Modification of the regulatory effects of thymocytes on hematopoiesis in irradiated mice as affected by heterologous immunoglobulin G and low-dose ionizing radiation

The administration of heterologous immunoglobulin G (IgG) and/or exposure of mouse thymocyte donors to 1 and 2 Gy radiation were shown to change the regulatory effects of thymus lymphocytes on the recovery of haemopoiesis in syngeneic recipients irradiated with a median lethal dose of 6 Gy. Thymocytes of exposed (2 Gy) donors produced a stimulatory effect on the restoration of the myelokaryocytes number and increased the number of endogenous splenic colonies and bone marrow CFUs in animals exposed to a median lethal dose, whereas the administration of IgG to thymocyte donors given 2 Gy eliminated the stimulatory effect of thymocytes on the number of myelokaryocytes, and the amount of CFUs in irradiated recipients decreased.     

The stimulating effect of preliminary irradiation of the hematopoietic stroma with small doses on the content of hematopoietic stem cells in the bone marrow in vivo and in a long-term culture system

Preirradiation of mouse recipients with a dose of 1-2 Gy 24 and 48 h before lethal irradiation (8 Gy) made CFUs content of femur increase upon transplantation of bone marrow from exposed and intact donors. The same was with the long-term bone marrow culture: preirradiation of a stromal sublayer increased the number of CFUs in the transplanted bone marrow preirradiated with 6 Gy radiation. Retransplantation of bone marrow to irradiated donors after 5 day cultivation, a sublayer being activated, increased the number of CFUs in the femur in comparison with donors which were injected with the bone marrow from the culture without activation of the sublayer by low-level radiation.     

Conservation of bone marrow CFUc in different phases of the cell cycle during cryopreservation

The effect of low temperature (-196 degrees C) preservation on the recovery of colon-forming units (CFUs) of bone marrow at different phases of the cell cycle before cryopreservation is dealt with. The intact bone marrow "enriched" with CFUs in S phase of the cell cycle and the bone marrow without colony-forming units in S phase were exposed to cryopreservation. After cryopreservation of the bone marrow enriched with CFUs in S phase and th bone marrow without colony-forming units in S phase the number of CFUs decreases by the same value as in the cryopreserved bone marrow obtained from intact mice.     

Elimination from bone marrow of a supplementary population participating in the proliferation of pluripotent hematopoietic stem cells using mouse brain antiserum

Changes in the number of spleen exo-colonies and post-radiation repopulation of hematopoietic organs were studied in recipients upon injection of bone marrow treated with anti-brain serum (ABS) with and without thymocytes on days 9-14. It was shown that on days 9-11 colony formation in mice injected bone marrow treated with ABS was much lower than the control level. However, by day 14 the number of colonies increased drastically as compared to the control. Thymocyte supplementation normalized colony formation at any time of observation. Similar pattern is noted in post-radiation repopulation of spleen and bone marrow of mice injected bone marrow pretreated with ABS with or without thymocytes. It is assumed that ABS inactivates bone marrow cells participating in the regulation of CFUs proliferation.     

Mechanism of action of the stem-cell inhibition factor on the formation of exogenous hemopoietic colonies in the spleen of mice

It was established by previous works that thymocytes treated with antilymphocyte serum secrete soluble factor capable of inhibiting exogenous colony formation in the spleen of lethally irradiated mice injected with bone marrow cells treated with the stem cell inhibition factor (SCIF). The purpose of the present investigation was to explore possible mechanisms of SCIF action. Regeneration of erythropoiesis (measured by 59Fe incorporation) in the spleen and bone marrow of mice injected with SCIF-treated bone marrow cells was inhibited as compared with control, while CFUs started proliferating with a 3-day delay. Two hours after SCIF treatment 60% of CFUs entered S phase as judged by hydroxyurea cell kill. The CFUs fraction treated with the SCIF was found to be diminished 3-4-fold as compared with control. The data obtained suggest that SCIF treatment makes CFUs enter 3 phase, which may account for the reduced capacity of CFUs to populate the spleen and to proliferate with a 3-day delay.     

