Synergistic hematopoietic growth factors

A number of growth factors acting on hematopoietic stem cells have now been purified and characterized. These include erythropoietin, granulocyte-macrophage colony-stimulating activity (GM-CSA), granulocyte colony-stimulating activity and colony-stimulating factor-1 (CSF-1). Factors which act in concert with these defined factors and appear to act relatively early in the hematopoietic stem cell lineage are currently under study. Interleukin 3 appears to have both the characteristics of a differentiating hormone and the ability to generate proliferation of relatively early stem cells. Interleukin 3 acts in concert with at least CSF-1 and erythropoietin to enhance their effect on stem cell proliferation and differentiation. A new class of hematopoietic growth factor activities termed synergizing activities also exist. These activities appear to have no intrinsic capacity to stimulate hematopoietic colony formation by themselves but enhance the effects of other differentiating hormones such as GM-CSA and CSF-1. Activities which appear to represent synergizing activities have now been found to evolve from a human bladder carcinoma line, a cell line derived from murine marrow adherent cells and normal murine marrow and thymic cells. These activities may act on very primitive hematopoietic progenitors to allow them to express receptors to various differentiating hormones or alternatively they may act as commitment factors in a commitment-progression model of stem cell regulation.     

Maintenance and proliferation control of primitive hemopoietic progenitors in long-term cultures of human marrow cells

Primitive, high-proliferative potential hemopoietic progenitors can be routinely maintained for many weeks in long-term marrow cultures (LTC) in the absence of added hemopoietic growth factors. Nevertheless, these progenitors are clearly responsive to both positive and negative regulatory control mechanisms that operate within the adherent layer as evidenced by cyclic changes in their proliferative activity each time the medium is replaced. The key event appears to be the addition of a constituent of fresh horse serum that is not found in fetal calf serum. Analogous primitive neoplastic progenitor cell types from CML or PV patients are insensitive to the negative arm of this proliferation control mechanism both in vitro and in vivo. A model to explain the progenitor cell cycle changes normally observed in the LTC system is proposed. This model suggests that perturbations of nonhemopoietic mesenchymal cells determine the net positive or negative influence that these regulatory cells exert on adjacent primitive hemopoietic cells, possibly by a mechanism involving direct cell contact. Recently, we have identified a number of cytokines that can simulate the transient positive effect of fresh horse serum, as well as another cytokine, that is, tumor growth factor-beta (TGF-beta), that can mimic the negative but reversible effect exerted by mesenchymal cells. These studies demonstrating the effects of positive and negative regulatory cytokines on the control of hemopoiesis in the adherent layer of LTC suggest new approaches for analyzing the basis of both normal and abnormal stem cell regulation by marrow stromal elements.     

Developments in modern hematology.

In the past 40 years our concepts about hemopoiesis have been changed dramatically. The results of bone marrow transplantation into lethally irradiated mice since the mid-fifties suggested the existence of a hemopoietic stem cell, which was initially identified as a spleen colony forming cell (CFU-S). Later experiments showed that the stem cell compartment is rather heterogeneous and that the most primitive stem cell, unlike the CFU-S, has the ability for long-term engraftment of an irradiated recipient. Daughter cells of such primitive quiescent stem cells lose their capacity for self-generation gradually with each mitosis and become more and more committed to a specific differentiation lineage. In vitro culture techniques in a serum-free semi-solid medium enabled the establishment and analysis of specific hemopoietic growth factors. Such factors, which are essential for the maintenance, proliferation and differentiation of progenitor cells and the functional activity of mature cells can now be produced with recombinant DNA techniques in pure form and large quantities. Hemopoiesis requires an appropriate microenvironment, consisting of various stromal cell types and an extracellular matrix. Intercellular contacts, adhesion of cells and growth factors to the matrix molecules seem essential in the regulating action of this hemopoietic microenvironment. In long-term bone marrow cultures the development of a stromal hemopoietic microenvironment can facilitate long-term maintenance of stem cells and hemopoietic differentiation. For bone marrow transplantation and infusion of hemopoietic growth factors many clinical indications are well established and our possibilities to interfere in the regulation of hemopoiesis are still growing.     

