Input:
High Antigen Doses and STAT4 Are Required for the In Vivo Generation of IL-10-Producing Th1 Cells
To address the mechanisms regulating IL-10 production by Th1 cells invivo, we transferred DO11.10 cells into BALB/c recipient mice and immunized the recipients with very high doses of OVA-protein with or without added lipopolysacharide (LPS). Tcells were recovered from the inguinal lymph nodes 3 days after priming and restimulated invitro with OVA peptide for 48 hr. This invivo immunization induced IL-10 and IFN-gamma production, and the amount of IL-10 production was enhanced by addition of LPS in the immunization (Figure3A) and with higher doses of OVA (3 muM versus 1 muM, data not shown). To test the role of STAT4 and STAT6 signaling in the invivo development of IL-10-producing Th1 cells, we transferred STAT4- or STAT6-deficient or WT DO11.10 cells into recipient BALB/c mice and immunized with OVA-protein plus LPS as before. In vivo expression of both IL-10 and IFN-gamma was markedly reduced but not completely abrogated in the absence of STAT4 signaling (Figures 3B and 3C), suggesting the existence of compensatory mechanisms that were absent in the invitro system. Signaling through STAT6 had no effect on IL-10 production by Th1 cells as shown by intracellular cytokine staining (ICS) and by immunoassay in STAT6-deficient Tcells (Figures 3B and 3C).

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IL-10 Production Is Maintained by High TCR Signal Strength and IL-12
We next investigated whether repeated strong TCR activation is a compensatory signal for IL-12-induced STAT4 signaling in the induction of IL-10 in Th1 cells. For this, CD4+ Tcells were differentiated for 2 consecutive weeks with high antigen doses in the presence or absence of IL-12 throughout (Figures 4A-4D). High antigen dose and IL-12 cooperated to induce maximal IL-10 production (Figures 4A and 4B), given that this combination resulted in the highest numbers of IL-10-producing Th1 cells. Repeated high antigen dose stimulation in the absence of exogenously added IL-12 resulted in the production of IL-10 by Th1 cells, suggesting that repeated strong TCR triggering may overcome the need for IL-12 for IL-10 induction (Figures 4C and 4D). However, IL-10 induction under these conditions was abrogated when IL-12p40-deficient DCs were used as APCs (Figure4E). Thus, IL-12 is essential during both primary and secondary antigenic stimulation for production of IL-10 by Th1 cells.
To determine the requirements for stability of the IL-10-producing Th1 cells, we differentiated CD4+ Tcells for 1 week with high antigen doses with or without IL-12 (Figures 4A and 4C), washed them, and then restimulated them for an additional week with a low antigen dose, in the absence or presence of IL-12 (Figure4F). Th1 cells induced in the first week to produce IL-10 by culture with high antigen doses and IL-12 lost their ability to express IL-10 when recultured with low doses of OVA, which could be compensated for, to some extent, by addition of IL-12 to the secondary cultures (Figure4F), again suggesting that antigen dose and IL-12 signals cooperate for the induction of IL-10. Finally, DO11.10 CD4+ cells that were exposed to low doses of antigen and IL-12 during the primary differentiation phase produced high amounts of IFN-gamma but little IL-10, but they could be induced to produce IL-10 when both high antigen dose and IL-12 were present during the recall phase (FigureS4). Thus, high antigen dose and IL-12 are required for sustaining the induction of IL-10 production by Th1 cells.

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IL-10 Production by Th1 Cells Requires ERK1 and ERK2 Activation
Our data showed that the maintenance of IL-10 induction in Th1 cells required stimulation with high antigen dose, which to some extent could be compensated for by the addition of IL-12. Signaling through the TCR with high doses of antigen induced stronger ERK1 and ERK2 activation than that induced by low antigen dose, not only in naive CD4+ Tcells (data not shown) as previously demonstrated (Jorritsma etal., 2003) but also in CD4+ Tcells restimulated with the same high and low antigen doses (Figure5A). Although the apparent peak and amount of ERK1 and ERK2 activation varied slightly between experiments, a consistent finding was that high antigen dose differentiated Th1 cells always showed enhanced and prolonged ERK1 and ERK2 activation in the presence of IL-12, regardless of whether they were restimulated with high or low antigen dose (Figure5B).
We then investigated whether ERK1 and ERK2 activation was required for the induction of IL-10 in Th1 cells by using U0126 (Figure5C), a compound that blocks downstream ERK activation. To ensure that only Tcell signaling was being affected by U0126, we used an APC-free system in which the Tcells were differentiated in the presence of increasing doses of anti-CD3 and a constant amount of IL-12. As in the APC-driven cultures, stronger TCR stimulation together with IL-12 led to higher percentages of cells producing both IL-10 and IFN-gamma after 1 week of culture (Figure5C). Addition of U0126 to the cultures abrogated the production of IL-10 at all doses of anti-CD3 (Figure5C). Because U0126 inhibits the MEK5-catalyzed activation of ERK5, as well as the MEK1- and MEK2-catalyzed activation of ERK1 and ERK2 (Bain etal., 2007; Mody etal., 2001), we also used the more specific, structurally unrelated MEK1 and MEK2 inhibitor PD184352 at concentrations in which it inhibits MEK1 and MEK2 but not MEK5 (Bain etal., 2007; Mody etal., 2001). PD184352 caused a similar inhibition of IL-10 production by Th1 cells in a dose-dependent fashion (Figure5D and FigureS5A). Upon addition of inhibitors to othersignaling pathways, including a p38 MAPK inhibitor, SB203580, or the GSK3beta inhibitor, CT99021 (Bain etal., 2007), no effect on IL-10 production was observed (FigureS5B). Our data thus suggested that IL-10 production by Th1 cells in response to high antigen dose and IL-12 requires ERK1 and ERK2 signaling, but not the activation of the p38 or the GSK3beta pathways.

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IL-10 Production by Th2 and Th17 Cells Also Requires ERK1 and ERK2 Activation
To address whether IL-10 production by Th2 and Th17 cells was also dependent on ERK1 and ERK2 activation, we differentiated these cells with anti-CD3 and anti-CD28 in the absence of APCs (Shoemaker etal., 2006; Veldhoen etal., 2009; Veldhoen etal., 2006), in the presence or absence of the MEK inhibitor (PD184352). We showed that ERK1 and ERK2 activation is a common pathway required for induction of IL-10 in different Th cell subsets because IL-10 production by both Th2 and Th17 cells was markedly inhibited in the presence of the MEK inhibitor (PD184352) (Figure5D and FigureS5B). In contrast, inhibitors of p38 MAPK or of GSK-3beta activation did not affect the expression of IL-10 by these subsets (FigureS5B). Activation of the ERK1 and ERK2 signaling pathway is therefore a common requirement for the induction of IL-10 production by Th1, Th2, and Th17 cells.

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c-maf Expression Correlates with IL-10 Production in Th1, Th2, and Th17 Cells
To investigate further the downstream factors involved in regulating IL-10 production, we differentiated DO11.10 CD4+ Tcells with increasing doses of OVA, in the presence or absence of IL-12, and quantified the expression of cytokines and transcription factors by real-time RT-PCR. Low-dose antigen resulted in transcription of Il4 and this was abrogated by both high antigen doses and IL-12 (Figure6A). A low amount of transcription of Ifngamma was induced by IL-12 when cells were differentiated with low antigen dose, but this effect of IL-12 was markedly upregulated with increasing doses of antigen (Figure6A). A low amount of Il10 transcription was observed at low doses of antigen accompanying Il4 expression (Th2 cell response), and this was abrogated by increased doses of antigen as was Il4 expression (Figure6A). At low doses of antigen, IL-12 had little effect to increase IL-10 mRNA expression (Figure6A) in keeping with the protein data (Figure1A). However, IL-12 induced a high amount of Il10 transcription as well as Ifngamma expression with increased antigen doses (Figure6A), again in keeping with the protein data (Figure1A).
CD4+ Tcells differentiated with increasing doses of antigen did not express high amounts of Tbx-21 (T-bet) mRNA, unless they were cocultured with IL-12 (Figure6B). In contrast, high amounts of GATA-3 mRNA expression were only observed under Th2 cell differentiation conditions (low-dose antigen) (Figure6B), and this expression was markedly downregulated by both increasing antigen dose and coculture in IL-12 (Figure6B). Differentiation of Tcells under low antigen dose led to expression of c-maf, in keeping with the Th2 cell profile (Ho etal., 1996), which was almost completely abrogated by increasing doses of antigen (Figure6B). Interestingly, IL-12 sustained the high expression of c-Maf mRNA even at the highest antigen dose (Figure6B). Moreover, IL-12 maintenance of c-maf expression required STAT4 activation (data not shown).
In Th17 cells that expressed IL-17a as well as IL-10 mRNA (Figure6C), T-bet and GATA-3 mRNA were undetectable (data not shown), whereas that of ROR-gammat was high (Figure6C) (Ivanov etal., 2007). Th17 cells also expressed high amounts of c-maf (Figure6C), confirming a recent report (Bauquet etal., 2009). c-Maf is therefore expressed in all IL-10-expressing Tcell populations tested (Figures 6B and 6C) and may not be just a Th2 cell-specific transcription factor as originally thought (Ho etal., 1996). We showed also that like Il10 expression, c-maf expression was inhibited in Th1 and Th17 cells in the presence of the MEK1 and MEK2 inhibitor (PD184352), whereas T-bet and RORgammat expression was hardly affected (Figure6D).

