High expression of the Thy-1 antigen on natural killer cells recently derived from bone marrow

The subset of murine natural killer (NK) cells that kills lymphoma targets contains about 50% cells expressing the Thy-1 antigen and this has been one of the reasons for assigning NK cells to the T-cell differentiation lineage. It has now been shown that the proportion of the Thy-1+ NK cells is not constant: ca. 90% of the NK cells appearing in the spleens of irradiated mice injected 10-14 days previously with bone marrow cells (anti-Thy-1 plus complement treated) express this antigen. The donor origin of these Thy-1+ NK cells was demonstrated by using semisyngeneic bone marrow cells in transfers but this same phenomenon could also be observed after entirely syngeneic transfers, excluding the possibilities that this Thy-1+ NK activity is due to activated T cells or to the effect of T-cell activation products on NK cells. Additionally, these early NK cells expressed the asialo-GM1 antigen, which is found on murine NK cells but not on cytotoxic T cells. These data suggest that the precursors for NK cells in the bone marrow are Thy-1-, and that the first splenic NK cells derived from these progenitors express this antigen.     

Pluripotential hematopoietic stem cells in post-5-fluorouracil murine bone marrow express the Thy-1 antigen

Expression of the Thy-1 alloantigen by hematopoietic stem and progenitor cells in post-5-fluorouracil (5-FU) murine bone marrow was investigated. FACS analysis of BDF1 bone marrow stained for Thy-1.2 with a triple-layer amplified labeling technique demonstrated that 35% of the total bone marrow population expressed Thy-1.2 (Thy-1.2+). Two distinct size subpopulations were observed in post-5-FU BDF1 marrow. Thy-1.2+ cells were present in both the large and the small subpopulations. FACS-separated bone marrow cells were also plated in methylcellulose cultures. Ninety percent of all colony-forming cells surviving in vivo administration of 5-FU were Thy-1.2+. Replating of primary hemopoietic colonies and morphologic examination of primary and secondary colonies demonstrated that the most primitive stem cells including "stem" (S) cells were Thy-1.2+. These cells (Thy-1.2+) were capable of self-renewal in vitro and exhibited multiple differentiation potentials in comparison to Thy-1.2-cells, which lacked significant self-renewal capability and were mono- or bipotent progenitor cells. Separation of Thy-1.2+ cells into large or small Thy-1.2+ subpopulations showed that only the large Thy-1.2+ colony-forming cells possessed significant self-renewal capacity. Treatment of BDF1 bone marrow with anti-Thy-1.2 plus complement reduced primary colony formation by 67% and eliminated those colony-forming cells which had extensive self-renewal properties. In the presence of PWMSCM, depletion and reconstitution of T lymphocytes had no effect on primary or secondary colony formation. These data demonstrate that Thy-1 is present on primitive hematopoietic stem cells in post-5-FU bone marrow. In addition, they show that the murine S cell is Thy-1+.     

Biosynthesis of mouse Thy-1 antigen

The biosynthesis and the maturation of Thy-1 antigen of mouse thymocytes have been studied by using a xenogeneic rabbit anti-mouse Thy-1 antibody. The earliest form of Thy-1 detected after a 5-min pulse with [35S]methionine and [35S]cysteine had an apparent m.w. of 26,500. During chase, this band converted to a molecular ratio (Mr) = 25,000 polypeptide, probably derived from the latter by trimming of glucose or mannose residues from the three high-mannose glycan units of Thy-1. Mature Thy-1 molecules were detected at the cell surface after a 15-min chase. At least one of the three N-linked oligosaccharide units was shown to be in the high mannose form at the cell surface, as indicated by its susceptibility to endo-beta-N-acetylglucosaminidase H digestion. Treatment of the early and late forms of Thy-1 antigen with endo-beta-N-acetylglucosaminidase F generated a single polypeptide of Mr = 13,500. The same precursor was obtained when cells were labeled in the presence of tunicamycin. This indicates the absence of O-linked glycan in the mature cell surface antigen. Finally, the resistance of Thy-1 antigen to trypsin digestion when associated with membranes confirmed that this molecule has no cytoplasmically oriented portion.     

