Structural analysis of the sequence coding for an inducible 26-kDa protein in human fibroblasts

When human fibroblast cells were stimulated with poly(I) X poly(C) in the presence of cycloheximide for the production of interferon-beta (IFN-beta), a 26-kDa protein could be immunoprecipitated by antiserum raised against partially purified human IFN-beta [Content, J., De Wit, L., Pierard, D., Derynck, R., De Clercq, E. & Fiers, W. (1982) Proc. Natl Acad. Sci. USA 79, 2768-2772]. In our hands this 26-kDa protein showed no antiviral activity. Other investigators have, however, reported the presence in the same conditions of a second type of IFN, a so-called beta 2 species [Weissenbach, J., Chernajovsky, Y., Zeevi, M., Shulman, L., Soreq, H., Nir, U., Wallach, D., Perricaudet, M., Tiollais, P. & Revel, M. (1980) Proc. Natl Acad. Sci. USA 77, 7152-7156] of which the mRNA structure and protein characteristics strongly suggests identity with the 26-kDa product. In this paper we describe the nucleotide sequence of the 26-kDa cDNA and part of the corresponding genomic clone. The cDNA clones were isolated from a library made with mRNA from induced human fibroblasts. As, however, the information thus obtained was still incomplete, genomic clones were isolated from a total human DNA library. In this way, the entire region coding for the 26-kDa protein was established, as well as the neighbouring sequences including the inducible promoter area. From the deduced polypeptide sequence a number of characteristics of the 26-kDa protein can be explained. It turns out that the 26-kDa protein gene and the so-called 'IFN-beta 2' gene are identical. However, extensive homology searches indicate that the 26-kDa protein does not show statistically significant sequence homology with any known interferon species. Hence, the question of whether the 26-kDa product represents a novel IFN species remains open.     

Induction of HLA class I mRNA by cytokines in human fibroblasts: comparison of TNF, IL-1 and IFN-beta.

Expression of HLA class I antigens is known to be regulated by various cytokines at both the mRNA and protein levels. We have examined the induction of HLA-B7 by tumor necrosis factor alpha (TNF), interleukin 1 alpha (IL-1) and interferon beta (IFN-beta) in normal human diploid FS-4 fibroblasts. Optimal induction of HLA-B7 by TNF at 24 h was shown to require a continuous presence of TNF. Since TNF also induces IFN-beta in these cells and the latter cytokine itself has the capacity to upregulate HLA class I expression, we investigated the role of autocrine IFN-beta in the induction of HLA-B7 by TNF. Experiments with neutralizing polyclonal antibodies to recombinant IFN-beta showed that the induction of HLA-B7 mRNA by TNF was partially dependent on autocrine IFN-beta. However, TNF and IFN-beta induced HLA-B7 mRNA with similar kinetics and treatment with saturating concentrations of both TNF and IFN-beta resulted in an additive or possibly synergistic response. The latter findings support the idea that induction of HLA class I by TNF is not mediated solely by autocrine IFN-beta produced in response to TNF. In addition, experiments with the protein synthesis inhibitor cycloheximide suggested that the induction of mRNAs for both the heavy and light (beta 2-microglobulin) chains of the HLA class I antigen by TNF did not require de novo protein synthesis. IL-1 was also shown to increase steady-state mRNA levels of HLA-B7 with kinetics similar to those of TNF and IFN-beta in FS-4 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

A possible role of autogenous IFN-beta for cytokine productions in human fibroblasts

