S in these congenic mice do not differ in their sensitivity to gamma-irradiation or stem cell-activating factor. While injection of +/+ mice with 10 μg of lipopolysaccharide-W (LPS) one day prior to irradiation led to a substantial increase in their survival, the survival of Sl_j/+ mice was only slightly increased. Irradiation induced a similar dose-related reduction in the numbers of CFU-S in the spleen and femora of LPS-injected Sl_j/+ mice compared to similarly treated +/+ mice when measured directly after irradiation. At Day 9 after irradiation, injection of LPS led to a significantly higher CFU-end formation and higher numbers of CFU-S and nucleated cells in the Sl_j/+ spleens compared to LPS-injected +/+ mice. No such differences in the radioprotective effect of LPS were observed in the +/+ and Sl_j/+ mice with respect to the splenic and femoral ⁵⁹Fe-incorporation and the femoral CFU-S numbers at Day 9. These data strongly suggest a contribution by immigrating CFU-S to the CFU-S numbers and endogenous colony formation in at least the Sl_j/+ spleen after LPS injection and subsequent sublethal irradiation. The observations also imply that the splenic organ stroma may play a mediatory role in the radioprotective action of LPS. In addition, the data represent an extreme example of a lack of correlation between animal survival and haemopoietic parameters. Caution should be taken when applying endogenous colony counts as a means of screening potential anti-radiation drugs.     

Colony formation in agar by adult bone marrow multipotential hemopoietic cells

Erythroid colony formation in agar cultures of CBA bone marrow cells was stimulated by the addition of pokeweed mitogen-stimulated spleen conditioned medium (SCM). Optimal colony numbers were obtained when cultures contained 20% fetal calf serum and concentrated spleen conditioned medium. By 7 days of incubation, large burst or unicentric erythroid colonies occurred at a maximum frequency of 40–50 per 10⁵ bone marrow cells. In CBA mice the cells forming erythroid colonies were also present in the spleen, peripheral blood, and within individual spleen colonies. A marked strain variation was noted with CBA mice having the highest levels of erythroid colony-forming cells. In CBA mice erythroid colony-forming cells were mainly non-cycling (12.5% reduction in colony numbers after incubation with hydroxyurea or 3H-thymidine). Erythroid colony-forming cells sedimented with a peak of 4.5 mm/hr, compared with CFU-S, which sedimented at 4.25 mm/hr. The addition of erythropoietin (up to 4 units) to cultures containing SCM did not alter the number or degree of hemoglobinisation of erythroid colonies. Analysis of the total number of erythroid colony-forming cells and CFU-S in 90 individual spleen colonies gave a correlation coefficient of r = 0.93 for these two cell types. In addition to benzidine-positive erythroid cells, up to 40% of the colonies contained, in addition, varying proportions of neutrophils, macrophages, eosinophils, and megakaryocytes. Taken together with the close correlation between the numbers of CFU-S in different adult hemopoietic tissues, including individual spleen colonies, the data indicate that the erythroid colony-forming cells expressing multiple hemopoietic differentiation are members of the hemopoietic multipotential stem cell compartment.     

The role of cells sensitive to anti-mouse brain serum in the regulation of CFU-S proliferation

CFU-S differentiation and regeneration kinetics in the spleen and femur was studied after treatment of bone marrow cells with RAMB serum. The effect of thymocytes on the rate of CFU-S regeneration was also investigated. It was found that CFU-S regeneration in the spleen was similar in RAMBS-treated and intact cell populations on days 4-14 after transplantation. On the contrary, the rate of CFU-S regeneration in the femur was slower in RAMBS-treated than in intact bone marrow cells. However, the growth rate in the femur could be restored to the normal level by the administration of freshly isolated syngeneic thymocytes to mice pre-injected with RAMBS-treated CFU-S population. The treatment of bone marrow suspension with RAMB serum did not affect the differentiation of spleen colonies. It is suggested that RAMBS eliminates cell population regulating CFU-S proliferation, without affecting its differentiation.     

The influence of IL-1 treatment on the reconstitution of the hemopoietic and immune systems after sublethal radiation

The influence of IL-1 administration on the recovery of the hemopoietic and immune systems from sublethal irradiation was assessed. Mice were irradiated (750 R) and injected twice daily with purified recombinant derived IL-1 beta (200 ng/injection). At various times after irradiation, the functional capacity of the hemopoietic and immune systems was determined. It was found that IL-1 therapy resulted in a significantly greater number of granulocyte-macrophage-CSF responsive colony-forming cells in the bone marrow of the irradiated mice on days 5 and 11 postirradiation but not at later times. In addition the radiation induced neutropenia recovered quicker in the IL-1-treated mice with significantly greater numbers of peripheral blood granulocytes being seen on days 15 and 20 after irradiation. The influence of IL-1 therapy on the recovery of the immune system was also assessed. Of note was the observation that mice receiving IL-1 therapy had chronically hypoplastic thymi. Although thymic cellularity increased with time after irradiation in the control mice, there was no such increase in the IL-1-treated mice. Similarly, the number of pre-B cells in the marrow of these mice was also diminished. Thus, in the IL-1-treated mice the regeneration of the peripheral immune function was retarded, characterized by a general lymphopenia and decreased splenic responses to mitogenic stimuli.     

