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Increased activity of various proteases is observed in both human and experimental pancreatitis; however, the information on the effects of specific protease inhibitors on the disease is limited. In this study we show that a novel elastase inhibitor, guamerin-derived synthetic peptide (GDSP), improves the parameters of cerulein-induced acute pancreatitis in the rat. The effects of GDSP on pancreatic weight, serum amylase and lipase, morphologic changes in the pancreas, neutrophil infiltration, and nuclear factor KB (NF-KB) activation were measured in rats infused with supramaximal dose of cerulein (5 (g/kg/h) for 6 h. The effects of GDSP were also measured on superoxide formation by activated human neutrophils. The effects of GDSP were compared with those of another elastase inhibitor, elastatinal. GDSP significantly inhibited edema formation, neutrophil infiltration, acinar cell damage, and plasma lipase and amylase increases caused by cerulein. GDSP also completely inhibited superoxide formation in the human neutrophils stimulated by N-formyl-methionine-leucine-phenyl-alanine (fMLP) or 12-O-tetradecanoylphorbol-13-acetate (TPA). Elastatinal had some of the same effects as GDSP but was less potent and effective. These results demonstrate a beneficial effect of GDSP, a novel specific elastase inhibitor, on the development of rat cerulein pancreatitis.

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Activated T-lymphocytes are found early in atherosclerosis lesions, but little is known about their role. Oxidized low-density lipoproteins (oxLDLs) are considered to be involved in the pathogenesis of the lesions, and we have previously demonstrated that oxLDLs inhibit not only interleukin (IL)-2-receptor expression on the surface of in vitro-activated T-lymphocytes but also their proliferation. We have now investigated the effect of oxLDLs on blast differentiation, on IL-2 synthesis and on the activation of the nuclear factor kappaB (NF-kappaB) system in activated lymphocytes. Mildly oxLDLs (50 and 100 microgram/ml) decreased the number of lymphoblasts and the level of IL-2 concentration in the culture supernatants after activation of lymphocytes by phytohaemagglutinin and PMA+ionomycin. The inhibition of IL-2 production was observed in the CD3(+) T-lymphocyte cytoplasm as early as 4 h after activation by PMA+ionomycin. The study of NF-kappaB showed that oxLDLs led to a decrease of activation-induced p65/p50 NF-kappaB heterodimer binding to DNA, whereas the presence of the constitutive nuclear form of p50 dimer was unchanged. This was correlated with an unchanged level of the active form of the cytosolic inhibitor protein IkappaB-alpha. Taken together, these observations

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Granulocyte-macrophage colony-stimulating factor (GM-CSF), supports proliferation, differentiation, and functional activation of hemopoietic cells by its interaction with a heterodimeric receptor. Although GM-CSF receptor is devoid of tyrosine kinase enzymatic activity, GM-CSF-induced peripheral blood polymorphonuclear leukocytes (PMN) functional activation is mediated by the phosphorylation of a large number of intracellular signaling molecules. We have previously shown that JAK2 becomes tyrosine-phosphorylated in response to GM-CSF in PMN. In the present study we demonstrate that also the signal transducers and activators of transcription (STAT) family members STAT1 p91 and STAT3 p92 and the product of the c-fps/fes protooncogene become tyrosine-phosphorylated upon GM-CSF stimulation and physically associated with both GM-CSF receptor beta common subunit and JAK2. Moreover GM-CSF was able to induce JAK2 and p93fes catalytic activity. We also demonstrate that the association of the GM-CSF receptor beta common subunit with JAK2 is ligand-dependent. Finally we demonstrate that GM-CSF induces a DNA-binding complex that contains both p91 and p92. These results identify a new signal transduction pathway activated by GM-CSF and provide a mechanism for rapid activation of gene expression in GM-CSF-stimulated PMN.

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Nuclear factor of activated T cells (NFAT) is a transcriptional activator that binds to sequences in the interleukin-2 (IL-2) promoter and Electrophoretic mobility shift assays (EMSA) showed that The B cell NFAT complex, however, was Competition with an AP-1 motif or with anti-Jun and anti-Fos antibodies abolished binding to the NFAT motif in both T and B cells, Purified recombinant Jun and Fos proteins failed to bind directly to the NFAT motif. However, when combined with unstimulated B or T cell extracts, full-length, but An NFAT oligonucleotide carrying mutations in the 5' purine-rich part of the NFAT sequence We therefore

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We report here that the nuclear factor of activated T cells (NFATp), a cyclosporin A (CsA)-sensitive factor that regulates the transcription of several cytokines, mediates CD16-induced activation of cytokine genes in human NK cells. CD16 (Fc gamma RIIIA)-induced expression of cytokine mRNA in NK cells occurs via a CsA-sensitive and Ca(2+)-dependent mechanism. Stimulation of NK cells with CD16 ligands induces NFAT-like DNA binding activity in the nuclear extracts from these cells, as detected in electrophoretic mobility shift assays. This occurs with fast kinetics after stimulation, via a CsA-sensitive and Ca(2+)-dependent mechanism that does NK cell NFAT is present in the cytosol of nonstimulated cells, migrates to the nucleus upon stimulation, and can associate with AP-1. Two distinct molecules, NFATp and NFATc, have been reported to mediate NFAT activity. The results of supershift assays using NFATp- and NFATc- specific antibodies NK cells do However, supershift assays using the available mAb recognizing the T cell NFATc revealed These results provide the first direct evidence that both CsA-sensitive transcription factors, NFATp and NFATc, are expressed in human NK cells, and that their activation and/or expression can be regulated in primary cells by a single stimulus, that, in the case of CD16 in NK cells, results in early activation of NFATp and subsequently induced expression of NFATc mRNA.

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Non-genomic effects of aldosterone on the sodium-proton-antiport have been shown in human mononuclear leukocytes In the present paper plasma membranes from human mononuclear leukocytes were covalently photolabeled with a [125I]-aldosterone derivative. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed significant aldosterone binding at a molecular weight of approximately 50000 Dalton which was absent with 1 microM cold aldosterone, but The presence of the sulfhydryl agent dithiothreitol did These data are the first to define the molecular weight of the membrane receptor for aldosterone.

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We describe the isolation of human LH-2, a putative transcription factor containing two cysteine-rich regions (LIM domains) and a homeobox (Hox) DNA-binding domain. High levels of hLH-2 expression were observed in all cases of chronic myelogenous leukaemia (CML) tested, regardless of disease status. hLH-2 was mapped to chromosome 9Q33-34.1, in the same region as the reciprocal translocation that creates the BCR-ABL chimera of the Philadelphia chromosome (Ph'), the hallmark of CML; hLH-2 was retained on the derivative 9 chromosome and is therefore centromeric of c-ABL. The proximity of hLH-2 to the breakpoint on chromosome 9 In addition to finding hLH-2 expression in all cases of CML, expression was observed in lymphoid malignancies and myeloid cell lines, but However, hLH-2

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The nuclear factor of activated T cells (NFAT) regulates cytokine gene expression in T cells through cis-acting elements located in the promoters of several cytokine genes. NFATx1, which is preferentially expressed in the thymus and peripheral blood leukocytes, is one of four members of the NFAT family of transcription factors. We have performed domain analysis of NFATx1 by examining the effects of deletion mutations. We found that NFATx1 DNA binding activity and interaction with AP-1 polypeptides were dependent on its central Rel similarity region and that transcriptional activation was reduced by deletions of either its N-terminal domain or its C-terminal domain, We also identified a potent inhibitory sequence within its N-terminal domain. We show that the inactivation of the inhibition was dependent on the activity of calcineurin, a calcium-calmodulin-dependent phosphatase. We also show that calcineurin associated with the N-terminal domain of NFATx1 at multiple docking sites and caused a reduction of size, indicative of dephosphorylation, in NFATx1. We have mapped the inhibitory activity to less than 60 residues, containing motifs that are conserved in all NFAT proteins. Finally, we demonstrate that deletion in NFATx1 of the mapped 60 residues leads to its nuclear translocation independent of calcium signaling. Our results support the model

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Thrombin stimulation of the T leukemic cell line Jurkat induced a transient increase in [Ca2+]i. Proteolytic activity of the enzyme was required for this effect since diisopropyl fluorophosphate-thrombin Furthermore, hirudin and anti-thrombin III inhibited the thrombin-induced [Ca2+]i rise in Jurkat T cells. A synthetic thrombin receptor agonist peptide (TRP) of 7 residues (SFLLRNP) was found to be as effective as thrombin for [Ca2+]i mobilization, and both agonists induced Ca2+ release exclusively from internal stores. Thrombin stimulated tyrosine phosphorylation of several proteins of molecular mass 40, 42, 70, 120, and 130 kDa. There was a good correlation between thrombin-induced tyrosine phosphorylation of the latter three proteins and Ca2+ mobilization. Thrombin and TRP also caused translocation of protein kinase C from the cytosol to the plasma membrane. As a Several cell lines of hematopoietic origin including the leukemic T cell line HPB.ALL and the erythroleukemic cell line K562 were responsive to thrombin, whereas others such as THP1, a myelomonocytic cell line, and BL2, a Burkitt lymphoma were refractory to thrombin or TRP stimulation. The magnitude of the thrombin response in the different cell types paralleled the expression of the thrombin receptor mRNA. We found that activation of Jurkat T cells by a combination of phytohemagglutinin and phorbol 12-myristate 13-acetate led to a dramatic inhibition of thrombin receptor mRNA expression and to a concomitant loss of the thrombin response. Finally, we demonstrate that thrombin and TRP enhanced CD69 expression and interleukin 2 production induced by T cell receptor cross-linking in both Jurkat T cells and peripheral blood lymphocytes. These findings highlight the role of thrombin as a potential regulator of T lymphocyte activation.

