RNA in the periphery of rapidly proliferating mouse lymphoid cells

RNA in the peripheries of various populations of lymph node cells (LNC) has been evaluated by measuring the electrophoretic mobilities of cells, before and after treatment with active or inactivated ribonucleases. Three different populations of LNC were studied: (1) “resting” normal age control LNC; (2) “syngeneic” LNC from irradiated (C3H × C57BL)F₁ or C3H mice four to six days following transplantation of syngeneic spleen cells; such cells were progeny of lymphopoietic progenitor cells of the spleen; and (3) “allogeneic” LNC from irradiated (C3H × C57BL)F₁ mice four to six days after grafting C3H (parental) spleen cells; such cells were progeny of lymphopoietic progenitor cells, but also alloantigen-sensitive cells of the spleen which proliferate in response to the host's alloantigens (a “graft-versus-host” immunological reaction). Whereas the normal LNC had no detectable peripheral RNA, the allogeneic and syngeneic LNC did, i.e., ribonuclease reduced their mean electrophoretic mobilities by 13.6 and 9.2 per cent, respectively. Since both allogeneic and syngeneic LNC had peripheral RNA, no specific correlation could be made with immunological activity. ³H-uridine and ¹⁴C-thymidine incorporation into lymph nodes was greatest in allogeneic, intermediate in syngeneic and least in age control lymph nodes, indicating a “population shift” in the spleen cell chimeras toward relatively immature, rapidly proliferating cells, which had a relatively high rate of RNA synthesis. Thus, rapidly proliferating lymphoid cells do have RNA in their peripheries, but its relation to specific immunological function has yet to be ascertained.     

Mitigation of Lethal Radiation Syndrome in Mice by Intramuscular Injection of 3D Cultured Adherent Human Placental Stromal Cells

Exposure to high lethal dose of ionizing radiation results in acute radiation syndrome with deleterious systemic effects to different organs. A primary target is the highly sensitive bone marrow and the hematopoietic system. In the current study C3H/HeN mice were total body irradiated by 7.7 Gy. Twenty four hrs and 5 days after irradiation 2×10⁶ cells from different preparations of human derived 3D expanded adherent placental stromal cells (PLX) were injected intramuscularly. Treatment with batches consisting of pure maternal cell preparations (PLX-Mat) increased the survival of the irradiated mice from ∼27% to 68% (P<0.001), while cell preparations with a mixture of maternal and fetal derived cells (PLX-RAD) increased the survival to ∼98% (P<0.0001). The dose modifying factor of this treatment for both 50% and 37% survival (DMF₅₀ and DMF₃₇) was∼1.23. Initiation of the more effective treatment with PLX-RAD injection could be delayed for up to 48 hrs after irradiation with similar effect. A delayed treatment by 72 hrs had lower, but still significantly effect (p<0.05). A faster recovery of the BM and improved reconstitution of all blood cell lineages in the PLX-RAD treated mice during the follow-up explains the increased survival of the cells treated irradiated mice. The number of CD45+/SCA1+ hematopoietic progenitor cells within the fast recovering population of nucleated BM cells in the irradiated mice was also elevated in the PLX-RAD treated mice. Our study suggests that IM treatment with PLX-RAD cells may serve as a highly effective “off the shelf” therapy to treat BM failure following total body exposure to high doses of radiation. The results suggest that similar treatments may be beneficial also for clinical conditions associated with severe BM aplasia and pancytopenia.     

The in vivo effects of interleukin 1. I. Bone marrow cells are induced to cycle after administration of interleukin 1

We have previously reported that interleukin 1 (IL-1) administration 20 hr before irradiation protects mice from lethal effects of radiation. The recovery of total nucleated bone marrow cells and of hematopoietic progenitor cells was enhanced in IL-1 treated, as compared to untreated, irradiated mice. This suggested that IL-1 administration may affect the cells in the bone marrow of normal mice. Intraperitoneal administration of recombinant IL-1 resulted in bone marrow cell enlargement and increased cycling of these enlarged cells. In addition, the capacity of bone marrow cells from IL-1 treated mice to proliferate in response to granulocyte macrophage-colony-stimulating factor (GM-CSF) in cell suspension cultures was enhanced. The above effects were not genetically restricted as C57BL/6, B6D2F1, C3H/HeN, and C3H/HeJ mice showed similar responses. A comparative study showed that 100 ng of IL-1 was much more effective in stimulating bone marrow cells by the above criteria than 5 micrograms GM-CSF. Since IL-1, unlike CSF, can not be demonstrated to have a direct in vitro stimulatory effect on bone marrow cells, the aforementioned in vivo effects of IL-1 are presumably mediated by other hematopoietic growth factors. We have previously shown that IL-1 induces the appearance of high titers of CSF in the serum. Consequently hematopoietic growth factors that are generated at local sites following IL-1 administration may mediate the observed cell cycling effect.     

