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Multiple Control Elements Mediate Activation of the Murine and Human Interleukin 12 p40 Promoters: Evidence of Functional Synergy between C/EBP and Rel Proteins
Abstract
Interleukin 12 (IL-12) is a heterodimeric cytokine whose activity is critical for T-helper 1 responses. The gene for the IL-12 p40 subunit is expressed in macrophages following induction by bacterial products, and its expression is augmented by gamma interferon. In this study, we performed a functional analysis of the murine and human p40 promoters in the murine macrophage cell line RAW 264.7. Transcription from the murine p40 promoter was strongly induced by lipopolysaccharide and heat-killed Listeria monocytogenes (HKLM), but promoter activity was not enhanced by gamma interferon. Multiple cis-acting elements involved in activated transcription were identified through an extensive mutant analysis. The most critical element, whose activity is conserved in mice and humans, is located between positions -96 and -88 relative to the murine transcription start site. This element exhibits functional synergy with a previously described NF-kappaB half-site which interacts with Rel proteins. DNase I footprinting and electrophoretic mobility shift assays demonstrated that C/EBP proteins interact with the critical element, but in nuclear extracts, cooperative binding of C/EBP and Rel proteins to their respective sites was not observed. Interestingly, promoter activity was induced by HKLM in the presence of cycloheximide, consistent with induction by posttranslational mechanisms. The results suggest that C/EBP and Rel proteins play important roles in the activation of IL-12 p40 transcription by bacteria. However, many complex interactions will need to be clarified to fully understand p40 regulation.
... IL-12p40 promoter has the binding sites for NF-B and transcription factors involved in p38 or JNK pathways. Human and mouse IL-12p40 promoters have been well characterized with regulatory elements such as C/EBP , NF-B, AP-1, ISRE and TATA box (Murphy et al., 1995;Plevy et al., 1997;Wang et al., 2000;Zhu et al., 2001). However, the porcine IL-12p40 promoter has not been identified yet. ...
... As expected, the porcine IL-12p40 promoter has a high similarity with human and mouse promoters, and the putative binding motifs for NF-B (TTGAAATTCCCCC) and AP-1 (AGTCAG) exist. The function of NF-B on IL-12p40 regulation is first revealed in murine cells and confirmed by following studies (Murphy et al., 1995;Plevy et al., 1997). While the activation of NF-B by PRSSV CH-1a infection has been revealed before (Fu et al., 2012), we showed a classical activation of NF-B signaling pathway by HP-PRRSV infection (data not shown). ...
... Consistent with the role of JNK in IL-12p40 induction, AP-1 inhibitor also significantly reduced the expression of IL-12p40 after PRRSV infection, suggesting that PAMs are able to produce IL-12p40 triggered by PRRSV through JNK-AP-1 and NF-B signaling pathways. The transcription of IL-12p40 is regulated by multiple routes, and other transcription factors such as C/EBP  and IRFs may also be involved in the regulation of PRRSV-induced IL-12p40 production (Murphy et al., 1995;Plevy et al., 1997;Wang et al., 2000;Zhu et al., 2001). Although p38 MAPK positively regulated the expression of IL-12p40, we did not find any transcription factors controlled by p38 in the porcine IL-12p40 promoter. ...
... C/EBPβ is a bZIP (basic leucin zipper) transcription factor and belongs to a larger family of C/EBP proteins. C/EBP-binding motifs have been found in the promoters of various genes, including genes encoding the inflammatory cytokines IL-6, IL-1β, TNF-α, IL-8 and IL-12b [11][12][13][14][15][16]. Our previous work has shown that MCPIP1 decreases the half-life of mRNA coding for C/EBPβ in HepG2 cells, and that this effect requires the presence of PIN domain [17]. ...
... Transcriptionally active isoforms of C/EBPβ are generally regarded as the key regulators of IL-6 signalling while other MCPIP1 targets, including NF-κB, coordinate signalling of IL-1β. Both transcription factors must act synergistically to activate genes coding for IL-6, IL-8, and IL-12 [15,16]. Interestingly, the mRNA of these proteins is also targeted by MCPIP1 [4,5,18]. ...
CCAAT/enhancer-binding protein beta (C/EBPβ) is a transcription factor controlling a broad range of genes essential for homeostasis, including genes related to immune functions, inflammation, metabolism and growth. Monocyte chemoattractant protein-1-induced protein 1 (MCPIP1) also called as Regnase-1 is an RNase and has been shown to decrease the stability of short-lived transcripts coding for inflammation-related proteins, including IL-1β, IL-6, IL-2, IL-8, IL-12b, IER-3, c-Rel. We found previously that the half-life of the C/EBPβ transcript is regulated by MCPIP. To understand the mechanism driving down-regulation of C/EBPβ by MCPIP1, we applied an in vitro cleavage assay, followed by a luciferase-reporter assay and RNA immunoprecipitation (RIP). We demonstrated that MCPIP1 recognizes regions of the 3’UTR of C/EBPβ mRNA and promotes its decay by introducing direct endonucleolytic cleavage.
... Recent data, however, suggest that IL-12 p40 is mainly regulated at the transcriptional level by both chromatin remodeling through Toll-like receptor signaling and promoter transactivation through regulatory transcription factors (12)(13)(14)(15). In particular, various studies have demonstrated the binding of strong transcriptional activators such as NF-κB, C/EBP-β, PU.1, and AP-1 to the p40 promoter region in monocytes and macrophages (12,13,(16)(17)(18)(19). Furthermore, a repressor element (denoted GA-12) was recently identified within the p40 promoter that modulates promoter activity in response to IL-4 and PGE 2 , suggesting that both positive and negative response elements tightly control p40 gene transcription (16). ...
... Second, producer cells of IL-12 p40 could be differentially activated through the local microenvironment. In this context, bacterial antigens and products such as LPS or CpG-DNA have been shown to be strong transcriptional activators of the IL-12 p40 promoter (12,13,16,19,44). Interestingly, FISH experiments using a specific probe for eubacteria demonstrated localization of bacteria in p40-expressing cells and endocytosis of bacteria by a subset (10%) of CD11c + LPDCs in the crypts of the terminal ileum but not the proximal parts of the small bowel. ...
... Steve Smale (UCLA), contains the -350 to +50 region of the p40 promoter-driving a firefly luciferase gene (156). The PRL-SV40 Renila luciferase plasmid was purchased from Promega (Madison , WI). ...
... 7 was used for these co-transfection experiments. An I L -12 p40 reporter construct, containing the 350 to +55 region of the I L -12 p40 promoter driving a firefly luciferase gene(156), was transiently transfected into RAW264. 7 cells. ...
p38 mitogen-activated protein (MAP) kinases represent a subgroup of MAP kinases that respond to environmental stress and inflammatory cytokines. p38 MAPK is activated by two upstream kinases, MKK3 and MKK6, by dual phosphorylation on threonine and tyrosine in conserved kinase subdomain VII. Until recently the relative roles of MKK3 and MKK6 have remained unclear. I have undertaken two strategies in an effort to understand the importance of MKK3 as a p38 MAPK activator. First, I cloned and characterized the murine mkk3 gene and determined the structure of the 5'-terminus. Comparison of the murine and human mkk3 genes revealed that the mouse gene encodes a single MKK3 isoform, MKK3b, and the human gene encodes two isoforms, MKK3a and MKK3b. Comparison of the mouse and human mkk3 genes suggests that expression of MKK3a and MKK3b is regulated from different promotors. Analysis of the mkk3 promoter demonstrates that muscle specific expression of murine MKK3b is controlled, in part, by the transcription factors MEF2 and MyoD. Second, I have utilized a gene targeting strategy to disrupt the murine mkk3 gene and to examine the effect on p38 MAPK signaling. I found that there is a p38-specific signaling defect in MKK3 deficient primary mouse embryo fibroblasts (MEF) which correlates with deficits in interleukin (IL)-1 and IL-6 production in response to tumor necrosis factor-α (TNFα) stimulation. In addition there is a defect in TNFα mediated expression of TNFα and macrophage inflammatory proteins (MIP) 1α, MIP1β and MIP2. p38 MAPK-specific signaling defects were also observed in lipopolysaccharide (LPS) stimulated mkk3 (-/-) macrophages. Additionally, mkk3 (-/-) macrophages exhibit defects in LPS and CD40-ligand (CD40L) stimulated IL-12 biosynthesis. Similar data were obtained from CD40L-stimulated mkk3 (-/-) dendritic cells. I also observe that interferon (Ifn)-γ production is diminished during T-helper-1 (TH1) differentiation of CD4+ T-cells derived from mkk3 (-/-) mice. Taken together these data demonstrate a crucial role for p38 MAPK activation by MKK3 in response to the inflammatory cytokine, TNFα and during a TH1 inflammatory response.
... It should be noted that as IL-12 is a key cytokine in Th1-mediated autoimmune responses, downregulation of IL-12 production by the compounds isolated from the soft coral S. maxima may ameliorate autoimmune diseases. 16,17 On the other hand, the bioactive IL-12 cytokine is a heterodimeric p70 molecule and both subunits are coexpressed in the same cells to generate the bioactive form. 14 The regulation of IL-12 p40 production has been investigated at the level of transcription, in which multiple regulatory elements have been implicated, including NF-jB, Ets-2, and members of the IFN regulatory factor (IRF) 15 and CCAAT enhancer binding protein (C/EBP) families. ...
... 14 The regulation of IL-12 p40 production has been investigated at the level of transcription, in which multiple regulatory elements have been implicated, including NF-jB, Ets-2, and members of the IFN regulatory factor (IRF) 15 and CCAAT enhancer binding protein (C/EBP) families. 17 Further studies are required to elucidate the anti-inflammatory actions of the active compounds dentified in the present study. ...
Chemical investigation of the soft coral Sinularia maxima resulted in the isolation of seven norditerpe-noids, including two new compounds, 12-hydroxy-scabrolide A (2) and 13-epi-scabrolide C (6). The structures of the isolated compounds were elucidated based on extensive spectroscopic evidence including Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) and both one-and two-dimensional nuclear magnetic resonance (1D and 2D NMR, respectively), in comparison with reported data. Compound 6 potently inhibited IL-12 and IL-6 production in LPS-stimulated bone marrow derived den-dritic (BMDCs) with IC 50 values of 5.30 ± 0.21 and 13.12 ± 0.64 lM, respectively. Compound 1 exhibited moderate inhibitory activity against IL-12 and IL-6 production with IC 50 values of 23.52 ± 1.37 and 69.85 ± 4.11 lM, respectively. Marine organisms are considered as a gold mine with respect to the diversity of their chemical metabolites and biological activities. Many marine metabolites have unique structures that are not found in terrestrial organisms. Soft corals are a group of colonial invertebrates which form a significant set of marine organisms occurring widely in the coral reefs throughout the world. 1,2 Among the Alcyonacean soft corals, genus Sinularia is one of the most widely distributed soft coral genera, constituting a dominant portion of the biomass in the tropical reef environment. Sinularia species are rich sources of structurally unique and biologically active diterpenoids. 1 To date, several novel norditerpenoids have been isolated and structurally elucidated from Sinularia species 1,3 with some exhibiting interesting biological activities including anti-cytomegalovirus, 3 anti-inflammatory, 3 and cytotoxic activities. 4,5 As a part of our investigations of the chemical constituents and biological activities of Vietnamese marine soft corals, this study involved the isolation and structure elucidation of seven norditerpe-noids (see Fig. 1) from the soft coral Sinularia maxima, as well as an evaluation of their in vitro anti-inflammatory effect. O H 7 1 R 1 = R 3 = H, R 2 = OH 2 R 1 = H, R 2 = R 3 = OH 3 7 , R 3 = H 1 15 16
... Recently, others and we have begun to define the transcriptional factors that control production of these proteins. [7][8][9]26,27,29,34,35 Analyses of animals that have deletion of these transcription factors are a powerful tool to define their relevance in host defense. C/EBP⑀Ϫ/Ϫ mice die of overwhelming infections after 3 to 5 months of birth, pointing to the importance of this transcription factor in innate immunity. ...
... Paradoxically, no difference or even higher expression of IL-12 and IL-18 was observed in the knockout macrophages after their treatment by LPS, suggesting that LPS stimulates IL-12 expression by a pathway independent of C/EBP⑀. [34][35][36] Remarkably, IL-10 mRNA and protein were absent in the peritoneal macrophages from C/EBP⑀Ϫ/Ϫ mice. IL-10 promotes the down-regulation of pro-inflammatory cytokine synthesis and the development of the Th2 response. ...
Members of the CCAAT/enhancer-binding protein (C/EBP) family are involved in the regulation of cellular differentiation and function of many tissues. Unlike the other members of the family, C/EBPe expression is restricted to granulocytes, macrophages, and lymphocytes. C/EBPe is highly conserved between human and rodents and is essential for terminal granulopoiesis in both species. To study the role that C/EBPe plays in macrophages, wild-type and C/EBPe–deficient (−/−) murine macrophages obtained from thioglycollate-elicited peritoneal lavages and differentiated bone marrow cells were compared. Although macrophage development occurred in both types of mice, the C/EBPe−/− cells had a lower expression of macrophage markers and a morphologic and ultrastructural appearance of immaturity. Phagocytic function, measured by calculating the percentage of internalized opsonized fluorescein isothiocyanate (FITC)–labeled yeast, was significantly impaired in the C/EBPe−/− macrophages compared with their wild-type counterparts. Furthermore, the differential expression of 26 macrophage-specific genes between wild-type and C/EBP−/− mice was analyzed. A subset of genes involved in differentiation, immune, and inflammatory responses was found down-regulated in the C/EBP−/− macrophages. Taken together, this study implicates the C/EBPe gene as an important transcription factor required for normal function and development of macrophages.
... The promoter region of the IRF-1 gene contains GAS/κB motifs [137], which involves in the combined transcriptional regulation by NF-κB and IRF-1 [138,139]. The induction of bioactive form of this proinflammatory cytokine IL-12p70 (p35 and p40 heterodimer) is coordinated by a number of transcription factors belonging to IRF-1 [140], NF-κB [141], CAAT/enhancer-binding protein(C/EBP), Rel proteins [142], gamma interferon consensus sequence binding protein (IRF-8) [143], AP-1, AP-2, CREB (CRE binding protein), SP-1 and the Pu.1 family that activated the IL-12p35 and IL-12p40 promoters were reported. NF-κB along with IRF-1, IRF-8, and ETS bind to IL-12p40 promoter in response to LPS and IFN-γ, and mutation in either ets or NF-kB abolish the IL-12p40 activity. ...
IFN-γ, a type 2 interferon and a cytokine, is critical for both innate and adaptive immunity. IFN-γ binds to the IFN-γRs on the cell membrane of macrophages, signals through JAK1-STAT-1 pathway and induces IFN-γ-stimulated genes (ISGs). As Leishmania amastigotes reside and replicate within macrophages, IFN-γ mediated macrophage activation eventuate in Leishmania elimination. As befits the principle of parasitism, the impaired IFN-γ responsiveness in macrophages ensures Leishmania survival. IFN-γ responsiveness is a function of integrated molecular events at multiple levels in the cells that express IFN-γ receptors. In Leishmania-infected macrophages, reduced IFN-γRα expression, impaired IFN-γRα and IFN-γRβ hetero-dimerization due to altered membrane lipid composition, reduced JAK-1 and STAT-1 phosphorylation but increased STAT-1 degradation and impaired ISGs induction collectively determine the IFN-γ responsiveness and the efficacy of IFN-γ induced antileishmanial function of macrophages. Therefore, parasite load is not only decided by the levels of IFN-γ produced but also by the IFN-γ responsiveness. Indeed, in Leishmania-infected patients, IFN-γ is produced but IFN-γ signalling is downregulated. However, the molecular mechanisms of IFN-γ responsiveness remain unclear. Therefore, we review the current understanding of IFN-γ responsiveness of Leishmania-infected macrophages.
... Il12b activation requires chromatin remodeling and recruitment of SWI/SNF complexes. In early works it was shown that activation Il12b requires C/EBP, AP-1 and NFAT factors, as well as c-Rel, which is part of the NF-κB family [47][48][49]. The critical c-Rel binding site is occupied by a nucleosome at position -30 to -175 bp upstream of the transcription initiation site. ...
... C/EBP transcription factors belong to the bZIP family of transcription factors and are involved in tissue-specific gene expression, proliferation, differentiation, and inflammation [27]. In particular, C/EBPβ has been shown to regulate inflammatory genes like the cytokines IL-6, IL-12 p40, and IL-36α [3,28,29], the chemokines IL-8 and macrophage inflammatory protein-1α [28,30] as well as the proinflammatory genes for inducible NO synthase (NOS2) and cyclooxygenase-2 [31,32] in macrophages. DNA binding and subsequent gene expression by C/EBPβ requires the formation of homodimers or heterodimers with other C/EBP family members or members of the CREB/ATF family via the bZIP domain [33]. ...
