TPA-induced differentiation and adhesion of U937 cells: changes in ultrastructure, cytoskeletal organization and expression of cell surface antigens

Incubation of the human promonocytic cell line U937 with 12-O-tetradecanoylphorbol-13-acetate (TPA) for 72 h resulted in differentiation into immature macrophage-like cells and was accompanied by marked morphological and functional changes. U937 cells which normally grow in suspension and show a smooth surface, extended pseudopodia and became adherent to each other and to the surface of the culture vessel. Concomitant with the TPA-induced adherence U937 cells ceased to proliferate. Our results show that phorbol ester-treated U937 cells exhibited markedly increased levels of fibronectin and of the cytoskeletal proteins actin, myosin and vimentin including a reorganization of actin and vimentin filaments. The induction of both cellular adherence and growth inhibition were accompanied by a significantly reduced level of cells expressing transferrin receptors and changes in cell surface antigen expression. Here, the expression of the leukocytefunction antigens (LFA-1), including CD11 and CD18 was markedly enhanced during phorbol ester-induced differentiation. TPA-treatment, however, failed to enhance the small amount of U937 cells expressing the monocyte/macrophage-specific CD14 antigen or expressing MHC class-II antigens. A detailed analysis of the CD14 cluster by 7 differential antibodies resulted in an induction of TM1, UCHM1, MEM15, My4, and 3C10, whereas the epitopes recognized by TM2 and Mo2 remained unaltered. Neither indomethacin nor interferon-gamma were capable of inducing a marked expression of these antigen epitopes in TPA-treated cells. Although these data demonstrate that during phorbol ester-induced differentiation U937 cells acquire many properties typically associated with macrophages, the failure to express marked levels of macrophage-specific cell surface antigens suggests a transition of U937 cells from a promonocytic to an immature macrophage intermediate state rather than into mature macrophage-like cells.     

Differentiation and retrodifferentiation of U937 cells: reversible induction and suppression of intermediate filament protein synthesis

Significant morphological and functional changes were observed when human monoblastoid U937 tumor cells growing in suspension were induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) for 72 h to differentiate along the monocyte/macrophage pathway. These include adherence of the cells to each other and to the substratum, alterations in cell-surface antigen expression and cessation of autonomous proliferation. In this study, we show by both, hybridization analysis of RNA and immunoblotting that an enhanced expression of the intermediate filament (IF) subunit proteins vimentin, lamin A and lamin C accompanied the TPA-induced differentiation process. After long-term culture of differentiated U937 cells in the absence of TPA (more than 28 days), however, the adherent cells retracted their pseudopodia, detached and started again to proliferate. This "retrodifferentiation" process, not previously described was paralleled by a rapid down-regulation of both, IF mRNA and protein synthesis back to the level of undifferentiated U937 control cells. These data suggest a functional relationship between the expression of vimentin and lamins A and C and the differentiation process taking place in these cells.     

Phenotypic differentiation patterns of the human monocyte/macrophage system

Summary In the present study phenotypic properties of non-stimulated and stimulated blood monocytes and of their normal macrophage derivatives were studied applying enzyme cytochemistry, isoenzyme analysis of acid esterase (EC 3.1.1.6), and immunohistochemical staining using a panel of newly established monoclonal antibodies specific for the monocyte/macrophage lineage. Certain marker profiles could be established for the various normal subpopulations within the monocyte/macrophage system, which were also observable in epithelioid cells and U-937 cell line considered as reactive and neoplastic differentiation variants of monocytes, respectively. Alveolar macrophages, in contrast to the other analysed monocyte/macrophage populations, showed a highly activated phenotype comparable to lymphokine stimulated blood monocytes and epithelioid cells. The results underline the concept that the adaptation of monocytes/macrophages to their particular microenvironment is of decisive importance for their definitive differentiation.     

