Interferon-gamma inhibits proliferation, but not commitment, of murine granulocyte-macrophage progenitors.

We investigated the effects of interferon gamma (IFN-gamma) on the growth of murine hematopoietic progenitors. IFN-gamma inhibited granulocyte colony-stimulating factor (G-CSF)- and interleukin-3 (IL-3)-dependent colony growth by granulocyte-macrophage (GM) progenitors derived from the bone marrow cells of normal mice. However, the number of IL-3-dependent GM colonies formed by the bone marrow cells of 5-fluorouracil (5-FU)-treated mice was not influenced by the addition of IFN-gamma. Replating experiments suggested that IFN-gamma suppressed GM colony growth directly and that it exerted an inhibitory effect on the proliferation, but not on the commitment, of GM progenitors. In contrast, IFN-gamma failed to suppress colony growth by mast cell progenitors. Erythroid and megakaryocytic progenitors exhibited different responses to IFN-gamma depending on mouse strains. These results suggest that potent negative regulators are not always inhibitors of hematopoietic progenitors.     

Development of rat anti-mouse interleukin 3 monoclonal antibodies which neutralize bioactivity in vitro

Several rat anti-mouse interleukin 3 (IL-3) monoclonal antibodies have been developed which inhibit the biologic activity of mouse IL-3. These antibodies were produced in rats immunized with preparations of purified, recombinant mouse IL-3, obtained from transiently transfected COS7 cell supernatant. Hybridomas secreting anti-IL-3 were selected initially either on the basis of their giving a positive signal in an indirect enzyme-linked immunosorbent assay, or for their ability to inhibit the proliferation of the IL-3-dependent mouse mast cell line, MC/9. Neutralizing rat monoclonal IgG1, IgG2a, and IgG2b antibodies have been identified; these also block IL-3-induced proliferation of the NFS-60 and IC2 cell lines. These antibodies also blocked the IL-3-induced proliferation of mouse bone marrow-derived colony-forming units-culture suggesting that the same epitopes on IL-3 influence receptor recognition for both the proliferation of factor-dependent cell lines as well as normal bone marrow cells. Fab fragments produced from certain of the IgG2a-neutralizing antibodies blocked as well as the parent IgG. Antibody cross-blocking studies identified one neutralizing antibody apparently recognizing an epitope that was spatially distinct from those recognized by the other blocking antibodies tested. The development of these neutralizing rat monoclonal antibodies to mouse IL-3 should facilitate further investigation on the role of this factor in hemopoietic regulation.     

Xyloside effects on in vitro hematopoiesis: functional and biochemical studies

Xyloside supplementation of long-term bone marrow cultures (LTBMCs) has been reported to result in greatly enhanced proliferation of hematopoietic stem cells. This was presumed to be the result of xyloside-mediated perturbation of proteoglycan synthesis by marrow-derived stromal cells. To investigate this phenomenon, we first studied the effects of xyloside supplementation on proteoglycan synthesis by D2XRadII bone marrow stromal cells, which support hematopoietic stem cell proliferation in vitro. D2XRadII cells were precursor labelled with 35S-sulfate, and proteoglycans separated by ion exchange chromatography, isopyknic CsCl gradient centrifugation, and gel filtration HPLC. Xyloside-supplemented cultures showed an approximately fourfold increase in total 35S incorporation, mainly as free chondroitin-dermatan sulfate (CS/DS) glycosaminoglycan chains in the culture media. Both xyloside supplemented and nonsupplemented cultures synthesized DS1, DS2, and DS3 CS/DS proteoglycans as previously described. In contrast to previous reports, xyloside was found to inhibit hematopoietic cell growth in LTBMC. Inhibitory effects were observed both in cocultures of IL-3-dependent hematopoietic cell lines with supportive stromal cell lines and in primary murine LTBMCs. Xyloside was found to have a marked inhibitory effect on the growth of murine hematopoietic stem cells and IL-3-dependent hematopoietic cell lines in clonal assay systems and in suspension cultures. In contrast, dialyzed concentrated conditioned media from LTBMCs had no such inhibitory effects. These findings suggest that xyloside-mediated inhibition of hematopoietic cell growth in LTBMC resulted from a direct effect of xyloside on proteoglycan synthesis by hematopoietic cells.     