Interleukin-36α is a novel member of the IL-1 cytokine family that is highly expressed in epithelial tissues and several myeloid-derived cell types after induction. The transcription factor (TF) C/EBPβ binds specifically to an essential half-CRE•C/EBP motif in the Il36a promoter to induce Il36a expression upon LPS stimulation. C/EBPs regulate gene expression by binding to recognition sequences that can contain 5′-cytosine-phosphate-guanine-3′ dinucleotides (CpG), whose methylation can influence TF binding and gene expression. Herein we show that the half-CRE•C/EBP element in the Il36a promoter is differentially methylated in the murine RAW264.7 macrophage cell line and in primary murine macrophages. We demonstrate that C/EBPβ binding to the half-CRE•C/EBP element in the Il36a promoter following LPS stimulation is insensitive to CpG methylation and that methylation of the CpG in the half-CRE•C/EBP element does not alter LPS-induced Il36a promoter activity which correlated with similar Il36a mRNA copy numbers and pro-IL-36α protein amount in both cell types. Taken together, our data indicate that C/EBPβ binding to the half-CRE•C/EBP element and subsequent gene activation occurs independently of the CpG methylation status of the half-CRE•C/EBP motif and underlines the potential of C/EBPs to recognize methylated as well as unmethylated motifs.
... NF-κB was previously thought to be the main transcription factor regulating expression of IL-6, IL-1β, and IL-18 [14][15][16]. It has been shown that C/ EBPβ and NF-κB have synergistic effects, and when both of the two transcription factors bind to gene sequences simultaneously, the transcriptional effects are enhanced [17,18]. Therefore, simultaneous intervention with C/EBPβ and NF-κB may be able to more strongly reduce expression of IL-6, IL-1β, and IL-18 at the transcriptional level. ...
Objective
CCAAT/enhancer binding protein β (C/EBPβ) plays an important role during atherogenesis. However, how C/EBPβ functions remains unclear. In this study, we explore the relationship between C/EBPβ and oxidized LDL-induced multiple pro-inflammatory cytokines released in monocytes.
Materials and methods
THP-1 cells (human monocyte cell line) were stimulated by ox-LDL, ChIP was used to detect the binding function of C/EBPβ to target genes, small interfering RNA was used to knock down the expression of C/EBPβ, Western Blot was used to detect protein expression, and ChIP-seq was used to detect different groups of C/EBPβ bound gene fragments. The integrative genomics viewer (IGV), model-based analysis of ChIP-seq (MACS) were used to visualize the results of ChIP-seq. GO (gene ontology), KEGG (Kyoto Encyclopedia of Genes and Genomes) and Reactome data bases enrichment analysis were performed by the ClusterProfiler software. Ingenuity pathway analysis (IPA) was used to analyze the results of ChIP-seq and to summarize the data within the database.
Results
We identified C/EBPβ as a key protein that regulated IL-1β, IL-6 through database. Then our results confirmed that C/EBPβ could bind directly to the gene of IL-18 and C/EBPβ plays a role in the increased expression and secretion of IL-18 protein after ox-LDL stimulation of THP-1. Using ChIP-seq, we found that the enhanced transcriptional function of C/EBPβ after ox-LDL treatment triggered changes in C/EBPβ-regulated downstream pathways. In the ChIP-seq results, we extracted inflammatory cytokines with significant expression differences, and by comparing them with the database of inflammatory cytokines that C/EBPβ directly regulated, we screened five inflammatory cytokines, CXCL8, IL17B, TNFSF11, CSF3, and CCL2, and the results showed that knockdown of C/EBPβ expression inhibited ox-LDL-induced secretion of CXCL8, TNFSF11, CSF3, and CCL2 by THP-1.
Conclusion
Our results suggest that ox-LDL stimulation enhances C/EBPβ-regulated transcription in THP-1 and C/EBPβ upregulate the release of multiple pro-inflammatory cytokines including IL-18, IL-1β, and IL-6 through direct binding to genes.
... C/EBP transcription factors belong to the bZIP family of transcription factors and are involved in tissue-specific gene expression, proliferation, differentiation, and inflammation (26). In particular, C/EBPβ has been shown to regulate inflammatory genes like the cytokines Il-6, Il-12 p40, and Il-36α (5,27,28), the chemokines Il-8 and macrophage inflammatory protein-1α (27,29) as well as the proinflammatory genes for inducible NO synthase (NOS2) and cyclooxygenase-2 (30,31) in macrophages. DNA binding and subsequent gene expression by C/EBPβ requires formation of homodimers or heterodimers with other C/EBP family members or members of the CREB/ATF family via the bZIP domain (32). ...
Interleukin-36α (Il-36α) is a member of the novel Il-1-like proinflammatory cytokine family that is highly expressed in epithelial tissues and several myeloid-derived cell types. We have recently shown that CCAAT enhancer binding protein β (C/EBPβ) binds specifically to an essential half cAMP response element (half-CRE)•C/EBP motif in the Il36A promoter to induce Il36A expression upon LPS stimulation. C/EBPs are transcription factors belonging to the basic leucine zipper (bZIP) family of transcriptional regulators. C/EBP proteins can form homo- and heterodimers and regulate gene expression by binding to C/EBP specific recognition sequences and composite sites that can contain 5′-cytosine-phosphate-guanine-3′ dinucleotides (CpG). CpG methylation of such elements has been shown to influence transcription factor binding and gene expression. Here we show that the half-CRE•C/EBP element in the Il36A promoter is differentially methylated in the murine RAW264.7 macrophage cell line and in primary murine macrophages. By using electrophoretic mobility gel shift and fluorescence polarization assays we demonstrate that C/EBPβ binding to the half-CRE•C/EBP element in the Il36A promoter following LPS stimulation is insensitive to CpG methylation. Transfection assays also show that methylation of the CpG in the half-CRE•C/EBP element does not alter LPS-induced Il36A promoter activity. A direct comparison of Il36A mRNA copy numbers as well as the pro-Il-36α protein level in RAW264.7 and primary macrophages revealed similar amounts in both cell types. Taken together, our data suggest that C/EBPβ binding to the half-CRE•C/EBP element and C/EBPβ mediated gene activation occurs independently of the CpG methylation status of the target DNA sequence and underline the potential of C/EBPβ to recognize methylated as well as unmethylated binding sites.
... Il12b 400bp pro-Luc plasmid was a gift from Dr. Stephen Smale (Addgene plasmid #20020; http://n2t.net/addgene:20020; RRID:Addgene_20020) (Plevy et al., 1997). ...
Turning non-inflamed (cold) tumors into inflamed (hot) tumors is important for maximizing the effect of immune checkpoint inhibitors (ICIs) against malignancies. We showed that lactate, a product of the Warburg effect, inhibited the efficacy of ICIs and suppressed IL-12 p40 expression in dendritic cells (DCs) through reducing NF-κB p65, p50 and c-Rel DNA-binding activity to the IL-12 p40 promoter. Additionally, lactate promoted the expression of early growth response protein 1 (EGR1), whose expression was increased in human invasive melanoma compared with non-invasive melanoma. We also found that EGR1 interacts with serum response factor (SRF) and represses the expression of CD80 in DCs. These findings suggest that lactate and its induced EGR1 are key factors that turn hot tumors into cold tumors and may represent targets in cancer treatment with ICIs.
... Additionally reports have shown that the EL-12 p40 gene promoter has NF-kB binding elements (Plevy et al., 1997;Ma et al., 1997). Further more a study showed that CD40 ligation on TH-Pl cells led to IL-12 p40 production via NF-KB (Yoshimoto et al., 1997). ...
Dendritic cells (DC) are known to be vital to the immune response, priming naive T cells to antigen specific proliferation. Dendritic cells in their immature state have a unique capacity to ingest large numbers of antigens via macopinocytosis. After receiving an appropriate signal they mature to an antigen presenting and T cell stimulatory state, displaying antigens bound to MHC molecules and up-regulating expression of co-stimulatory molecules. It is now well established that DC are matured by recognition of conserved molecular pattern on pathogenic organisms, often referred to as PAMPs (pathogen associated molecular patterns). In addition DC can mature in response to inflammatory cytokines such as TNF-a and IL-1 p. In this thesis an alternative DC maturation pathway is proposed, in which DC mature in response to interference with the ribosomal machinery, particularly 28S rRNA. The stress kinases are also proposed to signal any changes in 28S rRNA status within the DC. To test this hypothesis DC are incubated with ribotoxic compounds and maturation is measured by phenotype analysis, T cell stimulation and cytokine release. The results show that ribotoxic compounds can at low doses cause partial activation of DC. The proposal is then made that this route of DC activation may signal viral infection of DC, leading to maturation in response. In addition low level redox stress is shown to act in synergy with LPS to enhance T cell proliferation and cytokine release. Finally necrotic lymphocyte treated DC are shown to enhance T cell proliferation. Therefore the conclusion is made that stress per se acts as an auxiliary mechanism of enhancing immune responses, acting in synergy with receptor driven immune activation. The 28S rRNA pathway however is proposed as a major route of viral activation of DC.
... In addition, MDP + Tc CM induced substantial increase in IL-8 and minimal increase in IL-12 p40 and RANTES in MDP-treated monocytes. Therefore, Tc CM augments transcription of cytokines that are controlled by NF-B p65 (IL-1, IL-6, and IL-8) and not by c-Rel protein (IL-12 p40 and RANTES) or chemokines (RANTES) that require interferon regulatory transcription factors (IRF-3 or IRF-8) that cooperate with NF-B (40)(41)(42)(43). ...
Vaccine adjuvants containing analogs of microbial products activate pattern recognition receptors (PRRs) on antigen-presenting cells, including monocytes and macrophages, which can cause prostaglandin E 2 (PGE 2 ) release and consequently undesired inflammatory responses and fever in vaccine recipients. Here, we studied the mechanism of PGE 2 production by human monocytes activated with muramyl dipeptide (MDP) adjuvant, which activates cytosolic nucleotide-binding oligomerization domain 2 (NOD2). In rabbits, administration of MDP elicited an early increase in PGE 2 followed by fever. In human monocytes, MDP alone did not induce PGE 2 production. However, high amounts of PGE 2 and the proinflammatory cytokines IL-1β and IL-6 were secreted by monocytes activated with MDP in the presence of conditioned medium obtained from CD3 bead–isolated T cells (Tc CM) but not from those isolated without CD3 beads. Mass spectrometry and immunoblotting revealed that the costimulatory factor in Tc CM was glycoprotein Ib α (GPIbα). Antibody-mediated blockade of GPIbα or of its receptor, Mac-1 integrin, inhibited the secretion of PGE 2 , IL-1β, and IL-6 in MDP + Tc CM–activated monocytes, whereas recombinant GPIbα protein increased PGE 2 production by MDP-treated monocytes. In vivo, COX2 mRNA abundance was reduced in the liver and spleen of Mac-1 KO mice after administration of MDP compared with that of treated wild-type mice. Our findings suggest that the production of PGE 2 and proinflammatory cytokines by MDP-activated monocytes is mediated by cooperation between two signaling pathways: one delivered by MDP through NOD2 and a second through activation of Mac-1 by T cell–derived GPIbα.
... For example, IL-12 could excite Th1 T cell in response to colonization of gram-positive organisms. Cytokines trigger inflammation by recruitment of host immune cells and an antimicrobial defense that cause tissue injury or unwanted disease sometimes (Plevy et al. 1997). ...
Abstract Background Acne vulgaris is a common inflammatory skin disease, affecting adolescents across the globe. Recent evidences underline that Propionibacterium acnes (P. acnes) promotes acne through Toll-like receptor (TLR) activation. Especially, Toll-like receptor 2 (TLR2) has emerged as one of the major classes of pattern recognition receptors (PRRs) that are recognizing P. acnes in the epidermis and responsible for inflammation. Conclusions Although P. acnes has been known to be one of the major causes of acne vulgaris, an appropriate drug to alleviate acne pathogenesis is poorly developed. This review focuses on the molecular structure of TLR2 as well as mechanism how TLR2 recognize P. acnes to induce inflammatory cytokines, which is related to acne vulgaris pathogenesis. Rigorous study about P. acnes mediated by TLR2 activation may provide insight into novel therapeutic targets of acne vulgaris.
... Smale (Plevy et al., 1997). RAW 264.7 cells were co-transfected in duplicate with the Il6 or Il12b reporter plasmid and an expression plasmid encoding full length Hes1 ...
... 36 Signaling via this pathway leads in fact to the activation of NF-B and MAPKs, which ultimately regulate the transcription of IL-12p40, IL-23p19. [37][38][39] In In any case, the lack of type I IFN production would contribute to the explanation as to why human neutrophils do not express IL12A mRNA in response to either TLR8 or TLR4 activation. Another potential explanation for the inability of neutrophils to accumulate IL12A mRNA is that they do not express IRF8, a transcription factor that, in association with IRF1 at the IL12A locus, is essential for IL12A transcription in monocytes. ...
Human neutrophils contribute to the regulation of inflammation via the generation of a range of cytokines that affect all elements of the immune system. Here, we investigated their ability to express some of the members of the IL‐12 family after incubation with TLR8 agonists. Highly pure human neutrophils were thus incubated for up to 48 h with or without R848, or other TLR8 agonists, to then measure the expression levels of transcripts and proteins for IL‐12 family member subunits by RNA‐seq, reverse transcription quantitative PCR, and ELISA. We show a TLR8‐mediated inducible expression of IL‐12B and IL‐23A, but not IL‐12A, mRNA, which occurs via chromatin remodeling (as assessed by ChIP‐seq), and subsequent production of IL‐23 and IL‐12B, but no IL‐12, proteins. Induction of IL‐23 requires endogenous TNF‐α, as both mRNA and protein levels were blocked in TLR8‐activated neutrophils via a TNF‐α‐neutralizing Ab. We also show that supernatants from TLR8‐activated neutrophils, but not autologous monocytes, induce the differentiation of Th17 cells from naïve T cells in an IL‐23‐dependent fashion. This study unequivocally demonstrates that highly pure human neutrophils express and produce IL‐23, further supporting the key roles played by these cells in the important IL‐17/IL‐23 network and Th17 responses. This study shows that human neutrophils incubated with TLR8 agonists produce IL‐23, which promotes a Th17 polarization from naïve T cells.
... In addition, nuclear factor-kappa B (NF-κB), a protein complex composed of IκB bound to two proteins (p50 and p65), is the classical pathway in IL-1β production [21]. C/EBPβ has functional interaction with NF-κB p65 subunit, but the relationship of these two factors is still to be elucidated [22,23]. ...
Background/aims:
Interleukin-1β (IL-1β) is one of the critical inflammatory factors during atherogenesis. CCAAT/enhancer binding proteins β (C/EBPβ), a regulator of IL-1β production, recently been evidenced as a key player in the development of atherosclerosis. However, the mechanisms of how C/EBPβ regulates the production of IL-1β are unclear. In this study, we aimed to explore the role of C/EBPβ in regulating IL-1β production in macrophages after oxidized low-density lipoprotein (ox-LDL) exposure and the underlying mechanisms.
Methods:
RAW264.7 macrophages were treated with 0, 25, 50 or 100 μg/ml ox-LDL for 12, 24 or 48 h. Small interfering RNAs were used to silence related proteins. The gene and protein expression levels were determined by quantitative real-time polymerase chain reaction or western blot (WB). IL-1β secretion was assessed by enzyme-linked immunosorbent assay. The cytoplasmic and nuclear proteins were evaluated by nuclear fractionation followed by WB. Localization of p65 was observed by immunofluorescence. The binding activity of p65 to IL-1β was tested by dual-luciferase reporter assay.
Results:
Ox-LDL increased IL-1β production, accompanied with increasing C/EBPβ and p65 expression in a dose- and time-dependent manner. Moreover, C/EBPβ deficiency in macrophages blocked ox-LDL-induced increases in IL-1β expression, maturation as well as p65 activation. However, p65 deficiency inhibited the increase in IL-1β production, but not C/EBPβ expression. Dual-luciferase reporter results showed that overexpression of C/EBPβ significantly enhanced binding activity of p65 to IL-1β promoter. In addition, C/EBP 1β deficiency in macrophages abolished the ox-LDL-induced gene transcription increases of IL-1β, IL-6, p65 and caspase-1.
Conclusions:
Our results demonstrate that C/EBPβ acts upstream of NF-κB p65 subunit in ox-LDL-induced IL-1β production in macrophages and may regulate IL-1β maturation by promoting caspase-1. C/EBPβ may be a promising candidate for the prevention and treatment of atherosclerosis.
... DNase I footprinting. To further define the location of the protein-DNA interaction suggested by the substitution mutation studies, DNase I footprint analysis was performed according to established methods (35). Briefly, the pIgR-246 oligonucleotide ( Fig. 1) was kinased with [␥-32 P]dATP (6,000 Ci/mmol) and then used with the pGL3-3Ј primer to PCR amplify a fragment, using the mpIgR Ϫ246/ϩ44 /E-Luc vector as a template. ...
The regulatory elements that control basal and activated transcriptional expression of the polymeric IgA receptor gene ( pIgR) have not been defined. In this study, we performed functional analysis of the murine pIgR 5'-upstream region. Transient transfection studies identified the gene's minimal promoter to reside within 110 nucleotides upstream from the start of transcription. Substitution mutations of this region identified both a putative activator (-78 to -70) and a repressor (-66 to -52) element. DNase I footprint analysis confirmed an area of protection that spans from nucleotides -85 to -62. Mobility shift assays of the putative region confirmed binding of upstream stimulatory factor 1 (USF1) to an E box element at positions -75 and -70, representing the putative enhancer. Overexpression studies using various forms of USF suggest that both USF1 and USF2 enhance activity of the pIgR minimal promoter. We report the identification and characterization of the murine pIgR minimal promoter, as well as the critical role of USF in enhancing its basal level of transcription in Caco-2 cells.
