Homologous human macrophage hybridomas that produce a novel cytotoxic factor in their culture supernatants

Homologous human macrophage hybridoma cell lines were obtained by somatic cell fusion between peripheral blood monocyte-derived macrophages and a subclone of the myelomonocytic cell line, U937-F9. The hybridoma cell lines grown in vitro for more than a year were confirmed by manifestations of phagocytosis, adherence, nonspecific esterase, acid phosphatase, chromosome numbers and other cell surface antigens. Cell surface antigens on hybridomas were detected by flow cytometry analysis with monoclonal antibodies. With interclonal differences, a typical phenotype of hybridoma cells was CDw14+, OKM5+, Mac-1+ (equivalent to OKM1 and Mol), OKT9+, HLA-DR- and CD20+. After stimulation with lipopolysaccharide and calcium ionophore A23187, culture supernatants of clones c18A and c29A showed cytotoxic activity against human melanoma A375 Met-Mix and other cell lines which were resistant to the tumor necrosis factor, lymphotoxin and interleukin 1. This cytotoxic factor was found to be distinct from the tumor necrosis factor, lymphotoxin and interleukin 1 using the anti-tumor necrosis factor, anti-lymphotoxin and anti-interleukin 1 antisera.     

Effects of recombinant human colony stimulating factors (CSF) (granulocyte-macrophage CSF, granulocyte CSF, and CSF-1) on human monocyte/macrophage differentiation

Purified recombinant human granulocyte-macrophage (rhuGM)-CSF, rhuG-CSF, and rhuCSF-1 were evaluated for their capacity to influence the differentiation of U-937 cells and normal human monocytes. The human U-937 cell line represents an early stage of monocytic differentiation. It was found that rhuGM-CSF and rhuG-CSF, but not rhuCSF-1, induced phenotypic changes consistent with monocyte/macrophage differentiation in U-937 cells. After 3 days of culture in the presence of either rhuGM-CSF or rhuG-CSF, a small but significant proportion of U-937 cells were able to reduce nitroblue tetrazolium. Nitroblue tetrazolium reduction, however, was maximally induced when rhuGM-CSF and rhuG-CSF were added in combination. These changes were accompanied by increased alpha-naphthyl acetate esterase activity, acquisition of macrophage morphology, Mo-1 Ag expression, and decreased cell proliferation. rhuGM-CSF alone also induced expression of the c-fms proto-oncogene (CSF-1 receptor) in U-937 cells and this expression was enhanced by the combination of rhuGM-CSF and rhuG-CSF. In cultured normal human peripheral blood monocytes, representing a late stage of maturation, rhuGM-CSF and rhuCSF-1 differentially increased Mo-1 and My-4 Ag expression, respectively, whereas rhuG-CSF was without effect. Our results suggest that the interaction of GM-CSF, G-CSF, and CSF-1 may play a fundamental role in the early and late stages of the human monocyte/macrophage differentiation process.     

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.     

Regulation of complement factor H synthesis in U-937 cells by phorbol myristate acetate, lipopolysaccharide, and IL-1

The capacity of the human monocyte cell line U-937 to synthesize complement factor H was examined. The kinetics of secretion of factor H into cell culture supernatant were followed by a sensitive solid phase RIA capable of measuring 0.1 ng of protein. Daily secretion of factor H was low and almost linear and was approximately 3 ng of factor H per 10(6) cells. Factor H synthesis was inhibited by cycloheximide but returned to the levels seen in untreated cultures after removal of the inhibitor. LPS and IL-1 both effected a time- and dose-dependent enhancement of factor H synthesis. Induction of U-937 cells with PMA to differentiate into macrophage-like cells also resulted in increased factor H synthesis. RIA of cell lysates, immunofluorescence microscopy, as well as FACS analysis all revealed that factor H Ag was also associated with the U-937 cell membrane. The population of U-937 cells bearing membrane-associated factor H was decreased from 77 to 43% after stimulation for 48 h with LPS (1 microgram/ml). [35S]Methionine metabolic labeling and SDS-PAGE analysis of factor H immunoprecipitates from unstimulated and stimulated culture supernatants and cell lysates demonstrated a major polypeptide, m.w. 150,000, and a minor component, m.w. 42,000. Western blot analysis of factor H in fresh normal plasma also detected both 150,000 and 42,000 m.w. factor H proteins. This is in agreement with the recent demonstration of a 4.4- and 1.8-kb mRNA for factor H in human liver. These data demonstrate that U-937 cells synthesize factor H that is structurally and antigenically similar to factor H in normal plasma. The exact nature of the membrane-bound factor H and its functions and mechanism of attachment to the cell membrane remain to be elucidated.     