Stimulation by normal and leukemic mouse sera of colony formation in vitro by mouse bone marrow cells

Using a modification of the agar gel method for bone marrow culture, serum from various strains of mice has been tested for colony stimulating activity. Ninety percent of sera from AKR mice with spontaneous or transplanted lymphoid leukemia and 40–50% of sera from normal or preleukemic AKR mice stimulated colony formation by C57B1 bone marrow cells. Sera from 6% of C3H and 30% of C57B1 mice stimulated similar colony formation. The incidence of sera with colony stimulating activity rose with increasing age. All colonies were initially mainly granulocytic in nature but later became pure populations of mononuclear cells. Bone marrow cells exhibited considerable variation in their responsiveness to stimulation by mouse serum. Increasing the serum dose increased the number and size of bone marrow cell colonies and with optimal serum doses, 1 in 1000 bone marrow cells formed a cell colony. Preincubation of cells with active serum did not stimulate colony formation by washed bone marrow cells. The active factor in serum was filterable, non-dialysable and heat and ether labile.     

Histamine production by alloantigen-activated mouse bone marrow cells

Histamine's contribution to the manifestations associated with graft-versus-host disease (GVHD) and/or hybrid resistance is unknown. Thus, we initiated studies to see whether or not mouse bone marrow cells could produce histamine upon alloantigen stimulation. Irradiated allogeneic spleen cells were shown to stimulate bone marrow cells to produce and secrete high levels of histamine. During 7 days of culture there was only a marginal increase in cell-associated histamine while the amount of histamine in the supernatant increased 10- to 20-fold. Optimal histamine production was dependent upon Lyt 1+2+ T cells resident in the bone marrow. Further, bone marrow cells from Nude mice failed to produce high levels of histamine following alloantigen stimulation. Soluble factors produced by alloantigen-stimulated bone marrow cells or by Con A-stimulated rat spleen cells induced high levels of histamine production in bone marrow cells in the absence of alloantigen. We suggest that histamine production by alloantigen-activated bone marrow cells may modulate immune functions following bone marrow transplantation.     

Gene expression profile of mouse bone marrow stromal cells determined by cDNA microarray analysis

Bone marrow stromal cells (BMSC) have gained increased attention because of their multipotency and adult stem cell character. They have been shown to differentiate into other cell types of the mesenchymal lineage and also into non-mesenchymal cells. The exact identity of the original cells, which are isolated from bone marrow by their selective adherence to plastic, remains unknown to date. We have established and characterized mouse BMSC cultures and analyzed three independent samples by cDNA microarrays. The expression profile was compared with two previous expression studies of human BMSC and revealed a high degree of concordance between different techniques and species. To gain clues about the positional context and biology of the isolated cells within the bone marrow stroma, we searched our data for genes that encode proteins of the extracellular matrix, cell adhesion proteins, cytoskeletal proteins and cytokines/cytokine receptors. This analysis revealed a close association of BMSC with vascular cells and indicated that BMSC resemble pericytes.     

Immunoglobulin-positive cells in mouse bone marrow]

The content of Ig-bearing lymphocytes and their precursors in the mouse bone marrow was investigated 6 and 36 hours after the hydroxyurea treatment. Some increase of the B-cell content takes place in the trated bone marrow. Dividing and non-dividing B-cell precursors, except the stem cells, were practically absent.     

Differential expression of the mouse and human Thy-1 gene in embryonal carcinoma cells.

Mouse P19 embryonal carcinoma (EC) cells express on their surfaces a Thy-1 glycoprotein. The expression of Thy-1 at the mRNA and protein levels is down-regulated during differentiation induced by retinoic acid (RA). Thy-1 is also expressed in human NTERA-2 EC cells, but its expression is not down-regulated during RA-induced differentiation. As a first step towards understanding differential regulation of the mouse and human Thy-1 gene in EC cells, we have introduced genomic DNA fragments encompassing the mouse or human Thy-1 gene into NTERA-2 and P19-derived cells and analyzed surface properties of the transfectants. In the transient transfection assay, both mouse and human Thy-1 genes were expressed on cell surfaces at comparable levels. P19-derived stable transfectants exhibited great clonal variations in the expressions of the transfected Thy-1 gene products, which in part reflected copy numbers. There was no simple correlation between the expression of the transfected Thy-1 gene and two stem cell surface markers, TEC-1 and TEC-4. In the course of differentiation induced by RA several clones with a surface phenotype of EC cells exhibited a significant decrease in the expression of the transfected mouse Thy-1, whereas expression of the human Thy-1 was less efficiently down-regulated. The results suggest the presence of multiple cis- and trans-acting elements controlling expression of the mouse and human Thy-1 genes in P19 EC cells and their differentiated derivatives.     

