IL-8 Promoter Activity Research Articles

Human T cell leukemia virus type I Tax enhances IL-4 gene expression in T cells.

The human T cell leukemia virus type-1 (HTLV-1) is the etiologic agent of adult T cell leukemia (ATL). Since the HTLV-I-encoded transactivator Tax has been shown to activate many cellular genes including cytokine genes interleukin (IL-)1alpha, 2, 5, 6, 8, 10 and 15, we ask whether Tax also affects IL-4 expression. In this study, we show that addition of recombinant Tax proteins greatly enhances IL-4 secretion in human peripheral primary T cells. Transient transfection studies showed that ectopic expression of Tax significantly enhanced IL-4 promoter activity. The IL-4 promoter contains a strong NF-IL6 (PRE-I element) and a NF-AT/NF-kappaB overlapping site (P1 element). We show that expression of Tax stimulates NF-IL6 binding to the PRE-I element and, consequently, enhances PRE-I-mediated transcriptional activity. Using Jurkat T cell lines which stably express Tax fused to the hormone binding domain of the human estrogen receptor (ER), we show that Tax enhances endogenous IL-4 mRNA expression and increases IL-4 promoter activity in a hormone-dependent manner. Mutation analysis revealed that the IL-4 PRE-I (NF-IL6 site) and the P1 (NF-AT/NF-kappaB site) are involved in Tax-mediated transactivation. Our studies provide the first evidence of the functional involvement of Tax in IL-4 gene regulation.

Activation of nuclear factor-kappa b transcriptional activity in airway epithelial cells by thioredoxin but not by N-acetyl-cysteine and glutathione.

Increasing evidence indicates that intracellular redox status modulates the activity of various transcriptional factors, including nuclear factor (NF)-kappa B and activator protein-1. Our laboratory has been interested in characterizing the role thioredoxin (TRX) plays in regulating cellular redox status in airway epithelium. TRX is a small, ubiquitous protein with two redox-active half-cysteine residues, -Cys-Gly-Pro-Cys, in its active center. Using primary passage-1 human tracheobronchial epithelial cell cultures and an immortalized human bronchial epithelial cell line, HBE1, we observed that tumor necrosis factor (TNF)-alpha enhanced NF-kappa B transcriptional activity. This observation was based on gel mobility shift assays and interleukin (IL)-8 promoter-reporter gene transfection studies. TNF-alpha activation coincided with translocation of NF-kappa B p65 from the cytoplasm to the nucleus. Pretreatment with N-acetyl-cysteine (NAC) (1 to 10 mM) or glutathione (1 to 10 mM) inhibited TNF-alpha-induced activation of NF-kappa B transcriptional activity and IL-8 promoter-mediated reporter gene expression. In contrast, elevated TRX protein levels in cells enhanced TNF-alpha-dependent NF-kappa B transcriptional activity and IL-8 promoter activity. This observation was independent of the manner in which TRX was elevated in cells (e.g., by cotransfection with a FLAG-TRX expression clone, or by direct exposure to commercially available human TRX protein). Localization of TRX protein by anti-TRX antibody indicated an accumulation of TRX protein in the nucleus after TNF-alpha treatment. The nuclear localization phenomenon was different from the major cytosolic accumulation of glutathione and NAC. This is the first known report demonstrating movement of TRX into the nucleus of airway epithelial cells after an inflammatory stress. These results suggest a compartment effect of thiol chemicals in the regulation of redox-dependent transcriptional activity.