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In the immune system the first activation of a naive T cell by antigen is a key step in the shaping of the peripheral T cell specificity repertoire and maintenance of self-tolerance. In the present study, analysis of the interleukin-2 (IL-2) gene activation shows that naive human helper T cells (cord blood CD4+ T cells, adult CD4+CD45RO- T cells) regulate IL-2 transcription by a mechanism involving both a silencer and an activator acting on the purine-rich IL-2 promoter elements (NF-AT binding sites). By contrast, memory cells, either in vitro activated helper T cells reverting to a resting state, or CD4+ T ( memory ) clones, or CD4+CD45RO+ T cells isolated ex vivo, in resting memory T cells the activator is located in the cytoplasm and is inactive, whereas in stimulated cells it is functional in promoting transcription and now resides in the nucleus. Thus, the regulation of the gene coding for the main T cell growth factor changes irreversibly after the first encounter of T cells with antigen. It is most

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The immediate-early gene egr-1 encodes a transcription factor (EGR1) that links B-cell antigen receptor (BCR) signals to downstream activation events through the regulation of previously unidentified target genes. Here we identify the gene encoding the lymphocyte homing and migration protein CD44 as a target of EGR1 regulation in B cells. BCR-induced increases in CD44 mRNA expression and transcription levels are shown to occur in EGR1-expressing but Kinetics of egr-1 transcription and the appearance of nuclear EGR1 protein precede CD44 induction and occur within 30 min after stimulation in the EGR1-expressing subclone. A single EGR1 binding motif is demonstrated at bp -301 of the human CD44 promoter. Cotransfection of a CD44 promoter-chloramphenicol acetyltransferase reporter construct with an egr-1 expression vector resulted in a 6.5- to 8.5-fold induction of transcriptional activity relative to an empty expression vector. The EGR1 binding motif was shown to be necessary for stimulus-induced expression of a CD44 promoter-chloramphenicol acetyltransferase reporter construct in nontransformed B lymphocytes and was required for transactivation by an EGR1 expression vector in a B-cell line. These studies identify EGR1 as an intermediary linking BCR-derived signals to the induction of CD44. The relevance of these molecular events to BCR signal transduction and antigen-stimulated B-cell-mediated immune responses is discussed.

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Patients with one type of major histocompatibility complex class II combined immunodeficiency have mutations in a gene termed class II transactivator (CIITA), which coordinately controls the transcription of the three major human class II genes, HLA-DR, -DQ, and -DP. However, the experimentally derived B-lymphoblastoid cell line, clone 13, expresses high levels of HLADQ in the It was Alternatively, another factor, distinct from CIITA, We report here that ectopic expression of CIITA cDNAs derived by reverse transcriptase polymerase chain reaction from clone 13 do In addition, In contrast, somatic cell fusion between clone 13 and RJ2.2.5 restored expression of the HLA-DQ haplotype encoded by the RJ2.2.5 DQB gene. Taken together, these data demonstrate the existence of an HLA-DQ isotype-specific trans-acting factor, which functions independently of CIITA.

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The hierarchy of transcriptional control in B-cell development has recently been analyzed by targeted gene inactivation in the mouse. In this manner, the paired box containing gene Pax-5, encoding the B cell specific transcription factor BSAP, has been shown to play a key role in early B lymphopoiesis. Other experimental strategies have implicated BSAP in the control of cell proliferation, isotype switching and transcription of the immunoglobulin heavy-chain gene at late stages of B-cell differentiation.

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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 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 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.

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Induction of endothelial adhesion molecules by the cytokine tumor necrosis factor-alpha (TNF) can occur independently of protein kinase C and activation of a protein tyrosine kinase (PTK) has recently been implicated in the upregulation of vascular cell adhesion molecule 1 (VCAM-1) by interleukin-4 (IL-4) on endothelial cells. We demonstrate that the PTK inhibitors herbimycin A or genistein suppress induction of endothelial VCAM-1 and E-selectin, as well as subsequent monocytic cell adhesion to endothelial cells stimulated by TNF. Inhibition studies This

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Using human T lymphocytes and T cell lines, we show that progesterone, at concentrations found in the placenta, rapidly and reversibly blocks voltage-gated and calcium-activated K+ channels (KV and KCa, respectively), resulting in depolarization of the membrane potential. As a result, Ca2+ signaling and nuclear factor of activated T cells (NF-AT)-driven gene expression are inhibited. Progesterone acts distally to the initial steps of T cell receptor (TCR)-mediated signal transduction, since it blocks sustained Ca2+ signals after thapsigargin stimulation, as well as oscillatory Ca2+ signals, but K+ channel blockade by progesterone is specific; other steroid hormones had little or Progesterone effectively blocked a broad spectrum of K+ channels, reducing both Kv1.3 and charybdotoxin-resistant components of KV current and KCa current in T cells, as well as blocking several cloned KV channels expressed in cell lines. Progesterone had little or We

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The protooncogene p21ras, a monomeric G protein family member, plays a critical role in converting extracellular signals into intracellular biochemical events. Here, we report that nitric oxide (NO) activates p21ras in human T cells as evidenced by an increase in GTP-bound p21ras. In vitro studies using pure recombinant p21ras demonstrate that the activation is direct and reversible. Circular dichroism analysis reveals that NO induces a profound conformational change in p21ras in association with GDP/GTP exchange. The mechanism of activation is due to S-nitrosylation of a critical cysteine residue which stimulates guanine nucleotide exchange. Furthermore, we demonstrate that p21ras is essential for NO-induced downstream signaling, such as NF-kappa B activation, and that endogenous NO can activate p21ras in the same cell. These studies identify p21ras as a target of the same cell. These studies identify p21ras as a target of NO in T cells and

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Two different proteins which independently bound to neighboring sequences within the negative regulatory element (NRE) of human immunodeficiency virus type 1 (HIV-1) were detected in the nuclear extract of a virus-infected human T cell line. One of the factors bound to a novel dyad symmetrical sequence. This sequence is well conserved in various HIV-1 isolates and partial homology was found with the promoter region of the human retinoblastoma gene. Similar DNA binding activity was detected in a variety of virus-uninfected human T cell lines and HeLa cells by means of a gel mobility shift assay. The other factor bound to a However, this factor did The insertion of multiple copies of the binding site for the former or latter factor into a heterologous promoter reduced the promoter activity to one-tenth or one-third, respectively. Thus, each factor

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The NF-kappaB/Rel family of transcription factors regulates the inducible expression of many genes in activated human monocytes and endothelial cells. In this study, we examined the molecular mechanism by which agents that elevate intracellular cAMP inhibit the expression of the tumor necrosis factor alpha (TNFalpha), tissue factor, endothelial leukocyte adhesion molecule-1, and vascular cell adhesion molecule-1 genes. Both forskolin and dibutyryl cAMP, which elevate intracellular cAMP by independent mechanisms, inhibited TNFalpha and tissue factor expression at the level of transcription. Induction of NF-kappaB-dependent gene expression in transiently transfected human monocytic THP-1 cells and human umbilical vein endothelial cells was inhibited by elevated cAMP and by overexpression of the catalytic subunit of protein kinase A (PKA). Elevated cAMP did Functional studies demonstrated that transcriptional activation of a plasmid containing multimerized kappaB sites by p65 was inhibited by agents that elevate cAMP and by overexpression of the catalytic subunit of PKA. This study

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Human T and B lymphocytes demonstrate an early and transient hyperpolarization after ligand binding. Inasmuch as the change in membrane potential is dependent on elevation of free cytosolic calcium, the hyperpolarization is We have used charybdotoxin, a known inhibitor of Ca(2+)-dependent K+ channels, to study the role of these channels in lymphocyte activation and mitogenesis. We demonstrate that charybdotoxin inhibits the ligand-induced transient membrane hyperpolarization in B and T cells in a dose-dependent fashion, However, blockade of the Ca(2+)-activated K+ channel is These results

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IL-2-, IL-12-, and IFN-alpha-mediated signaling pathways were analyzed in primary NK cells and in the NK3.3 cell line. Gel mobility shift and immunoprecipitation analyses revealed that in addition to activating STAT3 (signal transducer and activator of transcription-3) and STAT5, IL-2 induced tyrosine and serine phosphorylation of STAT1 alpha, which formed IFN-gamma-activated sequence-binding complexes by itself and with STAT3. Although IL-2 and IFN-alpha activated STAT1 alpha and STAT5, IL-2 predominantly activated STAT5, while IFN-alpha predominantly activated STAT1 alpha. IL-2 induced less STAT1 alpha activation and IFN-alpha induced greater STAT5 activation in NK3.3 cells compared with preactivated primary NK cells. In NK3.3 cells, IL-2 induced comparable formation of c-fos promoter sis-inducible element IFN-gamma-activated sequence-binding complexes containing STAT3 alone with complexes containing STAT3 and STAT1 alpha, while in preactivated primary NK cells, it preferentially induced complexes containing STAT3 and STAT1 alpha. Thus, signaling in NK3.3 cells is not always identical with that in primary NK cells. In contrast to IL-2 and IFN-alpha, IL-12 induced strong tyrosine phosphorylation of STAT4 and variable weak phosphorylation of STAT3. However, supershift analyses using the c-fos promoter sis-inducible element probe showed that IL-12 activated STAT4, STAT1 alpha, and STAT3, and induced complexes containing STAT4 only, STAT4 with STAT1 alpha, STAT3 with STAT1 alpha, or STAT1 alpha only in preactivated primary NK cells. STAT1 alpha activation by IL-12 correlated with increased phosphorylation of serine, but Finally, IL-2 induced tyrosine phosphorylation of JAK1 and JAK3, while IL-12 induced phosphorylation of JAK2 and TYK2 in both preactivated primary NK and NK3.3 cells. Differential phosphorylation and consequent differential activation of both separate and overlapping STAT proteins by IL-2, IL-12, and IFN-alpha

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The two nuclear proteins NF-kappa B (consisting of subunits p50 andp65) and the DNA-binding subunit of NF-kappa B (p50) by itself, also called KBF1, are constitutively expressed and localized in the nucleus of the human T-cell line IARC 301.5. In order to define the roles of these two factors, which bind to the same kappa B enhancers, in transcription activation we have prepared somatic cell hybrids between IARC 301.5 and a murine myeloma. Most hybrids express both KBF1 and NF-kappa B in their nuclei, but one hybrid expresses only KBF1. The kappa B enhancer of the gene encoding the interleukin-2 (IL-2) receptor alpha chain (IL-2R alpha) is functional only in the hybrids expressing nuclear NF-kappa B. These findings show that nuclear NF-kappa B is necessary to activate the kappa B enhancer, while KBF1 by itself is We propose that KBF1 is a competitive inhibitor of NF-kappa B and discuss how

