Histiocytic U937 Cells Research Articles

Neisseria meningitidis serogroup B lipopolysaccharides induce a lower pro-inflammatory effect within the proteoliposome used in Cuban anti-meningococcal vaccines

Neisseria meningitidis outer membrane vesicles or proteoliposomes (PLs) have been used as vaccines and adjuvants. Despite the presence of potentially toxic amounts of lipopolysaccharide (LPS), they have been shown to be safe, well tolerated, and immunogenic. This suggests that LPS-PL may have reduced LPS toxicity. In this study, it is shown that the ability of PL to induce pro-inflammatory cytokine production in human U937 histiocytic cell line is significantly lower than that of an equivalent concentration of purified LPS. This demonstrates that certain components or physical properties of PL reduce the pro-inflammatory activity of their endogenous LPS. To investigate the mechanisms responsible for this protective effect, PLs were fractionated and an assessment was made of the ability of the resulting fractions to induce inflammatory cytokine expression. Several individual PLs fractions were more potent inducers of pro-inflammatory cytokine production than the non-fractionated PLs. The majority of the pro-inflammatory activities appeared to be mediated by the presence of LPS in the fractions, as shown by the ability of an anti-CD14 antibody to block it. However, in two PL fractions, the production of IL-8, and to a lesser extent IL-6, was not inhibited by anti-CD14 treatment, indicating that pro-inflammatory components other than LPS could also be present in PL. Eight proteins present in the fractions were identified by n-terminal sequencing. The results of this study suggest that two of them, PorB, and in particular, the RmpM protein, may also contribute to the pro-inflammatory activity of N. meningitidis PL. These results could support the development of PLs as vaccine adjuvant.

Neisseria meningitidis serogroup B lipopolysaccharides induce a lower pro-inflammatory effect within the proteoliposome used in Cuban anti-meningococcal vaccines

Neisseria meningitidis outer membrane vesicles or proteoliposomes (PLs) have been used as vaccines and adjuvants. Despite the presence of potentially toxic amounts of lipopolysaccharide (LPS), they have been shown to be safe, well tolerated, and immunogenic. This suggests that LPS-PL may have reduced LPS toxicity. In this study, it is shown that the ability of PL to induce pro-inflammatory cytokine production in human U937 histiocytic cell line is significantly lower than that of an equivalent concentration of purified LPS. This demonstrates that certain components or physical properties of PL reduce the pro-inflammatory activity of their endogenous LPS. To investigate the mechanisms responsible for this protective effect, PLs were fractionated and an assessment was made of the ability of the resulting fractions to induce inflammatory cytokine expression. Several individual PLs fractions were more potent inducers of pro-inflammatory cytokine production than the non-fractionated PLs. The majority of the pro-inflammatory activities appeared to be mediated by the presence of LPS in the fractions, as shown by the ability of an anti-CD14 antibody to block it. However, in two PL fractions, the production of IL-8, and to a lesser extent IL-6, was not inhibited by anti-CD14 treatment, indicating that pro-inflammatory components other than LPS could also be present in PL. Eight proteins present in the fractions were identified by n-terminal sequencing. The results of this study suggest that two of them, PorB, and in particular, the RmpM protein, may also contribute to the pro-inflammatory activity of N. meningitidis PL. These results could support the development of PLs as vaccine adjuvant.

ATP-Binding-Cassette A1 Regulates Extracellular Isopentenyl Pyrophosphate Release and Vγ9Vδ2 T-Cell Activation By Dendritic Cells