IL-3-mediated TNF production is necessary for mast cell development

Mouse mast cell development and survival are largely controlled by the cytokines IL-3 and stem cell factor (SCF). We have found that IL-3 stimulation of bone marrow cells induces the production of TNF via a PI3K- and MAPK kinase/ERK-dependent pathway. Specifically, Mac-1-positive cells were responsible for TNF production, which peaked on days 7-10 of culture and decreased rapidly thereafter. The importance of IL-3-induced TNF secretion was demonstrated by the failure of TNF-deficient bone marrow cells to survive for >3 wk when cultured in IL-3 and SCF, a defect that was reversed by the addition of soluble TNF. The development of human mast cells from bone marrow progenitors was similarly hampered by the addition of TNF-blocking Abs. Cell death was due to apoptosis, which occurred with changes in mitochondrial membrane potential and caspase activation. Apoptosis appeared to be due to loss of IL-3 signaling, because TNF-deficient cells were less responsive than their wild-type counterparts to IL-3-mediated survival. In vitro cultured mast cells from TNF-deficient mice also demonstrated reduced expression of the high affinity IgE receptor, which was restored to normal levels by the addition of soluble TNF. Finally, TNF-deficient mice demonstrated a 50% reduction in peritoneal mast cell numbers, indicating that TNF is an important mast cell survival factor both in vitro and in vivo.     

Structure and function of bone marrow environment

Bone marrow and spleen are the major hematopoietic tissue in adult mice. However, little is known about the specific mechanism regulating hematopoiesis within these tissues. Since Dexter et al. first described conditions to maintain bone marrow hematopoiesis, long term bone marrow culture (LTBMC) has been developed in order to analyze the mechanism of the maintenance of proliferation and differentiation of hematopoietic stem cells in vitro. Furthermore, several stromal cell lines which are able to support the growth and differentiation of hematopoietic lineage, has been established from LTBMC. Although it is well known that bone marrow stromal cell lines are able to produce colony stimulating factors, it has been suggested that the stromal cell factors which involve membrane bound moieties must have a key role in the regulation of hematopoiesis. We expect that monoclonal antibodies to the surface of bone marrow stromal cells could detect such a critical stroma-associated protein that bounds the cell surface of the bone marrow stroma.     

Transforming growth factor beta: possible roles in the regulation of normal and leukemic hematopoietic cell growth

We have recently demonstrated that transforming growth factor (TGF)-beta 1 and TGF-beta 2 are potent inhibitors of the growth and differentiation of murine and human hematopoietic cells. The proliferation of primary unfractionated murine bone marrow by interleukin-3 (IL-3) and human bone marrow by IL-3 or granulocyte/macrophage colony-stimulating factor (GM-CSF) was inhibited by TGF-beta 1 and TGF-beta 2, while the proliferation of murine bone marrow by GM-CSF or murine and human marrow with G-CSF was not inhibited. Mouse and human hematopoietic colony formation was differentially affected by TGF-beta 1. In particular, CFU-GM, CFU-GEMM, BFU-E, and HPP-CFC, the most immature colonies, were inhibited by TGF-beta 1, whereas the more differentiated unipotent CFU-G, CFU-M, and CFU-E were not affected. TGF-beta 1 inhibited IL-3-induced growth of murine leukemic cell lines within 24 h, after which the cells were still viable. Subsequent removal of the TGF-beta 1 results in the resumption of normal growth. TGF-beta 1 inhibited the growth of factor-dependent NFS-60 cells in a dose-dependent manner in response to IL-3, GM-CSF, G-CSF, CSF-1, IL-4, or IL-6. TGF-beta 1 inhibited the growth of a variety of murine and human myeloid leukemias, while erythroid and macrophage leukemias were insensitive. Lymphoid leukemias, whose normal cellular counterparts were markedly inhibited by TGF-beta, were also resistant to TGF-beta 1 inhibition. These leukemic cells have no detectable TGF-beta 1 receptors on their cell surface. Last, TGF-beta 1 directly inhibited the growth of isolated Thy-1-positive progenitor cells. Thus, TGF-beta may be an important modulator of normal and leukemic hematopoietic cell growth.     