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IL-12 is a 75-kDa heterodimeric cytokine composed of two covalently linked p35 and p40 chains. This pro-inflammatory cytokine plays a prominent role in the development of Th1 cell-mediated immune responses. Th1 cell-mediated immune responses have been implicated in the pathogenesis of chronic inflammatory autoimmune diseases. Thus, In this study, we investigated the effects of a commonly prescribed anti-inflammatory drug, acetyl salicylic acid (ASA), on IL-12 production and Th1 cell development. ASA was found to inhibit secretion of the IL-12 heterodimer as well as p40 monomer by human monocytic cells. This was associated with the down-regulation of IL-12p40 mRNA expression. Analysis of the regulation of the p40 gene promoter revealed that ASA inhibited NF-kappaB activation and binding to the p40-kappaB site in the p40 promoter, leading to transcriptional repression of the p40 gene. Addition of ASA to an in vitro T helper cell differentiation system, at concentrations compatible with plasma levels reached during anti-inflammatory therapy, resulted in reduced development of Th1 cells. These results

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Glucocorticoid receptor (GR) hormone-binding activity was studied by a whole-cell method in mononuclear cells (MNC) from peripheral blood of 7 patients during the hemodynamic compensatory phase of sepsis. 4 patients were receiving dopamine, which did The patients ' plasma cortisol concentrations were normal or slightly elevated. Despite a wide range, the mean GR count and affinity in MNC from septic patients did

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Activating protein-1 (AP-1) binding TPA responsive elements (TRE) are located downstream of the transcription initiation site in the U5 region of the HIV-1 long terminal repeat (LTR). These downstream sequence elements, termed DSE, can bind both AP-1 and CREB/ATF transcription factors. Recently, we demonstrated that the DSE are also cAMP-responsive elements (CRE), since they mediated activation signals elicited by cholera toxin (Ctx), a potent activator of the cAMP-dependent protein kinase A (PKA) signal transduction pathway. In the present study, we demonstrate that the HIV-1 DSE can mediate the transcriptional synergy elicited by the combination of Ctx and TNFalpha. Ctx combined with TNFalpha or IL-1beta to produce a synergistic increase in p24 antigen production in U1 promonocytic cells. Transfection studies of LTR reporter constructs indicated that mutation of the DSE sites abrogated the LTR-mediated synergy induced by Ctx and TNFalpha, whereas the synergy induced by Ctx and IL-1beta was unaffected, Because the DSE are also TRE sites, we assessed the effect of the agonist combinations on AP-1-dependent transcription. TNFalpha as well as IL-1beta cooperated with Ctx to produce a synergistic activation of AP-1-mediated transcription. These data Since the DSE and TRE sites Copyright 1997 Academic Press.

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Platelet thromboxane receptors are acutely and reversibly upregulated after acute myocardial infarction. To determine The exon-intron structure of the 5' portion of the thromboxane receptor gene was determined initially by comparing the nucleotide sequence of the 5' flanking genomic clone with that of a novel human uterine thromboxane receptor cDNA that extended the mRNA 141 bp further upstream than the previously identified human placental cDNA. A major transcription initiation site was located in three human tissues approximately 560 bp upstream from the translation initiation codon and 380 bp upstream from any previously identified transcription initiation site. The thromboxane receptor gene has Promoter function of the 5' flanking region of the thromboxane receptor gene was evaluated by transfection of thromboxane receptor gene promoter/chloramphenicol acetyltransferase (CAT) chimera plasmids into platelet-like K562 cells. Thromboxane receptor promoter activity, as assessed by CAT expression, was relatively weak but was significantly enhanced by phorbol ester treatment. Functional analysis of 5' deletion constructs in transfected K562 cells and gel mobility shift localized the major phorbol ester-responsive motifs in the thromboxane receptor gene promoter to a cluster of activator protein-2 (AP-2) binding consensus sites located approximately 1.8 kb 5' from the transcription initiation site. These studies are the first to determine the structure and organization of the 5' end of the thromboxane receptor gene and demonstrate that thromboxane receptor gene expression can be regulated by activation of protein kinase C via induction of an AP-2-like nuclear factor binding to upstream promoter elements. These findings strongly

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A novel B-cell derived (Bcd) oncogene has been isolated from the peripheral blood lymphocytes of one B-cell chronic lymphocytic leukemia (B-CLL) patient using DNA transfer and a mouse tumorigenicity assay. The Bcd proto-oncogene was activated by a truncation in the 5' UTR. It predicts for two open reading frames (ORFs). ORF1 consists of 240 bp that would encode 80 amino acids, while the major ORF2 consists of 648 bp capable of coding for 216 amino acids. Predicted peptide sequence of ORF2 contained a zinc finger domain which showed significant homology to GC box binding proteins BTEB2 and SP1. Transfection of an expression vector containing ORF2 but Bcd mRNA transcripts of < or = 2.6 kb were selectively expressed in PBL and testis of healthy individuals. Within the PBL, Bcd gene expression was restricted to CD19+ B-cells and absent from CD14+ monocytes and T-cells. Bcd transcripts were detected in all normal PBL samples tested but However, stimulation of B-cells from B-CLL patients under conditions which induced differentiation into plasma cells was associated with induction of Bcd gene expression. The Bcd gene

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Cross-linking of the CD3 and CD28 molecules on T lymphocytes represents one of the most effective signals for T lymphocyte activation and triggering of their cytotoxic effector function. To identify genes that are expressed in T cells after stimulation, mRNA from T lymphocytes that had been activated by the simultaneous stimulation of the CD3 and CD28 trigger molecules was transcribed for a differential mRNA display analysis into cDNA and was compared with cDNA from CD28- or CD3-activated or resting lymphocytes. Differential expression was confirmed subsequently by Northern blot analysis. One of the cDNA fragments expressed specifically in CD3- and CD28-activated T cells was designated RP1. The predictive protein-coding region of RP1 had a significant homology to members of the recently found adenomatous polyposis coli (APC) protein-binding EB1 gene family, which codes for yet unknown protein(s). Bacterially expressed RP1 protein revealed specific binding to wild-type but The rapid up-regulation of RP1 mRNA in properly activated T cells As the expression level of the RP1 gene in activated T cells and a spectrum of tumor-derived cell lines correlates with the proliferative status of the cells, members of the EB1-like gene family

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Interaction between the stage selector element (SSE) in the proximal gamma-globin promoter and hypersensitivity site 2 in the locus control region partly mediates the competitive silencing of the beta-globin promoter in the fetal developmental stage. We have now demonstrated that a second SSE-like element in the 5'-untranslated region of the gamma-gene also contributes to this competitive silencing of the beta-gene. Utilizing transient transfection assays in the fetal erythroid cell line, K562, we have shown that the core enhancer of hypersensitivity site 2 can preferentially interact with the proximal gamma-promoter in the Mutation of a 20-base pair sequence of the gamma-gene 5'-untranslated region (UTR) led to derepression of beta-promoter activity. A marked activation of gamma-promoter activity was also observed with this mutation, Fine mutagenesis dissected these activities to different regions of the 5'-UTR. The stage selector activity was localized to a region centered on nucleotides +13 to +15. Electromobility shift assays utilizing this sequence demonstrated binding of a fetal and erythroid-specific protein. The repressor activity of the 5'-UTR was localized to tandem GATA-like sites, These results

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Activation of the transcription factor nuclear factor-kappaB (NFkappaB) is impaired in T cells from patients with renal cell carcinomas (RCCs). In circulating T cells from a subset of patients with RCCs, the suppression of NFkappaB binding activity is downstream from the stimulus-induced degradation of the cytoplasmic factor IkappaBalpha. Tumor-derived soluble products from cultured RCC explants inhibit NFkappaB activity in T cells from healthy volunteers, despite a normal level of stimulus-induced IkappaBalpha degradation in these cells. The inhibitory agent has several features characteristic of a ganglioside, including sensitivity to neuraminidase but Indeed, we detected gangliosides in supernatants from RCC explants and Gangliosides prepared from RCC supernatants, as well as the purified bovine gangliosides G(m1) and G(d1a), suppressed NFkappaB binding activity in T cells and reduced expression of the cytokines IL-2 and IFN-gamma. Taken together, our findings

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Tissue factor (TF) is expressed rapidly by human monocytes exposed to bacterial endotoxin (lipopolysaccharide, or LPS). Transcriptional regulation is mediated by binding of c-Rel/p65 heterodimers to a kappa B-like site in the TF promoter. Nuclear translocation of cytosolic c-Rel/p65 heterodimers and other members of the NF-kappa B/Rel family requires dissociation and proteolytic degradation of the inhibitor protein, I kappa B alpha. The protease inhibitors N alpha-tosylphenylalanyl chloromethyl ketone (TPCK) and N alpha-tosyl-L-lysine chloromethyl ketone (TLCK) block activation of NF-kappa B/Rel proteins by preventing degradation of I kappa B alpha. To determine Both TPCK and TLCK inhibited LPS induction of TF protein, TF mRNA and TF promoter activity in a dose-dependent manner. These inhibitors specifically prevented degradation of I kappa B alpha and nuclear translocation of c-Rel/p65 heterodimers. In contrast, TPCK and TLCK did Taken together, these data

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In this study, the IFN-gamma induction of MHC class II gene expression in primary cultures of thymic epithelial cells (TEC) was analyzed. This cellular system offers the advantage that MHC class II induction is studied in a "physiologic" cell lineage that, as a result of this expression within the thymus, It was found that the MHC class II gene expression was associated with the de novo transcription of the gene encoding the CIITA trans-activator, a crucial MHC class II gene regulatory factor. Furthermore, the anatomy of interaction between the MHC class II DRA promoter and corresponding binding factors was analyzed by in vivo DNAse I footprint. It was found that treatment with IFN-gamma induces changes in the occupancy of the DRA gene regulatory sequences by nuclear factors. The resulting occupancy displays strong similarities with the one observed in the MHC class II-constitutive B cells, represented by both the Burkitt lymphoma line Raji and normal tonsil- derived B cells. However, some peculiar differences were observed between the TEC, either IFN-gamma-induced or These results

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The transactivator HTLV-I Tax activates the promoter of the gene coding for the interleukin 2 alpha-chain receptor (IL-2R alpha) via a kappa B site that can bind several protein species of the rel family. Tax1 strongly activates the enhancer activity of this motif, in both epithelial HeLa and lymphoid Jurkat cells. Overexpression of the p50, p65 and Rel proteins in these cells showed that significant activation of the IL-2R alpha kappa B site was observed only with Rel and Rel plus p65. Moreover, whereas both Tax and phorbol 12-myristate 13-acetate (PMA) are able to efficiently induce the binding of NF-kappa B to the IL-2R alpha kappa B site, PMA is functionally inactive. Using the DNA affinity precipitation assay, we observed that Tax1 is able to efficiently induce the binding of Rel, whereas PMA is This established a clear difference between both stimuli, We conclude from these results that the functional activity of members of the rel family is regulated by their interaction with DNA and that Rel

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IL-2 gene transcription is affected by several nuclear proteins. We asked Nuclear extracts from primary human T lymphocytes were analyzed by electrophoretic DNA mobility shift assays. Both Dex and CsA inhibited the binding of transcription factors AP-1 and NF-AT, but To correlate changes in nuclear factor binding in vitro with transcriptional activity in vivo and define the structural requirements for IL-2 promoter repression, we used transient DNA transfections. Jurkat cells were transfected with plasmids containing either the intact IL-2 promoter or its AP-1, NF-AT, and NF-kB motifs. Dex inhibited the IL-2 promoter and the AP-1, but In contrast, CsA inhibited the IL-2 promoter and the NF-AT, but These results We

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The rate of transcription initiation directed by the long terminal repeat (LTR) of HIV-1 increases in response to mitogenic stimuli of T cells. Here we show that The rate of LTR-directed gene expression increased in response to treatment with either a phorbol ester or tumor necrosis factor alpha if either the NFAT-1 or NF kappa B binding sites were deleted, but The HIV-1 mutant virus containing both NF kappa B and NFAT-1 deletion was able to replicate although at a much decreased growth rate, while the deletion of NFAT-1 alone increased the viral growth rate in Jurkat cells.