... Myctagged Batf2 was purchased from OriGene technologies (MR203728). Il12b 400-bp pro-Luc, p50 cFlag pcDNA3, and RelA cFlag pcDNA3 were gifts from Stephen Smale, Howard Hughes Medical Institute and Department of Microbiology and Immunology, UCLA School of Medicine, Los Angeles (Addgene plasmids 20020, 20018, and 20012, respectively) (41,42), and pmIL-6 FL was a gift from Gail Bishop, Department of Microbiology, University of Iowa, Iowa City, IA (Addgene plasmid 61286) (43). ...
Significance
The therapeutic activity of checkpoint blockers and toll-like receptor (TLR) agonists, which show some efficacy against malignancies, appears to at least partially result from the secretion of type-I IFNs. Thus, we hypothesized that type-I IFN-inducible transcription factors, such as basic leucine zipper transcription factor ATF-like 2 ( Batf2 ), might play a role in tumor immunity. Here, we investigated the role of Batf2 , especially its positive transcriptional activities, and evaluated its antitumor effect. This study shows that Batf2 has an antitumor effect through the up-regulation of IL-12 p40 in tumor-associated macrophages, which eventually induces the activation of CD8 ⁺ T cells and their accumulation within the tumor. Batf2 may be an important target in anticancer treatment with immune checkpoint blockers and TLR agonists.
... C/EBPβ has been found to induce the transcriptional activation of XBP1 (61,62) and, conversely, C/EBPβ is induced by XBP1 via an X-box present in human cells that is absent in rats and mice (63), as well as through the PERK route (64). In addition, C/EBPβ is fully active in unstimulated macrophages and poised to be recruited into enhanceosomes to team up with other factors to stimulate transcription, given its well-known function as a pioneer factor that promotes the opening of silent chromatin (49,65,66). The present results are of clinical relevance because DCs in tumor milieus show an activation of XBP1 that blunts the immune response and favors tumor cell progression (13). ...
Human monocyte-derived dendritic cells (DCs) exposed to pathogen-associated molecular patterns (PAMPs) undergo bioenergetic changes that influence the immune response. We found that stimulation with PAMPs enhanced glycolysis in DCs, whereas oxidative phosphorylation remained unaltered. Glucose starvation and the hexokinase inhibitor 2-deoxy-d-glucose (2-DG) modulated cytokine expression in stimulated DCs. Strikingly, IL23A was markedly induced upon 2-DG treatment, but not during glucose deprivation. Since 2-DG can also rapidly inhibit protein N-glycosylation, we postulated that this compound could induce IL-23 in DCs via activation of the endoplasmic reticulum (ER) stress response. Indeed, stimulation of DCs with PAMPs in the presence of 2-DG robustly activated inositol-requiring protein 1α (IRE1α) signaling and to a lesser extent the PERK arm of the unfolded protein response. Additional ER stressors such as tunicamycin and thapsigargin also promoted IL-23 expression by PAMP-stimulated DCs. Pharmacological, biochemical, and genetic analyses using conditional knockout mice revealed that IL-23 induction in ER stressed DCs stimulated with PAMPs was IRE1α/X-box binding protein 1-dependent upon zymosan stimulation. Interestingly, we further evidenced PERK-mediated and CAAT/enhancer-binding protein β-dependent trans-activation of IL23A upon lipopolysaccharide treatment. Our findings uncover that the ER stress response can potently modulate cytokine expression in PAMP-stimulated human DCs.
... Several transcriptional factors have been identified as important regulators of IFN-g-and LPS-induced Il12b transcription in human and mouse macrophages. These include NF-kB (25,26), C/EBPb (LAP) (27), Ets-2 (28), PU.1 (29), AP-1 (30), IFN regulatory factor 1) (31), IFN consensus sequence binding protein (32), and erythroid Kr€ uppel-like factor (33). The aim of the present study was to elucidate the molecular basis that mediates differential IL-12/IL-23 expression in a way dependent on the rs41292470 SNP within the Il12b promoter. ...
IL-12 and IL-23 are important host defense factors produced by APCs against certain intracellular and extracellular pathogens. Their dysregulation has also been implicated in several autoimmune diseases. The nucleotide polymorphism in the promoter region of Il12b (rs41292470 consisting of the long or short allele) encoding the shared subunit of IL-12 and IL-23, p40, has been reported to associate with susceptibility to infectious diseases and autoimmune disorders. How these genetic variants impact Il12b expression at the molecular level was unclear. We established an Il12b promoter-luciferase reporter system containing the long or short allele driving the reporter gene expression and found that the long allele (infection-resistant) displayed ∼2-fold higher transcriptional activity than the short allele (infection-susceptible), associated with a selective and differential nuclear binding activity to the two alleles in activated macrophages. DNA pull-down assays coupled with mass spectrometry analyses identified the specific DNA binding activity as poly(ADP-ribose) polymerase 1 (PARP-1). Small hairpin RNA-mediated knockdown of the endogenous PARP-1 expression resulted in reduced p40 mRNA expression and Il12b promoter activity. Bone marrow-derived macrophages from PARP-1-deficient mice had decreased p40 expression at both mRNA and protein levels. Furthermore, selective PARP-1 inhibitors resulted in impaired production of IL-12p40 and IL-23 in bone-marrow derived macrophages and PBMCs. Chromatin immunoprecipitation assay revealed that PARP-1 could bind specifically to Il12b in LPS-stimulated macrophages. Our study opens the way for further elucidating the molecular mechanism whereby allele-specific immune responses to foreign and self-antigens mediated by IL-12/IL-23 are controlled in an individually variable manner.
... IL-23 expression is induced through various MAPKs including p38, JNK and ERK [30], as well as NFкB. The p40 gene expression is regulated at the transcriptional level by binding of NFк-B, CCAAT/enhancer-binding protein (C/EBP), ets-2, PU.1, IRF1, IRF2, IRF5, IRF8 and activator protein 1 (AP-1) to the promoter region of p40 [31][32][33][34] upon stimulation with various ligands. The murine and human p19 promoter was also shown to contain three NFкB binding sites [30]. ...
... Overlapping or adjacent NF-B/CEBP binding sites are located within the promoter regions of IL-6, IL-8, IL-12, angiotensinogen, and serum amyloid A genes [Ray et al., 1995;Ruocco et al., 1996;Yoshimoto et al., 1996], but it is unknown whether rel/bZIP heteromers directly interact with such combined sites. C/EBP and rel proteins interact at an NF-B half-site in the IL-12 p40 subunit promoter to activate transcription [Plevy et al., 1997], but it remains unknown whether these proteins form heteromers in this event. ...
PGG-Glucan is a soluble β-glucan immuno-modulator which enhances a variety of leukocyte microbicidal activities without activating inflammatory cytoklnes. Although several different βglucan receptors have been described, the signal transduction pathway(s) utilized by soluble β-glucans have not been elucidated. Here we identify a novel transcription factor activated by PGG-Glucan in a mouse BMC2.3 macrophage cell line. We used probe competition, antibody competition, and immuno-precipitation experiments to demonstrate that the factor is a dimer containing one member of the rel family (rel-A, p65) attached to one member of the bZIP family (C/EBP-/3, p48). This heteromer is different than the p65/p50 rel/rel complex induced in these cells by lipopolysaccharide (IPS), thus our data demonstrate that PGG-Glucan utilizes signal transduction pathways different from those used by LPS, the latter which activates leukocyte microbicidal activities and inflammatory cytokines. We further show that PGG-Glucan treatment of the BMC2.3 cells increases the phosphorylation of inhibitor-kappa-B-alpha, and involves protein kinase C (PKC) and protein tyrosine kinase (PTK) pathways.
... Also, acetylation of p53 stimulates DNA binding (46), whereas deacetylation of p53 modulates its effect on cell growth and apoptosis (47). In addition, the transcription factor C/EBP, which upregulates IL-12p40 and IL-6 transcription, is also downregulated by TSA or butyrate, reducing both C/EBP mRNA and protein levels (48). NF-κB is another pivotal transcription factor that regulates the expression of several cytokine genes, including IFN-γ and IL-10 (49). ...
... The most common transcription factor binding sites present in the promoters of genes regulated similarly to IL12B were identified using the DAVID functional annotation tool [56]. Not surprisingly, binding sites for transcription factor families known to regulate IL12B were identified, including, Octomer-binding transcription factor (OCT), Nuclear Factor Kappa B (NFκB), Interferon Regulatory Factor (IRF), cAMP Response Element Binding protein (CREB), and CCAAT/Enhancer Binding Protein families [64][65][66][67][68] (Table 3). ...
Background:
Leishmania major infection induces robust interleukin-12 (IL12) production in human dendritic cells (hDC), ultimately resulting in Th1-mediated immunity and clinical resolution. The surface of Leishmania parasites is covered in a dense glycocalyx consisting of primarily lipophosphoglycan (LPG) and other phosphoglycan-containing molecules (PGs), making these glycoconjugates the likely pathogen-associated molecular patterns (PAMPS) responsible for IL12 induction.
Methodology/principal findings:
Here we explored the role of parasite glycoconjugates on the hDC IL12 response by generating L. major Friedlin V1 mutants defective in LPG alone, (FV1 lpg1-), or generally deficient for all PGs, (FV1 lpg2-). Infection with metacyclic, infective stage, L. major or purified LPG induced high levels of IL12B subunit gene transcripts in hDCs, which was abrogated with FV1 lpg1- infections. In contrast, hDC infections with FV1 lpg2- displayed increased IL12B expression, suggesting other PG-related/LPG2 dependent molecules may act to dampen the immune response. Global transcriptional profiling comparing WT, FV1 lpg1-, FV1 lpg2- infections revealed that FV1 lpg1- mutants entered hDCs in a silent fashion as indicated by repression of gene expression. Transcription factor binding site analysis suggests that LPG recognition by hDCs induces IL-12 in a signaling cascade resulting in Nuclear Factor κ B (NFκB) and Interferon Regulatory Factor (IRF) mediated transcription.
Conclusions/significance:
These data suggest that L. major LPG is a major PAMP recognized by hDC to induce IL12-mediated protective immunity and that there is a complex interplay between PG-baring Leishmania surface glycoconjugates that result in modulation of host cellular IL12.
... In addition to the production of IL-12, the upregulation of CD40 and toll like receptor 4 (TLR4) on DCs also serve as signal 3 in promoting a Th1 response and are critical for the production of IL-12 (15)(16)(17). DCs from CD40 knockout mice have reduced IL-12 production and are also defective in Th1 priming in response to intracellular pathogens (18,19). TLR-4 is the pattern recognition molecule associated with LPS and deleting this gene in mice abrogates TLR-4-mediated signaling and renders them unresponsive to TLR-4 and resistant to LPS-induced sepsis (20 ). ...
Dendritic cells (DCs) are integral to differentiation of T helper cells into Th1, Th2 and Th17 subsets. We have dissected two novel pathways in DCs that specifically regulate CD4 T cell responses. The first is the role of the c-Kit-Phosphatidyl inositol 3 kinase (PI3 kinase)-interleukin-6 (IL-6) axis and the second that of vascular endothelial growth factor (VEGF). IL-6 plays a central role in regulating CD4 T cell immune responses by limiting a Th1 response and promoting Th2 and Th17 responses. We investigate pathways in DCs that promote IL-6 production and show that the allergen house dust mite or the mucosal adjuvant cholera toxin but not Th1-inducing adjuvant, CpG oligodeoxynucleotide (ODN) promote cell surface expression of c-kit and its ligand, stem cell factor (SCF), in DCs. This dual upregulation of c-kit and SCF results in sustained PI3-kinase signaling promoting IL-6 secretion. Intranasal administration of antigen into c-kit mutant mice or neutralization of IL-6 blunted Th2 and Th17 but promoted Th1 responses in lung-draining lymph nodes. DCs lacking functional c-kit elicit diminished allergic airway inflammation when adoptively transferred into mice. Expression of the Notch ligand, Jagged-2, which has been associated with Th2 differentiation, was reduced in DCs from c-kit mutant mice. DCs generated from mice expressing a catalytically inactive form of the p110ƒÔ (p110D910A) subunit of PI3-kinase secrete lower levels of IL-6 upon stimulation with CT. These results collectively highlight the importance of the c-kit-PI3-kinase-IL-6 signaling axis in DCs in regulating T cell responses.We also investigated mechanisms underlying the production of VEGF, which has been recently shown to be a Th2-skewing cytokine and to promote allergic asthma. We found that CT-stimulated DCs secrete high levels of VEGF while LPS induces minimal VEGF production. Activation of iNOS, NF-ƒÛB and PI3 kinase enhanced production of VEGF in DCs whereas IL-12, a Th1-skewing cytokine, inhibited VEGF production. This mechanism highlights a critical but previously unknown role for DC-derived VEGF. Taken together, these findings broaden our understanding of diverse mechanisms in DCs that enable T cell polarization and offer novel targets for therapeutic interventions.
... Functional interaction, often synergistic, between C/EBPs and NF-κB has been observed for many genes (eg. (Plevy et al. 1997). This is not unexpected since NF-κB, mainly p65, and C/EBPs (C/EBPβ and C/EBPδ) are key regulators of cellular programs such as the acute-phase response and the pro-inflammatory gene expression program. ...
CCAAT/enhancer binding protein (C/EBP) β and C/EBPδ are transcription factors of the basic-leucine zipper class which share phylogenetic, structural and functional features. In this review we first describe in depth their basic molecular biology which includes fascinating aspects such as the regulated use of alternative initiation codons in the C/EBPβ mRNA. The physical interactions with multiple transcription factors which greatly opens the number of potentially regulated genes or the presence of at least five different types of post-translational modifications are also remarkable molecular mechanisms that modulate C/EBPβ and C/EBPδ function. In the second part, we review the present knowledge on the localization, expression changes and physiological roles of C/EBPβ and C/EBPδ in neurons, astrocytes and microglia. We conclude that C/EBPβ and C/EBPδ share two unique features related to their role in the CNS: whereas in neurons they participate in memory formation and synaptic plasticity, in glial cells they regulate the pro-inflammatory program. Because of their role in neuroinflammation, C/EBPβ and C/EBPδ in microglia are potential targets for treatment of neurodegenerative disorders. Any strategy to reduce C/EBPβ and C/EBPδ activity in neuroinflammation needs to take into account its potential side-effects in neurons. Therefore, cell-specific treatments will be required for the successful application of this strategy.
Copyright © 2015. Published by Elsevier Ltd.
... Because IRF1 was not reported to bind the mouse Il6 promoter before, we used EMSA and identified a 142-bp region (2344 to -203) in the mouse Il6 promoter that binds IRF1 (Supplemental Fig. 2F, 2G). Binding of JunB and C/EBPb to the mouse Il6 and Il12b promoters was reported previously (22)(23)(24). An earlier investigation using an Il12b promoter reporter assay demonstrated cooperation between the AP1 binding site and the adjacent C/EBP binding site (24). ...
Multiple pathogen-associated molecular pattern-induced TLR pathway cross-talk provokes proinflammatory cytokine synergy in macrophages, which is important for pathogen resistance and immune homeostasis. However, the detailed mechanisms are unclear. In this article, we demonstrate viral RNA analog-induced transcription synergy of Il6 and Il12b via IFN regulatory factor (IRF)1 (TLR3-TIR domain-containing adaptor inducing IFN-β [TRIF] responsive), C/EBPβ (TLR7-MyD88 responsive), and JunB (all responsive). Coactivation of the TLR3 and TLR7 pathways synchronizes the interaction of IRF1, JunB, and C/EBPβ with the Il6 and Il12b promoters, facilitating maximal gene expression. MyD88 pathway activation suppresses TRIF-induced IRF1 in a delayed manner, controlling the magnitude and timing of cytokine expression. Our findings provide novel mechanisms of cooperation of different TLR pathways to achieve optimal immune responses, with the potential for immunomodulatory strategies.
Copyright © 2015 by The American Association of Immunologists, Inc.
... Diese führt über ERK (extracellular signal-regulated kinase), p38-MAP-Kinase und JNK (Jun N-terminal kinase) zur Aktivierung der AP-1-Proteine Jun und Fos, die ebenfalls für die Cytokinproduktion benötigt werden [Aderem and Ulevitch, 2000]. Man geht davon aus, daß mikrobielle Liganden auch einen Signaltransduktionsweg in Gang setzen, der zur Aktivierung von C/EBP-Proteinen führt, da zum Beispiel das IL-12-p40-Gen durch Mitglieder der NF-κBund C/EBP-Familien reguliert wird [Murphy et al., 1995;Plevy et al., 1997]. Bisher wurden 9 Mitglieder der humanen TLR-Familie beschrieben [Rock et al., 1998] und ihr Homologes Toll setzt in Drosophila eine ähnliche Signaltransduktionskaskade in Gang, und führt dadurch zur Produktion antimikrobieller Peptide [Lemaitre et al., 1996]. ...
