In vitro and in vivo action of recombinant human GM-CSF (rhGM-CSF) in patients with myelodysplastic syndromes

The availability of rhGM-CSF has allowed the in vivo treatment of patients with cytopenia. Therefore, a phase I-II trial was initiated to study the effect of rhGM-CSF in patients with myelodysplastic syndromes who were not eligible for other kinds of therapy. rhGM-CSF has been tested in 10 patients in doses from 15 micrograms/m2 to 150 micrograms/m2 given intravenously over 8 hours for a cycle of 7 days followed by an interval of 14 days and a second 7-day treatment course. A dose-dependent increase in leukocyte count was observed in 9 of 10 patients. No change in reticulocyte numbers was seen and only one patient experienced an increase in platelet count. Toxicity mainly consisted of mild phlebitis at the site of infusion and sternal pain after bolus injection. An increase in blast cell counts in some patients necessitated the start of low-dose Ara-C therapy.     

Influence of small doses cytosine arabinoside (Ara-C) on leukemic cells in vitro and in vivo. Results in patients with leukemia and myelodysplastic syndrome

The authors reviewed the literature concerned with the action of low dose Ara-C on MDS and some forms of AL and demonstrated their own results in vitro and in vivo. In vitro bone marrow blast cells short-term liquid culture were performed with and without addition of low dose Ara-C. After 5 days culture morphological and also cytochemical changes could be observed often inconsistent with the original cellular type. In vivo 8 patients suffering from AL or MDS were treated with low dose Ara-C and among them marked improvement in 3 cases only was observed.     

Cell kinetics after high dose cytosine arabinoside in patients with acute myelocytic leukemia

In this study 10 patients with acute myelocytic leukemia (AML) each received a rapid intravenous injection of high dose cytosine arabinoside (HD Ara-C; 1 g/m2). Bone marrow aspirates were obtained before and after Ara-C administration to determine the percentage of cells in S-phase measured by flow cytometry. In 5 out of 10 cases synchronization of the leukemic cells in S-phase of the cell cycle was observed. However, the time of maximum synchronization turned out to be difficult to predict. Therefore, the strong correlation between percentage of cells in S-phase at diagnosis and the time of maximal accumulation of S-phase cells after Ara-C administration, as observed by others in childhood AML, could not be confirmed for adult AML patients. Although synchronization of AML cells after in vivo Ara-C administration could be demonstrated in at least half of the patients, the practical consequences are such that clinical application was hampered.     

Low dose cytosine arabinoside in refractory anemia with excess of blasts in transformation

Myelodysplastic syndromes (MDS) are heterogeneous diseases. Patients with blast counts of more than 20% of nucleated bone marrow cells have a high risk of short survival. We treated six patients with refractory anemia with excess of blast in transformation (RAEBiT) with low dose cytosine arabinoside (LD Ara-C). We had one partial remission (PR), surviving 16 weeks and two complete remissions (CR), surviving 22 and 55+ months. Myelosuppression was dominant in all patients, but was not as serious as with conventional remission-induction treatments for leukemias. Bone marrow aplasia occurred in all responding patients, but a differentiation effect is possible too. Maintenance therapy with LD Ara-C may be important for the two long-lasting CR.     

Cellular Intrinsic Mechanism Affecting the Outcome of AML Treated with Ara-C in a Syngeneic Mouse Model

The mechanisms underlying acute myeloid leukemia (AML) treatment failure are not clear. Here, we established a mouse model of AML by syngeneic transplantation of BXH-2 derived myeloid leukemic cells and developed an efficacious Ara-C-based regimen for treatment of these mice. We proved that leukemic cell load was correlated with survival. We also demonstrated that the susceptibility of leukemia cells to Ara-C could significantly affect the survival. To examine the molecular alterations in cells with different sensitivity, genome-wide expression of the leukemic cells was profiled, revealing that overall 366 and 212 genes became upregulated or downregulated, respectively, in the resistant cells. Many of these genes are involved in the regulation of cell cycle, cellular proliferation, and apoptosis. Some of them were further validated by quantitative PCR. Interestingly, the Ara-C resistant cells retained the sensitivity to ABT-737, an inhibitor of anti-apoptosis proteins, and treatment with ABT-737 prolonged the life span of mice engrafted with resistant cells. These results suggest that leukemic load and intrinsic cellular resistance can affect the outcome of AML treated with Ara-C. Incorporation of apoptosis inhibitors, such as ABT-737, into traditional cytotoxic regimens merits consideration for the treatment of AML in a subset of patients with resistance to Ara-C. This work provided direct in vivo evidence that leukemic load and intrinsic cellular resistance can affect the outcome of AML treated with Ara-C, suggesting that incorporation of apoptosis inhibitors into traditional cytotoxic regimens merits consideration for the treatment of AML in a subset of patients with resistance to Ara-C.     