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Discussion
IL-10 expression by cells of the innate and adaptive immune systems reflects the importance of this cytokine in the tight regulation of the immune response, to minimize pathology during infection. IL-10 expression by Th1 cells has been reported to regulate the immune response in leishmaniasis and toxoplasmosis. However, in many situations, IL-10 is not produced by Th1 cells in response to antigenic stimulation. Our goal was to address the molecular signals that determine whether Th1 cells develop to produce IL-10 (Trinchieri, 2007). Here, we showed that Th1 cells required high-antigen-dose-induced ERK1 and ERK2 phosphorylation and IL-12-induced STAT-4 activation to produce IL-10. Our findings that ERK1 and ERK2 activation was a common pathway required for the production of IL-10 by Th1, Th2, and Th17 cell subsets, which differentiate along distinct pathways, such that IL-10 provides a highly regulated feedback loop to avoid the extremes of excessive inflammation or chronic infections and also allow a protective response to diverse pathogens.
In certain viral or parasitic infections (Anderson etal., 2007; Brooks etal., 2006; Ejrnaes etal., 2006), high amounts of stimulation may lead to the chronic nonhealing infection shown to be regulated by IL-10. During the course of infection, after initial triggering with antigen, Tcells migrate to the tissue encountering high doses of antigen and factors produced by the innate immune response. Under these conditions, we speculate that Th1 cells will be induced to express high amounts of IL-10, in keeping with reports that IL-10-producing Th1 cells were found in CD4+ clones isolated from BAL but not blood of TB patients (Gerosa etal., 1999). Similarly, the immune response to a clinical isolate of L. major, which produces heavily infected nonhealing lesions, was found to be regulated by IL-10 derived from Foxp3- Th1 cells that coproduce IL-10 and IFN-gamma (Anderson etal., 2007), and the immune response during T. gondii infection was found also to be regulated by Foxp3- Th1 cells (Jankovic etal., 2007). It is likely that IL-10 production by Th1 cells is evoked under conditions of high inflammation and antigenic stimulation, whereas regulatory CD4+ Tcells producing IL-10 may operate to regulate the immune response under conditions in which the pathogen is clinically controlled, such as in infection with L. major (Friedlin strain) (Belkaid etal., 2002; Suffia etal., 2006). We now also reported that CD4+ Tcells cultured with high antigen dose and IL-12 differentiate into canonical Th1 effector cells , which, in addition to expressing large amounts of IFN-gamma and IL-10, lose their IL-2 expression as described before in certain chronic infection models (Sallusto etal., 2004). Our demonstration that loss of IL-2 is accompanied by production of IL-10 offers potential additional mechanisms whereby effector Tcell responses may be dampened during chronic disease.
Using an invivo transfer model of DO11.10 TCR transgenic cells (Castro etal., 2000), we showed that IL-10-producing Th1 cells were differentiated in the presence of high doses of OVA protein and LPS. We showed here that this induction of IL-10 inTh1 cells invivo was markedly, but not totally, reduced in STAT4-deficient Tcells as observed during T. gondii infection (Jankovic etal., 2002). A high antigenic activation during T. gondii infection or high antigen doses delivered in the presence of LPS, as seen in our system, may compensate for an absolute requirement for IL-12 in the induction of IL-10 by Th1 cells.
In our invitro system, repeated stimulation of Th1 cells with high antigen doses allowed the development of Th1 cells producing IL-10 in an IL-12-dependent manner. IL-10 production by Th1 cells induced by high antigen dose and IL-12 was independent of IFN-gamma, in keeping with previous findings (Jankovic etal., 2002). However, a role for IFN-gamma in mediating IL-10 reactivation by Th1 cells during secondary infection with T. gondii has been suggested (Shaw etal., 2006). We have found that CD4+ Tcells exposed to a high dose of antigen do not express IL-10 upon restimulation, but can be induced to produce IL-10 upon re-exposure to a high dose of antigen in the recall phase in the absence of added IL-12. However, this is dependent on the induction of IL-12 by antigen-presenting DCs. The combination of both high antigen dose and IL-12 resulted in the highest levels of IL-10 production and correlated with the high levels of ERK1 and ERK2 activation. The increased expression of IFN-gamma observed during the secondary phase will induce increased IL-12 production by DCs and suggests that repeated high-level TCR activation feeds back to upregulate IL-12 production by DC. It is thus likely that in T. gondii infection invivo (Shaw etal., 2006), the requirement for IFN-gamma to induce IL-10, was for feedback upregulation of IL-12 by DCs, which in turn induced IL-10 in the Th1 cells.
Although IL-10 may be differentially regulated in Th1 and Th2 cells as has been reported (Chang etal., 2007; Wang etal., 2005), some studies suggest the existence of common pathways, but the molecular basis for these is as yet unclear. Costimulatory OX-40 signals have been shown to negatively regulate IL-10 production (Ito etal., 2005) both in Th1 and Th2 cells, whereas ICOS signaling has been suggested to induce IL-10 (Ito etal., 2007; Witsch etal., 2002) in both Th1 and Th2 cells. However, in some cases, ICOS signaling also regulates IL-4 production and Th2 responses (Greenwald etal., 2005). We now provide a common mechanism of ERK1 and ERK2 activation for the regulation of IL-10 production in Th1, Th2, and Th17 cells, although each subset differentiates along a distinct and subset-specific transcriptional pathway. This reinforces the fact that IL-10 is not a Th cell-subset-specific cytokine, but instead is produced in a tightly regulated fashion during each differentiation pathway. Of note, a role for ERK1 and ERK2 activation in the induction of IL-10 production has already been described for macrophages and DC (Agrawal etal., 2006; Hacker etal., 1999).
Differential transcriptional regulation of IL-10 in Th1 and Th2 cells has been suggested (Chang etal., 2007; Wang etal., 2005), and extensive histone acetylation of the IL-10 gene is detectable in fully polarized Th2 cells, but not Th1cells (Chang etal., 2007). We provide evidence that IL-10 is produced in canonical Th1 cells and that its expression correlates with the expression of T-bet and the highest IFN-gamma production, in keeping with our observations that high-dose antigen stimulation and IL-12 signaling are required for IL-10 and IFN-gamma expression. It has also been shown that maintenance of IL-10 expression is conditional on IL-12 or IL-4 unless the IL-10 gene is imprinted by GATA-3 (Chang etal., 2007), which can remodel the IL-10 locus, thus explaining the highest amounts of IL-10 produced by Th2 cells (Chang etal., 2007; Shoemaker etal., 2006). We show here that high antigen dose and IL-12 drastically downregulate Gata-3 expression, suggesting that additional factors are in place to induce IL-10 expression in Th1 cells, albeit transiently. Expression of c-maf was greatly diminished by high antigen doses in Tcells and yet was unexpectedly maintained by IL-12 and present in Th17 cells. That c-maf expression is common to IL-10-producing Th1, Th2, and Th17 cells and, like IL-10, is dependent on ERK activation in Th1 and Th17 cells for its expression is of interest because c-Maf has been shown to be an essential transcription factor for IL-10 expression in macrophages (Cao etal., 2005).
In summary, we show that although Th1, Th2, and Th17 CD4+ Tcell subsets differentiate along distinct signaling and transcriptional pathways, they can all be induced to make IL-10. ERK1 and ERK2 activation is required for IL-10 production by all these Th cell subsets. With regard to the expression of IL-10 by Th1 cells, our data provide a mechanism for how IL-10 expression is induced and then amplified and regulated by the levels of antigen and IL-12 encountered in the environment. This provides a mechanism whereby a Th1 cell responds to extrinsic signals, reflecting increased inflammation in the tissue, to tightly regulate the production of IL-10 so as to allow a protective response to eradicate a pathogen with minimal damage to the host and also prevent chronic infection. Moreover, our findings have important implications for the regulation of IL-10 production during an inflammatory Th1 response in infection and may be of relevance for the design of vaccines and for strategies in immunotherapy in infectious diseases.

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Input:
Mice, Cytokines, Antibodies, and Other Reagents
BALB/c DO11.10 mice transgenic for OVA-specific TCR WT or crossed back with Rag1-, IL-4-,IFN-gamma-, STAT4-, and STAT6-deficient mice were used as a source of antigen-specific Tcells (Murphy etal., 1990; Ouyang etal., 1998; Shoemaker etal., 2006) and were bred and maintained under SPF conditions at the NIMR, London, Home Office, UK, Animals (Scientific Procedures) Act 1986 or at the Washington University School of Medicine. Female mice were used at 8-12 weeks old, and animal protocols were approved according to the Animals (Scientific Procedures) Act 1986, Home Office, UK. Reagents, including antibodies for Tcell and DC preparation, purification and culture, media, cytokines, and cytokine mAbs have been described (Hosken etal., 1995; Shoemaker etal., 2006; Veldhoen etal., 2009; Veldhoen etal., 2006). LPS (S. minnesota) was from Alexis, chicken ovalbumin protein (OVA protein) was from from Sigma-Aldrich, and ovalbumin peptide323-339 (OVA) (endotoxin-free) was from Biosynthesis. U0126 was from BioMol International. PD184352 (MEK inhibitors), SB203580 (p38 inhibitor), and CT99021 (GSK3beta inhibitor) were kind gifts from P. Cohen and N.Shpiro, University of Dundee, UK.

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Isolation of CD4+ T Cells and of Splenic DC and Cell Culture for T Cell Phenotype Differentiation
T cells were sorted for CD4+CD62Lhi, CD4+CD62LhiCD25-, or CD4+CD44loCD25- to >98% on a Moflo cytometer (Cytomation) as before (Shoemaker etal., 2006; Veldhoen etal., 2009). In most cases, experiments were reproduced with each type of purified CD4+ Tcell population with similar results obtained. Splenic DCs were prepared as described (Hosken etal., 1995), and sort purified CD11c+ cells were added to the Tcell culture. Purified DO11.10 CD4+ Tcells (1 x 105 cells/ml) were cultured as before (Hosken etal., 1995), in a total volume of 1 ml cRPMI medium in a 48-well plate, with splenic DCs (2 x 104 cells/ml), and varying amounts of OVA and of IL-12. APC-independent differentiation of naive CD4+ Tcells into Th1 and Th2 cells used stimulation with anti-CD3 and anti-CD28 and appropriate cytokine conditions, and control Th1 and Th2 cells were cultured as described before (Hosken etal., 1995; Shoemaker etal., 2006). Culture conditions for Th17 cells were as described before (Veldhoen etal., 2006). Importantly, Th1 and Th2 cells could be differentiated in cRPMI or IMDM (Hosken etal., 1995; Shoemaker etal., 2006; Veldhoen etal., 2006), but Th17 cells were only differentiated optimally in IMDM (Veldhoen etal., 2009). When indicated, U0126 or PD184352 (MEK inhibitors), SB203580 (p38 inhibitor), CT99021 (GSK3beta inhibitor), or a similar amount of DMSO were present in the culture. More details of specific culture conditions are provided in Figures S6 and S7.

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Cytokine Detection by ICS and ELISA
At day 5 or 7, cells were restimulated with immobilized anti-CD3 (2 mug/ml) and anti-CD28 (2 mug/ml) (4 hr with BrefeldinA [10 mug/ml] in the last 2 hr) or with PdBU and Ionomycin (5 hr with BrefeldinA [1 mug/ml]). After ICS FACS, data were collected on a FACSCalibur (Becton Dickinson) and analyzed with FlowJo (Tree Star). For ELISA, cells were similarly restimulated for 48 hr and supernatant was collected and analyzed for IL-4, IL-10, and IFN-gamma as described before (Shoemaker etal., 2006).


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In Vivo Studies
BALB/c mice were injected intravenously (i.v.) with a red blood cell-depleted single-cell spleen suspension (2.5 x 107 cells) (Castro etal., 2000) from DO11.10 WT or STAT-deficient mice. After 48 hr, they were injected subcutaneously with PBS or with OVA protein (5 mg) plus LPS (5 mug). The inguinal lymph nodes were removed 48 hr later. A single-cell suspension (1 x 106 cells) was restimulated for 24 or 48 hr with 1 muM or 3 muM of OVA and with BrefeldinA for the last 6 hr. Half of the supernatant was removed before the addition of BrefeldinA for use in an ELISA assay. The cells were fixed and stained as before. Those positive for KJ1-26-Bio and for CD4-PerCP were gated, and IFN-gamma and IL-10 staining was examined for this population and analyzed as before.