It has been already known that human diploid fibroblasts are able to produce not only high levels of IFN-beta but also various kinds of cytokines by poly rI: poly rC, and some inflammatory cytokines are induced by IFN-beta gene activation. We also obtained similar results. However, in our system, cytokine productions were extremely enhanced by treating the cells with a low dose of type 1 IFN and the priming effects on cytokine productions were blocked by cycloheximide similar to those on IFN-beta productions. Most of cytokines were produced later than IFN-beta and synthesis patterns of their mRNA showed the same phenomena. We made clear that cytokine productions by poly rI: poly rC are mediated by secreted IFN-beta at a protein level using a monoclonal antibody against human IFN-beta. Further, it was shown that intra-cellular IFN-beta which is not secreted might also participate in cytokine productions. Meanwhile, IL-1beta induced various kinds of cytokines in human fibroblasts and production time courses of these cytokines were similar to those of poly rI: poly rC induced cytokines. Although secreted IFN-beta was not detected in IL-1beta stimulated culture, expression of IFN-beta mRNA was augmented. These results showed that priming effects of type 1 IFN on cytokine productions by poly rI: poly rC might not be the direct action, but successive IFN-beta production might be essential in the production processes of other cytokines. Further, it was suggested that inducible IFN-beta might also take part in IL-1beta-induced cytokine productions.     

Cycloheximide induces expression of the human interferon beta 1 gene in mouse cells transformed by bovine papillomavirus-interferon beta 1 recombinants.

Mouse cells transformed by a bovine papillomavirus recombinant vector containing the human interferon (IFN) beta 1 (IFN-beta 1) gene could be induced to produce human as well as mouse IFNs. The optimal conditions for induction of human IFN and of its mRNA in these transformants resembled those needed for mouse IFN: high concentrations of DEAE-dextran and low concentrations of polyriboinosinic acid-polyribocytidylic acid. Superinduction by inhibitors of protein synthesis which strongly stimulate IFN-beta 1 induction in human cells had only a small effect on human IFN induction in bovine papillomavirus IFN-beta 1-transformed mouse cells. In contrast, cycloheximide without double-stranded RNA could induce significant levels of human IFN in the bovine papillomavirus IFN-beta 1 mouse transformants. After cycloheximide treatment, these cells contained IFN-beta 1 mRNA whose 5' ends originated in the authentic start site of the human IFN-beta 1 gene, as shown by S1 nuclease mapping. The transferred human gene, propagated extrachromosomally in the mouse cells, was, therefore, inducible under conditions different from those in human cells. The results also confirmed that the inhibitor of protein synthesis, cycloheximide, can induce expression of a human IFN gene.     

Critical role of a common transcription factor, IRF-1, in the regulation of IFN-beta and IFN-inducible genes.

Interferon regulatory factor 1 (IRF-1) is a protein that binds to cis-elements within the promoter of interferon (IFN)-beta and some IFN-inducible genes. We used a human fibroblast line, GM-637, to generate stable transfectants constitutively expressing IRF-1 mRNA in either the sense or antisense orientation. Upon induction with poly-(I).poly(C) or Newcastle disease virus, cells expressing sense IRF-1 mRNA produced significantly higher levels of IFN-beta mRNA and protein than control cells, whereas cells expressing antisense IRF-1 mRNA produced little or no IFN-beta mRNA and protein. Furthermore, clear differences were seen among the transfectants in the level of expression of two IFN-induced genes (2'-5'-oligoadenylate synthetase and class I HLA). Our data show that IRF-1 is essential for the induced expression of the IFN-beta gene. The results also indicate an important role of IRF-1 in the expression of IFN-inducible genes and suggest a role for IRF-1 in many other cytokine actions.     

Priming affects the activity of a specific region of the promoter of the human beta interferon gene.

Treatment of Daudi or HeLa cells with human interferon (IFN) alpha 8 before induction with either poly(I)-poly(C) or Sendai virus resulted in an 8- to 100-fold increase in IFN production. The extent of priming in Daudi cells paralleled the increase in the intracellular content of IFN-beta mRNA. IFN-alpha mRNA remained undetectable in poly(I)-poly(C)-treated Daudi cells either before or after priming. An IFN-resistant clone of Daudi cells was found to produce 4- to 20-fold more IFN after priming, indicating that priming was unrelated to the phenotype of IFN sensitivity. IFN treatment of either Daudi or HeLa cells transfected with the human IFN-beta promoter (-282 to -37) linked to the chloramphenicol acetyltransferase (CAT) gene resulted in an increase in CAT activity after induction with poly(I)-poly(C) or Sendai virus. A synthetic double-stranded oligonucleotide corresponding to an authentic 30-base-pair (bp) region of the human IFN-beta promoter between positions -91 and -62 was found to confer virus inducibility upon the reporter CAT gene in HeLa cells. IFN treatment of HeLa cells transfected with this 30-bp region of the IFN-beta promoter in either the correct or reversed orientation also increased CAT activity upon subsequent induction. IFN treatment alone had no detectable effect on the activity of either the 30-bp region or the complete human IFN promoter.     

