Interaction among hepatocyte-stimulating factors, interleukin 1, and glucocorticoids for regulation of acute phase plasma proteins in human hepatoma (HepG2) cells

Human hepatoma (HepG2) cells respond to unfractionated conditioned media of human squamous carcinoma (COLO-16) cells and lipopolysaccharide-stimulated human peripheral blood monocytes by increasing the synthesis of alpha 1-acid glycoprotein, haptoglobin, complement C3, alpha 1-antichymotrypsin, alpha 1-antitrypsin, and fibrinogen, while decreasing the synthesis of albumin. The regulation of the acute phase proteins is mediated by hepatocyte-stimulating factors (HSF) and interleukin 1 (IL-1) present in the conditioned medium. Purified HSF-I from COLO-16 cells stimulates preferentially alpha 1-acid glycoprotein synthesis, whereas COLO-HSF-II stimulates preferentially the synthesis of haptoglobin, fibrinogen, and alpha 1-antitrypsin. HSF from monocytes, which has been identified as interferon-beta 2 (B cell stimulating factor-2), displayed the same activity as COLO-HSF-II. Dexamethasone alone had no effect on acute phase plasma protein synthesis but enhanced the response to various HSF severalfold. IL-1 had a relatively low stimulatory activity on the synthesis of alpha 1-acid glycoprotein, haptoglobin, and alpha 1-antichymotrypsin but strongly reduced the basal expression of fibrinogen. The only synergistic action between IL-1 and HSF (or interferon-beta 2) was noted for the synthesis of alpha 1-acid glycoprotein. Tumor necrosis factor active on other hepatic cells failed to modulate significantly the expression of any plasma proteins in HepG2 cells. These studies showed that for an optimal HepG2-cell response a combination of HSF (or interferon-beta 2), IL-1, and dexamethasone is needed. This finding might indicate the identity of some of those hormones involved in regulation of the hepatic acute phase response in vivo.     

Herbal melanin modulates tumor necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6) and vascular endothelial growth factor (VEGF) production.

Recent studies have indicated that cytokines can enhance immunogenicity and promote tumor regression. However, the means for modulating cytokine production are not yet fully investigated. In this study we report the effects of a herbal melanin, extracted from Nigella sativa L., on the production of three cytokines [tumor necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6) and vascular endothelial growth factor (VEGF)], by human monocytes, total peripheral blood mononuclear cells (PBMC) and THP-1 cell line. Cells were treated with variable concentrations of melanin and the expression of TNF-alpha, IL-6 and VEGF mRNA in cell lysates and secretion of proteins in the supernatants were detected by RT-PCR and ELISA. Melanin induced TNF-alpha, IL-6 and VEGF mRNA expression by the monocytes, PBMC and THP-1 cell line. On the protein level, melanin significantly induced TNF-alpha and IL-6 protein production and inhibited VEGF production by monocytes and PBMC. In the THP-1 cell line melanin induced production of all three cytokine proteins. These observations raise the prospects of using N. sativa L. melanin for treatment of diseases associated with imbalanced cytokine production and for enhancing cancer and other immunotherapies.     

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.     

Limited effects of recombinant human and murine interleukin 1 and tumour necrosis factor on production of acute phase proteins by cultured rat hepatocytes

Albumin, fibrinogen, alpha 1-acid glycoprotein and cysteine proteinase inhibitor were determined by electroimmunoassay in the media of primary cultures of rat hepatocytes exposed to dialysed supernatants of rat, mouse and human macrophages or to recombinant human and murine interleukin 1 and tumour necrosis factor. Recombinant cytokines in the range of 1 to 1000 ng/ml caused only reduction of albumin synthesis and slight stimulation of alpha 1 acid glycoprotein production while crude preparations of macrophage cytokines elicited typical acute phase response. The results suggest that interleukin 1 or tumour necrosis factor are not likely the principal mediators responsible for the direct stimulation of normal rat hepatocytes to acute phase protein synthesis.     