Multilineage hemopoiesis induced by cloned stromal cells

Long-term hemopoiesis in culture depends upon the presence of an adherent layer composed of a variety of stromal cells. A subtype of endothelial-adipocytes from the bone marrow stroma (clone 14F1.1) was previously shown to induce long-term myelopoiesis and renewal of pluripotent stem cells. One of a series of stromal cell lines and clones from mouse thymus stroma (STAC-1.2) has now been found to support long-term hemopoiesis. These marrow- and thymus-derived stromal cell clones also have lymphopoietic activities: precursor T cells, or pre-B cells accumulated in co-cultures of thymus cells and the stromal clones, as indicated by cell surface markers, T cell receptor and immunoglobulin gene rearrangements. The predominance of a cell type in these cultures depended upon the serum used to supplement the medium. Recombinant interleukin 2 (IL-2) and the 14F1.1 clone synergistically promoted the proliferation of thymocytes, while a thymus hormone, THF-gamma 2, shifted the population to a relatively mature phenotype. It is proposed that one major function of stromal cells, whether from the bone marrow or thymus, is to restrain the maturation flow and preferentially support the accumulation of cells at early differentiation stages.     

Generation of murine stromal cell lines supporting hematopoietic stem cell proliferation by use of recombinant retrovirus vectors encoding simian virus 40 large T antigen.

The bone marrow is a complex microenvironment made up of multiple cell types which appears to play an important role in the maintenance of hematopoietic stem cell self-renewal and proliferation. We used murine long-term marrow cultures and a defective recombinant retrovirus vector containing the simian virus 40 large T antigen to immortalize marrow stromal cells which can support hematopoiesis in vitro for up to 5 weeks. Such cloned cell lines differentially supported stem cells which, when transplanted, allowed survival of lethally irradiated mice, formed hematopoietic spleen colonies in vivo, and stimulated lymphocyte proliferation in vitro. Molecular and functional analyses of these cell lines did not demonstrate the production of any growth factors known to support the proliferation of primitive hematopoietic stem cells. All cell lines examined produced macrophage colony-stimulating factor. The use of immortalizing retrovirus vectors may allow determination of unique cellular proteins important in hematopoietic stem cell proliferation by the systematic comparison of stromal cells derived from a variety of murine tissues.     

Polypeptide factors regulating osteogenesis and bone marrow repair

Osteogenic growth polypeptides regulate bone cell function in vitro and may act in vivo in an autocrine, paracrine, or endocrine manner. Several of these polypeptides are present in the blood in an inactive form. During postablation bone marrow regeneration these factors may be activated, released from the blood clot, and together with locally produced polypeptides mediate the initial intramedullary/systemic osteogenic phase of this process. Then, the same and/or other polypeptides expressed by stromal cells have the potential to promote the second phase of regeneration that consists of osteoclastogenesis, resorption of the transient intramedullary bone, and hemopoiesis. This may be an indirect influence since these polypeptides can regulate the stromal cell expression of some of the hemopoietic factors. Clinically, the osteogenic growth polypeptides that regulate osteogenesis and hemopoiesis have a potential role in osteoporosis therapy, implant bone surgery, and bone marrow transplantation. © 1994 Wiley-Liss, Inc.     

Stromal cell regulation of lymphoid and myeloid differentiation

In vitro microenvironmental influences seem to be critical for both B lymphocyte and myeloid differentiation. Studies on murine Dexter cultures and Whitlock-Witte lymphocyte cultures suggest the presence of two critical stromal regulatory cells: an alkaline-phosphatase-positive epithelioid cell and a macrophage. Further data suggest that these cells are capable of producing colony stimulating factor-1, granulocyte-macrophage CSF, a myeloid synergizing activity, and probably separate B cell growth factors. Isolation of a cell line from Dexter stroma was accomplished and this line produced CSF-1, GM-CSF, a pre-B cell and myeloid synergizing activity, and an activity acting on differentiated B cells. We speculate that the Dexter and Whitlock-Witte in vitro culture systems are regulated by factors produced by the two adherent cell types. A lineage nonspecific factor capable of inducing cells into the B lineage or synergizing with interleukin-3, GM-CSF, and CSF-1 is produced, which presumably acts on early stem cells. In addition, the cell line produces GM-CSF, CSF-1, and a factor acting on differentiated B cells. We speculate that in these culture systems, these "terminal differentiating hormones" regulate the final pathway of differentiation, whereas the pre-B-synergizing activity supports early stem cells that can then respond to the other differentiating hormones.     