THE INFLUENCE OF DIETARY PROTEIN DEFICIENCY ON HAEMOPOIETIC CELLS IN THE MOUSE

These experiments examined the effect of a diet limited only in protein (4% by weight) on haemopoietic stem cells in mice. This diet places severe restrictions on growth and cell proliferation and this was reflected in lower numbers of colony forming units (CFUs) and in vitro colony forming cells (CFCs). Differences were apparent in the response of different organs to this stress; for instance, the incidence of spleen CFUs fell sharply from around 40/mg spleen tissue to 1 -4/mg spleen tissue after 3 weeks on a low protein diet. This selective loss did not occur in bone marrow where total CFUs remained proportional to cellular content. Yet a third pattern was shown by thymus CFUs–although the numbers were low these increased from 16/thymus in normal mice to 132/thymus in deprived mice. This was the only organ examined which showed an increase. The effects of a return to a high protein (18 %) diet showed that the spleen was the most responsive organ. By day 5 after the return to 18% protein the spleen contained as many CFUs per million cells as the bone marrow. During this time the content of CFU in the spleen had increased some 50-fold whereas bone marrow CFUs only doubled. The spleen assumes the major reconstitutive role during the refeeding process.     

Ability of thymic lymphocytes to alter CFU kinetics in radiation chimeras.

It is known that the poor colony-forming ability of B6 bone marrow transplanted into B6D2F1 hybrids can be improved if B6 lymphocytes are given in addition. It was recently reported that the augmenting lymphocytes decrease the doubling time of differentiating hemopoietic cells. To determine whether thymus cells alter the self-renewal of CFUs in this parent leads to F1 combination, retransplantation and 3H-thymidine 'suicide' were employed as methods to determine the cell-division rate. We have observed that in the presence of thymocytes, parental bone marrow cells are seeded more efficiently in the spleen, and the lag phase of the CFUs growth curve is shortened. However, thymic lymphocytes do not increase the slope of the exponential growth phase of CFUs.     

The influence of dietary protein deficiency on haemopoietic cells in the mouse.

These experiments examined the effect of a diet limited only in protein (4% by weight) on haemopoietic stem cells in mice. This diet places severe restrictions on growth and cell proliferation and this was reflected in lower numbers of colony forming units (CFUs) and in vitro colony forming cells (CFCs). Differences were apparent in the response of different organs to this stress; for instance, the incidence of spleen CFUs fell sharply from around 40/mg spleen tissue to 1-4/mg spleen tissue after 3 weeks on a low protein diet. This selective loss did not occur in bone marrow where total CFUs remained proportional to cellular content. Yet a third pattern was shown by thymus CFUs--although the numbers were low these increased from 16/thymus in normal mice to 132/thymus in deprived mice. This was the only organ examined which showed an increase. The effects of a return to a high protein (18%) diet showed that the spleen was the most responsive organ. By day 5 after the return to 18% protein the spleen contained as many CFUs per million cells as the bone marrow. During this time the content of CFU in the spleen had increased some 50-fold whereas bone marrow CFUs only doubled. The spleen assumes the major reconstructive role during the refeeding process.     

ABILITY OF THYMIC LYMPHOCYTES TO ALTER CFU KINETICS IN RADIATION CHIMERAS

It is known that the poor colony-forming ability of B6 bone marrow transplanted into B6D2F₁ hybrids can be improved if B6 lymphocytes are given in addition. It was recently reported that the augmenting lymphocytes decrease the doubling time of differentiating hemopoietic cells. to determine whether thymus cells alter the self-renewal of CFUs in this parent F₁ combination, retransplan-tation and ³H-thymidine ‘suicide’ were employed as methods to determine the cell-division rate. We have observed that in the presence of thymocytes, parental bone marrow cells are seeded more efficiently in the spleen, and the lag phase of the CFUs growth curve is shortened. However, thymic lymphocytes do not increase the slope of the exponential growth phase of CFUs.     

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.     

Colony-forming activity of hematopoietic stem cells in tolerant mice as affected by antigens

The effect of sheep red blood cells (SRBC) and human red blood cells (HRBC) on the amount of CFUs in the bone marrow and spleen of (CBA X C57BL/6) FI SRBC-tolerant mice was studied. The increase in the number of bone marrow and spleen CFUs was demonstrated in SRBC-tolerant mice injected with HRBC. Using SRBC test injection the increase in CFUs amount was observed in the spleen, but not the bone marrow, where the amount of CFUs remained unchanged.     