人胎肝中一种35kD成分的造血调控研究

３－５月胎龄人肝脏造血异常活跃。其中造血干细胞约有３０－４０％处于细胞周期Ｓ期,远高于成年骨髓中约１０％的比例。胎肝中存在活性很高血的造血干细胞增殖刺激因子可能是这一活跃功能的分子基础。基于这种事实,本文用小鼠ＣＦＵ－Ｓ“自杀”率对这种活性进行了检测。经过多步分离纯化,获得一分子量约３５ｋＤ的单一活性组分,定名为ＦＬＳ－４。ＦＬＳ－４作用于脐带血ＣＤ３４细胞,使其＾３Ｈ－ＴｄＲ掺入率提高近１倍,与     

Thiol-sensitive mast cell lines derived from mouse bone marrow respond to a mast cell growth-enhancing activity different from both IL-3 and IL-4

A series of permanent IL-3-dependent cell lines have been established from normal BALB/c or C3H bone marrow using alpha-thioglycerol-supplemented culture medium and PWM-stimulated spleen cell-conditioned medium as a source of IL-3. The cell lines and derivatives cloned in agar resembled "mucosal type" mast cells with respect to phenotypic and functional properties. In this report we demonstrate that in vitro growth of these mast cell lines was not only dependent on IL-3 and synergistically enhanced by IL-4, but in addition regulated by alpha-thioglycerol which could be replaced by 2-ME or cysteamine. We show that these thiol-sensitive mast cell lines respond to a mast cell growth enhancing activity (MEA) present in spleen cell-conditioned medium and acting in concert with IL-3. Partially purified MEA was not able to stimulate the growth of IL-3-dependent 32Dcl.23 cells, IL-2-dependent CTLL-2 cells or the mouse T cell line F4/4K.6 (L3T4+) adapted to grow in purified IL-4. Moreover, 11B11 hybridoma-derived anti-IL-4 mAb specifically neutralizing mouse Il-4 were unable to abolish the bioactivity of MEA. PWM, CSF-1, GM-CSF, IL-1, IL-2, IL-5, IL-6, IL-7, IFN-gamma, TGF-alpha, TNF-alpha, NGF, or EPO did not substitute for MEA in our standard proliferation assay.     

Functional expression of neurokinin 1 receptors on mast cells induced by IL-4 and stem cell factor

It is widely accepted that neurokinin 1 (NK(1)) receptors are not generally expressed on mast cells but little is known about their expression in inflammation. The present study shows expression of NK(1) receptors on bone marrow-derived mast cells (BMMC) under the influence of IL-4 or stem cell factor (SCF). Highest expression was found when both cytokines are present. Six days of coculture with the cytokines IL-4 and SCF showed significant expression of NK(1) receptors (NK(1) receptor(+)/c-kit(+) BMMC; control: 7%, IL-4/SCF: 16%), while 12 days of cytokine coculture increased this expression to 37% positive cells. A longer coculture with IL-4 and SCF did not give an additional effect. Increased expression in IL-4/SCF-treated BMMC was further confirmed using Western blot analysis. Next, we demonstrated the functional relevance of NK(1) receptor expression for mast cell activation, resulting in an enhanced degranulation upon stimulation by substance P. BMMC activation was significantly diminished by the NK(1) receptor antagonist RP67580 (10 micro M) when stimulated with low concentrations of substance P. The inactive enantiomer RP65681 had no effect. In addition, BMMC cultured from bone marrow of NK(1) receptor knockout mice showed significantly decreased exocytosis to low concentrations of substance P. The present study clearly shows that NK(1) receptor-induced activation contributes significantly at low physiological substance P concentrations (<100 micro M). In conclusion, BMMC were shown to express NK(1) receptors upon IL-4/SCF coculture. This expression of NK(1) receptors has been demonstrated to be of functional relevance and leads to an increase in the sensitivity of BMMC to substance P.     