Defective transient endogenous spleen colony formation in S1/S1d mice.

WCB6F1 mice of the genotype S1/S1d did not form transient 5-day endogenous spleen colonies following midlethal irradiation, either spontaneously or in response to postirradiation bleeding. Their hematologically normal (+/+) littermates produced colonies equivalent in number and morphologic type to a normal strain (D2B6F1), as evaluated by both macroscopic and microscopic criteria. Bone marrow cells from S1/S1d mice, when transplanted into lethally irradiated +/+ mice, were able to generate equivalent numbers of transient endogenous spleen colonies (TE-CFUs), as compared to that obtained when syngeneic +/+ marrow cells were injected into lethally irradiated +/+ recipients. A defective growth of an early class of hematopoietic progenitor cells, resulting in the clinical course of the S1/S1d anemia is suggested and confirms previous reports on the microenvironmental nature of this abnormality.     

Foe turned friend: multiple functional roles attributable to hyper-activating stem cell factor receptor mutant in regeneration of the haematopoietic cell compartment

Objectives: Stem cell factor receptor, c‐kit, is considered to be the master signalling molecule of haematopoietic stem cells. It develops the orchestral pattern of haematopoietic cell lineages, seen by its varying degree of omnipresence in progenitors, lineage committed and mature cells. We have investigated the effect of over‐expressing c‐kit on early recovery of the haematopoietic compartment, in irradiated hosts. Materials and methods: Normal bone marrow cells (BMCs) were transfected with Kit_wt (wild‐type c‐kit) or its variant Kit_mu (asp814tyr) by electroporation. Lethally irradiated mice were transplanted with normal or transfected congeneic BMCs. The effect of ectopic expression of c‐kit on haematopoietic cell recovery was determined by analysing donor‐derived cells. Furthermore, effects of both types of c‐kit over‐expression on progenitor and lineage‐committed cells were examined by flow cytometric analysis of Sca‐1 and lineage‐committed (Lin⁺) cells respectively. Results: Hyper‐activating Kit_mu significantly improved recovery of the haematopoietic system in irradiated hosts. In vivo results showed that the donor‐derived c‐kit⁺ cell population was increased to more than 3‐fold in the case of Kit_mu‐transfected cells compared to normal and Kit_wt over‐expressing BMCs. In general, survival of progenitor and committed cell was improved in the Kit_mu over‐expressing system compared to the other two cohorts. Conclusion: These results suggest that recruitment of the hyper‐activating variant of c‐kit (Kit_mu) lead to early recovery of the bone marrow of lethally irradiated mice.     

Growth and senescence of antibody-forming cells

Studies were performed on the capacity of mice for hemagglutinating antibody production throughout their life-span. An in vivo culture method was used for assessment of primary and secondary antibody-forming potentials of spleen cells of mice ranging in age from 1 to 130 weeks. There was a marked growth of potential for antibody formation during neonatal and juvenile life followed by a gradual decline in potential with advancing age. It was possible to show that the changes in potential were principally due to changes in the number of competent progenitor cells and not to changes in their performance. Death of very old animals was correlated with decline in number of immunologically competent progenitor cells. The decay in number of progenitor cells during aging of mice was random. Loss of progenitor cells was not entirely attributable to either generative failure of the pool of progenitor cells or the capacity of the milieu of the animal to support such cells. Thus, spleen cells from aged animals displayed increasing capacity for primary antibody formation during a 3-week period of culture in young, irradiated mice; identical cultures in old, irradiated recipients failed in respect to growth of primary antibody-forming potential. Progressive imparirment of the milieu of aging animals was suggested by the fact that spleen cells from very old animals were “toxic” when infused into lightly irradiated recipients which were themselves of advanced age but far short of the senescent phase of their life-span. These results lead to the argument that senescence may be, to a major degree, the result of progressive loss of progenitor, or “stem,” cells which are normally utilized to replace terminally differentiated, dying cells.     