Interleukin-6 is the major regulator of acute phase protein synthesis in adult human hepatocytes

The three monokines interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF alpha), and interleukin-6 (IL-6) modulate acute phase plasma protein synthesis in adult human hepatocytes. Only IL-6 stimulates the synthesis of the full spectrum of acute phase proteins as seen in inflammatory states in humans, i.e. synthesis and secretion of C-reactive protein, serum amyloid A, fibrinogen, alpha 1-antitrypsin, alpha 1-antichymotrypsin and haptoglobin are increased while albumin, transferrin and fibronectin are decreased. IL-1 beta as well as TNF alpha, although having a moderate effect on the positive acute phase proteins and inhibiting the synthesis of fibrinogen, albumin and transferrin, fail to induce serum amyloid A and C-reactive protein. These data suggest that IL-6 plays the key role in the regulation of acute phase protein synthesis in human hepatocytes.     

Exposure to cigarette smoke, a major risk factor for sudden infant death syndrome: effects of cigarette smoke on inflammatory responses to viral infection and bacterial toxins

Exposure to cigarette smoke is a major risk factor for sudden infant death syndrome and also for respiratory infections in children. It has been suggested that toxigenic bacteria colonizing the respiratory tract might play a role in some cases of sudden infant death syndrome and nicotine has been demonstrated to enhance the lethality of bacterial toxins in a model system. Pyrogenic toxins of Staphylococcus aureus have been identified in tissues of infants who died of sudden infant death syndrome. It has been suggested that some of these deaths were due to induction of inflammatory mediators by infectious agents during a period when infants are less able to control these responses. The aim of this study was to assess the effects of a water-soluble cigarette smoke extract on the production of tumor necrosis factor alpha and nitric oxide from human monocytes in response to staphylococcal toxic shock syndrome toxin 1 or infection of the monocytes with respiratory syncytial virus. Cell culture supernatants were examined by a bioassay using mouse fibroblasts (L-929 cell line) for tumor necrosis factor alpha activity and by a spectrophotometric method for nitrite. Compared with monocytes incubated with medium only, monocytes incubated with any of the factors or their combinations tested in the study released higher levels of tumor necrosis factor alpha and lower levels of nitric oxide. Incubation with cigarette smoke extract increased tumor necrosis factor alpha from respiratory syncytial virus-infected cells while it decreased tumor necrosis factor alpha from cells incubated with toxic shock syndrome toxin. Incubation with cigarette smoke extract decreased the nitric oxide production from respiratory syncytial virus-infected cells while it increased the nitric oxide production from cells incubated with toxic shock syndrome toxin. Monocytes from a minority of individuals demonstrated extreme tumor necrosis factor alpha responses and/or very high or very low nitric oxide. The proportion of samples in which extreme responses with a very high tumor necrosis factor alpha and very low nitric oxide were detected was increased in the presence of the three agents to 20% compared with 0% observed with toxic shock syndrome toxin 1 or 4% observed with cigarette smoke extract or respiratory syncytial virus.     

Modulation of cytokine production by monocytes and developing-dendritic cells under the influence of leukemia and lymphoma cell products

Cytokines and other soluble factors released by tumor cells play an important role in modulating immune cells to favor tumor development. Monocyte differentiation into macrophages or dendritic cells (DCs) with specific phenotypes is deeply affected by tumor signals and understanding this context is paramount to prevent and propose new therapeutic possibilities. Hence, we developed a study to better describe the modulatory effects of leukemia and lymphoma cell products on human monocytes and monocyte-derived DCs secretion of cytokines such as interleukin (IL)-1β, tumor necrosis factor-α (TNF-α), IL-6, and IL-12. Except with the promyelocytic leukemia cell supernatants (HL-60), the other two tumor supernatants (chronic myeloid leukemia, K562 and Burkitt lymphoma, DAUDI) increased both TNF-α and IL-1β production by monocytes and monocytes undergoing differentiation. This effect was neither explained by alterations of cell number in culture nor by the high amount of vascular endothelial growth factor (VEGF) present in the tumor supernatants. Moreover, all supernatants used were able to induce drastic reduction of IL-12 secretion by cells induced to activation, suggesting a negative interference with Th1 antitumoral responses that should be a huge advantage for tumor progression.     

Interleukin 6 and tumor necrosis factor fully activate liver-specific gene expression of the alpha chain of C4b-binding protein.