Role of stromal microenvironment in the regulation of bone marrow hemopoiesis after curantyl administration

Role of the stromal microenvironment in regulation of bone marrow hemopoiesis at the administration of the thrombocyte disaggregant curantyl was studied by the method of heterotopic transplantation of the mice bone marrow. It is shown that the action of curantyl on hemopoiesis is realised through the stem stromal cells of the bone marrow. It is noted that the inhibitory action of the preparation on proliferation of osteogenic precursor-cells is followed by activation of bone resorption processes in regenerating ectopic hemopoietic organ. Under the action of curantyl at low bone marrow cellularity in the focus of heterotopic hemopoiesis and femur an increase of mitotic activity in hemopoietic elements is noted. It is revealed that a phenomenon of ineffective megakaryocytopoiesis with intramedullary destruction of megakaryocytes leads to the local excretion of the thrombocyte released growth factor (TRGF) which has a compensatory character.     

Embryonic and fetal hemopoiesis: an overview

Our current knowledge of embryonic and fetal hemopoiesis is critically reviewed in this article. In both murine and human systems, embryonic and fetal development is associated with multiple switching in the sites of hemopoiesis. The phenomenon is first extraembryonic, occurring in blood islands of the yolk sac. Hemopoietic stem cells (HSC) appear to derive from hemangioblasts that are of mesodermal origin. Yolk sac milieu is permissive only for erythropoiesis which proceeds synchronously and may be erythropoietin-insensitive. Yolk sac milieu is not permissive for the development of other cell lines. The final product is nucleated red cells. Yolk sac hemopoiesis is an example par excellence of primitive (as compared to definitive) form of hemopoiesis. HSC then seem to migrate via the bloodstream to the liver and spleen to seed these tissues, which then carry the burden of hemopoiesis until birth and for some time thereafter. Here also erythropoiesis predominates, but some granulopoiesis also occurs. Thus, the milieu is not totally impermissive. Hemopoiesis is in definitive form, lacking synchronicity of cell growth with the end product being anucleated cells and synthesized hemoglobin not limited to embryonic type. The site of hemopoiesis is finally transferred to the bone marrow, which is predominantly granulopoietic. Certain cellular and embryological features of these types of hemopoiesis in the context of more recent molecular understanding of stem cell homing are discussed.     

小鼠胎肝基质细胞造血刺激因子体外生物活性研究

本实验对基质细胞造血刺激因子－１（ＳＨＦ－１）的体外生物活性进行了研究。结果表明,ＳＨＦ－１可刺激小鼠骨髓ＣＦＵ－Ｅ、ＢＦＵ－Ｅ、ＣＦＵ－ＧＭ、ＣＦＵ－Ｍｉｘ集落的形成,它产生的这些广泛造血刺激作用是其自身所具活性的直接影响。正常小鼠骨髓细胞与ＳＨＦ－１在体外孵育４ｈ,其中ＣＦＵ－Ｓ的自杀率可提高约１０％,显示它对造血干细胞也有诱导增殖作用。     

Pre-CFU-f: young-type stromal stem cells in murine bone marrow following administration of DNA inhibitors

Occurrence of young-type stromal stem cells (defined here as "pre-CFU-f") in murine bone marrow is reported in this study. Two consecutive intraperitoneal (i.p.) cytosine arabinoside (ara-C) injections were administered to C57B1 mice (2 X 200 mg/kg at 6-h intervals). Two days later the bone marrow was collected and assayed for colony-forming units-fibroblastoid (defined here as "CFU-f"). In additional experiments, ara-C-treated marrow was exposed in vitro to hydroxyurea (HU; "hydroxyurea killing test"), prior to plating, to establish the cycling state of stromal stem cells. In separate cultures of ara-C-treated marrow, replating of adherent cells was carried out up to quaternary sub-cultures. The results indicate ara-C-treated marrow produces approximately 20% "huge" fibroblastoid colonies (approximately 5 mm diameter versus 0.5-2 mm normal size); most stromal stem cells producing huge colonies are cycling cells; and adherent cells from primary ara-C-treated marrow cultures replated to secondary cultures produce adherent layers with double the number of cells than in the control secondary cultures. We conclude that the ara-C-treated murine bone marrow contains certain young-type cycling stromal stem cells which we refer to as pre-CFU-f. These stem cells produce huge fibroblastoid colonies in culture, indicating that they probably go through more cell cycles than CFU-f during the culture period. Alternatively, pre-CFU-f may have a higher self-replicative capacity than CFU-f.     

Modulation of haematopoietic progenitor development by FLT-3 ligand.

