Phorbol-ester-induced stable changes in the regulation of DNA synthesis and intracellular pH are accompanied by altered expression of protein kinase C in the monoblastoid cell line U-937

12-O-tetradecanoylphorbol-13-acetate (TPA)-induced changes in cytoplasmic pH, cytoplasmic Ca2+-concentration, rate of DNA synthesis, and concentration and activity of protein kinase C (PKC) were studied in human monoblastoid cell lines. The cell line U-937 GTB was compared to the subline U-937 RES (adapted to growth in the presence of 10(-9) M TPA) and another subline U-937 RESREV (U-937 RES grown in TPA-free medium) established in order to analyze the stability of the TPA-induced differences. TPA induced half maximal inhibition of DNA synthesis in the wild-type U-937 GTB cell line at 10(-9) M, whereas 10 times higher concentrations of phorbol ester were needed for a corresponding inhibition of the U-937 RES and U-937 RESREV lines. Furthermore, the U-937 RES cells exhibited a decreased sensitivity to TPA, and the U-937 RESREV cells did not respond at all to this agent with regard to cytoplasmic alkalinization by an intracellular mechanism independent of Na+/H+ exchange. A Na+-dependent system for extrusion of protons, which was activated by the Ca2+ ionophore ionomycin, was also severely depressed as a result of TPA-adaptation. The concentration of PKC, measured by immunoblotting, was reduced by 34 and 24% in U-937 RES and U-937 RESREV cells, respectively, as compared to the wild-type U-937 GTB line. The corresponding reductions in PKC activity were 32 and 54% when histone III-S was used as substrate. The data suggest that adaptation to growth in the presence of TPA results in stable modifications of several parameters, which are assumed to be involved in the regulation of proliferation and differentiation. Furthermore, the data from the U-937 RESREV cells question a causal relationship between cytoplasmic alkalinization and control of proliferation.     

1-B-D arabinofuranosyl cytosine and all-trans retinoic acid in combination accelerates and increases monocyte differentiation of myeloid leukemic cells

The effect of the combination of two different agents on myeloid differentiation was studied. Human promyelocytic cells (HL-60) and monoblast-like cells (U-937) were treated with 1-B-D arabinofuranosyl cytosine (Ara-C) alone, or in combination with retinoic acid (trans-RA). The known dual potentiality of HL-60 promyelocytes was confirmed by their ability to mature into granulocytes following induction by retinoic acid and into monocyte-like cells following treatment with 1-B-D arabinofuranosyl cytosine. The U-937 cell line differentiated to monocyte-like cells with either of the two drugs. The differentiation induced by Ara-C involved an irreversible step after 24-h incubation with the drug, was concentration dependent, and far more superior on U-937 cells than on HL-60 cells. The outcome of these two cell lines after treatment with both Ara-C and trans-RA was also different: this combination maintained the monocyte differentiation in the HL-60 cell line, and resulted in a higher sensitivity of U-937 cells to Ara-C, as indicated by a cell response to low Ara-C concentrations and a more rapid expression of monocyte specific properties.     

Bone marrow matrix promotes differentiation and prolongs the cell cycle of U-937 cells.

The extracellular matrix influences the growth and differentiation of a variety of cell types. In this study, the effects of bone marrow extracellular matrix on U-937 cells, a human histiocytic lymphoma cell line, were assessed. Sixty percent of U-937 cells adhered to extracellular matrix, whereas only 1% adhered to uncoated plastic. U-937 cells grown on extracellular matrix released significantly more lysozyme into the medium (8.3 +/- 0.3 micrograms/10(6) cells) compared to those grown on plastic (4.2 +/- 0.5 micrograms/10(6) cells). FMLP (f-met-leu-phe) receptor expression was also enhanced suggesting a more mature phenotype in cells grown on matrix (2980 cpm/10(6) cells vs 230 cpm/10(6) cells on plastic). Furthermore, bone marrow extracellular matrix inhibited proliferation of U-937 cells. After four days in culture, there was a 65% inhibition of cell growth in matrix-coated flasks compared to uncoated flasks. Since an arrest in G0/G1 usually precedes mammalian cell differentiation, DNA histograms were performed on U-937 cells grown on matrix to detect such an arrest. However, the cell cycle distribution of U-937 cells grown on extracellular matrix or uncoated plastic for various time periods was similar. In contrast, bromodeoxyuridine pulse labeling revealed approximately a 5 hr prolongation in cycle length in cells grown on extracellular matrix. We conclude that bone marrow extracellular matrix induced macrophage-like differentiation and inhibited proliferation of U-937 cells with a prolongation of the cell cycle that was not G0/G1 phase specific.     