The role of interferon-beta 1 and the 26-kDa protein (interferon-beta 2) as mediators of the antiviral effect of interleukin 1 and tumor necrosis factor

This study confirms our earlier finding that human interleukin (IL)-1 beta exerts an antiviral effect on diploid fibroblasts and on MG-63 osteosarcoma cells. It also extends the observation in that a similar effect was noted on aged but not freshly trypsinized HEp-2 cells, and that not only IL-1 beta but also IL-1 alpha and tumor necrosis factor (TNF)-alpha exerted similar antiviral effects on cells. The antiviral effects of these cytokines were neutralized by addition to the assay system of an antibody that was specific for interferon (IFN)-beta 1, indicating that IFN-beta 1 or a structurally or functionally related substance is involved in the antiviral activity observed. Both IL-1 and TNF were able to induce production of the 26-kDa protein, also known as IFN-beta 2, hybridoma/plasmacytoma growth factor (HPGF) or B-cell stimulatory factor-2 (BSF-2) and previously proposed as an alternative to IFN-beta 1 for mediating the antiviral effect of TNF. However, no good correlation was found between the antiviral effects of TNF and its potential to induce production of the 26-kDa protein. Furthermore, the anti-IFN-beta 1 serum which neutralized the antiviral activity of IL-1 and TNF did not cross-react with the 26-kDa protein. Conversely, the antiviral effect of IL-1 and TNF was only weakly neutralized by an antibody that did react with the 26-kDa protein and showed low cross-reactivity with IFN-beta 1. These observations, together with the low specific activity of the 26-kDa protein as an antiviral agent (less than 10(5) U/mg protein) provide strong arguments against this protein and in favor of IFN-beta 1 (or still another IFN-beta 1-related molecule) as the ultimate mediator of the antiviral effect of IL-1 and TNF.     

Bacterial lipopolysaccharide and gamma interferon induce transcription of beta interferon mRNA and interferon secretion in murine macrophages

Bacterial lipopolysaccharide (LPS) induces interferon (IFN) secretion and an antiviral state in murine peritoneal macrophages (PM). These cells secrete predominantly IFN-beta, as shown by neutralization assays with monoclonal antibodies. Secretion of IFN-beta is also induced in PM by IFN-gamma. LPS and IFN-gamma synergistically stimulated PM to produce IFN in amounts almost comparable to those induced by infection with Newcastle disease virus. Low levels of IFN-beta mRNA can be detected in freshly harvested PM by hybridization assays. The accumulation of this mRNA is markedly increased in PM treated with LPS or IFN-gamma, and it is further enhanced in the presence of the inhibitor of protein synthesis, cycloheximide. Similar studies were carried out on the RAW 264.7 line of transformed macrophages. These cells are induced to secrete IFN-beta by LPS but not by IFN-gamma, suggesting that this cytokine may elicit such specific response only in PM. IFN-beta mRNA is undetectable in untreated RAW 264.7 cells, and accumulation of this mRNA is induced by LPS but not by IFN-gamma. The secretion of IFN induced by these agents in PM and by LPS in RAW 264.7 cells and the corresponding accumulation of IFN-beta mRNA are blocked by an inhibitor of protein kinase C, staurosporine. The activity of this kinase is apparently necessary to stimulate accumulation of IFN-beta mRNA. The induction of IFN-beta by IFN-gamma appears to be a characteristic response of PM and may be at least in part responsible for the resistance of these cells to viral infections.     

A cytokine network in human diploid fibroblasts: interactions of beta-interferons, tumor necrosis factor, platelet-derived growth factor, and interleukin-1.