Das proinflammatorische CC-Chemokin RANTES kann von einer Vielzahl von Geweben als Reaktion auf entsprechende Stimuli produziert werden. Die gewebespezifische Transkription ergibt sich aus der räumlichen Anordnung verschiedener cis-regulatorischer Elemente in der Promotorsequenz und der Anwesenheit von bestimmten Transkriptionsfaktoren. Humane Monocyten produzieren RANTES konstitutiv in geringen Mengen. Stimulation mit LPS führte in diesem Zelltyp zu einer schnellen und transienten Verstärkung der RANTES-Transkription. Im Rahmen dieser Arbeit wurden zwei Regionen untersucht, die an dieser Transkriptionsaktivierung beteiligt sind. Die Elemente wurden mit Hilfe von DNase I footprinting-Analysen und EMSA-Experimenten, sowie der transienten Transfektion von Reportergenkonstrukten charakterisiert.
Die Region E des RANTES-Promotors (R(E), -125/-99) zeigte in den Monocyten-Zellinien MM6 und THP-1 konstitutive Bindung von Mitgliedern der C/EBP-Familie. Die Mutation der C/EBP-Bindungssequenz in R(E) führte in beiden Zelltypen zu einer um 40-50 % verminderten LPS-induzierten Reporteraktivität.
Die Region R(AB) (-73/-34) besteht aus den NF-kB-ähnlichen Elementen R(A) und R(B). Diese beiden Elemente bilden zusammen ein LPS-induzierbares Promotor-Modul. R(A) bindet konstitutiv Sp1 und, nach Stimulation mit LPS, Rel-p50/p65-Heterodimere. Beide Faktoren können an R(A) transkriptionsaktivierend wirken. Für die Stimulation der RANTES-Transkription durch LPS ist zusätzlich das induzierte Binden von Rel-p50/p50-Homodimeren an R(B) erforderlich.
Die experimentell gewonnenen Daten wurden verwendet, um eine Reihe von Computermodellen zu erstellen. Diese Modelle sollten die Grundstruktur einer durch LPS regulierbaren Promotorsequenz beschreiben. Mit Hilfe dieser Modelle konnten in Datenbanksequenzen andere Promotorbereiche gefunden werden, die ebenfalls durch LPS reguliert werden können. Ferner wurden Kandidatengene für eine transkriptionelle Regulation durch LPS identifiziert.
... Therefore, the biological activity of IL-12 is regulated mainly by induction of the p40 subunit and is regulated primarily at the level of transcription (Murphy et al. 1995). Since IL-12 is a key cytokine in Th1-mediated autoimmune responses, downregulation of IL-12 production by the oleanane-type triterpenes and saponins from K. pictus may ameliorate autoimmune diseases (Bao et al. 2002;Plevy et al. 1997). ...
... Among its many biological activities, IL-12 provides an obligatory signal for the differentiation of effector T-helper 1 (Th1) cells and the secretion of Th1 cytokines, gamma interferon (IFN-γ) and IL-2. IL-12 plays an important role in the generation of a Th1 response against human pathogens [23,24]. Although the induction of IL-12 by intracellular organisms is necessary for a protective host Th1 response, overexpression of Th1 cytokines and IL-12 may contribute to the development and perpetuation of chronic inflammatory and autoimmune diseases. ...
Inflammation is important in biomedical research, because it plays a key role in inflammatory diseases including rheumatoid arthritis and other forms of arthritis, diabetes, heart disease, irritable bowel syndrome, Alzheimer's disease, Parkinson's disease, allergies, asthma, and even cancer. In the present study, we describe the inhibitory effect of crude extracts and steroids isolated from the starfish Astropecten polyacanthus on pro-inflammatory cytokine (Interleukin-12 (IL-12) p40, interleukin-6 (IL-6), and tumor necrosis factor α (TNF-α)) production in lipopolysaccharide (LPS)-stimulated bone marrow-derived dendritic cells (BMDCs). Among those tested, compounds 5 and 7 showed potent inhibitory effects on the production of all three pro-inflammatory cytokines with IC 50 values ranging from 1.82 ± 0.11 to 7.00 ± 0.16 μM. Potent inhibitory activities were also observed for compound 1 on the production of IL-12 p40 and IL-6 with values of 3.96 ± 0.12 and 4.07 ± 0.13 μM, respectively, and for compounds 3 and 4 on the OPEN ACCESS Mar. Drugs 2013, 11 2918 production of IL-12 p40 with values of 6.55 ± 0.18 and 5.06 ± 0.16 μM, respectively. Moreover, compounds 2 (IC 50 = 34.86 ± 0.31 μM) and 6 (IC 50 = 79.05 ± 2.05 μM) exhibited moderate inhibitory effects on the production of IL-12 p40, whereas compounds 3 (IC 50 = 22.80 ± 0.21 μM) and 4 (IC 50 = 16.73 ± 0.25 μM) moderately inhibited the production of TNF-α and IL-6, respectively.
... Protein concentration was determined using the Bradford assay. The electrophoretic mobility shift assay (EMSA) was performed on nuclear extracts using a NFκB DNA binding element probe, as previously described (197 Turner Designs Luminometer TD20/20. In RAW264.7 cells, transfection efficiency was assessed by βgalactosidase activity in the same cell lysates. ...
The human chronic inflammatory bowel diseases (IBD), Crohn's disease (CD) and ulcerative colitis (UC) are ostensibly disorders of innate immunity with an exaggerated inflammatory response and loss of tolerance to the normal enteric microbial flora. In this project, we have extensively characterized innate immune responses driven by Pathogen Associated Molecular Pattern Molecules (PAMPs) and the more recently recognized Damage Associated Molecular Pattern molecules (DAMPs). The prototype DAMP, a chromatin-associated protein, high mobility group box 1 (HMGB1), is released during cellular necrosis and is secreted from activated macrophages. Extracellularly, it binds the receptor for advanced glycation end products (RAGE), as well as toll-like receptor (TLR) 2 and TLR4, important in the recognition of PAMPs. PAMPs and DAMPs trigger inflammatory signaling pathways in neighboring cells through activation of the transcription factor family, NF-kappaB.Much attention has been given to the central role played by PAMPs in the form of the enteric bacterial flora in IBD pathogenesis. We hypothesize that DAMPs also play a pivotal role in this process. Accordingly, we have determined the significance of DAMPs and PAMPs in the mucosal inflammatory response in macrophages and in vivo in mouse models of IBD.We first investigated expression of TLRs in the gut to determine cell types in the intestinal epithelium that may respond to danger signals. TLR expression was most prominent on intestinal epithelial enteroendocrine cells (EEC). Using a murine EEC line, multiple functional consequences of TLR activation were demonstrated. Second, in IL-10 deficient (-/-) mice with chronic Th1-mediated enterocolitis, we demonstrate a role for HMGB1 in macrophage activation and IBD. Lastly, we examined an in vivo therapy targeted at inhibiting the prominent downstream effector of DAMP and PAMP signaling, NF-kappaB, in murine IBD. Inhibition of activated NF-kappaB with a short cell permeable peptide inhibited chronic enterocolitis in IL-10-/- mice.In summary, this dissertation provides new insight into our understanding of intestinal innate mucosal inflammatory responses. We demonstrate the relevance of TLRs on EECs and the contribution of DAMP and PAMP signaling in disease. These results also provide proof of concept for new therapeutic approaches in IBD.
... To normalize experiments for transfection efficiency, cells were cotransfected with a h-galactosidase reporter plasmid driven by cytomegalovirus promoter. The cells were harvested 24 h after transfection and extracted with reporter lysis buffer (Promega), and 20 AL of extract were used for the assay of luciferase activity as described (19). When indicated, heat-killed Lm was added to the culture for 6 to 12 h before harvest. ...
Aberrant fibroblast growth factor (FGF) signaling can promote tumor development by directly driving cancer cell proliferation and survival, and by supporting tumor angiogenesis. Multiple FGFs have been found upregulated in prostate cancers, including FGF1, FGF2, FGF6, FGF8 and FGF17, all of which can activate FGF receptor 4 (FGFR4). FGFR4 is overexpressed in prostate cancer (PCa) and positively associated with aggressive PCa. FGF19 is a distinct member of FGF family in that it predominantly binds to FGFR4 with high affinity. In this present study we aimed to study the role of FGF19 in human PCa progression, and to determine whether the targeted suppression of FGF19/FGFR4 signaling has potential therapeutic benefits in PCa.
Our results demonstrated that FGF19 is upregulated in human PCa compared to normal prostate tissues. FGF19 is expressed in an autocrine manner by all tested PCa cell lines. Exogenous FGF19 stimulates PCa cell proliferation, anchorage-independent growth, adhesion and invasion in vitro. The mRNAs of FGF19 co-receptors αKlotho and ßKlotho are expressed in 97.5% and 27.5% of PCa clinical samples, respectively; but αKlotho is expressed in only 57% normal prostate tissues, ßKlotho is barely detected in normal prostate tissues. Suppression of FGF19 by short hairpin RNA (shRNA) targeting FGF19 gene inhibits PCa cell proliferation, adhesion and invasiveness in vitro. Immunoprecipitation and western blot assays showed that FGF19 stimulates phosphorylation of FGFR4, FRS2α, Erk1/2 and p-38 MAPK, as well as MEK1/2 in both PC3 and DU145. Our data also revealed that FGF19 induced the serine/threonine protein kinase Akt phosphorylation in PCa cells.
Lentiviral shRNA delivery was used for stable FGFR4 gene silencing in PC3 and LNCaP cells. The targeted knockdown of FGFR4 in PC3 and LNCaP cells resulted in significantly inhibited cell proliferation, invasion and remarkably reduced Akt phosphorylation. The upregulated expression of activated Caspase 8 and activated Caspase 3 in FGFR4 knockdown cells indicates that FGFR4 signaling inhibits PCa cell apoptosis. To determine if FGFR4 suppression impacts prostate tumor growth and metastasis in vivo we generated a PCa orthotopic xenograft model in which PC3-sh-FGFR4 or PC3-shV cells are injected directly into the prostates of nude mice in each group. The results revealed that the FGFR4 suppression in PCa cells significantly inhibited tumorigenicity in this model (P=0.01). The primary tumor weight was decreased by 63% in sh-FRGR4 tumors (p<0.05) and the proportion of mice with lymph node metastasis was decreased from 93.3% to 50% (p<0.01). The Ki67 immunohistochemistry showed a significant decrease of Ki67 positive cell percentage in PC3-sh-FGFR4 xenograft tumors (19.85%) compared to control group (35.93%, p<0.001). Our data indicated that FGF19/FGFR4 signaling may be a promising target in PCa therapy.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1106. doi:10.1158/1538-7445.AM2011-1106
... Notably, GRA24 is a potent inducer of macrophage IL-12p40 secretion both in vitro and during acute infection in mice. We believe that GRA24 acts on cytokine synthesis by modulating the expression of C/EBP, which in cooperation with the rel/NF-kB complex was initially found to regulate the IL-12p40 gene expression (Plevy et al., 1997; Holscher, 2004; Rosowski et al., 2011). Along this line, GRA15 could contribute to this regulation by modulating synergistically the activity of NF-kB (Rosowski et al., 2011). ...
... Notably, GRA24 is a potent inducer of macrophage IL-12p40 secretion both in vitro and during acute infection in mice. We believe that GRA24 acts on cytokine synthesis by modulating the expression of C/EBP, which in cooperation with the rel/NF-kB complex was initially found to regulate the IL-12p40 gene expression (Plevy et al., 1997;Holscher, 2004;Rosowski et al., 2011). Along this line, GRA15 could contribute to this regulation by modulating synergistically the activity of NF-kB (Rosowski et al., 2011). ...
... Notably, GRA24 is a potent inducer of macrophage IL-12p40 secretion both in vitro and during acute infection in mice. We believe that GRA24 acts on cytokine synthesis by modulating the expression of C/EBP, which in cooperation with the rel/NF-kB complex was initially found to regulate the IL-12p40 gene expression (Plevy et al., 1997;Holscher, 2004;Rosowski et al., 2011). Along this line, GRA15 could contribute to this regulation by modulating synergistically the activity of NF-kB (Rosowski et al., 2011). ...
... Recent studies have emphasized the importance of the cytokine subunit IL-12 p40. IL-12 p40, expressed in macrophages and dendritic cells, plays a central role in bridging innate and adaptive immune responses [43,44]. IL-12 and IL-23 are involved in the induction and maintenance of Th1 responses and chronic inflammation [45,46]. ...
Inflammatory bowel disease (IBD), encompassing ulcerative colitis and Crohn's disease, are chronic, relapsing and remitting inflammatory disorders of the intestinal tract. Because the precise pathogenesis of IBD remains unclear, it is important to investigate the pathogenesis of IBD and to evaluate new anti-inflammatory strategies. Recent accumulating evidence has suggested that carbon monoxide (CO) may act as an endogenous defensive gaseous molecule to reduce inflammation and tissue injury in various organ injury models, including intestinal inflammation. Furthermore, exogenous CO administration at low concentrations is protective against intestinal inflammation. These data suggest that CO may be a novel therapeutic molecule in patients with IBD. In this review, we present what is currently known regarding the therapeutic potential of CO in intestinal inflammation. © 2015 S. Karger AG, Basel.
Aims:
The objective of this investigation is to evaluate the mechanisms Anisakis simplex employs to modify its host immune system, regarding the larval antigens interactions with Toll-Like-Receptors (TLRs).
Methods and results:
In a previous study, we described that the stimulation of bone marrow derived dendritic cells (BMDCs) with A. simplex larval antigens drive an acute inflammatory response in BALB/c mice, but a more discrete and longer response in C57BL/6J. Moreover, when A. simplex larval antigens were combined with TLR agonists (TLR 1/2-9), they modified mainly TLR2, TLR4 and TLR9 agonists responses in both mice strains, and also TLR3, TLR5 and TLR7 in BALB/c. Antigen-presenting ability was analyzed by the detection of CD11c + cells expressing surface markers (CD80-86, MHC I-II), intracellular cytokines (IL-10, IL-12, TNF-α) and intracellular proteins (Myd88, NF-κβ) by Flow Cytometry. Secreted IL-10 was measured by ELISA.
Conclusion:
Our findings confirm not only that the host genetic basis plays a role in the development of a Th2/Th1/Treg response, but also it states A. simplex larval antigens present specific mechanisms to modify the innate response of the host. As allergies share common pathways with the immune response against this particular helminth, our results provide a better understanding into the specific mechanisms of A. simplex allergy related diseases.
Leishmania parasites are the causative agents of human leishmaniases. They infect professional phagocytes of their mammalian hosts, including dendritic cells (DCs) that are essential for the initiation of adaptive immune responses. These immune functions strictly depend on the DC's capacity to differentiate from immature, antigen-capturing cells to mature, antigen-presenting cells—a process accompanied by profound changes in cellular phenotype and expression profile. Only little is known on how intracellular Leishmania affects this important process and DC transcriptional regulation. Here, we investigate these important open questions analyzing phenotypic, cytokine profile and transcriptomic changes in murine, immature bone marrow-derived DCs (iBMDCs) infected with antibody-opsonized and non-opsonized Leishmania amazonensis (L.am) amastigotes. DCs infected by non-opsonized amastigotes remained phenotypically immature whereas those infected by opsonized parasites displayed a semi-mature phenotype. The low frequency of infected DCs in culture led us to use DsRed2-transgenic parasites allowing for the enrichment of infected BMDCs by FACS. Sorted infected DCs were then subjected to transcriptomic analyses using Affymetrix GeneChip technology. Independent of parasite opsonization, Leishmania infection induced expression of genes related to key DC processes involved in MHC Class I-restricted antigen presentation and alternative NF-κB activation. DCs infected by non-opsonized parasites maintained an immature phenotype and showed a small but significant down-regulation of gene expression related to pro-inflammatory TLR signaling, the canonical NF-kB pathway and the NLRP3 inflammasome. This transcriptomic profile was further enhanced in DCs infected with opsonized parasites that displayed a semi-mature phenotype despite absence of inflammasome activation. This paradoxical DC phenotype represents a Leishmania-specific signature, which to our knowledge has not been observed with other opsonized infectious agents. In conclusion, systems-analyses of our transcriptomics data uncovered important and previously unappreciated changes in the DC transcription factor landscape, thus revealing a novel Leishmania immune subversion strategy directly acting on transcriptional control of gene expression. Our data raise important questions on the dynamic and reciprocal interplay between trans-acting and epigenetic regulators in establishing permissive conditions for intracellular Leishmania infection and polarization of the immune response.
