Role of growth hormone receptor signaling in osteogenesis from murine bone marrow progenitor cells

Growth hormone (GH) regulates many of the factors responsible for controlling the development of bone marrow progenitor cells (BMPCs). The aim of this study was to elucidate the role of GH in osteogenic differentiation of BMPCs using GH receptor null mice (GHRKO). BMPCs from GHRKO and their wild-type (WT) littermates were quantified by flow cytometry and their osteogenic differentiation in vitro was determined by cell morphology, real-time RT-PCR, and biochemical analyses. We found that freshly harvested GHRKO marrow contains 3% CD34 (hernatopoietic lineage), 43.5% CD45 (monocyte/macrophage lineage), and 2.5% CD106 positive (CFU-F/BMPC) cells compared to 11.2%, 45%, and 3.4% positive cells for (WT) marrow cells, respectively. When cultured for 14 days under conditions suitable for CFU-F expansion, GHRKO marrow cells lost CD34 positivity, and were markedly reduced for CD45, but 3- to 4-fold higher for CD106. While WT marrow cells also lost CD34 expression, they maintained CD45 and increased CD106 levels by 16-fold. When BMPCs from GHRKO mice were cultured under osteogenic conditions, they failed to elongate, in contrast to WT cells. Furthermore, GHRKO cultures expressed less alkaline phosphatase, contained less mineralized calcium, and displayed lower osteocalcin expression than WT cells. However, GHRKO cells displayed similar or higher expression of cbfa-1, collagen 1, and osteopontin mRNA compared to WT. In conclusion, we show that GH has an effect on the proportions of hematopoietic and mesenchymal progenitor cells in the bone marrow, and that GH is essential for both the induction and later progression of osteogenesis. (c) 2005 Elsevier Inc. All rights reserved.

Circulating bone marrow cells can contribute to neointimal formation

To examine the source of smooth muscle-like cells during vascular healing, C57BL/6 (Ly 5.2) female mice underwent whole body irradiation followed by transfusion with 10(6) nucleated bone marrow cells from congenic (Ly 5.1) male donors. Successful repopulation (88.4 +/- 4.9%) by donor marrow was demonstrated in the female mice by flow cytometry with FITC-conjugated A20.1/Ly 5.1 monoclonal antibody after 4 weeks. The arteries of the female mice were then subjected to two types of insult: (1) The iliac artery was scratch-injured by 5 passes of a probe causing severe medial damage. After 4 weeks, the arterial lumen was obliterated by a cell-rich neointima, with cells containing a smooth muscle actin present around the residual lumen. Approximately half of these cells were of male donor origin, as evidenced by in situ hybridization with a Y-chromosome-specific probe. (2) In an organized arterial thrombus formed by inserting an 8-0 silk suture into the left common carotid artery, donor cells staining with alpha smooth muscle actin were found in those arteries sustaining serious damage but not in arteries with minimal damage, Our results suggest that bone marrow-derived cells are recruited in vascular healing as a complementary source of smooth muscle-like cells when the media is severely damaged and few resident smooth muscle cells are available to effect repair. Copyright (C) 2001 S. Karger AG, Basel.

Neointimal formation by circulating bone marrow cells

The origin of smooth muscle cells involved in vascular healing was examined. Eighteen C57BL/6 (Ly 5.2) female mice underwent whole body irradiation followed by transfusion with 10(6) bone nucleated marrow cells from congenic (Ly 5.1) male donors. Successful repopulation by donor marrow was demonstrated after 4 weeks by flow cytometry with FITC-conjugated A20.1/Ly 5.1 monoclonal antibody. The iliac artery of six of the chimeric mice was scratch-injured by five passes of a probe, causing severe medial damage. After 4 weeks the arterial lumen was obliterated by a cell-rich neointima, with alpha-smooth muscle actin-containing cells present around the residual lumen. Approximately half of these cells were of male donor origin, as evidenced by in situ hybridization with a Y chromosome-specific probe. An organized arterial thrombus was formed in the remaining 12 chimeric mice by inserting an 8.0 silk suture into the left common carotid artery. Donor cells staining with alpha-smooth muscle actin were found in those arteries sustaining serious damage but not in arteries with minimal damage. Our results suggest that bone marrow-derived cells are recruited in vascular healing as a complementary source of smooth muscle-like cells when the media is severely damaged and few resident smooth muscle cells are available to effect repair.