The Role of the 90-kDa Heat Shock Protein in Cell Cycle Control and Differentiation of the Monoblastoid Cell Line U937

The human monoblastoid cell line, U937, has been widely used to study proliferation and differentiation in the monocyte–macrophage lineage. Recent evidence from other cell systems suggests that heat shock proteins (hsps) may participate in these processes. Therefore, we have examined expression of hsp and the effect of either increased or decreased expression of the hsp90 in U937 cells. Parental U937 cells express high levels of hsp90, hsp73, and hsp65, but little hsp72. Heat shock at 42°C for 30 min increased hsp72 levels but caused no change in hsp90. U937 cells transfected with the expression vector pBA.4 containing either an anti-sense or a sense hsp90 cDNA insert showed constitutive decrease, or increase, in expression of hsp90. Decreased hsp90 levels slowed the rate of cell division and levels of hsp90 correlated both with the responses to phorbol esters and with phenotypic changes: anti-sense-transfected cells expressed less CD50. Sense-transfected cells showed no change in cell cycle, but expressed less CD14 than controls. Thus, hsp90 plays a role in the monocyte–macrophage lineage, participating in proliferation and cell cycle control and in the acquisition of functional heterogeneity of the mature macrophage phenotype, with potential effects on the role of the macrophage in innate immunity.     

Cloning of a novel scavenger receptor cysteine-rich type I transmembrane molecule (M160) expressed by human macrophages

We report the cloning of a novel human type I cell surface Ag mainly expressed by macrophages. The primary structure was established by molecular cloning, which yielded a 4579-bp cDNA sequence encoding a polypeptide chain of 1453 amino acid residues with 16 potential N:-glycosylation sites. We designated this molecule M160. The domain organization features 12 scavenger receptor cysteine-rich domains followed by a transmembrane region and a cytoplasmic domain that occurs in two forms, a predominant form (M160-alpha) of 71 residues and an alternatively spliced form (M160-ss) of 39 residues. M160-alpha contains three possible phosphorylation sites, which are lost in the alternatively spliced form. RT-PCR analyses showed M160 to be expressed by alveolar macrophages and by the monocyte cell lines HL60, U937, and THP1, but not by Jurkat or Raji cells. Stimulation of U937 cells with phorbol ester resulted in an increased expression of M160 from day 5 onward. RT-PCR analysis of 19 different human tissues showed signals for M160-alpha of varying intensity in all tissues, whereas M160-ss was confined to the spleen. We conclude that M160 is a new member of the scavenger receptor cysteine-rich superfamily expressed by the monocyte/macrophage cell lineage.     

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.     

Dose-dependent regulation of macrophage differentiation by mos mRNA in a human monocytic cell line.

The proto-oncogene c-mos was expressed during differentiation of the human monocytic cell line U937 into macrophages. To investigate a possible role of the mos oncogene, we introduced the v-mos gene under an inducible promoter, MT-I, into U937 cells. The v-mos transformed cells expressed mos mRNA at an amount proportional to the concentration of Zn2+ ions. The induction of the v-mos gene caused growth inhibition and macrophage differentiation in these cells. The differentiation of v-mos transformed monocytes into macrophages required continuous expression of the v-mos gene. The extent of expression of phenotypic characteristics of macrophages, such as phagocytosis, cell surface antigens and typical morphology, depends on the amount of mos mRNA present. We were therefore able to demonstrate that the expression of only one oncogene, mos, determines monocyte differentiation into macrophages.     