Myelosuppressive effects in vivo of purified recombinant murine macrophage inflammatory protein-1 alpha.

Purified recombinant murine macrophage inflammatory protein-1 alpha (rmuMIP-1 alpha), a cytokine with myelopoietic activity in vitro, was assessed in vivo by injection into C3H/HeJ mice for effects on proliferation (percentage of cells in S phase DNA synthesis of the cell cycle) and absolute numbers of granulocyte-macrophage, erythroid, and multipotential progenitor cells in the femur and spleen, and on nucleated cellularity in the bone marrow, spleen, and blood. rmuMIP-1 alpha rapidly decreased cycling rates (at 2 to 10 micrograms/mouse i.v.) and absolute numbers (at 5 to 10 micrograms/mouse i.v.) of myeloid progenitor cells in the marrow and spleen. These effects were dose- and time-dependent and reversible. Suppressive effects were noted within 3 to 24 h for cell cycling and absolute numbers of progenitor cells in the marrow and spleen, and by 48 h for circulating neutrophils. A study comparing the effects of i.v. injection of rmuMIP-1 alpha versus rmuMIP-1 beta, a biochemically similar molecule but with no myelosuppressive effects in vitro, demonstrated myelosuppression in vivo by rmuMIP-1 alpha, but not by rmuMIP-1 beta. The results suggest that rmuMIP-1 alpha has myelosuppressive activity in vivo and offers the possibility that it may be a useful adjunct to treatments involving cytotoxic drugs because of its reversible suppressive effects on normal progenitor cell cycling.     

The potential benefits of nicaraven to protect against radiation-induced injury in hematopoietic stem/progenitor cells with relative low dose exposures

Nicaraven, a hydroxyl radical-specific scavenger has been demonstrated to attenuate radiation injury in hematopoietic stem cells with 5 Gy γ-ray exposures. We explored the effect and related mechanisms of nicaraven for protecting radiation injury induced by sequential exposures to a relatively lower dose γ-ray. C57BL/6 mice were given nicaraven or placebo within 30 min before exposure to 50 mGy γ-ray daily for 30 days in sequences (cumulative dose of 1.5 Gy). Mice were victimized 24 h after the last radiation exposure, and the number, function and oxidative stress of hematopoietic stem cells were quantitatively estimated. We also compared the gene expression in these purified stem cells from mice received nicaraven and placebo treatment. Nicaraven increased the number of c-kit⁺ stem/progenitor cells in bone marrow and peripheral blood, with a recovery rate around 60–90% of age-matched non-irradiated healthy mice. The potency of colony forming from hematopoietic stem/progenitor cells as indicator of function was completely protected with nicaraven treatment. Furthermore, nicaraven treatment changed the expression of many genes associated to DNA repair, inflammatory response, and immunomodulation in c-kit⁺ stem/progenitor cells. Nicaraven effectively protected against damages of hematopoietic stem/progenitor cells induced by sequential exposures to a relatively low dose radiation, via complex mechanisms.     

THYMIDINE SUICIDE IN VIVO AND IN VITRO OF SPLEEN COLONY FORMING AND AGAR COLONY FORMING CELLS OF MOUSE BONE MARROW

The ‘thymidine suicide’technique for indicating differences in the proliferation rate of early haemopoietic progenitor cells (spleen colony forming and agar colony forming cells) in C57BL mice has been evaluated. Special care was taken to use the same bone marrow cell suspension for the two progenitor cell assays. Both the in vivo and the in vitro techniques were employed. Following ³H-TdR in vivo, about 20% of both types of progenitor cell are killed in normal mice; however, after incubation in vitro with ³H-TdR, 35% of agar colony forming cells but only 4% of spleen colony forming cells are killed. Reasons for the difference between the in vivo and the in vitro results are discussed. With bone marrow from continuously irradiated animals, the thymidine suicide for both agar colony forming and spleen colony forming cells is in the range 42–50%, and there is no difference between in vivo and in vitro suicide. The in vivo results support the conclusion, based on the effect of proliferation dependent cytotoxic agents, that in C57BL mice agar colony forming and spleen colony forming cells are proliferating at the same rate in normal animals, and are speeded up to the same extent by continuous γ-irradiation. It is considered that in normal C57BL mice the in vitro method does not give a correct estimate of the proliferation rate of these progenitor cells. It would seem that the similarity in the proliferation rate of agar colony forming and spleen colony forming cells in C57BL mice is not true for other strains of mice: indeed using normal CBA and in vivo suicide, we have shown a significantly greater thymidine suicide for agar colony forming cells compared to spleen colony forming cells.     