Characterization of a species of non-specific cross-reacting antigen (NCA) exressed by human monocytic cell lines: Structure and expression during cell differentiation

It has been documented that human monocytes/macro-phages are reactive with antibodies directed to carcinoembryonic antigen (CEA) and non-specific cross-reacting antigens (NCAs), a group of glycoproteins antigenically cross-reactive with CEA, yet the molecules responsible for this antigenic activity have not been fully clarified. In the present study, among 7 myelomonocytic cell lines tested, 2 monoblastoid lines, U-937 and THP-I, were found to express NCA-50/90, a glycosyl-phosphatidylinositol-anchored cell-adhesion molecule chiefly expressed on granulocytes. The 2 cell lines showed a reaction pattern with 5 distinct anti-CEA and anti-NCA monoclonal antibodies, similar to that of CHO transfectants expressing recombinant NCA-50/90. Immunoprecipitation and SDS-PAGE analyses identified glycoproteins of about 95 and 55 kDa in U-937 and THP-1 cells, respectively. Deglycosylation of the 2 antigens with N-glycanase gave the same apparent molecular mass of about 45,000, which was also the same as that of the deglycosylated form of the recombinant NCA-50/90. Upon Northern-blot analysis, only one band of approximately 2.5 kb was detected in both cell lines with a cDNA probe for NCA-50/ 90, which has a broad specificity to the CEA gene family members. cDNA cloning demonstrated that the 2.5-kb clones encode the peptide of NCA-50/90. The expression of NCA-50/90 by U-937 and THP-1 was down-regulated at both the protein and mRNA levels during cell differentiation from monoblastoid to monocyte/macrophage-like cells induced by stimulation with phorbol 12-myristate 13-acetate. Our observations suggest that NCA-50/90 is a differentiation antigen of cells of the monocyte/macrophage lineage as well as of the granulocyte lineage.     

Differentiation of U-937 histiocytic lymphoma cells towards mature neutrophilic granulocytes by dibutyryl cyclic adenosine-3',5'-monophosphate

Treatment of U-937 cells with the cyclic nucleotide analog, dibutyryl cyclic adenosine-3',5'-monophosphate (dBcAMP) induced these cells to differentiate towards granulocytes. dBcAMP produced a dose- and time-dependent inhibition of U-937 cell growth reaching a maximum after 48-h treatment with 500 microM. At this concentration, dBcAMP had no effect on cell viability. Treatment with dBcAMP caused a rapid (within 24 h) decrease in the number of cells in the S phase of the cell cycle, with a concomitant increase in cells in the G0/G1 phase. dBcAMP also induced the appearance of f-met-leu-phe receptors on U-937 cells as well as the ability to produce hydrogen peroxide and superoxide anion. These data suggest that dBcAMP-treated U-937 cells were functionally mature. Using specific monoclonal antibodies and flow cytometry, we found that differentiated U-937 cells expressed the monocytic/granulocytic surface markers MY8 and MAC-1, but not the monocyte specific markers MO2 or MY4. In addition, dBcAMP-treated U-937 cells did not stain for nonspecific esterase, displayed less HLA-DR antibody binding than undifferentiated cells and appeared smaller and more granular. These are all characteristics of mature granulocytes. Taken together our studies indicate that differentiation of U-937 cells is not necessarily limited to the monocytic pathway of development.     

Synergism of prostaglandin E2 plus TNF in induction of differentiation of human monocytoid leukemic U-937 cells

Prostaglandin E2 (PGE2) induced differentiation of human monoblastic leukemia U-937 cells into cells with macrophage characteristics. PGE2 synergistically increased the differentiation, of U-937, ML-1 and HL-60 cells when combined with TNF. PGE1 and PGF2 also induced differentiation of U-937 cells; however, PGD2, deoxy-delta 9,12-13, 14-dihydro-PGD2 (delta 12-PGJ2), and PGI2 did not induce differentiation, either alone or in combination with TNF. PGE2 changed the dissociation constant of TNF for its receptors on U-937 cells only marginally, but it approximately doubled the number of binding sites.     