IntroductionPhosphorylated metabolites of mevalonate (Mev) pathway like isopentenyl-pyrophosphate (IPP) induce proliferation of Vγ9Vδ2 T cells. Endogenous IPP levels can be lowered or increased by aminobisphosphonates (NBPs) such as zoledronic acid (ZA). We have previously shown that ZA-treated dendritic cells (DC) produce and release high amounts of soluble IPP and are strong Vγ9Vδ2 T-cell activators. However, the mechanisms regulating IPP release in DC and other cells are still unknown.Methods"DC generation": purified CD14+ cells were cultured in standard culture medium at 0.5-1.5 x 106cells/ml supplemented with GM-CSF (1000 U/ml) and IL-4 (500 U/ml) in flat-bottomed 24-well plates for 24 h. After 24 h, DC were left untreated (ZA-) or treated for further 24 h with 1 µM ZA (ZA+). DC were washed after incubation with ZA and re-plated with fresh medium for an additional 24 and 48 h (washed DC [w-DC]). In selected experiments, supernatants were collected for quantification of extracellular IPP levels."Vγ9Vδ2 T-cell proliferation":on day 7, total counts of viable T cells were calculated with the trypan blue staining assay, and by flow cytometry. In selected experiments, PBMC from CTRL were stimulated for 7 days with supernatants obtained from autologous DCZA- and DCZA+and 10 IU/ml IL-2 to evaluate Vγ9Vδ2 T-cell proliferation."IPP efflux":cells were radiolabelled with 1 microCi/ml [3H]-acetate; after 24 h, cell supernatants were collected, [3H]-IPP was extracted and resolved by thin layer chromatography. After separation, the spot corresponding to IPP was cut and solubilized, and the radioactivity was quantified by liquid scintillation count."siRNA ABCA1": cells were incubated 96 h with a 20-25 nucleotide non targeting scrambled siRNA or specific ABCA1 siRNA (Accell Thermo Scientific), then lysed and subjected to the Western blot analysis of ABCA1 expression."Proximity ligation assay (PLA)":the ABCA1-BTN3A1 interaction was measured by the PLA method with the DuoLink In Situ kit (Sigma Chemicals Co.), using a mouse anti-human ABCA1 (Abcam) and a rabbit anti-human BTN3A1 (Sigma Chemicals Co.) antibody, respectively, as per the manufacturer's instructions. Nuclei were counterstained with 4',5-diamidino-2 phenylindole dyhydrochloride (DAPI). Cells were examined using a Leica TCS SP2 AOP confocal laser-scanning microscope (Leica Microsystem, Wetzlar, Germany).ResultsWe tested a variety of tumoral and non-tumoral cells for their ability to activate Vγ9Vδ2 T cells and correlated this ability with the release of extracellular IPP. We used samples from healthy donors included peripheral blood (PB) monocytes, monocyte-derived DCs, and bone marrow (BM) stromal cells; samples from cancer patients included myeloma cells, chronic lymphocytic leukemia (CLL) cells, and BM stromal cells from multiple myeloma (MM) and CLL patients; tumor cell lines included the monocytic THP1 and histiocytic U937 cells and the myeloma cell line SKMMI. We found that the activation of Vγ9Vδ2 T cells was proportional to the simultaneous efflux of IPP and to the activity/expression of ATP-binding cassette transporter A1 (ABCA1), a membrane transporter involved in the delivery of intracellular cholesterol to apolipoprotein A-I.We have demonstrated that the most potent IPP releaser cells had also the highest expression of ABCA1. Functional ABCA1 inhibition with probucol slightly increased intracellular IPP accumulation, but abrogated extracellular IPP release and Vγ9Vδ2 T-cell activation. Abca1 silencing experiments confirmed the key role played by ABCA1. ABCA1 is up-regulated by ZA viaLXRα transcriptional activation induced by intracellular IPP accumulation and inhibition of the PI3K/Akt/mTOR signaling pathway.Butyrophilin-3A1 (BTN3A1) is an immunoglobulin superfamily protein reported to be required for phosphoantigen presentation and target recognition by Vγ9Vδ2 T cells. We have also demonstrated that BTN3A1 is physically associated with ABCA1 and cooperates with ABCA1 in the activation of Vγ9Vδ2 T cells.ConclusionsThese results suggest that ABCA1 is involved in extracellular IPP release and Vγ9Vδ2 T-cell activation induced by ZA-treated DC and therefore play a crucial role as mediator of the immune response. DisclosuresBoccadoro:SANOFI: Honoraria, Research Funding; CELGENE: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Abbivie: Honoraria; Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Mundipharma: Research Funding; Amgen: Honoraria, Research Funding. Massaia:Roche: Other: advisory board, research support; Gilead: Other: advisory board; Janssen: Other: advisory board.

