The alpha chemokine, interleukin 8, inhibits the antiviral action of interferon alpha.

Interferon (IFN) exhibits a potent antiviral activity in vitro and plays a major role in the early defense against viruses. Like IFN, the proinflammatory chemokine, interleukin (IL)-8, is induced by viruses and appears in circulation during viral infections. In an in vitro cytopathic effect assay for IFN, we found that IL-8 can inhibit IFN-alpha activity in a dose-dependent manner. This action was reversed by specific monoclonal antibodies to IL-8. The chemokine was able to attenuate the IFN-mediated inhibition of viral replication as determined by measuring infectious virus yield. IL-8 also diminished the ability of IFN to inhibit an early stage of viral replication since IL-8 attenuated the inhibition of the formation of viral proteins. It appeared that IL-8 interfered with a late rather than an early step of IFN-mediated pathway such as early gene expression. The IL-8 inhibitory action on IFN-alpha antiviral activity was associated with reduced 2',5'-A oligoadenylate synthetase activity, a pathway well correlative with the anti- encephalomyocarditis virus action of IFN-alpha. Understanding pathways that antagonize IFN action may lead to novel approaches to potentiate endogenous and therapeutic IFN.

ARED: human AU-rich element-containing mRNA database reveals an unexpectedly diverse functional repertoire of encoded proteins.

The adenylate uridylate-rich elements (AREs) mediate the rapid turnover of mRNAs encoding proteins that regulate cellular growth and body response to exogenous agents such as microbes, inflammatory and environmental stimuli. However, the full repertoire of ARE-containing mRNAs is unknown. Here, we explore the distribution of AREs in human mRNA sequences. Computational derivation of a 13-bp ARE pattern was performed using multiple expectation maximization for motif elicitations (MEME) and consensus analyses. This pattern was statistically validated for the specificity towards the 3'-untranslated region and not coding region. The computationally derived ARE pattern is the basis of a database which contains non-redundant full-length ARE-mRNAs. The ARE-mRNA database (ARED; http://rc.kfshrc.edu.sa/ared) reveals that ARE-mRNAs encode a wide repertoire of functionally diverse proteins that belong to different biological processes and are important in several disease states. Cluster analysis was performed using the ARE sequences to demonstrate potential relationships between the type and number of ARE motifs, and the functional characteristics of the proteins.

Potential involvement of IL-8 in the pathogenesis of human cytomegalovirus infection.

The observations that several types of viruses induced interleukin (IL)-8 production prompted us to investigate the interrelationship between IL-8 and cytomegalovirus (CMV) infection. CMV infection caused IL-8 production in a human monocytic cell line, THP-1, in dose- and time-dependent manners. Moreover, CMV induced IL-8 gene expression by concurrently activating transcription factors, NF-kappaB and AP-1. Furthermore, CMV infection of human fibroblast cell lines increased gene expression of a specific receptor for IL-8, CXCR1. IL-8 in turn enhanced CMV replication in a human embryonic fibroblast, MRC-5, in dose- and time-dependent manners. Augmented replication eventually culminated in the increased production of infectious CMV virions. Moreover, IL-8 can attenuate the antiviral activity of interferon (IFN), particularly that of alpha-type against picornaviruses such as encephalomyocarditis virus and poliovirus. The inhibitory effects were associated with reduced 2',5'-A oligoadenylate synthetase activity. These results would imply that CMV can induce IL-8, which can augment CMV replication directly and indirectly by counteracting antiviral activity of IFN.

Type I interferons (IFN-alpha and -beta) suppress cytotoxin (tumor necrosis factor-alpha and lymphotoxin) production by mitogen-stimulated human peripheral blood mononuclear cell.

We studied the effect of the different types of interferons on the production of cytotoxin by human peripheral blood mononuclear cells (PBMCs) stimulated with the mitogen phytohemagglutinin (PHA). Maximum secreted levels of cytotoxin were observed at day 3 in culture and consisted of both tumor necrosis factor alpha (TNF-alpha) and lymphotoxin as determined by specific antibodies. Type I interferons (IFN-alpha and IFN-beta) consistently suppressed cytotoxin production. Both TNF-alpha and lymphotoxin were significantly suppressed. Mean suppression by IFN-alpha and IFN-beta (1000 U/ml) was 56 and 66%, respectively, in PBMCs from 18 different donors. The suppressive effects of IFN-alpha and IFN-beta on cytotoxin production were dose responsive over a range of 10 to 1000 U/ml. Type II interferon (IFN-gamma) did not have consistent significant effects. Pretreatment with IFN-alpha or IFN-beta for 24 or 48 h prior to PHA stimulation also resulted in significant suppression. Supplementation with interleukin-2 (10 U/ml) or IFN-gamma (1000 U/ml) did not overcome cytotoxin suppression by IFN-alpha or IFN-beta. Cytotoxin suppression by IFN-alpha and IFN-beta together appeared to be noninteractive. Suppression appeared not to be due to blockade of the cytotoxin release, since both cell-associated cytotoxin and secreted cytotoxin were suppressed to the same level. These results demonstrated that cytotoxin and lymphotoxin production by PHA-stimulated PBMCs could be down-regulated by type I interferons and that there is a substantial difference between the action of type I interferons and type II interferons (IFN-gamma) in modulating the biosynthesis of cytotoxins.