Monocytic origin of the human hematopoietic cell line U-937 and its convertibility to macrophages evidenced by isoenzyme mapping

U-937 represents a well established permanent human hematopoietic cell line. Electron microscopical and enzyme cytochemical studies as well as the analysis of surface glycoproteins have provided ample evidence for the monocytic origin of U-937. Upon stimulation with the tumour promotor 12-O-tetradecanoylphorbol-13-acetate (TPA), U-937 cells evolve into macrophage-like cells with phagocytic capacities. Since human blood monocytes (BM) are characterized by five acid esterase (AcE; EC 3.1.1.6) isoenzymes which are cell-specific in terms of isoelectric points (pI) and antigenicity, attempts were made in the present study to identify identical isoenzyme patterns in non- and TPA-stimulated U-937 cells. BM, cultured BM, non- and TPA-stimulated U-937 cells, as well as samples of resident human peritoneal macrophages (PM) as clearly-defined functional derivatives of BM, were subjected to isoenzyme analysis using isoelectric focusing (IEF). The five monocyte specific isoenzymes of AcE were identified in both populations of U-937. TPA-stimulated samples showed two additional bands, identical to those appearing in cultured BM after 4 days of glass-adherence and characteristic of resident human PM. Antisera raised against AcE of BM immunoprecipitated the two additional isoenzymes of TPA-stimulated U-937. It is concluded (1) the isoenzyme mapping of AcE documents the monocytic origin of U-937. (2) TPA-stimulation caricatures transformation of BM into resident tissue macrophages as far as pure morphology and AcE isoenzyme patterns are concerned. Thus, AcE isoenzyme mapping is apt for establishing reproducible and standardized criteria of different activation/differentiation states within the monocyte-macrophage lineage.     

Effect of endogenous and exogenous interferons on the differentiation of human monocyte cell line U937

The effect of interferons (IFNs) on the differentiation of hematopoietic cells was examined with the human monocyte cell line U937. The differentiation of U937 was induced by hydroxyvitamin D3 and was evaluated through the study of specific markers. The induction of the U937 differentiation was associated with a production of IFN and with a marked increase in (2'5') oligoadenylate synthetase. Addition of anti-IFN-alpha/beta antibodies inhibited the enhancement of (2'5') oligoadenylate synthetase and reduced the inhibitory effect of hydroxyvitamin D3 on cell growth. Nevertheless, neutralization of endogenous IFN excreted during U937 cell maturation did not modify the expression of the differentiation markers examined. Exogenous natural IFN-alpha, IFN-beta, or recombinant (r) IFN-gamma, when added to the culture medium, did not promote a "global" U937 differentiation. Most of the differentiation markers, except for reduction of nitroblue-tetrazolium, were not induced by IFN-alpha or -beta. However, rIFN-gamma was able to induce the appearance of several monocytic membrane markers at an extent comparable or slightly inferior to that elicited by hydroxyvitamin D3. Different effects on the expression of HLA antigens were obtained with these IFNs: IFN-alpha or -beta enhanced mainly class I HLA antigen expression, whereas rIFN-gamma increased selectively the expression of class II HLA DC1 but not HLA DR antigens. In contrast, phytohemagglutinin-leukocyte conditioned medium elicited a marked and selective enhancement of the expression of HLA-DR antigens. This induction of HLA DC1 antigens by rIFN-gamma was not observed in two other leukemic cell lines (HL60 and HEL). The present study shows that IFN-alpha or -beta may participate in the antiproliferative effect occurring during cellular differentiation, while IFN-gamma may be involved in the induction of the expression of specific monocytic markers involved in cellular immunoregulation.     