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Mammal pyruvate kinases are encoded by two genes. The L gene produces the erythroid (R-PK) or the hepatic (L-PK) isozymes by the alternative use of two promoters. We report the characterization of the cis- and trans-acting elements involved in the tissue-specific activity of the L gene erythroid promoter. A R-PK DNA fragment extending from -870 to +54 relative to the cap site confers erythroid specificity to a reporter gene. Within this region, we define a minimal promoter (-62 to +54) that displays erythroid-specific activity and contains two DNA binding sites. One, located at -50, binds members of the CCACC/Sp1 family and the other, located at -20, binds the erythroid factor GATA-1. Although the -20 GATA binding site (AGATAA) is also a potential TFIID binding site, it does Furthermore, the substitution of this GATA binding site by a canonical TFIID binding site suppresses the promoter activity. Mutations and deletions of both sites Finally, by co-transfection experiments, we study the elements involved in the hGATA-1 transactivation of the R-PK promoter in HeLa cells.

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Cell cycle progression initiated by interleukin-2 (IL-2) in T cells is critical for lymphoproliferation and an immune response. Phosphatidyl inositol 3-kinase (PI3K) is activated by IL-2. However, Here we identify the cell cycle regulator E2F as an IL-2 target in T lymphocytes and PI3K as the critical signaling pathway. We eliminate both Stat5 and Raf/MEK pathways from E2F regulation. Protein kinase B (PKB) is activated by IL-2 via PI3K. The expression of an active PKB is sufficient to induce E2F activity. Inhibition of PI3K inhibits phosphorylation of Rb, induction of cyclin D3, and degradation of p27kip1. These results establish a crucial PI3K/PKB-mediated link between the IL-2 teceptor and the cell cycle machinery.

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Since GH stimulates the development and function of granulocytes, we investigated the expression of GH in granulocyte subsets. By immunocytochemistry, 25 ± 7% of the human neutrophils were shown to express immunoreactive GH, whereas eosinophils were negative. Reversed transcription (RT)-PCR analysis demonstrated GH mRNA in neutrophils. Restriction analysis revealed that neutrophils express the GH-N gene but Furthermore, we demonstrated by western blot analysis that neutrophils express an alternatively spliced variant of the pituitary transcription factor Pit-1, designated Pit-1b.

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The chemokine RANTES has been implicated in the pathogenesis of allergic inflammatory diseases including asthma and rhinitis which are frequently treated with glucocorticoids. We observed that dexamethasone dramatically inhibited RANTES mRNA expression dose dependently in anti-CD3 activated Hut-78 T cells and human PBMCs. The down-regulation of RANTES expression by glucocorticoids in T cells

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Exposure of certain cell types to bacterial lipopolysaccharide (LPS) leads to activation of nuclear factor kappa B (NF-kappa B), an inducible transcription factor. One of NF-kappa B 's unique properties is its posttranslational activation via release of an inhibitory subunit, called inhibitor of NF-kappa B (I kappa B), from a sequestered cytoplasmic form. This event is also triggered under various other conditions of biomedical importance. Other bacterial toxins, tumor necrosis factor-alpha (TNF), interleukin-1 (IL-1), T cell mitogens, UV light, gamma rays and oxidative stress were reported to induce NF-kappa B. The activated form of NF-kappa B, which is rapidly taken up into nuclei, initiates transcription from immediate early genes in a wide variety of cell types. Most of the target genes for NF-kappa B are of relevance for the immune response and can be grouped into those encoding cytokines, cell surface receptors, acute phase proteins and viral genomes, such as that of human immunodeficiency virus type 1 (HIV-1). We will discuss recent experimental evidences This common pathway

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Two consensus sequences, called X and Y boxes, capable of binding nuclear proteins and regulating expression in B cells have been defined within the immediate upstream region of major histocompatibility complex (MHC) class II promoters. Unlike other class II promoters, the HLA-DR alpha (DRA) promoter also contains one element identical to the "octamer" motif of immunoglobulin variable region promoters that is responsible for B cell-specific transcription. In order to characterize the function of cis-acting elements, we have developed an in vitro system in which a DRA promoter construct is transcribed more efficiently in extracts from B cells than in extracts from class II-negative HeLa cells. 5' deletion constructs which Using supercoiled, but Demonstration of the complete dependence on the Y box in this system

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Interleukin-12 is produced in response to infection with bacteria or parasites or to bacterial constituents such as LPS in monocytes/macrophages and dendritic cells, and also generated by the interaction between activated T cells and antigen-presenting cells via CD40-CD40 ligand (CD40L). So far, transcriptional analyses of p40 have been carried out only using bacterial constituents such as LPS as stimuli. In the present study, we have characterized the transcriptional induction of p40 by CD40 ligation in a human B lymphoblastoid cell line, Daudi, and a human acute monocytic leukemia cell line, THP-1. These cells, stimulated by an agonistic monoclonal antibody against CD40 or by transfection with a CD40L expression vector, secreted p40 and showed enhanced p40 mRNA expression. Sequence analysis of the p40 promoter region identified two Electrophoretic mobility shift assay revealed that the Moreover, transfection of Daudi cells with the polymerized NF-kappaB binding sequence ligated to a thymidine kinase/chloramphenicol acetyltransferase (CAT) reporter plasmid greatly induced CAT activity, but transfection with the polymerized mutated NF-kappaB binding sequence did These results

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Productive infection of T cells with human immunodeficiency virus 1 (HIV-1) typically requires that the T cells be stimulated with antigens or mitogens. This requirement has been attributed to the activation of the transcription factor NF-kappa B, which synergizes with the constitutive transcription factor Sp1 to drive the HIV-1 promoter. Recently, we have found that vigorous replication of HIV-1 takes place in nonactivated memory T cells after syncytium formation with dendritic cells (DCs). These syncytia The expression and activity of NF-kappa B and Sp1 were, therefore, analyzed in isolated T cells and DCs from humans and mice. We have used immunolabeling, Western blot analysis, and electrophoretic mobility shift and supershift assays. T cells DCs express high levels of all known NF-kappa B and Rel proteins, with activity residing primarily within RelB, p50, and p65. However, DCs Coexpression of NF-kappa B and Sp1 occurs in the heterologous DC-T-cell syncytia that are induced by HIV-1. Therefore, HIV-1-induced cell fusion brings together factors that upregulate virus transcription. Since DCs and memory T cells frequently traffic together in situ, these unusual heterologous syncytia could develop in infected individuals and lead to chronic HIV-1 replication

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Triggering of HLA class II antigens by the anti-HLA-DR monoclonal antibody (mAb) L243 significantly (P < 0.05) and differentially enhanced the release of tumor necrosis factor alpha (TNF-alpha) by the non-Hodgkin's lymphoma cells Ri-I, Ci-I, and Sc-I, which are at a distinct stage of B-cell differentiation, and by the more mature Burkitt lymphoma cell Raji; in contrast, it did TNF-alpha release peaked at 24 h and decreased thereafter, and it was dose dependent and preceded by an increase of TNF-alpha mRNA detectable after 3 h of stimulation with mAb L243. Secreted TNF-alpha mediated the enhancement of nuclear factor kappa B (NF-kappa B) and activator protein-1 (AP-1) binding activity; in fact, the triggering of HLA-DR antigens in the presence of antihuman TNF-alpha-neutralizing antibodies did In contrast, released TNF-alpha was Altogether, our data demonstrate that: (a) the ability of B cells to release TNF-alpha after triggering of HLA-DR antigens depends on their stage of differentiation; (b)

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Most erythroleukemic cell lines established in vitro coexpress erythrocytic and megakaryocytic markers that often are associated with expression of Spi-1 and/or Fli-1 transcription factors known as transactivators of megakaryocyte-specific promoters. In the present study, we examined the possibility of establishing new cell lines keeping strictly erythroid-specific properties in vitro through the targeted and conditional immortalization of erythrocytic progenitors. For that purpose, we established several lines of transgenic mice displaying erythroid-specific expression of a thermosensitive SV40 T antigen. As expected, these transgenic mice developed splenomegaly due to the massive amplification of Ter 119 positive erythroid nucleated cells expressing T antigen. Despite this drastic effect in vivo, the in vitro immortalization of erythropoietin-dependent erythroid progenitors unexpectedly occurred at low frequency, and all four cell lines established expressed both erythrocytic (globins) and megakaryocytic markers (glycoprotein IIb, platelet factor 4) as well as Spi-1 and Fli-1 transcripts at permissive temperature. Switching the cells to the nonpermissive temperature led to a marked increase in globin gene expression and concomitant decrease in expression of Spi-1, Fli-1, and megakaryocytic genes in an erythropoietin-dependent manner. Interestingly, enhanced expression of Spi-1 and Fli-1 genes already was detected in the Ter 119 positive cell population of transgenic mice spleen in vivo. However, like normal Ter 119 erythroid cells, these Ter 119 positive cells from transgenic mice still expressed high levels of beta-globin and very low or undetectable glycoprotein IIb and platelet factor 4 megakaryocytic transcripts. Taken together, these data