Morphine directs T cells toward Th2 differentiation

Background. Failure of cell-mediated immunity is thought to increase the morbidity and mortality rates after trauma and major surgical procedures and to be the result, in part, of a redirection of CD4+ T cells toward Th2 differentiation. We tested the hypothesis that morphine treatment after injury promotes Th2 differentiation of precursor T cells through the μ-opioid receptor. Methods. Human peripheral blood mononuclear cells (PBMCs) or splenocytes from either wild type or μ-opioid receptor knock-out mice were treated in vitro with either vehicle or morphine and then stimulated with anti-CD3/anti-CD28. The supernatant was assayed for Th1 (interleukin-2 [IL-2], interferon γ [IFNγ]) and Th2 (IL-4, IL-5) cytokines (enzyme-linked immunosorbent assay). Morphine regulation of IL-4 transcription was investigated in PBMCs (IL-4 messenger RNA, nuclear factor of activated T-cells) and Jurkat T cells transfected with a murine IL-4 promoter-luciferase construct. Morphine-induced nuclear factor of activated T-cell (NFAT) binding was assayed with the electromobility shift assay in Jurkat T cells. Results. Morphine treatment of PBMCs decreases IL-2 and IFNγ and increases IL-4 and IL-5 as a function of morphine concentration. Morphine treatment in wild type splenocytes inhibited IFNγ and stimulated IL-4 protein synthesis. Changes in cytokine synthesis were abolished in μ-opioid receptor knockout mice. Morphine treatment increases IL-4 messenger RNA accumulation in PBMCs and increases IL-4 promoter activity in Jurkat T cells. Morphine increases NFAT nuclear protein binding to an NFAT DNA response element. Conclusions. We conclude that morphine treatment promotes Th2 differentiation through a μ-opioid receptor mechanism and that morphine treatment increases IL-4 transcription, in part, through an NFAT mechanism. (Surgery 2001;130:304-9.)

The effect of HIV-1 regulatory proteins on cellular genes: derepression of the IL-2 promoter by Tat.

In HIV-infected individuals dysregulation of the immune system is characterized by severe disorders of the cytokine network. Increase secretion of IL-2, the major T cell growth and differentiation cytokine, may play a decisive role in sensitization of T cells for activation induced apoptosis and indirect death of activated T cells through augmented virus replication. We investigated the cause of enhanced IL-2 secretion and found that the HIV Tat induces this effect. We demonstrate that increased IL-2 secretion is due to Tat-enhanced IL-2 promoter activation. Tat derepresses and activates the distal AP-1 site (position -185 to -177) in the IL-2 promoter. In nonstimulated T cells a repressor complex containing NF-IL6, JunB, c-Fos and Fra-1 is formed on the AP-1(IL-2/d) site and represses IL-2 promoter activity. After T cell activation, a heterodimeric activator containing p65 and c-Jun binds to the AP-1(IL-2/d) site. HIV Tat enhances activation of NF-kappaB and consequently, activates the AP-1(IL-2/d) site. Our data provide evidence for a novel mechanism by which HIV Tat dysregulates IL-2 production and therefore may contribute to the HIV-1 infection in a way yet to be clarified.

Regulation of IL-4 Gene Expression by Distal Regulatory Elements and GATA-3 at the Chromatin Level

Using a transgenic approach, we examined distal regulatory elements located in the IL-4 locus and the role of GATA-3 at these elements. The intergenic DNase I hypersensitive sites (HSS) showed strong enhancement, and the intronic enhancer (IE) and HS5/HS5a sites showed weaker enhancement of the IL-4 promoter. Elements in the 3′ region of the IL-4 gene contributed to Th2 specificity. All individual enhancers were T cell activation dependent but not Th2 specific, with the exception of IE. However, when these distal elements were combined into a “minilocus,” expression was strongly enhanced and Th2 specific. GATA-3 mediated strong enhancement of IL-4 promoter activity in Th1 cells when the promoter was embedded in the minilocus or linked to HSS and IE, demonstrating that GATA-3 acts through these elements to regulate IL-4 gene expression.

Selective inhibition of interleukin-4 gene expression in human T cells by aspirin