Low-dose Ara-C in the treatment of acute leukemia. Cytotoxicity or differentiation induction?

The differentiation inducing effect of low-dose Ara-C on human myeloid leukemic cells was studied in two patients with subacute myelocytic and subacute myelomonocytic leukemia in vivo and in vitro. By continuous i.v. administration of 10 mg Ara-C/m2 over 12 h daily for 12 or 20 days complete remissions were obtained in both patients with normalization of the incidence of the committed progenitor cells BFU-E and CFU-C in the marrow while the incidence of pluripotent CFU-GEMM remained subnormal. Parallel cultures of the patients' bone marrow cells in diffusion chambers (DC) implanted in mice demonstrated a clear cytotoxic effect of low-dose Ara-C. The greater increase of granulopoietic cells within DC in the Ara-C exposed group than in control mice after the end of drug administration is, in addition, an indication for differentiation induction by this kind of Ara-C therapy.     

Effects of IFN on human eosinophils in comparison with other cytokines. A novel class of eosinophil activators with delayed onset of action.

Extracellular killing is regarded as one of the main functions of eosinophils. Therefore, a cytotoxicity assay against antibody-coated Daudi-lymphoma cells was established to measure cytokine effects on peripheral blood eosinophils from healthy volunteers. Optimal time of exposure to cytokines and half optimal concentrations (EC50) were determined and the capability of various cytokines to enhance cytotoxicity of eosinophils was compared. Thus, after 24 h with cytokine, the highest activation of eosinophils was observed with recombinant human rhIFN-gamma (EC50 = 0.2 U/ml), followed by the known activators of eosinophils recombinant human granulocyte/macrophage CSF (rhGM-CSF), rhIL-3, and murine IL-5 (mIL-5). rhIFN-alpha and natural human IFN-beta (nhIFN-beta) enhanced cytotoxicity as well. On the other hand, in short term assays, eosinophils were not stimulated by IFN and the strongest stimulator was rhGM-CSF (EC50 = 0.2 U/ml), followed by rhIL-3, mIL-5, rhTNF, and rhIL-4. With rhTNF-alpha enhancement was more pronounced on freshly isolated eosinophils (EC50 = 0.6 U/ml) and declined with time. No significant stimulation was detected with rhG-CSF, rhIL-1 beta, rhIL-2, rhIL-6, and rhIL-8. On neutrophils, rhIL-8 enhanced cytotoxicity, but the stimulation was weak in relation to other neutrophil activators. Studies on the mechanism of cytotoxic activity revealed that cytotoxicity required opsonization of targets with specific antibody. FMF analysis was performed demonstrating that freshly isolated eosinophils express Fc-gamma RII (CD32), small amounts of Fc-gamma RIII (CD16), but not Fc-gamma RI (CD64). In experiments with blocking antibodies to Fc-gamma R cytotoxicity was restricted to Fc-gamma RII. Expression of Fc-gamma RII was not enhanced by rhGM-CSF, rhTNF-alpha, and mIL-5, but a significant increase in the number of positive cells was observed after incubation with rhIFN-gamma for 24 h (p less than 0.05). In addition, enhanced viability of eosinophils was observed when cultured in the presence of rhIFN-gamma, rhIFN-alpha, rhGM-CSF, and rhTNF-alpha, but not of rhG-CSF and rhIL-2. Thus, IFN appear to be another class of activators of eosinophils, characterized by their delayed type of action.     

Interactions between lymphocytes and hematopoietic progenitor cells in normal and leukemic human bone marrow (phase contrast observations)

Single cell observations of normal and of leukemic human bone marrow cells demonstrated cell-cell interactions of lymphocytes with hematopoietic progenitor cells. In all cases lymphocytes and target cells were from the same individual. Lymphocyte-target cell interactions occurred more frequently with normal committed progenitor cells and leukemic blast cells from acute myeloid leukemia than with precursor cells of the proliferative cell pool, including myeloblasts, promonocytes, erythroblasts and megakaryocytes. Both induction of mitosis and degeneration of the progenitor cells occurred after cell-cell interaction with almost the same frequency. Acute myeloid leukemic blast cells degenerated after contact with lymphocytes with the same frequency as normal progenitor cells (i. e. in 16% of cell contacts), but especially during mitosis. In contrast, normal and regenerating bone marrow progenitor cells from myeloproliferative diseases demonstrated no degeneration after cell-cell interaction with lymphocytes during mitosis. Otherwise the induction of mitoses by lymphocyte-target cell interactions was more frequently observed in normal progenitor cells than in leukemic blasts.     