Growth hormone receptor and insulin-like growth factor-I immunoreactivity in osteoclast-like cells during tooth eruption in the toothless (Osteopetrotic) rat following treatment with colony-stimulating factor-1

In the toothless (tl/tl) osteopetrotic rat, teeth form but fail to erupt. Treatment of tl/tl rats with colony-stimulating factor-1 (CSF-1) activates bone resorption by osteoclasts, permits tooth eruption, and up-regulates the immunoreactivity of bone marrow mononuclear cells to growth hormone receptor (GHr) and insulin-like growth factor (IGF)-I. This study examined the distribution of tartrate-resistant acid phosphatase (TRAP) and immunoreactivity for GHr and IGF-I in osteoclast-like cells located on the alveolar bone margin, adjacent to the lower first molar crown, in 14-day-old normal and tl/tl rats, following treatment with CSF-1. Osteoclast-like cells demonstrated a positive reaction for TRAP, GHr, and IGF-I in all groups. However, in tl/tl tissue, osteoclast-like cells were generally negative for GHr. There was no significant difference in the total number of TRAP, GHr, and IGF-I-positive osteoclast-like cells on the adjacent bone margin in normal, normal treated with CSF-1, and tl/tl rats. CSF-1 treatment of the tl/tl rat significantly increased the total number of osteoclast-like cells, which were positive for TRAP (p < 0.001), GHr (p < 0.05) and IGF-I (P < 0.01).

Granulocyte-colony-stimulating factor (G-CSF)-primed allogeneic bone marrow: significantly less graft-versus-host disease and comparable engraftment to G-CSF-mobilized peripheral blood stem cells

Prospective studies have shown rapid engraftment using granulocyte-colony-stimulating factor-mobilized peripheral blood stem cells (G-PBSCs) for allogeneic transplantation, though the risks for graft-versus-host disease (GVHD) may be increased. It was hypothesized that the use of G-CSF to prime bone marrow (GBM) would allow rapid engraftment without increased risk for GVHD compared with G-PBSC. Patients were randomized to receive G-BM or G-PBSCs for allogeneic stem cell transplantation. The study was designed (beta < .8) to detect a difference in the incidence of chronic GVHD of 33% ( < .05). The plan was to recruit 100 patients and to conduct an interim analysis when the 6-month follow-up point was reached for the first 50 patients. Fifty-seven consecutive patients were recruited (G-BM, n = 28; G-PBSC, n = 29). Patients in the G-PBSC group received 3-fold more CD34(+) and 9-fold more CD3(+) cells. Median times to neutrophil (G-BM, 16 days; G-PBSC, 14 days; P < .1) and platelet engraftment (G-BM, 14 days; G-PBSC, 12 days; P < .1) were similar. The use of G-PBSC was associated with steroid refractory acute GVHD (G-BM, 0%; G-PBSC, 32%; P < .001), chronic GVHD (G-BM, 22%; G-PBSC, 80%; P < .02), and prolonged requirement for immunosuppressive therapy (G-BM, 173 days; G-PBSC, 680 days; P < .009). Survival was similar for the 2 groups. Compared with G-PBSC the use of G-BM resulted in comparable engraftment, reduced severity of acute GVHD, and less subsequent chronic GVHD. (Blood. 2001;98:3186-3191) (C) 2001 by The American Society of Hematology.

Host B cells produce IL-10 following TBI and attenuate acute GVHD after allogeneic bone marrow transplantation

Host antigen-presenting cells (APCs) are known to be critical for the induction of graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (BMT), but the relative contribution of specific APC subsets remains unclear. We have studied the role of host B cells in GVHD by using B-cell-deficient mu MT mice as BMT recipients in a model of CD4-dependent GVHD to major histocompatlibility complex antigens. We demonstrate that acute GVHD is initially augmented in mu MT recipients relative to wild-type recipients (mortality: 85% vs 44%, P < .01), and this is the result of an increase in donor T-cell proliferation, expansion, and inflammatory cytokine production early after BMT. Recipient B cells were depleted 28-fold at the time of BMT by total body irradiation (TBI) administered 24 hours earlier, and we demonstrate that TBI rapidly induces sustained interleukin-110 (IL-10) generation from B cells but not dendritic cells (DCs) or other cellular populations within the spleen. Finally, recipient mice in which B cells are unable to produce IL-10 due to homologous gene deletion develop more severe acute GVHD than recipient mice in which B cells are wild type. Thus, the induction of IL-10 in host B cells during conditioning attenuates experimental acute GVHD.

