Synthesis of complement factor B by the human monocyte U-937 cell line. Augmentation by immunostimulatory agents

The human pre-monocytic cell line U-937 was shown to synthesize and to secrete increasing amounts of factor B in short term cultures in serum-free medium containing BSA. The kinetics of factor B production were higher on day 2 than on days 1 and 3. The production of factor B was reversibly inhibited by cycloheximide, indicating de novo synthesis. Metabolic labeling with [35S]-methionine and SDS-PAGE analysis revealed that both intracellular and secreted factor B were single-chain proteins with similar m.w. (90,000), which co-migrated with purified plasma factor B. Incubation of U-937 cells with the immunostimulants PMA, LPS, IFN-gamma, and IL-1 resulted in a dose-dependent augmentation of factor B production. A 24-h exposure to IL-1 was shown to be required for maximal stimulation. A combination of suboptimal doses of LPS and IFN-gamma was shown to exert a synergistic effect on factor B production. The U-937 cell line is thus a valuable model for the study of the regulation of the factor B gene expression.     

Interleukin-1 and tumour necrosis factor production by human monocytoid cells: study on a single cell level.

Human IL-1 beta and TNF alpha production by normal and transformed monocytoid cells was studied using biological assays, cytokine specific ELISA and by immunocytochemical methods on a single cell level. Quiescent human blood monocytes and cultured in vitro transformed human monocytoid cell lines U-937, THP-1 and HL-60 did not contain IL-1 beta and TNF alpha in their cytoplasm. IL-1 beta synthesis and secretion was induced by LPS stimulation in nearly 90% monocytes, 15-20% U-937, 3-5% THP-1 and in no HL-60 cells. Normal human blood monocytes had a more rapid kinetics of IL-1 beta synthesis. IL-1 beta positive cells stained with antibodies to human IL-1 beta appeared at 1-2 hours after LPS application, while in monocytic cell lines only after 4-6 hours. Using immunoperoxidase staining of U-937 cells pulse labelled with 3H-thymidine, it was shown that proliferating cells did not synthetize IL-1 beta. Instead of IL-1 beta, TNF alpha could be induced by LPS in U-937 cells only after preliminary differentiation with PMA. Recombinant IL-1 beta induced a very low level of TNF alpha production in PMA-treated cells. Similarly recombinant TNF alpha alone induced IL-1 beta synthesis only in a few U-937 cells.     

A structural characterization of the Mo3 activation antigen expressed on the plasma membrane of human mononuclear phagocytes

Mo3 is an activation Ag expressed by human monocytic cells after stimulation in vitro by PMA, LPS, certain cytokines, and muramyl dipeptide. The structural characterization of Mo3 has been made possible by the development of a mAb (anti-Mo3f) that immunoprecipitates Mo3 from Nonidet P-40 lysates of radiolabeled PMA-stimulated U-937 cells and LPS-activated monocytes. On SDS-PAGE (nonreducing conditions) of anti-Mo3f immunoprecipitates, U-937 Mo3 is a single broad band of 39 to 66 kDa, whereas monocyte Mo3 is smaller with an apparent molecular mass of 32 to 56 kDa. Under reducing conditions, there is an increase in the m.w. of both species of Mo3 suggesting the existence of internal disulfide bonds. Mo3 is a glycoprotein with carbohydrate of the N-linked complex type as evidence by a reduction in m.w. by 40 to 50% after treatment with endoglycosidase F or N-glycanase; neuraminidase treatment produces a 3-kDa reduction in m.w. Deglycosylated Mo3 isolated from U-937 and monocytes have similar m.w. suggesting that the molecular heterogeneity of the native Mo3 may be due to differences in glycosylation. Mo3 is sensitive to phosphatidylinositol-specific phospholipase C with the release of native Mo3 from the surface of PMA-stimulated U-937 cells. These results indicate that Mo3 is a member of the glycosylphosphatidylinositol-linked family of surface glycoproteins.     

