Human Long-term Bone Marrow Cultures Research Articles

Response kinetics of radiation-induced micronucleated reticulocytes in human bone marrow culture

The frequency of micronucleated reticulocytes (MN-RETs) in the bone marrow or peripheral blood is a sensitive indicator of cytogenetic damage. While the kinetics of MN-RET induction in rodent models following irradiation has been investigated and reported, information about MN-RET induction of human bone marrow after radiation exposure is sparse. In this report, we describe a human long-term bone marrow culture (LTBMC), established in three-dimensional (3D) bioreactors, which sustains long-term erythropoiesis. Using this system, we measured the kinetics of human bone marrow red blood cell (RBC) and reticulocyte (RET) production, as well as the kinetics of human MN-RET induction following radiation exposure up to 6 Gy. Human bone marrow established in the 3D bioreactor demonstrated an average percentage of RBCs among total viable cells peaking at 21% on day 21. The average percentage of RETs among total viable cells reached a maximum of 11% on day 14, and remained above 5% by day 28, suggesting that terminal erythroid differentiation was still active. Time- and dose-dependent induction of MN-RET by gamma radiation was observed in the human 3D LTBMC, with peak values occurring at approximately 3 days following 1 Gy irradiation. A trend towards delayed peak to 3–5 days post-radiation was observed with radiation doses ≥2 Gy. Our data reveal valuable information on the kinetics of radiation-induced MN-RET of human bone marrow cultured in the 3D bioreactor, a synthetic bioculture system, and suggest that this model may serve as a promising tool for studying MN-RET formation in human bone marrow, thereby providing opportunities to study bone marrow genotoxicity testing, mitigating agent effects, and other conditions that are not ordinarily feasible to experimental manipulation in vivo.

Tributyltin chloride induces adipocyte differentiation in long-term human bone marrow cultures

Tributyltin chloride induces adipocyte differentiation in long-term human bone marrow cultures

CCL18/PARC stimulates hematopoiesis in long-term bone marrow cultures indirectly through its effect on monocytes

AbstractChemokines play a role in regulating hematopoietic stem cell function, including migration, proliferation, and retention. We investigated the involvement of CCL18 in the regulation of bone marrow hematopoiesis. Treatment of human long-term bone marrow cultures (LTBMCs) with CCL18 resulted in significant stimulation of hematopoiesis, as measured by the total number of hematopoietic cells and their committed progenitors produced in culture. Monocytes/macrophages, whose survival was almost doubled in the presence of CCL18 compared with controls, were the primary cells mediating this effect. Conditioned media from CCL18-treated mature monocytes fostered colony-promoting activity that increased the number of colonies formed by hematopoietic progenitor cells. Gene expression profiling of CCL18-stimulated monocytes demonstrated more than 200 differentially expressed genes, including those regulating apoptosis (caspase-8) and proliferation (IL-6, IL-15, stem cell factor [SCF]). Up-regulation of these cytokines was confirmed on the protein expression level. The contribution of SCF and IL-6 in CCL18-mediated stimulatory activity for hematopoiesis was confirmed by SCF- and IL-6–blocking antibodies that significantly inhibited the colony-promoting activity of CCL18-stimulated conditioned medium. In addition to the effect on monocytes, CCL18 facilitated the formation of the adherent layer in LTBMCs and increased the proliferation of stromal fibroblast-like cells.

The Proteasome Inhibitor Bortezomib Affects Osteoblastogenesis and Bone Formation In Vitro and In Vivo in Multiple Myeloma Patients.