The Flt-3 receptor is expressed in primitive haematopoietic cells and its ligand exerts proliferative effects on these cells in vitro in synergy with other cytokines. To increase our knowledge of the functional properties of the human Flt-3 ligand (FL) as relating to in vitro expansion of haematopoietic stem cells, the effects on murine haematopoiesis of FL alone or in combination with other growth factors were studied. Analysis of Flk-2/Flt-3 mRNA expression indicated that Flk-2/Flt-3 was preferentially expressed in primitive haematopoietic cell populations. To examine the expression of the Flk-2/Flt-3 receptor on megakaryocyte progenitors (CFU-Meg), Flk-2/Flt-3 positive and negative CD34(+)populations were separated from human bone marrow and cultured in a plasma clot culture system. CFU-Meg colonies were found in the Flk-2/Flt-3 negative fraction. Myeloid (CFU-GM) derived colonies appeared in the presence of FL alone. Neither FL+IL-3 nor FL+IL-3+IL-6 had any effect on the generation of megakaryocyte colonies (CFU-MK), due to the lack of FL receptor expression on megakaryocyte progenitors. Bone marrow cells remaining after 5-fluorouracil (5-FU) treatment of mice represent a very primitive population of progenitors enriched for reconstituting stem cells. This cell population expressed FL receptors, as revealed by RT-PCR analysis. Addition of FL alone did not enhance the replication of such cells in liquid cultures as compared to controls. However, a significantly greater generation of myeloid progenitors (CFU-GM) in clonogenic assays was observed in the presence of FL+IL-3, FL+GM-CSF or FL+CSF-1. In addition, the effects of FL on in vitro expansion of murine haematopoietic stem cells were studied using lineage-negative (lin(-)) Sca-1 positive (Sca-1(+)) c-kit positive (c-kit(+)) marrow cells from 5-FU treated mice. FL enhanced the survival of primitive murine lin(-)Sca-1(+)c-kit(+)cells. FL and IL-6 were able to significantly expand murine progenitor stem cells in vitro and promote their survival. These studies strongly suggest that FL significantly and selectively enhanced the generation of myeloid progenitors in vitro and increased myeloid progenitor responsiveness to later acting growth factors. In addition, FL synergized with IL-6 to support in vitro expansion of haematopoietic progenitors and promoted the survival of lin(-)Sca-1(+)c-kit(+)cells.     

Differentiation of the IL-3-dependent NFS-60 cell line and adaption to growth in macrophage colony-stimulating factor

Macrophage CSF (M-CSF) induces responsive bone marrow precursors into rapid growth and differentiation to mature macrophages. Available cell lines that depend on M-CSF for growth are well differentiated and rather adherent. We investigated the effects of M-CSF on immature myeloid cell lines as models of the marrow precursors. The murine line NFS-60 requires IL-3 for growth and also responds to granulocyte-CSF and granulocyte-macrophage-CSF. Cultures of one NFS-60 subline, when switched from IL-3 to 10% L cell conditioned media, a source of M-CSF, or purified M-CSF, frequently acquired large, adherent cells. The adherent cells grew slowly in the presence of M-CSF, in contrast to the majority population of small, round, rapidly growing cells. The large cells had properties of differentiated macrophages that were absent in the nonadherent cells. Cells with macrophage phenotype were not observed in IL-3-supported cultures over many passages. A subline was derived from NFS-60 that grew rapidly and continuously in human or murine M-CSF as round, nonadherent cells. The line, called M-NFS-60, responded well to M-CSF and IL-3, weakly to granulocyte-CSF and not at all to murine granulocyte-macrophage-CSF, IL-4, or human IL-1. A mAb to human M-CSF specifically inhibited only M-NFS-60 proliferation induced by the human growth factor, whether produced by mammalian or bacterial cells. This study shows two effects of M-CSF on the IL-3-dependent NFS-60 line. Upon first exposure to M-CSF, cells may undergo global differentiation to slowly replicating macrophages in conditions we have not been able to define. The more common effect is rapid growth of immature myeloid cells like the bone marrow precursors, but with a block to differentiation. Thus, these cells may be useful as models of M-CSF-induced differentiation, and of permanently maintained macrophage precursors.     