Nanoceria protects from alterations in oxidative metabolism and calcium overloads induced by TNFα and cycloheximide in U937 cells: pharmacological potential of nanoparticles

The present study is aimed to determine the protective effect of a novel nanoparticle with antioxidant properties, nanoceria, on reactive oxygen species (ROS) production, and calcium signaling evoked by the tumor necrosis factor-alpha (TNFα) in combination with cycloheximide (CHX) on apoptosis in the human histiocytic lymphoma cell line U937. Our results show that treatment of U937 cells with 10ng/mL TNFα in combination with 1μg/mL CHX led to several Ca(2+) alterations. These stimulatory effects on calcium signals were followed by intracellular ROS production and mitochondria membrane depolarization, as well as a time-dependent increase in caspase-8 and -9 activities. Our results show that the pretreatment with well known antioxidants such as trolox and N-acetyl cysteine (NAC) partially reduced the apoptotic effects due to the administration of TNFα plus cycloheximide. Furthermore, nanoceria had a stronger protective effect than trolox or NAC. Our findings also suggest that TNFα plus cycloheximide-induced apoptosis is dependent on alterations in cytosolic concentration of calcium [Ca(2+)]c and ROS generation in human histiocytic U937 cells.

Antitumor activity of antimicrobial peptides against U937 histiocytic cell line.

We investigated cytotoxic activity of antimicrobial peptides of different origin (both naturally occurring and synthetic), structure and known mechanisms of action against human histiocytic lymphoma cell line U937. The strongest cytotoxic activity against U937 cell line was shown by Pexiganan MSI-78, followed by Citropin 1.1, Protegrin 1 and a synthetic lipopeptide, N-α-palmitoyl-L-lysyl-L-lysine amide (Pal-Lys-Lys-NH₂). The cytotoxic activity of the peptides was more dependent on the time of incubation than concentration. Only for the lipopeptide, whose mode of action was restricted to disruption of electric potential of the cell membrane, the correlation between cytotoxicity and concentration was almost linear. The high cytotoxicity of Pexiganan MSI-78, Protegrin 1 and the lipopeptide could be basically explained by their membranolytic activity leading to necrosis. However, in the case of Citropin 1.1, the cell membrane integrity was disrupted only slightly and independently of the peptide concentration. Therefore, some other mechanism of action might be responsible for its strong dose-dependent cytotoxic activity, e.g., membranolytic activity leading to apoptosis. Furthermore, TNF-α production due to LPS (lipopolysaccharide) stimulation was suppressed by the presence of Citropin 1.1, Pexiganan MSI-78 or Protegrin 1, but not by Buforin 2 or the lipopeptide. Our experiments have shown that cytotoxic activity is not limited to some specific molecular structure of a peptide, but rather to the length of the peptide chain as it is likely to affect the efficiency of the tumor cell membrane disruption and interaction with LPS.

Processing of the human transferrin receptor at distinct positions within the stalk region by neutrophil elastase and cathepsin G.

The ectodomain of the human transferrin receptor (TfR) is released as soluble TfR into the blood by cleavage within a stalk. The major cleavage site is located C-terminally of Arg-100; alternative cleavage sites are also present. Since the cleavage process is still unclear, we looked for proteases involved in TfR ectodomain release. In the supernatant of U937 histiocytic cells we detected alternatively cleaved TfR (at Glu-110). In membrane fractions of these cells we identified two distinct proteolytic activities responsible for TfR cleavage within the stalk at either Val-108 or Lys-95. Both activities could be inhibited by serine protease inhibitors, but not by inhibitors of any other class of proteases. Protein purification yielded a 28 kDa protein that generated the Val-108 terminus. The protease activity could be ascribed to neutrophil elastase according to the substrate specificity determined by amino acid substitution analysis of synthetic peptides, an inhibitor profile, the size of the protease and the use of specific antibodies. The results of analogous experiments suggest that the second activity is represented by another serine protease, cathepsin G. Thus, membrane-associated forms of neutrophil elastase and cathepsin G may be involved in alternative TfR shedding in U937 cells.