It is well established that osteoblast formation and function are profoundly impaired in multiple myeloma (MM) patients. Osteoblastic cells also regulate myeloma cell growth and increasing bone formation result in a reduction of tumoral burden in mice. Recent data suggest that ubiquitin-proteasome pathway, the major cellular degradative system and therapeutic target in myeloma cells, also regulates osteoblast differentiation. Further it has been demonstrated that different proteasome inhibitors may stimulate bone formation in mice. Finally, preliminary observations obtained in MM patients treated with the proteasome inhibitor Bortezomib show an increase of bone specific alkaline phosphatase in responder patients as compared to non-responder ones. Currently it is not know whether the proteasome inhibitor Bortezomib may have a direct effect on osteoblast and bone formation in vitro human cultures and in vivo in MM patients.To clarify this issue first we checked the effect of Bortezomib either on osteoblast differentiation and formation or on osteoblast proliferation, survival and function. In long-term human BM cultures we found that Bortezomib did not reduce the number of both early bone marrow (BM) osteoblast progenitors Colony Forming Unit-Fibroblast (CFU-F) and late ones Colony Forming Bone nodules (CFU-OB). On the other hand we found that Bortezomib (2–3 nM) significantly induced osteoblast phenotype in human mesenchymal cells incubated in presence of osteogenic factors. A stimulatory effect on osteoblast markers was observed after 24 hours of Bortezomib treatment. Consistently we found that Bortezomib significantly increased the activity of the transcription factor Runx2/Cbfa1 in human osteoblast progenitors without affecting the canonical WNT signaling pathway checked by the evaluation of nuclear and cytoplasmatic active beta-catenin levels. Using the human osteoblast like cells MG-63 and immortalized normal osteoblasts (HOBIT) we found that Bortezomib at concentration ranging between 2nM and 5nM did not inhibit osteoblast proliferation or induce osteoblast apoptosis. Similarly, Bortezomib did not affect the expression of osteoblast markers, Runx2/Cbfa1 activity and WNT signaling in both MG-63 and HOBIT cells.To extent our in vitro observation we have evaluated the potential effect of Bortezomib in vivo in MM patients. Bone histomorphometry as well as immunostainig for Runx2/Cbfa1 and beta-catenin was performed on BM biopsies obtained from 15 MM patients before and after 6–8 cycles of Bortezomib administrated in mono-therapy. A significant increase in the number of osteoblastic cells X mm2 of bone tissue and in the number of Runx2/Cbfa1 positive osteoblastic cells was observed only in responder patients showing an early increase of the serum alkaline phosphatase.In conclusion our data indicate that Bortezomib may increase osteoblast differentiation in human mesenchymal cells without affecting the proliferation, survival and function of mature osteoblasts. In vivo and in vitro observations support the hypothesis that both direct and indirect effects on bone formation process could occur during Bortezomib treatment.

Clozapine and its Major Stable Metabolites, N-desmethylclozapine and Clozapine N-oxide do not Affect Human Bone Marrow Stromal Cells in vitro

Treatment of refractory schizophrenia with the atypical antipsychotic drug clozapine is associated with life-threatening agranulocytosis, characterised by a drop in neutrophil count. Theoretically, toxicity may be accounted for by direct action of parent drug or one of its stable metabolites on bone marrow stroma given importance of these cells to neutrophil maturation. Effects of clozapine, N-desmethylclozapine and clozapine N-oxide on stromal cell viability were therefore assessed using human primary long-term bone marrow culture and stromal cell lines, HAS303 and LP101, to define cell-specificity of response. Clozapine, N-desmethylclozapine and clozapine N-oxide had no significant effect on bone marrow stromal, HAS303 and LP101 viability over a wide drug concentration range (10-20000 ng mLˉ1) compared with cells in absence of drug. Hence it is unlikely that parent drug or its stable metabolites are directly toxic to stroma under clinical conditions. Reduced capability of stroma to support myelopoiesis, however, cannot be excluded.

Fibroblast growth factor and ex vivo expansion of hematopoietic progenitor cells

Fibroblast growth factor (FGF) belongs to a family of heparin-binding polypeptides and shows multiple functions including cell proliferation, differentiation, survival and motility. The expression of FGF receptors is widely distributed on different hematopoietic progenitor cells and stromal cells, and FGFs play an important role in hematopoietic stem cell homeostasis. FGFs have been shown to sustain the proliferation of hematopoietic progenitor cells, maintaining their primitive phenotype. Basic FGF (bFGF, FGF-2) stimulates the formation of an adherent stromal cell layer in human long-term bone marrow cultures, and promotes hematopoietic cell development. FGF-2 has also been shown to synergize with other hematopoietic growth factors to enhance in vitro colony formation by several classes of hematopoietic progenitor cells. Results of ex vivo expansion and clinical trials to date suggest that hematopoietic cells cultured under stroma-free cytokine combination conditions may be insufficient to restore hematopoiesis after a myeloablative conditioning regimen, although some recent trials demonstrated an improvement in engraftment and a reduction of the period of pancytopenia, especially neutrophils and platelets, after transplantation. A recent study by our group demonstrated that FGF-2 is effective in supporting the generation of megakaryocytic progenitor cells during ex vivo expansion. These observations could be useful in reducing the long period of severe thrombocytopenia that occurs frequently after umbilical/placental cord blood transplantation. The development of more effective amplifying systems for hematopoietic stem/progenitor cells can be expected since FGFs have multiple functions in hematopoiesis.

