Role of Humoral Factors in the Regulation of Hemopoiesis during Immobilization Stress

Experiments were performed on CBA/CaLac mice with hyperplasia of hemopoiesis induced by 10-h immobilization. Erythropoietic and colony-stimulating activity increased under the influence of hemopoietic growth factors and other humoral substances, which plays a major role in the increase in bone marrow cellularity during immobilization stress. Erythropoietin stimulated proliferation of erythroid colony-forming units, while granulocyte colony-stimulating factor accelerated maturation of granulocyte precursors.     

Ligand-activated natural killer T lymphocytes promptly produce IL-3 and GM-CSF in vivo: relevance to peripheral myeloid recruitment

Natural killer (NK) T cells are prominent for their prompt IL-4 and IFN-gamma production upon TCR ligation that enables them to influence acquired immune responses. In the present study we provide evidence that the regulatory functions of this particular T cell subset extend to the myeloid compartment of bone marrow and spleen through its production of hematopoietic growth factors. Bone marrow and spleen NKT cells responded to a single injection of their specific ligand alpha-galactosylceramide (alpha-GalCer) by producing both IL-3 and granulocyte-macrophage colony stimulating factor (GM-CSF), whose colony-stimulating activity became detectable in the serum as early as 1 h post treatment. These cytokines were not produced in mice lacking NKT cells (CD1d-/-), whose exclusive involvement in this biological activity was further confirmed by intracellular immuno-staining. Growth factor production was accompanied by significant changes in the myeloid compartment of treated mice, namely mobilization of myeloid progenitors (colony-forming unit cells, CFU-C) and neutrophils from the bone marrow to the periphery. Taken together, our data support the notion that activated NKT cells influence innate immune responses by recruiting myeloid progenitors and granulocytes to the periphery through their production of hematopoietic growth factors.     

Anatomical compartments modify the response of human hematopoietic cells to a mitogenic signal

Methods for specifically regulating transplanted cells have many applications in gene and cell therapy. We examined the response of human cord blood CD34+ cells to a specific mitotic signal in vivo. Using a conditional signaling molecule (F36VMpl) that is specifically activated by an artificial ligand called a chemical inducer of dimerization (CID), human hematopoietic cells transplanted into immune deficient mice were induced to proliferate. Only differentiating erythroid precursors and multipotential and erythroid progenitors (colony-forming unit [CFU]-mix and burst forming unitserythroid [BFUe]) responded; however, the nature of the response differed markedly between bone marrow and spleen. In the marrow, F36VMpl induced a 12- to 17-fold expansion of differentiated erythroid precursors and a loss of CFU-mix and BFUe. In the spleen, F36VMpl induced a marked rise in BFUe and CFU-mix and, relative to marrow, a much less prominent rise in more mature red cells. Clonal analysis was most consistent with the interpretation that the spleen and bone marrow differentially regulate the response of human progenitors to a mitotic signal, possibly influencing progenitor expansion versus differentiation. These findings establish CIDs as in vivo growth factors for human hematopoietic cells.     

Reactions of the blood system and stem cells in bleomycin-induced model of lung fibrosis

On the model of toxic diffuse pulmonary fibrosis induced by intratracheal administration of bleomycin, we studied reactions of the blood system, content of stem cells, committed hemopoietic and stromal progenitor cells in the bone marrow, spleen and peripheral blood of C57Bl/6 mice. It was shown that the development of diffuse pulmonary fibrosis was accompanied by hyperplasia of bone marrow hemopoiesis and leukocytosis in the peripheral blood. Activation of the erythroid and granulocytic hemopoietic stems was related to stimulation of hemopoietic stem cells (polypotent cells, granulocyte/erythroid/macrophage/megakaryocyte precursor cells) and committed erythroid and myeloid progenitor cells in the bone marrow. At the same time, the number of stromal precursors increased. Bleomycin increased the count of hemopoietic stem cells the peripheral blood and spleen and reduced the content of mesenchymal stem cells in the spleen and bone marrow.     