A comparison of the effect of cytotoxic agents on agar colony forming cells, spleen colony forming cells, and the erythrocytic repopulating ability of mouse bone marrow

Three assays for bone marrow progenitor cells have been used to determine the effect of single doses of two cytotoxic agents, cyclophosphamide and vinblastine. The assays employed were the agar colony forming and spleen colony forming assays and the crythroid repopulating ability. In normal mice, there was little difference between the response of the progenitor cells assayed by the three methods, following cyclophosphamide: and no detectable difference following vinblastine. Bone marrow from continuously irradiated mice and bone marrow regenerating seven days following transplantation was also studied: in both these situations the proliferation rate of the progenitor cells is increased. Cyclophosphamide was found to be only slightly proliferation dependent with each assay. However, vinblastine was strikingly proliferation dependent. In irradiated mice and also in regenerating marrow the agar colony forming cells were many times more sensitive to this agent than were the other progenitor cells. These results show that under some but not all circumstances the agar colony forming and spleen colony forming cells behave similarly in C57BL mice, but are not a single population of cells.     

Effect of a neutrophilia-inducing tumor on hemopoietic stem cells in mice

The influence of neutrophilic stimulation on hemopoietic stem cells was studied in mice with tumor-induced neutrophilia. Transfusions of marrow cells from normal and neutrophilic tumor-bearing mice into lethally irradiated normal and tumor-bearing mice were performed. The number and the erythroid:granuloid (E:G) ratio of day 7 colonies in the recipient spleens and bones as well as the size of spleen colonies of recipient animals were determined. The E:G ratio of spleen and bone marrow colonies between normal and tumor-bearing mouse recipients and the number of spleen colonies did not differ significantly in either experiment. However, spleen colonies which developed in tumor-bearing irradiated mice were significantly larger than those which developed in normal recipients in both experiments. These studies indicated that while the line of differentiation taken by hemopoietic stem cells was not affected by the neutrophilic influence of the tumor, the tumor-bearing host environment appeared to enhance proliferation of transfused stem cells and/or their descendants. The stimulators of granulocytopoiesis in this model of neutrophilia appear to act on a population of progenitor cells more mature than the stem cells capable of forming 7-day colonies in the spleen and bone marrow of irradiated recipient mice.     

Post-irradiation thymocyte regeneration after bone marrow transplantation. II. Physical characteristics of thymocyte progenitor cells

Bone marrow cells were separated according to buoyant density, velocity sedimentation and cell surface charge. Fractionated (C3H x AKR)F1 bone marrow cells were transplanted into lethally-irradiated C3H recipients. In all fractions, the CFUs content and the capacity to restore the thymus cell population were determined. For all the physical parameters tested, the thymocyte progenitor cells show the same distribution as CFUs. The relationship between number of thymocyte progenitor cells and number of CFUs is dependent on density. Bone marrow progenitors of PHA responsive cells are of low buoyant density and show a distribution which resembles the distribution of the progenitors of Thy 1 positive cells. After transplantation of large numbers of bone marrow cells into irradiated mice, no significant change in the CFUs content of the thymus was observed.     

Post-Irradiation Thymocyte Regeneration After Bone Marrow Transplantation Ii. Physical Characteristics of Thymocyte Progenitor Cells

Bone marrow cells were separated according to buoyant density, velocity sedimentation and cell surface charge. Fractionated (C3H × AKR)F₁ bone marrow cells were transplanted into lethally-irradiated C3H recipients. In all fractions, the CFUs content and the capacity to restore the thymus cell population were determined. For all the physical parameters tested, thymocyte progenitor cells show the same distribution as CFUs. the relationship between number of thymocyte progenitor cells and number of CFUs is dependent on density. Bone marrow progenitors of PHA responsive cells are of low buoyant density and show a distribution which resembles the distribution of the progenitors of Thy 1 positive cells. After transplantation of large numbers of bone marrow cells into irradiated mice, no significant change in the CFUs content of the thymus was observed.     