Lymphokine production by human T cell hybridomas

Human T cell hybridomas were generated by hybridization of SH9 cells, the 6-thioguanine-resistant variant of human T lymphoma Hut102-B2, with concanavalin A-stimulated human peripheral blood lymphocytes. The hybrid nature of the established cell lines was documented by the difference in D14S1 restriction fragment length polymorphism between SH9 and the hybridoma cell DNA, and by the expression of OKT11 antigen on hybrid cells. T cell growth factor, macrophage growth factor (MGF), and interferon (IFN) activities have been demonstrated in the supernatants of different hybrid cultures, but not in SH9 cell cultures. Substantial quantities of MGF were secreted by several hybrids including the L23 line. MGF activity was dose-dependent, heat-labile, and synergistic with indomethacin. High titers of IFN activity were found in the cultures of hybridoma L415 and its subclones. Neutralization with specific antisera showed the IFN synthesized by L415 clones was immune interferon (IFN-gamma). Like the parental SH9 line, all of the hybridomas producing these lymphokines exhibited a cell surface phenotype typical for helper T cells. The hybridoma system therefore shows potential for the study of various lymphokines produced by human helper T lymphocytes.     

Effects of combinations of transforming growth factor-beta 1 and tumor necrosis factor on induction of differentiation of human myelogenous leukemic cell lines

Effects of transforming growth factor-beta 1 (TGF-beta 1), either alone or in combination with TNF, on the induction of differentiation of human myelogenous leukemic cell lines were examined. TGF-beta 1 alone induced differentiation of a human monocytic leukemia U-937 line into the cells with macrophage characteristics. When combined with TNF, TGF-beta 1 synergistically or additively induced differentiation associated properties. A human myeloblastic leukemia cell line, ML-1, differently responded to TGF-beta 1 in induction of differentiation. FcR activity and phagocytic activity induced by TNF were suppressed by TGF-beta 1. However, nitroblue tetrazolium reducing activity was synergistically induced by combinations of TGF-beta 1 and TNF. Scatchard analysis of TNF receptors indicated that the number of binding sites and dissociation constant of TNF for its receptors on U-937 or ML-1 cells were not changed by treatment with TGF-beta 1. Although IFN-gamma, IL-6, granulocyte CSF, and granulocyte-macrophage CSF-induced nitroblue tetrazolium reducing activity of U-937 cells, only IFN-gamma, and TNF induced it synergistically in combination with TGF-beta 1. Synergism between TGF-beta 1 and TNF was also observed in inhibition of growth of U-937 and ML-1 cells. Although TGF-beta 1 induction of differentiation of other monocytoid leukemic THP-1 cells was similar to that of U-937 cells, TGF-beta 1 only slightly induced differentiation of promyelocytic leukemic HL-60 cells, either alone or in combination with TNF. Our observations indicate that TGF-beta 1 strongly modulates differentiation and proliferation of human myelogenous leukemia cells, macrophage precursors.     

Functional analysis of cloned macrophage hybridomas. VII. Modulation of suppressor T cell-inducing activity

We have previously characterized a macrophage hybridoma clone, termed clone 59, which induced immunity but consistently failed to induce Ts responses. Macrophage 59 cells were cultured with supernatants from several activated T cell clones to determine if lymphokines could modulate the activity of this macrophage hybridoma to generate effector Ts. Culture supernatants from Th1 clones and from one atypical IL-4 and IFN-gamma-producing T cell clone successfully modulated clone 59 cells to induce effector Ts cells. In contrast, supernatants from activated Th2 cells failed to generate Ts-inducing activity in macrophage 59 cells. Culture with recombinant derived IFN-gamma was sufficient to cause modulation of Ts-inducing activity in macrophage 59 cells. The data imply that the differential functional activities ascribed to various macrophage hybridoma clones reflect macrophage heterogeneity instead of independent macrophage lineages. The suppression induced by clone 59 macrophages was genetically restricted to the putative I-J region. The ability of IFN-gamma containing supernatants to endow macrophage 59 with the capacity to induce effector suppressor cells was specifically abrogated by addition of an anti-IJk-idiotype antibody, which also reacts with IJ-interaction molecules, indicating that the mechanism of modulation most likely involves expression of IJ-interaction molecule determinants on antigen presenting cells.     