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Stimulation of highly purified human T-cells via CD2 and CD28 adhesion molecules induces and maintains proliferation for more than 3 weeks. This potent interleukin 2 ( IL-2 ) -dependent activation does Long-lasting IL-2 receptivity is associated with high-level expression of the inducible IL-2 receptor alpha chain (IL-2R alpha) gene that is regulated at both transcriptional and posttranscriptional levels. Increase of IL-2R alpha gene transcription involves the enhanced binding of the transcription factor NF-kappa B to its consensus sequence in the 5'-regulatory region of the IL-2R alpha gene. To dissect the molecular basis for the unusually persistent transcription of the IL-2R alpha gene, we analyzed nuclear NF-kappa B binding to a radiolabeled IL-2R alpha kappa B-specific oligonucleotide probe during the time course of CD2 + CD28 activation. Resting T-cell nuclear extracts contained KBF1/p50 homodimer. After stimulation, two new kappa B-specific complexes were identified as NF-kappa B p50-p65 heterodimer and Both inducible complexes persisted for at least 3 weeks. Their relative levels were very similar for the duration of proliferation. In parallel, CD2 + CD28 activation triggered a significant intracellular thiol decrease, Finally, micromolar amounts of pyrrolidine dithiocarbamate, an oxygen radical scavenger that efficiently blocked the nuclear appearance of NF-kappa B in T-lymphocytes, also inhibited IL-2 secretion, IL-2R alpha cell surface expression, and T-cell proliferation. Together, these results

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The helix-loop-helix (HLH) proteins are a family of transcription factors that include proteins critical to differentiation and development in species ranging from plants to mammals. Five members of this family (MYC, SCL, TAL-2, LYL-1 and E2A) are implicated in oncogenic events in human lymphoid tumors because of their consistent involvement in chromosomal translocations. Although activated in T cell leukemias, expression of SCL and LYL-1 is low or undetectable in normal T cell populations. SCL is expressed in erythroid, megakaryocyte and mast cell populations (the same cell lineages as GATA-1, a zinc-finger transcription factor). In addition, both SCL and GATA-1 undergo coordinate modulation during chemically induced erythroid differentiation of mouse erythroleukemia cells and are down-modulated during myeloid differentiation of human K562 cells, thus However, in contrast to GATA-1, SCL is expressed in the developing brain. Studies of the function of SCL

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Binding of 3H-dexamethasone and 3H-aldosterone by peripheral lymphocyte receptors was investigated in healthy persons and hypertensive patients before and after 2-week captopril treatment. The number of glucocorticoid and mineralocorticoid binding sites was increased in hypertensives vs normotensives. The treatment with the ACE inhibitor captopril led to activation of hormone-receptor interactions. There was a more marked rise of the number of receptors in middle-aged (44-55 years) hypertensives vs elderly (61-80 years) subjects after captopril treatment.

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The CD4 coreceptor interacts with non-polymorphic regions of major histocompatibility complex class II molecules on antigen-presenting cells and contributes to T cell activation. We have investigated the effect of CD4 triggering on T cell activating signals in a lymphoma model using monoclonal antibodies (mAb) which recognize different CD4 epitopes. We demonstrate that CD4 triggering delivers signals capable of activating the NF-AT transcription factor which is required for interleukin-2 gene expression. Whereas different anti-CD4 mAb or HIV-1 gp120 could all trigger activation of the protein tyrosine kinases p56lck and p59fyn and phosphorylation of the Shc adaptor protein, which mediates signals to Ras, they differed significantly in their ability to activate NF-AT. The results identify functionally distinct epitopes on the CD4 coreceptor involved in activation of the Ras/protein kinase C and calcium pathways.

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During the course of serious bacterial infections, lipopolysaccharide (LPS) interacts with monocyte/macrophage receptors, resulting in the generation of inflammatory cytokines. Transcription factor NF-kappaB is crucial in activating the transcription of genes encoding proinflammatory cytokines. In this paper, we demonstrate that the activation of NF-kappaB by LPS in a promonocytic cell line (U937) followed a rather slow kinetics, depending on the rate of IkappaB-alpha inhibitor hydrolysis. The transduction pathway leading to NF-kappaB activation in U937 cells involved the intracellular generation of reactive oxygen species (ROS), as demonstrated by the concomitant inhibitory effects of antioxidants on NF-kappaB activation and the emission of a fluorescent probe reacting intracellularly with hydrogen peroxide. This ROS pathway was also characterized by the use of other inhibitors. This finding However,

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Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia. We examined the effect of Tax activity on the growth of the interleukin-2 (IL-2)-dependent T-cell line CTLL-2. Stable expression of Tax in CTLL-2 transformed cell growth from being IL-2 dependent to IL-2 independent. Tax stimulated transcription through NF-kappaB and the cyclic AMP-responsive element-like sequence in the HTLV-1 promoter. The finding of Tax mutants segregating these two pathways However, both pathways were necessary for another transformation-related activity (colony formation in soft agar) of CTLL-2/Tax. Our results show that Tax has at least two distinct activities on T cells, and

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Interferons (IFNs) inhibit induction by IL-4 of multiple genes in human monocytes. However, IL-4 activates gene expression by inducing tyrosine phosphorylation, homodimerization, and nuclear translocation of the latent transcription factor, STAT6 (signal transducer and activator of transcription-6). STAT6-responsive elements are characteristically present in the promoters of IL-4-inducible genes. Because STAT6 activation is essential for IL-4-induced gene expression, we examined the ability of type I and type II IFNs to regulate activation of STAT6 by IL-4 in primary human monocytes. Pretreatment of monocytes with IFN-beta or IFN-gamma, but This inhibition was associated with decreased tyrosine phosphorylation and nuclear translocation of STAT6 and was Furthermore, inhibition by IFN could be blocked by cotreatment with actinomycin D and correlated temporally with induction of the JAK/STAT inhibitory gene, SOCS-1. Forced expression of SOCS-1 in a macrophage cell line, RAW264, markedly suppressed trans-activation of an IL-4-inducible reporter as well as IL-6- and IFN-gamma-induced reporter gene activity. These findings demonstrate that IFNs inhibit IL-4-induced activation of STAT6 and STAT6-dependent gene expression, at least in part, by inducing expression of SOCS-1.

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The expression of the hematopoietic transcription factors GATA-1, GATA-2, and GATA-3 was studied in eosinophils and basophils. Eosinophils express mRNA for GATA-1, GATA-2, and GATA-3. Basophils express GATA-2 and GATA-3. Treatment of HL-60 eosinophilic sublines with either interleukin-5 or butyric acid increased the expression of GATA-1 mRNA concomitant with the expression of eosinophil-specific genes, whereas levels of GATA-2 mRNA remained relatively constant. The presence of mRNA for these proteins in eosinophils and basophils

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Activation of T cells by antigen, lectin, or a combination of phorbol-12-myristate acetate (PMA) and calcium ionophore (A23187) leads to the induction of genes for a set of lymphokines, including granulocyte-macrophage colony-stimulating factor (GM-CSF). We demonstrated in earlier studies that the upstream region of the mouse GM-CSF promoter at positions between -95 and -73 is essential for transcriptional activation in response to PMA/A23187. This region contains two DNA-binding motifs, GM2 and GC-box. The GM2 sequence (GGTAGTTCCC) is recognized by an inducible factor NF-GM2; the other (CCGCCC) by constitutive factors A1, A2, and B. To elucidate the mechanism of GM-CSF gene activation, we have purified the inducible factor NF-GM2 from the nuclear extract of stimulated Jurkat cells on the basis of specific DNA-binding activity. The purified NF-GM2 consists of 50 (p50) and 65 kDa (p65) polypeptides and has a binding activity specific for both the GM-CSF and immunoglobulin kappa (GGAAAGTCCC) enhancers. Electrophoretically purified p50 alone can form a protein-DNA complex, but in the mixture, p50 associates preferentially with p65 to form the NF-GM2 complex. In addition, p65 gave per se, with low affinity, a protein-DNA complex that migrated more slowly than native NF-GM2 complex. Furthermore, an antiserum against KBF1 (identical to 50 kDa NF-kappa B protein) reacted with the p50 of NF-GM2, The purified NF-GM2 activated in vitro transcription from the kappa B enhancer, while it This

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The CD20 ( B1 ) gene encodes a B cell-specific protein involved in the regulation of human B cell proliferation and differentiation. Studies with 5' deletion CD20 promoter-CAT constructs have previously revealed two regions of the promoter between bases -186 and -280 and between bases -280 and -454 which contained positive regulatory elements. In this study we identified a sequence element present in the most proximal region located between bases -214 and -201, TTCTTCTAATTAA, which is important in the high constitutive expression of CD20 in mature B cells and the induction of CD20 in pre-B cells. This sequence element was referred to as the BAT box and its deletion significantly reduced the activity of a CD20 promoter-CAT construct in B cells. Mobility shift assays with various mutant probes and B cell nuclear extracts demonstrated that the core sequence TAAT was essential for binding to this site. Cross competition experiments with an octamer sequence from the Ig heavy chain promoter, the BAT box, and a TA-rich sequence present in the CD21 promoter revealed that all three sequences bound the same nuclear proteins Southwestern blotting and UV cross-linking studies confirmed that the BAT box binding proteins were Oct-1 and Oct-2. The affinity of the BAT box binding proteins for the BAT box was approximately 25-fold less than for the octamer sequence and the BAT box binding proteins dissociated from the BAT box 10-fold more rapidly than from the octamer sequence. Despite this lower affinity, a trimer of the BAT box sequence was as efficiently transactivated by an Oct-2 expression vector as was a trimer of the octamer sequence in HeLa cells. The BAT box and Oct-2 were also implicated in the induction of CD20 in the pre-B cell line, PB-697, via phorbol esters. The induction of CD20 mRNA was temporally associated with induction of Oct-2 mRNA and a BAT box-deleted CD20-CAT construct, in contrast to the wild type, was poorly induced by phorbol esters. Together these results

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The Id family of helix-loop-helix proteins function as negative regulators of DNA binding, basic helix-loop-helix proteins in the regulation of cell growth and differentiation. We report here on the identification of a 17 kDa variant of the 14 kDa Id-3 protein termed Id-3L (long version) which possesses a unique 60 amino acid carboxy-terminus generated by read through of a ' coding intron ' and alternative splicing. Northern analysis revealed expression of a minor 1.1 kb Id-3L transcript together with the predominant 0.95 kb Id-3 transcript in the majority of adult human tissues analysed. The variant Id-3L protein is functionally distinguishable from conventional Id-3 since in in vitro DNA mobility shift assays, it was greatly impaired in its ability to abrogate binding of the basic helix-loop-helix protein, E47, to an E box recognition sequence.