Dendritic cells (DC) from diabetes‐prone NOD mice and patients with type 1 diabetes (T1D) produce excess IL‐12 that drives development of β‐cell‐destroying IFN‐γ‐producing T cells. The molecular mechanisms that control IL‐12 production in T1D are unclear. In this study, we report that β‐catenin, a multifunctional protein involved in inflammation, is dramatically increased in DC from NOD mice. We further investigated the mechanisms leading to accumulation of β‐catenin in NOD DC and its role in the inflammatory pathogenic responses associated with T1D. Hyperphosphorylation of β‐catenin at a stabilizing residue, serine 552, mediated by activation of Akt, appears to lead to β‐catenin accumulation in NOD DC. Elevated β‐catenin in DC correlated with IL‐12 production and induction of IFN‐γ‐producing CD4 cells. On the one hand, knockdown/inhibition of β‐catenin significantly reduced NOD DC production of IL‐12 and their ability to induce IFN‐γ‐producing CD4 cells. On the other hand, overexpression of β‐catenin in control DC resulted in increased IL‐12 production and induction of IFN‐γ‐production in T cells. Additionally, we found that β‐catenin inhibitors decreased NF‐κB activation in NOD DC and IFN‐γ production by NOD T cells in vivo. These data strongly suggest that accumulation of β‐catenin in DC from NOD mice drives IL‐12 production, and consequently, development of pathogenic IFN‐γ‐producing T cells. Targeting the defect responsible for β‐catenin accumulation and subsequent overproduction of pro‐inflammatory cytokines by NOD DC could be an effective therapeutic strategy for the prevention and/or treatment of T1D. Stabilizing phosphorylation of β‐catenin at Ser552 leads to its accumulation in NOD DC and subsequent inflammatory response.
Since the initial observation that bacterial DNA is recognized by and activates cells of the immune system (reviewed in 1), substantial progress has been made with respect to the understanding of the molecular mechanisms involved. This bears upon both sides, the immunostimulatory DNA as a ligand and the immune cell with its receptor and signaling systems. In the meantime, it has been well established that the stimulatory capacity of bacterial DNA depends on short sequences with a central, unmethylated CG, called the CpG-motif (2,3). This stimulatory information can be transferred to single-stranded oligonucleotides (ODN). So far as it is known, all stimulatory activities of bacterial DNA are reflected in such ODNs. Therefore, these single-stranded ODNs might be regarded as the active principle of immunostimulatory DNA. It is however important to note that—as worked out with sophisticated arrays of ODNs—there is a clear species specificity in respect to the sequences active in mouse vs humans. Hence, it might be speculated that double-stranded bacterial DNA represents a pool of various stimulatory sequences that are recognized by specific but species-dependent receptor systems. DNA that harbors immunostimulatory capacity owing to CpG-motifs is collectively referred to as CpG-DNA.
Interleukins are critical cytokines that are ubiquitously present in both vertebrates and invertebrates and constitute the front line of host innate immunity. Here, we identified and analyzed IL-12p40 from the Chinese sea bass Lateolabrax maculatus (LmIL-12p40). The LmIL-12p40 gene is expressed as a 1386-base pair transcript that encodes a polypeptide of 321 amino acids. Transcriptional expression analysis indicated that LmIL-12p40 mRNA was ubiquitously expressed in all tested tissues and had a comparatively high expression level in immune-associated tissues (head-kidney and intestines). Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) experiments showed that, after Vibro harveyi and Streptococus agalactiae infection, LmIL-12p40 mRNA expression was significantly up-regulated in the spleen, liver and head-kidney. To further clarify the immune function of LmIL-12p40 after bacterial challenge, the recombinant LmIL-12p40 protein was acquired using a prokaryotic expression method. Furthermore, the LmIL-12p40 dimer (LmIL-12p80) could be produced via protein-protein interactions by incubating p40 monomer expressed from the pET28a vector (pET28a-LmIL-12p40) with p40 monomer expressed from the pGEX4T-1 vector (pGEX4T-1-LmIL-12p40). The antimicrobial activity of the purified LmIL-12p40 and LmIL-12p80 proteins were further studied in vitro using a bacterial growth inhibition test (for both liquid and solid cultures) and in vivo (using a bacterial growth inhibition test with the head-kidney tissues). Furthermore, BL21 (DE3) E. coli cells transformed with the recombinant pET28a-LmIL-12p40 vector were dramatically protected in response to metal toxicity and H2O2-related oxidative stress. In summary, this study will provide foundational information regarding the role of LmIL-12p40 in defending against various biotic and abiotic stresses in fishes, which should help to further clarify the functional mechanism of interleukins.
Growing evidence has revealed that the transcription factor basic leucine zipper transcription factor ATF-like 2 (BATF2) has unique transcriptional activities, including regulating cytokines via TLR signals in macrophages, which affect mortality due to infection and cancer. Based on genome-wide analyses using the chromatin immunoprecipitation-sequencing technique, we found that dual-specificity phosphatase 2 (Dusp2) had a significantly lower acetyl-histone status in Batf2-/- bone marrow-derived macrophages (BMDMs) compared with wild-type (WT) BMDMs. The phosphatase DUSP2 has been reported to play a critical role in inflammatory responses. Therefore, we evaluated the BATF2 transcriptional activities on the Dusp2 promoter. We found that the DUSP2 and IL-12 p40 expression levels were significantly lower in Batf2-/- BMDMs than in WT controls following their stimulation with TLR7 ligands. Further in vitro studies revealed that phospho-STAT3 was upregulated and NF-κB p50/p65 were downregulated in Batf2-/- BMDMs compared with their levels in WT controls. Additionally, Th1 immunity was impaired in Batf2-/- mice following their stimulation with TLR7 ligands. We also found that BATF2 interacts with NF-κB p65 and promotes DUSP2 expression through the NF-κB-binding site in the Dusp2 promoter at -203 to -121. Collectively, our findings suggest that BATF2 activates DUSP2 gene expression and upregulates NF-κB activity via phospho-STAT3 dephosphorylation.
Interleukin-12 is a heterodimeric cytokine produced primarily by pathogen-activated antigen-presenting cells, particularly macrophages and dendritic cells, during encountering with intracellular microbes. IL-12 plays a key role in the activation of natural killer cells and CD4+ T helper cells in both innate and adaptive immune responses against infectious agents and immunosurveillance against endogenous malignancies. However, the potency of IL-12 makes it a target for stringent regulation. Indeed, the temporal, spatial, and quantitative expression of IL-12 during an immune response in a microenvironment contributes critically to the determination of the type, extent, and ultimate resolution of the reaction. Breaching of the delicate control and balance involving IL-12 frequently leads to autoimmune inflammatory disorders and pathogenesis. Thus, a better understanding of the regulatory mechanisms in the production and control of this cytokine is both scientifically significant and clinically beneficial. Here we provide an update on the research that has been conducted on this subject particularly in the last 10 years since the publication of a major thesis of this nature.
Interleukin-10 and Interleukin-12 are produced primarily by pathogen-activated antigen-presenting cells, particularly macrophages and dendritic cells. IL-10 and IL-12 play very important immunoregulatory roles in host defense and immune homeostasis. Being anti- and pro-inflammatory in nature, respectively, their functions are antagonistically opposing. A comprehensive and in-depth understanding of their immunological properties and signaling mechanisms will help develop better clinical intervention strategies in therapy for a wide range of human disorders. Here, we provide an update on some emerging concepts, controversies, unanswered questions, and opinions regarding the immune signaling of IL-10 and IL-12.
Interleukin (IL)-36α - one of the novel members of the IL-1 family of cytokines - is a potent regulator of dendritic and T cells and plays an important role in inflammatory processes like experimental skin inflammation in mice and in mouse models for human psoriasis. Here, we demonstrate that C/EBPβ, a transcription factor required for the selective expression of inflammatory genes, is a key activator of the Il36A gene in murine macrophages. RNAi-mediated suppression of C/EBPβ expression in macrophages (C/EBPβ(low) cells) significantly impaired Il36A gene induction following challenge with LPS. Despite of the presence of five predicted C/EBP binding sites, luciferase reporter assays demonstrated that C/EBPβ confers responsiveness to LPS primarily through a half-CRE•C/EBP element in the proximal Il36A promoter. Electrophoretic mobility shift assays showed that C/EBPβ but not CREB proteins interact with this critical half-CRE•C/EBP element. In addition, overexpression of C/EBPβ in C/EBPβ(low) cells enhanced expression of Il36A whereas CREB-1 had no effect. Finally, chromatin immunoprecipitation confirmed that C/EBPβ but neither CREB-1, ATF-2 nor ATF4 is directly recruited to the proximal promoter region of the Il36A gene. Together, these findings demonstrate an essential role of C/EBPβ in the regulation of Il36A gene via the proximal half-CRE•C/EBP element in response to inflammatory stimuli.
Copyright © 2015. Published by Elsevier B.V.
Nine new diterpenoids, termed sinumaximols A–I (1–3, 5–9, 12), together with three known compounds (4, 10, 11), were isolated from the methanol extract of the soft coral Sinularia maxima. Their structures were elucidated on the basis of extensive spectroscopic methods, including one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). The isolated compounds were evaluated for their inhibitory effects on the lipopolysaccharide (LPS)-stimulated production of pro-inflammatory cytokines in bone marrow-derived dendritic cells (BMDCs). Among them, compounds 2, 3, and 11 were potent inhibitors of LPS-stimulated interleukin-12 (IL-12) p40 with half-maximal inhibitory concentration (IC 50) values ranging from 4.350.12 to 18.040.21 µM. Compounds 2, 3, and 11 showed moderate inhibitory activity on IL-6 production with IC 50 values ranging from 17.720.31 to 59.772.34 µM. Soft corals (Cnidaria: Octocorallia: Alcyonacea: Alcyo-niina) are distributed worldwide and are important components of coral reef ecosystems, especially in the Indo-Pacific region. 1) These marine invertebrates are a rich source of secondary metabolites, including terpenoids and hydroxylated sterols. Many soft corals contain a very high diterpene level; in some species of Sarcophyton, diterpenes can comprise up to 10% of their dry weight. Large amounts of these secondary metabolites may play an important role in the survival of soft coral with respect to defensive, competitive, reproductive, and possibly pheromonal functions. 2)
Activation of murine peritoneal macrophages or the macrophage cell line RAW264 with IFN-g and bacterial lipopolysaccharide promotes a transient up-regulation of c-fos family gene expression following inducible NO synthase (iNOS) production. Since introduction of a double mutation into the two AP-1-binding sites in the iNOS promoter region reduced the promoter activity to 25% of the authentic one in activated RAW264 cells, the induced c-Fos/AP-1 may promote iNOS expression in activated macrophages. Surprisingly, overexpression of c-fos in activated macrophages completely suppressed the production of iNOS, but not that of IL-6 and IL-1b. The regulatory effect was also observed by overexpression of c-fos ,c -jun or fosB on the promoter activity as deduced from transfection experiments. However, the mutation of AP-1-binding sites in the promoter region did not abrogate the regulatory effect of c-fos and the effect of c-fos was diminished by co-transfection with c-jun, but not with fosB, suggesting no relation between the regulatory effect and a c-Fos/AP-1 complex. Expression of NF-IL6 (C/EBPb), whose gene product can make a non-functional heterodimer with c-Fos family proteins, was transiently induced in activated macrophages. Overexpression of NF-IL6 in activated RAW264 cells augmented iNOS promoter activity and reduced the regulatory effect of c-fos overexpression. Thus, overproduction of c-Fos family proteins acts as a dominant-negative-type regulator on iNOS expression in activated macrophages.
To understand the mechanisms by which large increases in serum amyloid A (SAA) occur during the acute phase response, human hepatoma cells were transfected with SAA2 gene reporter plasmids and stimulated with combinations of cytokines. Although interleukin-1 (IL-1) and interleukin-6 (IL-6) stimulated transcription from this promoter individually, addition of both mediators produced a response between two and nine times greater than the expected additive response. This synergistic activation was dependent on the integrity of at least two cis-acting sequences in the SAA2 enhancer. The SAA2 NF-kappaB site was required functionally for the response to both IL-1 and IL-6 alone as well as for synergistic activation; however, IL-6 did not directly induce binding of nuclear proteins to the NF-kappaB sequence. A NF-IL-6 site was required for full induction by IL-1 and IL-6, and also mediated strong transactivation by recombinant NF-IL6. Furthermore, transfected NF-IL6 synergized strongly with co-transfected NF-kappaB, particularly with RelA (p65). However synergy between IL-1 and IL-6 was only partly reduced by mutation of the NF-IL6 site, indicating further levels of interaction in addition to the NF-kappaB/NF-IL6 cooperativity.
The promoter regions of three IL-6 Inducible genes, hemopexin (Hpx), haptoglobin (Hp) and C-reactive protein (CRP) contain
cs-acting IL-6 responsive elements (IL-6RE) which are necessary and sufficient to induce IL-6 transcription activation. Transcription
factors of the CEBP family interact with IL-6REs. Among these, IL-6DBP NF-IL6 plays a key role in IL-6 signal transduction
because its frans-activation potential Is induced by IL-6 in the human hepatoma cell line Hep3B. We show here that a different
CEBP related factor, C/EBP/ NF L6β, is the major IL-6 induced protein interacting with IL-6REs in the nuclei of Hep3B cells.
In contrast to IL-6DBPδNF/-IL6, whose activity in Hep3B cells is modulated by IL-6 via a post translational mechanism, CEBδPS/NF-IL6β
is transcriptionally induced by IL-6. Another contrasting feature is that the CEBP6 cDNA transfected In Hep3B cells activates
transcription from an IL-6RE synthetic promoter in a constitutive manner which is not further enhanced by IL-6. Therefore,
in Hep3B cells, two distinct members of the CEBP family are recruited In the IL-6 signal transductlon pathway via different
mechanisms.
The levels of interferon mRNA as a function of interferon induction by poly(rI) . poly(rC) in human fibroblast cells were determined by RNA hybridization using a cloned beta interferon cDNA and by translation in Xenopus oocytes. Whereas previous studies analyzed mixtures of interferons, the availability of the cloned beta interferon cDNA and the antiserum to purified beta interferon enabled us to focus on the expression of only one class (beta) of interferon genes. The induction of interferon synthesis depends primarily on the accumulation of interferon beta mRNA in the cells, and the interferon beta mRNA rapidly disappears several hours after its appearance in the cytoplasm. No detectable interferon beta mRNA sequences are present in uninduced cells. The degradation of interferon beta mRNA in the induced cells requires ongoing protein synthesis; accumulation of interferon beta mRNA was observed in the continuous presence of cycloheximide. The interferon beta mRNA detected at the early stages of induction is 1100 nucleotides long and its size progressively decreases with time. By both the hybridization and the translational assay in Xenopus oocytes, only one size of interferon beta mRNA and one species of beta interferon could be identified.
Natural killer cell stimulatory factor (NKSF), or interleukin 12 (IL-12), is a 70-kD heterodimeric cytokine composed of two covalently linked chains, p40 and p35. NKSF/IL-12 has multiple effects on T and NK cells and was originally identified and purified from the supernatant fluid of Epstein-Barr virus (EBV)-transformed human B lymphoblastoid cell lines. We have produced a panel of monoclonal antibodies against both chains of NKSF/IL-12. Some of these antibodies have neutralizing activity, and several combinations of them have been used to establish sensitive radioimmunoassays detecting the free p40 chain, the free p35 chain, or the p70 heterodimer. Using these reagents, we have determined that most EBV-transformed human B lymphoblastoid cell lines constitutively produce low levels of the p70 heterodimer and an excess of the free p40 chain, whereas Burkitt lymphoma-derived, T, myeloid, and many solid tumor-derived cell lines produce neither. Production of both p40 and p70 is increased several-fold upon stimulation of the EBV-transformed cell lines with phorbol diesters. The ability of supernatant fluids from unstimulated and phorbol diester-stimulated cell lines to induce interferon gamma (IFN-gamma) production from T and NK cells, one of the effects of NKSF/IL-12, parallels the levels of production of the p70 heterodimer, known to be the biologically active form of NKSF/IL-12. Staphylococcus aureus Cowan I strain (SAC) and other stimuli induce accumulation of p40 mRNA and production of both p40 and p70 by peripheral blood mononuclear cells (PBMC). The producer cells appear to include both adherent cells and nonadherent lymphocytes, possibly B cells. The supernatant fluids from SAC-stimulated PBMC mediate the typical functions of NKSF/IL-12 (i.e., IFN-gamma induction, mitogenic effects on T/NK blasts, enhancement of NK cell cytotoxicity) at concentrations of p70 similar to those at which recombinant NKSF/IL-12 mediates the same functions. Moreover, these activities are significantly inhibited by anti-NKSF/IL-12 antibodies. The neutralizing anti-NKSF/IL-12 antibodies also inhibit 85% of the IFN-gamma production in response to SAC, an NKSF/IL-12 inducer, and approximately 50% of the IFN-gamma production in response to non-NKSF/IL-12-inducers such as IL-2, phytohemagglutinin, and anti-CD3 antibodies. These results indicate that induced or constitutively produced NKSF/IL-12 has a major role in facilitating IFN-gamma production by peripheral blood lymphocytes.(ABSTRACT TRUNCATED AT 400 WORDS)
Members of the CCAAT/enhancer binding protein (C/EBP) family have been shown to regulate the terminal differentiation of adipocytes and hepatocytes. In these cell lineages, high levels of C/EBP alpha are found only in mature, nondividing cells. Using Western blotting and immunohistochemical staining, we have determined the temporal order of expression for C/EBP alpha, C/EBP beta, and C/EBP delta in differentiating myelomonocytic marrow cells. These studies show a unique temporal pattern of C/EBP isoform expression in the myeloid lineage. In particular, C/EBP alpha expression is very high in proliferative myelomonocytic cells, and diminishes during phenotypic maturation. While we have detected C/EBP alpha, C/EBP beta, and C/EBP delta in multiple myeloid leukemia cell lines, and C/EBP alpha in normal myeloid cells and in de novo human myeloid leukemias, we have not detected these C/EBP isoforms in either erythroid or lymphoid cells. Finally, we show that C/EBP alpha, C/EBP beta, and C/EBP delta protein and messenger RNA levels correlate in maturing granulocytic cells. The formation of tissue-specific combinations of C/EBP homodimers and heterodimers may allow this family of transcription factors to regulate different sets of genes in adipocytes, hepatocytes, and myelomonocytes.