The effect of cytokines on the ploidy of megakaryocytes

The effects of recombinant cytokines on the ploidy of human megakaryocytes derived from megakaryocyte progenitors were studied using serum-free agar cultures. Nonadherent and T cell-depleted marrow cells were cultured for 14 days. Megakaryocyte colonies were identified in situ by the alkaline phosphatase anti-alkaline phosphatase technique, using monoclonal antibody against platelet IIb/IIIa. The ploidy of individual megakaryocytes in colonies was determined by microfluorometry with DAPI (4',6-diamidino-2-phenylindole) staining. Recombinant human interleukin 3 (rhIL-3) and recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) supported megakaryocyte colony formation in a dose-dependent manner. However, both rhIL-3 and rhGM-CSF had no definite ability to increase the ploidy values. Recombinant human erythropoietin (rhEpo) or recombinant human macrophage colony-stimulating factor (rhM-CSF) by itself did not stimulate the growth of megakaryocyte progenitors. rhEpo or rhM-CSF, however, stimulated increases in the number, size and ploidy values of megakaryocyte colonies in the presence of rhIL-3 or rhGM-CSF. Recombinant human interleukin 6 (rhIL-6) showed no capacity to generate or enhance megakaryocyte colony formation when added to the culture alone or in combination with rhIL-3. rhIL-6, however, increased the ploidy values in colonies when added with rhIL-3. These results show that rhEpo, rhM-CSF and rhIL-6 affect endomitosis and that two factors are required for megakaryocyte development.     

Prolonged Treatment with DNMT Inhibitors Induces Distinct Effects in Promoters and Gene-Bodies

Treatment with the demethylating drugs 5-azacytidine (AZA) and decitabine (DAC) is now recognised as an effective therapy for patients with Myelodysplastic Syndromes (MDS), a range of disorders arising in clones of hematopoietic progenitor cells. A variety of cell models have been used to study the effect of these drugs on the methylation of promoter regions of tumour suppressor genes, with recent efforts focusing on the ability of these drugs to inhibit DNA methylation at low doses. However, it is still not clear how nano-molar drug treatment exerts its effects on the methylome. In this study, we have characterised changes in DNA methylation caused by prolonged low-dose treatment in a leukemic cell model (SKM-1), and present a genome-wide analysis of the effects of AZA and DAC. At nano-molar dosages, a one-month continuous treatment halved the total number of hypermethylated probes in leukemic cells and our analysis identified 803 candidate regions with significant demethylation after treatment. Demethylated regions were enriched in promoter sequences whereas gene-body CGIs were more resistant to the demethylation process. CGI methylation in promoters was strongly correlated with gene expression but this correlation was lost after treatment. Our results indicate that CGI demethylation occurs preferentially at promoters, but that it is not generally sufficient to modify expression patterns, and emphasises the roles of other means of maintaining cell state.     

Synergistic and antagonistic effects of recombinant human interleukin (IL) 3, IL-1 alpha, granulocyte and macrophage colony-stimulating factors (G-CSF and M-CSF) on the growth of GM-CSF-dependent leukemic cell lines

Three human leukemia cell lines (TALL-101, AML-193, and MV4-11) that require granulocyte/macrophage-colony stimulating factor (GM-CSF) for growth in a chemically defined medium were examined for their response to recombinant human (rh) cytokines. Either rh interleukin (IL)-3 or rhGM-CSF alone supported the long term growth of all three cell lines, and the two growth factors acted synergistically to stimulate the proliferation of the early T lymphoblastic leukemia (TALL-101) and of the monocytic leukemia (AML-193) cells. However, IL-3 antagonized the proliferation of the biphenotypic B-myelomonocytic leukemia (MV4-11) cells in the presence of GM-CSF when both factors were used at very low concentrations. The rh granulocyte (G)-CSF independently supported the long and short term growth of AML-193 and MV4-11, respectively, and synergized with GM-CSF in inducing proliferation of these cells. By contrast, G-CSF did not stimulate TALL-101 cell growth and antagonized the effect of GM-CSF such that proliferation was arrested. Although neither rh macrophage (M)-CSF nor rhIL-1 alpha independently promoted proliferation of the three leukemia cell lines, these cytokines were able to either up- or down-regulate the GM-CSF-dependent growth of these cells. Taken together, these data demonstrate that leukemic cells often require the synergistic action of several cytokines for optimal growth, whereas other combinations of factors may be growth-inhibitory. This raises the possibility that multiple hemopoietic growth factors sustain or control leukemic cell proliferation also in vivo. In addition, the observation the G-CSF, M-CSF, and IL-1 alpha can, in some cases, arrest cell proliferation without inducing differentiation suggests that the programs of proliferative arrest and differentiation in leukemic cells can be dissociated.     