Previous studies indicated that aspirin (acetylsalicylic acid [ASA]) can have profound immunomodulatory effects by regulating cytokine gene expression in several types of cells. This study is the first in which concentrations of ASA in the therapeutic range were found to significantly reduce interleukin (IL)-4 secretion and RNA expression in freshly isolated and mitogen-primed human CD4+ T cells. In contrast, ASA did not affect IL-13, interferon-gamma, and IL-2 expression. ASA inhibited IL-4, but not IL-2, promoter-driven chloramphenicol acetyltransferase expression in transiently transfected Jurkat T cells. The structurally unrelated nonsteroidal anti-inflammatory drugs indomethacin and flurbiprofen did not affect cytokine gene expression in T cells, whereas the weak cyclo-oxygenase inhibitor salicylic acid was at least as effective as ASA in inhibiting IL-4 expression and promoter activity. The inhibitory effect of ASA on IL-4 transcription was not mediated by decreased nuclear expression of the known salicylate target nuclear factor (NF)-kappaB and was accompanied by reduced binding of an inducible factor to an IL-4 promoter region upstream of, but not overlapping, the NF of activated T cells- and NF-kappaB-binding P1 element. It is concluded that anti-inflammatory salicylates, by means of a previously unrecognized mechanism of action, can influence the nature of adaptive immune responses by selectively inhibiting the expression of IL-4, a critical effector of these responses, in CD4+ T cells.

IL-4 gene expression up-regulated by mercury in rat mast cells: a role of oxidant stress in IL-4 transcription.

In the Brown Norway (BN) rat, chemical compounds [mercuric chloride (HgCl2), D-penicillamine or gold salts] induce a T(h)2-dominated autoimmune syndrome with tissue injury in the form of a vasculitis and arthritis. An early phase of vasculitis in the model occurs within 24 h of an injection of HgCl2, is alphabeta T cell independent and involves the mast cell. In addition, HgCl2 induces IL-4 mRNA in mast cells from BN rats. Our recent work has demonstrated that the balance of oxidative/antioxidative influences plays an important role in the modulation of mast cell function (degranulation) in chemically induced autoimmunity. The aim of this study was to determine, in mast cells, whether oxidative status influences IL-4 transcription and translation, which is required for the development of a T(h)2 response. Exposure of the mast cell line RBL-2H3 to HgCl2 enhanced both IL-4 mRNA and its promoter activity. Oxidative stress by hydrogen peroxide mimicked the effects of HgCl2 in enhancing IL-4 promoter activity. The enhancement of IL-4 gene expression by HgCl2 was significantly reduced by antioxidants (both sulphydryl and non-sulphydryl containing). The same pattern of regulation was also observed on IL-4 protein expression in the mast cells. These data suggest a novel mechanism of IL-4 transcriptional up-regulation by oxidative stress. Our results provide evidence to support our hypothesis that alterations in intracellular reactive oxygen species production modulate both IL-4 gene expression and mast cell function.

Identification and Characterization of a Critical CP2-binding Element in the Human Interleukin-4 Promoter

Expression of cytokine genes in T cells is thought to result from a complex network of antigen- and mitogen-activated transcriptional regulators. CP2, a factor homologous to Drosophila Elf-1 and previously found to be a critical regulator of several viral and cellular genes in response to developmental signals, is rapidly activated in T helper (Th) cells in response to mitogenic stimulation. Here we show that overexpression of CP2 enhances interleukin (IL)-4 promoter-driven chloramphenicol acetyltransferase expression, while repressing IL-2 promoter activity, in transiently transfected Jurkat cells. A CP2-protected element, partially overlapping the nuclear factor of activated T cell-binding P2 sequence, was required for IL-4 promoter activation in CP2-overexpressing Jurkat cells. This CP2-response element is the site of a cooperative interaction between CP2 and an inducible heteromeric co-factor(s). Mutation of conserved nucleotide contacts within the CP2-response element prevented CP2 binding and significantly reduced constitutive and induced IL-4 promoter activity. Expression of a CP2 mutant lacking the Elf-1-homology region of the DNA-binding domain inhibited IL-4 promoter activity in a dominant negative fashion in transiently transfected Jurkat cells. Moreover, overexpressed CP2 markedly enhanced, while its dominant negative mutant consistently suppressed, expression of the endogenous IL-4 gene in the murine Th2 cell line D10. Taken together, these findings point to CP2 as a critical IL-4 transactivator in Th cells.

T cell-specific adapter protein inhibits T cell activation by modulating Lck activity.

