Factors controlling the recirculation of hematopoietic stem cells. IV. The effect of thymosin on the migration and differentiation of hematopoietic stem cells

Thymosine, a thymus hormone, restores the thymectomy induced deterioration of the routine pathways of migration and differentiation ofhemopoietic stem cells in mice. Administration of thymosine together with bone marrow cells from thymectomized mice to irradiated recipients also restores the level of migration and differentiation of hemopoietic stem cells. The inducing effect of thymosine on the maturation of T-lymphocyte precursors, which in their turn restore the usual rate of migration and differentiation of hemopoietic stem cells, has been suggested.     

Different effect of testosterone on polypotential stem hematopoietic stem cells and immunocompetent B-lymphocytes]

A research was made to study the dynamics of the proliferative, colony-forming and migration capacity of stem hemopoietic cells in (CBA X C57Bl) F1 hybrid mice under the influence of testosterone propionate, 10 mg/100 g, as well as the migration of immunocompetent B lymphocytes from the bone marrow to the spleen and the accumlation of their progeny, antibody-producing cells, in the spleen. The immunodepressive effect of testosterone was manifested by a decrease in the migration of B cells and the number of antibody-producing cells in the spleen. On the contrary, testosterone had a stimulating effect on the functional activity of stem hemopoietic cells, increasing their proliferation and migration. Under conditions of the suppressed erythropoietic differentiation of multipotent stem hemopoietic cells the injection of testosterone resulted in an increase in the number of antibody-producing cells in the spleen. This suggests that the stimulation of erythropoiesis and immunosuppression, induced by testosterone, are interconnected and determined by the direct action of the hormone on the cellular cycle of the stem cells, as well as by their prevailing differentiation towards the erythroid series, resulting in the decrease of their differentiation into B cells.     

Preparation of radioactive iodinated cholylhistamine for use in the radioimmunoassay of cholic acid

A major handicap in the development of simple and accurate radioimmunoassay procedures for bile acids has been the lack of a radioactive standard of high specific activity. To provide such a compound, we first synthesized cholylhistamine using the carbodiimide reaction. The hypothesized structure was confirmed by elemental analysis, thin-layer chromatography, infrared and mass spectral analysis. The cholylhistamine was then iodinated with 125I, using the choloramine-T method. The 125I-cholylhistamine was bound by antisera raised against a cholic acid-bovine serum albumin conjugate. This procedure should prove useful in preparing radioactive conjugates for all of the bile acids.     

Effect of various factors on the differentiation of hematopoietic stem cells]

The treatment in vitro of bone marrow cells in mice by phytohemagglutinin, concanavaline, or antilimphocytic globulin resulted in the suppression of exogenous hemopoietic colonies in the spleen of lethally irradiated (830r) syngenic recipients, whereas lipopolysaccharide, tuberculin, anti-theta serum or nati-gamma-globulin serum exerted no influence on the colony-forming function of hemopoietic stem cells. The morphological analysis of the ratio and cell composition of hemopoietic colonies has revealed no marked differences between the experimental and control groups. The suppression of hemopoietic stem cells by mitogens might be due both to their direct effect and indirect one, possibly, through a humoral factor.     

Studies on the kinetics of hemopoietic stem cells and immune responses to the hapten-carrier conjugate.

The correlation between the kinetics of hemopoietic stem cells and immune responses to the hapten-carrier conjugate was investigated. The numbers of both pluripotent stem cells (CFU-S) and myeloid stem cells (CFU-C) in the spleen from mice immunized with the hapten-carrier conjugate were significantly greater than those of the control and the activity of colony-stimulating factor (CSF) in the serum of these mice was markedly elevated. The supernatant of short-term incubation of splenic T lymphocytes from these mice, when stimulated with carrier protein, had high levels of both activities of CSF and helper T cell factors. The study by gel chromatography showed that these factors are similar m.w. substances of 35,000 to 45,000 daltons. But analysis by ion-exchange chromatography demonstrated that they do not have identical biochemical properties. The present studies suggest that biologically active factors produced by T cells stimulated with carrier protein may induce the enhancing effect on the proliferation and differentiation of hemopoietic stem cells and immune responses to the hapten-carrier conjugate.     