Using peripheral blood circulating DNAs to detect CpG global methylation status and genetic mutations in patients with myelodysplastic syndrome

Myelodysplastic syndrome (MDS) is a hematopoietic stem cell disorder. Several genetic/epigenetic abnormalities are deeply associated with the pathogenesis of MDS. Although bone marrow (BM) aspiration is a common strategy to obtain MDS cells for evaluating their genetic/epigenetic abnormalities, BM aspiration is difficult to perform repeatedly to obtain serial samples because of pain and safety concerns. Here, we report that circulating cell-free DNAs from plasma and serum of patients with MDS can be used to detect genetic/epigenetic abnormalities. The plasma DNA concentration was found to be relatively high in patients with higher blast cell counts in BM, and accumulation of DNA fragments from mono-/di-nucleosomes was confirmed. Using serial peripheral blood (PB) samples from patients treated with hypomethylating agents, global methylation analysis using bisulfite pyrosequencing was performed at the specific CpG sites of the LINE-1 promoter. The results confirmed a decrease of the methylation percentage after treatment with azacitidine (days 3-9) using DNAs from plasma, serum, and PB mono-nuclear cells (PBMNC). Plasma DNA tends to show more rapid change at days 3 and 6 compared with serum DNA and PBMNC. Furthermore, the TET2 gene mutation in DNAs from plasma, serum, and BM cells was quantitated by pyrosequencing analysis. The existence ratio of mutated genes in plasma and serum DNA showed almost equivalent level with that in the CD34+/38- stem cell population in BM. These data suggest that genetic/epigenetic analyses using PB circulating DNA can be a safer and painless alternative to using BM cells.     

A Zebrafish Model of Myelodysplastic Syndrome Produced through tet2 Genomic Editing

The ten-eleven translocation 2 gene (TET2) encodes a member of the TET family of DNA methylcytosine oxidases that converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) to initiate the demethylation of DNA within genomic CpG islands. Somatic loss-of-function mutations of TET2 are frequently observed in human myelodysplastic syndrome (MDS), which is a clonal malignancy characterized by dysplastic changes of developing blood cell progenitors, leading to ineffective hematopoiesis. We used genome-editing technology to disrupt the zebrafish Tet2 catalytic domain. tet2^m/m (homozygous for the mutation) zebrafish exhibited normal embryonic and larval hematopoiesis but developed progressive clonal myelodysplasia as they aged, culminating in myelodysplastic syndromes (MDS) at 24 months of age, with dysplasia of myeloid progenitor cells and anemia with abnormal circulating erythrocytes. The resultant tet2^m/m mutant zebrafish lines show decreased levels of 5hmC in hematopoietic cells of the kidney marrow but not in other cell types, most likely reflecting the ability of other Tet family members to provide this enzymatic activity in nonhematopoietic tissues but not in hematopoietic cells. tet2^m/m zebrafish are viable and fertile, providing an ideal model to dissect altered pathways in hematopoietic cells and, for small-molecule screens in embryos, to identify compounds with specific activity against tet2 mutant cells.     

Lysis of fresh leukemic blasts by interferon-activated human natural killer cells