In vitro biosynthesis of complement protein D by U937 cells

Preliminary studies demonstrating the secretion of antigenic D by blood monocytes/macrophages led us to study the biosynthesis of D by U937 cells, a human monocyte cell line. The kinetics of secretion of D into cell culture supernatants were followed by a solid-phase radioimmunoassay and by hemolytic assay. Daily synthesis of antigenic D was nearly linear (mean +/- 1 SD = 5.3 +/- 2.2 ng D/10(6) cells) over a 6-day period. The D produced after day 2 was hemolytically active, with a specific hemolytic activity greater than (although in the same range as) D in normal serum. Cycloheximide (10(-7) M) inhibited D synthesis, which returned to the levels found in untreated cells after removal of the inhibitor. Supernatants and lysates of cells grown in the presence of [35S]methionine were incubated with rabbit anti-D serum or FD10-1, a monoclonal anti-D antibody, bound to protein A-agarose. Autoradiograms of SDS-PAGE analysis of the precipitates demonstrated a main band of an approximate m.w. of 24,000, co-migrating with purified 125I-D. Identity of this band with D was established by blocking with excess purified D. Pulse-chase studies with the use of [35S]cysteine demonstrated a single D band both intra and extracellularly. Both forms of D had the same apparent m.w. which was approximately 3000 heavier than control 125I-D. These data demonstrate that U937 cells synthesize functionally active D, which appears to be structurally and antigenically similar to D in serum.     

Biosynthesis of the third component of complement (C3) by the human monocytic-cell line U-937. Induction by phorbol myristate acetate.

Phorbol myristate acetate (PMA)-stimulated human monocyte-like cells (U-937) were found to synthesize the third component of complement (C3), as shown by enzyme-linked immunosorbent assay and immunoprecipitation from [35S]methionine-labelled culture supernatants. C3 synthesis occurred at a rate of about 160 ng of C3/24 h per 10(6) cells on day 7 after addition of PMA; it was blocked by cycloheximide treatment and was restored after removal of the inhibitor. SDS/polyacrylamide-gel-electrophoretic analysis of the immunoprecipitated protein showed that the size and subunit structure of the newly synthesized C3 were identical with those of plasma C3, and that a single-chain intracellular precursor was present in the cell lysates. Haemolytic assays showed that the synthesized C3 fully expressed functional activity in early culture within 4 h. After longer culture, a loss of haemolytic activity was observed. The possibility that newly secreted C3 is cleaved by U-937 cells themselves was suggested.     

A human macrophage hybridoma producing a cytotoxic factor distinct from TNF,LT, and IL-1

Summary A stable human macrophage hybridoma was established by somatic cell fusion between human peripheral blood monocyte-derived macrophages and an 8-azaguanine resistant clone of a human histiocytic lymphoma cell line U-937 (clone U-937-F9). The hybrid cell line (F9P) exhibited typical macrophage-like morphology and had 30 more chromosomes than U-937-F9 cells. Its macrophage characteristics were confirmed by the manifestation of intracellular nonspecific esterase, the detection of Mo-2 and LEU-M3 antigens on the cell surface, and the demonstration of phagocytic activity. Furthermore, when stimulated with lipopolysaccharide (LPS), this cell line could secrete a considerable amount of a cytotoxic factor (CTF). Distinct from the hybrid cell line, the parental U-937-F9 cells expressed neither Mo-2 nor LEU-M3 antigens on the cell surface, did not show phagocytic activity, and their culture supernatants did not show cytotoxic activity even after LPS stimulation. The activity of CTF in the culture supernatant of the LPS-stimulated hybrid cells could not be neutralized with anti-tumor necrosis factor, anti-interleukin-1, or anti-lymphotoxin antibodies. The CTF had a relative molecular mass of 45–60×10³ daltons as determined by gel filtration on a column of Superose 12, and an isoelectric point of 5.1. The cytotoxic activity was also induced when the hybrid cells were stimulated with the concentrated supernatants of a human T-cell hybridoma containing macrophage activating factor for cytotoxicity or with LP3 tumor cells which were used as target cells.     

C-reactive protein receptors on the human monocytic cell line U-937. Evidence for additional binding to Fc gamma RI.

C-reactive protein (CRP) is an acute-phase protein that binds to components of damage tissue, activates C, and stimulates phagocytic cells. CRP binding to receptors on monocytic and polymorphonuclear phagocytes has been shown. Recently, CRP-binding proteins of 38 to 40 kDa and 57 to 60 kDa have been identified on the human promonocyte cell line U-937 and the mouse macrophage cell line PU5 1.8, respectively. However, analysis of CRP binding to these cells and to peripheral blood leukocytes suggests that additional CRP receptor sites may be present. Because many studies have shown interactions between CRP binding and IgG binding to leukocytes, we have examined further the CRP binding sites on U-937 cells and determined their relationship to the FcR for IgG (Fc gamma R) expressed on these cells. Our results demonstrate specific saturable binding of CRP to peripheral blood monocytes and U-937 cells, which is readily inhibited by aggregated IgG. Monomeric IgG, which binds specifically to Fc gamma RI, inhibited a maximum of 20% of CRP binding to these cells. mAb 197 and mAb IV.3, which block IgG binding to Fc gamma RI and Fc gamma RII, respectively, failed to inhibit CRP binding to U-937 cells. Two CRP-binding molecules were identified by precipitation of lysates from surface-labeled U-937 cells and cross-linking experiments. One of these had a molecular mass of 43 to 45 kDa, similar to the molecule previously described as the CRPR on U-937 cells. The other had the same mobility by SDS-PAGE as Fc gamma RI. The identity of this protein with Fc gamma RI was confirmed by the ability of both IgG-Sepharose and CRP-Sepharose to preclear the protein from cell lysates and by inhibition of binding to both IgG-Sepharose and CRP-Sepharose by anti-Fc gamma RI mAb 197.     