Characteristics of lymphocytoid cells detected during post-radiation bone marrow recovery]

As a result of complex study of lymphocyte-like cells which arise in the regenerating bone marrow of sublethally irradiated mice, it was established that: (1) X-cells precipitate in the 2nd and, partially, 3rd fractions of the albumin gradient, i. e. in the same fractions as the stem cells of intact animals; (2) X-cells do not belong to polypotent hemopoietic stem cells; (3) X-cells belong to B-lymphocytes, i. e. to thymus-independent lymphocytes.     

Characteristics of the differentiation of hematopoietic stem cells of the bone marrow in experimental lesions of the liver

The liver lesion in the CBA mice has been induced by administration of one of three agents five times every day; gamma-globulin fraction of antihepatocytotoxic serum in doses of 4.8 and 7.7 mg of protein per 100 g of body mass; gamma-globulin fraction of normal rabbit serum and bovine serum albumin in a dose of 4.8 mg of protein; three- four- or five-fold introduction of carbon tetrachloride in a dose of 0.5 ml per 100 g of body mass with oil (1:1) each three days; calibrated stenosis of the portal vein was produced. The total number of hemopoietic stem cells in the bone marrow was estimated by the colony-forming unit/spleen assay. Histological analysis of the colony-forming units was applied. The liver lesion was accompanied by a decrease in the ratio of the erythroid/granulocytic colonies.     

Density gradient centrifugation of hemopoietic colony-forming cells

Buoyant density distributions of hemopoietic colony-forming units (CFU) from normal mouse marrow were determined by equilibrium density gradient centrifugation in bovine serum albumin (BSA) gradients. The distributions were compared with those obtained for the total population of nucleated cells from normal mouse marrow. The buoyant density distribution for CFU was found to differ from the density distribution for the total nucleated cell population, and the portion of the total cell population with densities much less than the mean value was found to contain up to a 30-fold greater proportion of CFU than an uncentrifuged control. These results provide a preliminary approach to the purification and characterization of normal hemopoietic colony-forming stem cells.     

Effect of mouse age on the ability of hematopoietic stem cells to interact with thymus cells]

The effect of the thymus cells of the C57BL/6 mice on the colony forming ability of the stem hemopoietic cells of the embryonic liver and bone marrow of young (3 months) and old (2 years) mice was studied their joint transplantation into the mice (CBAXXC57BL/6) F1. The stimulating effect of the thymus cells on the colony forming ability of the stem hemopoietic cells of different age depends both on the dose of the stem hemopoietic cells of embryonic liver and the dose of T-lymphocytes. A suggestion is put forward that the stimulating effect of the thymus cells on the colony formation is due to their interaction with the stem cells in the G2 phase of the mitotic cycle.     

Relationship between hematopoietic stem differentiation and the functional state of the thymus]

The direction of differentiation of the stem cells with respect to the physiological activity of thymus determined by the age of an animal was studied by means of histological analysis of hemopoietic colonies in the spleen of lethally irradiated mice. The immaturity of thymus of its involution are characterized by the inhibition of differentiation of the stem cell along the granuloid path. An analysis of the data on differentiation of the stem cells in mice of different age, as well as in thymectomized mice allows to draw a conclusion that the process of differentiation of the hemopoietic stem cells is thymus-dependent.     

The positive regulatory effect of TGF-beta2 on primitive murine hemopoietic stem and progenitor cells is dependent on age, genetic background, and serum factors

TGF-beta is considered a negative regulator of hemopoietic stem and progenitor cells. We have previously shown that one TGF-beta isoform, TGF-beta2, is, in fact, a positive regulator of murine hemopoietic stem cell function in vivo. In vitro, TGF-beta2, but not TGF-beta1 and TGF-beta3, had a biphasic dose response on the proliferation of purified lin-Sca1(++)kit(+) (LSK) cells, with a stimulatory effect at low concentrations, which was subject to mouse strain-dependent variation. In this study we report that the stimulatory effect of TGF-beta2 on the proliferation of LSK cells increases with age and after replicative stress in C57BL/6, but not in DBA/2, mice. The age-related changes in the TGF-beta2 effect correlated with life span in BXD recombinant strains. The stimulatory effect of TGF-beta2 on the proliferation of LSK cells requires one or more nonprotein, low m.w. factors present in fetal calf and mouse sera. The activity of this factor(s) in mouse serum increases with age. Taken together, our data suggest a role for TGF-beta2 and as yet unknown serum factors in the aging of the hemopoietic stem cell compartment and possibly in organismal aging.     

