Modulation of interleukin 1 beta gene expression by the immediate early genes of human cytomegalovirus.

The immediate early (IE) genes of human cytomegalovirus (HCMV) can be expressed in monocytes/macrophages and are known to regulate other viral genes. The purpose of these studies was to determine if HCMV IE gene products also modulate expression of a monocyte/macrophage-derived gene, interleukin 1 (IL-1) beta. Steady-state cell-derived IL-1 beta mRNA was increased in lipopolysaccharide (LPS)-stimulated THP-1 cells when transfected with the HCMV IE1 + 2 genes, when compared to cells transfected with a control DNA. LPS-stimulated THP-1 cells also exhibited approximately 30-fold higher IL-1 CAT activity when cotransfected with IE1 + 2 than was observed for the same cells cotransfected with IL-1 CAT and a control plasmid containing the IE promoter alone. LPS increased IL-1 CAT activity in the absence of HCMV genes only twofold. IE1, by itself, increased IL-1 CAT activity in LPS-stimulated cells, whereas, IE2, by itself, caused no change in IL-1 CAT activity. These studies show that the IE1 gene of HCMV can regulate IL-1 beta gene expression. The observations further suggest that some of the inflammatory processes associated with HCMV infection may be due to an effect of HCMV IE genes on cell-derived genes, such as the IL-1 beta gene.

Lipopolysaccharide-mediated signal transduction through phospholipase D activation in monocytic cell lines.

Lipopolysaccharide (LPS)-induced phospholipase D (PLD) activation was investigated in undifferentiated monocytic leukemic cell lines THP-1 and U-937. Treatment of THP-1 or U-937 cells labelled with [32P]orthophosphate, [32P]acyl GPC or [3H]alkyl GPC with LPS, in the presence of 0.5% ethanol, resulted in the accumulation of labelled phosphatidylethanol (PEt) through PLD activation. LPS-mediated PLD activation of THP-1 or U-937 was inhibited by staurosporine (2 microM) and by protein kinase C (PKC) down-regulation with 12-O-tetradecanoylphorbol 13-acetate (TPA) suggesting a role for PKC. In addition to LPS, TPA, ionomycin and cell-permeant analogs of diacylglycerol also stimulated [3H]PEt accumulation. The TPA-induced PEt accumulation was also completely abolished by staurosporine or down-regulation of PKC (> 95% inhibition). Furthermore, the LPS-mediated [32P]PEt formation was attenuated by either depletion of extracellular Ca2+ with EGTA (5 mM) or chelation of intracellular Ca2+ by BAPTA (30 microM). These results indicate that an increase in intracellular Ca2+ is necessary for LPS-mediated PLD activation. Further support for PKC activation by LPS was obtained by determining PKC activity in an in vitro assay of histone H1 phosphorylation using [gamma-32P]ATP. In untreated THP-1 cells, approximately 64% of the PKC activity was localized in the cytosol and 36% in the membrane fraction. Treatment of the cells with LPS (10 micrograms/ml, for 2 h) resulted in an increase of 10% of the membrane-associated PKC activity and a corresponding decrease in the cytosol fraction. These data provide evidence that one of the mechanisms of LPS-mediated signal transduction in human monocytic cell lines involves activation of PLD.

Cytomegalovirus immediate early genes upregulate interleukin-6 gene expression.

BACKGROUND: The immediate early genes (IE) of human cytomegalovirus (CMV) can be expressed in monocytic cells and are known to regulate viral and cellular genes. Interleukin-6 (IL-6) plays a central role in numerous inflammatory and immune processes. Interleukin-6 levels are increased in lung transplant patients clinically diagnosed with CMV pneumonitis. The regulation of IL-6 is dependent on various stimuli that include lipopolysaccharide (LPS), viruses, and other cytokines. These studies examined the ability of CMV IE gene products to modulate IL-6 production. METHODS: THP-1 cells, a monocytic cell line, were transfected with the CMV IE genes. Interleukin-6 protein and IL-6 mRNA were measured in control and CMV immediate early transfected cells. Cotransfection of CMV IE genes and IL-6 chloramphenicol acetyl transferase (CAT) or IL-6 luciferase constructs were used to study IL-6 promoter activity. RESULTS: Interleukin-6 protein and mRNA production were significantly increased in cells transfected with the CMV IE genes and stimulated with LPS compared to LPS-stimulated control cells. Cytomegalovirus IE gene products significantly enhanced LPS stimulation of IL-6 promoter activity in both IL-6 CAT and IL-6 luciferase assays. A deletion construct that contains a NF-kappa B site but is missing the multiple response region demonstrated a continued increase in IL-6 luciferase activity in LPS-stimulated CMV transfected cells. CONCLUSION: Cytomegalovirus immediate early gene products significantly enhanced expression of IL-6 in LPS-stimulated cells. The increase in IL-6 luciferase activity occurs in the absence of the multiple response region, the area of the IL-6 promoter responsive to IL-1, TNF alpha, cyclic amp, and phorbol 12-myristate 13-acetate. The ability of CMV IE gene products to enhance IL-6 production may play an important role in immune inflammatory states associated with CMV infection.