Gene expression profiling in the inductive human hematopoietic microenvironment

Human hematopoietic stem cells (HSCs) and their progenitors can be maintained in vitro in long-term bone marrow cultures (LTBMCs) in which constituent HSCs can persist within the adherent layers for up to 2 months. Media replenishment of LTBMCs has been shown to induce transition of HSCs from a quiescent state to an active cycling state. We hypothesize that the media replenishment of the LTBMCs leads to the activation of important regulatory genes uniquely involved in HSC proliferation and differentiation. To profile the gene expression changes associated with HSC activation, we performed suppression subtractive hybridization (SSH) on day 14 human LTBMCs following 1-h media replenishment and on unmanipulated controls. The generated SSH library contained 191 differentially up-regulated expressed sequence tags (ESTs), the majority corresponding to known genes related to various intracellular processes, including signal transduction pathways, protein synthesis, and cell cycle regulation. Nineteen ESTs represented previously undescribed sequences encoding proteins of unknown function. Differential up-regulation of representative genes, including IL-8, IL-1, putative cytokine 21/HC21, MAD3, and a novel EST was confirmed by semi-quantitative RT-PCR. Levels of fibronectin, G-CSF, and stem cell factor also increased in the conditioned media of LTBMCs as assessed by ELISA, indicating increased synthesis and secretion of these factors. Analysis of our library provides insights into some of the immediate early gene changes underlying the mechanisms by which the stromal elements within the LTBMCs contribute to the induction of HSC activation and provides the opportunity to identify as yet unrecognized factors regulating HSC activation in the LTBMC milieu.

Differential in vivo and in vitro expression of ED-B+ fibronectin in adult human hematopoiesis

Fibronectin in general is involved in adhesion and maturation of the erythroid lineage, in megakaryopoiesis and in the differentiation of human multipotent hematopoietic progenitor cells. However, little information exists about the expression of the oncofetal fibronectin isoform containing the ED-B domain (ED-B+ fn) in adult human hematopoiesis. The study was aimed to analyze the ED-B+ fn expression in normal human bone marrow cells by immunocytochemistry, flow cytometry and reverse-transcriptase polymerase chain reaction. The in vivo results were compared to ED-B+ fn expression in human long-term bone marrow cultures (LTBMC), cytokine supported expansion cultures of CD34+ peripheral blood progenitor cells (PBPC) and in leukemic cell lines with megakaryocytic characteristics (K562, CMK). ED-B+ fn protein was immunocytochemically demonstrated in normal bone marrow megakaryocytes as well as in megakaryocytic progenitor/precursor cells generated ex vivo from PBPC but we failed to detect ED-B+ fn mRNA. It was strongly expressed in LTBMC (RNA and protein). Analysis of human bone marrow mononuclear cells by flow cytometry and confocal microscopy revealed only cytoplasmic ED-B+ fn. The SCF/TPO induced megakaryocytic differentiation of ED-B+ fn negative CMK cells is associated with an increase of large megakaryocytes followed by an intracellular accumulation of ED-B+ fn mRNA and protein. We conclude that in normal human hematopoiesis ED-B+ fn protein expression and intracellular accumulation is restricted to differentiation of megakaryocytes. Low-abundant synthesis, intracellular accumulation and failure of membrane exposure might be due to a function during early events of wound healing (formation of a platelet-rich provisional extracellular matrix).

Hepatitis A virus suppresses monocyte-to-macrophage maturation in vitro.