Stem cells and T- and B-lymphocytes in acute hypoxia

During the stepped rapid training of mice for hypoxia the number of colony-forming units in the blood and bone marrow increases and that in the spleen falls down. In acute hypoxic hypoxia there is an enhancement of the migration of stem hemopoietic cells and B-lymphocytes from the bone marrow and T-lymphocytes from the thymus.     

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.     

Mock retroviral infection alters the developmental potential of murine bone marrow stem cells.

Retroviral vectors were used to introduce an activated ras gene into murine pluripotent hemopoietic stem cells. We attempted to reconstitute the hemopoietic system of lethally irradiated mice with isolated spleen colonies obtained in vivo after injection of infected bone marrow cells. Spleen colonies derived from infected bone marrow were inefficient in promoting long-term survival of irradiated hosts. This loss of reconstitutive capacity of spleen colonies was not due to the retroviral infection per se but to the in vitro culture of spleen colony precursors. Incubation for 24 h in the presence of fetal calf serum and interleukin-3 without virus-producing cells was sufficient to abolish completely the reconstitutive capacity of spleen colonies while maintaining both self-renewal and pluripotential capacities of spleen colony precursors. These results show that the in vitro manipulation of stem cells that is included in current protocols for retroviral infection can modify the developmental potential of these cells. This finding clearly indicates that the use of retroviral vectors can introduce a bias in the analysis of hemopoiesis.     

The origin of hematopoietic stem cells in the ontogenesis of vertebrates

A review of the origin of stem blood cells in ontogeny of vertebrates is presented. The comparative analysis of the data on laying, determination and migration of the hemopoietic precursor cells during embryogenesis in various taxonomic groups (teleosteans, urodeleans, anurans, avians and mammals) is performed. The change of the hemopoietic site and erythroid cells populations has been described. The data on sources of blood cell precursors and the origin of hemopoietic cells in the primordiums of hemopoietic organs were classified. A conclusion has been reached that in the course of evolution the hemopoietic anlage is gradually divided into two parts: one part migrates to the extraembryonic (ventral) mesoderm and another one remains intraembryonically and gives rice to the predecessors of definitive hemopoietic stem cells.     

Binding of histamine- and other ligand-conjugated macromolecules to lymphocytes

The presence of histamine receptors on lymphocyte membranes was investigated using conjugates of histamine and macromolecules tritiated or iodinated with I-125. Histamine-RSA conjugate binds to lymphocytes and causes patching and capping of the bound conjugate. It was found, however, that free histamine did not inhibit the binding of histamine-rabbit serum albumin to mouse lymphocytes, nor did His-RSA interfere with the binding of free histamine. In addition conjugates between RSA and other small molecules, such as ethylamine, ethanolamine, tyramine and glycine, were found to bind to the same sites on lymphocyte membrane as did His-RSA. Ethylamine-RSA like His-RSA when coupled to Sepharose, was capable of removing antibody producing cells from spleen cells of mice immunized against sheep red blood cells. In addition, when spleen cells from such immunized mice were passed through ethylamine or histamine-RSA-Sepharose and the unbound cells were subsequently injected into X-irradiated mice, a 1.8 fold increase in the immunological response was noted. We conclude that the selective binding to lymphocytes of the various ligand-macromolecular conjugates may be due to some general properties of the cell membrane and not to any specific receptors. Nevertheless, these conjugates can be used as a tool to remove selectively antibody producing cells as well as some regulatory cells.     