Chloroquine decreases cell-surface expression of tumour necrosis factor receptors in human histiocytic U-937 cells.

Proinflammatory cytokine tumour necrosis factor (TNF) mediates its diverse effects through cell surface receptors. A variety of inflammatory signals are known to modulate TNF activities by changing expression and shedding of cell-surface TNF receptors. We have examined the effects of anti-rheumatic drug chloroquine on the expression of cell surface and soluble TNF receptors in human histiocytic U-937 cells. Chloroquine partially reduced production of soluble p55 and p75 TNF receptors in cells stimulated with phorbol 12-myristate 13-acetate (PMA). In these cells, induction of both TNF receptor mRNA was not changed and the levels of cell-associated TNF receptors were rather increased by chloroquine. Flow cytometric analysis revealed that chloroquine does not inhibit the PMA-triggered shedding of TNF receptors from cell surface, while it was suppressed by a metalloproteinase inhibitor BB-3103. Treatment of U-937 cells with chloroquine significantly reduced the level of cell surface TNF receptors and a similar effect was observed with human peripheral blood monocytes. Other weak-base amines, including hydroxychloroquine, ammonium chloride and methylamine, also induced reduction of cell surface TNF receptors, whereas lysosomal proteinase inhibitor, leupeptin, and BB-3013 were without effect. Our results suggest that chloroquine down-regulates cell surface TNF receptors by retarding their transport to the cell surface, while cleavage of cell surface receptors is not inhibited by chloroquine.

In vivo demethylation of a MoMuLV retroviral vector expressing the herpes simplex thymidine kinase suicide gene by 5' azacytidine.

We constructed a functional MoMuLV-based bicistronic retroviral vector encoding the herpes simplex virus type I thymidine kinase gene, which induces sensitivity to the prodrug ganciclovir (gcv), and the reporter beta-galactosidase gene (MFG-tk-IRES-lacZ). The U937 histiocytic cell line was transduced with this vector, and a clone (VB71) with high-level transgene expression was selected. Severe combined immunodeficient (SCID) mice were injected with VB71 cells to evaluate the role of long terminal repeat methylation in transgene silencing in vivo and to see whether 5-azacytidine (5' aza-C) demethylating agent prevented it. We found 5' aza-C maintained gene expression at high level in vitro. In vivo, time to tumor onset was significantly longer in SCID mice receiving the VB71 cells, 5' aza-C, and gcv compared with animals treated with either 5' aza-C or gcv alone. The number of injected tumor cells influences tumor onset time and the efficacy of 5' aza-C and gcv treatment. The standard gcv treatment schedule (10 mg/kg from d + 1 until the onset of tumor) controlled tumor onset better than short-term treatment with high doses. In conclusion, the results extend our previous findings that transgene methylation in vivo may be prevented with an appropriate schedule of 5' aza-C and gcv.

Modulation of monocyte chemotactic protein-1 production by hyperoxia: importance of RNA stability in control of cytokine production.

Pulmonary oxygen toxicity occurs after prolonged administration of increased fractions of inspired oxygen. Lung damage in this setting manifests as diffuse alveolar damage. In animals exposed to hyperoxia, increased numbers of alveolar macrophages are noted 72 h after initiation of high concentrations of oxygen. Monocyte chemotactic protein-1 (MCP-1) is a cytokine released by a number of cell types that has potent chemotactic activity for monocytes, precursor cells for alveolar macrophages. In the current study, we examined whether MCP-1 production was increased in response to hyperoxia. We used the monocyte/ histiocytic U937 cell line and exposed these cells to hyperoxia for variable amounts of time, then determined MCP-1 concentrations by enzyme-linked immunosorbent assay and MCP-1 mRNA levels by Northern blot analysis. We also examined the effects of dexamethasone on the response of U937 cells to hyperoxia. Finally, as a potential mechanism for regulation of U937 MCP-1 production, we examined effects of hyperoxia on MCP-1 mRNA stability. The results demonstrate that hyperoxia stimulates MCP-1 production after 6 and 24 h of exposure. MCP-1 mRNA levels are also increased after initiation of hyperoxia in part through effects on MCP-1 transcript stability. Dexamethasone significantly reduces MCP-1 production and mRNA levels also in part through effects on transcript stability. These studies suggest monocytes may be attracted to hyperoxia-exposed lungs through enhanced MCP-1 production. MCP-1 production appears to be upregulated in part through post-transcriptional processes in this setting.