Stromal cells of hemopoietic origin

Hemopoiesis is a multistep process involving stem cell renewal, commitment, differentiation, maturation and consequent positioning of the cells within the tissue. Stromal cells are a major component of the hemopoietic microenvironment. The in vitro culture of cloned stromal cells has enabled detailed analysis of their functions and has provided answers relating to the contribution of stromal cells to the control of hemopoiesis. Cultured stromal cells were found to support the renewal of stem cells through a mechanism that did not seem to involve already known cytokines. Cloned stromal cells from both marrow and thymus supported the in vitro accumulation of myeloid as well as T and B lymphoid cells. Thus, cloned stromal cells had the ability to induce multilineage hemopoiesis, irrespective of the organ from which they were derived. Invariably, stromal cells tended to select in culture for hemopoietic cells at early differentiation stages and restricted the accumulation of mature cells. These functions may be part of the mechanism that protects the stem cell pool from excess differentiation.     

Effect of cytokines on the proliferation/differentiation of stroma-initiating cells

A culture system that identifies the precursor of murine bone marrow fibroblastic stromal cells (stroma-initiating cells, SIC) has been developed. In this system, mature fibroblasts are depleted by adherence to plastic dishes and the nonadherent cells are seeded at a low density, which results in the formation of colonies composed of fibroblastic cells. Macrophage colony-stimulating factor (M-CSF) has been shown to accelerate the colony formation in the system. In this study, we examined the stroma-inducing activity of a number of cytokines. Neither granulocyte-CSF, stem cell factor, interleukin (IL)-1, IL-6, transforming growth factor, epidermal growth factor, insulin-like growth factor, platelet-derived growth factor, nor fibroblast growth factor showed the activity. Similarly, tumor necrosis factor (TNF) did not show any stroma-inducing activity, but the factor inhibited the stromal colony formation induced by M-CSF. In this study, we found that granulocyte/macrophage-CSF (GM-CSF) and IL-3, as well as M-CSF had the stroma-inducing activity. Neither an additive nor synergistic effect was observed when the three factors were assayed in various combinations. The stroma-inducing activity of M-CSF, GM-CSF and IL-3 was observed even if lineage-negative bone marrow cells were used as target cells, suggesting that mature hematopoietic cells such as macrophages and granulocytes were not involved in the induction of stromal colony formation by these factors. Our results raise the possibility that GM-CSF and IL-3 as well as M-CSF stimulate the proliferation or differentiation of the precursor of bone marrow fibroblastic stromal cells.     

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-1 inhibits B cell differentiation in long term bone marrow cultures

There is evidence that stromal cells are responsive to changes in their external milieu and that this can affect their function. IL-1 has been identified as one mediator that can affect stromal cells by increasing their secretion of CSF. The monokine has also been reported to be a B cell differentiation factor. The purpose of this study was to test the effects of IL-1 on the pattern of hemopoietic cell differentiation by adding IL-1 alpha to myeloid long term bone marrow cultures (MBMC) at the time of their transfer to lymphoid bone marrow culture conditions. This usually results in the cessation of myelopoiesis and the induction of B lymphopoiesis. The addition of 50 U/ml of rIL-1 alpha, but not 10 U/ml, to MBMC at the time of their transfer to lymphoid conditions resulted in a complete inhibition of B cell differentiation and sustained myelopoiesis. To determine whether adherent layer cells contributed to this effect, conditioned medium (CM) was collected from adherent layers treated previously with the antibiotic mycophenolic acid. This depletes the hemopoietic cells from the cultures and retains a purified population of stromal cells. CM from mycophenolic acid- treated adherent layers exposed for 24 h to 50 U/ml of IL-1 was added at volume concentrations of 5, 10, and 25% to MBMC at the time of transfer to lymphoid bone marrow culture conditions and at each feeding thereafter. Expression of the B lineage associated 14.8 Ag and IgM was inhibited on a dose dependent basis, and myelopoiesis was sustained in cultures to which 25% CM had been added. Induction of B lymphopoiesis occurred in cultures to which adherent cell CM not exposed to IL-1 had been added. The CM from the IL-1-treated adherent cells contained CSF, because it promoted the growth of myeloid colonies from fresh marrow or MBMC cells and stimulated the granulocyte-macrophage-CSF sensitive FDC-P1 cell line to proliferate. IL-3 was not present in the CM, because stimulation of the IL-3 sensitive 32D cell line was not observed. The CM from the IL-1-treated adherent cells stimulated thymocytes to proliferate in the presence of PHA. This raised the possibility that the induced CSF may have required IL-1 to mediate their effects in the cultures. However, B lymphopoiesis was inhibited and myelopoiesis maintained upon addition of recombinant granulocyte-, macrophage-, and granulocyte-macrophage-CSF to cultures, indicating that IL-1 or other non-CSF molecules induced by it need not be present.(ABSTRACT TRUNCATED AT 400 WORDS)     