Modulation of CD157 expression in multi-lineage myeloid differentiation of promyelocytic cell lines

CD157/BST-1 is expressed on mature myeloid cells but not on their precursors in vivo. Also CD38, a homologous gene to CD157, is upregulated in promyelocytic HL-60 cells by the monocyte and granulocyte differentiation-inducing 1alpha,25dihydroxyvitamin D3 (VD3) and all-trans retinoic acid (ATRA), respectively. We have examined whether CD157 expression is upregulated when the promyeloid HL-60 and/or U937 cells are induced to differentiate into mature phenotypes in vitro. VD3 treatment irreversibly upregulated the expression of CD157 in HL-60 cells but not in U937 cells in a time- and concentration-dependent manner when analyzed by flow cytometry, immunoblotting and/or RT-PCR. Different monocyte and granulocyte lineage inducers induced CD157 expression to varying extents while the macrophage differentiation-inducing phorbol 12-myristate 13-acetate (PMA) induced its down-regulation. Time-kinetics of VD3 treatment of HL-60 cells showed that the appearance of CD157 and CD11b (a differentiation marker) antigens were not substantial up to 24 hours but increased subsequently although the appearance of CD38 became significant within 6 hours. Two-color staining of VD3-treated HL-60 cells displayed an apparently linear correlation between CD157 and CD11b expression. Dibutyryl cAMP (cAMP agonist) and forskolin (cAMP-increasing agent) augmented the VD3-dependent induction of CD157 and CD11b expression while PGE1 (cAMP-decreasing agent) inhibited it, suggesting the involvement of a cAMP-dependent mechanism in VD3-induced CD157 upregulation. Co-treatment of HL-60 cells with VD3 plus TNF-alpha or ara-C produced an additive effect on CD157 upregulation. The upregulated CD157 in the VD3-differentiated HL-60 cells was able to activate CD157-dependent tyrosine kinase signal when cross-linked with anti-CD157 antibody.     

Anti-Leu-3/T4 antibodies react with cells of monocyte/macrophage and Langerhans lineage

Anti-Leu-3a, anti-Leu-3b, OKT4, and anti-T4 murine monoclonal antibodies react with a membrane component expressed by mature peripheral blood helper T cells and certain thymocyte subsets. Using a variety of immunologic staining techniques, we have demonstrated the reactivity of these antibodies with other cell types. Normal and neoplastic cells of monocyte/macrophage lineage bear the Ia+/Leu-6-/Leu-3+ phenotype, whereas histiocytosis X cells bear the Ia+/Leu-6+/Leu-3+ phenotype. The Ia+/Leu-6- cells of malignant histiocytosis and the Ia+/Leu-6+ epidermal Langerhans cells were variably Leu-3+. Normal monocyte/macrophage reactivity with anti-Leu-3/T4 appears to be primarily intracytoplasmic, whereas on U937 monocyte tumor cells, marked membrane reactivity is also observed. These results strongly suggest that certain cells other than helper T cells and thymocytes can express and, at least in some cases, synthesize a component previously regarded as T-lineage specific.     

The arachidonate-dependent cytoprotective signaling evoked by peroxynitrite is a general response of the monocyte/macrophage lineage

U937, THP-1, and J774 cells or human monocytes and macrophages display similar levels of sensitivity to peroxynitrite and exposure to an otherwise non-toxic concentration of the oxidant in the presence of a phospholipase A(2) inhibitor was invariably associated with the onset of mitochondrial permeability transition (MPT)-dependent toxicity. These events were prevented by exogenous arachidonic acid (AA). In general, the protective concentrations of AA were greater in those cell types releasing more AA. Thus, non-toxic concentrations of peroxynitrite commit cells belonging to the monocyte/macrophage lineage to MPT-dependent toxicity that is however prevented by endogenous AA.     