辐射对造血微环境损伤在放疗中的意义

本文观察了500～3000rad、局部照射后一年内骨髓中CFU-S数的变化动态,同时了解造血微环境支持造血的功能之演变过程。实验发现,500rad照射后局部骨髓中CFU-S含量明显减少,恢复不稳定,同时造血微环境支持造血的功能亦有相类似的波形起伏的损伤修复过程。1000rad局部照射的骨髓中CFU-S有更显著的降低,恢复缓慢而不稳定,造血微环境支持造血的功能早期明显受损,以后虽有修复但不能恢复到正常水平。2000rad以上的X线照射可导致局部骨髓长期再生不良,造血微环境亦见剧烈而持久的功能缺陷,这一结果表明:局部照射后,屏蔽区正常造血干细胞不能在照射部位骨髓中正常种植增殖,其原因与局部造血微环境的功能障碍密切相关。     

A comparative study of the kinetic changes of hemopoietic stem cells in mice infected with lethal and non-lethal malaria.

The kinetic changes of hemopoietic stem cells in bone marrow and spleen were compared between lethal Plasmodium berghei- and non-lethal P. yoelii 17x-infected mice. P. yoelii 17x-infected mice showed more severe splenomegaly than those infected with P. berghei. P. yoelii 17x-infected mice also showed a greater degree of sustained increase in number of multipotent hemopoietic stem cells (colony-forming units in spleen: CFU-S) and committed stem cells for granulocytes and macrophages (CFU-GM) and for erythrocytes (CFU-E) than P. berghei-infected mice. Such an increase was predominantly seen in the spleen of P. yoelii 17x-infected mice. In P. berghei-infected mice, the number of CFU-S, CFU-GM and also CFU-E only transiently increased and then decreased to a subnormal level at the late stage of infection. The proportion of cycling CFU-S was higher in P. berghei-infected mice than in P. yoelii 17x-infected mice. The IL-3 producing activity per spleen was much higher in P. yoelii 17x-infected than in P. berghei-infected mice at any point in time during the infection. Thus, hemopoietic changes seen after malaria infection seem to be closely related to the pathogenicity of the malaria parasite.     

Effects of 10-day long exposure to gamma irradiation at low doses on bone marrow cells in mice

Effects of ten day long exposure to gamma-irradiation at low doses (mean dose rate of 1.5-2.0 m Gy/day, total dose of 15 m Gy) on hemopoietic (CFU-S) and stromal (CFU-F) progenitor cells from murine bone marrow were examined. The CFU-F content measured as in vitro fibroblastic colony number showed 1.5-4.5-fold increase. Additionally, the size of ectopic marrow transplants evaluated by counting myelokariocytes and CFU-S numbers also increased. No significant changes of CFU-S proliferation rate were found.     

PROPERTIES OF HAEMOPOIETIC STEM CELLS IN PHENYLHYDRAZINE TREATED MICE

Recovery of erythropoiesis was fast in Balb/c mice irradiated 700 R 5 days after initiation of phenylhydrazine treatment and took place predominantly in the spleen, which showed numerous large frequently confluent endogenous colonies. Post irradiation phenylhydrazine induced anaemia did not accelerate recovery of erythropoiesis; it did, however, produce a slight but significant rise in endogenous colony formation.
Radiosensitivity of spleen CFU-S from phenylhydrazine treated mice was similar to that of CFU-S in normal mouse spleen.
Spleen CFU-S in mice 5 days after initiation of phenylhydrazine treatment were sensitive to the lethal action of Hydroxyurea, while bone marrow CFU-S were not.
The self-renewal capacity of CFU-S in the endogenously repopulated spleen of phenylhydrazine pretreated 700 R X-irradiated mice was low when compared to that of spleen exogenously repopulated by cells from normal mouse bone marrow, normal and phenylhydrazine treated mouse spleen. CFU circulating in blood of phenylhydrazine treated mice had a low self-renewal capacity.
The marked strain differences in self-renewal capacity of spleen CFU-S, and of the capacity of spleen CFU-S to increase by proliferation are discussed.