MTS interferon assay: a simplified cellular dehydrogenase assay for interferon activity using a water-soluble tetrazolium salt.

MTS, a tetrazolium dye, is reduced by hydrogenases in living cells to a water-soluble formazan. When it is added to the medium at the end of a cytopathic effects (CPE) inhibition interferon assay, the formazan formed diffuses into the medium; the resultant optical density directly and quantitatively measures how much cellular damage has been produced by the challenge virus in the presence of different amounts of interferon. The use of MTS has considerable advantages in that after it is added, no further steps, such as washing of the cells, extraction of dye, or other manipulations, are needed.

Effect of deficiency of the double-stranded RNA-dependent protein kinase, PKR, on antiviral resistance in the presence or absence of ribonuclease L: HSV-1 replication is particularly sensitive to deficiency of the major IFN-mediated enzymes.

Control of viral replication by interferon (IFN) is thought to be principally mediated by the 2',5'-oligoadenylate synthetase (OAS)/RNAse L, double-stranded dependent protein kinase (PKR), and myxovirus resistance protein (Mx) pathways. In this study, we monitored the constitutive and IFN-induced antiviral activity in mouse embryo fibroblasts lines derived from mice with targeted disruption of either PKR or PKR/RNAse L genes. At high multiplicity of infection (moi = 10), the absence of PKR had no effect on replication of vesicular stomatitis virus (VSV) but moderately enhanced encephalomyocarditis virus (EMCV) growth and greatly increased replication of herpes simplex virus-1 (HSV-1). Replication of EMCV, HSV-1, and VSV was modestly higher in PKR-/- RNAse L-/- fibroblasts when compared with control cells. Although the antiviral action of IFN-alpha was unaffected by the absence of PKR, IFN action was significantly impaired in the double knockout cells but was dependent on the stage of the virus cycle. At early stages, it appeared that anti-EMCV and anti-HSV-1 action of IFN-alpha was significantly compromised, although weak residual antiviral activity was seen. The action of IFN-alpha against VSV was specifically compromised at a late stage of virus replication. The results showed that PKR is an important mediator in constitutive resistance against HSV-1 and that RNAse L is also necessary for the full antiviral activity of IFN against a variety of viruses. These results supported the existence of novel pathways aimed toward specific stages of the virus life cycle.

WEHI-13VAR: a stable and sensitive variant of WEHI 164 clone 13 fibrosarcoma for tumor necrosis factor bioassay.

Bioassaying tumor necrosis factor (TNF) relies not only on a sensitive but also on a stable cell line towards TNF action. A variant of WEHI 164 clone 13, WEHI-13VAR, was characterized by us. This variant demonstrated both stability and high sensitivity towards rTNF-alpha or rTNF-beta when assayed in the presence of actinomycin-D (AcD). Fifteen subclones were generated from WEHI-13VAR (rTNF-alpha; ED50 range: 0.005-0.065 ng/ml). No clones were found more sensitive than the AcD-treated WEHI-13VAR cell line. An important feature of this line is its stable sensitivity. The stability of the cell line sensitivity towards rTNF-beta was demonstrated in the presence of AcD over a period of 16 months with an average ED50 of 0.046 +/- 0.004 ng/ml. In conclusion, unlike other TNF-sensitive cell lines in which the sensitivity is lost or the stability is unknown, WEHI-13VAR provides a sensitive, reliable and stable bioassay system to detect cytotoxin, TNF-alpha, and TNF-beta.

The RNA-binding zinc-finger protein tristetraprolin regulates AU-rich mRNAs involved in breast cancer-related processes.