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We describe an enhancer site in the third intron of tumor necrosis factor alpha (TNF-alpha). A reporter construct containing the 5'-flanking region of the mouse TNF-alpha gene displayed weak activity when transfected into RAW264.7 macrophage-like cells. The addition of the third intron of TNF-alpha to this construct resulted in an enhancement of CAT protein. This enhancement was eliminated if a conserved 20-bp sequence was removed from the intron or if a dominant-negative ets-binding factor was co-transfected with the reporter gene. Mutations of this site that destroyed The major transcription factor that bound to the oligonucleotide was confirmed to be GABP by supershift and competition analysis. In RAW264.7 cells, the binding was constitutive, however, in bone marrow-derived macrophages binding activity was shown to be interferon-gamma inducible. This

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The Thy-1 gene promoter resembles a " housekeeping " promoter in that it is located within a methylation-free island, Using transgenic mice, we show that this promoter does It can only be activated in a tissue-specific manner by elements that lie downstream of the initiation site. We have analyzed the functional domains of the minimal Thy-1 promoter and show that the dominant promoter elements consist of multiple binding sites for the transcription factor Sp1, an inverted CCAAT box, and sequences proximal to the transcription start site. DNase I and gel mobility shift assays show the binding of a number of nuclear factors to these elements, including Sp1 and CP1. Our results show that the structure of this promoter only permits productive interactions of the two transcription factors Sp1 and CP1 with the basal transcription machinery in the presence of enhancer sequences.

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The transcription factor GATA-1 is essential for normal erythropoiesis. By examining in vitro-differentiated embryonic stem cells, we showed previously that in the Here we report that in erythroid cells, GATA-1 strongly induces the expression of the anti-apoptotic protein bcl-xL, but Consistent with a role for bcl-xL in mediating GATA-1-induced erythroid cell survival, in vitro-differentiated bcl-xL-/- embryonic stem cells In addition, we show that erythropoietin, which is also required for erythroid cell survival, cooperates with GATA-1 to stimulate bcl-xL gene expression and to maintain erythroid cell viability during terminal maturation. Together, our data show that bcl-xL is essential for normal erythroid development and

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Human T-cell leukemia virus type 1 (HTLV-1) is an etiological agent of adult T-cell leukemia (ATL). We showed here by mobility-shift assay that T-cell lines transformed with the virus contained high levels of AP-1 activities. Consistent with this result, these cell lines expressed increased levels of mRNAs encoding the AP-1 proteins, c-Fos, Fra-1, c-Jun, JunB, and JunD. Previously, transcription of the c-fos gene has been reported to be transactivated by the viral transcription factor, Tax1. By using the human T-cell line (JPX-9), in which expression of the Tax1 is inducible, we showed that expression of mRNAs for Fra-1, c-Jun, and JunD was also transactivated by Tax1. Moreover, Tax1 activated expression of two other transcription factors having zinc finger motifs, Egr-1 and Egr-2, in the same cells. The Tax1-inducible transcription factors identified here are encoded by the members of immediate early genes under the control of growth signals. Thus ,

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We have investigated the effect of adenovirus 2 (Ad2) infection on human monocytes and monocyte-derived macrophages with regard to expression of TNF-alpha and IL-1 beta. In monocytes, the virus was bound to the surface On the other hand, Ad2 was internalized by macrophages. Ad2 infection induced transient increase in the mRNA levels for TNF-alpha and IL-1 beta in both monocytes and in macrophages, although the kinetics of the transcription was slightly different. The production of both cytokines, measured by ELISA tests, was enhanced in monocytes. In macrophages, a slight enhancement of TNF-alpha production was seen, whereas The data

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In this study the genetic control of major histocompatibility complex (MHC) class II gene expression during the transition from B cell to plasma cell has been analyzed. Class II molecules are We show here that the plasma cell-specific repressor function, designated SIR (suppressor of immune response genes), does This was unambiguously demonstrated by the fact that plasmacytoma x B cell hybrids carrying an AIR-1 locus derived from CIITA-expressing cells do Transfection of a cDNA containing the human CIITA coding sequence under the control of an heterologous promoter restores expression of human MHC class II genes in the hybrids and is responsible for de novo expression of mouse MHC class II genes in both the mouse plasmacytoma cell line and the hybrids. These results confirm and extend the notion of the functional conservation of the AIR-1 gene product across species barriers. Interestingly, in CIITA-transfected cell hybrids, This result was These findings further support our previous observations on the distinct regulation of expression of the human HLA-DQ class II subset, which

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Stimulation of resting human T cells with the CD28-specific mAb BW 828 induces proliferation and cytokine synthesis This observation prompted us to postulate that signal 2 (costimulatory signal) alone To test Stimulation of T cells with BW 828 induced an increase in intracellular Ca2+, but did This pathway resulted in the induction of the transcription factors NF-kappa B, NF-AT, and proteins binding to the CD28 response element of the IL-2 promoter. On the other hand, stimulation of T cells with mAb 9.3 increased the level of intracellular Ca2+ and triggered the activation of p56(lck) and c-Raf-1, but In contrast to the differential signaling of BW 828 and 9.3 in resting T cells, the two mAbs exhibited a similar pattern of early signaling events in activated T cells and Jurkat cells (p56(lck) activation, association of phosphatidylinositol 3-kinase with CD28), These data support the view that stimulation through CD28 can induce some effector functions in T cells and

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The binding of natural or synthetic ligands to nuclear receptors is the triggering event leading to gene transcription activation or repression. Ligand binding to the ligand binding domain of these receptors induces conformational changes that are evidenced by an increased resistance of this domain to proteases. In vitro labeled receptors were incubated with various synthetic or natural agonists or antagonists and submitted to trypsin digestion. Proteolysis products were separated by SDS-PAGE and quantified. The amount of trypsin-resistant fragments was proportional to receptor occupancy by the ligand, and allowed the determination of dissociation constants (kDa). Using the wild-type or mutated human retinoic acid receptor alpha as a model, kDa values determined by classical competition binding assays using tritiated ligands are in agreement with those measured by the proteolytic assay. This method was successfully extended to human retinoic X receptor alpha, glucocorticoid receptor, and progesterone receptor, thus providing a basis for a new, faster assay to determine simultaneously the affinity and conformation of receptors when bound to a given ligand.

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The human T-cell leukemia virus type I (HTLV-I) and HTLV-II Tax proteins are potent transactivators of viral and cellular gene expression. Using deletion mutants, the downstream parathyroid hormone-related protein (PTHrP) promoter is shown to be responsive to both HTLV-I and HTLV-II Tax as well as the AP1/c-jun proto-oncogene. Transactivation of PTHrP by Tax was seen in T cells but A carboxy terminal Tax deletion mutant was deficient in transactivation of both the PTHrP and IL2R alpha promoters but Exogenous provision of NFkB rescued IL2R alpha expression but Thus, HTLV-I Tax, HTLV-II Tax, and c-jun transactivate PTHrP and

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We have cloned a group of cDNAs representing mRNAs that are rapidly induced following adherence of human monocytes. One of the induced transcripts (MAD-3) encodes a protein of 317 amino acids with one domain containing five tandem repeats of the cdc10/ankyrin motif, which is 60 % similar (46 % identical) to the ankyrin repeat region of the precursor of NF-kappa B/KBF1 p50. The C-terminus has a In vitro translated MAD-3 protein was found to specifically inhibit the DNA-binding activity of the p50/p65 NF-kappa B complex but The MAD-3 cDNA encodes an I kappa B-like protein

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The molecular mechanisms of the effects of IL-4 and IL-13 on HIV infection in human monocytes as they matured into monocyte-derived macrophages over 7 days were investigated using HIV-1(BaL), and low passage clinical strains. IL-4 and IL-13 up-regulated the expression of both genomic and spliced HIV mRNA in monocytes cultured on Teflon, as determined by Northern analysis and p24 Ag assay. Using a nuclear run-on assay, IL-4 stimulation was shown to enhance transcription by two- to threefold. IL-4 stimulated nuclear factor-kappaB nuclear translocation and binding before enhancement of HIV RNA expression. Conversely, IL-4 and IL-13 markedly and significantly inhibited HIV replication at the transcriptional level in monocyte-derived macrophages, and this occurred whether these cytokines were added before or after HIV infection. The reversal from stimulation to inhibition occurred after 3 to 5 days of adherence to plastic. IL-4 had The effect of both cytokines on the monocyte maturation/differentiation (CD11b, CD13, and CD26) and other macrophage markers (CD14 and CD68) was examined. IL-4 enhanced CD11b, but inhibited CD26 expression and delayed CD13 loss. IL-13 had similar effects on CD11b and CD13, but Hence, these cytokines do not simply enhance monocyte differentiation, but have complex and slightly divergent effects that impact on HIV replication

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The expression of tissue factor (TF), the principal initiator of coagulation, is increased during inflammation and atherosclerosis. Both conditions are promoted by lysophosphatidylcholine (lysoPC). We observed in the present study that lysoPC (1 to 10 micromol/L) dose-dependently reduced TF activity in human monocytes, as elicited by lipopolysaccharide (LPS). Lysophosphatidylethanolamine (lysoPE) and other lysophospholipids did TF antigen expression as elicited by LPS was also lowered by lysoPC. Phospholipid analyses indicated a selective increase in the lysoPC content of the monocytes after preincubation with the lysophospholipid. LysoPC inhibited the TF activity of Mono Mac-6 cells to a similar extent as in the monocytes. In contrast, LPS-mediated nuclear binding of nuclear factor-kappaB/Rel to a TF-specific kappaB site was inhibited by lysoPC. Induction of TF mRNA expression by LPS tended to be partially reduced by the lysophospholipid. Preincubation with lysoPC increased monocytic cAMP levels. Inhibition of adenylyl cyclase by pretreatment with 2'-deoxy-3'-adenosine monophosphate partially reversed the inhibition of TF activity promoted by lysoPC. In conclusion, lysoPC markedly decreases LPS-mediated TF expression of human monocytes, the effect LysoPC

