Increase in endogenous spleen colonies without recovery of blood cell counts in radioadaptive survival response in C57BL/6 mice

The radioadaptive survival response induced by a conditioning exposure to 0.45 Gy and measured as an increase in 30-day survival after mid-lethal X irradiation was studied in C57BL/6N mice. The acquired radioresistance appeared on day 9 after the conditioning exposure, reached a maximum on days 12-14, and disappeared on day 21. The conditioning exposure 14 days prior to the challenge exposure increased the number of endogenous spleen colonies (CFU-S) on days 12-13 after the exposure to 5 Gy. On day 12 after irradiation, the conditioning exposure also increased the number of endogenous CFU-S to about five times that seen in animals exposed to 4.25-6.75 Gy without preirradiation. The effect of the interval between the preirradiation and the challenge irradiation on the increase in endogenous CFU-S was also examined. A significant increase in endogenous CFU-S was observed when the interval was 14 days, but not 9 days. This result corresponded to the increase in survival observed on day 14 after the challenge irradiation. Radiation-inducted resistance to radiation-induced lethality in mice appears to be closely related to the marked recovery of endogenous CFU-S in the surviving hematopoietic stem cells that acquired radioresistance by preirradiation. Preirradiation enhanced the recovery of the numbers of erythrocytes, leukocytes and thrombocytes very slightly in mice exposed to a sublethal dose of 5 Gy, a dose that does not cause bone marrow death. There appears to be no correlation between the marked increase in endogenous CFU-S and the slight increase or no increase in peripheral blood cells induced by the radioadaptive response. The possible contribution by some factor, such as Il4 or Il11, that has been reported to protect irradiated animals without stimulating hematopoiesis is discussed.     

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.     

Administration of IL-7 to normal mice stimulates B-lymphopoiesis and peripheral lymphadenopathy.

Normal mice were injected with IL-7 (500 ng, twice daily) for various periods of time up to 6 days and the cellularity and phenotypic composition of the thymus, spleen, lymph node, and bone marrow was assessed. After 6 days of treatment, significant increases in the cellularity of the spleen, lymph node, and bone marrow were observed which returned to the normal range within 6 days after cessation of treatment. After 3 days of IL-7 treatment, increased numbers of B220+/surface(s) IgM- bone marrow cells were observed. After 6 days of treatment, these numbers were still further increased and a significant population of B220+/sIgM- cells were observed in the spleen. The numbers of c mu+/sIgM- cells were also increased in the IL-7-treated mice. Analysis of the expression of B220 and BP-1 on the sIgM- bone marrow cells revealed that the B220+/BP-1+ population was dramatically increased after IL-7 treatment and the size of the B220+/BP-1- population did not differ from control mice. The pre-B cell numbers declined rapidly after the cessation of IL-7 treatment. After 6 days of IL-7 treatment, a twofold increase in the number of B cells in the spleen and lymph node was observed. The B cell numbers declined to normal values within 6 days after the cessation of IL-7 administration. In the spleens of the IL-7-treated mice, there was a significant increase in the number of B cells with an immature phenotype (e.g., sIgMhi/sIgDlo, decreased levels of Ia and FcR expression). The numbers of CD8+ and CD4+ T cells were also increased in the lymph node and spleen of the IL-7-treated mice. These numbers declined to normal levels after the cessation of IL-7 treatment.     

Sustained engraftment of mice transplanted with IL-1-primed blood-derived stem cells.

IL-1 is considered the primary mediator of the acute phase response. One of the characteristic manifestations of this response is early neutrophilia that is probably caused by release of mature neutrophils from the bone marrow into the peripheral blood. In the present study, we assessed whether IL-1 had a similar releasing effect on the number of circulating progenitor cells and stem cells. Female BALB/c mice were injected i.p. with increasing (0.1-1.0 micrograms/mouse) concentrations of rhu-IL-1 alpha. IL-1 injection resulted in a marked dose-dependent increase in the number of polymorphonuclear neutrophils, granulocyte-macrophage colony-forming units (CFU-GM), and cells forming spleen colonies (CFU-S day 8 and day 12). The maximal increase was found at 4 to 8 h after injection of 1 micrograms IL-1 per mouse, yielding a mean fivefold elevation in neutrophil count, and a mean 30-fold and 10-fold increase in the number of circulating CFU-GM and CFU-S, respectively. In a subsequent series of experiments, lethally irradiated (8.5 Gy) female recipient animals were transplanted with 5 x 10(5) blood mononuclear cells derived from male IL-1-treated animals. Long-term survival was obtained in 68% of mice transplanted with peripheral blood cells derived from donor animals at 6 h after a single injection of 1 micrograms IL-1. The mean number of circulating CFU-GM in these donor animals was 557/ml blood. At 6 mo after transplantation, greater than 95% of the bone marrow cells were of male origin, as determined using in situ hybridization with a Y-chromosome specific probe. In contrast, long-term survival was reached in less than 10% of mice transplanted with an equal number of blood cells derived from saline-treated controls or donor animals treated with a dose of 0.1 micrograms IL-1. These results indicate that a single injection of IL-1 induces a shift of hematopoietic progenitor cells and marrow repopulating cells into peripheral blood and that these cells can be used to rescue and permanently repopulate the bone marrow of lethally irradiated recipients.     