Tristetraprolin (TTP or ZFP36) is a tandem CCCH zinc-finger RNA-binding protein that regulates the stability of certain AU-rich element (ARE) mRNAs. Recent work suggests that TTP is deficient in cancer cells when compared with normal cell types. In this study we found that TTP expression was lower in invasive breast cancer cells (MDAMB231) compared with normal breast cell lines MCF12A and MCF-10. TTP targets were probed using a novel approach by expressing the C124R zinc-finger TTP mutant that functions as dominant negative and increases target mRNA expression. In contrast to wild-type TTP, C124R TTP was able to increase certain ARE-mRNA expressions in serum-stimulated breast cancer cells. Using an ARE-gene microarray, novel targets of TTP regulation were identified, namely, urokinase plasminogen activator (uPA), uPA receptor and matrix metalloproteinase-1, all known to have prominent roles in breast cancer invasion and metastasis. Expression of these targets was upregulated in tumorigenic types, particularly in highly invasive MDAMB231. The mRNA half-lives of these TTP-regulated genes were increased in TTP-knockout embryonic mouse fibroblasts, as assessed using real-time polymerase chain reaction, whereas forced restoration of TTP by transfection led to a reduction in their mRNA levels. RNA immunoprecipitation confirmed an association of TTP, but not C124R, with these target transcripts. Moreover, TTP reduced, whereas the mutant C124R TTP increased, the activity of reporter constructs fused to target ARE. As a result of TTP regulation, invasiveness of MDAMB231 cells was reduced. The data suggest that TTP, in a 3' untranslated region-and ARE-dependent manner, regulates an important subset of cancer-related genes that are involved in cellular growth, invasion and metastasis.

RNase L downmodulation of the RNA-binding protein, HuR, and cellular growth.

Ribonuclease L (RNase L) is an intracellular enzyme that is vital in innate immunity, but also is a tumor suppressor candidate. Here, we show that overexpression of RNase L decreases cellular growth and downmodulates the RNA-binding protein, HuR, a regulator of cell-cycle progression and tumorigenesis. The effect is temporal, occurring in specific cell-cycle phases and correlated with the cytoplasmic localization of RNase L. Both cellular growth and HuR were increased in RNASEL-null mouse fibroblast lines when compared to wild-type cells. Moreover, the stability of HuR mRNA was enhanced in RNASEL-null cells. The HuR 3' untranslated region (UTR), which harbors U-rich and adenylate-uridylate-rich elements, was potently responsive to RNase L when compared to control 3' UTR. Our results may offer a new explanation to the tumor suppressor function of RNase L.

Elevated levels of interleukin-8 in serum are associated with hepatitis C virus infection and resistance to interferon therapy.

Hepatitis C virus (HCV), a major cause of liver disease worldwide, is frequently resistant to the antiviral alpha interferon (IFN). We have recently found that the HCV NS5A protein induces expression of the proinflammatory chemokine IL-8 to partially inhibit the antiviral actions of IFN in vitro. To extend these observations, in the present study we examined the relationship between levels of IL-8 in serum, HCV infection, and biochemical response to IFN therapy. Levels of IL-8 were significantly elevated in 132 HCV-infected patients compared to levels in 32 normal healthy subjects and were also significantly higher in patients who did not respond to IFN therapy than in patients who did respond to therapy. This study suggests that HCV-induced changes in levels of chemokine and cytokine expression may be involved in HCV antiviral resistance, persistence, and pathogenesis.

Analysis and examination of cytokine interactions by the median-effect model: an example with antiviral action of tumor necrosis factor and interferon-gamma.

A mathematical model based on the median effect principle was used to demonstrate its usefulness in studying cytokine interactions. The inhibition of cytopathological effects due to encephalomyocarditis virus (EMCV) by interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF) in WISH cells using a microtiter assay was employed. The model was used to analyze the characteristics of the dose-effect relationships of IFN-gamma, TNF, and their combination. The method also was used to define synergism as opposed to additive interaction. This application of the median-effect model proved to be easy and accurate and could be useful in the study of interactions in cytokine research.

Nigella sativa: effect on human lymphocytes and polymorphonuclear leukocyte phagocytic activity.

The effects of Nigella sativa (N. sativa) seeds and their soluble fractions were studied in vitro on lymphocyte response to different mitogens and on polymorphonuclear leukocyte phagocytic activity. No stimulatory effect of N. sativa was detected on lymphocyte response to phytohemagglutinin, concanavalin-A or pokeweed mitogen. A stimulatory effect of N. sativa was noticed on the lymphocyte response to pooled allogeneic cells. This effect was more pronounced when the low molecular weight (< 10 kDa) fraction was used and varied from one normal individual to another (25% to 825%). N. sativa enhanced the production of interleukin-3 by human lymphocytes when cultured with pooled allogeneic cells or without any added stimulator. N. sativa did not, however, enhance or suppress interleukin-2 secretion by mitogen activated peripheral blood mononuclear cells. Interestingly, N. sativa increased interleukin-1 beta, suggesting therefore, that it has an effect on macrophages. It also suppressed the leukocyte chemiluminescence activity using phorbol myristate acetate and Zymosan as stimulants. No effect of N. sativa or its fractions was, however, noticed on bacterial phagocytosis or killing when Staphylococcus aureus was used, indicating that the decrease in chemiluminescence activity in the presence of N. sativa is not relevant to the bactericidal activity.