Dexamethasone induces the expression of the mRNA of lipocortin 1 and 2 and the release of lipocortin 1 and 5 in differentiated, but not undifferentiated U-937 cells.

The effect of dexamethasone on mRNA and protein synthesis of lipocortins (LCT) 1, 2 and 5 has been investigated in U-937 cells. A constitutive expression of both mRNAs and proteins was detected in undifferentiated U-937 cells. This constitutive level was increased time- and dose-dependently by incubation with phorbol myristate acetate (PMA). In U-937 cells differentiated by 24 h incubation with 6 ng/ml PMA, dexamethasone (DEX) (1 microM for 16 h) caused an increased synthesis of the mRNA level of LCT-1 and 2, but not of LCT-5, over the level induced by PMA. DEX had no effect in undifferentiated cells. Moreover, DEX stimulated the extracellular release of LCT-1 and 5, but not of LCT-2, and inhibited the release of PGE2 and TXB2 only in the differentiated U-937 cells. These results suggest that the responsiveness of these cells to glucocorticoids is dependent on the phase of cell differentiation. The selective release of lipocortins by differentiated U-937 cells may explain, at least in part, the inhibition by DEX of the prostanoid release.     

The human myelomonocytic cell line U-937 as a model for studying alterations in steroid-induced monokine gene expression: marked enhancement of lipopolysaccharide-stimulated interleukin-1 beta messenger RNA levels by 1,25-dihydroxyvitamin D3.

The active metabolite of vitamin D, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], is a potent regulator of human monocyte/macrophage function in vitro. To establish a model for 1,25-(OH)2D3 regulation of human monocyte monokine synthesis, three human cell lines (U-937, THP-1, and HL-60) were examined for: 1) the presence of functional 1,25-(OH)2D3 receptors; 2) the accumulation of interleukin-1 beta (IL-1 beta) mRNA and IL-1 beta protein in response to lipopolysaccharide (LPS); and 3) the regulation of this response by 1,25-(OH)2D3. All three cell lines expressed vitamin D receptor and had increased levels of IL-1 beta mRNA in response to LPS. Preincubation of cells with 1,25-(OH)2D3 augmented IL-1 beta mRNA levels only in U-937 and HL-60 cells. From these data, and taking into consideration their state of differentiation and relative ease of culture, U-937 was chosen over HL-60 and THP-1 as the cell line we further characterized. In U-937 cells, optimum time and dose of pretreatment with 1,25-(OH)2D3 were determined to be 12-24 h at a receptor saturating concentration of 1,25-(OH)2D3 (10 nM). Preincubation of cells with 1,25-(OH)2D3 had no effect on the time course of IL-1 beta mRNA appearance in response to LPS. However, exposure of U-937 cells to 1,25-(OH)2D3 increased by 200% the level of IL-1 beta mRNA detected and decreased by three orders of magnitude the concentration of LPS required to achieve steady state mRNA levels equivalent to those observed in U-937 cells not preincubated with the hormone.2+o     

Evidence for an intracellular pool of a migration inhibitory factor-associated, activation antigen of human mononuclear phagocytes, Mo3e