To analyze the pathogenetic mechanism of hematopoietic dysregulation associated with hepatitis A virus (HAV) infections, we studied the influence of HAV on monocyte (MO)-to-macrophage (MAC) maturation in vitro. Exposure of peripheral blood-derived mononuclear cells (MNC) to HAV led to diminished adherence of MO to plastic. Furthermore, HAV inhibited the ability of peripheral blood MO to differentiate toward MAC. Freshly isolated and 14-day-old MO cultures demonstrated reduced differentiation and decreased phagocytic capacity after challenge with HAV. Viral replication in MO/MAC cultures was confirmed by titration of infectious virus. We also determined the influence of HAV on the MO/MAC population in human long-term bone marrow cultures (LTBMCs). Inoculation of bone marrow MNC with HAV suppressed the establishment of an adherent stromal layer containing a reduced number of MAC. Furthermore, increased MO numbers in the nonadherent fraction of HAV-challenged LTBMCs are indicative of the disturbance of MO adherence. These findings suggest that HAV infection leads to a disorder of the mononuclear phagocytic system which may contribute to functional abnormalities of the bone marrow stroma.

Effect of bone morphogenetic protein-6 on haemopoietic stem cells and cytokine production in normal human bone marrow stroma.

Normal human bone marrow stroma cells include stem cells for both haemopoietic and osteochondrogenic lineages and express both bone morphogenetic protein (BMP) type I and type II receptors. As a member of the TGF-beta super-family, BMP-6 binds to both BMP type I and type II receptors and is involved in the developmental processes of renal and hepatic systems as well as of human foetal intestine. Also, BMP-6 induces osteoblastic differentiation of pluripotent mesenchymal cells and is an autocrine stimulator of chondrocyte differentiation. The present study was carried out to investigate the effect of BMP-6 on human cobblestone-area-forming cells (CAFC), that represent the functional primitive repopulating haemopoietic stem cell in long-term bone marrow culture. Also, the effect of BMP-6 on marrow stroma production of interleukin-6, -11 and their common receptor gp130 that is expressed in haemopoietic stem cells and is indispensable for their proliferation and tri-lineage differentiation was examined. Moreover, the effect of BMP-6 on marrow stroma release of soluble adhesion molecule VCAM-1 mediating the primitive haemopoietic stem cell adhesion to marrow stroma was examined. The number of CAFC was significantly reduced after BMP-6 treatment from 88+/-10 per 10(5)cells in control cultures in a dose dependent manner to only 48+/-3 per 10(5)cells in 50 ng/ml BMP-6-treated cultures, P< 0.01. Quantitative ELISA measurement revealed 50 ng/ml BMP-6 was able to significantly reduce IL-6 and IL-11 production from marrow stroma, P< 0.01. Also, BMP-6 significantly increased soluble gp130 release by 7.4-fold in 50 ng/ml BMP-6-treated marrow stroma cultures. The profound rapid increase in this natural antagonist of human IL-6 cytokine family may reduce the gp130 signaling. Also, the soluble VCAM-1 released increased by two-fold in 50 ng/ml BMP-6-treated marrow stroma cultures. The marked increase in the soluble form may exert an antagonist effect on the function of VCAM-1 (ligand for VLA4). Recently, blocking the VLA4/VCAM-1 adhesion pathway was shown to mobilise haemopoietic CD34 positive cells in normal individuals. Also, we previously observed a significantly lower expression of VLA4 (CD49d) on G-CSF-mobilised blood CD34 positive cells than on bone marrow CD34 positive cells before mobilisation in the same normal donors. Since BMP are currently being used in clinical trials for bone repair and fracture healing, the present results suggest a possible role for BMP-6 in mobilising CD34 positive cells for transplantation. Further in vitro tests are required to evaluate this potential mobilising role of BMP-6 in human long-term bone marrow culture.