IL-33 induces granzyme C expression in murine mast cells via an MSK1/2-CREB-dependent pathway

Granzymes comprise a group of proteases involved in the killing of infected or cancerous cells by the immune system. Although best studied in T cells and natural killer (NK) cells, they are also expressed in some innate immune cells. Granzymes B and C are encoded in the mouse chymase locus that also encodes a number of mast cell-specific proteases. In line with this, mast cells can express granzyme B, although how this is regulated and their ability to express other granzymes is less well studied. We therefore examined how IL-33, a cytokine able to activate mast cells but not induce degranulation, regulated granzyme B and C levels in mast cells. Granzyme C, but not B, mRNA was strongly up-regulated in bone marrow-derived mast cells following IL-33 stimulation and there was a corresponding increase in granzyme C protein. These increases in both granzyme C mRNA and protein were blocked by a combination of the p38α/β MAPK inhibitor VX745 and the MEK1/2 inhibitor PD184352, which blocks the activation of ERK1/2. ERK1/2 and p38α activate the downstream kinases, mitogen and stress-activated kinases (MSK) 1 and 2, and IL-33 stimulated the phosphorylation of MSK1 and its substrate CREB in an ERK1/2 and p38-dependent manner. The promoter for granzyme C contains a potential CREB-binding site. Bone marrow-derived mast cells from either MSK1/2 double knockout or CREB Ser133Ala knockin mice were unable to up-regulate granzyme C. Together these results indicate that IL-33-induced granzyme C expression in mast cells is regulated by an MSK1/2-CREB-dependent pathway.     

Bone marrow mononuclear cells up-regulate toll-like receptor expression and produce inflammatory mediators in response to cigarette smoke extract

Several reports link cigarette smoking with leukemia. However, the effects of cigarette smoke extract (CSE) on bone marrow hematopoiesis remain unknown. The objective of this study was to elucidate the direct effects of cigarette smoke on human bone marrow hematopoiesis and characterize the inflammatory process known to result from cigarette smoking. Bone marrow mononuclear cells (BMCs) from healthy individuals when exposed to CSE had significantly diminished CFU-E, BFU-E and CFU-GM. We found increased nuclear translocation of the NF-κB p65 subunit and, independently, enhanced activation of AKT and ERK1/2. Exposure of BMCs to CSE induced IL-8 and TGF-β1 production, which was dependent on NF-κB and ERK1/2, but not on AKT. CSE treatment had no effect on the release of TNF-α, IL-10, or VEGF. Finally, CSE also had a significant induction of TLR2, TLR3 and TLR4, out of which, the up-regulation of TLR2 and TLR3 was found to be dependent on ERK1/2 and NF-κB activation, but not AKT. These results indicate that CSE profoundly inhibits the growth of erythroid and granulocyte-macrophage progenitors in the bone marrow. Further, CSE modulates NF-κB- and ERK1/2-dependent responses, suggesting that cigarette smoking may impair bone marrow hematopoiesis in vivo as well as induce inflammation, two processes that proceed malignant transformation.     

Effects of endotoxin on proliferation of human hematopoietic cell precursors

In examining the effects of corticosteroids on hematopoiesis in vitro, we observed that results were highly dependent on the lot of commercial fetal calf serum (FCS) utilized. We hypothesized that this variability correlated with the picogram (pg) level of endotoxin contaminating the FCS. Randomly obtained commercial lots of FCS contained 0.39 to 187 pg/ml of lipopolysaccharide (LPS). Standard FCS concentrations in hematopoietic precursor proliferation assays (granulocyte-marcrophage colony forming units [CFU-GM]) resulted in final LPS levels as high as 40 pg/ml. LPS (2–5 pg/ml) added to essentially endotoxin-free cultures, induced human mononuclear cell release of interleukin (IL)-1, IL-6 and granulocyte colony stimulating factor (G-CSF). Lots of FCS induced the release of IL-1, IL-6, and G-CSF from human mononuclear cells and the release of these factors correlated with the level of contaminating LPS. Human bone marrow CFU-GM proliferation, in response to granulocyte-macrophage colony stimulating factor (GM-CSF), positively correlated with the level of LPS contaminating the FCS and the FCS-induced release of IL-6 from mononuclear cells. CFU-GM proliferation of human bone marrow cluster of differentiation (CD) 34+CD14-cells were not affected by the presence of endotoxin. These data suggest that LPS at 2–5 pg/ml may induce bone marrow accessory cell release of hematopoietic growth factors, thus altering proliferative response of hematopoietic precursors and confounding the study of exogenously added cytokines to culture systems. This revised version was published online in July 2006 with corrections to the Cover Date.     