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Real-Time Quantitative RT-PCR
Cells were harvested and restimulated in the presence of immobilized anti-CD3 (2 mug/ml) plus anti-CD28 (2 mug/ml) for 3 hr or immediately lysed. RNA was extracted and reverse-transcribed and cDNA was analyzed for the expression of cytokines and transcription factors by real-time PCR assay as before (Shoemaker etal., 2006). Target gene mRNA expression was quantified either with SYBR Green (Applied Biosystems) or with Master Mix (Applied Biosystems) and normalized to ubiquitin or HPRT mRNA levels, respectively.

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Immunoblotting
Differentiated CD4+ Tcells were rested for 5 hr in 1% FCS-containing medium and restimulated as described for specific experiments. Cell lysates were prepared, equal amounts of protein were separated by SDS-PAGE, and phosphorylated or total ERK and actin were detected as described before (Beinke etal., 2004).


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Reactive oxygen intermediate-dependent NF-kappaB activation by interleukin-1beta requires 5-lipoxygenase or NADPH oxidase activity.
We previously reported that the role of reactive oxygen intermediates (ROIs) in NF-kappaB activation by proinflammatory cytokines was cell specific. However, the sources for ROIs in various cell types are yet to be determined and might include 5-lipoxygenase (5-LOX) and NADPH oxidase. 5-LOX and 5-LOX activating protein (FLAP) are coexpressed in lymphoid cells but not in monocytic or epithelial cells. Stimulation of lymphoid cells with interleukin-1beta (IL-1beta) led to ROI production and NF-kappaB activation, which could both be blocked by antioxidants or FLAP inhibitors, confirming that 5-LOX was the source of ROIs and was required for NF-kappaB activation in these cells. IL-1beta stimulation of epithelial cells did not generate any ROIs and NF-kappaB induction was not influenced by 5-LOX inhibitors. However, reintroduction of a functional 5-LOX system in these cells allowed ROI production and 5-LOX-dependent NF-kappaB activation. In monocytic cells, IL-1beta treatment led to a production of ROIs which is independent of the 5-LOX enzyme but requires the NADPH oxidase activity. This pathway involves the Rac1 and Cdc42 GTPases, two enzymes which are not required for NF-kappaB activation by IL-1beta in epithelial cells. In conclusion, three different cell-specific pathways lead to NF-kappaB activation by IL-1beta: a pathway dependent on ROI production by 5-LOX in lymphoid cells, an ROI- and 5-LOX-independent pathway in epithelial cells, and a pathway requiring ROI production by NADPH oxidase in monocytic cells.

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Input:
Activation-dependent transcriptional regulation of the human Fas promoter requires NF-kappaB p50-p65 recruitment.
Fas (CD95) and Fas ligand (CD95L) are an interacting receptor-ligand pair required for immune homeostasis. Lymphocyte activation results in the upregulation of Fas expression and the acquisition of sensitivity to FasL-mediated apoptosis. Although Fas upregulation is central to the preservation of immunologic tolerance, little is known about the molecular machinery underlying this process. To investigate the events involved in activation-induced Fas upregulation, we have examined mRNA accumulation, fas promoter activity, and protein expression in the Jurkat T-cell line treated with phorbol myristate acetate and ionomycin (P/I), pharmacological mimics of T-cell receptor activation. Although resting Jurkat cells express Fas, Fas mRNA was induced approximately 10-fold in 2 h upon P/I stimulation. Using sequential deletion mutants of the human fas promoter in transient transfection assays, we identified a 47-bp sequence (positions -306 to -260 relative to the ATG) required for activation-driven fas upregulation. Sequence analysis revealed the presence of a previously unrecognized composite binding site for both the Sp1 and NF-kappaB transcription factors at positions -295 to -286. Electrophoretic mobility shift assay (EMSA) and supershift analyses of this region documented constitutive binding of Sp1 in unactivated nuclear extracts and inducible binding of p50-p65 NF-kappaB heterodimers after P/I activation. Sp1 and NF-kappaB transcription factor binding was shown to be mutually exclusive by EMSA displacement studies with purified recombinant Sp1 and recombinant p50. The functional contribution of the kappaB-Sp1 composite site in P/I-inducible fas promoter activation was verified by using kappaB-Sp1 concatamers (-295 to -286) in a thymidine kinase promoter-driven reporter construct and native promoter constructs in Jurkat cells overexpressing IkappaB-alpha. Site-directed mutagenesis of the critical guanine nucleotides in the kappaB-Sp1 element documented the essential role of this site in activation-dependent fas promoter induction.

interacting receptor-ligand pair

binding

binding

binding

binding

expression

Fas

expression

Fas

express

Fas

mutually exclusive

binding

dependent

regulation

requires

regulation

recruitment

recruitment

recruitment

upregulation

expression

upregulation

Fas

induced

upregulation

upregulation

Fas

induced

Fas

required

upregulation

upregulation

fas

inducible

binding

activation

fas

promoter

essential role

induction

induction

fas

promoter

regulation

Fas

promoter


Input:
RFLAT-1: a new zinc finger transcription factor that activates RANTES gene expression in T lymphocytes.
RANTES (Regulated upon Activation, Normal T cell Expressed and Secreted) is a chemoattractant cytokine (chemokine) important in the generation of inflammatory infiltrate and human immunodeficiency virus entry into immune cells. RANTES is expressed late (3-5 days) after activation in T lymphocytes. Using expression cloning, we identified the first "late" T lymphocyte associated transcription factor and named it "RANTES Factor of Late Activated T Lymphocytes-1" (RFLAT-1). RFLAT-1 is a novel, phosphorylated, zinc finger transcription factor that is expressed in T cells 3 days after activation, coincident with RANTES expression. While Rel proteins play the dominant role in RANTES gene expression in fibroblasts, RFLAT-1 is a strong transactivator for RANTES in T cells.

expression

RANTES

expressed

RANTES

expressed

expression

RANTES

expression

RANTES

phosphorylated

activates

expression

dominant role

expression

transactivator

RANTES


Input:
Interleukin-10 inhibits expression of both interferon alpha- and interferon gamma- induced genes by suppressing tyrosine phosphorylation of STAT1.
Interleukin-10 (IL-10) helps maintain polarized T-helper cells in a T-helper lymphocyte 2 (Th2) phenotype. Part of this process involves the prevention of the development of Th1 cells, which are a primary source of interferon gamma (IFNgamma), a potent activator of monocytes and an inhibitor of Th2 proliferation. Because monocytes and macrophages are important mediators of Th1-type responses, such as delayed-type hypersensitivity, we sought to determine if IL-10 could directly mediate inhibition of IFNgamma- and IFNalpha-induced gene expression in these cells. Highly purified monocytes were incubated with IL-10 for 60 to 90 minutes before the addition of IFNgamma or IFNalpha. IL-10 preincubation resulted in the inhibition of gene expression for several IFN-induced genes, such as IP-10, ISG54, and intercellular adhesion molecule-1. The reduction in gene expression resulted from the ability of IL-10 to suppress IFN-induced assembly of signal transducer and activator of transcription (STAT) factors to specific promoter motifs on IFNalpha- and IFNgamma-inducible genes. This was accomplished by preventing the IFN-induced tyrosine phosphorylation of STAT1, a component of both IFNalpha- and IFNgamma-induced DNA binding complexes. Therefore, IL-10 can directly inhibit STAT-dependent early response gene expression induced by both IFNalpha and IFNgamma in monocytes by suppressing the tyrosine phosphorylation of STAT1. This may occur through the ability of IL-10 to induce expression of the gene, suppressor of cytokine signaling 3 (SOCS3).

primary source

IFNgamma

expression

expression

expression

intercellular adhesion molecule-1

expression

suppressing

phosphorylation

inhibition

expression

preventing

induced

suppressing

phosphorylation

phosphorylation

tyrosine

phosphorylation

tyrosine

phosphorylation

tyrosine

induced

induced

induced

intercellular adhesion molecule-1

resulted

inhibition

accomplished

resulted

preventing

induced

phosphorylation

induced

IFNgamma

induced

induce

expression


Input:
The Megakaryocyte/Platelet-specific enhancer of the alpha2beta1 integrin gene: two tandem AP1 sites and the mitogen-activated protein kinase signaling cascade.
The alpha2beta1 integrin, a collagen receptor on platelets and megakaryocytes, is required for normal platelet function. Transcriptional regulation of the alpha2 integrin gene in cells undergoing megakaryocytic differentiation requires a core promoter between bp -30 and -92, a silencer between bp -92 and -351, and megakaryocytic enhancers in the distal 5' flank. We have now identified a 229-bp region of the distal 5' flank of the alpha2 integrin gene required for high-level enhancer activity in cells with megakaryocytic features. Two tandem AP1 binding sites with dyad symmetry are required for enhancer activity and for DNA-protein complex formation with members of the c-fos/c-jun family. The requirement for AP1 activation suggested a role for the mitogen-activated protein kinase (MAPK) signaling pathway in regulating alpha2 integrin gene expression. Inhibition of the MAP kinase cascade with PD98059, a specific inhibitor of MAPK kinase 1, prevented the expression of the alpha2 integrin subunit in cells induced to become megakaryocytic. We provide a model of megakaryocytic differentiation in which expression of the alpha2 integrin gene requires signaling via the MAP kinase pathway to activate two tandem AP1 binding sites in the alpha2 integrin enhancer.

complex formation

c-fos

complex formation

c-jun

expression

alpha2 integrin

expression

alpha2 integrin

expression

alpha2 integrin

inhibitor

MAPK kinase 1

prevented

expression

requires

Transcriptional regulation

required

complex formation

requirement

regulating

role

regulating

requires

expression

Transcriptional regulation

alpha2 integrin

regulating

expression


Input:
Differential expression and phosphorylation of CTCF, a c-myc transcriptional regulator, during differentiation of human myeloid cells.
CTCF is a transcriptional repressor of the c-myc gene. Although CTCF has been characterized in some detail, there is very little information about the regulation of CTCF activity. Therefore we investigated CTCF expression and phosphorylation during induced differentiation of human myeloid leukemia cells. We found that: (i) both CTCF mRNA and protein are down-regulated during terminal differentiation in most cell lines tested; (ii) CTCF down-regulation is retarded and less pronounced than that of c-myc; (iii) CTCF protein is differentially phosphorylated and the phosphorylation profiles depend on the differentiation pathway. We concluded that CTCF expression and activity is controlled at transcriptional and post-transcriptional levels.

