Endosteal cells of the bone marrow stroma in human iliac bone

The data on histological and electron microscopical investigation of the endosteal cells of the human iliac bone are presented. Three types of stromal elements in the endosteum, differing in their ultrastructural organization have been revealed. In the endosteal areas young hemopoietic cells are present, they are closely connected with the stroma. A suggestion is made on an important role of the endosteum in processes of proliferation and differentiation of hemopoietic predecessors.     

Sinusoids of the bone marrow stroma in the human iliac bone

Results of histological and ultramicroscopic investigations of sinusoid vessels are presented. Interrupted structure of the basal membrane and continuous endothelial lining are revealed. Endotheliocytes of two types are described. At places of blood cells migration functional activity of endothelium is increased and the basal membrane is absent. A suggestion is made that the endothelial cells directly influence hemopoiesis.     

Resistance of bone marrow stroma to genotoxic preconditioning is determined by p53

Transplantation of bone marrow (BM) is made possible by the differential sensitivity of its stromal and hematopoietic components to preconditioning by radiation and/or chemotherapeutic drugs. These genotoxic treatments eliminate host hematopoietic precursors by inducing p53-mediated apoptosis but keep the stromal niche sufficiently intact for the engraftment of donor hematopoietic cells. We found that p53-null mice cannot be rescued by BM transplantation (BMT) from even the lowest lethal dose of total body irradiation (TBI). We compared structural changes in BM stroma of mice differing in their p53 status to understand why donor BM failed to engraft in the irradiated p53-null mice. Irradiation did not affect the general structural integrity of BM stroma and induced massive expression of alpha-smooth muscle actin in mesenchymal cells followed by increased adiposity in p53 wild-type mice. In contrast, none of these events were found in p53-null mice, whose BM stroma underwent global structural damage following TBI. Similar differences in response to radiation were observed in in vitro-grown bone-adherent mesenchymal cells (BAMC): p53-null cells underwent mitotic catastrophe while p53 wild-type cells stayed arrested but viable. Supplementation with intact BAMC of either genotype enabled donor BM engraftment and significantly extended longevity of irradiated p53-null mice. Thus, successful preconditioning depends on the p53-mediated protection of cells critical for the functionality of BM stroma. Overall, this study reveals a dual positive role of p53 in BMT: it drives apoptotic death of hematopoietic cells and protects BM stromal cells essential for its functionality.Subject terms: Haematopoietic stem cells, Stem-cell research     

The function of adipocytes in the bone marrow stroma

The fibroblasts and adipocytes of the bone marrow stroma provide the cytokines and extracellular matrix proteins required for the maturation and proliferation of the circulating blood cells. Due to the complexity of the bone marrow as an organ, the normal physiology of these stromal cells is not well understood. In particular, the role of adipocytes in the bone marrow remains controversial. Cloned bone marrow stromal cell lines provide an in vitro model for analysis of the lympho-hematopoietic microenvironment. These cells may be capable of multiple differentiation pathways, assuming the phenotype of adipocytes, chondrocytes, myocytes, and osteocytes in vitro. Characterization of these cell lines and recent in vivo experiments give new insight into the normal physiology of the bone marrow.     

Rescue of myeloid lineage-committed preprogenitor cells from cytomegalovirus-infected bone marrow stroma.

The effect of murine cytomegalovirus on myelopoiesis was studied in long-term bone marrow culture to find an in vitro correlate for the lethal virus interference with bone marrow reconstitution (W. Mutter, M. J. Reddehase, F. W. Busch, H.-J. Bühring, and U. H. Koszinowski, J. Exp. Med. 167:1645-1658, 1988). The in vitro generation of granulocyte-monocyte progenitors (CFU-GM) discontinued after infection of the stromal cell layer, whereas the proliferation and differentiation of CFU-GM to granulocyte-monocyte colonies remained unaffected. A protocol was established to probe the functional integrity of earlier hematopoietic cells. Pre-CFU-GM (the progenitors of the CFU-GM) could be recovered from an infected bone marrow donor culture by transfer onto an inductive recipient stromal cell layer. Thus, at least in vitro, infection of bone marrow stroma appears to be the only cause of the defect in myelopoiesis.     