We investigated the gene expression of the alpha chain of C4b-binding protein (C4bp alpha) in a variety of tissues, and in liver cell and hepatoma lines. C4bp alpha mRNA was detected in the liver, but not in the other tissues examined. The constitutive gene expression of C4bp alpha by a hepatoma line, HepG2, was significantly augmented by treatment with monocyte-conditioned medium (MoCM), 12-O-tetradecanoylphorbol-13-acetate (TPA), interleukin-6 (IL6) and tumor necrosis factor (TNF) but not by a calcium ionophore (A23187) or interleukin-1 beta (IL1 beta).     

A stable long-term hepatocyte culture system for studies of physiologic processes: cytokine stimulation of the acute phase response in rat and human hepatocytes.

Prior studies on the in vitro hepatic acute phase response have involved either hepatoma cell lines or conventional short-term cultures of primary hepatocytes. No data are available on the response of primary hepatocytes in stable long-term culture systems. In this study, the acute phase response of rat and human hepatocytes in a new long-term culture system was examined in response to interleukin-6 (IL-6), interleukin-1 beta (IL-1 beta), and tumor necrosis factor alpha (TNF-alpha). The cultured cells were sandwiched between two layers of collagen in a (double-gel) configuration which has been shown to preserve both hepatocyte function and morphology over prolonged periods of time. The stability of this culture configuration enabled us to investigate, for the first time, the temporal aspects of the response in addition to the effects of the mediators on protein secretion. Exposure of rat hepatocytes to IL-6 after culture for 16 days resulted in a 2-fold reduction of albumin secretion and a 15-fold increase in the secretion rates of fibrinogen and alpha 2-macroglobulin. In all instances, the peak response occurred at 48 h after IL-6 exposure, and all protein secretion rates returned to pretreatment values within 5 days posttreatment. Changes in the mRNA levels of these proteins in response to IL-6 corresponded with those changes seen with the secreted products, indicating pretranslational regulation. Administration of IL-1 beta to rat hepatocyte produced a similar decline of albumin secretion and a 5-fold increase of fibrinogen secretion, whereas alpha 2-macroglobulin secretion remained undisturbed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential induction of interleukin-6 production in monocytes, endothelial cells and smooth muscle cells.

Studying the production of IL-6 (interleukin-6) by monocytes, endothelial cells and smooth muscle cells we observed that cytokine inducers like IL-1, TNF alpha (tumor necrosis factor alpha), LPS (lipopolysaccharide), SAC (Staphylococcus Aureus Cowan 1) and PMA could be divided roughly into two categories. Bacterial products such as LPS or SAC have a potent IL-6 inducing effect on monocytes and minor or no effect on endothelial- and smooth muscle cells. The other category comprising IL-1, TNF alpha and PMA induces IL-6 production in endothelial- and smooth muscle cells. Only IL-1 induces IL-6 production in monocytes as well as in endothelial cells and smooth muscle cells. In addition to IL-6, also IL-1 and TNF alpha are produced by monocytes however with different kinetics. None of the stimuli had any inhibitory effect on IL-6 production with the exception of PMA. Whereas PMA induced IL-6 production in endothelial cells and it potentiated the induction of IL-6 by IL-1 in these cells, it inhibited LPS-stimulated IL-6 production in monocytes. In line with the effects of PMA, staurosporin induced IL-6 production in monocytes and it inhibited IL-1 driven IL-6 production by endothelial cells.     

Ionizing radiation enhances IL-6 and IL-8 production by human endothelial cells

Irradiation exposure is known to induce an inflammatory reaction. Endothelial cells play a crucial role both in the inflammatory process and in radiation damage. Therefore, supernatants and cell lysates of (60)Co-irradiated human umbilical vein endothelial cells (HUVEC) have been assessed for the presence of pro-inflammatory cytokines. After gamma irradiation, interleukin (IL)-1alpha, IL-1beta and tumor necrosis factor (TNF)-alpha remained undetectable in both cell supernatants and cell lysates. However, a dose-dependent increase in the production of IL-6 and IL-8 has been demonstrated up to 6 days after exposure. These data indicate that the pro-inflammatory cytokines IL-6 and IL-8 may be involved in the inflammatory response of vascular endothelium induced by exposure to ionizing radiation.     