expression

CTCF

expression

CTCF

expression

CTCF

transcriptional repressor

CTCF

c-myc

down-regulated

CTCF

down-regulation

CTCF

down-regulation

c-myc

phosphorylation

CTCF

phosphorylation

CTCF

phosphorylated

CTCF

depend

phosphorylated

transcriptional regulator

CTCF

c-myc

regulation

CTCF

controlled at transcriptional and post-transcriptional levels

CTCF

controlled at transcriptional and post-transcriptional levels

expression


Input:
T-cell expression of the human GATA-3 gene is regulated by a non-lineage-specific silencer.
The GATA-3 transcription factor is required for development of the T-cell lineage and Th2 cytokine gene expression in CD4 T-cells. We have mapped the DNase-I-hypersensitive (HS) regions of the human GATA-3 gene in T-cells and non-T-cells and studied their transcriptional activities. HS I-III, located 5' from the transcriptional initiation site, were found in hematopoietic and non-hematopoietic cells, whereas HS IV-VII, located 3' from the transcriptional start site, were exclusively observed in T-cells. Among these hypersensitive sites, two transcriptional control elements were found, one in the first intron of the GATA-3 gene and the other between 8.3 and 5.9 kilobases 5' from the GATA-3 transcriptional initiation site. The first intron acted as a strong transcriptional activator in a position-dependent manner and with no cell-type specificity. The upstream regulatory element could confer T-cell specificity to the GATA-3 promoter activity, and analysis of this region revealed a 707-base pair silencer that drastically inhibited GATA-3 promoter activity in non-T-cells. Two CAGGTG E-boxes, located at the 5'- and 3'-ends of the silencer, were necessary for this silencer activity. The 3'-CAGGTG E-box could bind USF proteins, the ubiquitous repressor ZEB, or the basic helix-loop-helix proteins E2A and HEB, and we showed that a competition between ZEB and E2A/HEB proteins is involved in the silencer activity.

bind

ZEB

bind

bind

HEB

expression

inhibited

promoter

competition

bind

ZEB

confer

promoter

necessary

inhibited

regulated

expression

involved

inhibited

competition

transcriptional


Input:
AML and Ets proteins regulate the I alpha1 germ-line promoter.
The immunoglobulin heavy chain (IgH) class switch recombination of B lymphocytes preferentially targets unrearranged IgH genes that have already been rendered transcriptionally active. Transcription of the germ-line IgH genes is controlled by intervening (I) regions upstream of their switch regions. The I alpha1 promoter activates transcription of the human germ-line C alpha1 gene for IgA1 and mediates the transforming growth factor (TGF)-beta1 responsiveness of this locus. Here we show that the I alpha1 promoter contains several binding sites for the AML/PEBP2/CBF family of transcription factors and that AML and Ets proteins are major regulators of the basal and TGF-beta-inducible promoter activity. Our data constitute a starting point for studies to elucidate the molecular mechanism by which TGF-beta regulates IgA production.


Input:
Peripheral blood mononuclear cells isolated from patients with diabetic nephropathy show increased activation of the oxidative-stress sensitive transcription factor NF-kappaB.
Increased oxidative stress and subsequent activation of the transcription factor NF-kappaB has been linked to the development of late diabetic complications. To determine whether oxidative stress dependent NF-kappaB activation is evident in patients with diabetic nephropathy we used an Electrophoretic Mobility Shift Assay based semiquantitative detection system which enabled us to determine NF-kappaB activation in ex vivo isolated peripheral blood mononuclear cells. We examined 33 patients with diabetes mellitus (Type I and Type II). Patients with diabetic nephropathy showed higher NF-kappaB binding activity in Electrophoretic Mobility Shift Assays and stronger immunohistological staining for activated NF-kappaBp65 than patients without renal complications. NF-kappaB binding activity correlated with the degree of albuminuria (r = 0.316) and with thrombomodulin plasma concentrations (r = 0.33), indicative for albuminuria associated endothelial dysfunction. In a 3 day intervention study in which 600 mg of the antioxidant thioctic acid (alpha-lipoic acid) per day were given to nine patients with diabetic nephropathy oxidative stress in plasma samples was decreased by 48% and NF-kappaB binding activity in ex vivo isolated peripheral blood mononuclear cells by 38%. In conclusion, activation of the transcription factor NF-kappaB in ex vivo isolated peripheral blood mononuclear cells of patients with diabetes mellitus correlates with the degree of diabetic nephropathy. NF-kappaB activation is at least in part dependent on oxidative stress since thioctic acid (alpha-lipoic acid) reduced NF-kappaB binding activity.

stronger

activated

activated


Input:
Activation of human immunodeficiency virus type 1 expression by Gardnerella vaginalis.
Bacterial vaginosis (BV) is associated with an increased rate of sexual transmission of human immunodeficiency virus (HIV) type 1, and Gardnerella vaginalis is frequently isolated from the genital tracts of women with BV. G. vaginalis lysates were found to significantly stimulate HIV expression in monocytoid cells. Stimulation was significantly higher when lysates were heated at 100 degrees C for 5 min but was reduced by treatment with lysozyme or protease. G. vaginalis lysates also activated HIV expression in certain T cell lines. G. vaginalis lysates activated HIV long-terminal repeat transcription in HIV-infected cells and increased NF-kappaB binding activity, indicating an effect by G. vaginalis on HIV transcription. The activation of HIV production by G. vaginalis suggests that genital tract infection with G. vaginalis increases the risk of HIV transmission by increasing HIV expression in the genital tract. This may explain, at least in part, the increased rate of HIV transmission in women with BV.


Input:
Interferon-alpha activates multiple STAT proteins and upregulates proliferation-associated IL-2Ralpha, c-myc, and pim-1 genes in human T cells.
Interferon-alpha (IFN-alpha) is a pleiotropic cytokine that has antiviral, antiproliferative, and immunoregulatory functions. There is increasing evidence that IFN-alpha has an important role in T-cell biology. We have analyzed the expression of IL-2Ralpha, c-myc, and pim-1 genes in anti-CD3-activated human T lymphocytes. The induction of these genes is associated with interleukin-2 (IL-2)-induced T-cell proliferation. Treatment of T lymphocytes with IFN-alpha, IL-2, IL-12, and IL-15 upregulated IL-2Ralpha, c-myc, and pim-1 gene expression. IFN-alpha also sensitized T cells to IL-2-induced proliferation, further suggesting that IFN-alpha may be involved in the regulation of T-cell mitogenesis. When we analyzed the nature of STAT proteins capable of binding to IL-2Ralpha, pim-1, and IRF-1 GAS elements after cytokine stimulation, we observed IFN-alpha-induced binding of STAT1, STAT3, and STAT4, but not STAT5 to all of these elements. Yet, IFN-alpha was able to activate binding of STAT5 to the high-affinity IFP53 GAS site. IFN-alpha enhanced tyrosine phosphorylation of STAT1, STAT3, STAT4, STAT5a, and STAT5b. IL-12 induced STAT4 and IL-2 and IL-15 induced STAT5 binding to the GAS elements. Taken together, our results suggest that IFN-alpha, IL-2, IL-12, and IL-15 have overlapping activities on human T cells. These findings thus emphasize the importance of IFN-alpha as a T-cell regulatory cytokine.

binding

GAS elements

binding

GAS elements

binding

GAS elements

binding

GAS elements

binding

GAS elements

binding

GAS elements

binding

GAS elements

binding

GAS elements

binding

GAS elements

binding

GAS elements

binding

GAS elements

binding

GAS elements

binding

GAS elements

binding

GAS elements

binding

GAS elements

binding

expression

expression

c-myc

expression

expression

expression

c-myc

expression

phosphorylation

tyrosine

phosphorylation

tyrosine

phosphorylation

tyrosine

phosphorylation

tyrosine

phosphorylation

tyrosine

upregulates

c-myc

upregulates

upregulates

induction

induction

c-myc

induction

upregulated

expression

induced

binding

enhanced

phosphorylation

induced

binding


Input:
Anti-rheumatic compound aurothioglucose inhibits tumor necrosis factor-alpha-induced HIV-1 replication in latently infected OM10.1 and Ach2 cells.
NF-kappaB is a potent cellular activator of HIV-1 gene expression. Down-regulation of NF-kappaB activation is known to inhibit HIV replication from the latently infected cells. Gold compounds have been effectively used for many decades in the treatment of rheumatoid arthritis. We previously reported that gold compounds, especially aurothioglucose (AuTG) containing monovalent gold ion, inhibited the DNA-binding of NF-kappaB in vitro. In this report we have examined the efficacy of the gold compound AuTG as an inhibitor of HIV replication in latently infected OM10.1 and Ach2 cells. Tumor necrosis factor (TNF)-alpha-induced HIV-1 replication in OM10.1 or Ach2 cells was significantly inhibited by non-cytotoxic doses of AuTG (>10 microM in OM10.1 cells and >25 F.M in Ach2 cells), while 25 microM of the counter-anion thioglucose (TG) or gold compound containing divalent gold ion, HAuCl3, had no effect. The effect of AuTG on NF-kappaB-dependent gene expression was confirmed by a transient CAT assay. Specific staining as well as electron microscopic examinations revealed the accumulation of metal gold in the cells, supporting our previous hypothesis that gold ions could block NF-kappaB-DNA binding by a redox mechanism. These observations indicate that the monovalent gold compound AuTG is a potentially useful drug for the treatment of patients infected with HIV.


Input:
Evidence for suppressed activity of the transcription factor NFAT1 at its proximal binding element P0 in the IL-4 promoter associated with enhanced IL-4 gene transcription in T cells of atopic patients.
Allergen-specific T cells in atopic patients are polarized IL-4-producing Th2 cells, promoting IgE synthesis by B cells. The molecular basis for increased IL-4 gene expression in atopy is not fully understood. IL-4 gene regulation in general involves the nuclear factor of activated T cells (NFAT) family of transcription factors, of which NFAT1 and NFAT2 are most prominent in peripheral T cells. Recently, a unique inhibitory role of NFAT1 in IL-4 gene control was shown in the mouse. In a series of electrophoretic mobility shift assays with protein extracts of highly polarized Th2 clones from atopics and Th1 clones from controls we compared DNA-binding activities at the two NFAT-binding elements P0 and P1 of the crucial proximal human IL-4 promoter. At the most proximal P0 site, NFAT-containing complexes devoid of NFAT2 were readily inducible in the Th1 clones, but hardly or not in the Th2 clones. In contrast, both in Th1 and Th2 clones NFAT-containing complexes were strongly inducible at the P1 site, consisting of NFAT2 and a P0-compatible NFAT activity, without apparent differences between Th1 and Th2 clones. Like in Th2 clones, suppressed NFAT-P0 complex formation was observed also at the polyclonal level in peripheral blood mononuclear cells (PBMC) of three of five severe atopic dermatitis patients with strongly elevated serum IgE levels, but not in control PBMC. These findings suggest that high-level IL-4 production in atopic Th2 cells is associated with selective reduction of suppressive NFAT1 activity at the IL-4 P0 element and that some patients with this multifactorial disease may have a putative systemic disorder at this level.

binding

proximal binding element P0

complexes

complexes

producing

synthesis

IgE

expression

levels

IgE

production

suppressed

binding

inhibitory role

control

enhanced

transcription

promoting

producing

synthesis

increased

expression

inducible

complexes

elevated

levels

high-level

production

regulation

involves

regulation

involves

regulation

control

transcription


Input:
Nuclear factor-kappa B activity in T cells from patients with rheumatic diseases: a preliminary report.
OBJECTIVE: The NF-kappa B/Rel family of transcription factors regulates the expression of many genes involved in the immune or inflammatory response at the transcriptional level. The aim of this study was to determine whether distinctive patterns of NF-kappa B activation are seen in different forms of joint disease. METHODS: The DNA binding activity of these nucleoproteins was examined in purified synovial and peripheral T cells from patients with various chronic rheumatic diseases (12: four with rheumatoid arthritis; five with spondyloarthropathies; and three with osteoarthritis). RESULTS: Electrophoretic mobility shift assays disclosed two specific complexes bound to a NF-kappa B specific 32P-labelled oligonucleotide in nucleoproteins extracted from purified T cells isolated from synovial fluid and peripheral blood of patients with rheumatoid arthritis. The complexes consisted of p50/p50 homodimers and p50/p65 heterodimers. Increased NF-kappa B binding to DNA in synovial T cells was observed relative to peripheral T cells. In non-rheumatoid arthritis, binding of NF-kappa B in synovial T cells was exclusively mediated by p50/p50 homodimers. CONCLUSION: Overall, the results suggest that NF-kappa B may play a central part in the activation of infiltrating T cells in chronic rheumatoid arthritis. The activation of this nuclear factor is qualitatively different in rheumatoid synovial T cells to that in other forms of non-rheumatoid arthritis (for example, osteoarthritis, spondyloarthropathies).