Adipocytes and reticular cells of the bone marrow stroma in the human iliac bone

The results of the histological and electron microscopic investigation of adipose and reticular cells and their interconnections with blood cells are presented in the material of trephine biopsies of the iliac bone. A possibility for development of adipocytes from the adventitial reticular cells is demonstrated. Close contacts are revealed between pre-adipocytes and young hemopoietic cells. Two types of the reticular cells are characterized, they differ in their position, structural organization and interconnection with the young hemopoietic elements. The peculiarities revealed in the morphofunctional state of the microenvironmental structures demonstrate functional variegation of the stromal elements, and also attest an essential importance of intercellular contacts of the hemopoietic predecessors and the stromal cells in maintaining the hemopoietic function of the bone marrow.     

Platelet-derived growth factor-stimulated versican synthesis but not glycosaminoglycan elongation in vascular smooth muscle is mediated via Akt phosphorylation

Proteoglycans are associated with the initiation of atherosclerosis due to their binding of apolipoproteins on lipid particles leading to retention in the vessel wall. The signaling pathways through which growth factors regulate the synthesis and structure of proteoglycans are potential therapeutic targets. Platelet-derived growth factor (PDGF) is present in atherosclerotic plaques and activates phosphorylation of the serine/threonine kinase Akt. We have investigated the role of Akt in the signaling pathways for proteoglycan core protein expression and elongation of glycosaminoglycan chains on proteoglycans secreted by human vascular smooth muscle cells. The pharmacological inhibitor of Akt phosphorylation, SN30978, blocked PDGF stimulated phosphorylation of Akt. SN30978 caused concentration dependent inhibition of PDGF stimulated radiosulfate incorporation into secreted proteoglycans and the response was blocked by the PDGF receptor antagonists Ki11502 and imatinib. Analysis of the size of the biglycan molecules by SDS-PAGE showed that PDGF increased the apparent size of biglycan but this effect on glycosaminoglycan chain elongation was blocked by Ki11502 but not by SN30978. PDGF also stimulated total protein core protein synthesis assessed as ³⁵S-methionine/cysteine incorporation and specifically the expression of versican mRNA. Both of these responses were blocked by SN30978. This data shows that PDGF-stimulated proteoglycan core protein synthesis but not glycosaminoglycan chain elongation is mediated via Akt phosphorylation. These data identify potential pathways for the development of agents which can pharmacologically regulate individual components of the synthesis of proteoglycans.     

Age-related changes in the capacity of bone marrow cells to differentiate in thymic organ cultures

The capacity of the bone marrow to give rise to T cells in advanced age was studied in vitro by reconstituting fetal thymic lobes from 14-day C57BL/Ka (Thy-1.1) mice with bone marrow cells from old (24-month) or young (3-month) C57BL/6 (Thy-1.2) mice. The use of these congenic strains enabled distinguishing between donor and host contribution to the developing T cells. We found that bone marrow cells from aged mice maintained their capacity to reconstitute fetal thymic explants and to differentiate into various T-cell subsets as assessed by distinct T-cell-specific surface markers (Thy-1, Lyt-1, Lyt-2, and L3T4) and functions (concanavalin A-induced proliferative and cytotoxic responses). However, when mixtures of old and young bone marrow cells reconstituted fetal thymic explants, the cells of old mice were less efficient than those of young in their capacity to give rise to T cells. These results indicate that bone marrow cells from aged mice can reconstitute the thymus and differentiate into T cells; however, their reconstituting capacity is inferior to that of bone marrow cells from young mice.     

Role of bone marrow stroma in the phenomenon of hybrid resistance]

In the site of hemopoiesis produced by implantation of an irradiated bone marrow plug beneath the kidney capsule all dividing hemopoietic cells were of recipient origin, as shown by karyologic analysis. In such implants hybrid resistance was of the donor's type. It is suggested that the migrating cells of hemopoietic origin, including lymphocytes and macrophages, do not participate in producing the hybrid resistance.     

Reduced gap strains induce changes in bone regeneration during distraction.

A bilateral New Zealand white rabbit model of distraction osteogenesis (DO) was used to investigate the relationship between strain environment and bone regeneration during limb lengthening. In seven (n = 7) rabbits, a stiffener was applied to the fixator on one side to reduce strains within the gap tissue after lengthening was completed. Animals were euthanized six days later and their distraction zones were harvested and analyzed for changes in new bone volume and architecture. Nonlinear finite element analyses (FEA) were performed to predict changes in the gap strain environment. FEA results predicted a nearly uniform sevenfold decrease in average strain measures within the distraction zone. No change in total average new bone volume and significant decreases in both bone volume fraction (BV/TV) and trabecular thickness (Tb.Th) were observed in tibiae in which gap strains were reduced experimentally, compared to contralateral controls. These results suggest that fixator stiffening influenced the architecture but not the amount of newly formed bone. This animal model of distraction might be used to study the mechanisms by which strain fields affect events in bone repair and regeneration, such as cell proliferation, precursor tissue differentiation, and altered growth factor and nutrient delivery to tissues.     