Population balance model of in vivo neutrophil formation following bone marrow rescue therapy

In this paper, we develop a simple four parameter population balance model of in vivo neutrophil formation following bone marrow rescue therapy. The model is used to predict the number and type of neutrophil progenitors required to abrogate the period of severe neutropenia that normally follows a bone marrow transplant. The estimated total number of 5 billion neutrophil progenitors is consistent with the value extrapolated from a human trial. The model provides a basis for designing ex vivo expansion protocols.

Blood vessels from bone marrow

Lengths of silastic tubing were inserted into the peritoneal cavity of rats or rabbits. By two weeks the free-floating implants had become covered by a capsule consisting of several layers of macrophage-derived myofibroblasts and collagen matrix overlaid by a single layer of mesothelial cells. The tubing was removed from the harvested implant and the tissue everted. This now resembled an artery with an inner lining of mesothelial cells (the intima), a media of myofibroblasts, and an outer collagenous adventitia. The tube of living tissue was grafted by end-to-end anastomoses into the transected carotid artery or abdominal aorta of the same animal in which the tissue had been grown, where it remained parent for four months and developed structures resembling elastic lamellae, The myofibroblasts developed a high volume fraction of myofilaments and became responsive to contractile and relaxing agents similar to smooth muscle cells of the adjacent artery wall.

Persistence of recipient lymphocytes in NOD mice after irradiation and bone marrow transplantation

The non-obese diabetic (NOD) mouse is a unique and invaluable model of autoimmune disease, in particular type I diabetes. Bone marrow transplantation as a therapy for type I diabetes has been explored in NOD mice. NOD mice require higher doses of conditioning irradiation for successful allogeneic bone marrow transplantation, suggesting that NOD hematopoietic cells are radioresistant compared to those of other mouse strains. However, studies of hematopoietic reconstitution in NOD mice are hampered by the lack of mice bearing a suitable cell-surface marker that would allow transferred cells or their progeny to be distinguished. In order to monitor hematopoietic reconstitution in NOD mice we generated congenic NOD mice that carry the alternative allelic form of the pan-leukocyte alloantigen CD45. Following irradiation and congenic bone marrow transplantation, we found that the myeloid lineage was rapidly reconstituted by cells of donor origin but substantial numbers of recipient T lymphocytes persisted even after supra-lethal irradiation. This indicates that radiation resistance in the NOD hematopoietic compartment is a property primarily of mature T lymphocytes. (C) 2004 Elsevier Ltd. All rights reserved.

The mononuclear phagocyte system revisited

The mononuclear phagocyte system (MPS) was defined as a family of cells comprising bone marrow progenitors, blood monocytes, and tissue macrophages. In this review, we briefly consider markers for cells of this lineage in the mouse, especially the F4/80 surface antigen and the receptor for macrophage colony-stimulating factor. The concept of the MPS is challenged by evidence that there is a separate embryonic phagocyte lineage, the blurring of the boundaries between macrophages and other cells types arising from phenotypic plasticity and transdifferentiation, and evidence of local renewal of tissue macrophage populations as opposed to monocyte recruitment. Nevertheless, there is a unity to cells of the MPS suggested by their location, morphology, and shared markers. We discuss the origins of macrophage heterogeneity and argue that macrophages and antigen-representing dendritic cells are closely related and part of the MPS.

Expression of activated mutants of the human interleukin-3/interleukin-5 granulocyte-macrophage colony-stimulating factor receptor common beta subunit in primary hematopoietic cells induces factor-independent proliferation and differentiation

To date, several activating mutations have been discovered in the common signal-transducing subunit (h beta c) of the receptors for human granulocyte-macrophage colony-stimulating factor, interleukin-3, and interleukin-5. Two of these, Fl Delta and 1374N, result in a 37 amino acid duplication and a single amino acid substitution in the extracellular domain of h beta c, respectively. A third, V449E, results in a single amino acid substitution in the transmembrane domain, Previous studies comparing the activity of these mutants in different hematopoietic cell lines imply that the transmembrane and extracellular mutations act by different mechanisms and suggest the requirement for cell type-specific molecules in signalling. To characterize the ability of these mutant hpc subunits to mediate growth and differentiation of primary cells and hence investigate their oncogenic potential, we have expressed all three mutants in primary murine hematopoietic cells using retroviral transduction. It is shown that, whereas expression of either extracellular hpc mutant confers factor-independent proliferation and differentiation on cells of the neutrophil and monocyte lineages only, expression of the transmembrane mutant does so on these lineages as well as the eosinophil, basophil, megakaryocyte, and erythroid lineages, Factor-independent myeloid precursors expressing the transmembrane mutant display extended proliferation in liquid culture and in some cases yielded immortalized cell lines. (C) 1997 by The American Society of Hematology.