Input:
N-acetyl-L-cysteine inhibits primary human T cell responses at the dendritic cell level: association with NF-kappaB inhibition.
N-acetyl-L-cysteine (NAC) is an antioxidant molecule endowed with immunomodulatory properties. To investigate the effect of NAC on the induction phase of T cell responses, we analyzed its action on human dendritic cells (DC) derived from adherent PBMC cultured with IL-4 and granulocyte-macrophage CSF. We first found that NAC inhibited the constitutive as well as the LPS-induced activity of the transcription factor NF-kappaB. In parallel, NAC was shown to down-regulate the production of cytokines by DC as well as their surface expression of HLA-DR, CD86 (B7-2), and CD40 molecules both at the basal state and upon LPS activation. NAC also inhibited DC responses induced by CD40 engagement. The inhibitory effects of NAC were not due to nonspecific toxicity as neither the viability of DC nor their mannose receptor-mediated endocytosis were modified by NAC. Finally, we found that the addition of NAC to MLR between naive T cells and allogeneic DC resulted in a profound inhibition of alloreactive responses, which could be attributed to a defect of DC as APC-independent T cell responses were not inhibited by NAC. Altogether, our results suggest that NAC might impair the generation of primary immune responses in humans through its inhibitory action on DC.

engagement

expression

expression

down-regulate

expression

down-regulate

activation

activation

expression


Input:
Bacterial lipopolysaccharide activates nuclear factor-kappaB through interleukin-1 signaling mediators in cultured human dermal endothelial cells and mononuclear phagocytes.
Bacterial lipopolysaccharide (LPS)-mediated immune responses, including activation of monocytes, macrophages, and endothelial cells, play an important role in the pathogenesis of Gram-negative bacteria-induced sepsis syndrome. Activation of NF-kappaB is thought to be required for cytokine release from LPS-responsive cells, a critical step for endotoxic effects. Here we investigated the role and involvement of interleukin-1 (IL-1) and tumor necrosis factor (TNF-alpha) signal transducer molecules in LPS signaling in human dermal microvessel endothelial cells (HDMEC) and THP-1 monocytic cells. LPS stimulation of HDMEC and THP-1 cells initiated an IL-1 receptor-like NF-kappaB signaling cascade. In transient cotransfection experiments, dominant negative mutants of the IL-1 signaling pathway, including MyD88, IRAK, IRAK2, and TRAF6 inhibited both IL-1- and LPS-induced NF-kappaB-luciferase activity. LPS-induced NF-kappaB activation was not inhibited by a dominant negative mutant of TRAF2 that is involved in TNF signaling. LPS-induced activation of NF-kappaB-responsive reporter gene was not inhibited by IL-1 receptor antagonist. TLR2 and TLR4 were expressed on the cell surface of HDMEC and THP-1 cells. These findings suggest that a signal transduction molecule in the LPS receptor complex may belong to the IL-1 receptor/toll-like receptor (TLR) super family, and the LPS signaling cascade uses an analogous molecular framework for signaling as IL-1 in mononuclear phagocytes and endothelial cells.

expressed

expressed


Input:
Selective activation and functional significance of p38alpha mitogen-activated protein kinase in lipopolysaccharide-stimulated neutrophils.
Activation of leukocytes by proinflammatory stimuli selectively initiates intracellular signal transduction via sequential phosphorylation of kinases. Lipopolysaccharide (LPS) stimulation of human neutrophils is known to result in activation of p38 mitogen-activated protein kinase (MAPk); however, the upstream activator(s) of p38 MAPk is unknown, and consequences of p38 MAPk activation remain largely undefined. We investigated the MAPk kinase (MKK) that activates p38 MAPk in response to LPS, the p38 MAPk isoforms that are activated as part of this pathway, and the functional responses affected by p38 MAPk activation. Although MKK3, MKK4, and MKK6 all activated p38 MAPk in experimental models, only MKK3 was found to activate recombinant p38 MAPk in LPS-treated neutrophils. Of p38 MAPk isoforms studied, only p38alpha and p38delta were detected in neutrophils. LPS stimulation selectively activated p38alpha. Specific inhibitors of p38alpha MAPk blocked LPS-induced adhesion, nuclear factor-kappa B (NF-kappaB) activation, and synthesis of tumor necrosis factor-alpha (TNF-alpha). Inhibition of p38alpha MAPk resulted in a transient decrease in TNF-alpha mRNA accumulation but persistent loss of TNF-alpha synthesis. These findings support a pathway by which LPS stimulation of neutrophils results in activation of MKK3, which in turn activates p38alpha MAPk, ultimately regulating adhesion, NF-kappaB activation, enhanced gene expression of TNF-alpha, and regulation of TNF-alpha synthesis.

detected

detected

synthesis

TNF-alpha

synthesis

TNF-alpha

expression

TNF-alpha

synthesis

TNF-alpha

inhibitors

p38alpha MAPk

blocked

inhibitors

synthesis

Inhibition

p38alpha MAPk

loss

Inhibition

synthesis

activated

results

activation

activation

activates

results

p38alpha MAPk

enhanced

activates

expression

regulation

activates

synthesis


Input:
Inhibition of IL-4-inducible gene expression in human monocytes by type I and type II interferons.
The Th2-type cytokines, interleukin-4 (IL-4) and interleukin-13 (IL-13), induce expression of a distinct subset of genes in human monocytes, including FcepsilonRIIb (CD23), 15-lipoxygenase, IL-1 receptor antagonist (IL-1ra), and type I and type II IL-1 receptors (IL-1R). Type I interferons (IFN-alpha and IFN-beta) and type II interferon (IFN-gamma) inhibit induction of these genes by IL-4 and IL-13. However, the mechanism by which IFNs mediate this inhibition has not been defined. In this overview, we discuss the role of the transcription factor, STAT6 (signal transducer and activator of transcription-6) in mediating IL-4- and IL-13-induced gene expression in monocytes. We also discuss our recent findings that type I and type II IFNs suppress IL-4/IL-13-inducible gene expression by inhibiting tyrosine phosphorylation and nuclear translocation of STAT6. The ability of type I and type II IFNs to inhibit IL-4/IL-13-induced STAT6 activity is dose- and time-dependent, and is not unique to monocytes because IFNs induce the same effects in fibroblasts. Inhibition of STAT6 activity is not evident unless cells are preincubated with IFN for at least 1 h before IL-4 stimulation. Furthermore, inhibition can be blocked by actinomycin D, indicating a requirement for de novo transcription. We propose a model in which stimulation of monocytes by IFN activates de novo synthesis of an inhibitory factor, possibly one or more members of the SOCS/ SSI/CIS gene family, capable of suppressing activation of STAT6 by IL-4 and IL-13. Because STAT6 activation plays an essential role in IL-4/IL-13-induced gene expression, the ability of IFN-beta and IFN-gamma to inhibit STAT6 activity provides an explanation for how IFNs can suppress IL-4/IL-13-inducible gene expression.

expression

expression

expression

translocation

nuclear

inhibit

IFN-beta

induction

inhibit

IFN-gamma

induction

inhibit

induction

inhibiting

phosphorylation

inhibit

induced

Inhibition

blocked

Inhibition

suppressing

activation

inhibit

IFN-beta

inhibit

IFN-gamma

phosphorylation

tyrosine

induce

expression

induce

expression

induction

induction

induction

induction

induction

induction

mediate

inhibit

induced

induced

requirement

Inhibition

activation

activation

activation


Input:
Involvement of NF-kappaB p50/p65 heterodimer in activation of the human pro-interleukin-1beta gene at two subregions of the upstream enhancer element.
A region between-3134 and -2729 bp upstream from the transcription site of the human pro-interleukin 1beta (proIL-1beta) gene was identified as an LPS-responsive enhancer element. In this study, the influence of the sequences located between -3134 and -2987 on the transcriptional activity of the proIL-1beta gene in LPS-stimulated Raw 264.7 cells was examined in detail. The results obtained by transient transfection of fos -CAT constructs that contained serial 5'-deletion mutations showed that the region between -3134 and -3059 appears to be required for the induction of transcription by LPS. Gel shift assay studies with synthetic oligonucleotides corresponding to partial sequences of the latter region and nuclear extracts from stimulated cells revealed specific protein binding sites between -3110 and -3090 and between -3079 and -3059. These specific bindings were time and LPS dose dependent. The results of supershift analysis using specific antibodies against transcription factors suggested that both binding complexes contained the NF-kappaB components p50 and p65, and did not contain other NF-kappaB proteins (p52, c-Rel, Rel B), AP-1 proteins (c-Fos, C-Jun), CREB or C/EBPbeta (NF-IL6). Mutation of either of the putative NF-kappaB-binding sites in the enhancer element decreased the LPS-stimulated transcriptional activity. These data indicated that two NF-kappaB-binding sites, which are located between -3134 and -3059, are critical for the activation of proIL-1beta gene transcription. Copyright 1999 Academic Press.

decreased

stimulated

Involvement

activation

Involvement

activation

activation

induction

transcriptional activity

stimulated

transcriptional activity

critical

activation

activation

transcription

influence

transcriptional activity

transcriptional activity

transcription


Input:
Phosphorylation of TRAF2 inhibits binding to the CD40 cytoplasmic domain.
TRAF2 is a signal transducing adaptor molecule which binds to the CD40 cytoplasmic domain. We have found that it is phosphorylated, predominantly on serine residues, when transiently overexpressed in 293 cells. The phosphorylation appears to be related to the signaling events that are activated by TRAF2 under these circumstances, since two nonfunctional mutants were found to be phosphorylated significantly less than the wild-type protein. Furthermore, the phosphorylation status of TRAF2 had significant effects on the ability of the protein to bind to CD40, as evidenced by our observations that the CD40 cytoplasmic domain interacted preferentially with underphosphorylated TRAF2 and that phosphatase treatment significantly enhanced the binding of TRAF2 to CD40. We conclude from these studies that the phosphorylation of TRAF2 is likely to play an important role in regulating signaling by virtue of its ability to influence the CD40-TRAF2 interaction. Copyright 1999 Academic Press.