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The activation of transcriptional factor c-Fos/c-Jun AP-1 is essential for normal T cell responsiveness and is often impaired in T cells during aging. In the present study, we investigated Whereas T cells from young subjects stimulated with cross-linked anti-CD3epsilon mAb OKT3 plus PMA or with the lectin PHA plus PMA demonstrated considerable increases in c-Fos protein expression, the expression of c-Fos but In addition, RNase protection assays revealed that anti-CD3/PMA-stimulated T cells from a substantial proportion of elderly subjects exhibited decreased levels of c-fos and/or c-jun mRNA compared to T cells from young subjects. Using electrophoretic mobility shift assays, the levels of nuclear regulatory proteins recognizing the AP-1 consensus TRE motif, the proximal c-jun TRE-like promoter element, and the c-fos serum response element (SRE) were determined in resting and stimulated T cells. Although the stimulation of T cells from young subjects resulted in coordinated increases of nuclear protein complexes binding the AP-1 TRE, c-jun TRE, and c-fos SRE DNA sequence motifs, age-related reductions in the activation of AP-1 were accompanied by decreased levels of c-jun TRE and c-fos SRE binding complexes. Furthermore, the nuclear protein complexes binding the SRE motif induced in activated T cells of young and elderly subjects contained serum response factor and Elk-1 pointing toward age-related defects in the activation of transcriptional regulatory proteins distinct from c-jun/AP-1. These results

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Terminal differentiation of the leukemic cell lines U-937 and HL-60 by 12-O-tetradecanoylphorbol-13-acetate is accompanied by marked changes in gene expression. In this study, we demonstrate that the expression of jun and fos gene family members is induced with variable kinetics during 12-O-tetradecanoylphorbol-13-acetate induced differentiation, with c-jun expression best paralleling differentiation. The generation of AP-1 complexes, as measured by DNA binding activity, closely parallels morphological differentiation. Furthermore, the ability of these complexes to regulate gene expression is demonstrated by increased transcription from an AP-1 driven reporter construct and marked increases in the expression of endogenous AP-1 regulated genes. Differentiation assays using water soluble phorbol esters reveal that differentiation becomes irreversible soon after AP-1 appears. This tight correlation between c-jun expression, the generation of AP-1 activity, and differentiation

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In 1991, we demonstrated , using electrophoretic mobility shift assays, that 3 different factors (termed B1, B2 and B3) with affinity for the KB-enhancer target sequence were specifically detected in nuclear extracts from HIV1-infected monocytes and macrophages. The B2 factor was induced in the nuclei of these cells only upon HIV1 infection. The B3 factor was only slightly evident in nuclei of uninfected cells but was readily detectable in nuclei of infected monocytes. Its expression remained very low in nuclei of HIV1-infected macrophages. In this paper, we demonstrate that the B2 factor is expressed in the cytosol of monocytes and macrophages as a DNA-binding protein, This factor remained clustered in the cytosol and was translocated to the nuclei only after HIV1 infection. The B3 factor is detected in the cytosol only when cells are HIV1-infected. The role of HIV1 infection in the expression and the translocation of these factors is discussed.

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Double-stimulation was used to demonstrate that, in a T lymphocytic cell line (CEM), phorbol myristate acetate (PMA) rapidly induced NF-kappa B through a signaling pathway which did Since these latter compounds were known to activate NF-kappa B translocation in a redox-sensitive way, we have demonstrated that NF-kappa B activation by PMA was resistant to antioxidant N-acetyl-L-cysteine (NAC) and sensitive to kinase inhibitors staurosporine and H7 while activation by H2O2 or TNF-alpha were

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The expression of major histocompatibility complex (MHC) class II antigens is constitutive in professional antigen presenting cells (APCs) but can also be induced by interferon-gamma (IFN-gamma) on the majority of the non professional APCs (e.g. fibroblasts). We have recently characterised a new factor called IK which is an efficient inhibitor of IFN-gamma induction of MHC class II antigens expression. Here, we demonstrate a novel role for IK in MHC class II expression since over-expression of this protein by stable transfection into human B cells led to a total disappearance of constitutive MHC class II mRNA expression. The class II transactivator (CIITA) is necessary for both constitutive and IFN-gamma induced MHC class II expressions. Examination of CIITA mRNA in IK stably transfected clones revealed a marked reduction of CIITA mRNA transcription. Taken together these results demonstrate that the IK protein plays a key role in the constitutive expression of MHC class II antigens and that inhibition induced by IK is upstream of CIITA in this regulatory pathway.

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Complications after lung transplantation include the development of rejection and an increased incidence of infection, particularly with cytomegalovirus (CMV). Several recent studies have In addition, IL-6 Because CMV is also associated with the development of bronchiolitis obliterans after transplantation, we determined We demonstrated that CMV infection increased both IL-6 protein and mRNA in peripheral blood mononuclear cells. We also demonstrated that the CMV immediate early 1 gene product increased expression of the IL-6 promoter. This effect of the CMV immediate early 1 gene product was dependent upon the presence of specific transcription factor binding sites in the IL-6 promoter. These studies demonstrate that CMV

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Ig heavy chain class switching is directed by cytokines inducing transcription from unrearranged CH genes. Subsequently, such primed cells can undergo switch recombination to express the selected new isotype. In the case of IgE class switching, IL-4 activates the IgE germline promoter by inducing the interaction of the transcription factor STAT6 (IL-4STAT) with a responsive DNA element in the proximal region of the promoter. This study describes the characterization of two additional cis-acting elements that interact with members of the NF kappa B/rel transcription factor family in an IL-4-independent fashion. Electrophoretic mobility shift assays show that the nucleoprotein complex formed on the upstream site (NF kappa B1) contains the classical p50/p65 heterodimer. IgE germline promoter reporter gene constructs carrying point mutations in the NF kappa B2 site were largely unresponsive to IL-4 stimulation in transient transfection experiments, while plasmids with similar mutations in the NF kappa B1 site responded to cytokine stimulation better than the wild-type promoter. The NF kappa B2 effect was dependent on the presence of the STAT6 binding site, demonstrating that the NF kappa B2 motif is necessary but In addition, the combination of a NF kappa B/rel binding site and the STAT6 response element conferred IL-4 inducibility to a heterologous minimal promoter, while the individual sites had The available data

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A previously unrecognized element, located downstream of the start site of transcription in the first exon of the DR alpha gene, has been defined that enhances promoter activity up to eightfold in a position-dependent manner. Mutations in this DNA-binding site abolished binding of a nuclear factor in human B cell nuclear extract and decreased the activity of the DR alpha promoter to a basal level. Significant sequence homology of this element was found in the DNA of the DR beta, DP alpha and -beta, and DQ alpha genes, always located downstream of the transcriptional start site. The nuclear factor binds to the DR alpha and DP alpha element but It was identified as NF-E1 (YY1).

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Cell-to-cell contact between peripheral blood lymphocytes and transfected human colonic carcinoma cell line HT29 activates transcription of the long terminal repeats (LTR) of human immunodeficiency virus. HIV-1 LTR transcription is controlled by a complex array of virus-encoded and cellular proteins. Using various constructs expressing a lacZ reporter gene under the control of the intact or three deleted forms of HIV-1 LTR, we obtained evidence that the kappaB regulatory elements located in the U3 region are involved in cell-to-cell activation of HIV-1 LTR. Cell-to-cell contact activates in vitro binding of the nuclear factor kappaB (NF-kappaB) p50/p65 heterodimer to an HIV-1 kappaB oligonucleotide. Cell-to-cell contact activation of NF-kappaB was only partially inhibited by 100 microM pyrrolidine dithiocarbamate and NF-kappaB nuclear activation was

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Tissue factor (TF) expression by cells of monocyte/macrophage lineage represents an important mechanism underlying the initiation of fibrin deposition at sites of extravascular inflammation. Recent evidence The effect of antioxidant treatment on lipopolysaccharide (LPS)-induced TF expression was examined in murine peritoneal macrophages and human monocytes. Both pyrrolidine dithiocarbamate, an oxidant scavenger, and N-acetyl-cysteine, a precursor of the endogenous antioxidant glutathione, inhibited stimulation of macrophage procoagulant activity by LPS. Northern blot analysis showed that Immunofluorescence studies of human monocytes using polyclonal anti-TF antibody showed that N-acetyl-cysteine treatment prevented the characteristic plasmalemmal localization of TF antigen that occurs in response to LPS. Western blot analysis showed that N-acetyl-cysteine reduced the accumulation of the 47-kD mature glycoprotein in LPS-treated cells, a finding consistent with the results of the immunofluorescence studies. Furthermore, these conditions did When considered together, these data The posttranscriptional effect of antioxidants on TF expression data

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We previously showed that granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) stimulate the differentiation of human monocytes into two phenotypically distinct types of macrophages. However, in vivo, not only CSF but also many other cytokines are produced under various conditions. Those cytokines In the present study, we showed that CD14+ adherent human monocytes can differentiate into CD1+relB+ dendritic cells (DC) by the combination of GM-CSF plus interleukin-4 (IL-4) and that they differentiate into tartrate-resistant acid phosphatase ( TRAP ) -positive osteoclast-like multinucleated giant cells (MGC) by the combination of M-CSF plus IL-4. However, the monocyte-derived DC were they could still convert to macrophages in response to M-CSF. Tumor necrosis factor-alpha (TNF-alpha) stimulated the terminal differentiation of the DC by downregulating the expression of the M-CSF receptor, cfms mRNA, and aborting the potential to convert to macrophages. In contrast to IL-4, interferon-gamma (IFN-gamma) had Rather, IFN-gamma antagonized the effect of IL-4 and suppressed the DC and MGC formation induced by GM-CSF + IL-4 and M-CSF + IL-4, respectively. Taken together, these results provide a new aspect to our knowledge of monocyte differentiation and provide evidence that human monocytes are flexible in their differentiation potential and are precursors not only of macrophages but also of CD1+relB+DC and TRAP-positive MGC. Such a diverse pathway of monocyte differentiation

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The renin-angiotensin system A common feature of all stages of atherosclerosis is inflammation of the vessel wall. The transcription factor nuclear factor-kappaB (NF-kappaB) participates in most signaling pathways involved in inflammation. This study therefore examined the effect of angiotensin (ANG) II on NF-kappaB activation in monocytic cells, a major cellular component of human atheroma, by electrophoretic mobility shift assay. ANG II, like TNFalpha, caused rapid activation of NF-kappaB in human mononuclear cells isolated from peripheral blood by Ficoll density gradient. This ANG II effect was blocked by the angiotensin AT1 receptor antagonist losartan. Specificity of ANG II-induced NF-kappaB activation was ascertained by supershift and competition experiments. Moreover, ANG II stimulated NF-kappaB activation in human monocytes, but Together, the data demonstrate the ability of the vasoactive peptide ANG II to activate inflammatory pathways in human monocytes. Copyright 1999 Academic Press.