髓母细胞瘤中脑肿瘤干细胞的分离培养及鉴定

背景与目的:长期以来,脑肿瘤等实体肿瘤中是否存在肿瘤干细胞一直存在争论,最近已经有报道从胶质瘤和髓母细胞瘤等脑肿瘤及其它系统实体肿瘤中成功分离出肿瘤干细胞。本研究旨在利用简化的无血清培养基和悬浮培养方法,从人髓母细胞瘤中分离培养脑肿瘤干细胞,观察其在体外的增殖、分化,鉴定其特异性标志物CD133和Nestin的表达,验证肿瘤组织切片中CD133阳性细胞的存在,为脑肿瘤干细胞的进一步研究打下基础。方法:术中取11例患者髓母细胞瘤组织,分离成单细胞悬液,接种于含表皮生长因子、成纤维生长因子以及B27添加剂的无血清培养基中悬浮培养,以分离其中的脑肿瘤干细胞;对其增殖形成的脑肿瘤干细胞球连续传代培养;并进行单克隆形成实验,以确定原代肿瘤组织中肿瘤干细胞的百分率,观察脑肿瘤干细胞球的形成过程。然后,将脑肿瘤干细胞球接种于含血清培养基,观察脑肿瘤干细胞的分化现象。最后,利用免疫荧光法检测脑肿瘤干细胞特异性标志物CD133和Nestin在脑肿瘤干细胞球中的表达;利用免疫组化法检测组织切片中CD133阳性细胞的分布,并计算CD133阳性细胞百分率。结果:本组11例髓母细胞瘤中均仅有少数肿瘤细胞能够在无血清培养基中存活并悬浮生长、增殖形成克隆性脑肿瘤干细胞球,传代后脑肿瘤干细胞仍保持很强的自我更新和增殖能力。单克隆形成实验表明原代肿瘤细胞中具有单克隆形成能力的肿瘤干细胞百分率为(31.18±6.18)%。在含血清培养基中脑肿瘤干细胞发生贴壁分化,产生具有多种细胞形态的分化细胞。脑肿瘤干细胞表达神经干细胞的特异性标志物CD133和Nestin;肿瘤组织切片中CD133阳性细胞呈巢状或散在分布,占全部肿瘤细胞的(33.06±8.57)%。结论:人髓母细胞瘤组织中存在一定量的具有自我更新和增殖能力、表达CD133和Nestin的脑肿瘤干细胞,并能在体外将其分离、培养和诱导分化。     

Regulation of TNF-alpha, IL-1 and IL-6 synthesis in differentiating human monoblastoid leukemic U937 cells

The human monoblastoid tumor cell line U937 was induced to differentiate along the monocyte/macrophage lineage by treatment with 5 x 10(-9) M 12-O-tetradecanoyl phorbol-13-acetate (TPA). Between 2 h and 4 h following TPA-treatment U937 cells started to release significant amounts of TNF-alpha which remained detectable until 8-10 days. A significant IL-1 beta release was measured 24 h-48 h post stimulation and increased levels of IL-1 beta persisted until 20-22 days of culture. In contrast no release of either IL-1 alpha or IL-6 could be detected with 5 x 10(-9) M TPA during the whole time course of the experiments. The sequential induction of TNF-alpha and IL-1 beta appeared to be independently regulated since TNF-alpha release was not required for the onset of IL-1 beta production. Northern-blot analysis confirmed the sequential induction and the long term expression of TNF-alpha and IL-1 beta mRNAs. Western-blot analysis predominantly showed a high molecular weight IL-1 beta protein of about 35 kD. Further investigations on the regulation of cytokine production and release by TPA-differentiated U937 cells revealed that TNF-alpha and IL-1 beta synthesis was not influenced by exogenously added rhTNF-alpha or PGE2, whereas rh gamma-IFN specifically enhanced the IL-1 beta production. Thus, the regulation and intracellular processing of cytokines generated by differentiating U937 cells shows some differences when compared to mature monocytes/macrophages which may be related to the tumorigenic origin of U937 cells or to an incomplete differentiation.     

Idiotypic immunoglobulin secretion by human B cell non-Hodgkin's lymphomas is related to the expression of the interleukin-2 receptor

Tumor cells from 5 human B cell non-Hodgkin's lymphoma (B-NHL) patients were investigated for proliferative activity and idiotypic (Id+) immunoglobulin (Ig) secretion in serum-free medium without deliberate addition of B cell growth or differentiation factors (BCDF). These data were compared with cell surface marker expression, notably of activation antigens such as 4F2 and interleukin-2 (IL-2) receptor. Cells from all patients became 4F2 positive at the end of the 6-day culture period. Freshly drawn cells from 3 out of 5 patients expressed the IL-2 receptor (CD25; Tac antigen) or acquired this marker during culture in vitro and secreted relatively high levels of Id+ Ig in vitro. This correlated with elevated serum Id levels (greater than or equal to 0.5 micrograms/ml in vitro versus greater than or equal to 20 micrograms/ml in vivo). In the 2 CD25 (Tac)- B-NHL patients serum Id levels were below the detection limit and the amount of Id+ Ig secreted in vitro did not surpass 50 ng/ml. Only the B-NHL cells from a single patient were initially CD25 (Tac) positive and only these cells proliferated in serum-free culture. To test whether IL-2 receptor expression in the 3 CD25 (Tac)+ patients was functional, recombinant IL-2 (rIL-2) either alone or in conjunction with BCDF and recombinant IL-4 (rIL-4) was added to the cultures. In 2 out of 3 CD25 (Tac)+ patients rIL-2 was capable of enhancing proliferation or Ig secretion. In addition rIL-2 was found to enhance BCDF-mediated but not rIL-4 mediated responses. The third CD25 (Tac)+ B-NHL population was resistant to any of these lymphokines. Thus, this serum-free culture system may accurately reflect patient serum Id levels. IL-2 appears to regulate not only the in vitro but also the in vivo Ig secretion by neoplastic B cells.     