binding

cytoplasmic domain

binds

cytoplasmic domain

bind

interacted

cytoplasmic domain

binding

interaction

overexpressed

inhibits

Phosphorylation

binding

underphosphorylated

underphosphorylated

Phosphorylation

phosphorylated

serine residues

phosphorylation

underphosphorylated

phosphorylation

when

phosphorylated

overexpressed

overexpressed

overexpressed

enhanced

binding

effects

phosphorylation

bind

influence

phosphorylation

interaction


Input:
Cobalt chloride-induced signaling in endothelium leading to the augmented adherence of sickle red blood cells and transendothelial migration of monocyte-like HL-60 cells is blocked by PAF-receptor antagonist.
In response to hypoxia, sickle red blood cells (SS RBC) and leukocytes exhibit increased adherence to the vascular endothelium, while diapedesis of leukocytes through the blood vessel increases. However, the cellular signaling pathway(s) caused by hypoxia is poorly understood. We utilized CoCl2 as a mimetic molecule for hypoxia to study cellular signaling pathways. We found that in human umbilical vein endothelial cells (HUVEC), CoCl2 at 2 mM concentration induced the surface expression of a subset of CAMs (VCAM-1) and activation of transcription factor NF-kappaB in the nuclear extracts of HUVEC. Furthermore, CoCl2 also caused time-dependent tyrosine phosphorylation of mitogen-activated protein (MAP) kinase isoform ERK2 without significantly affecting ERK1, indicating ERK2 is the preferred substrate for upstream kinase of the MAPK pathway. Inhibitors of MAP kinase (PD98059) or platelet-activating factor (PAF)- receptor antagonist (CV3988) inhibited the CoCl2-induced NF-kappaB activation and VCAM-1 expression. Augmented expression of VCAM-1 led to increased SS RBC adhesion, inhibitable by a VCAM-1 antibody. Additionally, CoCl2 caused a two- to threefold increase in the rate of transendothelial migration of monocyte-like HL-60 cells and a twentyfold increase in phosphorylation of platelet endothelial cell adhesion molecules (PECAM-1). The transendothelial migration of monocytes was inhibited by an antibody to PECAM-1. Both phosphorylation of PECAM-1 and transendothelial migration of monocytes in response to CoCl2 were inhibited by protein kinase inhibitor (GF109203X) and augmented by protein phosphatase inhibitor (Calyculin A). Our data suggests that CoCl2-induced cellular signals directing increased expression of VCAM-1 in HUVEC involve downstream activation of MAP kinase and NF-kappaB, while the phosphorylation of PECAM-1 occurs as a result of activation of PKC. We conclude that PAF-receptor antagonist inhibits the CoCl2- or hypoxia-induced increase in the adhesion of SS RBC, PECAM-1 phosphorylation, and the concomitant transendothelial migration of monocytes.

expression

expression

expression

expression

inhibited

induced

inhibited

phosphorylation

inhibits

increase

phosphorylation

tyrosine

phosphorylation

phosphorylation

phosphorylation

phosphorylation

induced

expression

caused

phosphorylation

for

phosphorylation

induced

expression

Augmented

expression

caused

increase

increase

phosphorylation

in response

phosphorylation

augmented

phosphorylation

induced

directing

directing

increased

increased

expression

occurs

phosphorylation

increase

phosphorylation

affecting


Input:
Molecular mechanisms of neutrophil-endothelial cell adhesion induced by redox imbalance.
Previous studies have implicated a role for intracellular thiols in the activation of nuclear factor-kappaB and transcriptional regulation of endothelial cell adhesion molecules. This study was designed to determine whether changes in endothelial cell glutathione (GSH) or oxidized glutathione (GSSG) can alter neutrophil adhesivity and to define the molecular mechanism that underlies this GSSG/GSH-induced adhesion response. Treatment of human umbilical vein endothelial cell (HUVEC) monolayers for 6 hours with 0.2 mmol/L diamide and 1 mmol/L buthionine sulfoximine (BSO) decreased GSH levels and increased the ratio of GSSG to GSH without cell toxicity. These redox changes are similar to those observed with anoxia/reoxygenation. Diamide plus BSO-induced thiol/disulfide imbalance was associated with a biphasic increase in neutrophil adhesion to HUVECs with peak responses observed at 15 minutes (phase 1) and 240 minutes (phase 2). N-Acetylcysteine treatment attenuated neutrophil adhesion in both phases, which indicated a role for GSH in the adhesion responses. Interestingly, phase 1 adhesion was inversely correlated with GSH levels but not with the GSSG/GSH ratio, whereas phase 2 neutrophil adhesion was positively correlated with GSSG/GSH ratio but not with GSH levels. Intercellular adhesion molecule-1 and P-selectin-specific monoclonal antibodies attenuated the increased neutrophil adhesion during both phases, whereas an anti-E-selectin monoclonal antibody also attenuated the phase 2 response. Pretreatment with actinomycin D and cycloheximide or with competing ds-oligonucleotides that contained nuclear factor-kappaB or activator protein-1 cognate DNA sequences significantly attenuated the phase 2 response, which implicated a role for de novo protein synthesis. Surface expression of intercellular adhesion molecule-1, P-selectin, and E-selectin on HUVECs correlated with the phase 1 and 2 neutrophil adhesion responses. This study demonstrates that changes in endothelial cell GSSG/GSH cause transcription-independent and transcription-dependent surface expression of different endothelial cell adhesion molecules, which leads to a 2-phase neutrophil-endothelial adhesion response.

expression

intercellular adhesion molecule-1

expression

P-selectin

expression

E-selectin


Input:
Decreased proteasome-mediated degradation in T cells from the elderly: A role in immune senescence.
Induction of NFkappaB is a highly regulated process requiring phosphorylation, ubiquitination, and proteasome-mediated degradation of the cytosolic inhibitor IkappaBalpha. Analyses of the regulation of IkappaBalpha in TNF-alpha-treated T lymphocytes from young and elderly donors revealed severely compromised degradation of IkappaBalpha in T cells from the elderly. Examination of activation-induced phosphorylation and ubiquitination of IkappaBalpha did not demonstrate any significant age-related alterations. However, examination of proteasome activity in these T cells using fluorogenic peptide assays revealed a significant age-related decline in chymotryptic activity. These results suggest that a decline in proteasome activity results in a failure to fully degrade IkappaBalpha in the elderly. This failure to degrade IkappaBalpha may underlie both the observed decrease in NFkappaB induction and the IL-2 receptor expression in TNF-treated T cells during aging. Thus, decreased proteasome-mediated degradation may be central to immune dysfunction that accompanies aging. Copyright 1999 Academic Press.

phosphorylation

IkappaBalpha

mediated

degradation

induced

phosphorylation

results

degrade

degradation

IkappaBalpha

degradation

IkappaBalpha

degrade

IkappaBalpha

degrade

IkappaBalpha

regulation

IkappaBalpha

alterations

induced


Input:
Identification of upstream regulatory elements that repress expression of adult beta-like globin genes in a primitive erythroid environment.
Our investigations have focused on localizing cis-elements responsible for the down regulation of the adult beta-like globin genes (delta and beta) in immature, or primitive erythroid tissues. We studied their activity after transfection into K562 cells, an erythroleukemia cell line with an embryonic-fetal phenotype. Analyzed DNA sequences included delta and beta 5' flanking regions extending from approximately -500 to +50bp (promoter regions), truncated delta and beta 5' flanking regions extending from approximately -250 to +50 bp, and chimeric promoter constructions, which consisted of a distal delta or beta fragment fused to a proximal beta or delta sequence. In CAT reporter constructions no appreciable level of CAT activity was supported by the beta globin promoter, and only low level activity by the delta promoter. Truncation of the beta globin promoter led to a 2-3 fold increase in promoter activity. In contrast, deletion of the upstream portion of the delta promoter led to a 10 fold decrease in expression. Coupling of the upstream beta globin sequence from approximately -500 to -250 bp to the truncated delta promoter fragment led to complete extinction of transcription activity, consistent with a negative regulatory effect of the beta globin gene upstream element(s). Fusion of the upstream portion of the delta promoter to the truncated beta globin promoter yielded a modest increase in promoter strength relative to the truncated beta gene promoter, indicating the presence of a positive transcriptional element(s) in the upstream delta globin regulatory region. Site-directed mutagenesis of binding sites for the repressor proteins BP1 and BP2 in the upstream portion of the beta globin gene flanking region led to a 4-6 fold increase in promoter activity. DNase I footprinting of the upstream delta-globin region revealed protected sequences corresponding to consensus binding sites for GATA-1 and BP2. These results confirm that sequences in the upstream promoter region of the adult beta globin gene contribute to its factor-mediated suppression early in development and then may modulate its expression at a later stage.

expression

CAT

expression

beta globin

down regulation

delta

down regulation

beta

decrease

expression

suppression

beta globin

supported

CAT

beta globin

promoter

supported

CAT

delta

promoter

increase

beta globin

promoter

increase

beta globin

promoter

contribute

upstream promoter region

beta globin

suppression

responsible

down regulation

modulate

contribute

expression


Input:
Reduction of tumour necrosis factor alpha expression and signalling in peripheral blood mononuclear cells from patients with thalassaemia or sickle cell anaemia upon treatment with desferrioxamine.
Recent evidence indicates that the rate of progression of the HIV-1 disease is significantly reduced in thalassaemia major patients upon treatment with high doses of desferrioxamine (DFX). The authors have previously demonstrated that in vitro exposure of mononuclear cells to DFX decreases the bioavailability of tumour necrosis factor alpha (TNF-alpha) which has a stimulatory effect on HIV-1 replication. In this study, therefore, TNF-alpha bioavailability from mononuclear cells isolated from 10 patients with thalassaemia or sickle cell anaemia given DFX as compared to 10 untreated subjects has been evaluated. Evidence is presented showing that DFX treatment reduces TNF-alpha bioavailability (P<0.05) by inhibiting its steady state (P<0.05) and by enhancing its inactivation through binding to soluble TNF-alpha receptor type II (P<0.05). We also show that DFX treatment limits the in vivo activation of NF-kappaB, a transcription factor involved in both TNF-alpha gene transcription and TNF-alpha signalling (P<0.005). We conclude that TNF-alpha bioavailability and signalling are impaired in patients upon DFX treatment. This mechanism may contribute to delayed progression of the HIV-1 infection in vivo. Copyright 1999 Academic Press.