Stromal cells provide signals different from cytokines for STAT5 activation in hematopoietic cells

After detachment from the stromal cells, hematopoietic stem cells are thought to differentiate to the cytokine-dependent stages where their growth and differentiation are promoted by these cytokines. To examine the stromal regulation of hematopoietic stem cells, we previously established a primitive hematopoietic stem-like cell line, THS119, whose growth was dependent on the bone marrow stromal cell line, TBR59, and from which IL-3- (THS119/IL-3) or IL-7- (THS119/IL-7) dependent cell lines were then generated. Using these cell lines, we examined the difference in signals mediated by the stromal cells and cytokines. The cytokine-dependent cell lines (THS119/IL-3 and THS119/IL-7) showed induction of STAT5 phosphorylation and target genes for STAT5 such as CIS, pim-1, p21 and bcl-xL upon addition of IL-3 or IL-7. IL-3 or IL-7 also induced STAT5 phosphorylation and STAT5 target genes of the stromal cell-dependent cell line, THS119, in the absence of stromal cells at levels similar to the cytokine-dependent cell lines. However, quite interestingly, TBR59 stromal cells could not induce STAT5 phosphorylation of THS119 cells, although they did induce STAT5 target genes in THS119 cells. In addition, the mRNAs for STAT5 target genes in THS119 cells on the stromal cells seemed to be more stable than those in the cytokine-dependent cell lines. Expression of the antiapoptotic genes bcl-2 and bcl-xL was higher in the stromal cell-dependent cell line than in the cytokine-dependent cell lines. These results suggested that stromal cells and cytokines may provide different signals for growth and differentiation of the hematopoietic cells.     

Persistent production of colony-stimulating factor (CSF-1) by cloned bone marrow stromal cell line D2XRII after X-irradiation

The adherent stromal layer in long-term bone marrow cultures (LTBMC) provides the cellular environment necessary for the in vitro proliferation and differentiation of pluripotential hematopoietic stem cells. The role of humoral hematopoietic growth factors, colony-stimulating factors (CSF) in the regulation of hematopoietic cell production in this system is poorly understood. We have recently isolated and cloned an adherent cell line, D2XRII, derived from murine LTBMC. Plateau phase 25 cm2 cultures of 2 X 10(6) D2XRII cells in 8.0 ml produced CSF-1 (M-CSF) at around 100-150 units/0.1 ml medium. Following X-irradiation there was a dose-dependent decrease in the production of CSF-1 to a plateau of 50% of control levels at 10,000 rad. Higher doses did not produce a further decrease. The X-ray dose reducing CSF-1 production to 50% was 100-fold above the lethal dose as measured by clonagenic survival following trypsinization and replating. Trypsinized replated viable adherent but nondividing X-irradiated D2XRII cells were maintained for up to 8 weeks after irradiation and demonstrated continuous production of CSF-1. The data indicate significant divergence of two biologic effects of X-irradiation on plateau-phase marrow stromal cells: physiologic function of adherence and CSF-1 production, versus proliferative integrity. This divergence of effects may be very relevant to understanding the mechanism of X-irradiation-associated marrow suppression and leukemogenesis.     

Plasma cell survival is mediated by synergistic effects of cytokines and adhesion-dependent signals

Recent results suggest that plasma cell longevity is not an intrinsic capacity, but depends on yet unknown factors produced in their environment. In this study, we show that the cytokines IL-5, IL-6, TNF-alpha, and stromal cell-derived factor-1alpha as well as signaling via CD44 support the survival of isolated bone marrow plasma cells. The cytokines IL-7 and stem cell factor, crucially important for early B cell development, do not mediate plasma cell survival, indicating that plasma cells and early B cells have different survival requirements. As shown in IL-6-deficient mice, IL-6 is required for a normal induction, but not for the maintenance of plasma cell responses in vivo, indicating that the effects of individual survival factors are redundant. Optimal survival of isolated plasma cells requires stimulation by a combination of factors acting synergistically. These results strongly support the concept that plasma cell survival depends on niches in which a combination of specific signals, including IL-5, IL-6, stromal cell-derived factor-1alpha, TNF-alpha, and ligands for CD44, provides an environment required to mediate plasma cell longevity.