In a comprehensive study of 30 leukemia patients, it was found that a measurable fraction of fresh leukemic blasts from 8 of 8 adult patients with chronic myelogenous leukemia (CML) in blast crisis and 10 of 11 pediatric patients with childhood acute lymphocytic leukemia (ALL) were efficiently lysed by human peripheral blood natural killer (NK) cells as measured in 4-hour chromium release assays. The observed lysis of these fresh, noncultured, neoplastic blasts was mediated by a population of interferon-augmentable, FcR-positive, non-adherent large granular lymphoid cells from normal donors, which were also able to kill the 'standard' NK target K562. It was of further interest that all 8 of the patients with blast crisis CML exhibited myeloid type morphology. Furthermore, neoplastic lymphoblasts from 9 of 10 patients with NK-susceptible childhood ALL lacked easily detectable B or T cell markers and were of 'null' cell type. In marked contrast to the lytic susceptibility of fresh leukemic blasts from patients with ALL and CML in blast crisis, fresh neoplastic granulocytes from 5 patients with chronic phase CML (2 of which eventually progressed to myeloid type blast crisis), as well as leukemic blasts from 8 patients with acute myeloid leukemias (AML, AMMoL, and AMoL) were resistant to lysis as mediated by human NK cells from normal donors. The clinical implications of these findings are discussed.     

Shear stress regulates adhesion and rolling of CD44+ leukemic and hematopoietic progenitor cells on hyaluronan

Leukemic cells and human hematopoietic progenitor cells expressing CD44 receptors have the ability to attach and roll on hyaluronan. We investigated quantitatively the adhesion behavior of leukemic cell lines and hematopoietic progenitor cells on thin films of the polysaccharides hyaluronan and alginate in a microfluidic system. An applied flow enhances the interaction between CD44-positive cells and hyaluronan if a threshold shear stress of 0.2 dyn/cm² is exceeded. At shear stress ∼1 dyn/cm², the cell rolling speed reaches a maximum of 15 μm/s. Leukemic Jurkat and Kasumi-1 cells lacking CD44-expression showed no adhesion or rolling on the polysaccharides whereas the CD44-expressing leukemic cells KG-1a, HL-60, K-562, and hematopoietic progenitor cells attached and rolled on hyaluronan. Interestingly, the observations of flow-induced cell rolling are related to those found in the recruitment of leukocytes to inflammatory sites and the mechanisms of stem-cell homing into the bone marrow.     

Pathophysiological Scenario of Hematopoietic Disorders: A Comparative Study of Aplastic Anemia,Myelodysplastic Syndrome and Leukemia in Experimental Animals

The conversion of physiology to pathophysiology in hematological disorders viz: aplastic anemia, myelodysplastic syndrome (MDS) and leukemia in murine models was the subject of study in the present programme. Peripheral blood hemogram, spleno-somatic index, bone marrow smear study, cytochemical staining of marrow, cell release kinetics study during marrow explants culture, hematopoietic niche assessment, chromosomal aberration study, plasma membrane stability study of marrow cells, lysosomal membrane and mitochondrial membrane stability study and innate immune parameters were performed in the aplastic anemia, leukemia and MDS mouse model. In bone marrow aplasia, peripheral blood pancytopenia, marrow hypocellularity, decreased marrow cellular viability, deterioration of bone marrow hematopoiesis as well as hematopoietic microenvironment and extramedullary hematopoiesis were noticed. In addition, disruption of mitochondrial and lysosomal membrane integrity along with reduction of innate immune parameters were found in the hematopoietic suppressed condition. Surprisingly, no noticeable chromosomal aberration was found in the aplastic condition. Ineffective marrow hematopoiesis together with the disruption of hematopoietic microenvironment was observed in MDS. Also, extramedullary hematopoiesis, increased marrow cellular death, chromosomal aberration and loss of innate immunity were the common events. During leukemia, the number of functionally and structurally immature cells in the peripheral blood and bone marrow was increased together with malignant conversion of hematopoietic cells in the presence of malignancy supportive stromal microenvironment. Chromosomal aberration, decrease of cell mediated immunity with least mitochondrial apoptotic damage were also found in leukemic condition as well.     

Towards the understanding of the local hematopoietic bone marrow renin-angiotensin system