We previously reported the isolation of a cDNA encoding a T cell-specific adapter protein (TSAd). Its amino acid sequence contains an SH2 domain, tyrosines in protein binding motifs, and proline-rich regions. In this report we show that expression of TSAd is induced in normal peripheral blood T cells stimulated with anti-CD3 mAbs or anti-CD3 plus anti-CD28 mAbs. Overexpression of TSAd in Jurkat T cells interfered with TCR-mediated signaling by down-modulating anti-CD3/PMA-induced IL-2 promoter activity and anti-CD3 induced Ca2+ mobilization. The TCR-induced tyrosine phosphorylation of phospholipase C-gamma1, SH2-domain-containing leukocyte-specific phosphoprotein of 76kDa, and linker for activation of T cells was also reduced. Furthermore, TSAd inhibited Zap-70 recruitment to the CD3zeta-chains in a dose-dependent manner. Consistent with this, Lck kinase activity was reduced 3- to 4-fold in COS-7 cells transfected with both TSAd and Lck, indicating a regulatory effect of TSAd on Lck. In conclusion, our data strongly suggest an inhibitory role for TSAd in proximal T cell activation.

MHC Class II Transactivator Inhibits IL-4 Gene Transcription by Competing with NF-AT to Bind the Coactivator CREB Binding Protein (CBP)/p300

The MHC class II transactivator (CIITA) activates the expression of multiple genes involved in Ag presentation, but inhibits Th2-type cytokine production, including IL-4, during Th1 cell differentiation. Th1 cells derived from CIITA-deficient mice produce both Th1- and Th2-type cytokines, and the introduction of CIITA to Th2 cells down-regulates Th2-type cytokine gene transcription. Here we show that the IL-4 promoter is regulated by multiple protein-protein interactions among CIITA, NF-AT, and coactivator CBP/p300. The introduction of CBP/p300 and NF-AT enhances the IL-4 promoter activity, and this activation was repressed by CIITA. Furthermore, our data show that CIITA competes with NF-AT to bind CBP/p300 and that this competition dramatically influences transcriptional activation of the IL-4 promoter. We identified two domains of CIITA that interact with two distinct domains of CBP/p300 that are also recognized by NF-AT. CIITA mutants that retain the ability to interact with CBP/p300 are sufficient to inhibit NF-AT-mediated IL-4 gene expression.

C/EBPbeta enhances IL-4 but impairs IL-2 and IFN-gamma induction in T cells.

C/EBP transcription factors have been described to control the activity of the human IL-4 promoter. The C/EBP binding sites within the IL-4 promoter overlap with composite NF-AT and AP-1 binding motifs. We show here that similar binding sites are part of the murine IL-4 promoter. Retroviral overexpression of C/EBPbeta in murine EL-4 thymoma cells led to a strong induction of endogenous IL-4 and a reduction in IL-2 and IFN-gamma expression. Similarily, in primary murine T cells C/EBPbeta induction resulted in an increase in IL-4 levels, whereas in human Jurkat T cells a decrease in IL-2 RNA was detected. Like AP-1, C/EBP factors belong to the large class of basic leucine zipper proteins. However, unlike AP-1, C/EBPbeta does not act in synergy with NF-AT in the induction of the murine IL-4 promoter. Instead, both factors compete in their binding to the P4/Pu-bD site, one of the most important sequence elements of the IL-4 promoter. Whereas NF-AT factors require high levels of free Ca2+ and calcineurin activity for induction, C/EBP induction in T cells is Ca2+/calcineurin independent. These observations suggest that various induction conditions lead to the activation of transcription factors, inducing IL-4 promoter activity at specific developmental stages of T cells.

Yersinia enterocolitica invasin protein triggers IL-8 production in epithelial cells via activation of Rel p65-p65 homodimers.