Thy-1 antigen expression by murine high-proliferative capacity hematopoietic progenitor cells. I. Relation between sensitivity to depletion by Thy-1 antibody and stem cell generation potential

Hematopoietic stem cells of high proliferative potential such as the giant macrophage colony-forming cell HPP-CFC, were present in the marrow of mice treated with high dose 5-fluorouracil (5Fu) (150 mg/kg i.v.), whereas most committed granulocyte-macrophage progenitors, GM-CFU-C, were depleted. Enrichment of primitive stem cells in post 5-Fu bone marrow (5FuBM) was reflected in an enhanced capacity to proliferate in suspension cultures stimulated by the mixture of lymphokines present in Con A spleen-conditioned medium supernatant (Con A CM) when compared to normal bone marrow. The population of blast-like cells harvested at 5 days from suspension cultures of 5FuBM with Con A CM showed marked increases in stem cells GM-CFU-C and HPP-CFC. For this reason, 5FuBM was utilized to study the cell surface characteristics of putative pluripotential stem cells capable of giving rise to committed stem cells in suspension cultures. Treatment of 5FuBM (BDF1 mice) before suspension culture with a high concentration of either of two cytotoxic monoclonal antibodies directed against the Thy-1.2 surface antigen in the presence of rabbit complement reduced or abrogated the generation of stem cells HPP-CFC and GM-CFU-C in suspension cultures, even though the input content of HPP-CFC and GM-CFU-C in treated 5FuBM compared with control 5FuBM showed little reduction by the antibody plus complement treatment. The Thy-1+ cell required for generation of stem cells was not a T cell, because reconstitution of Thy-1.2-depleted 5FuBM with spleen nylon nonadherent (T) cells did not reconstitute the generation of stem cells, even though T cells did grow in the suspension cultures. In addition, depletion from 5FuBM of cells expressing Lyt-1 and Lyt-2 antigens, unambiguous markers of T cell-thymocyte differentiation, did not ablate the generation of HPP-CFC and GM-CFU-C. Rather, performance of Thy-1 cell depletion at lower efficiency, which still abrogated T cell function, ablated generation of HPP-CFC but did not affect the generation of GM-CFU-C. It was concluded that 5FuBM contains distinct Thy-1+ primitive stem cells expressing different amounts of Thy-1 antigen correlating with their respective generation potentials. Some of these Thy-1+ progenitor cells may be pluripotential.     

Identifying superoxide-sensitive progenitor cells in mouse bone marrow

Macrophage progenitor cells in bone marrow, that develop into attached colonies in liquid culture medium, contain a fraction of cells sensitive to photochemically generated superoxide radicals. This fraction varies from one animal to another. Populations of cells containing the superoxide-sensitive fraction show a greater sensitivity to X-rays than do populations in which this fraction has been photochemically inactivated. The change in radiosensitivity was proportional to the superoxide-sensitive fraction.     

Formation of eosinophilic-like granulocytic colonies by mouse bone marrow cells in vitro

Formation of granulocytic and macrophage colonies in agar cultures of mouse marrow or spleen cells was stimulated by the addition of medium from pokeweed mitogen-stimulated cultures of mouse spleen cells (PKW-CM). Approximately 5% of the colonies developing were large, dispersed granulocytic colonies (DG-colonies) composed of cells with eosinophilic cytoplasmic granules. The capacity to stimulate DG-colonies was shown by media conditioned by PKW-treated lymphoid and peritoneal cells but not by other cells or organ fragments. Velocity sedimentation studies indicated that cells generating DG-colonies were separable from cells generating regular granulocytic or macrophage colonies. DG-colonies did not survive if transfered to cultures containing other forms of CSF. The active colony stimulating factor in pokeweed mitogen-conditioned medium which stimulates DG-colony formation was antigenically distinct from the factor stimulating granulocytic and macrophage colony formation, was separable electrophoretically from the latter factor and on gel filtration had an apparent molecular weight of 50,000. Although the cells in DG-colonies have not been established to be eosinophils, DG-colonies represent an interesting new system for analysing further aspects of the control of growth and differentiation in hemopoietic populations.     