The classical view of the renin-angiotensin system (RAS) as a circulating endocrine system has evolved to organ- and tissue-based systems that perform paracrine/autocrine functions. Angiotensin II (Ang II), the dominant effector peptide of the RAS, regulates cellular growth in a wide variety of tissues in (patho)biological states. In 1996, we hypothesized that there exists a locally active RAS in the bone marrow affecting the growth, production, proliferation and differentiation of hematopoietic cells. Evidences supporting this hypothesis are growing. Ang II, through interacting with Ang II type 1 (AT1) receptor stimulates erythroid differentiation. This stimulatory effect of Ang II on erythropoiesis was completely abolished by a specific AT1 receptor antagonist, losartan. AT1a receptors are present on human CD34(+) hematopoietic stem cells. Ang II increases hematopoietic progenitor cell proliferation and this effect was also blocked by losartan. Angiotensin-converting enzyme (ACE) is involved in enhancing the recruitment of primitive stem cells into S-phase in hematopoietic bone marrow by degrading tetrapeptide AcSDKP. ACE inhibitors modified the circulating hematopoietic progenitors in healthy subjects. RAS may also affect pathological/neoplastic hematopoiesis. Renin has been isolated from leukemic blast cells. Higher bone marrow ACE levels in acute leukemic patients suggested that ACE is produced at higher quantities in the leukemic bone marrow. In this review, the 'State of the Art' of the local bone marrow RAS is summarized. A local RAS in the bone marrow can mediate, in an autocrine/paracrine fashion, some of the principal steps of hematopoietic cell production. To show a causal link between the components of RAS and the other regulatory hematopoietic growth factors is not only an academic curiosity. Elucidation of such a local bone marrow system may offer novel therapeutic approaches in pathologic/neoplastic conditions.     

Essential thrombocythemia: impaired regulation of megakaryocyte progenitors

In this paper, the in vitro growth of bone marrow early (megakaryocyte burst-forming units, BFU-meg) and late (megakaryocyte colony-forming units, CFU-meg) progenitors was evaluated in 18 essential thrombocythemia (ET) patients and 22 normal control subjects. BFU-meg clonality was demonstrated both in normal and ET bone marrows, cultivating these primitive progenitors at limiting dilutions in plasma clot assay: 1 to 7 BFU-meg/2.5 x 10(4) mononuclear non-adherent cells were observed, with a strong correlation in ET [r = 0.955 stimulated by recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) plus recombinant human interleukin (rhIL) 3], as well as in normal controls (r = 0.969). In order to clearly elucidate the in vitro response of ET megakaryocyte (meg) progenitors to recombinant growth factors, the interference of accessory cells (i.e., monocytes, T lymphocytes, and natural killer cells) and human serum were avoided by performing experiments on CD34+ cells in a serum-free fibrin clot assay. The number of both early and late meg progenitors in ET was significantly increased in response to rhIL-3, rhIL-3 plus rhIL-6, and rhIL-3 plus rhGM-CSF, but not in response to rhGM-CSF alone. Furthermore, both meg progenitors were investigated for their response to rh transfer growth factor (TGF)-beta 1, tested at concentrations from 0.01 to 10 ng/ml. rhTGF-beta 1 was able to inhibit CFU-meg and BFU-meg in a dose-response manner normal, whereas ET CFU-meg appeared less sensitive to the lower doses investigated (p less than 0.05) and ET BFU-meg were slightly reduced in number only at the higher concentrations of rhTGF-beta 1 (p less than 0.01). Our data suggest that the increased thrombopoiesis in ET may depend on an increased sensitivity of meg progenitors to some of the physiological growth factors and to a disrupted sensitivity to at least one negative regulator of megakaryocytopoiesis. Since these abnormalities involve both meg progenitors, this can be considered a demonstration that the neoplastic event hits the most primitive hemopoietic progenitors.     

IL-6 stimulates osteoclast-like multinucleated cell formation in long term human marrow cultures by inducing IL-1 release

IL-6 enhances the differentiation of pluripotent hematopoietic stem cells but predominantly affects the differentiation of hematopoietic cells in the granulocyte-macrophage lineage. We have previously shown that multinucleated cells (MNC) with many features of the osteoclast phenotype form in long term human marrow cultures. Addition of rhIL-6 (10 to 100 pg/ml) to these cultures significantly increased MNC formation, with greater than 80% of the MNC expressing an Ag that cross-reacts with the mAb 23c6. This antibody preferentially binds to osteoclasts. rhIL-6 did not enhance MNC formation in marrow cultures treated with 1,25 dihydroxyvitamin D3, a potent stimulator of MNC formation, but significantly increased the percentage of MNC that cross-reacted with the 23c6 mAb. Addition of antihuman IL-1 to cultures treated with rhIL-6 totally inhibited the increase in MNC formation stimulated by rhIL-6. In contrast, anti-IL-1 did not affect MNC formation stimulated by 1,25 dihydroxyvitamin D3. Further, conditioned media from marrow cultures exposed to rhIL-6 contained elevated levels of IL-1 beta (500 pg/ml compared to 23 pg/ml in control cultures 15 h after IL-6 addition). These results suggest that the capacity of rhIL-6 to stimulate formation of MNC which cross-react with 23c6 is mediated by induction of release of IL-1 beta.