Heterogeneous nature of the acute phase response. Differential regulation of human serum amyloid A, C-reactive protein, and other acute phase proteins by cytokines in Hep 3B cells

Because a number of different cytokines have been reported to regulate the synthesis of human, murine, and rat acute phase proteins (APP), we studied the effect of cytokines on production of several major human APP in a single system, the human hepatoma cell line Hep 3B. Conditioned medium (CM) prepared from human blood monocytes activated with LPS in the presence of dexamethasone led to substantial induction of serum amyloid A (SAA) and C-reactive protein (CRP) synthesis whereas the defined cytokines IL-1 beta, TNF alpha, and medium from a human keratinocyte cell line (COLO-16), containing hepatocyte-stimulating factor activity, failed to induce these two major APP. Induction of SAA and CRP was accompanied by an increase in concentration of their specific mRNA. Size fractionation of CM from activated monocytes by fast protein liquid chromatography indicated that SAA- and CRP-inducing activity eluted as a single peak with a Mr of approximately 18 kDa. alpha 1-Antitrypsin, which also failed to respond to IL-1 beta or TNF alpha, was induced by both CM and medium from COLO-16 cells. The induction of AT by CM was accompanied by an increase in specific mRNA. Induction of ceruloplasmin and alpha 1-antichymotrypsin and decrease in the synthesis of albumin was achieved by both CM and IL-1 beta. Ceruloplasmin and albumin responded in a comparable fashion to both TNF alpha and medium from COLO-16 cells; the response of ACT to these cytokines was not evaluated. These results indicate that human SAA and CRP are induced in Hep 3B cells by products of activated monocytes but not by IL-1 beta, TNF-alpha, or some hepatocyte-stimulating factor preparations and that a group of heterogeneous mechanisms are involved in the induction of the various human APP.     

Induction of inflammatory cytokine release from cultured human monocytes by C-reactive protein.

The human acute phase protein, C-reactive protein (CRP), is capable of specifically binding to and modulating the function of mononuclear phagocytes. To investigate whether CRP can also affect the capacity of these cells to produce inflammatory cytokines, enzyme immunoassays and Western blot techniques were used to quantitate interleukin 1 beta (IL-1 beta), interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) produced by freshly-isolated normal human monocytes. CRP induced the rapid release of each cytokine, with significantly elevated levels in culture supernatants at 4 hours and maximal levels of TNF-alpha at 8 hours, and of IL-1 beta and IL-6 at 16 hours of culture. The effects of CRP were dose-dependent; greater than 10-fold increases of each cytokine were observed following culture with greater than or equal to 50 micrograms/ml CRP, concentrations which are often found in the presence of moderate to severe inflammation or tissue injury. The induction of cytokine release by CRP was unaffected by inclusion of 25 micrograms/ml polymyxin-B in culture media, but was completely abrogated by prior boiling of the CRP, a procedure which had no effect on induction of monocyte cytokine release by lipopolysaccharide. The dose-dependent induction of inflammatory cytokines by CRP provides further support for the hypothesis that interaction with mononuclear phagocytes constitutes an important biological role for this acute phase protein.     

Induction of human monocyte IL-1 mRNA and secretion during anti-CD3 mitogenesis requires two distinct T cell-derived signals.

The T cell signals that regulate the induction of human monocyte IL-1 during primary immune activation were investigated by using anti-CD3 mitogenesis. The induction of monocyte IL-1 alpha and beta mRNA during anti-CD3 mitogenesis was rapid (less than or equal to 1 h) and required the presence of both T cells and anti-CD3. The addition of T cells plus a nonmitogenic anti-CD5 antibody failed to induce IL-1 alpha or beta mRNA, indicating that IL-1 mRNA induction by anti-CD3 required T cell activation. Experiments using double chamber culture wells revealed that the major initial phase of IL-1 alpha and beta mRNA induction (1 to 12 h) required direct cell contact between monocytes and T cells. A subsequent minor late phase (greater than or equal to 12 h) of IL-1 mRNA was induced independently of cell contact in monocytes that received only soluble factors generated during anti-CD3 mitogenesis and was temporally associated with the appearance in culture supernatants of the late phase IL-1-inducing cytokines, IL-2, IFN-gamma, and TNF-alpha. Metabolic inactivation of T cells using paraformaldehyde demonstrated that the ability of T cells to induce IL-1 mRNA via cell contact was acquired only after activation of T cells via solid phase anti-CD3. Furthermore, pretreatment of T cells with the protein synthesis inhibitor emetine had no effect on T cell-mediated induction of monocyte IL-1 mRNA or cell-associated IL-1 alpha and beta, indicating that the expression of the IL-1 inductive signal did not require protein synthesis. Despite their ability to induce monocyte IL-1 alpha and beta mRNA, activated T cells treated with paraformaldehyde or emetine were no longer able to induce monocytes to secrete IL-1 beta into culture supernatants. However, supernatants from purified T cells that were activated with solid-phase anti-CD3 restored the ability of paraformaldehyde or emetine-treated T cells to induce IL-1 secretion. These studies provide evidence that supports a two-signal model of monocyte IL-1 production during primary immune activation. The first signal leads to the induction of monocyte IL-1 mRNA and is mediated by direct contact with activated T cells, and the second signal is provided by soluble T cell factors and results in IL-1 secretion.     