Interferon-gamma regulation of interleukin 6 in monocytic cells.

The regulation of interleukin 6 (IL-6) is dependent on many factors that include numerous stimuli such as lipopolysaccharide (LPS), viruses, and other cytokines. These studies demonstrate the ability of interferon-gamma (IFN-gamma) to significantly enhance IL-6 mRNA and protein production in LPS-stimulated monocytes and THP-1 cells. IL-6 protein production was increased sevenfold in LPS-stimulated cells with the addition of IFN-gamma. IL-6 mRNA production was increased in a dose-dependent fashion up to 15-fold in LPS-stimulated cells with the addition of IFN-gamma. The enhanced production of IL-6 mRNA that occurs with the addition of IFN-gamma to LPS-stimulated monocytic cells was due to increased transcription of IL-6 mRNA. The ability of IFN-gamma to enhance IL-6 production may play an important role in many inflammatory processes.

Modulation of accessory cell function and interleukin-6 production by the HIV-1 tat gene.

Manifestations of HIV-1 infection such as fever, hypergammaglobulinemia, and interstitial pneumonitis may be due to increased production of inflammatory cytokines such as interleukin-1 and interleukin-6 (IL-6). Monocytes/macrophages of HIV-1-infected individuals have been noted to produce increased amounts of IL-6, as well as to have enhanced accessory cell function. These studies examined the ability of HIV-1 tat, an important HIV-1 regulatory gene, to modulate monocyte/macrophage function. In these experiments, HIV-1 tat-transfected THP-1 cells, a monocytic cell line, enhanced THP-1 immune accessory cell function in the presence of pokeweed mitogen and concanavalin A. HIV-1 tat-transfected cells also increased production of lipopolysaccharide-stimulated IL-6 mRNA and IL-6 protein. The ability of monocytes/macrophages to support HIV-1 production while exhibiting little or no cytopathic effects allows these cells to serve as a reservoir for the virus. The ability of HIV-1 tat to regulate cellular function in monocytes/macrophages may play an important part in the pathogenesis of HIV-1 infection.

Hyperthermia enhances cytomegalovirus regulation of HIV-1 and TNF-alpha gene expression.

The immediate-early (IE) genes of human cytomegalovirus (CMV) can be expressed in monocytic cells and are known to regulate viral and cellular genes. Reactivation of human immunodeficiency virus (HIV-1) may be stimulated by a variety of factors including other viruses and inflammatory cytokines. These studies examine the role of hyperthermia and CMV in the regulation of HIV-1 and tumor necrosis factor (TNF)-alpha. THP-1 cells were transfected with the CMV IE genes. HIV-1 and TNF-alpha transcription were assessed with chloramphenicol acetyltransferase promoter constructs. Hyperthermia sufficient to stimulate production of heat shock proteins was used to stimulate the cells. Hyperthermia significantly enhances the effect of CMV IE gene products on the expression of HIV-1 and TNF-alpha. The increases in HIV-1 transcription appear to be in part due to increases in TNF-alpha. Heat shock proteins induced by hyperthermia may play an important role in the viral regulation of monocytic function by CMV.

Role of cytokines in alveolar macrophage accessory cell function in HIV-infected individuals.

Mononuclear phagocytes, including alveolar macrophages (AM), can be chronically infected with HIV and thus serve as a reservoir for the virus. Acting as AC during the generation of an immune response, HIV-infected mononuclear phagocytes can facilitate viral T cell infection by several mechanisms, including direct contact of T cells with HIV-infected macrophages as well as cytokine-induced up-regulation of latent T cell infection. Our laboratory has shown that AM from HIV-infected individuals have enhanced AC function compared to normal AM. In this study we explored AM production and secretion of IL-1 beta and IL-6, two cytokines critical for optimal AC function, in normal volunteers and HIV-infected patients. Cultured AM supernatants and lysates were generated in the presence and absence of LPS and standard mitogens. In initial mixing experiments HIV AM supernatants enhanced mitogen-induced T cell proliferation using normal AM as AC significantly more than normal AM supernatants, suggesting that HIV AM secreted more T cell stimulatory factors than normal AM. Neither group could enhance T cell proliferation induced by HIV AM suggesting these cells already secreted optimal levels of these factors. AM from HIV+ individuals produced and secreted more IL-1 beta (measured by ELISA) and IL-6 (measured in a B9 bioassay and by immunoprecipitation) than normal AM both spontaneously and in the presence of low LPS concentrations and mitogens. In some cases depleting HIV AM supernatants of IL-1 beta and IL-6 on immunoaffinity columns abrogated their enhancement properties indicating that these cytokines were important in the observed enhancement. However, in other patients different factors must also be involved as depletion of IL-1 beta and IL-6 in their AM supernatants had no effect on enhancement function. These results show that HIV AM secretory products are important in the enhanced AC function demonstrated by these cells. However, although augmented IL-1 beta and IL-6 secretion likely contribute significantly to this enhancement, other AC secretory factors and/or functions must also be involved.