Macrophage colony-stimulating factor (M-CSF), as well as granulocyte colony-stimulating factor (G-CSF), accelerates neovascularization

It has been reported that bone marrow cells (BMCs) differentiate into endothelial cells of blood vessels, and that granulocyte colony-stimulating factor (G-CSF) mobilizes progenitors in the BMCs to the peripheral blood, while macrophage colony-stimulating factor (M-CSF) augments the production of monocytes. We examined whether M-CSF augments the differentiation of BMCs into endothelial cells of blood vessels using a hindlimb-ischemic model. Either G-CSF or M-CSF, or both, was administered to the hindlimb-ischemic mice for 3 days. Both M-CSF and G-CSF augmented the differentiation of BMCs into endothelial cells of blood vessels through vascular endothelial cell growth factor (VEGF), resulting in early recovery of blood flow in the ischemic limbs.     

Antigen-induced proliferation in vitro of bone marrow precursors of granulocytes and macrophages

The injection of polymerized flagellin into C57BL mice increased the number of granulocyte cluster-forming cells and macrophage colony-forming cells in the bone marrow. The addition of polymerized flagellin to agar cultures of bone marrow cells from normal or antigen-injected mice increased the number and growth rate of macrophage colonies developing when mouse serum was used as the source of colony stimulating factor (CSF). Polymerized flagellin had no potentiating effect on colony growth when urine or medium conditioned by bone marrow, spleen, lymph node or thymic cells were used as the source of CSF. Antigens may stimulate the proliferation of granulocyte and macrophage precursor cells by an indirect mechanism involving the production by bone marrow cells of CSF, or a similar growth promoting factor.     

Enhanced hematopoietic activity accompanies parasite expansion in the spleen and bone marrow of mice infected with Leishmania donovani

In this study, we have analyzed hematopoietic activity in the spleen, bone marrow, and blood of BALB/c and scid mice infected with Leishmania donovani. Our analysis demonstrates that infection induces a rapid but transient mobilization of progenitor cells into the circulation, associated with elevated levels of granulocyte/macrophage colony-stimulating factor (GM-CSF) and MIP-1alpha. From 14 to 28 days postinfection, when parasite expansion begins in the spleen and bone marrow, both the frequency and cell cycle activity of hematopoietic progenitors, particulary CFU-granulocyte, monocyte, are dramatically increased in these organs. This is associated with increased accumulation of mRNA for GM-CSF, M-CSF, and G-CSF, but not interleukin-3. Our data also illustrate that hematopoietic activity, as assessed by changes in the frequency of progenitor cell populations and their levels of cell cycle activity, can be regulated in both a T-cell-independent and T-cell-dependent, as well as in an organ-specific, manner. Collectively, these data add to our knowledge of the long-term changes which occur in organs in which L. donovani is able to persist.     

Murine schistosomiasis mansoni: experimental analysis of bone marrow and peripheral myelopoiesis

In schistosomiasis a systemic hyperplasia of the monomacrophagic cell lineage is associated with its mild modifications in myelograms and hemograms. We monitored the in vitro proliferation of myeloid precursors obtained from bone marrow, blood, spleen, and liver. The macrophage colony-forming unit (M-CFU) numbers were stable in bone marrow but increased progressively in spleen and in liver, reaching in each organ the values equivalent to one femur. The bone marrow had an increased production and enhanced capacity to release M-CFU. Their quantitative increase in blood and in peripheral tissues of schistosome-infected mice was associated with their qualitative modifications: augmented proliferative capacity, enhanced adhesion, and accelerated differentiation. The accelerated release of monomacrophage progenitors and their enhanced proliferation in peripheral tissues potentially account for the relatively low involvement of the bone marrow and for an efficient in situ production of phagocytes, which participate in host reactions to parasites. Received: 29 October 1997 / Accepted: 15 February 1998     

The use of colony stimulating factors in clinical bone marrow transplantation.

The use of colony stimulating factors in clinical bone marrow transplantation is rapidly evolving. Both granulocyte colony stimulating factor and granulocyte macrophage colony stimulating factor have shown efficacy in bone marrow transplant patients in accelerating the rate of myeloid recovery. The use of colony stimulating factor primed peripheral blood progenitor cells appears to accelerate platelet recovery as well as myeloid recovery.     