The c-fos serum response element (SRE) is a multifunctional regulatory region of the c-fos promoter that responds to a variety of inducers. Recently, we have demonstrated that the SRE binds the C/EBP-related transcription factor rat NFIL-6 (rNFIL-6). In this study we show that rNFIL-6 is regulated by the cAMP second messenger pathway in the rat pheochromocytoma PC12 cell line. Following forskolin treatment, rNFIL-6 binding to the SRE is increased, and the factor becomes phosphorylated and undergoes a trans-location to the nucleus. In transient cotransfection assays, rNFIL-6 is capable of trans-activating the c-fos promoter in a manner dependent on the SRE. These data show that rNFIL-6 undergoes a novel activation in which cAMP-induced nuclear trans-location allows rNFIL-6 to bind to the SRE and contribute to c-fos activation. We propose that rNFIL-6 is an additional regulatory component of the c-fos gene, which provides cAMP responsiveness to the multifunctional SRE.
We have developed a procedure for preparing extracts from nuclei of human tissue culture cells that directs accurate transcription
initiation in vitro from class II promoters. Conditions of extraction and assay have been optimized for maximum activity using
the major late promoter of adenovirus 2. The extract also directs accurate transcription initiation from other adenovirus
promoters and cellular promoters. The extract also directs accurate transcription initiation from class III promoters (tRNA
and Ad 2 VA).
The promoter regions of three IL-6 inducible genes, hemopexin (Hpx), haptoglobin (Hp) and C-reactive protein (CRP) contain cis-acting IL-6 responsive elements (IL-6REs) which are necessary and sufficient to induce IL-6 transcription activation. Transcription factors of the C/EBP family interact with IL-6REs. Among these, IL-6DBP/NF-IL6 plays a key role in IL-6 signal transduction because its trans-activation potential is induced by IL-6 in the human hepatoma cell line Hep3B. We show here that a different C/EBP-related factor, C/EBP delta/NF-IL6 beta, is the major IL-6 induced protein interacting with IL-6REs in the nuclei of Hep3B cells. In contrast to IL-6DBP/NF-IL6, whose activity in Hep3B cells is modulated by IL-6 via a post-translational mechanism, C/EBP delta/NF-IL6 beta is transcriptionally induced by IL-6. Another contrasting feature is that the C/EBP delta cDNA transfected in Hep3B cells activates transcription from an IL-6RE synthetic promoter in a constitutive manner which is not further enhanced by IL-6. Therefore, in Hep3B cells, two distinct members of the C/EBP family are recruited in the IL-6 signal transduction pathway via different mechanisms.
The promoter region of the mouse gene for macrophage-inducible nitric oxide synthase (mac-NOS; EC 1.14.13.39) has been characterized. A putative TATA box is 30 base pairs upstream of the transcription start site. Computer analysis reveals numerous potential binding sites for transcription factors, many of them associated with stimuli that induce mac-NOS expression. To localize functionally important portions of the regulatory region, we constructed deletion mutants of the mac-NOS 5' flanking region and placed them upstream of a luciferase reporter gene. The macrophage cell line RAW 264.7, when transfected with a minimal promoter construct, expresses little luciferase activity when stimulated by lipopolysaccharide (LPS), interferon gamma (IFN-gamma), or both. Maximal expression depends on two discrete regulatory regions upstream of the putative TATA box. Region I (position -48 to -209) increases luciferase activity approximately 75-fold over the minimal promoter construct. Region I contains LPS-related responsive elements, including a binding site for nuclear factor interleukin 6 (NF-IL6) and the kappa B binding site for NF-kappa B, suggesting that this region regulates LPS-induced expression of the mac-NOS gene. Region II (position -913 to -1029) alone does not increase luciferase expression, but together with region I it causes an additional 10-fold increase in expression. Together the two regions increase expression 750-fold over activity obtained from a minimal promoter construct. Region II contains motifs for binding IFN-related transcription factors and thus probably is responsible for IFN-mediated regulation of LPS-induced mac-NOS. Delineation of these two cooperative regions explains at the level of transcription how IFN-gamma and LPS act in concert to induce maximally the mac-NOS gene and, furthermore, how IFN-gamma augments the inflammatory response to LPS.
C/EBP beta is considered a key element of interleukin-6 (IL-6) signalling as well as an important transcriptional regulator of the IL-6 gene itself. We describe here how mice lacking C/EBP beta develop a pathology similar to mice overexpressing IL-6 and nearly identical to multicentric Castleman's disease in human patients, with marked splenomegaly, peripheral lymphadenopathy and enhanced haemopoiesis. Humoral, innate and cellular immunity are also profoundly distorted, as shown by the defective activation of splenic macrophages, the strong impairement of IL-12 production, the increased susceptibility to Candida albicans infection and the altered T-helper function. Our data show that C/EBP beta is crucial for the correct functional regulation and homeostatic control of haemopoietic and lymphoid compartments.
Peripheral blood mononuclear cells (PBMC) from human immunodeficiency virus (HIV)-infected patients, asymptomatic or with acquired immunodeficiency virus, produced 10-fold less interleukin 12 (IL-12) free heavy chain and fivefold less biologically active IL-12 heterodimer than PBMC from uninfected healthy donors when challenged in vitro with the common human pathogen Staphylococcus aureus. In contrast, PBMC from HIV-infected individuals and uninfected control donors produced similar levels of tumor necrosis factor alpha, IL-1 beta, and IL-10, and PBMC from HIV-infected individuals produced three- to fourfold more IL-6 compared with PBMC from uninfected control donors. The defect in IL-12 production is not due to hyperproduction of IL-10, a cytokine exerting an autocrine-negative feedback on IL-12 production, but was directly related to HIV infection, as suggested by the reduced ability of monocytes infected in vitro with HIV to produce IL-12. IL-12 deficiency may be an important component of the immunodeficiency associated with HIV infection.
Activated macrophages contribute to chronic inflammation by the secretion of cytokines and proteinases. Tumor necrosis factor alpha (TNF alpha) is particularly important in this process because of its ability to regulate other inflammatory mediators in an autocrine and paracrine fashion. The mechanism(s) responsible for the cell type-specific regulation of TNF alpha is not known. We present data to show that the expression of TNF alpha is regulated by the transcription factor C/EBP beta (NF-IL6). C/EBP beta activated the TNF alpha gene promoter in cotransfection assays and bound to it at a site which failed to bind the closely related protein C/EBP alpha. Finally, a dominant-negative version of C/EBP beta blocked TNF alpha promoter activation in myeloid cells. Our results implicate C/EBP beta as an important regulator of TNF alpha by myelomonocytic cells.
NF-kappa B and C/EBP represent distinct families of transcription factors that target unique DNA enhancer elements. The heterodimeric NF-kappa B complex is composed of two subunits, a 50- and a 65-kDa protein. All members of the NF-kappa B family, including the product of the proto-oncogene c-rel, are characterized by their highly homologous approximately 300-amino-acid N-terminal region. This Rel homology domain mediates DNA binding, dimerization, and nuclear targeting of these proteins. C/EBP contains the bZIP region, which is characterized by two motifs in the C-terminal half of the protein: a basic region involved in DNA binding and a leucine zipper motif involved in dimerization. The C/EBP family consist of several related proteins, C/EBP alpha, C/EBP beta, C/EBP gamma, and C/EBP delta, that form homodimers and that form heterodimers with each other. We now demonstrated the unexpected cross-coupling of members of the NF-kappa B family three members of the C/EBP family. NF-kappa B p65, p50, and Rel functionally synergize with C/EBP alpha, C/EBP beta, and C/EBP delta. This cross-coupling results in the inhibition of promoters with kappa B enhancer motifs and in the synergistic stimulation of promoters with C/EBP binding sites. These studies demonstrate that NF-kappa B augments gene expression mediated by a multimerized c-fos serum response element in the presence of C/EBP. We show a direct physical association of the bZIP region of C/EBP with the Rel homology domain of NF-kappa B. The cross-coupling of NF-kappa B with C/EBP highlights a mechanism of gene regulation involving an interaction between distinct transcription factor families.
Natural killer cell-stimulatory factor or interleukin-12 (NKSF/IL-12) was originally identified and purified from the conditioned medium of Epstein-Barr virus (EBV)-transformed B-cell lines. Phorbol diesters were observed to be potent stimulators of NKSF/IL-12 production from the B-cell lines. Although monocytes were found to be the major producers of NKSF/IL-12 in peripheral blood (PB) in response to lipopolysaccharide (LPS) or to Staphylococcus aureus, several myeloid leukemia cell lines tested did not produce detectable NKSF/IL-12 either constitutively or upon stimulation with phorbol diesters. However, three lines, ML-3, HL-60, and THP-1, responded to LPS with significant levels of NKSF/IL-12 production, whereas S aureus was effective only on THP-1 cells. When the cell lines were preincubated with compounds known to induce them to differentiate, production of tumor necrosis factor alpha (TNF alpha) and IL-1 beta was in most cases maximal in cells with differentiated characteristics, whereas NKSF/IL-12 production in response to LPS in all three producing cell lines was significantly enhanced only by pretreatment with dimethylsulfoxide (DMSO) for 24 hours, or by costimulation with interferon gamma (IFN gamma). The efficiency of DMSO enhancement of NKSF/IL-12 production decreased after 2 to 5 days of incubation, when the cells acquired differentiated characteristics. Unlike DMSO, IFN gamma enhanced NKSF/IL-12 production, and IL-10 and dexamethasone inhibited it in cell lines and PB mononuclear cells stimulated by either LPS or S aureus. The ability of the cell lines to respond to these mediators of possibly physiologically relevant function provides a tissue-culture model for studying their mechanism of action.
Members of the CCAAT/enhancer binding protein (C/EBP) family have been shown to regulate the terminal differentiation of adipocytes and hepatocytes. In these cell lineages, high levels of C/EBP alpha are found only in mature, nondividing cells. Using Western blotting and immunohistochemical staining, we have determined the temporal order of expression for C/EBP alpha, C/EBP beta, and C/EBP delta in differentiating myelomonocytic marrow cells. These studies show a unique temporal pattern of C/EBP isoform expression in the myeloid lineage. In particular, C/EBP alpha expression is very high in proliferative myelomonocytic cells, and diminishes during phenotypic maturation. While we have detected C/EBP alpha, C/EBP beta, and C/EBP delta in multiple myeloid leukemia cell lines, and C/EBP alpha in normal myeloid cells and in de novo human myeloid leukemias, we have not detected these C/EBP isoforms in either erythroid or lymphoid cells. Finally, we show that C/EBP alpha, C/EBP beta, and C/EBP delta protein and messenger RNA levels correlate in maturing granulocytic cells. The formation of tissue-specific combinations of C/EBP homodimers and heterodimers may allow this family of transcription factors to regulate different sets of genes in adipocytes, hepatocytes, and myelomonocytes.
The purpose of this study was to determine whether the female hormones estradiol-17β (E2) and progesterone (P4) influence inducible nitric oxide synthase (iNOS) and the production of nitric oxide (NO) by interferon-γ(IFN-γ)- and lipopolysaccharide (LPS)-activated mouse macrophages. Treatment with P4 alone caused a time- and dose-dependent inhibition of NO production by macrophage cell lines (RAW 264.7, J774) and mouse bone marrow culture-derived macrophages as assessed by nitrite accumulation. RAW 264.7 cells transiently transfected with an iNOS gene promoter/luciferase reportergene construct that were stimulated with IFN-γ/LPS in the presence of P4 displayed reduced luciferase activity and NO production. Analysis of RAW 264.7 cells by Northern blot hybridization revealed concurrent P4-mediated reduction in iNOS mRNA. These observations suggest that P4-mediated inhibition of NO may be an important gender-based difference within females and males that relates to macrophage-mediated host defense.
The expression of B lineage associated genes during early B cell differentiation stages is not firmly established. Using cell surface markers and multiparameter flow cytometry, bone marrow (BM) cells can be resolved into six fractions, representing sequential stages of development; i.e., pre-Pro-B, early Pro-B, late Pro-B/large Pre-B, small Pre-B, immature B, and mature B cells. Here we quantitate the levels of several B lineage associated genes in each of these fractions by RT-PCR, demonstrating different patterns of expression. We find that expression of terminal deoxynucleotidyl transferase (TdT), lambda 5, and VpreB is predominantly restricted to the Pro-B stages. Rag-1 and Rag-2 expression is also tightly regulated, and is found largely in the Pro-B through small Pre-B stages. Mb-1 is present from Pro-B throughout the pathway at high levels. Finally, Bcl-2 is expressed at high levels only at the pre-Pro-B and mature B stages, whereas it is low during all the intermediate stages. We also correlate this expression data with an analysis of the onset of Ig gene rearrangement as assessed by amplifying D-JH, VH-DJH, and VK-JK. Finally, we report differences in gene expression during B lymphopoiesis at two distinct ontogenic timings, in fetal liver and adult BM: both TdT and the precursor lymphocyte regulated myosin-like light chain are expressed at high levels in the Pro-B cell stage in bone marrow, but are absent from the corresponding fraction in fetal liver. In contrast, lambda 5, VpreB, Rag-1, and Rag-2 are expressed at comparable levels.
Members of the CCAAT/enhancer binding protein (C/ EBP) family have been shown to regulate the terminal differentia- tion of adipocytes and hepatocytes. In these cell lineages, high levels of C/EBPa are found only in mature, nondividing cells. Using Western blotting and immunohistochemical stain- ing, we have determined the temporal order of expression for C/EBPa, C/EBPp, and C/ EBPG in differentiating myelomono- cytic marrow cells. These studies show a unique temporal pattern of C/EBP isoform expression in the myeloid lineage. In particular, C/EBPa expression is very high in proliferative myelomonocytic cells, and diminishes during phenotypic maturation. While we have detected C/EBPa, C/EBPp, and HE CCAAT/enhancer-binding protein (C/EBP) fam- T ily of transcription factors consists of several proteins with highly homologous dimerization and DNA contact domains. The founding member of this family, C/EBPa, was originally purified from rat This protein binds DNA as an obligate homodimer via a basic region-leucine zipper (bZIP) domain.'-6 The C/EBP family also includes C/EBPP (also known as NF-IL6, LAP, IM-DBP, AGP/ EBP, and CRP2),7-12 CIEBPG (also known as CRP3),"J2 and C/EBPy (also known as Ig/EBP-1).I3 The various C/EBP family members can homodimerize, but can also heterodimerize and maintain DNA-binding activity.11-13 Members of the C/EBP family, especially C/EBPu, have been implicated in regulating the terminal differentiation of several mammalian cells. Although present at low levels in hepatoma cell lines, C/EBPa is expressed at high levels in mitotically quiescent hepatocytes, in which it is believed to regulate a variety of hepatocyte-specific genes.14J5 Simi- larly, although absent from 3T3-Ll preadipocytes, C/EBPa is expressed at high levels when these cells differentiate into nondividing adipocytes.11J6 C/EBPa is capable of activating several "fat-specific'' genes in 3T3-Ll cells, including steroyl- CoA desaturase and the insulin-responsive glucose trans- p~rter.'~J* Selective inhibition of C/EBPa expression in differentiating adipocytes by expression of antisense C/EBPa RNA reduced the ability of these cells to form lipid droplets, a phenotype of their terminally differenti- ated ~tate.'~,~~ In contrast to C/EBPa, C/EBPP and C/EBPG are expressed highest in the early stages of adipocyte formation." The ability of C/EBP family members to heterodimerize may allow complex regulation of hepato- cyte and adipocyte development.21 In both hepatocytes and adipocytes, high levels of C/EBPa expression have been found only in nondividing cells. Moreover, when C/EBPa is ectopically expressed in divid- ing preadipocytes, mitotic proliferation ceases.22 Therefore, it has been proposed that C/EBPu may regulate a genetic program that blocks cell divi~ion.~~J2 We have now discovered that members of the C/EBP family are also expressed in myelomonocytic cells of human and rodent bone marrow. Using the myelomonoblastic murine cell line 32D C13, a valuable model of granulocytic myeloid differentiati0n,2~,~~ we have examined the expres- sion profile of C/EBPa, C/EBPP, and C/EBPS as a C/EBPG in multiple myeloid leukemia cell lines, and C/EBPa in normal myeloid cells and in de novo human myeloid leukemias, we have not detected these C/EBP isoforms in either erythroid or lymphoid cells. Finally, we show that C/EBPa. C/EBPp, and C/EBPS protein and messenger RNA levels correlate in maturing granulocytic cells. The formation of tissue-specific combinations of C/EBP homodimers and heterodimers may allow this family of transcription factors to regulate different sets of genes in adipocytes, hepatocytes, and myelomonocytes. o 1992 by The American Society of Hematology. function of granulocytic differentiation. Although variabil- ity of this cell line in culture resulted in some quantitative differences between experiments, a surprisingly different pattern from that described for adipocyte development was evident. Most notably, C/EBPa was found to be expressed at a high level in dividing myeloblasts and to diminish to low levels during their terminal differentiation into polymorpho- nuclear leukocytes (PMNs). We will discuss the implica- tions of this novel temporal pattern of C/EBP isoform expression.