Induction of immune resistance against L1210 lymphatic leukemia in mice after lethal irradiation and reconstruction with fetal liver cells.

The immunohematopoietic potential of syngeneic fetal liver cells (SFLC) was examined and compared with syngeneic bone marrow cells (SBMC). SFLC generated about 3 times less 12th-day spleen colonies (CFU-S) than adult SBMC did. To test the SFLC ability for reconstitution of the immune system, mice were lethally total body irradiated (TBI) and transplanted i.v. with 3 x 10(7) SFLC or 1 x 10(7) SBMC. Thus, injected hematopoietic cells contained the same number of CFU-S. On days 28, 35, 42, and 49 after transplantation the mice were injected i.p. with 10(6) immunogenic L1210-Maf cells (L1210 leukemia cells treated in vitro with mafosfamide for inhibition of their growth in vivo) to test the ability for generation of immune response against L1210 leukemia. On day 56 the animals were challenged with 10(3) L1210 leukemia cells. Strong resistance against the leukemia was induced in TBI + SFLC and TBI + SBMC mice, suggesting that the SFLC similarly as SBMC are able to reconstitute immune system of the TBI host.     

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.     

The development of the hemopoietic system: colony-forming splenic units in mouse embryogenesis

Localization and time of appearance as well as dynamics of quantitative changes of splenic colony-forming units (CFU-S) in mouse (C57BL/6 X CBA)F1 embryos were studied. Cells taken from the whole embryo (day 8), yolk sac and embryo per se (day 9), and also liver (day 10) were injected into the lethally irradiated syngenic mice. 7-8 days after the injection the spleens were fixed and the number of macrocolonies was counted. Statistically significant number of CFU-S was detected starting from day 10 of development, first in the embryo (30-33 somites), then in yolk sac and blood (37-38 somites) and liver (after the 40 somites stage). Rapid increase of CFU-S number during days 11-12 (two-fold increase in about 4.6 hours) suggest that not only active proliferation of CFU-S but also maturation of CFU-S precursors take place.     

Protective effect of swerchirin on hematopoiesis in 60Co-irradiated mice.

The protective effect of swerchirin, a purified xanthone isolated from whole herb of Swertia calycina Franch. on hematopoiesis was investigated. A significant increase of colony formation in the spleen (colony forming unit in spleen = CFU-S) of mice irradiated with 550 rad 60Co gamma-rays and an enhancement of proliferative response of BMC to rmGM-CSF treated with swerchirin [10 mg/kg, 3 time/wk, i.p.] was observed. After introduction of swerchirin [10 mg/kg, i.p. once] a significant increase in the number of peripheral blood leukocytes and a rise in the serum of colony stimulating factor (CSF) were also confirmed. The types of CSF in serum were M-CSF and other hematopoietic growth factors, which were confirmed using McAb of IL-3, GM-CSF and PcAb of M-CSF. These beneficial effects of swerchirin on hematopoiesis may be related to its activity inducing CSFs and other hematopoietic growth factors, and warrant further evaluation of its usefulness.     

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.     