Interleukin 1 release by human alveolar macrophages and blood monocytes.

These studies utilized a sensitive and specific radioimmunoassay for interleukin 1 beta (IL-1 beta) to compare release of IL-1 by human alveolar macrophages and blood monocytes. The studies demonstrate that alveolar macrophages release amounts of antigenic IL-1 beta that are similar to that of blood monocytes. The amounts of IL-1 released were similar for both cell types at early (4 h) and late (24 h) time points and with differing amounts of stimuli [endotoxin; lipopolysaccharide (LPS)]. In addition, alveolar macrophages actually produced more total IL-1 (intracellular IL-1 plus released IL-1) than did blood monocytes. Alveolar macrophages that were stimulated with LPS released significantly more prostaglandin E2 (PGE2, an inhibitor of IL-1) than did blood monocytes. These studies demonstrate that human alveolar macrophages are not defective in their capacity to release IL-1.

Cigarette smoking decreases interleukin 1 release by human alveolar macrophages.

To determine whether alveolar macrophages from smokers have an abnormal interleukin 1 beta (IL-1) release, we obtained macrophages by bronchoalveolar lavage (BAL) of otherwise healthy volunteers in three groups: nonsmokers (NS; n = 11), light smokers (LS, less than 10 pack-yr smoking history; n = 4) and heavy smokers (HS, greater than 10 pack-yr smoking history; n = 9). After 24 h in culture, unstimulated macrophages (from each group) released negligible amounts of IL-1. Lipopolysaccharide (LPS) (1 micrograms/ml) caused release of 21.77 +/- 4.33 ng IL-1/10(6) cells at 24 h from NS macrophages; IL-1 release from HS macrophages was significantly decreased (5.52 +/- 1.66 ng/10(6) cells; P less than 0.05), whereas LS macrophages released intermediate amounts (15.07 +/- 6.15 ng/10(6) cells). Release of IL-1 from HS macrophages was also decreased after 48 and 72 h in culture and was observed over a wide range of concentrations of LPS. The decreased amount of IL-1 in HS macrophage supernatants appeared to be due to a defect in release of IL-1 from the cells and not due to a defect in production of the mediator, since total IL-1 (IL-1 present in the cell lysates plus that in the cell supernatants) was similar in the NS and HS groups. In addition, after 24 h in culture, LPS-stimulated HS macrophages released significantly less prostaglandin E2 (PGE2) (which can suppress IL-1 production) than did NS macrophages; in the presence of indomethacin, which abolished macrophage PGE2 release, no augmentation of LPS-stimulated IL-1 release was observed. Cell viability, as measured by lactate dehydrogenase release, was not different between HS and NS macrophages under any conditions. We conclude that there is a defect in release but not production of IL-1 from the alveolar macrophages of chronic smokers.

Production of interferon-gamma by lung lymphocytes in HIV-infected individuals.

A CD8(+) lymphocytic alveolitis occurs in up to 60% of asymptomatic human immunodeficiency virus (HIV)-infected individuals. Early in HIV infection, lymphocytes consist predominantly of cytotoxic T lymphocytes directed against HIV-infected targets. As HIV disease progresses, they are replaced by CD8(+)CD57(+) suppressor cells. Virus-specific cytotoxic T lymphocytes secrete interferon-gamma (IFN-gamma), an important cytokine in upregulating immune responses, primarily through macrophage activation. We examined the ability of lung and blood lymphocytes from HIV-positive patients at various stages of HIV infection to secrete IFN-gamma spontaneously and in response to phytohemagglutinin A. IFN-gamma production and secretion were determined with ELISA, Western blot, immunoprecipitation, and Northern blot techniques. Lung lymphocytes from HIV-infected individuals secreted large amounts of IFN-gamma. However, this ability was lost in patients with late-stage disease. Correlation between blood and lung lymphocyte IFN-gamma secretion was poor, suggesting regional differences in lymphocyte function. These data suggest that lung levels of IFN-gamma are high until late in HIV disease. These findings support the concept of administering exogenous IFN-gamma to patients with late-stage HIV disease and opportunistic infections.