Succinoyl trehalose lipid induced differentiation of human monocytoid leukemic cell line U937 into monocyte-macrophages

A novel type of succinoyl trehalose lipid (STL-1) prepared from n-hexadecane-culture ofRhodococcus erythropolis SD-74 markedly inhibited the growth of a human monocytoid leukemic cell line, U937, and induced its morphological alteration along a monocyte-macrophage lineage. STL-1 markedly increased differentiation-associated characteristics in macrophage, such as nitroblue tetrazolium reducing ability, appearance of Fc receptor, phagocytic activities in U937. Furthermore, U937 cells, which were activated with STL-1 exhibited cytotoxic activity against human lung carcinoma cell line A549. However, STL-1 did not affect growth of a normal human fetal lung cell line TIG-1. The individual components of STL-1, neither sugar moiety nor fatty acids in the free form, were effective at inducing the differentiation of U937 cell. From these results, we concluded that STL-1 has low cytotoxicity against normal human cells and the ester molecule itself is responsible for the activity of inducing differentiation of human monocytoid leukemic cell line U937 into monocyte-macrophage which results in the stimulation of the production of some cytotoxic substances.     

Phenytoin induces interleukin-1 production in vitro

Human adherent mononuclear cells and subcloned cell lines established from the human histiocytic cell line U-937 were cultured with phenytoin (PHT) and/or lipopolysaccharide (LPS) purified from Bacteroides fragilis. After the cultivation period, the cell-free supernatants were tested for interleukin-1 (Il-1) activity. The results showed that PHT induces Il-1 activity and potentiates LPS-induced Il-1 production. In the monocytic cell line U-937, the induced Il-1 production was found to be clonally distributed indicating that the response to PHT may be exerted by a subpopulation of monocytes. The PHT-induced Il-1 activity may be of importance in the development of gingival overgrowth. The induced Il-1 could also contribute to other known side effects such as dermatologic reactions accompanied by transient fever seen in patients medicating the drug.     

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     

Effect of fibroblast-derived differentiation inducing factor on the differentiation of human monocytoid and myeloid leukemia cell lines

A fibroblast-derived differentiation inducing factor (F-DIF) purified from medium conditioned by a human fibroblast cell line (WI-26VA4) induced differentiation of human monocytic leukemia cell lines (U-937, THP-1) into cells with macrophage characteristics. F-DIF alone induced the differentiation of ML-1 cells only marginally, but it synergistically increased the differentiation when combined with TNF. Interferon-gamma, tumor necrosis factor, GM-CSF, interleukin-1 and interlukin-4 synergistically enhanced the differentiation of U-937 cells when combined with F-DIF.     

Detection of viral surface antigens on HIV-2ben infected human tumor cell lines by flow cytometry.

The human monocytic cell line U-937 clone 2 and two T-cell lines CEM and MOLT-4 clone 8 were infected with HIV-2ben, a recent isolate of HIV-2. Infection and subsequent antigen expression on the cell surface was monitored by flow cytometry using a rabbit-anti-serum against tween-ether-treated HIV-2ben and a fluorescein-isothiocyanate-conjugated IgG against rabbit-IgG. The sensitivity of the three cell lines to infection with HIV-2ben correlated with the percentages of CD4-expressing cells but not with the levels of CD4-expression on the cell. The appearance of viral surface antigens preceded the formation of syncytia and correlated closely with the infecting virus dose. After 1-2 weeks in culture, 20-85% of the cells of each line expressed viral surface antigens. The variation depended on the cell type and cell culture conditions. The MOLT-4 clone 8 and the U-937 clone 2 cells died around 10 or 20 days, respectively, after HIV-2ben infection. Only HIV-2ben infected CEM cells grew permanently. Flow cytometry was an appropriate method to monitor the expression of viral proteins on the cell surface of HIV-infected cell lines. Flow cytometry proved to be more sensitive than determination of RT activity in supernatants of HIV-infected cells and more precise than light microscopy examinations.     