Earlier studies demonstrated the induction of beta 2-interferon (IFN-beta 2) in human diploid fibroblasts (FS-4 strain) exposed to tumor necrosis factor (TNF). These studies suggested that IFN-beta 2 mediates an antiviral effect in TNF-treated cells and exerts a feedback inhibition of the mitogenic effect of TNF. Here we demonstrate that the expression of the antiviral action of TNF can be enhanced by prior exposure of FS-4 cells to trace amounts of IFN-beta 1. IFN-beta 1, at a higher concentration, can directly increase the expression of IFN-beta 2. Exposure of cells to TNF enhanced IFN-beta 2 (but not IFN-beta 1) mRNA expression in response to poly(I).poly(C), an IFN inducer which is also known to stimulate FS-4 cell growth. Platelet-derived growth factor and interleukin-1 also led to the increased expression of IFN-beta 2. However, platelet-derived growth factor and interleukin-1 could override the antiviral effect of TNF and also that of exogenously added IFN-beta 1. Our data suggest that a complex network of interactions that involves the endogenous production of IFN-beta 2 is triggered by several growth-modulatory cytokines. Cellular homeostasis is likely to represent a balance between the induction of IFN-beta 2 by these cytokines and their ability to override the inhibitory actions of IFN-beta 2.     

Quantitative multiplex real-time PCR for the sensitive detection of interferon beta gene induction and viral suppression of interferon beta expression

Interferon-beta (IFN-beta) protein and activity can be detected by enzyme immunoassays and biological assays. However, precise quantification of low IFN-beta mRNA concentrations, which is advantageous for investigating IFN-beta gene expression in small tissue samples or during the early stage of a virus infection, remains a challenge. Therefore, we established a quantitative real-time PCR (qPCR) for IFN-beta and the housekeeping gene porphobilinogen deanimase (PBGD) in separated assays as well as in a multiplex procedure. Sensitivity for both the templates was less than 20 copies with an intra- and interassay variability of less than 5%. IFN-beta qPCR was utilized to optimize IFN-beta induction with dsRNA polyinosic-polycytidylic acid (poly I:C), delivered by a liposomal transfection agent for reproducible but low, non-cell-toxic IFN-beta concentrations. For studying coxsackievirus B3 (CVB3) interference with IFN-beta expression, CVB3 infected fibroblasts were induced with poly I:C. A significant reduction of IFN-beta mRNA but not PBGD mRNA was demonstrated 5 h after CVB3 infection, indicating a specific inhibition of IFN-beta expression by CVB3 on the mRNA level, in addition to previously reported effects on the translation/post-translation level. In conclusion, sensitive IFN-beta/PBGD multiplex qPCR proved to be a useful tool to study viral interaction with IFN-beta expression.     

TRAF6 and MEKK1 play a pivotal role in the RIG-I-like helicase antiviral pathway

Type I interferons (IFN-alpha/beta) are essential for immune defense against viruses and induced through the actions of the cytoplasmic helicases, RIG-I and MDA5, and their downstream adaptor molecule IPS-1. TRAF6 and the downstream kinase TAK1 have been shown to be essential for the production of proinflammatory cytokines through the TLR/MyD88/TRIF pathway. Although binding of TRAF6 with IPS-1 has been demonstrated, the role of the TRAF6 pathway in IFN-alpha/beta production has not been fully understood. Here, we demonstrate that TRAF6 is critical for IFN-alpha/beta induction in response to viral infection and intracellular double-stranded RNA, poly(I:C). Activation of NF-kappaB, JNK, and p38, but not IRF3, was impaired in TRAF6-deficient mouse embryo fibroblasts in response to vesicular stomatitis virus and poly(I:C). However, TAK1 was not required for IFN-beta induction in this process, since normal IFN-alpha/beta production was observed in TAK1-deficient mouse embryo fibroblasts. Instead, another MAP3K, MEKK1, was important for the activation of the IFN-beta promoter in response to poly(I:C). Forced expression of MEKK1 in combination with IRF3 was sufficient for the induction of IFN-beta, whereas suppression of MEKK1 expression by small interfering RNA inhibited the induction of IFN-beta by poly(I:C). These data suggest that IPS-1 requires TRAF6 and MEKK1 to activate NF-kappaB and mitogen-activated protein kinases that are critical for the optimal induction of type I interferons.     