Peripheral blood mononuclear cells (PBMCs) from many asymptomatic individuals infected with human immunodeficiency virus-type 1 (HIV) are unresponsive as measured by in vitro T cell proliferation and interleukin-2 (IL-2) production to influenza virus and synthetic peptides of HIV envelope (Env). Strong influenza virus- and Env-stimulated IL-2 responses and T cell proliferation were restored when cultures were stimulated in the presence of IL-12. Interferon-gamma production by PBMCs from HIV seropositive (HIV^+) patients was also restored with IL-12. Furthermore, in vitro antigen-specific production of IL-2 and proliferation of PBMCs from HIV^- donors were suppressed by antibody to IL-12, but were not enhanced by addition of exogenous IL-12. Thus, IL-12 may be limiting in PBMCs from HIV^+ but not HIV^- individuals. These findings demonstrate that IL-12 can restore HIV-specific cell-mediated immunity in vitro in HIV-infected individuals and suggest a potential use of IL-12 in augmenting the diminished immunologic functions associated with HIV infection.
We have analyzed the interleukin-l beta (IL-1 beta) promoter region near the cap site. Specific DNA sequences required for lipopolysaccharide (LPS) induction within this region were identified using transfection of reporter plasmids that contained portions of the proximal IL-1 beta 5 '-flanking sequence. An LPS-responsive activation area was localized between nucleotides -50 to -100, and down-regulating sequences were present between nucleotides -100 and -2,111. A NFIL-6 site between -92 and -84 was identified in the functionally active region. Base substitutions within this single NFIL-6 site in the context of a 4.1-kb IL-1 beta promoter segment resulted in dramatic reduction of LPS-induced gene transcription. Introduction of multimers of this NFIL-6 sequence immediately 5 ' to minimal homologous or heterologous promoters conferred LPS inducibility in each case. Anti-C/EBP beta (NFIL-6) and anti-C/EBP delta (NFIL-6 beta) antibodies identified both of these proteins in complexes formed between the NFILd site and mononuclear cell nuclear extracts. These data show that the proximal NFIL-6 site is required for the activation of murine IL-1 beta gene expression by endotoxin.
Human IL-12 (NK cell stimulatory factor, cytotoxic lymphocyte maturation factor) is a heterodimeric cytokine that can act as a growth factor for activated human T and NK cells, enhance the lytic activity of human NK/lymphokine-activated killer cells, and stimulate the production of IFN-gamma by resting human PBMC. Because in our hands, human IL-12 did not elicit similar responses in murine lymphocytes, we have cloned and expressed the murine IL-12 subunit cDNA in order to obtain recombinant protein for murine studies. Comparison of the predicted amino acid sequences of the murine subunits with their human counterparts revealed that the p40 subunits are more highly conserved than the p35 subunits (70% vs 60% identity, respectively). The sizes of the p35 and p40 subunit mRNA were estimated to be 1.5 kb and 2.6 kb, respectively. RNA blot analysis showed that p35 mRNA was expressed in lymphoid tissues (spleen, thymus) and nonlymphoid tissues (lung, brain), whereas p40 mRNA expression was only detected in lymphoid cells. Incubation of splenocytes with pokeweed mitogen did not significantly affect p35 mRNA levels, however, it resulted in a decrease of p40 mRNA. Coexpression of the murine p35 and p40 cDNA clones in COS cells resulted in the secretion of IL-12, which was active in human and mouse T cell proliferation, murine NK cell activation, and murine IFN-gamma induction assays. Transfection of each subunit cDNA alone did not result in measurable secreted IL-12 activity. A hybrid heterodimer consisting of murine p35 and human p40 subunits retained bioactivity on murine cells; however, the combination of human p35 and murine p40 was completely inactive on murine cells. These results indicate that the observed inability of human IL-12 to act on murine cells is largely determined by the p35 subunit.
The NF-kappa B-p50 polypeptide, a member of the Rel family of transcription factors, was produced as a fusion protein containing amino-terminal peptide additions that facilitate purification and detection with a monoclonal antibody and specific radiolabeling by phosphorylation in vitro. The 32P-labeled NK-kappa B-p50 fusion polypeptide was used as the probe in Western blotting experiments and in screenings of a bacteriophage expression library to isolate cDNAs encoding interacting protein domains. As expected, cDNAs encoding proteins of the Rel family were identified. Surprisingly, the 32P-labeled NF-kappa B protein also specifically bound to proteins encoded by cDNAs for the human NF-IL6 transcription factor. The NF-kappa B-p50 and NF-IL6 proteins directly interact, and the Rel homology domain and leucine-zipper motif, respectively, are important for this interaction. Since induction of the NF-kappa B and NF-IL6 factors are important events in immune and acute-phase responses, this interaction could permit coregulation of genes.
Previously we have reported the purification and characterization of a novel cytokine from an EBV-transformed B cell line, RPMI 8866. This factor, termed natural killer cell stimulatory factor (NKSF), possessed pleiotropic activities including the induction of IFN-gamma from PBL, enhancement of cytotoxicity by NK cells, and stimulation of the proliferation of PBL. Purified NKSF was found to be a disulfide-linked heterodimeric protein composed of 35-kDa and 40-kDa subunits (p35 and p40). We now report the molecular cloning of cDNA for both subunits of NKSF from RPMI 8866 cellular RNA. The cDNA sequences indicate that both genes are novel, and Southern blot analysis confirmed that both cDNA are of human genomic origin. [35S]Methionine labeling indicated that cos-1 cells transfected with either p35 or p40 cDNA produced unique protein species of appropriate size. Methionine labeling of cos-1 cells cotransfected with p35 plus p40 cDNA yielded a broad band migrating between 70 and 90 kDa on a nonreducing gel. Reduction of this high molecular weight material yielded bands correlating with p35 and p40 gene products. Only culture supernatant from cotransfected cos-1 cells had a high level of NKSF biologic activity. That the high molecular weight material was responsible for this activity was indicated by the observation that biologic activity in the culture supernatant migrated at 70 to 90 kDa in a nonreducing gel. Furthermore, anti-p40 serum was able to block the biologic activities of both recombinant and natural NKSF, which indicates that it is a component of the active protein. In contrast, no activity could be detected in the supernatants of cos-1 cells transfected with p40 or p35 cDNA alone. The spectrum of biologic activity produced by cotransfected cos-1 cells was the same as NKSF purified to homogeneity from the RPMI 8866 cell line. A synergistic augmentation of some of these responses was found by the addition of IL-2 or the co-stimulators PHA or phorbol diester. The synergistic stimulation by NKSF plus IL-2 of T and NK function supports the possibility that these cytokines might prove useful in cancer therapy.
Cytotoxic lymphocyte maturation factor (CLMF) is a disulfide-bonded heterodimeric lymphokine that (i) acts as a growth factor for activated T cells independent of interleukin 2 and (ii) synergizes with suboptimal concentrations of interleukin 2 to induce lymphokine-activated killer cells. We now report the cloning and expression of both human CLMF subunit cDNAs from a lymphoblastoid B-cell line, NC-37. The two subunits represent two distinct and unrelated gene products whose mRNAs are coordinately induced upon activation of NC-37 cells. Coexpression of the two subunit cDNAs in COS cells is necessary for the secretion of biologically active CLMF; COS cells transfected with either subunit cDNA alone do not secrete bioactive CLMF. Recombinant CLMF expressed in mammalian cells displays biologic activities essentially identical to natural CLMF, and its activities can be neutralized by monoclonal antibodies prepared against natural CLMF. Since this heterodimeric protein displays the properties of an interleukin, we propose that CLMF be given the designation interleukin 12.
In an effort to identify protein factors that play a regulatory role in the differentiation of adipocytes, we have isolated two genes that encode polypeptides related to CCAAT/enhancer-binding protein (C/EBP; hereafter termed C/EBP alpha). The proteins encoded by these C/EBP-related genes, termed C/EBP beta and C/EBP delta, exhibit similar DNA-binding specificities and affinities compared with C/EBP alpha. Furthermore, C/EBP beta and C/EBP delta readily form heterodimers with one another as well as with C/EBP alpha. The transcriptional activating capacity of these two newly identified C/EBP isoforms was demonstrated by transient transfection experiments in which expression vectors encoding C/EBP beta and C/EBP delta were observed to induce transcription from the promoter of the serum albumin gene in cultured hepatoma cells. The mRNAs encoding C/EBP beta and C/EBP delta were detected in a number of tissues, most of which corresponded to sites of expression of C/EBP alpha. The expression pattern of C/EBP beta and C/EBP delta during adipose conversion of 3T3-L1 cells was examined by Western and Northern blotting assays. In contrast to the expression profile of the gene encoding C/EBP alpha, whose product is not detectable until the late phase of adipocyte differentiation, the c/ebp beta and c/ebp delta genes were actively expressed very early during adipocyte differentiation. Moreover, transcription of the c/ebp beta and c/ebp delta genes was observed to be induced directly by adipogenic hormones. The accumulation of C/EBP beta and C/EBP delta reached a maximal level during the first 2 days of differentiation and declined sharply before the onset of C/EBP alpha accumulation. The temporal pattern of expression of these three C/EBP isoforms during adipocyte differentiation may reflect the underpinnings of a regulatory cascade that controls the process of terminal cell differentiation.
We have studied transcriptional control of the murine terminal deoxynucleotidyltransferase (TdT) gene, which is activated
specifically in immature B and T lymphocytes. This analysis has led to the identification and purification of a 50-kDa sequence-specific
DNA-binding protein, LyF-1, that interacts with the approximate consensus sequence PyPyTGGGAGPu and is enriched in cells at
most stages of B- and T-cell differentiation. LyF-1 binds tightly to an element in the TdT promoter that we show is required
for transcription in lymphocytes. LyF-1 also interacts with an element in the immunoglobulin mu enhancer, called microB, that
was recently shown to be important for lymphocyte-specific enhancer activity. Moreover, LyF-1 binds to the promoters for the
lymphocyte-specific genes lambda 5, VpreB, and lck, all of which we speculate have additional features in common with the
TdT promoter. Thus, LyF-1 may be a general transcriptional activator for genes whose expression is restricted to the B- and/or
T-lymphocyte lineages.
LAP, a transcriptional activator, and LIP, a transcriptional repressor, are translated from a single mRNA species by using two AUGs within the same reading frame. These two proteins share the 145 C-terminal amino acids that contain the basic DNA-binding domain and the leucine zipper dimerization helix. Probably owing to its higher affinity for its DNA cognate sequences, LIP can attenuate the transcriptional stimulation by LAP in substoichiometric amounts. As revealed by transient transfection experiments, a moderate increase in the LAP/LIP ratio results in a significantly higher transcriptional activation of an appropriate target gene. The LAP/LIP ratio increases about 5-fold during terminal rat liver differentiation and is thus likely to modulate the activity of LAP in the intact animal.
Lymphokine production is regulated both at the transcriptional and the posttranscriptional level. To date, it has been shown that the protein synthesis inhibitor cycloheximide (CHX) up-regulates IL-2 expression in T cells by stabilizing its mRNA. In this report we have examined the effect of CHX on IL-2 at the transcriptional level. We have found that CHX has a positive regulatory function in IL-2 transcription, which is dependent on prior activation of this gene. This is not due to posttranslational conversion of inactive NFkB into its active form by CHX, because a clustered mutation in the kB-like sequence in the IL-2 enhancer that abrogates NFkB binding does not affect the up-regulation of IL-2 transcription. These results favor the hypothesis that, in addition to positive factors, negative elements regulate IL-2 transcription. Furthermore, we have tested the effect of CHX on IL-4 and granulocyte-macrophage-CSF transcription of both lymphokines. These results suggest that transcriptional up-regulation by CHX may be specific for IL-2 with respect to lymphokine expression.
Transcription of the lymphocyte-specific terminal deoxynucleotidyltransferase gene begins at a single nucleotide, but no TATA box is present. We have identified a 17 bp element that is sufficient for accurate basal transcription of this gene both in vitro and in vivo. This motif, the initiator (Inr), contains within itself the transcription start site. Homology to the Inr is found in many TATA-containing genes, and specific mutagenesis influences both the efficiency and accuracy of initiation. Moreover, in the presence of either a TATA box or the SV40 21 bp repeats, a greatly increased level of transcription initiates specifically at the Inr. Thus, the Inr constitutes the simplest functional promoter that has been identified and provides one explanation for how promoters that lack TATA elements direct transcription initiation.
The latent period of AIDS is influenced by factors which activate human immunodeficiency virus (HIV) replication in different cell types. Although monocytic cells may provide a reservoir for virus production in vivo, their regulation of HIV transcription has not been defined. We now report that HIV gene expression in the monocyte lineage is regulated by NF-kappa B, the same transcription factor known to stimulate the HIV enhancer in activated T cells; however, control of NF-kappa B and HIV in monocytes differs from that observed in T cells. NF-kappa B-binding activity appears during the transition from promonocyte to monocyte in U937 cells induced to differentiate in vitro and is present constitutively in mature monocytes and macrophages. In a chronically infected promonocytic cell, U1, differentiation is associated with HIV-1 replication as well as NF-kappa B binding activity. These findings suggest that NF-kappa B binding activity is developmentally regulated in the monocyte lineage, and that it provides one signal for HIV activation in these cells.
Overlap extension represents a new approach to genetic engineering. Complementary oligodeoxyribonucleotide (oligo) primers and the polymerase chain reaction are used to generate two DNA fragments having overlapping ends. These fragments are combined in a subsequent 'fusion' reaction in which the overlapping ends anneal, allowing the 3' overlap of each strand to serve as a primer for the 3' extension of the complementary strand. The resulting fusion product is amplified further by PCR. Specific alterations in the nucleotide (nt) sequence can be introduced by incorporating nucleotide changes into the overlapping oligo primers. Using this technique of site-directed mutagenesis, three variants of a mouse major histocompatibility complex class-I gene have been generated, cloned and analyzed. Screening of mutant clones revealed at least a 98% efficiency of mutagenesis. All clones sequenced contained the desired mutations, and a low frequency of random substitution estimated to occur at approx. 1 in 4000 nt was detected. This method represents a significant improvement over standard methods of site-directed mutagenesis because it is much faster, simpler and approaches 100% efficiency in the generation of mutant product.
The immunoglobulin kappa light chain gene contains a lymphoid-specific enhancer that includes several short protein-binding sequences. The sequence that binds the nuclear factor NF-kappa B was tested for its ability to act independently as an enhancer element by inserting it into test plasmids containing the chloramphenicol acetyltransferase gene. When analyzed for activity by transient transfection into lymphoid and nonlymphoid cells, a single copy of the NF-kappa B binding site could act as a tissue-specific upstream activating element. Two copies (dimer) showed 10-fold higher activity than did one copy and could act as an enhancer element 2.5 kilobases downstream of the transcriptional start site. The enhancer activity of this sequence was correlated with the presence of the cognate binding protein, NF-kappa B. This sequence acted as an inducible enhancer under conditions that induce NF-kappa B binding activity. Thus, the NF-kappa B binding site acts by itself as a tissue-specific and inducible enhancer element, and two copies show cooperative interaction.
To investigate the role of NF-IL6 in vivo, we have generated NF-IL6 (-/-) mice by gene targeting. NF-IL6 (-/-) mice were highly susceptible to infection by Listeria monocytogenes. Electron microscopic observation revealed the escape of a larger number of pathogens from the phagosome to the cytoplasm in activated macrophages from NF-IL6 (-/-) mice. Furthermore, the tumor cytotoxicity of macrophages from NF-IL6 (-/-) mice was severely impaired. However, cytokines involved in macrophage activation, such as TNF and IFN gamma, were induced normally in NF-IL6 (-/-) mice. Nitric oxide (NO) formation was induced to a similar extent in macrophages from both wild-type and NF-IL6 (-/-) mice. These results demonstrate the crucial role of NF-IL6 in macrophage bactericidal and tumoricidal activities as well as the existence of a NO-independent mechanism of these activities. We also demonstrate that NF-IL6 is essential for the induction of G-CSF in macrophages and fibroblasts.
We have analysed the molecular basis for the function of the C/EBP alpha transactivation domain. We have previously found that the three C/EBP alpha transactivation elements (TEs) synergistically activate transcription in mammalian cells. We now report that two of these elements, TE-I and -II, co-operatively mediate in vitro binding of C/EBP alpha to TBP and TFIIB, two essential components of the RNA polymerase II basal transcriptional apparatus. The TBP and TFIIB binding elements of C/EBP alpha coincide, and require amino acid motifs conserved between the activating members of the C/EBP family. These same motifs are necessary for the transcription activation function of TE-I and -II in both yeast and mammalian cells. Our data demonstrate a biochemical basis for the modular buildup of transactivation domains, and indicate that this modularity is conserved in eukaryote evolution. We also show that the same amino acid motifs in a cellular activator can co-operate to mediate contacts between the activator and two distinct basal transcription factors. These results suggest that domains of TBP and TFIIB that interact with activating surfaces are functionally similar and may be structurally related, and support the idea that the same amino acid motifs in an activator carry out multiple functions during the initiation process.