Proliferation inhibition of murine pluripotent haemopoietic stem cells by interferon or poly I:C

The effect of mouse serum interferon (IF) in vitro and an inducer in vivo on the proliferation of a pluripotent stem cell population with high turnover rate was studied. Proliferation rate was characterized by the number of CFUs in the S phase of the cell cycle. Increased proliferation of bone marrow stem cell populations was produced either by irradiating the donor mice with 3.36 Gy (336 rad) 60Co-gamma rays 7 days before the experiment or by incubating normal bone marrow cells with 10(-11) M concentration of isoproterenol. IF considerably reduced the number of CFUs in S phase in both cases without reducing the CFUs content of the samples. Injection of IF inducer (4 mg/kg poly I:C) into regenerating mice also inhibited the proliferation of CFUs without decreasing the femoral CFUs level. Regeneration kinetics of CFUs from irradiated poly I:C-treated mice ran parallel with that of irradiated untreated animals but showed a characteristic delay corresponding to approximately one CFUs doubling. A transient, non-cytotoxic proliferation inhibitory effect of IF or IF inducer is, therefore, proposed.     

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.     

Hematopoietic precursor cells in radiation chimeras restored by bone marrow from thymectomized mice]

Radioprotective capacity of bone marrow CFUs of adult thymectomized mice was studied. Lethally irradiated mice were inoculated with bone marrow of mice thymectomized 8-11 months before. The colony forming capacity and proliferative rate of CFUs were studied 1-7.5 months after obtaining the radiation chimeras. It has been shown that proliferative capacity of bone marrow of adult thymectomized mice was reduced in comparison with that of normal animals. It is related to the decrease (4-fold) of the proliferative rate of bone marrow of thymectomized mice which was inoculated into lethally irradiated recipients 1 month before. We also found that the content of CFUs in bone of those chimeras was reduced later--after 7.5 months. In this period (1-7.5 months) the cellularity of bone marrow did not change.     

Proliferation Inhibition of Murine Pluripotent Haemopoietic Stem Cells By Interferon Or Poly I:C

The effect of mouse serum interferon (IF) in vitro and an inducer in vivo on the proliferation of a pluripotent stem cell population with high turnover rate was studied. Proliferation rate was characterized by the number of CFUs in the S phase of the cell cycle. Increased proliferation of bone marrow stem cell populations was produced either by irradiating the donor mice with 3·36 Gy (336 rad) ⁶⁰Co-gamma rays 7 days before the experiment or by incubating normal bone marrow cells with 10–11 M concentration of isoproterenol. IF considerably reduced the number of CFUs in S phase in both cases without reducing the CFUs content of the samples. Injection of IF inducer (4 mg/kg poly I:C) into regenerating mice also inhibited the proliferation of CFUs without decreasing the femoral CFUs level. Regeneration kinetics of CFUs from irradiated poly I:C-treated mice ran parallel with that of irradiated untreated animals but showed a characteristic delay corresponding to approximately one CFUs doubling. A transient, non-cytotoxic proliferation inhibitory effect of IF or IF inducer is, therefore, proposed.     

Colony-forming units of the bone marrow and spleen of immunodeficient mice after stimulation of the cells with thymalin

It has been previously demonstrated by the authors that histological characteristics of colony-forming units (CFUs) in normal mice prove a certain shift in their differentiation in erythroid direction comparing to the bone marrow CFUs. Thymectomy of mature animals is accompanied with weakening growth of granular colonies at cloning of the bone marrow CFUs and with loss of stability in direction of splenic CFUs differentiation. Polypeptide preparation of the thymus--thymalin stimulates growth of the granulocytic colonies from the splenic CFUs in thymectomized mice both in in vivo and in vitro experiments. Differentiation of the bone marrow CFUs is normalized under the effect of thymalin in in vivo experiment only. The data obtained confirm the suggestion made by R. V. Petrov on existence of T-cell clone, enhancing CFUs differentiation in granulocytic direction. Activation of this clone in the spleen is revealed at thymectomy and stimulation of the cells with thymalin both in in vivo and in vitro experiments. Thus, affirmations are obtained on differences of clonic T-cell regulation of the CFUs differentiation in the bone marrow and in the spleen.     