Amplification of prostaglandin I2 production by thapsigargin.

Rat liver cells (the C-9 cell line) are synergistically stimulated to produce prostaglandin I2 (PGI2) when incubated in the presence of thapsigargin and several recombinant human growth factors or tumor promoters, including basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), transforming growth factor-alpha (TGF-alpha), 12-O-tetradecanoylphorbol-13-acetate (TPA), teleocidin and aplysiatoxin, but not acidic fibroblast growth factor (aFGF). The production of PGI2 by exogenous arachidonic acid in the presence of thapsigargin is not amplified. The effects of varying levels of bFGF on PGI2 production in the presence of thapsigargin are biphasic: at low levels (0.05 nM) bFGF's effect is synergistic; at high levels (24 nM) it is not.     

Characterization of the amnion-derived AV3 cell line for use as a model for investigation of prostaglandin-cytokine interactions in human amnion

Increased production of prostaglandins and cytokines by amnion, particularly prostaglandin (PG) E2, interleukin (IL)-6 and IL-8, is thought to be an important event in infection-associated preterm labour. We characterized the amnion-derived AV3 cell line to determine its appropriateness as a model for investigation of the regulation of amnion cytokine and PG production. Amnion-derived AV3 cells were treated with tumour necrosis factor-alpha (TNF-alpha, interleukin-1beta (IL-1beta), epidermal growth factor (EGF) and phorbol 12-myristate 13-acetate (PMA) and IL-6, IL-8 and prostaglandin production was determined by immunoassay. Production of IL-6 and IL-8 rose dramatically with all treatments. PGE2, but not PGF2alpha or 6-keto-PGF1alpha, biosynthesis was also increased in a concentration-dependent manner with all treatments. A rapid increase in PGHS-2 (but not PGHS-1) mRNA expression was observed in response to TNF-alpha and IL-1beta. We conclude that the AV3 cell line inflammatory response profile is similar to those observed in primary amnion and other amnion-derived cell lines, and is an appropriate model for human amnion.     

Regulation of A + U-rich element-directed mRNA turnover involving reversible phosphorylation of AUF1

Proteins binding A + U-rich elements (AREs) contribute to the rapid cytoplasmic turnover of mRNAs containing these sequences. However, this process is a regulated event and may be accelerated or inhibited by myriad signal transduction systems. For example, monocyte adherence at sites of inflammation or tissue injury is associated with inhibition of ARE-directed mRNA decay, which contributes to rapid increases in cytokine and inflammatory mediator production. Here, we show that acute exposure of THP-1 monocytic leukemia cells to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate mimics several features of monocyte adherence, including rapid induction and stabilization of ARE-containing mRNAs encoding interleukin-1 beta and tumor necrosis factor alpha. Additionally, TPA treatment alters the activity of cytoplasmic complexes that bind AREs, including complexes containing the ARE-specific, mRNA-destabilizing factor, AUF1. Analyses of AUF1 from control and TPA-treated cells indicated that post-translational modifications of the major cytoplasmic isoform, p40AUF1, are altered concomitant with changes in RNA binding activity and stabilization of ARE-containing mRNAs. In particular, p40AUF1 recovered from polysomes was phosphorylated on Ser83 and Ser87 in untreated cells but lost these modifications following TPA treatment. We propose that selected signal transduction pathways may regulate ARE-directed mRNA turnover by reversible phosphorylation of polysome-associated p40AUF1.