binding

TNF-alpha receptor type II

expression

tumour necrosis factor alpha

Reduction

expression

decreases

TNF-alpha

reduces

TNF-alpha

inactivation

TNF-alpha receptor type II

impaired

TNF-alpha

enhancing

inactivation

binding

involved

transcription

transcription

TNF-alpha


Input:
Inhibition of cyclooxygenase-2 expression by 4-trifluoromethyl derivatives of salicylate, triflusal, and its deacetylated metabolite, 2-hydroxy-4-trifluoromethylbenzoic acid.
The therapeutic potential of drugs that block the induction of cyclooxygenase-2 has been emphasized. When two 4-trifluoromethyl salicylate derivatives [2-acetoxy-4-trifluoromethyl-benzoic acid (triflusal) and its deacetylated metabolite 2-hydroxy-4-trifluoromethylbenzoic acid (HTB)] were compared with aspirin and sodium salicylate as cyclooxygenase-2 (COX-2) inhibitors, we observed that in bacterial lipopolysaccharide-activated human blood, triflusal, aspirin, and HTB, but not sodium salicylate, inhibited COX-2-mediated prostaglandin E2 (PGE2) production (IC50 = 0.16, 0.18, 0.39, and >10 mM, respectively). However, only triflusal and aspirin inhibited purified COX-2 enzyme. To test this apparent discrepancy, we realized that HTB and triflusal (but neither aspirin nor salicylate) produced a concentration-dependent inhibition of COX-2 protein expression in peripheral human mononuclear cells. This observation was further confirmed in a rat air pouch model in vivo, in which both aspirin and triflusal inhibited PGE2 production (ID50 = 18.9 and 11.4 mg/kg p.o., respectively) but only triflusal-treated animals showed a decrease in COX-2 expression. This different behavior may be, at least in part, due to the ability of HTB and triflusal to block the activation of the transcription factor nuclear factor-kappaB to a higher extent than aspirin and sodium salicylate. Thus, in addition to inhibiting the COX-2 activity at therapeutic concentrations, triflusal is able to block through its metabolite HTB the expression of new enzyme, and hence the resumption of PGE2 synthesis. Triflusal and HTB may exert beneficial effects in processes in which de novo COX-2 expression is involved and, in a broader sense, in pathological situations in which genes under nuclear factor-kappaB control are up-regulated.

expression

expression

expression

expression

expression

Inhibition

expression

inhibited

inhibition

expression

decrease

expression

block

expression

produced

inhibition


Input:
Expression of IkappaBalpha in the nucleus of human peripheral blood T lymphocytes.
According to current models the inhibitory capacity of I(kappa)B(alpha) would be mediated through the retention of Rel/NF-kappaB proteins in the cytosol. However, I(kappa)B(alpha) has also been detected in the nucleus of cell lines and when overexpressed by transient transfection. To gain better insight into the potential role of nuclear I(kappa)B(alpha) in a physiological context we have analysed its presence in the nucleus of human peripheral blood T lymphocytes (PBL). We demonstrate the nuclear localization of I(kappa)B(alpha) in PBL by different techniques: Western blot, indirect immunofluorescence and electron microscopy. Low levels of nuclear I(kappa)B(alpha) were detected in resting cells whereas a superinduction was obtained after PMA activation. The nuclear pool of I(kappa)B(alpha) showed a higher stability than cytosolic I(kappa)B(alpha) and was partially independent of the resynthesis of the protein. Unexpectedly, the presence of nuclear I(kappa)B(alpha) did not inhibit NF-kappaB binding to DNA and this phenomenon was not due to the presence of IkappaBbeta at the nuclear level. Immunoprecipitation experiments failed to demonstrate an association between nuclear I(kappa)B(alpha) and NF-kappaB proteins. Our results demonstrate that in resting and PMA-activated human PBL, I(kappa)B(alpha) is present in the nucleus in an apparently inactive form unable to disrupt NF-kappaB binding from DNA.

Expression

IkappaBalpha

detected

I(kappa)B(alpha)

resynthesis

I(kappa)B(alpha)

localization

nuclear

I(kappa)B(alpha)

presence

nuclear

I(kappa)B(alpha)

presence

IkappaBbeta

nuclear

present

I(kappa)B(alpha)

nucleus

inhibitory capacity

I(kappa)B(alpha)

mediated

inhibitory capacity

detected

detected

overexpressed

overexpressed

I(kappa)B(alpha)

detected

I(kappa)B(alpha)

super

induction

induction

I(kappa)B(alpha)

higher stability

I(kappa)B(alpha)

presence

presence

presence


Input:
PGG-glucan, a soluble beta-(1,3)-glucan, enhances the oxidative burst response, microbicidal activity, and activates an NF-kappa B-like factor in human PMN: evidence for a glycosphingolipid beta-(1,3)-glucan receptor.
PGG-Glucan, a soluble beta-(1,6)-branched beta-(1,3)-linked glucose homopolymer derived from the cell wall of the yeast Saccharomyces cerevisiae, is an immunomodulator which enhances leukocyte anti-infective activity and enhances myeloid and megakaryocyte progenitor proliferation. Incubation of human whole blood with PGG-Glucan significantly enhanced the oxidative burst response of subsequently isolated blood leukocytes to both soluble and particulate activators in a dose-dependent manner, and increased leukocyte microbicidal activity. No evidence for inflammatory cytokine production was obtained under these conditions. Electrophoretic mobility shift assays demonstrated that PGG-Glucan induced the activation of an NF-kappaB-like nuclear transcription factor in purified human neutrophils. The binding of 3H-PGG-Glucan to human leukocyte membranes was specific, concentration-dependent, saturable, and high affinity (Kd approximately 6 nM). A monoclonal antibody specific to the glycosphingolipid lactosylceramide was able to inhibit activation of the NF-kappaB-like factor by PGG-Glucan, and ligand binding data, including polysaccharide specificity, suggested that the PGG-Glucan binding moiety was lactosylceramide. These results indicate that PGG-Glucan enhances neutrophil anti-microbial functions and that interaction between this beta-glucan and human neutrophils is mediated by the glycosphingolipid lactosylceramide present at the cell surface.


Input:
[Corticoids and allergy]
Inflammation is constantly observed in allergic reactions. Corticosteroids are most effective in preventing the late phase of allergic reaction. The action of glucocorticosteroids is mediated through glucocorticoid receptors present in the cellular cytoplasm. When activated, glucocorticoid receptors form a dimer and bind to DNA after migration into the nucleus. Interaction to DNA induces changes in the transcription rate, leading to either gene induction or gene repression. Glucocorticoid receptors are also able to interact with transcriptional factors such as AP-1 (activator protein-1) of NF-kappa B (nuclear factor-kappa B). Through these actions glucocorticosteroids are susceptible to modify functions of cells involved in the allergic inflammatory response. They are in particular able to inhibit most of the pro-inflammatory functions of the eosinophils.


Input:
Activation of NF-kappaB in Mycobacterium tuberculosis- induced interleukin-2 receptor expression in mononuclear phagocytes.
Soluble interleukin-2 receptor-alpha (IL-2Ralpha) has been reported to be increased in the sera of patients with advanced tuberculosis, and levels decline after therapy in accordance with improvement of radiologic findings. We investigated expression of the IL-2Ralpha in bronchoalveolar lavage (BAL) cells in active pulmonary tuberculosis, and evaluated the mechanism Mycobacterium tuberculosis induces in the IL-2Ralpha using the THP-1 mononuclear phagocyte cell line. We found IL-2Ralpha expression to be increased in BAL cells from involved sites of active pulmonary tuberculosis. Expression of the alpha-chain of IL-2Ralpha on peripheral blood monocytes (PBM) was induced by M. tuberculosis by flow cytometry evaluation. Northern analysis demonstrated increased IL-2Ralpha gene expression after stimulation with M. tuberculosis which was further induced by interferon-gamma (IFN-gamma). The IL-2Ralpha promoter containing the nuclear factor kappa B (NF-kappaB) site was transcriptionally induced by M. tuberculosis and this NF-kappaB site could confer inducibility to a heterologous herpes thymidine kinase (TK) promoter by M. tuberculosis. Electrophoretic mobility shift assays (EMSAs) revealed specific binding of nuclear protein to the NF-kappaB site upon induction with M. tuberculosis. Using antibodies against the p50 and p65 subunits of NF-kappaB in EMSAs, the involvement of both p50 and p65 proteins was further demonstrated. Functional expression of the IL-2Ralpha on mononuclear phagocytes in M. tuberculosis infection may play an important immunomodulatory role in the host response.

expression

expression

Expression

expression

expression

decline

increased

increased

induces

increased

expression

induced

Expression

increased

expression

induced

expression

IFN-gamma

induced

promoter


Input:
Regulation of the megakaryocytic glycoprotein IX promoter by the oncogenic Ets transcription factor Fli-1.
Glycoprotein (GP) IX is a subunit of the von Willebrand receptor, GPIb-V-IX, which mediates adhesion of platelets to the subendothelium of damaged blood vessels. Previous characterization of the GPIX promoter identified a functional Ets site that, when disrupted, reduced promoter activity. However, the Ets protein(s) that regulated GPIX promoter expression was unknown. In this study, transient cotransfection of several GPIX promoter/reporter constructs into 293T kidney fibroblasts with a Fli-1 expression vector shows that the oncogenic protein Fli-1 can transactivate the GPIX promoter when an intact GPIX Ets site is present. In addition, Fli-1 binding of the GPIX Ets site was identified in antibody supershift experiments in nuclear extracts derived from hematopoietic human erythroleukemia cells. Comparative studies showed that Fli-1 was also able to transactivate the GPIbalpha and, to a lesser extent, the GPIIb promoter. Immunoblot analysis identified Fli-1 protein in lysates derived from platelets. In addition, expression of Fli-1 was identified immunohistochemically in megakaryocytes derived from CD34(+) cells treated with the megakaryocyte differentiation and proliferation factor, thrombopoietin. These results suggest that Fli-1 is likely to regulate lineage-specific genes during megakaryocytopoiesis.

binding

expression

GPIX

identified

expression

reduced

GPIX

promoter

transactivate

GPIX

promoter

transactivate

GPIIb

promoter

transactivate

GPIbalpha

promoter

Regulation

glycoprotein IX

regulated

expression


Input:
Studies into the effect of tyrosine phosphatase inhibitor phenylarsine oxide on NFkappaB activation in T lymphocytes during aging: evidence for altered IkappaB-alpha phosphorylation and degradation.
Nuclear Factor kappa B (NFkappaB) is a critical regulator of several genes involved in immune and inflammatory responses. Treatment of T cells with a variety of stimuli, including TNF-alpha, leads to the translocation of the active p65-50 heterodimer to the nucleus, albeit at a lower level in T cells from the elderly. We demonstrate here that pretreatment with PAO results in the inhibition of NFkappaB induction in TNF-alpha treated T cells, suggesting a role for PAO-sensitive phosphatase in the activation of the NFkappaB via this pathway in human T cells. Furthermore, it demonstrates that aging does not influence the sensitivity of this phosphatase. Treatment with DMP prior to treatment with PAO and TNF abolishes the inhibition induced by PAO, in T cells from both young and old donors, alike. Finally, we demonstrate that a failure to degrade IkappaB-alpha in cytosols of TNF-treated T cells pretreated with PAO is due to its interference with the phosphorylation of IkappaB-alpha and not due to its inhibitory effect on proteasomal degradation. These data collectively suggest that PAO interferes with the phosphorylation and the regulated degradation of IkappaB-alpha, induced by TNF, without affecting the chymotryptic activity of the proteasome, independent of age.