Cytotoxicity in feline leukemia virus subgroup-c infected fibroblasts is mediated by adherent bone marrow mononuclear cells

Summary The pathogenesis of retrovirus-induced erythroid aplasia in cats is unknown. In studies to define mechanisms of cytotoxicity associated with retroviral infections, bone marrow mononuclear cells (BMMC) from healthy specific pathogenfree cats were co-cultured with uninfected feline embryonic fibroblasts (FEA cells) and FEA cells infected with feline leukemia virus (FeLV) of subgroup A (FEA-A) or subgroup C (FEA-C). Moderate to marked cytotoxicity (CPE) developed in co-cultures of BMMC and FEA-C cells on Days 5 to 7 of incubation but not in co-cultures of BMMC and FEA-A or BMMC and uninfected cells (FEA-CT). Cytotoxicity was associated with adherent cells of light density (1.056) from bone marrow and peripheral blood, which were positive for alpha naphthyl butyrate esterase activity. Stimulation of adherent cells with phorbol ester or addition of recombinant human tumor necrosis factor-alpha (rhTNF-α) caused similar CPE in FEA-CT cells. The TNF-α concentrations in the culture supernatants of BMMC+FEA-C were higher than those of BMMC+FEA-A or BMMC+FEA-CT, and addition of anti-TNF antibodies to the cultures blocked the CPE. These data support the hypothesis that macrophages exposed to FeLV-C cause CPE in co-cultures of BMMC and FEA cells by a mechanism involving TNF-α. It is suggested that TNF-α may be involved in the suppression of hematopoiesis in cats which develop FeLV-C induced erythroid aplasia.     

The proliferative capacity of pure red cell aplasia bone marrow cells.

Pure red cell aplasia (PRCA) is a heterogeneous disorder. Immunologic abnormalities have recently been uncovered suggesting that both cell-mediated and humoral immune mechanisms may be of etiological importance in PRCA. Utilizing a technique for the cloning of bone marrow erythroid precursors, we determined the in vitro proliferative capacity of erythroid cells obtained from 21 patients with PRCA. Bone marrow cells from one group of patients produced normal or increased numbers of erythroid colonies while the in vitro proliferative capacity of bone marrow cells from a second group was characterized by subnormal erythroid colony formation. Sera obtained from the former group was frequently associated with demonstrable serum inhibitors of erythropoiesis, while PRCA in the latter group was probably the consequence of intrinsic erythroid stem cell defects or pathologic cellular interactions with nonerythroid regulatory cells. This survey of a relatively large population of patients with PRCA provides evidence for the multiple causative mechanisms that can be operative in the production of PRCA.     

Salinity-induced inhibition of leaf elongation in maize is not mediated by changes in cell wall acidification capacity

The physiological mechanisms underlying leaf growth inhibition under salt stress are not fully understood. Apoplastic pH is considered to play an important role in cell wall loosening and tissue growth and was demonstrated to be altered by several growth-limiting environmental conditions. In this study we have evaluated the possibility that inhibition of maize (Zea mays) leaf elongation by salinity is mediated by changes in growing cell wall acidification capacity. The kinetics of extended apoplast pH changes by leaf tissue of known expansion rates and extent of growth reduction under stress was investigated (in vivo) and was found similar for non-stressed and salt-stressed tissues at all examined apoplast salinity levels (0.1, 5, 10, or 25 mM NaCl). A similar rate of spontaneous acidification for the salt and control treatments was demonstrated also in in situ experiments. Unlike growing cells that acidified the external medium, mature nongrowing cells caused medium alkalinization. The kinetics of pH changes by mature tissue was also unchanged by salt stress. Fusicoccin, an enhancer of plasmalemma H(+)-ATPase activity level, greatly stimulated elongation growth and acidification rate to a similar extent in the control and salt treatments. That the ability of the growing tissue to acidify the apoplast did not change under same salt stress conditions that induced inhibition of tissue elongation rate suggests that salinity does not inhibit cell growth by impairing the acidification process or reducing the inherent capacity for cell wall acidification.     