Molecular mechanisms governing tumor-necrosis-factor-mediated regulation of plasminogen-activator inhibitor type-2 gene expression.

Plasminogen-activator inhibitor type 2 (PAI-2), a serine protease inhibitor involved in the regulation of urokinase-dependent proteolysis, is also implicated in the inhibition of tumor-necrosis-factor-(TNF)-mediated apoptosis. The PAI-2 gene is one of the most TNF-responsive genes known and is also highly induced by the phorbol ester phorbol 12-myristate 13-acetate (PMA) and the phosphatase inhibitor, okadaic acid, in both HT-1080 fibrosarcoma and U-937 histiocytic cells. We sought to identify and characterize regulatory cis-acting DNA elements and trans-acting factors which mediate basal and inducible PAI-2 gene transcription. A series of promoter deletion mutants (nucleotides -1859 to -91) fused to the chloramphenicol acetyl transferase (CAT) reporter gene were transfected into HT-1080 cells. Two repressor regions were identified; one distally between positions -1859 and -1100, and one proximally between positions -259 and -219. Cells transfected with constructs harboring more than 259 bp promoter sequence produced a 10-15-fold increase in CAT activity when treated with PMA or okadaic acid, but produced only a minimal (2.5-fold) increase in response to TNF. Removal of the proximal repressor by deletion to position -219, or by internal deletion from the -1100 PAI-2 CAT construct, resulted in a selective increase in TNF responsiveness, suggesting that induction of PAI-2 gene transcription by TNF is associated with derepression. Detailed analysis of the proximal repressor utilizing the electrophoretic mobility shift assay (EMSA), identified two novel and distinct protein-binding sites (A and B). Site A is located within the 40-bp proximal repressor while site B is situated immediately adjacent to the 3' boundary. Treatment of cells with PMA or okadaic acid produced no change in the binding activity of proteins recognising sites A or B. However, treatment of cells with TNF results in a profound selective reduction in site-B-binding activity, suggesting that this site plays a significant role in TNF-mediated regulation of PAI-2 gene expression. Our findings suggest that TNF-mediated induction of PAI-2 gene expression involves derepression and is associated with cis-acting and trans-acting factors located within and adjacent to the proximal repressor region.

Increase in Cytosolic Calcium Upregulates the Synthesis of Type 1 Plasminogen Activator Inhibitor in the Human Histiocytic Cell Line U937

Abstract In the U937 histiocytic cell line, we investigated the effect of calcium-mobilizing agents with or without tumor necrosis factor-alpha (TNF) on the regulation of the synthesis of plasminogen activator inhibitor-type 1 (PAI-1). Cultured U937 cells were stimulated with ionophore A23187 and thapsigargin with or without TNF. The response was analyzed in terms of cytosolic calcium mobilization, PAI-1 accumulation in the medium, and PAI-1 mRNA expression. The study was extended to urokinase (uPA) secretion and surface expression of its receptor (uPAR). Using Fluo-3 as a calcium-indicator dye to measure cytosolic calcium mobilization, we showed by flow cytometry that both agents mobilized calcium in a dose-dependent manner. TNF provoked a slight calcium mobilization that was also observed by digital imaging microscopy. Association of TNF with the calcium-mobilizing agents potentiated the calcium mobilization. Both calcium-mobilizing agents induced at 18 hours a dose-dependent accumulation of PAI-1 in culture medium, whereas uPA was not affected. TNF alone induced a more marked accumulation of PAI-1 than of uPA. Association of TNF with the agents induced a PAI-1 response that was more than additive of the two, whereas the secretion of uPA was not enhanced. Membrane expression of uPAR, measured by flow cytometry, tended to be slightly augmented by the calcium-mobilizing agents only. All the treatments resulted in a significant increase in PAI-1 mRNA level at 3 hours after the stimulation, which was very marked when calcium-mobilizing agents were present. Incubation of U937 cells in a calcium-free medium totally prevented both the mRNA expression and accumulation of PAI-1 induced by calcium-mobilizing agents and, to lesser extent, that induced by TNF. The increase in PAI-1 mRNA expression did not require de novo protein synthesis, as cycloheximide did not suppress the increase in PAI-1 mRNA induced by calcium-mobilizing agents. It is concluded that, in U937 cells, calcium triggers a pathway that upregulates PAI-1 synthesis and positively interacts with the TNF-induced pathway that stimulates PAI-1 synthesis. As uPA and uPAR were differently affected, it is suggested that an increase in cytosolic calcium leads to a reduced pericellular proteolysis.