Enteropathogenic Yersinia bacteria trigger the production of the proinflammatory chemokine IL-8, an important chemokine for the recruitment of polymorphonuclear leukocytes (PMN). Yersinia is resistant to phagocytosis by PMN, and the recruitment of these cells is thought to be part of a pathogenic strategy of Yersinia to establish infection by allowing the pathogen to gain access to, and disseminate within, host tissue. We report here that Yersinia expressing the outer membrane protein invasin triggers IL-8 production in epithelial cells. The 195 carboxyl-terminal amino acids of invasin when linked to latex beads are sufficient to trigger IL-8 production. By means of IL-8 promoter reporter gene assays and electrophoretic mobility shift assay experiments, the minimal optimal region of the IL-8 promoter responsive to invasin was identified and invasin-responsive control elements were characterized. Invasin-induced activation of the IL-8 promoter was found to be mediated through a previously identified NF-kappaB element. This NF-kappaB binding site preferentially binds Rel p65-p65 homodimers as well as some p50-p65 heterodimers in response to stimulation by invasin. Invasin-induced NF-kappaB activation correlated with degradation of IkappaBalpha and the inhibition of NF-kappaB by specific inhibitors of IkappaB activation blocked invasin-induced IL-8 secretion. Invasin-triggered IL-8 production does not depend on invasin-triggered uptake of bacteria, and is independent of a functional PI3-kinase. This report is the first to demonstrate the molecular basis of IL-8 production triggered by enteropathogenic bacteria. Together, these data elucidate the possible early pathomechanisms operating in Yersinia infection and may have implications for the design of novel therapeutics directed against this enteropathogen.

Repression of IL-2 promoter activity by the novel basic leucine zipper p21SNFT protein.

IL-2 is the major autocrine and paracrine growth factor produced by T cells upon T cell stimulation. The inducible expression of IL-2 is highly regulated by multiple transcription factors, particularly AP-1, which coordinately activate the promoter. Described here is the ability of the novel basic leucine zipper protein p21SNFT to repress AP-1 activity and IL-2 transcription. A detailed analysis of the repression by p21SNFT repression on the IL-2 promoter distal NF-AT/AP-1 site demonstrates that it can bind DNA with NF-AT and Jun, strongly suggesting that it represses NF-AT/AP-1 activity by competing with Fos proteins for Jun dimerization. The importance of this repression is that p21SNFT inhibits the trans-activation potential of protein complexes that contain Jun, thereby demonstrating an additional level of control for the highly regulated, ubiquitous AP-1 transcription factor and the IL-2 gene.

A novel mechanism of retinoic acid-enhanced interleukin-8 gene expression in airway epithelium.

A 3- to 8-fold stimulation of interleukin (IL)-8 gene expression by all-trans-retinoic acid (ATRA) was demonstrated in primary cultures of human and monkey tracheobronchial epithelial cells and BEAS-2B serum-sensitive cell line. The effect of ATRA on IL-8 gene expression is dose- and time-dependent. Using cycloheximide, it was observed that new protein synthesis was required for the stimulation. ATRA had no effect on IL-8 messenger RNA stability. A difference in nuclear run-on activity suggests that a transcriptional mechanism is involved in ATRA-enhanced IL-8 gene expression. Promoter-reporter gene transfection studies demonstrated ATRA enhanced IL-8 promoter activity, especially when cells were cotransfected with retinoic acid nuclear receptor-alpha expression vector. Deletion and site-directed mutagenesis analysis revealed the involvement of nuclear factor (NF)-kappaB binding site of the IL-8 gene in ATRA-enhanced promoter activity. Electrophoretic mobility shift assay (EMSA) demonstrated that ATRA enhanced DNA-NF-kappaB complex formation, especially with the p65 subunit. Western blot analysis demonstrated that ATRA did not enhance the protein amount of both the p50 and the p65 subunits in the nuclei. Because ATRA also enhances thioredoxin (TRX) gene expression, the effect of TRX on IL-8 gene expression was examined. IL-8 promoter activity was enhanced in transfected cells by the addition of TRX protein. Treatment of nuclear extracts with TRX also enhanced DNA- NF-kappaB complex formation as observed by EMSA, particularly the p65 subunit. Taking these data together, a novel mechanism is proposed in which ATRA activates promoter activity of IL-8 gene through TRX-dependent NF-kappaB activation.

The role of high-mobility group I(Y) proteins in expression of IL-2 and T cell proliferation.