Activation of T and B thymus cells to recognize histocompatibility antigens

Lethally irradiated (A × CBA) F₁ or (A × C57BL/6) F₁ mice were injected with 10⁷ A strain thymus cells in attempts to activate donor cells to recognize CBA or C57BL/6 histocompatibility antigens, respectively. Activation could be revealed by injecting activated thymus cells (day 5 irradiated F₁ hybrid spleen cells) into corresponding unirradiated F₁ hybrid hosts. The alloantibody titers formed by these cells and the antirecognition structure (anti-RS) antibody titers induced by them were similar to those observed after injection of normal parental strain spleen cells, indicating that thymus cells had become endowed with recognition structures (RS). Alloantibodies, but no anti-RS antibodies, were present in the serum of F₁ mice given activated thymus cells treated with anti-θ and complement. It, therefore, appeared that activated thymus cells contained sufficient B cells differentiated into antibody-forming cells to give a measurable alloantibody response. On the other hand, receptors responsible for anti-RS antibody induction presumably were located on T cells. Specificity and restriction of antigenic recognition were revealed by negative results obtained when activated thymus cells were injected into F₁ hosts not containing the antigens against which activation had been directed.     

Nicaraven Attenuates Radiation-Induced Injury in Hematopoietic Stem/Progenitor Cells in Mice

Nicaraven, a chemically synthesized hydroxyl radical-specific scavenger, has been demonstrated to protect against ischemia-reperfusion injury in various organs. We investigated whether nicaraven can attenuate radiation-induced injury in hematopoietic stem/progenitor cells, which is the conmen complication of radiotherapy and one of the major causes of death in sub-acute phase after accidental exposure to high dose radiation. C57BL/6 mice were exposed to 1 Gy γ-ray radiation daily for 5 days in succession (a total of 5 Gy), and given nicaraven or a placebo after each exposure. The mice were sacrificed 2 days after the last radiation treatment, and the protective effects and relevant mechanisms of nicaraven in hematopoietic stem/progenitor cells with radiation-induced damage were investigated by ex vivo examination. We found that post-radiation administration of nicaraven significantly increased the number, improved the colony-forming capacity, and decreased the DNA damage of hematopoietic stem/progenitor cells. The urinary levels of 8-oxo-2′-deoxyguanosine, a marker of DNA oxidation, were significantly lower in mice that were given nicaraven compared with those that received a placebo treatment, although the levels of intracellular and mitochondrial reactive oxygen species in the bone marrow cells did not differ significantly between the two groups. Interestingly, compared with the placebo treatment, the administration of nicaraven significantly decreased the levels of the inflammatory cytokines IL-6 and TNF-α in the plasma of mice. Our data suggest that nicaraven effectively diminished the effects of radiation-induced injury in hematopoietic stem/progenitor cells, which is likely associated with the anti-oxidative and anti-inflammatory properties of this compound.     

A requirement for helper T cells in the induction of delayed-type hypersensitivity

This paper describes a model system for studying the role of helper T cells in the induction of delayed-type hypersensitivity (DTH). Cyclophosphamide- (CP) treated mice sensitized with antigen 3 days later develop high levels of delayed-type immunity; however, DTH cannot be demonstrated in mice that are sensitized with antigen 1 day after drug treatment. The inability to respond to antigen 1 day after CP treatment can be restored if either normal or low-dose primed spleen cells are transferred at the time of sensitization. Although irradiated (1500 rad) normal spleen cells are unable to restore DTH, such treatment has no effect on the primed spleen cell population. The lymphocytes responsible for restoring the DTH response were identified as T cells, in that treatment with anti-Thy-1.2 serum and C abrogated their effect. Furthermore, restoration of the DTH response was dependent on the presence of antigen at the time of lymphocyte transfer; irradiated primed cells could not transfer DTH alone. The DTH effector cells in reconstituted mice were identified as originating from the host and not from the transferred cell population. This was accomplished by using anti-H-2 serum to identify the source of the DTH effector cells after transferring parental (H-2b) irradiated primed spleen cells into CP-treated F1 mice (H-2b,k). Thus, the irradiated transferred cells are behaving as helper T cells and promoting the development of DTH effector cells in the host.     