Activity of Acetyl-Ser-Asp-Lys-Pro (AcSDKP) on Human Hematopoietic Progenitor Cells in Short-Term and Long-Term Bone Marrow Cultures

The tetrapeptide acetyl-Ser-Asp-Lys-Pro (AcSDKP) is a potent inhibitor of hematopoietic stem cell proliferation. We examined the effects of AcSDKP on the production of granulocyte-macrophage colony-forming cells (CFU-GM) and high proliferative potential colony-forming cells (HPP-CFC) in human long-term bone marrow (LTBM) cultures and CFU-GM and erythroid burst-forming cells (BFU-e) in short-term liquid cultures. The addition of AcSDKP in short-term bone marrow cultures resulted in a maximum depression of the total number of progenitor cells as well as the number of progenitor cells entering cell cycle following culture with 10(-12) to 10(-14) M AcSDKP and 10(-14) M AcSDKP when exogenous cytokines (GM-CSF, IL-3, or SCF) were added. AcSDKP was added daily to LTBM cultures at various concentrations (10(-8) M to 10(-16) M) for up to 5 weeks. In these LTBM culture studies, AcSDKP inhibited the entry of nonadherent progenitor cells into S phase and decreased the number of nonadherent progenitor cells with peak activity at 10(-12) M. In contrast, AcSDKP had no effect on the number of adherent CFU-GM, HPP-CFC, or cellularity per culture or percent of adherent progenitor cells in S phase. These studies indicate that the concentration of the tetrapeptide is critical to the activity of AcSDKP on human hematopoietic progenitor cells. Furthermore, we report that the presence of cytokines or stromal cells also affects the response of progenitor cells to AcSDKP. These results will aid in determining kinetic properties of AcSDKP for the development of clinical protocols to protect normal human hematopoietic stem and progenitor cells following cycle-specific chemotherapy agents.

Evaluation of mmp and timp expression in the hematopoietic microenvironment

It is now recognized that the extracellular matrix (ECM) of bone marrow (BM) plays a role not only in supporting BM structure but also in regulating cellular functions. Matrix metalloproteinases (MMPs) and their nature tissue inhibitors (TIMPs) regulate ECM turnover and TIMPs also regulate cell growth. As human long-term bone marrow culture (LTMC) mimics the hematopoietic microenvironment, we used this model to examine the secretion of MMPs and TIMPs. To evaluate the contribution of individual lineages we examined the expression of MMP-2, MMP-9, MT-1 MMP, MT-4 MMP and TIMPs-1, -2 and -3 by hematopoietic cells of erythroid, myeloid and megakaryocytic lineages (expanded in vitro from human normal BM CD34+ cells) and by BM stromal cells (CFU-F, endothelial cells). We found that MMP-9 is expressed and secreted by precursors of all hematopoietic lineages while MMP-2 and MT-1 MMP are expressed mainly by stromal cells and cells of myeloid and megakaryocytic but not erythroid lineages. BM stromal cells expressed and secreted high levels of all the TIMPs tested. This pattern of secretion of MMPs and TIMPs was reflected in the LTMC system: during the first week of LTMC MMP-9 levels were high and then decreased with reduced number of hematopoietic cells while levels of MMP-2, TIMP-1 and TIMP-2 increased with establishment of an adherent layer and expansion of stromal cells. TIMP-3 was released from the ECM matrix by the end of week 8–9 when the production of CFU-GM by adherent and nonadherent layers decreased significantly. Thus we present clear evidence that MMPs and TIMPs are constituents of the hematopoietic microenvironment and we are now investigating their role in hematopoiesis.

Control of pH in human long-term bone marrow cultures with low-glucose medium containing zwitterion buffer lengthens the period of haemopoietic activity.

Primary long-term bone marrow cultures grown in 40 mM HEPES-buffered McCoy's 5A medium produced granulocyte-macrophage colony-forming units (CFU-GM) for a median of 9 weeks compared with 7 weeks with CO2/bicarbonate-buffered cultures. Reducing the medium glucose concentration (from 12.5 to 2.75 mM) extended the culture longevity to 17 weeks. The median period of erythroid colony detection increased from 6 to 8 weeks. Secondary cultures (5 x 106 cord blood mononuclear cells seeded on irradiated stroma) showed statistically similar myeloid and erythroid longevity to primary cultures. Improved control of medium pH significantly improved the capacity of long-term stromal layers to maintain stem cells in vitro.