The high-mobility group I(Y) (HMGI(Y)) family of proteins plays an important architectural role in chromatin and have been implicated in the control of inducible gene expression. We have previously shown that expression of HMGI antisense RNA in Jurkat T cells inhibits the activity of the IL-2 promoter. Here we have investigated the role of HMGI(Y) in controlling IL-2 promoter-reporter constructs as well as the endogenous IL-2 gene in both Jurkat T cells and human PBL. We found that the IL-2 promoter has numerous binding sites for HMGI(Y), which overlap or are adjacent to the known transcription factor binding sites. HMGI(Y) modulates binding to the IL-2 promoter of at least three transcription factor families, AP-1, NF-AT and NF-kappaB. By using a mutant HMGI that cannot bind to DNA but can still interact with the transcription factors, we found that DNA binding by HMGI was not essential for the promotion of transcription factor binding. However, the non-DNA binding mutant acts as a dominant negative protein in transfection assays, suggesting that the formation of functional HMGI(Y)-containing complexes requires DNA binding as well as protein:protein interactions. The alteration of HMGI(Y) levels affects IL-2 promoter activity not only in Jurkat T cells but also in PBL. Importantly, we also show here that expression of the endogenous IL-2 gene as well as proliferation of PBL are affected by changes in HMGI(Y) levels. These results demonstrate a major role for HMGI(Y) in IL-2 expression and hence T cell proliferation.

Identification of Two Major Sites in the Type I Interleukin-1 Receptor Cytoplasmic Region Responsible for Coupling to Pro-inflammatory Signaling Pathways

Type I interleukin-1 receptor is the prototype for a family of proteins, which play a central role in early responses to injury and infection. The similarity of function across the family is reflected in similarity in signaling: all members tested couple to activation of NFkappaB and stress kinases. The coupling to these pathways is mediated by a 200-residue intracellular domain (the Toll/interleukin-1 receptor domain), in which sequence conservation is primarily confined to three short motifs (boxes 1, 2, and 3) located at amino acid residue positions 10 (box 1), 60 (box 2), and 170 (box 3). We have analyzed the contribution of these motifs to function by alanine scanning mutagenesis of the human interleukin-1 receptor type I. Mutant receptors were tested for expression, ligand binding, activation of receptor-associated kinase(s), NFkappaB, stress kinases, and transcription. Mutations in all three motifs led to low cell surface expression. Mutants in box 3 were, however, wild type for signaling, whereas mutants in boxes 1 and 2 were defective. We conclude that the conserved motifs box 1 and box 2 mediate the coupling of molecules in the family to inflammation signaling pathways.

Glucocorticoids Inhibit Calcium- and Calcineurin-Dependent Activation of the Human IL-4 Promoter

The mechanism by which glucocorticoids (GC) inhibit IL-4 gene expression is currently unknown. In T lymphocytes, IL-4 gene expression is regulated at the level of transcription by increases in intracellular calcium concentration and by the calcium-activated phosphatase calcineurin. In this paper we report that dexamethasone (Dex) inhibits calcium ionophore-induced activation of the human IL-4 promoter in transiently transfected Jurkat T cells. Inhibition of the promoter by Dex is dependent on expression of the GC receptor (GR), because it does not occur in GR-deficient cells. Dex also represses activation of the promoter induced by cotransfecting cells with a constitutively active mutant of calcineurin. Using a series of deletion constructs, we show that the proximal 95 bp of the IL-4 promoter contain a Dex-sensitive regulatory element. This region contains the P1 sequence, a proximal binding site for NF-AT. A calcium-induced but Dex-inhibited nuclear complex containing NF-AT binds to the P1 element in EMSA. Using immunoprecipitation under nondenaturing conditions, we found that the GRalpha isoform coprecipitates with NF-ATc in nuclear extracts of calcium ionophore- and Dex-treated cells. Taken together, our results show that GC inhibit IL-4 gene expression by interfering with NF-AT-dependent transactivation of the proximal human IL-4 promoter.