Mo3e is a protease-sensitive Ag (p75,50) selectively expressed by human monocytic cells stimulated in vitro by exposure to various activating factors including PMA. Here, we report the existence of a large intracellular pool of Mo3e Ag in addition to that expressed on the surface of activated U-937 cells. As detected by quantitative immunofluorescence analysis, permeabilization of unstimulated and PMA-stimulated U-937 cells revealed a latent pool of Mo3e Ag that was 75-fold and 9-fold greater, respectively, than the magnitude of Mo3e Ag expressed on the surface. PMA stimulation not only induced an increase in the relative proportion of Mo3e antigen expressed on the surface membrane, but also stimulated a 1.8-fold increase in "total" Mo3e detectable in permeabilized cells. Trypsin treatment of intact PMA-stimulated U-937 cells eliminated surface Mo3e expression but had little measureable effect on the total Mo3e pool. Permeabilization also uncovered a sequestered compartment of Mo3e Ag in I-937 cells, a variant of U-937 that is surface Mo3e negative. Although the PMA-induced surface Mo3e expression of U-937 was abrogated by cycloheximide, the total pool of Mo3e detectable in permeabilized PMA-stimulated cells was only partially reduced; cycloheximide treatment caused no reduction in the intracellular Mo3e compartment of unstimulated U-937 cells. Detergent lysates of PMA-stimulated U-937 cells exhibited undiminished quantities (relative to untrypsinized cells) of p75 and p50 proteins immunoreactive with anti-Mo3e mAb as detected by Western blotting. This trypsin-sequestered intracellular Mo3e Ag may serve as a reservoir for the up-regulated surface expression of Mo3e that occurs as a result of mononuclear phagocyte activation.     

Internalization, lysosomal degradation and new synthesis of surface membrane CD4 in phorbol ester-activated T-lymphocytes and U-937 cells.

Protein kinase C activating phorbol esters downregulated membrane CD4 by endocytosis in U-937 and human T-cells. Half-time for internalization (approximately 15 min at 50 ng/ml PMA) was determined by FACS. CD4-bound 125I-labeled anti-CD4 mAb was rapidly degraded in PMA-activated cells, whereas degradation was low in resting cells. Endocytosis and/or degradation of anti-CD4 mAb was suppressed by H7, and by inhibitors of membrane traffic (Monensin) and lysosome function (methylamine, chloroquine). Immunocytochemistry localized CD4 to the surface of unstimulated T-cells. Upon PMA stimulation occasional labeling was seen in endosomes but whole cell CD4 decreased dramatically. However, methylamine-treated PMA blasts showed accumulation of CD4 in lysosomes and accordingly, pulse-chase experiments in biolabeled cell cultures suggested a manifest reduction of CD4 half-life in response to PMA. Despite their low surface CD4 density, PMA blasts exhibited uptake and accelerated degradation of anti-CD4 mAb. Also, inhibitors of protein synthesis enhanced the PMA-induced downregulation, and membrane CD4 reappeared on fully activated as well as unstimulated cells treated with trypsin. Ongoing CD4 synthesis in activated cells was further evidenced by metabolic labeling and Northern blot analysis demonstrating unaltered or slightly increased CD4 protein and mRNA levels resulting from PMA. Our findings demonstrate that phorbol esters downregulate the cellular CD4 pool by endocytosis and subsequent lysosomal degradation of membrane CD4. Transport of CD4 to the cell surface and CD4 synthesis is unaffected by activation.     

Radioimmunossay for detection of antigen and antibodies to Newcastle disease virus.

When Newcastle disease virus (NDV) is treated with NP-40 and ether a membrane fraction of 150,000 m.w. is obtained. This fraction which is composed of two polypeptides with m.w. of 56,000 and 76,000 was used in a radioimmunoassay (RIA). The assay was developed for both antigen and antibody and was found to be reproducible, specific, and highly sensitive. Titers of 1:51,200 were determined by RIA as compared to 1:4 by agar gel diffusion and 1:200 by hemaglutination inhibition (HI). As little as 5 ng of viral protein were detected by RIA inhibition technique. Labeled antigen could be stored in the presence of serum, KCI and Triton X-100 at -20 degrees C for as long as 6 weeks and retained similar reactivity as fresh reagent.     

Switch of histamine receptor expression from H2 to H1 during differentiation of monocytes into macrophages

It is known that histamine suppresses gene expression and synthesis of tumor necrosis factor alpha (TNF-alpha) induced by lipopolysaccharide (LPS) in human peripheral blood mononuclear monocytes (HPM) or alveolar macrophages via histamine H2 receptors. We investigated the effect of histamine and differentiation in macrophages on the expression and secretion of TNF-alpha, TNF-alpha-converting enzyme (TACE), and histamine H1 and H2 receptors by use of a leukemia cell line, U937, and HPM. Differentiation of U937 and HPM cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) enhanced the H1 receptor expression and rather suppressed the H2 receptor, resulting in up-regulation of the histamine-induced expression and secretion of TNF-alpha, modulated via TACE. Therefore, histamine failed to inhibit up-regulated expression of TNF-alpha induced by LPS in macrophages. The switch from H2 to H1 receptors during differentiation in the monocyte/macrophage lineage could participate in the pathogenic processes of atherosclerosis and inflammatory reactions in the arterial wall.     