Interleukin 12 (IL-12) is an inducible cytokine composed of 35- and 40-kDa subunits that is critical for promoting T helper type 1 development and cell-mediated immunity against pathogens. The 40-kDa subunit, expressed by activated macrophages and B cells, is induced by several pathogens in vivo and in vitro and is augmented or inhibited by gamma interferon (IFN-gamma) or IL-10, respectively. Control of IL-12 p40 expression is therefore important for understanding resistance and susceptibility to a variety of pathogens, including Leishmania major and perhaps human immunodeficiency virus. In this report, we provide the first characterization of IL-12 p40 gene regulation in macrophages. We localize inducible activity of the promoter to the sequence -122GGGGAATTTTA-132 not previously recognized to bind Rel family transcription factors. We demonstrate binding of this sequence to NF-kappa B (p50/p65 and p50/c-Rel) complexes in macrophages activated by several p40-inducing pathogens and provide functional data to support a role for NF-kappa B family members in IL-12 p40 activation. Finally, we find that IFN-gamma treatment of cells enhances this binding interaction, thus potentially providing a mechanism for IFN-gamma augmentation of IL-12 production by macrophages.
Mice with the severe combined immunodeficiency (SCID) possess an IFN-gamma-dependent mechanism of resistance to the intracellular pathogens Toxoplasma gondii and Listeria monocytogenes that is dependent on IL-12-induced production of IFN-gamma by NK cells. In this report we demonstrate that IL-1 beta is required for IL-12 to stimulate production of IFN-gamma by NK cells, and that IL-1 is important in IL-12-mediated resistance to T. gondii in vivo. Stimulation of SCID mouse splenocytes with tachyzoites of T. gondii resulted in production of IFN-gamma. Addition of neutralizing Ab specific for IL-1 beta to these cultures inhibited completely the production of IFN-gamma. Similar results were obtained when LPS or L. monocytogenes were used to stimulate production of IFN-gamma by SCID mouse splenocytes. Addition of a neutralizing Ab to IL-1 alpha did not affect production of IFN-gamma by SCID mouse splenocytes stimulated with T. gondii, L. monocytogenes, or LPS. Stimulation of SCID mouse splenocytes with IL-1 beta or IL-1 alpha did not result in production of IFN-gamma but enhanced remarkably the ability of T. gondii or IL-12 to stimulate production of IFN-gamma. Furthermore, production of IFN-gamma by SCID mouse splenocytes stimulated with IL-12 plus TNF-alpha was completely ablated by anti-IL-1 beta, but not by anti-IL-1 alpha. Analysis of the culture supernatants of spleen cells from SCID mice stimulated with T. gondii or IL-12 plus TNF-alpha detected low levels of IL-1 beta; addition of a neutralizing Ab to IFN-gamma resulted in a 5- to 10-fold increase in levels of IL-1 beta. Furthermore, stimulation of SCID mouse splenocytes with IL-12, in the presence of anti-IFN-gamma, resulted in an increase in detectable levels of IL-1 beta. To determine the in vivo relevance of our in vitro data, SCID mice were infected with T. gondii and treated with IL-12 alone or IL-12 in combination with an Ab specific for the type I IL-1 receptor. This Ab reduced production of IFN-gamma by SCID mouse splenocytes stimulated with either T. gondii, LPS, L. monocytogenes, or IL-12 plus IL-1 beta. In vivo administration of this Ab antagonized significantly the ability of exogenous IL-12 to delay the time to death of SCID mice infected with T. gondii.(ABSTRACT TRUNCATED AT 400 WORDS)
We have characterized the transcriptional response to IFN-gamma in two maturationally distinct macrophage populations: the mature RAW 264.7 cell line, phenotypically identical to thioglycollate-elicited peritoneal macrophages, and the less mature WEHI-3 cell line. We first investigated the use of two IFN-gamma-responsive regulatory elements, the interferon-stimulated response element (ISRE) and the gamma-activated sequence (GAS), in these cells. Transient transfection assays revealed that synthetic promoter constructs containing either the ISRE or GAS regulatory motif fused to a luciferase reporter gene were transcriptionally inactive in the WEHI-3 cell line. We then analyzed the expression in the two cell lines of a panel of known IFN-gamma-responsive genes that are transcriptionally controlled by different regulatory elements. RT-PCR analysis revealed that both cell lines responded to IFN-gamma treatment by up-regulating genes that are transcriptionally controlled by kappa B or W box DNA binding motifs. However, genes regulated by ISRE or GAS elements were induced by IFN-gamma only in the RAW 264.7 cell line. Kinetic analysis of the transcriptional activity of synthetic promoter constructs in the RAW 264.7 cell line showed rapid IFN-gamma induction through both the ISRE and GAS motifs, indicating that both elements are utilized early after IFN-gamma stimulation in mature macrophages. These results suggest that cis-acting DNA response element utilization, and the subsequent profiles of IFN-gamma-induced gene expression, differ in macrophages at different stages of maturation.
Interleukin-12 (IL-12) is a heterodimeric cytokine produced mostly by phagocytic cells in response to bacteria, bacterial products, and intracellular parasites, and to some degree by B lymphocytes. IL-12 induces cytokine production, primarily of IFN-gamma, from NK and T cells, acts as a growth factor for activated NK and T cells, enhances the cytotoxic activity of NK cells, and favors cytotoxic T lymphocyte generation. In vivo IL-12 acts primarily at three stages during the innate resistance/adaptive immune response to infection: 1. Early in the infection, IL-12 is produced and induces production from NK and T cells of IFN-gamma, which contributes to phagocytic cell activation and inflammation; 2. IL-12 and IL-12-induced IFN-gamma favor Th1 cell differentiation by priming CD4+ T cells for high IFN-gamma production; and 3. IL-12 contributes to optimal IFN-gamma production and to proliferation of differentiated Th1 cells in response to antigen. The early preference expressed in the immune response depends on the balance between IL-12, which favors Th1 responses, and IL-4, which favors Th2 responses. Thus, IL-12 represents a functional bridge between the early nonspecific innate resistance and the subsequent antigen-specific adaptive immunity.
We show that the transactivating COOH terminus of the p65 subunit of human transcription factor NF-kappa B directly binds the general transcription factors TFIIB and TATA-binding protein (TBP) in vitro. Interaction of p65 with TFIIB required the most COOH-terminal sequence repeat within TFIIB. A functional interaction of TFIIB with p65 was evident from assays in yeast cells. Cotransfection experiments in COS cells revealed that only overexpression of TBP was able to further stimulate p65-dependent transactivation of a reporter gene. The coexpression of neither TBP nor TFIIB was able to relieve squelching, indicating the involvement of additional factors in transactivation by p65. A cell-free assay using highly purified factors revealed a specific transcriptional stimulation through the COOH-terminal activation domain of NF-kappa B by at least one cofactor, PC1, isolated from HeLa cells. These data show that the potent acidic transactivation domains in the COOH terminus of p65 are able to functionally recruit various components of the basic transcription machinery as well as coactivators.
Distinct patterns of T cell cytokine production have been shown to influence the outcome of infection in mouse models and humans. Th1 or Type 1 cytokines, interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) are generally associated with resistance to infection, whereas Th2 or Type 2 cytokines, IL-4 and IL-10 are associated with progressive disease. Leprosy is a useful model for studying the role of cytokines in modulating T cell responses in human infectious disease. Infection by Mycobacterium leprae results in disease manifestations that encompass an immunological spectrum. Tuberculoid patients are able to restrict the growth of the pathogen and mount strong T cell responses to M. leprae. In contrast, lepromatous patients manifest disseminated infection and their T cells weakly respond to M. leprae. We have found that tuberculoid leprosy lesions have a predominance of CD4+ T cells producing the Type 1 cytokine pattern. Secondly, IL-12 mRNA was expressed at 10-fold higher levels in tuberculoid lesions as compared to lepromatous lesions and that IL-12 promotes the selective expansion of the Type 1 cytokine producing cells. In contrast, lepromatous lesions contain CD8+ IL-4-producing cells that suppress antigen-specific T cell responses and promote the outgrowth of additional suppressor T cells. IL-10, also expressed at higher levels in lepromatous as compared to tuberculoid lesions, was found to be produced by macrophages, effectively inhibiting cytokine production and macrophage activity.
Using the 3T3-F442A preadipocyte line as a model of GH-dependent differentiation, early changes in the DNA-binding affinity of transcription factors in response to GH addition were investigated. Addition of 50 ng/ml human GH to cells in chemically defined medium led to a rapid increase in binding activity of activator protein 1 (AP-1) and CCAAT enhancer-binding protein (C/EBP), which was significant at 30 min and reached maximal induction by 2 h (3-fold for AP-1, 2.5-fold for C/EBP). Induction in AP-1 DNA binding correlates with a concomitant GH trans-activation of c-jun and c-fos genes described previously. Using specific antibodies in electrophoretic mobility shift assays and Western blots, it was shown that the increase in activity of C/EBP is the result of an increase in synthesis of two alternatively translated forms of C/EBP beta: 40-C/EBP beta and 23-C/EBP beta. This increase in protein was not accompanied by alteration in mRNA level and could be blocked by a Janus kinase 2 tyrosine kinase inhibitor and a C kinase inhibitor at concentrations shown to inhibit GH-dependent activation of microtubule-associated protein (MAP) kinases. Concomitant with the translationally activated increase in C/EBP beta, a GH-dependent increase was observed in C/EBP delta transcription. This was accompanied by an increase in mRNA for C/EBP delta, which was superinduced by cycloheximide and, unlike the increase in C/EBP beta protein, was not observed with insulin. Thus GH exerts its effects on C/EBP isoforms at two levels: transcriptional activation of C/EBP delta and translational activation of C/EBP beta. It is proposed that GH-dependent phosphorylation results in the efficient translation of 40-C/EBP beta and 23-C/EBP beta (the mouse homolog of the inhibitor liver-enriched inhibitory protein), and that together with the induction of C/EBP delta, these may be involved in initiating the adipocyte differentiation program.
Peripheral blood mononuclear cells (PBMCs) from many asymptomatic individuals infected with human immunodeficiency virus-type
1 (HIV) are unresponsive as measured by in vitro T cell proliferation and interleukin-2 (IL-2) production to influenza virus
and synthetic peptides of HIV envelope (Env). Strong influenza virus- and Env-stimulated IL-2 responses and T cell proliferation
were restored when cultures were stimulated in the presence of IL-12. Interferon-gamma production by PBMCs from HIV seropositive
(HIV+) patients was also restored with IL-12. Furthermore, in vitro antigen-specific production of IL-2 and proliferation
of PBMCs from HIV- donors were suppressed by antibody to IL-12, but were not enhanced by addition of exogenous IL-12. Thus,
IL-12 may be limiting in PBMCs from HIV+ but not HIV- individuals. These findings demonstrate that IL-12 can restore HIV-specific
cell-mediated immunity in vitro in HIV-infected individuals and suggest a potential use of IL-12 in augmenting the diminished
immunologic functions associated with HIV infection.
Protection induced by vaccination depends on the capacity of the vaccine to elicit an appropriate immune response. In leishmaniasis,
protection requires leishmanial-specific CD4+ T helper (TH) cells. Vaccination of BALB/c mice with leishmanial antigens and
interleukin-12 (IL-12) promoted the development of leishmanial-specific CD4+ TH1 cells. These mice were resistant to subsequent
infection with Leishmania major. Thus, IL-12 is an effective adjuvant for the initiation of protective cell-mediated immunity
against leishmaniasis and may be an important component in other vaccines that need to induce cell-mediated immunity.
Interleukin-12 (IL-12) is necessary for the production of IFN-gamma by NK cells during the generation of innate immunity and by T cells for the development of the Th1 response during specific cell-mediated immunity. Here we demonstrate that the endogenous production of IL-12 is critical to the survival of both immunocompromised SCID mice and normal C.B-17 control mice during a primary infection with Listeria monocytogenes. When IL-12 is neutralized in vivo, both strains of mice die at a normally sublethal dose of Listeria. Anti-IL-12 antibody-treated mice showed a decrease in macrophage I-Ad expression and an increase Listeria burden in the spleen. Furthermore, as has been demonstrated in vitro, these effects of IL-12 in vivo were predominantly regulated through the production of IFN-gamma. Administration of IFN-gamma simultaneously with neutralizing antibodies to IL-12 restored macrophage I-Ad expression, limited the spread of the infection, and resulted in the survival of SCID mice. Thus, IL-12 is critical for resistance to infection with Listeria monocytogenes, and this resistance is mediated through stimulation by IL-12 of IFN-gamma production. Concomitant experiments confirmed that anti-TNF antibodies also resulted in uncontrolled infection and a decrease in macrophage I-Ad expression. However, administration of IFN-gamma restored the levels of I-Ad in macrophages but did not limit Listeria growth.
Natural killer cell-stimulatory factor or interleukin-12 (NKSF/IL-12) was originally identified and purified from the conditioned medium of Epstein-Barr virus (EBV)-transformed B-cell lines. Phorbol diesters were observed to be potent stimulators of NKSF/IL-12 production from the B-cell lines. Although monocytes were found to be the major producers of NKSF/IL-12 in peripheral blood (PB) in response to lipopolysaccharide (LPS) or to Staphylococcus aureus, several myeloid leukemia cell lines tested did not produce detectable NKSF/IL-12 either constitutively or upon stimulation with phorbol diesters. However, three lines, ML-3, HL-60, and THP-1, responded to LPS with significant levels of NKSF/IL-12 production, whereas S aureus was effective only on THP-1 cells. When the cell lines were preincubated with compounds known to induce them to differentiate, production of tumor necrosis factor alpha (TNF alpha) and IL-1 beta was in most cases maximal in cells with differentiated characteristics, whereas NKSF/IL-12 production in response to LPS in all three producing cell lines was significantly enhanced only by pretreatment with dimethylsulfoxide (DMSO) for 24 hours, or by costimulation with interferon gamma (IFN gamma). The efficiency of DMSO enhancement of NKSF/IL-12 production decreased after 2 to 5 days of incubation, when the cells acquired differentiated characteristics. Unlike DMSO, IFN gamma enhanced NKSF/IL-12 production, and IL-10 and dexamethasone inhibited it in cell lines and PB mononuclear cells stimulated by either LPS or S aureus. The ability of the cell lines to respond to these mediators of possibly physiologically relevant function provides a tissue-culture model for studying their mechanism of action.
Interleukin 12 (IL-12), a heterodimeric cytokine composed of p40 and p35 chains, has potent immunologic effects in vitro. We used tuberculous pleuritis as a model to study the immunoregulatory potential of IL-12 in vivo at the site of human infectious disease. Messenger RNAs for p40 and p35 were detected in pleural fluid from six of six patients by reverse-transcription polymerase chain reaction. By using an ELISA that detected both free p40 and heterodimeric IL-12, we found that mean concentrations were 585 +/- 89 pg/ml in pleural fluid of patients with tuberculous pleuritis, which were significantly higher than those in serum of the same patients (54 +/- 36 pg/ml), or in malignant pleural effusions (123 +/- 35 pg/ml). By using an ELISA specific for heterodimeric IL-12, we found that mean concentrations in pleural fluid of patients with tuberculous pleuritis were 165 +/- 28 pg/ml and undetectable in serum of the same patients, or in malignant pleural effusions. Bioactive IL-12 was detectable in five of five supernatants of pleural fluid cells stimulated with Mycobacterium tuberculosis. Addition of anti-IL-12 antibodies suppressed proliferative responses of pleural fluid cells to M. tuberculosis by 36 +/- 7%. These data indicate that IL-12 may play a role in the human immune response to infectious agents in vivo. We hypothesize that IL-12 contributes to the antimycobacterial immune response by enhancing production of interferon-gamma, facilitating development of Th1 cells and augmenting cytotoxicity of antigen-specific T cells and natural killer cells.
In vitro and in vivo studies were performed to assess the involvement of IL-12 in resistance to acute and chronic infection with an avirulent strain of Toxoplasma gondii. Our previous findings implicated macrophages as a major source of parasite-induced IL-12. This finding was confirmed by showing that peritoneal macrophages exposed to either live parasites or soluble tachyzoite Ags produce IL-12 protein. In mice, increased expression of IL-12 (p40) mRNA in both spleen and peritoneal cells was detected as early as 2 days postinfection. Treatment with neutralizing mAbs against IL-12 increased the susceptibility of C57BL/6, BALB/c, and severe combined immunodeficient (SCID) mice to acute infection, which resulted in 100% mortality within the first 15 days after parasite inoculation. In contrast, neutralization of endogenously produced IL-12 had no effect when given during chronic infection. In agreement with the survival data, treatment with anti-IL-12 resulted in decreased IFN-gamma and enhanced Th2 (IL-4 and IL-10) cytokine synthesis by splenocytes when given during acute, but not chronic, toxoplasmosis. Sorting experiments on spleen cells from acutely infected mice indicated that both CD4+ lymphocytes and NK1.1+/CD3- cells contribute to the early IFN-gamma response. In contrast, CD4+ cells were found to be the major source of the cytokine during chronic disease. Together, these results suggest that the stimulation of macrophage-derived IL-12 plays a major role in both the induction of resistance and Th1 cell subset selection in acute T. gondii infection, but may not be required to maintain established immunity.