Clastogenic effects of acrylamide in mouse bone marrow cells

Acrylamide, known to induce dominant-lethal mutations (Shelby et al., 1986; Smith et al., 1986) and heritable translocations (Shelby et al., 1987) in rodent germ cells, was hitherto a questionable clastogen in rodent bone marrow (Shiraishi, 1978). Therefore, it was tested for chromosomal aberrations in mouse bone marrow cells, spermatogonia and by the micronucleus test. The intraperitoneally injected doses ranged from 50 to 150 mg/kg. In the chromosomal bone marrow test and the micronucleus assay positive results were obtained with acrylamide, and in the latter test the effect increased linearly with dose. Chromosomal aberrations were not induced in differentiating spermatogonia by the acute acrylamide treatment. Cisplatin was used as a positive control and gave the expected positive response in all 3 tests. The present results demonstrate that acrylamide is no exception among clastogens. It breaks chromosomes not only in mammalian germ cells but also in somatic cells.     

Identification of parathyroid hormone-regulated proteins in mouse bone marrow cells by proteomics

The ability of parathyroid hormone (PTH) to enhance bone formation has recently been exploited in the treatment of osteoporosis. Several studies have suggested that the activation of bone marrow stromal cells could be preceded to show the anabolic effect of PTH on bone formation, but little is known of PTH-regulated proteins in bone marrow cells. Therefore, protein profiling in the intermittent PTH-treated bone marrow cells was evaluated using proteomics. Daily treatment for 5 days consisting of subcutaneous injection of either 150 microg/kg per day of mouse PTH (1-84) or vehicle (0.9% normal saline) was performed on the ICR mouse. At the end of the treatment period, bone marrow cells were separated and used in proteomics. The expression levels of seven proteins including vimentin were decreased, but those of four proteins including calreticulin and thioredoxin domain containing 7 protein (Txnde7) were increased. Among these, the decrease of vimentin and the increase of both calreticulin Txnde7 in mRNA levels were confirmed by semi-quantitative RT-PCR. In PTH-treated mouse MC3T3-E1 osteoblast cells, mRNA expression levels were not totally consistent with the results observed in proteomics. In conclusion, the differentially expressed proteins in bone marrow cells depending on PTH could be highly linked to the differentiation of osteoprogenitor cells in the bone marrow into preosteoblast cells.     

Stimulation of host bone marrow stromal cells by sympathetic nerves promotes breast cancer bone metastasis in mice

Bone and lung metastases are responsible for the majority of deaths in patients with breast cancer. Following treatment of the primary cancer, emotional and psychosocial factors within this population precipitate time to recurrence and death, however the underlying mechanism(s) remain unclear. Using a mouse model of bone metastasis, we provide experimental evidence that activation of the sympathetic nervous system, which is one of many pathophysiological consequences of severe stress and depression, promotes MDA-231 breast cancer cell colonization of bone via a neurohormonal effect on the host bone marrow stroma. We demonstrate that induction of RANKL expression in bone marrow osteoblasts, following β2AR stimulation, increases the migration of metastatic MDA-231 cells in vitro, independently of SDF1-CXCR4 signaling. We also show that the stimulatory effect of endogenous (chronic stress) or pharmacologic sympathetic activation on breast cancer bone metastasis in vivo can be blocked with the β-blocker propranolol, and by knockdown of RANK expression in MDA-231 cells. These findings indicate that RANKL promotes breast cancer cell metastasis to bone via its pro-migratory effect on breast cancer cells, independently of its effect on bone turnover. The emerging clinical implication, supported by recent epidemiological studies, is that βAR-blockers and drugs interfering with RANKL signaling, such as Denosumab, could increase patient survival if used as adjuvant therapy to inhibit both the early colonization of bone by metastatic breast cancer cells and the initiation of the "vicious cycle" of bone destruction induced by these cells.