Synergism among interleukin 1, interleukin 3, and interleukin 5 in the production of eosinophils from primitive hemopoietic stem cells

The in vitro production of eosinophils from committed progenitor cells is influenced by interleukin (IL)-5 (eosinophil differentiation factor) and to a lesser extent by IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF). In primary suspension cultures of marrow cells taken from eosinophilic mice, IL-3 induced a modest stimulation of eosinophil production compared to IL-5. In contrast, IL-3 was sevenfold more effective than IL-5 in generating eosinophil progenitors (eosinophil colony-forming units (CFU-eo] from more primitive precursors present in the marrow of normal mice. Pre-incubation of marrow cells in suspension culture with IL-3, but not IL-5, increased the recovery of myeloid precursors responsive to G-CSF, GM-CSF, CSF-1, or IL-3 two- to fourfold while eosinophil progenitor cells responsive to IL-5 were increased by more than 70-fold. Similarly, pre-incubation of bone marrow cells under clonal conditions with IL-3, but not IL-5, resulted in a more than 50 fold increase in CFU-eo responsive to IL-5 over input values. Bone marrow from mice pre-treated with 5-fluorouracil is greatly depleted of progenitor cells directly responsive to IL-3 or IL-5. IL-1 which synergistically interacts with various CSF species to confer a clonogenic response by primitive stem cells present in 5-fluorouracil-treated marrow also failed to stimulate eosinophil production. A marked synergism was observed when IL-1 and IL-3 were combined in the suspension pre-culture phase with a more than sixfold recovery of CFU-eo than induced by either factor alone. Furthermore, pre-culture of 5-fluorouracil-treated marrow cells with a combination of IL-1 and IL-3 resulted in a more than 260-fold increase of CFU-eo over input numbers. These data suggest that the concatenate action of IL-1, IL-3, and IL-5 is an absolute requirement for the in vitro generation of eosinophils from primitive hemopoietic stem cells.     

Cell surface peptidase CD26/dipeptidylpeptidase IV regulates CXCL12/stromal cell-derived factor-1 alpha-mediated chemotaxis of human cord blood CD34+ progenitor cells

CD26/dipeptidylpeptidase IV (DPPIV) is a membrane-bound extracellular peptidase that cleaves dipeptides from the N terminus of polypeptide chains. The N terminus of chemokines is known to interact with the extracellular portion of chemokine receptors, and removal of these amino acids in many instances results in significant changes in functional activity. CD26/DPPIV has the ability to cleave the chemokine CXCL12/stromal cell-derived factor 1alpha (SDF-1alpha) at its position two proline. CXCL12/SDF-1alpha induces migration of hemopoietic stem and progenitor cells, and it is thought that CXCL12 plays a crucial role in homing/mobilization of these cells to/from the bone marrow. We found that CD26/DPPIV is expressed by a subpopulation of CD34(+) hemopoietic cells isolated from cord blood and that these cells have DPPIV activity. The involvement of CD26/DPPIV in CD34(+) hemopoietic stem and progenitor cell migration has not been previously examined. Functional studies show that the N-terminal-truncated CXCL12/SDF-1alpha lacks the ability to induce the migration of CD34(+) cord blood cells and acts to inhibit normal CXCL12/SDF-1alpha-induced migration. Finally, inhibiting the endogenous CD26/DPPIV activity on CD34(+) cells enhances the migratory response of these cells to CXCL12/SDF-1alpha. This process of CXCL12/SDF-1alpha cleavage by CD26/DPPIV on a subpopulation of CD34(+) cells may represent a novel regulatory mechanism in hemopoietic stem and progenitor cells for the migration, homing, and mobilization of these cells. Inhibition of the CD26/DPPIV peptidase activity may therefore represent an innovative approach to increasing homing and engraftment during cord blood transplantation.     

Effect of erythrocyte breakdown products on mast cells and erythropoietin formation]

Experiments were conducted on CBA mice and albino rats. A study was made of the effect of erythrocyte destruction products (EDP) on the content of hemopoietic colony-forming units (CFU), differentiation of stem cells and the erythropoietin production. It was shown that 3 or 4 EDP injections to normal mice or to lethally irradiated (1000 rad) mice after the transplantation of bone marrow cells caused no changes in the CFU level of stem cells differentiation. In case of a daily (for 3 days) administration of EDP to mice before the irradiation (1000 rad) and bone marrow transplantation there was observed an increase of the colonies count in the recipients' spleen on account of the erythroid colonies. EDP injection caused no changes in the erythropoietic activity of the blood serum. A possible role of erythrocyte destruction products in the mechanism of erythropoiesis autoregulation is discussed.     

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.