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。     

Orally administered IL-6 induces elevated intestinal GM-CSF gene expression and splenic CFU-GM

Orally administered interleukin (IL)-6 has been shown to be of benefit in eliminating Campylobacter infection and in preventing sepsis following hemorrhage. In related experiments, it was seen that proliferating cells were found in the spleens of untreated mice given IL-6 by oral gavage. Injection of the DNA label, BrdU, showed that significant proliferation began at 4 h and peaked at 24 h in the splenic red pulp of animals given oral IL-6. Mice given saline showed no increase in splenic BrdU uptake. Histological analysis suggested a hematopoietic lineage for these cells. Clonogenic assays performed on spleen cells taken from mice given oral IL-6 revealed that increased granulocyte-macrophage colony forming units (GM-CFU) were present at 24 h post-IL-6 administration. No increase in GM colonies occurred in mice fed IL-3, granulocyte-colony stimulating factor (G-CSF) or granulocyte-macrophage (GM)-CSF. RT-PCR analysis of intestinal mRNA from treated mice revealed that GM-CSF mRNA was elevated at 4 h after oral IL-6 administration, but not in mice fed other cytokines. It is suggested that oral administration of IL-6 induces both proliferation and a brief elevation of GM-CFU in the hematopoietic spleens of mice. This increase appears to be the result of increased GM-CSF mRNA production in the intestines of mice fed IL-6.     

Pluripotent (Cfu-S) and Granulocyte-Committed (Cfu-C) Stem Cells In Intact and 89Sr Marrow-Ablated S1/S1D Mice

Peripheral blood values, femur cell counts, spleen weights, pluripotent (CFU-S) and granulocyte progenitor cell (CFU-C) concentrations and total content of spleens and femurs have been evaluated in intact (non-marrow-ablated) and ⁸⁹Sr marrow-ablated S1/S1^d and +/+ mice. ⁸⁹Sr-irradiated mice were studied 6 and 11 days after the administration of ⁸⁹Sr. In intact S1/S1^d mice the femur CFU-S concentration, total femur CFU-S, femur CFU-C concentration and total femur CFU-C were 84, 54, 105 and 68% that of +/+ mice femurs respectively; the respective values for the spleens of S1/S1^d mice were 40,46,61 and 69%. These are the first simultaneous determinations of CFU-S and CFU-C concentrations, and content of spleens and marrows, of S1/S1^d and +/+ mice. In ⁸⁹Sr marrow-ablated mice, 11 days after injection of the radionuclide: (a) the total content of marrow CFU-C and CFU-S was about 1% of that found in the marrows of intact mice for both +/+ and S1/S1^d groups; (b) the spleens of +/+ mice increased in weight to 162% of the control, but the spleens of S1/S1^d mice did not increase in weight; and (c) the spleens of +/+ mice had a total content of CFU-C and CFU-S of 800% and 260% of the control, respectively, whereas the respective values for the S1/S1^d mice were 120% and 76% of the control. Thus the S1/S1^d spleen fails to compensate for marrow ablation by housing additional CFU-S and has an impaired ability to compensate by housing additional CFU-C.     

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.     

Stimulator of proliferation of spleen colony-forming cells in T-cell deprived mice treated with cyclophosphamide or irradiation

A role for T-cells in the regulation of CFU-S proliferation was investigated by determining the presence and activity of CFU-S proliferation stimulator (CFU-S stimulator) in adult mouse bone marrow after irradiation or cyclophosphamide (Cy) treatment. CBA mice previously deprived of T-cells by thymectomy, irradiation and bone marrow reconstitution (TIR) were thereafter treated with 4.5 Gy irradiation or 200 mg/kg Cy. Regenerating bone marrow cells of TIR and corresponding control mice after irradiation or Cy treatment produced CFU-S stimulator. The dose dependent increase in cytosine arabinoside cell death of normal bone marrow day 8 CFU-S was found when both CFU-S stimulators obtained after irradiation of TIR or corresponding control animals were tested. CFU-S stimulator activity in the bone marrow of TIR-Cy treated mice was also detected, but the effect was not dose-dependent. This was not related to the presence of an inhibitor of CFU-S proliferation. It appears that the CFU-S stimulator activity is not related to IL-6, IL-1 or IL-2, or to an inhibitor of IL-6 or IL-1 activity. The results demonstrate the existence of CFU-S proliferation stimulator unrelated to the two major monokines in the bone marrow of immunosuppressed mice.     

In vitro production of CFU-S and cells with erythropoiesis repopulating ability by 5-fluorouracil treated mouse bone marrow

Mouse bone marrow, obtained from donors three days after treatment with 5-fluorouracil, had a very low ability to form macroscopic spleen colonies in irradiated mice at 10 days after transplantation of the cells (CFU-S10); such marrow also had no detectable erythropoiesis repopulating ability but did have near normal marrow repopulating ability and spleen megakaryocyte repopulating ability. Incubation of this marrow in vitro for 7 days with medium containing growth factor preparations (a) pregnant mouse uterus extract plus human spleen conditioned medium or (b) mouse spleen conditioned medium, resulted in marked increases in CFU-S10 and in cells with erythropoietic repopulating ability together with maintenance of cells with marrow repopulating ability. These responses were not observed in cultures with control medium alone. Spleen megakaryocyte repopulating ability was also maintained in the presence of the factor preparations.     