Studies of delayed hypersensitivity to L. Monocytogenes in mice: nature of cells involved in passive transfers

C3Hf/Umc mice were immunized by an intravenous injection of a sublethal dose of live Listeria monocytogenes. The animals developed delayed-type hypersensitivity (DH) concomitant with infectious immunity to this organism. Delayed hypersensitivity could be transferred to normal lethally irradiated mice with spleen cells from immune animals. The immune cells cells responsible for transfer of adoptive immunity were susceptible to in vitro cytolytic action of anti-theta iso-antibody and complement, since such treatment rendered these cells incapable of further passive transfer of specific immunity to Listeria. The acquired DH to Listeria persisted in mice after 900 R lethal irradiation, provided normal syngeneic bone marrow cells were also administered, thus indicating the persistance of a cell population in the immune irradiated mice, resistant to effects of radiation. The radio resistant nature of this immune cell population was further demonstrated by passive transfer with spleen cells, derived from preimmunized lethally irradiated mice to normal syngeneic mice or to lethally irradiated nonimmunized hosts reconstituted with normal bone marrow which then responded to antigenic challenge with DH.Treatment of the immune radio resistant spleen cells in vitro with anti-theta and complement eliminated passive transfers of DH by these cells; however, this effect was less obvious than similar treatment of the immune, nonirradiated, spleen cells.     

Transient erythropoietic spleen colonies: effects of erythropoietin in normal and genetically anemic W/Wv mice.

Properties of the cells (TE-CFU) that give rise within four to six days to transient endogenous erythropoietic spleen colonies in irradiated mice have been investigated. The results obtained indicate that (1) erythropoietic maturation within such colonies is highly erythropoietin-dependent, (2) the population size of TE-CFU is not erythropoietin-dependent, (3) initial exposure to a high dose of erythropoietin followed by continuing exposure to lower doses is required for maximal efficiency of colony formation by TE-CFU, (4) successful transplantation of TE-CFU has not been achieved, but they appear among the progeny of transplanted hemopoietic cells, (5) TE-CFU are defective in mice of genotype W/Wv. These findings are consistent with the view that the TE-CFU assay detects a class of early erythropoietin-sensitive progenitor cells committed to erythropoietic diffferentiation, rather than "abortive" colony formation by pluripotent stem cells.     

THE EFFECT OF CYTOSINE ARABINOSIDE IN VITRO ON AGAR COLONY FORMING CELLS AND SPLEEN COLONY FORMING CELLS OF C57BL MOUSE BONE MARROW

This study reports the effect of cytosine arabinoside in culture on two classes of bone marrow progenitor cells in C57BL mice, agar colony forming cells (ACU) and spleen colony forming cells (CFU). Both normal cells and rapidly proliferating cells were studied. The results show that in normal mice, 23 % of ACU but only 7 % of CFU are killed following 1 hr incubation with the drug. With longer periods of incubation, the survival of ACU in the controls is poor, and the results for the drug-treated cultures suggest that the cells are held up in cycle. In continuously irradiated mice, the proportion of ACU and CFU killed after 1 hr incubation with drug is increased to 43–54%, confirming previous results that these cells are proliferating more rapidly than in normal mice. In mice treated with myerlan, 54 % of ACU are killed by 1 hr in vitro exposure to cytosine arabinoside, again confirming that ACU are rapidly proliferating. However, the proportion of CFU killed is lower (23 %). These results are compared with other studies of the effect of cytosine arabinoside in vivo and also with thymidine suicide in the same strain of mice. The results show that cytosine arabinoside has the same effect as tritiated thymidine, and also that the proportion of CFU killed by these agents in vitro is lower than when the agents are injected in vivo. It is suggested that the conditions in culture have an adverse effect on CFU, which cease DNA synthesis, and are protected from the killing effect of cytosine arabinoside and tritiated thymidine. Since cytosine arabinoside in vitro has an effect similar to tritiated thymidine in vitro on bone marrow progenitor cells in C57BL mice, in vitro incubation with cytosine arabinoside could be an alternative method to thymidine suicide for measuring differences in cell proliferation rate.