The unique myelopoiesis strategy of the amphibian Xenopus laevis

Myeloid progenitors reside within specific hematopoietic organs and commit to progenitor lineages bearing megakaryocyte/erythrocyte (MEP) or granulocyte/macrophage potentials (GMP) within these sites. Unlike other vertebrates, the amphibian Xenopus laevis committed macrophage precursors are absent from the hematopoietic subcapsular liver and instead reside within their bone marrow. Presently, we demonstrate that while these frogs’ liver-derived cells are unresponsive to recombinant forms of principal X. laevis macrophage (colony-stimulating factor-1; CSF-1) and granulocyte (CSF-3) growth factors, bone marrow cells cultured with CSF-1 and CSF-3 exhibit respectively archetypal macrophage and granulocyte morphology, gene expression and functionalities. Moreover, we demonstrate that liver, but not bone marrow cells possess erythropoietic capacities when stimulated with a X. laevis erythropoietin. Together, our findings indicate that X. laevis retain their MEP within the hematopoietic liver while sequestering their GMP to the bone marrow, thus marking a very novel myelopoietic strategy as compared to those seen in other jawed vertebrate species.     

Different effect of granulocyte colony-stimulating factor or bacterial infection on bone-marrow cells of cyclophosphamide-treated or irradiated mice

In the present study, the effect of treatment with granulocyte colony-stimulating factor (G-CSF) on cellular composition of the bone marrow and the number of circulating leucocytes of granulocytopenic mice, whether or not infected with Staphylococcus aureus, was assessed. With two monoclonal antibodies, six morphologically distinct cell populations in the bone marrow could be characterised and quantitated by two-dimensional flow cytometry. Granulocytopenia was induced by cyclophosphamide or sublethal irradiation. Cyclophosphamide predominantly affected the later stages of dividing cells in the bone marrow resulting in a decrease in number of granulocytic cells, monocytic cells, lymphoid cells and myeloid blasts. G-CSF administration to cyclophosphamide-treated mice increased the number of early blasts, myeloid blasts and granulocytic cells in the bone marrow, which indicates that this growth factor stimulates the proliferation of these cells in the bone marrow. During infection in cyclophosphamide-treated mice the number of myeloid blasts increased. However, when an infection was induced in cyclophosphamide and G-CSF-treated mice, the proliferation of bone-marrow cells was not changed compared to that in noninfected similarly treated mice. Sublethal irradiation affected all bone-marrow cell populations, including the early blasts. G-CSF-treatment of irradiated mice increased only the number of myeloid blasts slightly, whereas an infection in irradiated mice, whether or not treated with G-CSF, did not affect the number of bone-marrow cells. Together, these studies demonstrated that irradiation affects the early blasts and myeloid blasts in the bone marrow more severely than treatment with cyclophosphamide. Irradiation probably depletes the bone marrow from G-CSF-responsive cells, while cyclophosphamide spared G-CSF responsive cells, thus enabling the enhanced G-CSF-mediated recovery after cyclophosphamide treatment. Only in these mice, bone marrow recovery is followed by a strong mobilisation of mature granulocytes and their band forms from the bone marrow into the circulation during a bacterial infection.     

Effect of hyaluronidase immobilized using electron-beam synthesis nanotechnology on sensitivity of progenitor cells to regulatory factors

In vitro experiments demonstrated increased colony-forming capacity of erythroid, granulomonocytic, and mesenchymal progenitors of the bone marrow and parenchymal progenitor elements of the liver after treatment with immobilized hyaluronidase. Increased sensitivity of these progenitor cells to erythropoietin, granulocyte colony-stimulating factor, fibroblast growth factor, and stem cell factor, respectively, was demonstrated. Immobilized hyaluronidase enhanced the formation of tissue-specific hepatic CFU against the background of reduced yield of stromal precursors in liver tissue culture containing insulin.     

Pregnancies modulate B lymphopoiesis and myelopoiesis during murine ageing

We recently reported that pregnancy affects age-related changes in the distribution of lymphoid and macrophage populations in the spleen of C57Bl/6 mice. In the present study, we examined the influence of pregnancies on the generation of various developmental B-cell subsets and granulocyte/macrophage lineage cells during murine ageing. Using flow cytometry, changes in lymphoid (mature and early B-cell precursors: B220high, B220low, surface immunoglobulin M (sIgM) mu chain +/-) and myeloid (monocyte/macrophage Mac-1/CD11b, granulocyte Gr-1/Ly-6G) compartments were monitored in the bone marrow of young (2 months) and 15- and 23-month-old mice including male, multiparous and virgin female mice. Pregnancies delayed the age-related decline in murine B lymphopoiesis and maintained B-cell reserve capacity during ageing. We also found an increased production of myeloid cells induced by pregnancies at middle (15 months) and advanced (23 months) ages. This comparative study provides new information on changes in marrow lymphopoiesis and myelopoiesis with age. Our data emphasizes that the onset, magnitude and kinetics of age-related changes in the haematopoietic marrow are parity dependent. These changes could influence the incidence of age-related diseases and may account for the greater longevity of females.     