Cytokine production by different population of macrophages following radiation or combined radiation injury

Male mice (CBA x C57BL6)F1 were used for the experiments throughout this study. The levels of spontaneous and LPS-stimulated cytokines production (IL-1 beta, IL-6 and TNF-alpha) by peritoneal, splenic, and bone marrow macrophages were evaluated by means of enzyme-linked immunosorbent assay at 3, 6, 24, 48 and 72 hours after irradiation alone or combined injury (irradiation + thermal burn). The results suggest that macrophages, harvested from the main mice hematopoietic organs (bone marrow, spleen), did not increase cytokines production within the first three days following the 7 Gy gamma-irradiation or combined injury. Peritoneal macrophages revealed a capacity to enhance IL-6 and IL-1 production versus normal healthy mice. There were no significant differences of cytokine-producing activity if macrophages were harvested from irradiated or combined injured mice.     

A comparison of stem cell assays using early or late spleen colonies

The distribution of spleen colony diameters was determined 5.5, 8.0, 10.5 and 13.0 days after injection of normal bone marrow cells to lethally irradiated recipients. A relative lack of small colonies on day 8.0, as compared with days 5.5, 10.5 and 13.0, argued against a time continuum in colony appearance. The spleen colonies observed after 10 days or more probably represented a mixture of colonies which developed from the originally transplanted CFU-S and those arising from secondary CFU-S. Thus, late appearing spleen colonies may not necessarily identify a different, less mature, population of CFU-S. Administration of increasing amounts of bone marrow cells was used for comparing the linearity of the CFU-S assay for colonies observed after 8 days or after 12 to 13 days. The influence of overlapping colonies on the results was considerably augmented if large spleen colonies were observed after 12 or 13 days. Subsequently the CFU-S assay lost much of its quantitative character. We believe that some previously published data might have been misinterpreted by neglecting the important differences between 'early' and 'late' CFU-S assays.     

Radioprotective effects of ginsan,an immunomodulator

We previously reported that ginsan, a purified polysaccharide isolated from Panax ginseng, had a mitogenic activity, induced LAK cells, and increased levels of several cytokines. In an effort to identify other immunostimulatory effects, we evaluated the protective effects of ginsan injected in vivo against radiation by measuring its effects on the CFU-S bone marrow cells and spleen cells. Ginsan was found to significantly increase the number of bone marrow cells, spleen cells, granulocyte-macrophage colony-forming cells (GM-CFC), and circulating neutrophils, lymphocytes and platelets in irradiated mice. In addition, ginsan induced the endogenous production of cytokines such as Il1, Il6, Ifng and Il12, which are required for hematopoietic recovery, and was able to enhance Th1 function while interfering with the Th2 response in irradiated mice. We demonstrated that pretreatment with ginsan protected mice from the lethal effects of ionizing radiation more effectively than when it was given immediately after or at various times after irradiation. A significant increase in the LD(50/30) from 7.54 Gy for PBS injection to 10.93 Gy for mice pretreated with 100 mg/kg ginsan was observed. These findings indicate that ginsan may be a useful agent to reduce the time necessary for reconstituting hematopoietic cells after irradiation.     

Activation of IL-4 and IL-6 secreting cells by antigen

The number, antigenic specificity and phenotype of cells secreting IL-4 and IL-6 in mice immunized with ovalbumin or keyhole limpet haemocyanin (KLH) in Freund's adjuvant (FA) was studied. The frequency of cells producing either of these cytokines began to rise 6 days post immunization, peaked at 11–14 days post-immunization, and fell to background by 21 days. The number of spleen cells secreting IL-6 was higher than the number producing IL-4 at all time points. Boosting elicited an anamnestic response characterized by a significant increase in the number of cytokine secreting cells within 4 days. Cytokine production was induced in multiple strains of normal mice, and was critically dependent on the use of Complete FA in addition to antigen. Immunization induced IL-4 and IL-6 production in vivo while ‘priming’ additional cells to release these cytokines when reexposed to soluble antigen in vitro. The latter response was antigen specific and was dominated by non-B/non-T cells. Those cells may serve to boost the immune response in cases of persistent or repeated antigenic challenge.