Human macrophage hybrid forming spontaneous giant cells

Thymidine kinase-deficient clones of the human monocyte/macrophage cell line U-937 were established and used for fusion experiments with separated normal human blood monocytes. A hybrid (H 29) was generated during HAT-selection procedure, about 50% of which formed spontaneous giant cells, as shown by morphological, immunocytochemical, and chromosomal analyses. It is concluded that giant cells originate from monocytes and display mitotic activity. Macrophage hybrids are the basic requirement for the elucidation of monocyte/macrophage heterogeneity and immortalization of their functional properties.     

Granulocyte-macrophage colony stimulating factor modulates the production of TNF alpha by differentiated U937 cells infected with Leishmania major

In this work, the production of tumor necrosis factor alpha (TNF alpha) during interaction of human phagocytes with the intracellular parasite Leishmania major was further investigated. The human monocytic cell line U937, differentiated with a combination of 1 alpha, 25 dihydroxyvitamin D3 (VD) and retinoic acid (RA), or with granulocyte macrophage colony stimulating factor (GM-CSF) was used. Differentiated U937 cells were infected with Leishmania major promastigotes, and TNF alpha was assayed in cell culture supernatants. It was found that the cytokine was produced only by U937 cells differentiated with VD/RA and further incubated with GM-CSF and LPS or interferon gamma (IFN gamma). L. major induced TNF alpha production only in the presence of GM-CSF. No direct relationship was found, however, between production of TNF alpha and resistance of differentiated U937 cells to infection with L. major.     

Isolation and characterization of monoclonal antibodies reactive with endothelial cells

Monoclonal antibodies were generated to antigens on cultured human umbilical vein endothelial cells. Spleen cells from BALB/c mice, immunized with low passage cultures of human umbilical vein endothelial cells, were fused with the non-secretory myeloma line, P3 x 63Ag 8.653. Hybridoma supernatants were screened for the desired immunological reactivity using ELISA binding assays. Hybridomas secreting antibodies reacting with the immunizing endothelial cells, but not with peripheral blood mononuclear cells, were cloned by limiting dilution and three stable clones were chosen for study. Further testing by ELISA revealed that each antibody displayed a unique pattern of reactivity. One antibody, 14E5, reacted with the macrophage-like cell line DHL-2, cultured macrophages derived from peripheral blood monocytes, and macrophages derived from malignant effusions. The antibody failed to react with fibroblasts or bovine endothelial cells. The second antibody, 12C6, reacted with human and primate fibroblasts and endothelial cells derived from bovine arteries, but not with mature macrophages. The third clone, 10B9, reacted only with the immunizing endothelial cells and the immature-macrophage line U-937. All three antibodies failed to react with long-term human B or T lymphoblastoid cell lines, leukemic cell lines, or murine macrophage lines. None of the antibodies reacted with a battery of human epithelial derived cell lines or primary cultures of human epithelial cells. Indirect immunofluorescence assays revealed that the antigens were expressed on the cell surface. These antibodies should prove useful as differentiation markers of human endothelial cells and in studies of endothelial cell function.     

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.     

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.     

Mouse macrophage hybridomas secreting a cytotoxic factor and interleukin 1

Stable mouse macrophage hybridomas were produced by somatic cell fusion between proteose peptone-elicited peritoneal macrophages and NS-1 myeloma cells. Three cloned hybrid cell lines, designated as N/P-5-3, -6-2, and -7-1, exhibited typical macrophage-like morphology. Moreover, their macrophage characteristics were confirmed by the manifestation of intracellular nonspecific esterase, the detection of Mac-1 antigens and Fc-receptors on the cell surface, and the demonstration of phagocytic and antigen-presenting activities. Furthermore, these cell lines, stimulated with LPS, secreted considerable amounts of a cytotoxic factor and interleukin 1. Cultured cells of various tumor cell lines were sensitive to the cytotoxic factor, but normal thymocytes, spleen cells, and liver cells were not killed by the factor.     

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.