Maturation of antigen-presenting cells is compromised in HLA-G transgenic mice

The human MHC class Ib antigen HLA-G is thought to regulate maternal immune responses during pregnancy. Here we show that expression of HLA-G in transgenic mice diminished cellular immunity by inhibiting maturation of myelomonocytic cells into functional antigen-presenting cells (APC). Skin allografts applied to HLA-G transgenic mice survived longer and resultant T cell responses were less potent compared to control mice. T cells from HLA-G mice responded normally to allogeneic APC and immunohistological analyses of spleen revealed no marked abnormalities. However, spontaneous outgrowths of myeloid cells were observed when bone marrow or splenocytes from HLA-G mice were cultured in vitro, but functionally competent APC did not develop spontaneously in bone marrow cultures supplemented with granulocyte macrophage colony stimulating factor (GM-CSF). Addition of lipopolysaccharide (LPS) to GM-CSF-derived bone marrow cultures rescued APC maturation. Studies using HLA-G tetrameric reagents revealed that HLA-G-specific binding activity was associated with CD11c(+) myelomonocytic cells, while binding to lymphoid and NK cell subsets was undetectable. These data show that spontaneous maturation of functionally competent dendritic cells (DC) is compromised in HLA-G mice. We hypothesize that HLA-G inhibits maturation of DC via receptor-mediated interactions with myelomonocytic precursors, which render immature DC precursors unable to receive signals from activated T cells.     

Abnormal differentiation of tissue macrophage populations in ''osteopetrosis'' (op) mice defective in the production of macrophage colony-stimulating factor.

Examination of the op/op mouse disclosed marked reduction and abnormal differentiation of osteoclasts in the bones and of tissue-specific macrophages in various visceral organs and tissues. Most of these macrophages were immature as judged by ultrastructural criteria. In co-cultures of normal mouse bone marrow cells with fibroblast cell lines prepared from the lungs of the op/op mice, a defective differentiation of monocytes into macrophages was confirmed, supporting previous evidence that the fibroblast cell lines of the mutant mouse failed to produce functional macrophage colony-stimulating factor (M-CSF/CSF-1). In such co-cultures, however, a small number of macrophages apparently mature under the influence of granulocyte/macrophage colony-stimulating factor (GM-CSF) produced by the op/op fibroblast cell lines. In the mutant mice, the numbers of macrophages in the uterine wall and ovaries were severely reduced. Compared with the tissues of normal littermates, those of the mutants contained about 60% fewer macrophages in many tissues. This suggests that an M-CSF-independent population of macrophages is derived from granulocyte/macrophage-colony-forming cells (GM-CFC) or earlier hematopoietic progenitors.     

Increased production of cytotoxic macrophage progenitors by Lactobacillus casei in mice

Heat-killed Lactobacillus casei YIT9018 (LC9018), when administered intravenously to normal mice, induced increase in Mac-1+ cells and Mac-2+ cells but not in Mac-3+ cells in spleen. The number of both populations changed in the same time course and was maximal 14 d after the administration. To know the effect of LC9018 on hematopoietic progenitor level, we examined the number of macrophage colony-forming cells (M-CFC), granulocyte-macrophage CFC (GM-CFC), and colony-forming units in spleen (CFU-S) in bone marrow 3 d after the administration. LC9018 stimulated the proliferation of M-CFC but not that of GM-CFC and CFU-S. LC9018-induced M-CFC were similar to normal M-CFC in dependence on macrophage colony-stimulating factor (M-CSF) and buoyant density. M-CFC-derived macrophages cultured in the presence of M-CSF expressed Mac-1 and Mac-2 but not Mac-3. They showed cytotoxic activity against syngenic tumor cells, Meth A, via direct contact, when assayed by using an in vitro colony inhibition assay or an in vivo Winn test. These results indicate that LC9018 stimulates the proliferation of cytotoxic macrophage progenitors in bone marrow and induces their differentiation in spleen. These effects may be one of the ways in which LC9018 suppresses tumor growth.     