interference

phosphorylation

phosphorylation

IkappaB-alpha

phosphorylation

IkappaB-alpha

phosphorylation

IkappaB-alpha

due

degrade

interference

degradation

IkappaB-alpha

degrade

IkappaB-alpha

degradation

IkappaB-alpha

altered

phosphorylation

altered

degradation

due

degrade


Input:
A novel lipopolysaccharide-induced transcription factor regulating tumor necrosis factor alpha gene expression: molecular cloning, sequencing, characterization, and chromosomal assignment.
Lipopolysaccharide (LPS) is a potent stimulator of monocytes and macrophages, causing secretion of tumor necrosis factor alpha (TNF-alpha) and other inflammatory mediators. Given the deleterious effects to the host of TNF-alpha, it has been postulated that TNF-alpha gene expression must be tightly regulated. The nature of the nuclear factor(s) that control TNF-alpha gene transcription in humans remains obscure, although NF-kappaB has been suggested. Our previous studies pertaining to macrophage response to LPS identified a novel DNA-binding domain located from -550 to -487 in the human TNF-alpha promoter that contains transcriptional activity, but lacks any known NF-kappaB-binding sites. We have used this DNA fragment to isolate and purify a 60-kDa protein binding to this fragment and obtained its amino-terminal sequence, which was used to design degenerate probes to screen a cDNA library from THP-1 cells. A novel cDNA clone (1.8 kb) was isolated and fully sequenced. Characterization of this cDNA clone revealed that its induction was dependent on LPS activation of THP-1 cells; hence, the name LPS-induced TNF-alpha factor (LITAF). Inhibition of LITAF mRNA expression in THP-1 cells resulted in a reduction of TNF-alpha transcripts. In addition, high level of expression of LITAF mRNA was observed predominantly in the placenta, peripheral blood leukocytes, lymph nodes, and the spleen. Finally, chromosomal localization using fluorescence in situ hybridization revealed that LITAF mapped to chromosome 16p12-16p13.3. Together, these findings suggest that LITAF plays an important role in the activation of the human TNF-alpha gene and proposes a new mechanism to control TNF-alpha gene expression.

binding

DNA-binding domain

TNF-alpha

gene expression

tumor necrosis factor alpha

gene expression

TNF-alpha

gene expression

TNF-alpha

secretion

TNF-alpha

Inhibition

expression

resulted in a reduction

Inhibition

transcripts

causing

secretion

induction

LITAF

high level

expression

activation

TNF-alpha

regulating

gene expression

regulated

gene expression

control

gene transcription

dependent

induction

role

LITAF

activation

control

LITAF

gene expression

gene transcription

TNF-alpha

expression

LITAF

transcripts

TNF-alpha

expression

LITAF


Input:
Engagement of natural cytotoxicity programs regulates AP-1 expression in the NKL human NK cell line.
NK cell cytotoxicity is a fast and efficient mechanism of target cell lysis. Using transcription analysis, such as multiplex messenger assays, we show here that natural cytotoxicity exerted by the human NKL cell line correlates with mRNA accumulation of very early activator protein (AP)-1 transcription factor genes such as JunB, FosB and c-Fos. In addition, DNA-binding activities of Jun-Fos heterodimers were observed by electrophoretic mobility shift assays during the course of natural cytotoxicity. Interaction between immunoglobulin-like transcript-2/leukocyte Ig-like receptor 1 on NKL cells and HLA-B27 on target cells leads to an impairment of NKL natural cytotoxicity, which correlates with an absence of JunB, FosB, and c-Fos transcription, as well as an absence of their DNA-binding activity. Our studies thus indicate that, despite the rapidity of NK cell-mediated lysis, AP-1 transcription factor is activated during the early stage of NK cell cytolytic programs and that engagement of NK cell inhibitory receptors for MHC class I molecules impairs the very early activation of AP-1.

Interaction

immunoglobulin-like transcript-2

binding activity

JunB

binding activity

FosB

binding activity

c-Fos

absence

transcription

absence

binding activity

mRNA accumulation

JunB

mRNA accumulation

FosB

mRNA accumulation

c-Fos

transcription

JunB

transcription

FosB

transcription

c-Fos


Input:
Human cytomegalovirus binding to human monocytes induces immunoregulatory gene expression.
To continue our investigation of the cellular events that occur following human CMV (HCMV) infection, we focused on the regulation of cellular activation following viral binding to human monocytes. First, we showed that viral binding induced a number of immunoregulatory genes (IL-1beta, A20, NF-kappaB-p105/p50, and IkappaBalpha) in unactivated monocytes and that neutralizing Abs to the major HCMV glycoproteins, gB (UL55) and gH (UL75), inhibited the induction of these genes. Next, we demonstrated that these viral ligands directly up-regulated monocyte gene expression upon their binding to their appropriate cellular receptors. We then investigated if HCMV binding also resulted in the translation and secretion of cytokines. Our results showed that HCMV binding to monocytes resulted in the production and release of IL-1beta protein. Because these induced gene products have NF-kappaB sites in their promoter regions, we next examined whether there was an up-regulation of nuclear NF-kappaB levels. These experiments showed that, in fact, NF-kappaB was translocated to the nucleus following viral binding or purified viral ligand binding. Changes in IkappaBalpha levels correlated with the changes in NF-kappaB translocation. Lastly, we demonstrated that p38 kinase activity played a central role in IL-1beta production and that it was rapidly up-regulated following infection. These results support our hypothesis that HCMV initiates a signal transduction pathway that leads to monocyte activation and pinpoints a potential mechanism whereby HCMV infection of monocytes can result in profound pathogenesis, especially in chronic inflammatory-type conditions.

production

production

release

inhibited

induction

induced

induced

induced

induced

induced

IkappaBalpha

induction

induction

induction

induction

induction

IkappaBalpha

resulted

production

resulted

release

role

production

Changes

IkappaBalpha


Input:
Signal transduction pathways activated in endothelial cells following infection with Chlamydia pneumoniae.
Chlamydia pneumoniae is an important respiratory pathogen. Recently, its presence has been demonstrated in atherosclerotic lesions. In this study, we characterized C. pneumoniae-mediated activation of endothelial cells and demonstrated an enhanced expression of endothelial adhesion molecules followed by subsequent rolling, adhesion, and transmigration of leukocytes (monocytes, granulocytes). These effects were blocked by mAbs against endothelial and/or leukocyte adhesion molecules (beta1 and beta2 integrins). Additionally, activation of different signal transduction pathways in C. pneumoniae-infected endothelial cells was shown: protein tyrosine phosphorylation, up-regulation of phosphorylated p42/p44 mitogen-activated protein kinase, and NF-kappaB activation/translocation occurred within 10-15 min. Increased mRNA and surface expression of E-selectin, ICAM-1, and VCAM-1 were noted within hours. Thus, C. pneumoniae triggers a cascade of events that could lead to endothelial activation, inflammation, and thrombosis, which in turn may result in or may promote atherosclerosis.

expression

E-selectin

expression

expression

up-regulation

up-regulation

p44 mitogen-activated protein kinase

occurred

up-regulation

Increased

expression

expression

E-selectin

expression

expression


Input:
Extracellular-regulated kinase 1/2, Jun N-terminal kinase, and c-Jun are involved in NF-kappa B-dependent IL-6 expression in human monocytes.
In the present study we investigated the possible involvement of the mitogen-activated protein kinase family members extracellular-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) in mediating IL-6 gene expression in human monocytes, in particular their role in enhancing NF-kappa B activity. Freshly isolated monocytes treated with the protein phosphatase inhibitor okadaic acid secreted high levels of IL-6 protein, which coincided with enhanced binding activity of NF-kappa B as well as with phosphorylation and activation of the ERK1/2 and JNK proteins. The ERK pathway-specific inhibitor PD98059 inhibited IL-6 secretion from monocytes. Transient overexpression of inactive mutants of either Raf-1 or JNK1 showed that both pathways were involved in kappa B-dependent IL-6 promoter activity. By using PD98059, we demonstrated that the Raf1/MEK1/ERK1/2 pathway did not affect the DNA binding of NF-kappa B but, rather, acted at the level of transcriptional activity of NF-kappa B. Interestingly, it was shown that NF-kappa B-mediated gene transcription, both in the context of the IL-6 promoter as well as on its own, was dependent on both serine kinase activity and interaction with c-Jun protein. We conclude that okadaic acid-induced IL-6 gene expression is at least partly mediated through the ERK1/2 and JNK pathway-dependent activation of NF-kappa B transcriptional capacity. Our results suggest that the JNK pathway may regulate NF-kappa B-mediated gene transcription through its phosphorylation and activation of c-Jun.

interaction

c-Jun

expression

expression

secreted

expression

secretion

inhibited

secretion

phosphorylation

JNK

phosphorylation

c-Jun

dependent

expression

mediating

expression

high levels

secreted

activation

JNK

dependent

transcription

interaction

dependent

transcription

induced

expression

mediated

induced

phosphorylation

JNK

phosphorylation

activation

JNK

c-Jun

dependent

promoter

transcription


Input:
Differential induction of interferon (IFN)-inducible protein 10 following differentiation of a monocyte, macrophage cell lineage is related to the changes of nuclear proteins bound to IFN stimulus response element and kappaB sites.
We examined chemokine gene expression following the differentiation of a monocyte, macrophage cell lineage. The human monoblastic cell line, U937 was differentiated to macrophages by the treatment with either phorbol 12-myristate 13-acetate (PMA), retinoic acid (RA), or vitamin D3 (VitD3). The gene expression of interferon (IFN)-inducible protein 10 (IP-10) (a CXC chemokine) was markedly augmented by the IFNgamma treatment in PMA- or RA-differentiated U937 cells, but only marginally in undifferentiated or VitD3-treated cells. In contrast, another inducible gene expression of monocyte chemotactic protein-1 (a CC chemokine) and the activation of the transcriptional factor (FcRFgamma) bound to the gamma response region were similarly or less abundantly induced by IFNgamma treatment in PMA- or RA-differentiated U937 cells, indicating that increased IP-10 mRNA induction was not due to the augmented ability of the cells to respond to the presence of IFNgamma. Increased expression of IFNgamma-induced IP-10 mRNA following the differentiation of U937 cells was mediated largely by augmented transcriptional activity of the gene and was related to differentiation-dependent changes of the proteins bound to IFN stimulus response element (ISRE) and kB sites, suggesting that these nuclear proteins may determine the IP-10 mRNA inducibility by IFNgamma.

gene expression

expression

monocyte chemotactic protein-1

induction

interferon (IFN)-inducible protein 10

following

induction

augmented

gene expression

IFNgamma

induced

expression

IFNgamma

increased

induction

induction

due

increased

Increased

induced

induced

expression

IFNgamma

mediated

Increased

augmented

augmented

transcriptional activity

transcriptional activity

inducibility

IFNgamma

determine

inducibility

expression