The role of monocyte/macrophages as vehicles of dissemination of Simkania negevensis: an in vitro simulation model

Exposure to Simkania negevensis (Sn), an intracellular microorganism that has been associated with respiratory tract infections in infants and adults, is prevalent. Sn can multiply within free-living amoebae and has been detected in domestic water supplies, which may constitute a source of infection with the organism. Its path of transport from its portal of entry to the body to its target organs is unknown. In this study, the possibility that monocytes/macrophages may serve as vehicles of transmission was examined. In vitro cocultivation of Sn-infected Acanthamoeba polyphaga with the monocyte/macrophage cell line U937 resulted in the death of the amoebae and infection of the U937 cells. Sn entered and multiplied in U937 cells within short periods of time, and the microorganism could be transferred from U937 cells to cell cultures of various origins. Uninfected monocyte/macrophages could become infected when in contact with either actively or persistently Sn-infected cell cultures. Persistently infected cultures in contact with uninfected U937 cells became actively infected. The results of this study provide a basis for determination of the molecular mechanisms of monocyte/macrophage-cell interactions in transfer of infection and may contribute to a better understanding of the pathogenesis of Sn infections in vivo.     

Promonocytic U937 subclones expressing CD4 and CXCR4 are resistant to infection with and cell-to-cell fusion by T-cell-tropic human immunodeficiency virus type 1.

Different strains of human immunodeficiency virus type 1 (HIV-1) vary markedly in the ability to infect cells of the monocyte/macrophage (M/M) lineage. M/M are generally resistant to infection with T-cell-tropic (T-tropic) strains of HIV-1. Recently, the chemokine receptors CCR5 and CXCR4 were identified as cofactors for fusion/entry of macrophage- and T-tropic strains of HIV-1, respectively. To investigate the mechanisms of resistance of M/M to T-tropic HIV-1 infection, we examined a number of subclones of the U937 promonocytic cell line. We found that certain subclones of U937 (plus clones) could, while others (minus clones) could not, support replication of T-tropic strains of HIV-1. We demonstrate that (i) both minus and plus clones support HIV-1 replication when transfected with an infectious molecular cDNA clone of a T-tropic HIV-1; (ii) minus clones do not, but plus clones do, efficiently support fusion with cells expressing HIV-1 IIIB Env; (iii) both plus and minus clones (with the exception of one clone) express physiologically functional CXCR4 protein as well as CD4 on the cell surface; (iv) introduction of CXCR4 into the CXCR4-negative clone does not restore fusogenicity with or susceptibility to T-tropic HIV-1; and (v) a ligand (stromal cell-derived factor 1) for or a monoclonal antibody (12G5) to CXCR4 does not effectively inhibit HIV-mediated cell-to-cell fusion of U937 cells. These data indicate that resistance to T-tropic HIV-1 infection of U937 minus clones occurs at fusion/ entry events and that expression of functional CXCR4 and CD4 is not a sole determinant for susceptibility to T-tropic HIV-1 infection; furthermore, they suggest that other factors are positively or negatively involved in HIV-mediated cell-to-cell fusion in U937 promonocytic cells.     

The nanomechanical properties of rat fibroblasts are modulated by interfering with the vimentin intermediate filament system

The contribution of the intermediate filament (IF) network to the mechanical response of cells has so far received little attention, possibly because the assembly and regulation of IFs are not as well understood as that of the actin cytoskeleton or of microtubules. The mechanical role of IFs has been mostly inferred from measurements performed on individual filaments or gels in vitro. In this study we employ atomic force microscopy (AFM) to examine the contribution of vimentin IFs to the nanomechanical properties of living cells under native conditions. To specifically target and modulate the vimentin network, Rat-2 fibroblasts were transfected with GFP-desmin variants. Cells expressing desmin variants were identified by the fluorescence microscopy extension of the AFM instrument. This allowed us to directly compare the nanomechanical response of transfected and untransfected cells at high spatial resolution by means of AFM. Depending on the variant desmin, transfectants were either softer or stiffer than untransfected fibroblasts. Expression of the non-filament forming GFP-DesL345P mutant led to a collapse of the endogenous vimentin network in the perinuclear region that was accompanied by localized stiffening. Correlative confocal microscopy indicates that the expression of desmin variants specifically targets the endogenous vimentin IF network without major rearrangements of other cytoskeletal components. By measuring functional changes caused by IF rearrangements in intact cells, we show that IFs play a crucial role in mechanical behavior not only at large deformations but also in the nanomechanical response of individual cells.     