Possible role of the T-cells in regulating in situ hematopoiesis

The aim of the study was to reveal the possible role of T cells in the negative regulation of hematopoiesis. The main experimental approach included incubation of bone marrow cells obtained from mice of different strains with the anti-serum against a specific marker of suppressor T cells--antigen I-J. Anti-I-Jk serum-treated cells and cells treated with nontoxic normal mouse serum or non-treated cells (controls) were further incubated with complement and tested for their CFUs content, using Till & McCulloch exocolonization technique. Treatment with anti-I-Jk serum had a stimulating effect on the CFUs colony formation in mice of the appropriate haplotype (CBA, AKR, A/Sn) bearing I-Jk, but not I-Jb (CC57Br) allele. The same results were obtained in transfer experiments using spleen cells; only in this case stimulating effect was observed in 7-8-day CFUs, while with the marrow transplant augmentation it was seen both 7-8 and 11-12 days following grafting. The seeding efficiency of CFUs was not changed after incubation with anti-I-J serum. The data prove that indigenous for the spleen and bone marrow of mice cells expressing I-J determinants are involved in the negative regulation of hematopoiesis in situ.     

Sialic acid: A specific role in hematopoietic spleen colony formation

Vibrio cholerae neuraminidase (VCN) treatment of donor bone marrow cells results in a reduction in the number of hematopoietic colonies (CFUs) formed in the spleens of lethally irradiated mice. Treatment of marrow cells with sodium periodate under mild conditions, known to preferentially oxidze sialic acid, also reduced CFUs while subsequent potassium borohydride reduction restored CFUs to 80% of control levels. Innoculum viability as measured by in vitro incorporation of tritiated precursors into proteins, nucleic acids, and oligosaccharides was unaffected by VCN treatment. The ability of bone marrow cells in culture to respond to the hormone erythropoietin, as measured by the incorporation of ⁵⁹Fe into cyclohexanone-extractable heme, was also not affected by neuraminidase, making a cytotoxic effect of the VCN preparation unlikely. Incubation of VCN-treated marrow with either β-galactosidase or trypsin had no effect on the VCN-induced reduction in CFUs. These results are consistent with the idea that membrane sialic acid plays a direct and specific role in the implantation and development of CFUs.     

The distribution of terminal deoxynucleotidyl transferase (TdT) among subsets of thymocytes in the rat.

Terminal deoxynucleotidyl transferase (TdT), a unique DNA-polymerizing enzyme,has been shown to be present in a moderately dense subpopulation of rat thymocytes separated on discontinuous Ficoll density gradients. This subpopulation has been characterized by using antigenic and functional markers to identify directly and quantify cortical and medullary thymocytes. The TdT-positive thymocytes are depleted by cortisone administration, lack responsiveness to phytohemagglutinin, concanavalin-A, and histocompatibility alloantigens, bear surface antigens characteristic of cortical thymocytes (bone marrow lymphocyte antigen) and lack surface antigens characteristic of medullary thymocytes (rat-masked thymocyte antigen and histocompatibility antigens). The results indicate that TdT is present exclusively (or in markedly higher concentrations) in a subset of cells which comprised about 65% of cortical thymocytes. Two other major subsets of cortical thymocytes were identified which appeared to be TdT-negative. A minor subset of very low density cortical thymocytes was also defined. These observations have provided insight into the possible pathways of thymocyte ontogeny.     

Effect of Hyperthyroidism On Haemopoietic Stem Cell Kinetics In Mice

Changes in the pool of haemopoietic colony-forming units (CFUs) of bone marrow and spleen were studied in experiments with mice fed dried thyroid gland (TH) for 21 days, and during the 13 days that followed feeding. After HU treatment, the number of CFUs in DNA synthesis was estimated. As early as the second day of TH treatment, the pool of CFUs is gradually increased, leading to an increase in the total number of splenic and bone marrow CFUs persisting after TH treatment for the period examined. Simultaneously, the numbers of nucleated cells in the bone marrow and spleen are increased. During TH feeding and following its termination, the total number of erythrocytes and the haematocrit values did not change significantly, whereas an increased number of leucocytes was observed in the peripheral blood after TH treatment. Elevation of the proliferative activity of CFUs occurred early in the period of TH treatment, with the maximum attained by end of the first week of TH feeding. This suggests a rapid response of the haemopoietic stem cell compartment to the administration of TH hormones. the participation of humoral factors controlling CFUs compartments in the mechanism of the stimulatory effect of TH hormones on haemopoietic stem cells is discussed.