Growth-factor-dependent migration of human lung-cancer cells.

Human lung tumors express different types of growth-factor receptors and corresponding ligands that might modulate several biological functions such as proliferation, differentiation, adhesion, and chemotaxis. In the present study, we have investigated the expression of different growth-factor receptors and their ligands in 5 established human lung-cancer cell lines. Using RT-PCR, we found that IGF-II/mannose-6-phosphate (M6P), c-met, EGF and c-kit receptors are expressed in 5/5 human lung-cancer cell lines. In order to investigate the biological function of these receptors, we performed Boyden-chamber assays using various growth factors as chemo-attractants. Human non-small-cell-lung-cancer cells (non-SCLC) migrated to recombinant human (rh)IGF I and IGF II at concentrations ranging from 1 to 1000 ng/ml, to HGF at 10 to 100 ng/ml, to EGF at 1 to 100 ng/ml and SCF at 1 to 50 ng/ml. In addition, we performed Boyden-chamber assays using U-1810-, U-1752- and Wart-derived serum-free conditioned medium as chemo-attractants. Serum-free conditioned medium stimulated migration of producer cells in a dose-dependent manner. The autocrine motility stimulating effect of U-1810-derived serum-free conditioned medium could be inhibited by 50% in the presence of neutralizing ahIGF-II antibodies in the assay, suggesting a possible autocrine motility loop in vitro.     

Purification and characterization of human fibroblast derived differentiation inducing factor for human monoblastic leukemia cells identical to interleukin-6.

A differentiation inducing factor for human monocytic leukemia cells was purified to homogeneity from conditioned medium of WI-26VA4, a human fibroblast cell line. The purification scheme consisted of micro bead silica gel chromatography, hydroxyapatite chromatography, gel filtration chromatography, chromatofocusing and reverse phase high performance liquid chromatography. The purified protein was almost homogeneous when determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and N-terminal sequence analysis. The protein has a molecular weight of approximately 27,000 and an isoelectric point of 5.4. The sequence of the first 13 N-terminal amino acid residues was consistent with that of B-cell stimulatory factor 2 (interleukin-6) except for the absence of the N-terminal proline. The purified factor induced differentiation of human monocytic leukemia U-937 cells into a monocyte/macrophage pathway.     

Differentiation induction in non-lymphocytic leukemia cells upon treatment with mycophenolate mofetil

Inosine 5'-monophosphate (IMP) dehydrogenase catalyzes the rate-limiting reaction of guanine nucleotide biosynthesis and has been implicated in the reaction of cell growth and differentiation. We investigated the ability of mycophenolate mofetil, a prodrug of mycophenolic acid, to induce differentiation in HL-60 and U937 leukemic cells as well as in fresh leukemia cells from patients with non-lymphocytic leukemia. Treatment with mycophenolate mofetil reduced the intracellular guanosine 5'-triphosphate (GTP) levels and induced morphologic and functional differentiation in HL-60 and U937 cells dose-dependently. HL-60 and U937 cells developed macrophage-like cytoplasm as well as the expression of CD11b and CD14 antigens and the ability to oxidize nitroblue tetrazorium (NBT). These changes became evident when the intracellular GTP levels decreased to approximately 20-30% of the untreated control level and were abrogated by the addition of guanosine. In the fresh leukemic cells, differentiation induction was shown in the cells derived from seven of 13 patients. The fresh leukemia cells responding to mycophenolate mofetil revealed significant higher positivity to CD11b, CD14, and NBT before treatment and significantly reduced intracellular GTP levels after treatment compared to the non-responding cells. These findings suggest that mycophenolate mofetil induces differentiation in HL-60 and U937 cells and some fresh leukemia cells with moderate tendency to maturation, by causing a decrease in the intracellular GTP levels. Mycophenolate mofetil could be a promising differentiation inducer in vivo.     