Kit signaling through PI 3-kinase and Src kinase pathways: an essential role for Rac1 and JNK activation in mast cell proliferation.

The receptor tyrosine kinase Kit plays critical roles in hematopoiesis, gametogenesis and melanogenesis. In mast cells, Kit receptor activation mediates several cellular responses including cell proliferation and suppression of apoptosis induced by growth factor deprivation and gamma-irradiation. Kit receptor functions are mediated by kinase activation, receptor autophosphorylation and association with various signaling molecules. We have investigated the role of phosphatidylinositol 3'-kinase (PI 3-kinase) and Src kinases in Kit-mediated cell proliferation and suppression of apoptosis induced both by factor deprivation and irradiation in bone marrow-derived mast cells (BMMC). Analysis of Kit-/- BMMC expressing mutant Kit receptors and the use of pharmacological inhibitors revealed that both signaling pathways contribute to these Kit-mediated responses and that elimination of both pathways abolishes them. We demonstrate that the PI 3-kinase and Src kinase signaling pathways converge to activate Rac1 and JNK. Analysis of BMMC expressing wild-type and dominant-negative mutant forms of Rac1 and JNK revealed that the Rac1/JNK pathway is critical for Kit ligand (KL)-induced proliferation of mast cells but not for suppression of apoptosis. In addition, KL was shown to inhibit sustained activation of JNK induced by gamma-irradiation and concomitant irradiation-induced apoptosis.     

Unique pathway of IL-3-driven hemopoietic differentiation

The results of this study support a proposed sequence of IL-3-induced hemopoietic cell proliferation and differentiation. Specifically, IL-3 uniquely induces the transient expression of Thy-1 Ag on Thy-1- bone marrow cells during a 2-wk culture period. Thy-1 Ag is expressed on immature myeloid cells that are undergoing lineage restrictions to granulocytes, macrophages, and mast cells. Flow microfluorimetry-separated Thy-1+ cells require the addition of IL-3 or granulocyte/macrophage-CSF to the culture medium for continued growth and, as these cells divide and undergo terminal differentiation they gradually lose Thy-1 Ag expression. The loss of Thy-1 expression is not strictly correlated with cellular proliferation since the expression of Thy-1 decreases on proliferating cells. Last, IL-3 does not maintain the Thy-1- stem cell population that can give rise to Thy-1+ cells in vitro. The relevance of this scheme of differentiation to normal hemopoiesis and to differentiation-arrested IL-3-dependent leukemic cell populations is discussed.     

Modification of expression of stem cell factor by various cytokines.

The local production of stem cell factor (SCF) may be an important mechanism for regulating proliferation, differentiation, and migration of various cells bearing c-kit receptors, and might be susceptible to the cytokines that serve in inflammation and tissue repair. We have demonstrated that in three murine cell lines, Balb/3T3A31, MC3T3-E1, and C3H-2K, which constitutively produced SCF with different quantity, the SCF mRNA expression was greatly enhanced in response to basic fibroblast growth factor (bFGF) or transforming growth factor beta1 (TGF-beta1). The study was carried out by in situ hybridization utilizing nonradioactive oligonucleotide probes and quantitative image analysis. Leukemia inhibitory factor (LIF) or interleukin-4 (IL-4) moderately increased SCF mRNA in all cell lines, but IL-3 did not. The dot-blot enzyme-linked immunosorbent assay (ELISA) further confirmed that SCF protein production in these cell lines and bone marrow stromal cells was markedly enhanced by TGF-beta1, although TGF-beta1 suppressed the proliferation of all these cells. bFGF also enhanced the SCF production in these cell lines, but did not in bone marrow stromal cells, suggesting a difference in their susceptibility to the cytokine. Our results suggest that TGF-beta1 and bFGF potentially modulate the biological function of cells bearing c-kit receptors through the modulation of SCF production in fibroblasts.     