New aspects in the synthesis and secretion of lysozyme by cultured human monocyte cell lines

The main purpose of this study was to investigate lysozyme synthesis and secretion in three human monocyte cell lines: U-937, HL-60, and THP-1, using sensitive fluorescence-based assay of lysozyme activity. PMA and hIFN-γ were evaluated for inducing lysozyme activity. Using well-defined cell lines from the cell culture collection, no lysozyme activity could be detected in the cultured U-937 cells either with or without addition of the inducing factors. These data suggested, contrary to previous reports, that U-937 cell line cannot synthesize or secrete active lysozyme. THP-1 and HL-60 cells were proved to produce enzymatically active lysozyme in increasing amounts with the time course. PMA and hIFN-γ had no significant inducing effect on the production or the release of active lysozyme in THP-1 and HL-60 cells. We showed inhibiting effect of PMA and hIFN-γ on the lysozyme activity, particularly in HL-60 cell line.     

Bacterial lipopolysaccharide, phorbol myristate acetate, and muramyl dipeptide stimulate the expression of a human monocyte surface antigen, Mo3e

Exposure of mononuclear phagocytes to bacterial lipopolysaccharide (LPS), phorbol myristate acetate (PMA), or muramyl dipeptide (MDP) is known to stimulate a variety of cellular activities that include increases in phagocytosis, oxidative metabolism, synthesis and secretion of monokines, and cytotoxicity of microbes and tumor cells. We now report that culture of human peripheral blood monocytes in medium containing LPS, phorbol compounds, or MDP also results in the acquired expression of a plasma membrane antigen. Mo3e, as identified by a murine monoclonal antibody. Mo3e is barely detectable (by immunofluorescence flow cytometry) on freshly isolated monocytes, but is expressed in high antigen density after exposure of cells to E. coli, Salmonella minnesota, or Serratia marcescens LPS (at concentrations exceeding 0.1 ng/ml), PMA (and other biologically active phorbol compounds) (0.5 to 1 X 10(-8) M), or MDP (0.01 to 1 X 10(-6) M). Mo3e expression stimulated by LPS is prevented by pretreatment of LPS with polymyxin B, suggesting that the lipid A portion of LPS is responsible for Mo3e induction (polymyxin B has no effect on Mo3e expression stimulated by PMA or MDP). Culture of monocytes in medium containing protein synthesis inhibitors (or at 4 degrees C) blocks the acquisition of Mo3e. Recombinant IFN-gamma, which is also known to "activate" mononuclear phagocytes, does not stimulate Mo3e expression, although both LPS and IFN induce enhanced expression of monocyte Ia antigen. Analogous to their stimulatory effect on monocytes, LPS and PMA induce Mo3e expression by the human monocytic cell line, U-937. On the basis of these observations, Mo3e may represent an immunologic marker for monocyte activation stimulated in vitro by LPS, PMA (and related compounds), and MDP.     

Serglycin and secretion in human monocytes

The human monocytic cell line U-937 has been widely used as a model system for human monocytes. The subclone U-937-B has been adapted to serum-free conditions. This particular U-937 clone and its parent clone U-937-1 were used to investigate the role of the proteoglycan serglycin in human monocytes. For this purpose cells were treated with hexyl-β-D-thioxyloside to abrogate proteoglycan expression. U-937-B cells expressed and secreted exclusively chondroitin sulphate proteoglycans, and after treatment with this xyloside they only expressed and released free chondroitin sulphate chains. Western blotting showed that serglycin core protein was present in conditioned medium of control cells, but absent in medium from xyloside-treated cells. Also, serglycin core protein could be detected in the cell fractions of control cells, but not in the cell fractions from xyloside-treated cells. Furthermore, less proteoglycan-associated proteins could be detected in medium from cells incubated with xyloside, suggesting that the absence of secreted sergycin affects the secretion of such proteins. Cells incubated in the presence of xyloside were analyzed by transmission electron microscopy and shown to contain numerous large empty vesicles. The lack of serglycin, the dominant proteoglycan in U-937 monocyte-like cells, consequently, leads to effects on vesicle formation and secretion of some low molecular weight proteins, suggesting that this particular proteoglycan is of importance for secretory processes in human monocytes.     

cDNA for Mo3, a monocyte activation antigen, encodes the human receptor for urokinase plasminogen activator.