Modification of the proliferative capacity of transplanted bone marrow colony forming units by changes in the host environment

Regulation of the proliferation of transplanted colony forming units (CFUs) was investigated in lethally irradiated mice, pretreated by methods known to accelerate hemopoietic recovery after sublethal irradiation. Prospective recipients were exposed to either hypoxia, vinblastine or priming irradiation and at different intervals thereafter lethally irradiated and transplanted with bone marrow. Repopulation of CFUs was determined by counting the number of splenic colonies in primary recipients or by retransplantation. Regeneration of grafted CFUs was greatly accelerated and their self-renewal capacity increased in mice grafted within two days after hypoxia. Also the number of splenic colonies formed by grafted syngeneic CFUs as well as by C57BL parent CFUs growing in BC3F1 hosts was significantly increased. The effect was not dependent on the seeding efficiency of CFUs and apparently resulted from hypoxia induced changes in the hosts physiological environment. Proliferative capacity of grafted CFUs increased remarkably in hosts receiving vinblastine two or four days prior to irradiation. Priming irradiation given six days before main irradiation accelerated, given two days before impaired regeneration of CFUs. The increased rate of regeneration was not related to the cellularity of hemopoietic organs at the time of transplantation. The growth of CFUs in diffusion chambers implanted into posthypoxic mice was only slightly improved which does indicate that the accelerated regeneration of CFUs in posthypoxic mice is mainly due to the changes in the hemopoietic microenvironment. A short conditioning of transplanted CFUs by host factor(s) was sufficient to improve regeneration. The results might suggest that the speed of hemopoietic regeneration depends on the number of CFUs being induced to proliferate shordy after irradiation, rather than on the absolute numbers of CFUs available to the organism.     

Cellular changes in bone marrow of malaria-infected mice. III. Chemotaxis of granulocytes

The number of bone marrow cells and their chemotactic activity was studied during malaria infection. Two days after infection of Balb/c mice with Plasmodium berghei, an increase in granulocyte number was observed in the blood. A modified Boyden chamber chemotaxis assay was employed to investigate the mechanism of granulocyte accumulation in the blood. Bone marrow cells from normal mice, from mice during a primary lethal infection and from immune mice after challenge were compared. The complement factor C5a showed chemotactic activity for bone marrow cells; a significant decrease of chemotaxis was only observed after 6 days of primary infection. Extracts of spleen, liver and infected erythrocytes lacked chemotactic activity, or caused inhibition of cell migration. Serum from mice with a 2-day primary infection contained chemotactic activity. The active component was heat labile, protease sensitive and had an estimated molecular weight of 250,000.     

Estimation of effective eosinopoiesis and bone marrow eosinophil reserve capacity in normal man.

The eosinophil reserve capacity of the post-mitotic granulocyte compartment in the bone marrow and the effective eosinopoiesis in three haematologically normal men have been quantified by means of kinetic parameters of [3H]thymidine flash-labelled peripheral blood eosinophils. From (a) the time of the appearance in the blood of labelled eosinophils after the tracer injection, (b) the inflow characteristics of the labelled eosinophils in the blood and (c) the magnitude of the eosinophil granulocyte pool in the venous blood, the effective eosinopoiesis (i.e. the eosinophil turnover) was calculated to range between 0.014 and 0.031 x 10(9) cells/kg body weight per day (mean 0.22 x 10(9) cell/kg per day). The post-mitotic eosinophil reserve capacity of the bone marrow ranged from 0.09 to 0.20 x 10(9) cells/kg body weight (mean 0.14 x 10(9) cells/kg). The large reserve pool and the high turnover rate may contribute to sudden rises of the peripheral blood oesinophil counts in some cases of eosinophilia.     

The effect of aluminium upon the activity of selected bone marrow enzymes in rats.

This study was carried out on male Wistar rats. The bone marrow activities of acetylcholinesterase (AchE-EC.3.1.1.7.), glutathione reductase (GR-EC.1.6.4.2.) and glucose-6-phosphate dehydrogenase (G6PD-EC.1.1.1.49.) were determined as affected by aluminium. The present experiments have revealed that within the first days there will be an increase in enzyme activity in the bone marrow. Simultaneously a decline in the activity of bone marrow could be observed which occurred later in the first course of the experiment.