Immunological and regenerative aspects of hepatic mast cells in liver allograft rejection and tolerance

The precise roles of mast cells in liver allograft rejection and tolerance are still unknown. This study aimed to explore the roles of mast cells in immune regulation and liver regeneration for tolerance induction by using rat models of orthotopic liver transplantation (OLT). Stem cell factor (SCF) and its receptor c-Kit, which are critical to the migration and development of not only stem cells but also mast cells, significantly increased in the tolerogenic livers as compared with rejected livers. The significant elevation of mast cell tryptase, high-affinity IgE receptor, and histamine suggested the activation of mast cells in liver allografts at the tolerogenic phase after OLT. Immunohistochemical analysis using confocal microscope clearly showed colocalization of mast cells, Foxp3+ Tregs, γδ T cells, and recipient-derived hepatic progenitor cells with higher expression of SCF, IL-9, IL-10, TGF-β1, and IL-17 related to immunoregulation and liver regeneration in the donor grafts of a tolerogenic OLT model. Cross-talk among mast cells and other cells was evaluated by in vitro studies demonstrating that syngeneic bone marrow-derived mast cells (BMMCs) co-cultured with na?ve splenocytes or primary hepatocytes significantly increased the population of splenic γδ T cells by mitogen stimulation or by mast cell degranulation, and also significantly induced the hepatocyte proliferation, respectively. Our results suggested that mast cells in the donor grafts may play important roles in the induction/maintenance of immune tolerance and liver regeneration resulting in the replacement of hepatic cells from donor to recipient.     

Systematic analysis of the ability of stromal cell lines derived from different murine adult tissues to support maintenance of hematopoietic stem cells in vitro.

Hematopoietic stem cells interact with a complex microenvironment both in vivo and in vitro. In association with this microenvironment, murine stem cells are maintained in vitro for several months. Fibroblast-like stromal cells appear to be important components of the microenvironment, since several laboratories have demonstrated that cloned stromal cell lines support hematopoiesis in vitro. The importance of the tissue of origin of such cell lines remains unknown, since systematic generation of stromal cell lines from adult tissues has never been accomplished. In addition, the capacity of stromal cell lines to support reconstituting stem cell has not been examined. We have previously described an efficient and rapid method for the immortalization of primary bone marrow stromal cell lines (Williams et al., Mol. Cell. Biol. 8:3864-3871, 1988) which can be used to systematically derive cell lines from multiple tissues of the adult mouse. Here we report the immortalization of primary murine lung, kidney, skin, and bone marrow stromal cells using a recombinant retrovirus vector (U19-5) containing the simian virus large T antigen (SV40 LT) and the neophosphotransferase gene. The interaction of these stromal cells with factor-dependent cells Patterson-Mix (FDCP-Mix), colony forming units-spleen (CFU-S), and reconstituting hematopoietic stem cells was studied in order to analyze the ability of such lines to support multipotent stem cells in vitro. These studies revealed that stromal cell lines from these diverse tissues were morphologically and phenotypically similar and that they quantitatively bound CFU-S and FDCP-Mix cells equally well. However, only those cell lines derived from bone marrow-supported maintenance of day 12 CFU-S in vitro. One lung-derived stromal cell line, ULU-3, supported the survival of day 8 CFU-S, but not the more primitive CFU-S12. A bone marrow-derived stromal cell line, U2, supported the survival of long-term reconstituting stem cells for up to 3 weeks in vitro as assayed by reconstitution 1 year post-transplant. These studies suggest that adherence of HSC to stromal cells is necessary but not sufficient for maintenance of these stem cell populations and that bone marrow provides specific signals relating to hematopoietic stem cell survival and proliferation.