Increase in cytosolic calcium upregulates the synthesis of type 1 plasminogen activator inhibitor in the human histiocytic cell line U937

In the U937 histiocytic cell line, we investigated the effect of calcium-mobilizing agents with or without tumor necrosis factor-alpha (TNF) on the regulation of the synthesis of plasminogen activator inhibitor-type 1 (PAI-1). Cultured U937 cells were stimulated with ionophore A23187 and thapsigargin with or without TNF. The response was analyzed in terms of cytosolic calcium mobilization, PAI-1 accumulation in the medium, and PAI-1 mRNA expression. The study was extended to urokinase (uPA) secretion and surface expression of its receptor (uPAR). Using Fluo-3 as a calcium-indicator dye to measure cytosolic calcium mobilization, we showed by flow cytometry that both agents mobilized calcium in a dose-dependent manner. TNF provoked a slight calcium mobilization that was also observed by digital imaging microscopy. Association of TNF with the calcium-mobilizing agents potentiated the calcium mobilization. Both calcium-mobilizing agents induced at 18 hours a dose-dependent accumulation of PAI-1 in culture medium, whereas uPA was not affected. TNF alone induced a more marked accumulation of PAI-1 than of uPA. Association of TNF with the agents induced a PAI-1 response that was more than additive of the two, whereas the secretion of uPA was not enhanced. Membrane expression of uPAR, measured by flow cytometry, tended to be slightly augmented by the calcium-mobilizing agents only. All the treatments resulted in a significant increase in PAI-1 mRNA level at 3 hours after the stimulation, which was very marked when calcium-mobilizing agents were present. Incubation of U937 cells in a calcium-free medium totally prevented both the mRNA expression and accumulation of PAI-1 induced by calcium-mobilizing agents and, to lesser extent, that induced by TNF. The increase in PAI-1 mRNA expression did not require de novo protein synthesis, as cycloheximide did not suppress the increase in PAI-1 mRNA induced by calcium-mobilizing agents. It is concluded that, in U937 cells, calcium triggers a pathway that upregulates PAI-1 synthesis and positively interacts with the TNF-induced pathway that stimulates PAI-1 synthesis. As uPA and uPAR were differently affected, it is suggested that an increase in cytosolic calcium leads to a reduced pericellular proteolysis.