Human cytomegalovirus induces interleukin-8 production by a human monocytic cell line, THP-1, through acting concurrently on AP-1- and NF-kappaB-binding sites of the interleukin-8 gene.

Cytomegalovirus (CMV) infection induced interleukin-8 (IL-8) gene transcription in a human monocytic cell line, THP-1 cells, leading to IL-8 secretion. The functional analysis of the IL-8 gene revealed that both AP-1- and NF-kappaB factor-binding elements were involved in conferring the responsiveness to CMV. Moreover, electrophoretic mobility shift assays demonstrated that CMV induced the formation of NF-kappaB and AP-1 complexes. These results suggest that CMV activates these transcriptional factors, resulting in IL-8 gene expression.

Circulating endotoxin and cytokines after cardiopulmonary bypass: differential correlation with duration of bypass and systemic inflammatory response/multiple organ dysfunction syndromes.

Cardiopulmonary bypass constitutes an injury that may cause postoperative pathophysiological changes due to systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS). These complications include coagulopathy, hypotension, capillary leakage, and multiple organ injury. To investigate the role of endotoxin and cytokines in the response to bypass injury, we measured plasma levels of endotoxin and proinflammatory cytokines in 20 pediatric patients before and after bypass. Clinical data, including duration of injury and tests indicative of SIRS/MODS, were collected. Levels of endotoxin, TNF-alpha, IL-6, and IL-8 but not IL-1 beta were significantly increased after bypass. Most of the cytokines have been found to correlate with each other. Endotoxin did not correlate with duration of bypass, cytokines, or SIRS/MODS. In contrast, TNF-alpha and IL-8 correlated with duration of bypass and were associated with SIRS/MODS. Certain clinical complications were associated with specific cytokines. Understanding the role of cytokinemia in SIRS/MODS may lead to better prognostic assessment and therapeutic modalities.

A carbocyclic nucleoside analogue is a TNF-alpha inhibitor with immunosuppressive action: role of prostaglandin E2 and protein kinase C and comparison with pentoxifylline.

Tumor necrosis factor-alpha (TNF-alpha) is associated with several acute and chronic inflammatory conditions. New therapies directed at inhibiting TNF-alpha will be important in treating pathological processes mediated by TNF-alpha. In this study, we studied and compared the effect of the carbocyclic nucleoside analogue (9-[(1R, 3R)-trans-cyclopentan-3-ol] adenine) with pentoxifylline on modulating TNF-alpha production. The carbocyclic nucleoside analogue inhibited TNF-alpha production in a dose-dependent manner (1 microM-1 mM) by stimulated peripheral blood mononuclear cells and cell lines of both monocyte (THP-1) and T-lymphocyte phenotypes (CEM x 174). The drug potently inhibited TNF production in cells stimulated by endotoxin, the superantigen (staphylococci enterotoxin A), the mitogen (phytohemagglutinin), and the protein kinase C activator (phorbol myristate acetate) with ED50 ranging from 5 to 30 microM. At moderate concentrations, the carbocyclic nucleoside analogue inhibited superantigen (ED50 = 300 microM) and alloantigen (mixed lymphocyte reaction) T cell proliferative responses (ED50 = 150 microM). The involvement of protein kinase C and prostaglandin E2 (PGE2), mediators that regulate TNF-alpha production, was also investigated. Unlike PTX, the nucleoside analogue did not upregulate PGE2 production. The inhibition of TNF-alpha production appeared to be mediated at least partly by PKC, since the nucleoside analogue caused suppression of PKC activity in stimulated cells. The results show that the carbocyclic nucleoside analogue is a TNF-alpha inhibitor that may be appropriate in the therapy of TNF-alpha-associated complications. The suppressive properties of the carbocyclic nucleoside analogue on antigen and alloantigen (mixed lymphocyte reaction) responses may be appropriate in disease conditions in which inhibiting both TNF-alpha and T-cell reactivity is desirable.

Hepatitis C virus nonstructural 5A protein induces interleukin-8, leading to partial inhibition of the interferon-induced antiviral response.