We have cloned the cDNA for Mo3, an activation Ag expressed by human monocytes and myelomonocytic cell lines after stimulation by PMA, LPS, muramyl dipeptide, certain cytokines, and cAMP agonists. We have previously shown that Mo3 expression in vivo is associated predominantly with macrophages in inflammatory sites. Mo3 is a highly glycosylated protein of about 50 kDa in monocytes and U-937 cells and is anchored to the plasma membrane by glycosyl-phosphatidylinositol linkage. We purified Mo3 protein by cleavage from the U-937 cell surface with phosphatidylinositol-specific phospholipase C, followed by affinity chromatography using a mAb. An internal peptide sequence was determined and used to design oligonucleotide probes for screening an expression cDNA library. Nucleotide sequencing indicated that the complete coding sequence encodes 335 amino acids, including a predicted signal peptide of 22 residues and a hydrophobic C-terminal portion that is probably cleaved during formation of the GPI linkage. The resulting mature protein of about 290 amino acids is consistent with the 29-kDa molecular mass of deglycosylated Mo3. A Northern blot of RNA from U-937 cells revealed a 1.5-kb band that was induced by PMA treatment. Mo3 cDNA was transfected into Cos cells and surface expression of Mo3 was detected by ELISA using various anti-Mo3 mAb. We performed a computer search of the National Biomedical Research Foundation database and found that Mo3 is identical to the human receptor for the urokinase plasminogen activator (uPA-R). Purified soluble Mo3, as well as anti-Mo3 antibodies, were able to block uPA binding to its receptor on U-937 cells, indicating that Mo3 is indeed uPA-R. The use of these anti-Mo3 antibodies may be helpful in assessing the role of uPA-R in processes such as inflammation and tumor invasion.     

Synthesis of C1 inhibitor (C1-INA) by a human monocyte-like cell line, U937

Human monocytes are known to synthesize many of the components of complement, including C1-INA. In this report we demonstrate that the human monocyte-like cell line U937 is also capable of synthesizing functional C1-INA. This was shown in several ways, including 1) incorporation of tritiated amino acids into antigenic C1-INA, immunoprecipitation, and detection by fluorography; 2) a sensitive ELISA, which allowed quantitation of antigenic C1-INA in cell lysates, and 3) a C2-dependent hemolytic assay in which the functional activity of U937 C1-INA was assayed. Data from the ELISA indicate that U937 cells contain between 2.1 to 12.8 ng of C1-INA per 1 X 10(6) cells. Furthermore, fluorescence-activated cell sorter analysis revealed that approximately 16% of U937 cells carry C1-INA as a surface bound antigen. Other proteins found to be synthesized by U937 cells include C1r, C8, and possibly alpha-2-macroglobulin. These results suggest that the U937 cell line could be a convenient and valuable model for the study of monocyte C1-INA synthesis and physiology.     

Ion channels in human macrophages compared with the U-937 cell line

Summary The human cell line U-937 has been used extensively to model many macrophage functions. We have examined the cell membranes of human monocyte-derived macrophages (HMDM) and U-937 cells to compare membrane properties as expressed by single ion channel currents. The patch-clamp technique was applied to isolated, nonactivated, inside-out patches of cell membranes obtained from HMDM and from the U-937 cell line. Voltage-gated potassium channels of similar conductance but different kinetics are present in both types of cells, and a calcium-activated potassium channel is present only in the HMDM. These differences in ion channel properties suggest fundamentally different behavior between these two cell types at the level of the cell membrane.     

Establishment of a macrophagelike cell line derived from U-937, human histiocytic lymphoma,grown serum-free

Summary A human macrophagelike cell line which grows in serum-free medium was established from a histiocytic lymphoma cell line, U-937. U-937 cells failed to differentiate into macrophagelike cells in serum-free medium plus 12-O-tetradecanoyl phorbol 13-acetate (TPA). Fibronectin and albumin in serum were necessary for differentiation of U-937 ceds into macrophagelike cells in enriched RDF medium supplemented with insulin, transferrin, ethanolamine, selenite, egg yolk lipoprotein (eRDF-ITESL medium). The established cell line exhibited several characteristic properties of macrophage such as nitroblue tetrazolium reduction, phagocytic and α-naphthylbutyrate-esterase activities, and tumor necrosis factor and interleukin 1 production. At present the cells have been continuously maintained in eRDF-ITESL medium through over 150 passages.