Interferon-beta from melanoma cells suppresses the proliferations of melanoma cells in an autocrine manner

Interferon (IFN)-alpha and IFN-beta have been utilized in the treatment of melanoma as a form of cytokine therapy. While previous studies have demonstrated that melanocytes and melanoma cells produce a number of cytokines, it remains unclear whether or not melanocytes and melanoma cells per se produce IFN-alpha or IFN-beta. In the present study, we investigated the expression of IFN-alpha or IFN-beta in human melanocytes and five melanoma cell lines: G-361, C32TG, MMAc, MEWO and VMRC-MELG at both mRNA and protein levels. Both IFN-alpha and IFN-beta mRNA were detected in normal human melanocytes. Likewise, IFN-alpha mRNA was detected in all five melanoma cell lines. However, IFN-beta mRNA was only detected in one melanoma cell line, VMRC-MELG. When melanocytes and melanoma cells were treated with a potent IFN inducer, polyinosinic:polycytidylic acid (poly I:C), the mRNA expression of both IFN-alpha and IFN-beta was significantly upregulated. Poly I:C was not able to induce melanocytes or melanoma cells to produce detectable amounts of IFN-alpha protein, but able to induce a significant amount of IFN-beta in melanocytes and two of the melanoma cell lines: MMAc and VMRC-MELG. Moreover, similar to exogenous IFN-alpha and IFN-beta, poly I:C significantly inhibited the proliferation of all five melanoma cell lines. This suppressive effect was partially blocked by anti-IFN-beta antibody treatment in the IFN-beta-producing melanoma cell lines: MMAc and VMRC-MELG, but not in the non-IFN-beta-producing cell lines: G-361, C32TG and MEWO. Collectively, these studies have demonstrated for the first time that human melanocytes and melanoma cells produce IFN-beta. Furthermore, melanoma cells are capable of suppressing their own proliferation via secretion of endogenous IFN-beta. This finding may have important implications for melanoma therapy.     

Innate immunity in the human female reproductive tract: antiviral response of uterine epithelial cells to the TLR3 agonist poly(I:C)

The objective of this study was to examine the expression of TLR by human primary uterine epithelial cells (UEC) and to determine whether exposure to the TLR agonist poly(I:C) would induce an antiviral response. The secretion of several cytokines and chemokines was examined as well as the mRNA expression of human beta-defensin-1 and -2 (HBD1 and HBD2), IFN-beta, and the IFN-beta-stimulated genes myxovirus resistance gene 1 and 2',5' oligoadenylate synthetase. The expression of TLR1-9 by UEC was demonstrated by RT-PCR, with only TLR10 not expressed. Stimulation of UEC with the TLR3 agonist poly(I:C) induced the expression of the proinflammatory cytokines TNF-alpha, IL-6, GM-CSF, and G-CSF, as well as the chemokines CXCL8/IL-8, CCL2/MCP-1, and CCL4/MIP-1beta. In addition, poly(I:C) exposure induced the mRNA expression of HBD1 and HBD2 by 6- and 4-fold, respectively. Furthermore, upon exposure to poly(I:C) UEC initiated a potent antiviral response resulting in the induction of IFN-beta mRNA expression 70-fold and myxovirus resistance gene 1 and 2',5' oligoadenylate synthetase mRNA expression (107- and 96-fold), respectively. These results suggest that epithelial cells that line the uterine cavity are sensitive to viral infection and/or exposure to viral dsRNA released from killed epithelial cells. Not only do UEC release proinflammatory cytokines and chemokines that mediate the initiation of an inflammatory response and recruitment of immune cells to the site of infection, but they also express beta-defensins, IFN-beta, and IFN-beta-stimulated genes that can have a direct inhibiting effect on viral replication.