IL-1β mediates diethyldithiocarbamate-induced granulocyte colony-stimulating factor production and hematopoiesis

Diethyldithiocarbamate (DDTC) exhibits chemoprotective effects via reduced myelosuppression in mice treated with various chemotherapeutic agents. The mechanism of DDTC-mediated chemoprotection is believed to involve the induction and release of several cytokines, including interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), and granulocyte colony-stimulating factor (G-CSF). In the present study the roles of IL-1β and TNF-α in DDTC-mediated G-CSF induction were examined using human long-term bone marrow cultures (hLTBMCs). Administration of IL-1 receptor antagonist (IL-1ra) to DDTC-treated hLTBMCs obviated the G-CSF induction profile and blocked the resultant colony proliferation, indicating that IL-1β mediates DDTC-induced G-CSF release and hematopoiesis. IL-1β mRNA levels were increased threefold over control following DDTC treatment of hLTBMCs, implying that DDTC induces IL-1β at the level of transcription. Conversely, studies involving inhibition of DDTC-induced TNF-α synthesis, with the inhibitor MNX 160, had no effect on DDTC-induced G-CSF release or colony proliferation. These findings taken together strongly suggest that IL-1β mediates the chemoprotective effects of DDTC.

Two different functions for CD44 proteins in human myelopoiesis.

CD44 is important during myelopoiesis, although the contributions of variant CD44 proteins are unclear. We show here that in human long-term bone marrow culture antibodies recognizing a CD44 NH2-terminal epitope (mab 25-32) or a CD44v6 epitope (mab VFF18) inhibit myelopoiesis. However, mab 25-32 but not mab VFF18 affects myeloid colony formation. These data suggest that an early precursor cell compartment is the target for the 25-32 antibody, whereas the mab VFF18 targets later stages in myelopoiesis. Since the bulk of hemopoietic precursor cells are negative for the v6 epitope and only a minor subset of myeloid cells express the v6 epitope, we have used several human myeloid progenitor cell lines to unravel the function of different CD44 proteins. These cell lines produce variant CD44 proteins, predominantly a new variant CD44v4-v10, when stimulated towards myeloid differentiation. Features that can be acquired by the expression of CD44v4-v10 are an increased hyaluronate (HA) and a de novo chondroitin sulphate A (CS-A) binding. Although, the expression of CD44v4-v10 per se is necessary for HA and CS-A binding, the protein backbone seems to require appropriate glycosylation. HA binding results in CD44-mediated cellular self-aggregation and adhesion to the stromal cell line MS-5. In summary, our data suggest that different CD44 proteins are important for at least two different steps in myelopoiesis.

Growth hormone stimulates multinucleated cell formation in long-term bone marrow cultures

Although the effects of growth hormone on bone metabolism are well-documented, their role in the regulation of immune responses such as the inflammatory process has not been thoroughly explored. This study investigated the formation of multinucleated cells (MNC) in long-term human bone marrow cultures. Experiments using 1 and 100 ng/ml of human recombinant growth hormone (hGH) and 10(-7) M of 1,25 dihydroxyvitamin D3 (VD3) showed that hGH increased the total number and nucleation of MNC. The effects of hGH were generally greater than those observed with VD3. Cytological and immunological characterization of MNC revealed several macrophage polykaryon features. MNC did not respond to calcitonin in a cyclic adenosine monophosphate assay and failed to resorb dentin slices. These results demonstrate that MNC formed in the presence of hGH and VD3 present an essentially macrophage polykaryon phenotype. In this context, growth hormone may be involved in the inflammatory process through upmodulation of macrophage polykaryon formation.

Increased hematopoietic progenitor cell maintenance in long-term bone marrow cultures containing minimal numbers of contaminating breast cancer cells.