Hepatitis C virus (HCV), a major cause of liver disease worldwide, is frequently resistant to the antiviral alpha interferon (IFN). The HCV nonstructural 5A (NS5A) protein has been implicated in HCV antiviral resistance in many studies. NS5A antagonizes the IFN antiviral response in vitro, and one mechanism is via inhibition of a key IFN-induced enzyme, the double-stranded-RNA-activated protein kinase (PKR). In the present study we determined if NS5A uses other strategies to subvert the IFN system. Expression of full-length NS5A proteins from patients who exhibited a complete response (FL-NS5A-CR) or were nonresponsive (FL-NS5A-NR) to IFN therapy in HeLa cells had no effect on IFN induction of IFN-stimulated gene factor 3 (ISGF-3). Expression of mutant NS5A proteins lacking 110 (NS5A-DeltaN110), 222 (NS5A-DeltaN222), and 334 amino-terminal amino acids and mutants lacking 117 and 230 carboxy-terminal amino acids also had no effect on ISGF-3 induction by IFN. Expression of FL-NS5A-CR and FL-NS5A-NR did not affect IFN-induced STAT-1 tyrosine phosphorylation or upregulation of PKR and major histocompatibility complex class I antigens. However, NS5A expression in human cells induced interleukin 8 (IL-8) mRNA and protein, and this effect correlated with inhibition of the antiviral effects of IFN in an in vitro bioassay. NS5A induced transcription of a reporter gene driven by the IL-8 promoter, and the first 133 bp of the IL-8 promoter made up the minimal domain required for NS5A transactivation. NS5A-DeltaN110 and NS5A-DeltaN222 stimulated the IL-8 promoter to higher levels than did the full-length NS5A protein, and this correlated with increased nuclear localization of the proteins. Additional mutagenesis of the IL-8 promoter suggested that NF-kappaB and AP-1 were important in NS5A-DeltaN222 transactivation in the presence of tumor necrosis factor alpha and that NF-IL-6 was inhibitory to this process. This study suggests that NS5A inhibits the antiviral actions of IFN by at least two mechanisms and provides the first evidence for a biological effect of the transcriptional activity of the NS5A protein. During HCV infection, viral proteins may induce chemokines that contribute to HCV antiviral resistance and pathogenesis.

Interleukin-8 selectively enhances cytopathic effect (CPE) induced by positive-strand RNA viruses in the human WISH cell line.

Interleukin-8 (IL-8), a proinflammatory chemokine, is induced by viruses and appears in circulation during viral infections. We found that IL-8 enhanced cytopathic effect induced by the positive strand RNA virus, encephalomyocarditis virus (EMCV), in the human WISH cell line. The enhancement was dependent on IL-8 dose and virus dose and was reversible by specific monoclonal antibodies to IL-8. The chemokine was also able to increase EMC viral RNA synthesis and infectious virus yield. This IL-8 enhancing action was not observed in the case of the negative strand RNA virus, vesicular stomatitis virus (VSV), in WISH cells. We examined the activity of constitutive 2',5'-oligoadenylate synthetase (OAS), a pathway that was implicated in protection from EMCV but not VSV. The IL-8 action in EMCV-infected cells, unlike VSV-infected cells, was associated with decreased OAS activity in a manner that was independent of OAS gene expression. Understanding mechanisms of cytokine enhancement of viral activity may lead to novel ways to control viral infections.

The immediate early gene 1 product of human cytomegalovirus is sufficient for up-regulation of interleukin-8 gene expression.

We previously observed that human cytomegalovirus (CMV) infection induced a massive production of a chemokine with potent neutrophil chemotactic activity, interleukin-8 (IL-8). Hence, we examined the effect of CMV immediate early (IE) gene products on IL-8 production by the human astrocytoma cell line, U373MG. Transient or stable transfection with a CMV IE1 gene expression vector, but not with a IE2 gene expression vector, significantly augmented IL-8 protein secretion and IL-8 mRNA expression. Luciferase activity was enhanced in U373MG cells when the cells were cotransfected with CMV IE1 and chimeric firefly luciferase reporter genes driven by the transcriptional regulatory region of the human IL-8 gene. Moreover, IE1 gene-mediated enhancement of luciferase activity was abolished by the introduction of mutations into the AP-1 or NF-kappa B factor binding elements in the regulatory region of the IL-8 promoter. Furthermore, electrophoretic mobility shift assays demonstrated that CMV IE1 gene products induced the formation of NF-kappa B or AP-1 complexes. Finally, Western blotting analysis demonstrated that the CMV IE1 gene product increased the amount of NF-kappa B complexes translocated into the nucleus. Collectively, CMV IE1 gene expression may be sufficient to activate AP-1 and NF-kappa B, resulting in IL-8 gene expression.