Infectivity of Trypanosoma rangeli in a Promonocytic Mammalian Cell Line

The existence of an intracellular stage of Trypanosoma rangeli in the vertebrate host was evaluated by experimental infection of the U937 histiocytic cell line with the San Agustín strain and the Ub66-5b clone. The identity of the parasites at the beginning and end of the experiments was confirmed through biological behavior in the vector and mammal hosts, isoenzymes, polymerase chain reaction (PCR), and monoclonal antibodies. Infectivity to U937 cells of T. rangeli obtained from culture and salivary glands was evaluated under different experimental conditions. These included 34 C vs. 37 C, opsonized vs. nonopsonized parasites, and 2, 4, 6, 24, 48, and 72 hr of cell-parasite contact. Trypanosoma rangeli adopted a characteristic nondividing amastigote-like form within U937 cells, which was different in size (P = 0.001) from Trypanosoma cruzi amastigotes. Culture forms of T. rangeli were more infective than parasites from salivary glands (P = 0.049) but were less infective than T. cruzi (P = 0.0001). Variations in temperature (34-37 C) and complement opsonization did not affect infectivity. Viability of intracellular forms was confirmed by feeding Rhodnius prolixus with T. rangeli-infected cells. Resistance of T. rangeli to the intracellular milieu could be an important mechanism in producing chronic infections in mammals and in the infection of triatomines.

Significance of cytokines and CD68-positive microparticles in immune thrombocytopenic purpura.

We investigated the significance of cytokines (soluble interleukin-2 receptor, granulocyte-macrophage colony-stimulating factor, interleukin-6, and interferon-gamma) and CD68-positive microparticles in immune thrombocytopenic purpura. Cytokines were measured by enzyme-linked immunosorbent assay and microparticles were detected by flow cytometry. CD68 expression by histiocytic U937 cells incubated with lipopolysaccharide or cytokines was also assessed in a control study. The level of CD68-positive microparticles was significantly higher in the patients with thrombocytopenia than in normal controls (p < 0.01). The soluble interleukin-2 receptor level was also significantly higher in patients than in controls (p < 0.01), but the other cytokines did not show a significant difference. However, patients with severe thrombocytopenia (platelet count > 20,000/microliters) had significantly higher levels of granulocyte-macrophage colony-stimulating factor and interleukin-6 than the controls (p < 0.05). When opsonized platelets were incubated with activated U937 cells, lipopolysaccharide and granulocyte-macrophage colony-stimulating factor caused an increase of CD68-positive microparticles in the supernatant. These results suggest that granulocyte-macrophage colony-stimulating factor is released by activated T cells in immune thrombocytopenic purpura and activates monocyte/macrophage phagocytosis, resulting in an increase of circulating CD68-positive microparticles and enhanced platelet destruction.

Hyperoxia stimulates interleukin-8 release from alveolar macrophages and U937 cells: attenuation by dexamethasone.

Pulmonary oxygen toxicity is associated with histological evidence of polymorphonuclear neutrophil (PMN) infiltration into lung parenchyma. What guides infiltration of these cells is unknown. A number of chemoattractants for PMN have been documented including interleukin-8 (IL-8), a cytokine released by alveolar macrophages (AM) and other cell types. The purposes of this study were to 1) determine whether human AM and the histiocytic U937 cell line release IL-8 in response to hyperoxia, 2) assess whether hyperoxia results in increased IL-8 steady-state mRNA levels in U937 cells and 3) establish whether dexamethasone could attenuate noted effects of hyperoxia. Our study shows that hyperoxia stimulates human AM and U937 cell release of IL-8. Hyperoxia also increases IL-8 mRNA levels in U937 cells. IL-8 released in response to hyperoxia by AM was biologically active as evidenced by ability to induce PMN chemotaxis. A polyclonal antibody to IL-8 partially attenuated this chemotactic activity. Finally, dexamethasone at concentrations of 10 microM, 1 microM, and 100 nM markedly reduced hyperoxia-induced IL-8 release and mRNA synthesis by U937 cells. We conclude that IL-8 may be important in the pathogenesis of pulmonary oxygen toxicity and that therapeutic concentrations of dexamethasone can suppress production of this cytokine.