Nestin promotes the phosphorylation-dependent disassembly of vimentin intermediate filaments during mitosis

The expression of the intermediate filament (IF) protein nestin is closely associated with rapidly proliferating progenitor cells during neurogenesis and myogenesis, but little is known about its function. In this study, we examine the effects of nestin expression on the assembly state of vimentin IFs in nestin-free cells. Nestin is introduced by transient transfection and is positively correlated with the disassembly of vimentin IFs into nonfilamentous aggregates or particles in mitotic but not interphase cells. This nestin-mediated disassembly of IFs is dependent on the phosphorylation of vimentin by the maturation/M-phase-promoting factor at ser-55 in the amino-terminal head domain. In addition, the disassembly of vimentin IFs during mitosis appears to be a unique feature of nestin-expressing cell types. Furthermore, when the expression of nestin is downregulated by the nestin-specific small interfering RNA in nestin-expressing cells, vimentin IFs remain assembled throughout all stages of mitosis. Previous studies suggest that nonfilamentous vimentin particles are IF precursors and can be transported rapidly between different cytoplasmic compartments along microtubule tracks. On the basis of these observations, we speculate that nestin may play a role in the trafficking and distribution of IF proteins and potentially other cellular factors to daughter cells during progenitor cell division.     

Oxysterol-induced up-regulation of MCP-1 expression and synthesis in macrophage cells

To investigate the proinflammatory potential of cholesterol and cholesterol oxidation products (oxysterols), which are present in oxidized low-density lipoproteins, foam cells, and fibrotic plaque, we used an in vitro model mimicking the challenge of macrophage cells by the cholesterol accumulating within the central core of atheroma. A biologically representative oxysterol mixture was shown to be potentially able to sustain a chronic inflammatory process within the vascular wall by up-regulating the expression of defined proinflammatory genes. In particular, expression and synthesis of the major chemokine for monocytes/macrophages, namely monocyte chemotactic protein-1 (MCP-1), were consistently increased when cells of the macrophage lineage (U937 cell line) were incubated with this mixture. On the contrary, an identical concentration of unoxidized cholesterol in no case modified expression or synthesis of the chemokine. Up-regulated expression and synthesis of MCP-1 by the oxysterol mixture was clearly dependent on a net increment of phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and nuclear factor kappaB (NF-kappaB) nuclear binding. The results indicate that cholesterol may contribute to the progression of atherosclerotic lesions by strongly up-regulating crucial proinflammatory factors like MCP-1, but only after having been oxidized to oxysterols.     

Synthetic peptides of human CD4 enhance binding of Ig to monocyte/macrophage cells. I. Characterization and mapping studies.

Human T cell glycoprotein CD4 binds to class II MHC molecules and to HIV envelope protein gp120. We have shown that CD4 and synthetic peptides corresponding to amino acid residues 21-49 of the first extracellular domain of CD4, also bind Ig and, with greater avidity, antibody:Ag complex. We investigated the effect of CD4 synthetic peptides on the binding and uptake of human Ig by monocyte/macrophage U937 cells. We found that a synthetic peptide corresponding to amino acid residues 21-49 enhanced binding to U937 cells of both aggregated and nonaggregated Ig. The enhancement was concentration dependent, occurred both in normal and low ionic strength conditions, and varied with the time and the temperature of the preincubation step. The enhancement was maximal after preincubation for 3 h at 37 degrees C. A peptide concentration of 20 micrograms/ml was sufficient for optimal binding of both nonaggregated and aggregated Ig. CD4 peptide 21-49 also enhanced binding of Ig to Staphylococcus aureus protein A. These studies open a new perspective in the way monocyte/macrophage cells handle Ig, antibody:Ag or Id:anti-Id complex, in particular when present at threshold amounts in a nonprecipitating form.