The maintenance of hematopoietic progenitor cells as assayed in the mixed colony (CFU-GEMM) assay in human long-term bone marrow cultures was compared between normal allogeneic marrow transplantation donor collections and those from candidates for high-dose therapy and autologous bone marrow transplantation (ABMT). To be eligible for ABMT, patients were required to have a histologically normal appearing bone marrow and therefore any tumor contamination was at minimal levels and detectable only after evaluation of the cultured harvests. Marrow from 15 normal donors, 36 patients with breast cancer, and 30 patients with Hodgkin's disease was evaluated. The number of mononuclear cells placed in culture was standardized. In all groups, significantly more progenitor cells were recovered at 4-6 weeks of culture that at 12-14 weeks. At 4-6 and 12-14 weeks, there were no significant differences in the number of progenitor cells recovered from the cultures of normal donors and tumor negative cultures of breast cancer or Hodgkin's disease patients. However, following 4-6 and 12-14 weeks of culture, progenitor cell numbers of cultures which contained breast cancer cells were significantly higher than the pooled values for cultures from the concurrent normal controls, and those from breast cancer and Hodgkin's disease patients with tumor negative cultures. These results suggest that minimal breast cancer cell contamination of the bone marrow can influence the production of marrow progenitor cells. Exposure to prior chemotherapy or radiation therapy does not appear to be the cause of this effect. The most likely mechanism is the local production of cytokines by the tumor cells, although a process involving direct adhesive contact of the tumor cells with hematopoietic cells, which is sometimes observed in semisolid cultures, cannot be excluded.

UPMODULATION OF MULTINUCLEATED CELL FORMATION IN LONG-TERM HUMAN BONE MARROW CULTURES BY LEUKAEMIA INHIBITORY FACTOR (LIF)

Inflammatory processes are mediated by many cellular events involving different cell types (leukocytes, monocytes, stromal cells, etc.). Numerous soluble mediators regulate these reactions, including leukaemia inhibitory factor (LIF), a cytokine which may play an important role in inducing acute-phase protein synthesis by hepatocytes during inflammation. This study was designed to determine the effects of LIF on the human monocyte/macrophage lineage and provide a better definition of its behaviour during systemic inflammation. In-vitro exposure of human long-term bone marrow cultures to recombinant human LIF significantly increased (about two-fold) the number of multinucleated cells (MNC) formed after three weeks of culture. These LIF-induced MNC expressed tartrate-resistant acid phosphatase, and LIF increased this intracellular activity by about 50%. MNC displayed phagocytotic activity but were unable to degrade sperm whale dentin or respond to human calcitonin. They did not possess the main characteristics of osteoclasts and were in fact macrophage polykaryons. Our results demonstrate for the first time that LIF can induce macrophage polykaryon formation from human bone marrow culture, suggesting that this factor not only produces leukocytes but also has a direct influence on the monocyte/macrophage lineage.

Possible involvement of bone marrow stromal cells in agranulocytosis caused by vesnarinone treatment.

Vesnarinone, an oral therapeutic agent for cardiac failure, causes agranulocytosis as a side effect. To elucidate the mechanism of occurrence of the agranulocytosis, we examined the effect of vesnarinone on granulopoiesis using an in vitro human long-term bone marrow culture system. Addition of vesnarinone to the culture decreased the total number of hematopoietic cells, mainly composed of mature granulocytes and macrophages, but increased the number of granulocyte-macrophage progenitor cells (CFU-GM) and CD33-CD34+ cells as compared with an untreated control. Differentiation of CFU-GM was induced by removing the agent from the culture medium, indicating that the effect of vesnarinone was reversible. The agent did not directly affect CFU-GM in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). Furthermore, treatment of stromal cells with vesnarinone repressed the production of G, GM, M-CSF, suggesting that the agent may cause a hematopoietic disorder, agranulocytosis, through the impairment of stromal cell function.

Heterogeneity of Expression of CD68 and Other Macrophage-associated Antigens in Human Long-term Bone Marrow Culture

The authors have performed an immunohistochemical study of intact human long-term bone marrow cultures (hLTBMC) grown directly onto glass slides. Between 4 and 12 weeks of growth, such cultures consist of a complex stromal layer supporting foci of haemopoietic cells which undergo granulocytic and monocytic differentiation. As part of a large panel of antibodies employed to characterize monocytes and macrophages within hLTBMC, we included six different anti-CD68 reagents and three antibodies representing a putative new CD group recognizing a macrophage-associated antigen of 130 kDa molecular weight. These gave heterogeneous immunostaining patterns with macrophages and stromal myofibroblastic cells.