Lack of correlation between an assay used to determine early marrow allograft rejection and long-term chimerism after murine allogeneic bone marrow transplantation: effects of marrow dose.

The acute rejection of bone marrow (BM) allografts by host effectors can occur within a short period after BM transplantation (BMT) in lethally irradiated mice. Common assays used to ascertain engraftment/resistance involve measuring the growth of granulocyte/monocyte progenitors (colony-forming unit-granulocyte-macrophage) in vitro or splenocyte proliferation assessed by radioisotope incorporation in vivo 5 to 8 days after BMT. However, the correlation of the long-term outcome of BMT with the kinetics of recovery by using the dose of allogeneic BM cells (BMCs) that leads to early rejection as determined by the in vitro assessment has not been extensively studied. Thus, to investigate whether the early rejection of donor BMCs is an indication of a long-term engraftment failure, C57BL/6 (H2b) mice were lethally irradiated and transplanted with various doses of BALB/c (H2d) BMCs. The short-term engraftment of donor precursors (colony-forming unit-granulocyte-macrophage), the kinetics of hematopoietic cell recovery, the extent of donor chimerism, and the proportion of the recipients with long-term survival were determined. The results show that the kinetics and extent of hematopoietic cell recovery were significantly delayed in mice receiving limiting doses of BMCs that were rejected or severely resisted at day 8 after BMT. However, a proportion of these mice survived up to 98 days after BMT with mixed chimerism or donor chimerism. This study demonstrates that early rejection of BM precursors, as assessed by measurement of myeloid progenitors in the spleen after BMT, does not always correlate with the long-term outcome of the marrow allograft and that significant variability is inherent in the extent of chimerism when threshold amounts of BMCs are used.     

Bendamustine with Total Body Irradiation Limits Murine Graft-versus-Host Disease in Part Through Effects on Myeloid-Derived Suppressor Cells

Graft-versus-host disease (GVHD) remains a significant challenge in allogeneic hematopoietic cell transplantation (HCT). An underinvestigated strategy to reduce GVHD is the modification of the preparative conditioning regimen. In the present study, we aimed to evaluate GVHD associated with bendamustine (BEN) conditioning in conjunction with total body irradiation (TBI) as an alternative to the standard myeloablative regimen of cyclophosphamide (CY) and TBI. We demonstrate that BEN-TBI conditioning, although facilitating complete donor chimerism, results in significantly less GVHD compared with CY-TBI. In BEN-TBI-conditioned mice, suppressive CD11b⁺Gr-1^high myeloid cells are increased in the blood, bone marrow, spleen, and intestines. When Gr-1^high cells are depleted before transplantation, the beneficial effects of BEN-TBI are partially lost. Alternatively, administration of granulocyte colony-stimulating factor, which promotes CD11b⁺Gr-1⁺ myeloid cell expansion, is associated with a trend toward increased survival in BEN-TBI-conditioned mice. These findings indicate a potential role of myeloid-derived suppressor cells in the mechanism by which BEN allows engraftment with reduced GVHD. BEN-TBI conditioning may present a safer alternative to CY-TBI conditioning for allogeneic HCT.     

Bone marrow and blood findings after marrow transplantation and rhGM-CSF therapy.

Sequential bone marrow biopsy specimens and peripheral blood findings were evaluated from patients treated with recombinant human granulocyte-macrophage colony-stimulating factor after autologous bone marrow transplantation for recurrent non-Hodgkin's lymphoma. Parallel increases were found in white blood cell count and marrow cellularity and in absolute neutrophil count, monocyte count, and marrow myeloid:erythroid ratio. Platelet count recovery and the reappearance of megakaryocytes occurred later than granulocyte/monocyte recovery. Elevated peripheral eosinophil counts and eosinophilic hyperplasia in the marrow were noted during recombinant human granulocyte-macrophage colony-stimulating factor administration, as was the appearance of distinctive, prominently granulated and/or vacuolated neutrophils and neutrophilic precursors in the blood and bone marrow. No detrimental effects of recombinant human granulocyte-macrophage colony-stimulating factor administration in the marrow or peripheral blood were observed.