THE ROLE OF DIFFERENTIAL CLASS II ANTIGEN EXPRESSION IN STIMULATION OF ALLOGENEIC MIXED LYMPHOCYTE REACTIONS BY HUMAN MONOCYTE HYBRIDOMAS

The recognition of foreign class II antigens on accessory cells is the crux of an alloreactive immune response. This phenomenon is clearly demonstrated in the primary mixed lymphocyte reaction, which correlates with the type and density of expressed gene products of the HLA-D region. We have generated a series of human monocyte hybridomas by fusing monocytes with the hypoxanthine guanine phosphoribosyl transferase (HGPRT)-deficient, HLA-D antigen-negative U937 histiocytic cell line. Clones bearing combination of HLA-DQ, -DP with or without HLA-DR have been isolated, allowing for the functional assessment of these molecules. In contrast to the U937 cells, the HLA-DR+DQ+DP+ clone 16.1 was capable of stimulating a primary allogeneic MLR. Interestingly, the DR- but DP+DQ+ clones 13 and 15 were also capable of stimulating alloreactive T cells, and the addition of anti-DQ or -DP but not -DR was associated with significant inhibition of the MLR response. Furthermore, gamma-IFN was found to have diverse effects on class II antigen expression in the U937 cells and the hybrids. gamma-IFN down-regulated the expression of HLA-DQ, -DP without altering -DR on clone 16.1, and this was associated with a significant reduction in its MLR stimulatory capacity. The MLR generated by this gamma-IFN-stimulated hybridoma (HLA-DR+DQ-DP-) was now unaffected by the addition of anti-DQ or -DP mAbs. In contrast, up-regulation of DQ and DP antigens on the U937 cells by gamma-IFN now rendered these cells stimulatory in MLR. These data are consistent with the concept that DQ and DP are both important allostimulatory determinants. Our results stress the potential importance of all D-region molecules in acute allograft rejection or successful engraftment.

Protein kinase C is implicated in the modulation of TNF resistance in U937 histiocytic cells and A U937 monocytic hybridoma A10: [formula omitted] Division of Immunobiology, NIBSC, Blanche Lane, South Mimms, Potters Bar, Herts. EN6 3QG

Protein kinase C is implicated in the modulation of TNF resistance in U937 histiocytic cells and A U937 monocytic hybridoma A10: [formula omitted] Division of Immunobiology, NIBSC, Blanche Lane, South Mimms, Potters Bar, Herts. EN6 3QG

TNF-α Signal Transduction Involves Distinct Phosphorylation Events in U937 Histiocytic Cells

TNF-α Signal Transduction Involves Distinct Phosphorylation Events in U937 Histiocytic Cells

Induction of differentiation of human leukemia cells by various combinations of cytokines and low-molecular-weight inducers.

To explore agents for differentiation therapy of leukemias, various combinations of cytokines and low-molecular-weight inducers were examined for differentiation-inducing activity toward three kinds of human leukemia-derived cell lines. The strongest differentiation inducing activity on promyelocytic HL60 cells and histiocytic U937 cells was obtained by combining recombinant tumor necrosis factor (rTNF), interferon-gamma (IFN-gamma), retinoic acid (RA), and 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3). For myeloblastic ML1 cells, the combination of rTNF, IFN-gamma, and RA had the strongest differentiation-inducing activity.

Pseudomonas aeruginosa alkaline protease degrades human gamma interferon and inhibits its bioactivity.

This study was performed to determine the effect of Pseudomonas aeruginosa on gamma interferon (IFN-gamma) production by antigen-stimulated human T-cell clones. Crude bacterial filtrates prepared from certain strains of P. aeruginosa inhibited IFN-gamma production by T cells and reduced the antiviral activity of preformed IFN-gamma. Bacterial filtrates prepared from mutant strains that did not produce the exoenzyme alkaline protease (AP) did not inhibit IFN-gamma activity. The inhibitory activity of bacterial filtrates was heat and trypsin sensitive and was neutralized by an antiserum to AP. Crystalline AP mimicked the effects of the bacterial filtrates, and an inactive filtrate from a protease-deficient mutant strain was reconstituted by the addition of AP. AP-treated recombinant IFN-gamma showed altered migration on Western blots (immunoblots) of polyacrylamide gels, and this modification correlated with a dose-dependent loss of antiviral activity. The ability of recombinant IFN-gamma to elevate the expression of Fc receptors on cells of the U-937 histiocytic cell line was also diminished by AP treatment. These results indicate that the Pseudomonas protease AP can inhibit the antiviral and immunomodulatory activities of IFN-gamma.