NF-κB and AP-1 are responsible for inducibility of the IL-6 promoter by ionizing radiation in HeLa cells

Purpose : To investigate the mechanisms leading to initiation by ionizing radiation of IL-6 transcription in HeLa cells. Materials and methods : HeLa cells were irradiated with X-rays at a dose rate of approximately 1 Gy/min or treated with TPA (100 ng/ml). Transient transfection analysis with truncated IL-6 promoter CAT constructs was used to identify the radiation-sensitive region within the IL-6 promoter/enhancer. Results : For basal expression of the IL-6 gene in unirradiated control cells the presence of the binding site for the nuclear factor kappa B (NF- κB) and the multiple response elements (MRE) were necessary. After deletion of either the activator protein (AP)-1 or the MRE site, radiation-induced IL-6 promoter CAT activity was significantly reduced, whereas after deletion of the NF-kappaB site it was completely abolished. Maximal radiation-induced IL-6 promoter CAT activity was observed when the AP1, NF κB and MRE motifs were present. In electrophoretic mobility shift analyses (EMSA), X-ray-inducible activity was found for NF-kappaB and AP-1 at the MRE constitutive, but no inducible activities were detectable. The nuclear factor IL-6 (NFIL6) element showed no specific radiation-responsive activity. Conclusions : These results demonstrate that NF- κB plays a major role in X-ray-inducible IL-6 expression in HeLa cells. The fact that IL-6 promoter activity was dramatically enhanced in the presence of the MRE and distal AP-1 binding motif is indicative of a cooperative mode of transcriptional activation involving all three transcription factor systems. These data provide new insights into the prodromal events of radiation-induced inflammation of epithelial cells and putatively the cutaneous radiation syndrome.

Catecholamines induce IL-10 release in patients suffering from acute myocardial infarction by transactivating its promoter in monocytic but not in T-cells.

The anti-inflammatory cytokine IL-10 is up-regulated in response to TNF-alpha suggesting a control mechanism of inflammation. In addition, we recently found systemic IL-10 release in response to acute stress reactions in the absence of any systemic inflammation. In vitro and in vivo studies in experimental models suggest that catecholamines induce IL-10 release via a cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) dependent pathway. Here we studied patients for plasma IL-10 after acute myocardial infarction, a very stressful event without significant signs of systemic inflammation. In fact, the activation of the sympathetic system initiated by cardiac infarction was accompanied by a temporary systemic release of IL-10. Catecholamine induced IL-10 may be released by different cells. Recently, we demonstrated that catecholamines directly stimulate the IL-10 promoter/enhancer via a cAMP/PKA pathway in monocytic cells. A cAMP responsive element (CRE) was identified as major target. Here we show that there is no influence of catecholamines on the IL-10 promoter activity in T-cells. In contrast to monocytic cells, in T-cells cAMP-induced PKA-dependent phosphorylation of the CRE-binding protein 1 (CREB-1) seems to play a marginal role in IL-10 induction, which was reflected by a low cAMP-dependent IL-10-promoter/enhancer stimulation in reporter gene assays. Thus, catecholamines are directly involved in the regulation of IL-10 expression in monocytic but not in T-cells after acute stressful conditions.

Cutting Edge: SLP-76 Cooperativity with FYB/FYN-T in the Up-Regulation of TCR-Driven IL-2 Transcription Requires SLP-76 Binding to FYB at Tyr595 and Tyr651

SLP-76 (Src homology (SH) 2-domain-containing leukocyte protein of 76 kDa) and FYB/SLAP (FYN-T-binding protein/SLP-76-associated protein) are two hemopoietic cell-specific adaptor proteins downstream of TCR-activated protein tyrosine kinases. SLP-76 has been implicated as an essential component in T cell signaling. FYB is selectively phosphorylated by FYN-T, providing a template for the recruitment of FYN-T and SLP-76 SH2 domains. Coexpression of FYN-T, FYB, and SLP-76 can synergistically up-regulate IL-2 production in T cells upon TCR ligation. In this report, we show that two tyrosines, Tyr595 and Tyr651, of FYB are major sites of phosphorylation by FYN-T and mediate binding to SLP-76 in Jurkat T cells. Furthermore, the synergistic up-regulation of IL-2 promoter activity in the FYN-T-FYB-SLP-76 pathway is contingent upon the interaction between FYB and SLP-76, but not the interaction between FYB and FYN-T. These observations define a pathway by which SLP-76 interacts with downstream components in the up-regulation of T cell cytokine production.