Reexpression of the major histocompatibility complex (MHC) class-I antigen h-2k(b) by m1 (b16-f10) murine melanoma-cells

We monitored the expression of MHC class I antigens in a clonal isolate (M1) of B16-F10 murine melanoma cells, under various condition of growth in vitro. We found that, compared to exponentially growing cultures, cells from confluent M1 cultures expressed higher levels of H-2D(b) and H-2K(b) surface antigens. Expression of MHC class I antigens could be enhanced further by incubation of M1 cells in serum-free medium for 24-48 hours. Northern blot analyses indicated that the up-regulation of MHC class I antigen expression was associated with increased levels of H-2K(b) and H-2D(b) mRNAs, without a concomitant decrease in c-myc expression. Analyses of the cell cycle distribution of M1 cells stained for MHC class I antigens failed to show any differential segregation of H-2D(b)- or H-2K(b)-positive cells in any phase of the cell cycle. Enhanced MHC class I antigen expression appeared not to be due to the release into the medium of known cytokines like IFN-alpha, -beta, -gamma, TNF-alpha, IL-6 (IFN-beta(2)), and IL-1 by the M1 cells either at confluence, or after growth in serum-free medium. Taken together, our experimental results indicate that expression of MHC class I genes by M1 cells can be greatly enhanced by manipulating culture conditions; these observations are particularly important for the re-expression of the H-2K(b) gene, which was assumed to be phenotypically silent in B16 cells. These data also suggest that re-expression of H-2K(b) antigen in M1 cells at confluence may be related to cell-cell contact, consistent with the concept that H-2K(b) molecules can play a nonimmune role in cellular communication and growth control.     

mda7-/IL2-4和IL2-4delE5诱导急性髓系白血病细胞向单核细胞分化

目的:研究人黑素瘤分化相关基因-7(melanoma differentiation associated gene-7,mda-7;又称IL-24)及其剪接体IL-24 delE5与急性髓系白血病(acute myeloid leukemia,AML)细胞分化的关系。方法:十四烷酰佛波醇-乙酯(12-O-tetrade-canoylphorbol-13-acetate,TPA)处理人急性髓系白血病细胞系U937、HL-60,real-time PCR及Western blotting检测细胞中mda-7/IL-24和IL-24 delE5的表达,FACS检测细胞表面CD11b、CD14和CD115的表达。siRNA干扰U937、HL-60细胞中mda-7/IL-24和IL-24 delE5的表达后,再以TPA诱导细胞分化,FACS检测CD11b、CD14和CD115的表达。用前期构建的mda-7/IL-24和IL-24 delE5载体转染U937、HL-60及AML-M5患者的原代白血病细胞,观察异位过表达mda-7/IL-24和IL-24 delE5能否诱导白血病细胞向单核细胞分化。结果:TPA在诱导U9...     

Synergistic regulatory effects of interleukin 6 and interleukin 1 on the growth and differentiation of human and mouse myeloid leukemic cell lines

We analyzed the effect of recombinant human interleukin 6 (IL-6), in combination with human recombinant interleukin 1 alpha (IL-1 alpha), on the growth and differentiation of several human and mouse myeloid leukemic cell lines, specifically U937, HL-60, M1, and its subclone M1-3b-N, into macrophage-like cells. IL-6 and IL-1 inhibited the growth of U937, M1, and M1-3b-N in a dose-dependent manner. Treatment of these cells with both IL-6 and IL-1 resulted in either an additive or a synergistic growth inhibition. IL-6 alone induced moderate differentiation of U937 and M1-3b-N, but the combination of IL-6 and IL-1 synergistically augmented this differentiation. In M1, only the combination of IL-1 and IL-6 resulted in differentiation. These two cytokines, whether alone or in combination, did not influence the growth and differentiation of HL-60. Therefore IL-6 in conjunction with IL-1 can induce differentiation in several human and mouse myeloid leukemic cell lines, although this effect varies with cell type. IL-6 did not stimulate the expression of IL-1 mRNA or IL-1 activity in U937 cells. IL-1 also failed to stimulate IL-6 production. Furthermore, the differentiation of U937 cells induced by IL-6 was not neutralized by antibody against either IL-1 alpha or IL-1 beta. The minimal differentiative effect of IL-1 was not affected by anti-IL-6 antibody. Therefore IL-6 and IL-1 appear to provide distinct signals for differentiation.