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The chemiluminescent (CL) response of interferon-gamma-treated U937 (IFN-U937) cells to sensitized target cells has been used to detect red cell, platelet and granulocyte antibodies. A clone of U937 cells was selected which expressed Fc receptor I (Fc gamma RI) and which, after incubation with IFN-gamma for 72 h, was capable of generating high levels of lucigenin-enhanced CL. The CL responses of IFN-U937 cells and peripheral blood human monocytes to sensitized red cells, platelets or granulocytes were then compared. Assays using monocytes or IFN-U937 cells were of comparable sensitivity for detection of antibodies against all three types of target cell. In addition, the use of IFN-U937 cells reduced interassay variation and simplified assay performance. In addition, monocytes and IFN-U937 cells both responded to red cells sensitized with antibodies against a variety of specificities of In contrast, monocytes and IFN-U937 cells responded only weakly to red cells sensitized with either anti-D in sera from mothers of babies unaffected by HDN, or with antisera containing high titre antibodies with specificities

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The PEBP2 alpha A and PEBP2 alpha B genes encode the DNA-binding subunit of a murine transcription factor, PEBP2, which is implicated as a T-cell-specific transcriptional regulator. These two related genes share the evolutionarily conserved region encoding the Runt domain. PEBP2 alpha B is the murine counterpart of human AML1, which is located at the breakpoints of the 8;21 and 3;21 chromosome translocations associated with acute myeloid leukemia. Northern (RNA) blots of various adult mouse tissues revealed that the levels of expression of both genes were most prominent in the thymus. Furthermore, transcripts of PEBP2 alpha A and mouse AML1/PEBP2 alpha B were detected in T lymphocytes in the thymuses from day 16 embryos and newborns, as well as 4-week-old adult mice, by in situ hybridization. The expression of the genes persisted in peripheral lymph nodes of adult mice. The transcripts were detected in all the CD4- CD8-, CD4+ CD8+, CD4+ CD8-, and CD4- CD8+ cell populations. The results Transcripts of mouse AML1/PEBP2 alpha B were also detected in day 12 fetal hematopoietic liver and in the bone marrow cells of newborn mice. The implication of mouse AML1/PEBP2 alpha B expression in hematopoietic cells other than those of T-cell lineage is discussed in relation to myeloid leukemogenesis.

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Tepoxalin, a dual enzyme inhibitor of cyclooxygenase and 5-lipoxygenase has been shown to inhibit T-cell activation. Its immunosuppressive property is distinct from cyclosporin because only tepoxalin, but Here we report that tepoxalin selectively inhibits intercellular adhesion molecule-1 ( ICAM-1 , CD54 ) /MAC-1 ( CD11b/CD18 ) dependent adhesion of polymorphonuclear cells to IL-1 activated human umbilical vein endothelial cells. The mechanism of inhibition is related to the surface expression of several cell adhesion molecules. Flow cytometry analyses on cultured cells that were treated with tepoxalin or antisense oligonucleotides to the P65/p50 subunit of NF-kappa B, and then stimulated with PMA, revealed a reduced expression of CD11b/CD18 on monocytic HL60 cells, and endothelial adhesion molecule-1 (CD62E) and vascular adhesion molecule-1 (CD106) on human umbilical vein endothelial cells. Expression of other adhesion molecules such as lymphocyte function associated-antigen-1 (CD11a/CD18) and CD54 were unaffected. Tepoxalin also inhibited the secretion of a NF-kappa B regulated chemokine, IL-8, a known inducer of CD11b/CD18 expression. Thus the suppression of CD11b/CD18 expression by tepoxalin Our results

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Thrombin plays a critical role in platelet activation, hemostasis, and thrombosis. Cellular activation by thrombin leads to the phosphorylation of multiple proteins, most of which are unidentified. We have characterized several 29-kDa proteins that are rapidly phosphorylated following exposure of intact human platelets to thrombin. A murine monoclonal antibody raised to an unidentified estrogen receptor-related 29-kDa protein selectively recognized these proteins as well as a more basic, unphosphorylated 27-kDa protein. Cellular activation by thrombin led to a marked shift in the proportion of protein from the 27-kDa unphosphorylated form to the 29-kDa phosphoprotein species. Using this antibody, we isolated and sequenced a human cDNA clone encoding a protein that was identical to the mammalian 27-kDa heat shock protein (HSP27), a protein of uncertain function that is known to be phosphorylated to several forms and to be transcriptionally induced by estrogen. The 29-kDa proteins were confirmed to be phosphorylated forms of HSP27 by immunoprecipitation studies. Thus, the " estrogen receptor-related protein " is HSP27, and the three major 29-kDa proteins phosphorylated in thrombin-activated platelets are forms of HSP27. These data

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The problem of the neuro-immuno interactions on the level of the protein trans-factors, stimulating interleukin-2 (IL-2) gene expression was discussed. The physico-chemical and functional parameters of the low molecular nuclear proteins (SP and BP- 14, 18, 19 kDs) isolated from splenic and brain cells of immunized rats were studied. The binding of these proteins to the regulatory region of IL-2 gene in vitro and stimulation of the IL-2mRNA synthesis in splenic T-lymphocytes culture in normal conditions were shown. The protective effect of SP and BP on the IL-2mRNA synthesis in stressful conditions and by the T-cells treatment with the CsA was demonstrated.

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In the present study we investigated the 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 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

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Nuclear factor of activated T cells (NF-AT) is a transcriptional activator involved in the induction of IL-2 gene expression. The response element for NF-AT is a sequence localized between -285/-254 in the IL-2 regulatory region. We demonstrate that , in normal human T cells, an AP-1 protein is a component of the NF-AT protein complex. This was evidenced by the ability of the AP-1 site to compete with the NF-AT site for binding to NF-AT and by the capacity of immobilized anti-Jun and anti-Fos antibodies to deplete NF-AT-binding activity from nuclear extracts of activated T cells. There was The presence of an AP-1 protein in the NF-AT protein complex

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In a Jurkat cell model of T-cell activation an interleukin-2 promoter/reporter gene construct was activated by antigen receptor agonism in combination with the lymphokine interleukin-1. Antigen receptor signals could be mimicked by suboptimal activation of protein kinase C (PKC) with phorbol esters in combination with calcium mobilization by an ionophore. In cotransfection experiments, oncogenic rats obviated the need for PKC stimulation but did Activated ras expression also replaced the requirement for PKC stimulation in activation of the T-cell transcription factor NF-AT. A dominant inhibitory ras mutant specifically blocked antigen receptor agonism, In addition, an inhibitor of PKC blocked both activated ras and phorbol ester stimulation,

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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 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.

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We previously identified the P sequence as a critical regulatory element of the human IL-4 promoter. In the mouse IL-4 promoter, there are five elements homologous to the human P sequence designated conserved lymphokine element 0 (CLE0), P, P2, P3 and P4. To characterize the role of these P-like elements and their binding factors in the native promoter, we did transient transfection and electrophoretic mobility shift assays (EMSA). Transfection of EL-4 cells with the IL-4 promoter-reporter constructs carrying mutated P-like elements showed that four P-like elements, CLE0, P, P2 and P4, but EMSA showed that both constitutive and inducible complexes bound to CLE0, P, P2 and P4, whereas only a constitutive complex bound to P3. In competition and antibody supershift assays in EMSA, complexes formed with P or P2 proved to contain nuclear factor of activated T cells (NFAT) family proteins as major components. Activator protein (AP)-1 family proteins interacted with CLE0, P, P2 and P4. NFAT/AP-1 complex formed only with P and P2. Cross-competition assays among the P-like elements revealed element-specific and common complexes. Six tandem repeats of the P element linked to the SV40 promoter responded to phorbol 12-myristate 13-acetate, while that of other elements did It

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1. In this study we 2. Our study involved a group of 60 male and female subjects who were exposed to MTBE and benzene-contaminated water concentrations up to 76 PPB for MTBE and 14 PPB for benzene, for a period of 5 to 8 years. For comparison, we recruited a control group consisting of 32 healthy males and females with similar age distribution and 3. Peripheral blood lymphocytes (PBL) of both groups were tested for the percentage of apoptotic cells and cell cycle progression using flow cytometry. 4. When apoptotic lymphocytes from exposed individuals were compared to apoptotic lymphocytes from the control group, statistically-significant differences between each mean group were detected (26.4 ± 1.8 and 12.1 ± 1.3, respectively), indicating an increased rate of apoptosis in 80.5% of exposed individuals (P < 0.0001, Mann-Whitney U-Test). MTBE and benzene-induced apoptosis is attributed to a discrete block within the cell cycle progression. Because cell cycle analysis showed that in PBL from chemically-exposed individuals, between 20-50% of cells were accumulated at the S-G2/M boundaries. 5. One of the signaling molecules which mediates programmed cell death is nuclear factor Kappa-B (NF-kappa B). NF-kappa B was examined as one of the many molecular mechanisms for mediating cell death by MTBE and benzene. Indeed, addition of inhibitors of NF-kappa B activation pyrrolidine dithiocarbamate (PDTC), to the lymphocytes of the chemically-exposed group was capable of inhibiting programmed cell death by 40%. This reversal of apoptosis almost to the control level by inhibitor of NF-kappa B activation

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The induction of immediate early genes in cells of the immune system is critical to determining the ultimate outcome of exposure to antigen. The importance of many of these genes relates to the role their transcription factor products play in dictating patterns of expression of downstream, function-related genes. Evidence from several systems Recently, the egr-1 promoter has been shown to be highly responsive to the diverse biochemical signals generated by antigen and cytokines in cells of the immune system. Furthermore, an important role for egr-1 in determining the differentiation pathway of myeloid cell precursors has been recently elaborated. Finally, potential targets of regulation by the zinc-finger transcription factor encoded by egr-1 include the interleukin-2, CD44, ICAM-1, and tumor necrosis factor genes. The role of egr-1 in regulation of the immune response will be discussed in the context of these recent studies.

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T lymphocytes from patients with renal cell carcinoma (RCC) show reduced immune function and impaired activation of the transcription factor, NF-kappaB. We determined the mechanism of NF-kappaB suppression in T cells of RCC patient and determined The pattern of kappaB-binding activity in T cells of RCC patient was altered as compared to that seen in T cells obtained from normal volunteers. In some patients, IkappaBalpha was degraded normally following stimulation in both normal controls and T cells from RCC patients. RCC-S did These results show that RCC-S can induce in normal T cells the same phenotype of impaired NF-kappaB activation that is detected in T cells of RCC patient. It also

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Prostaglandins of the E series are immunomodulatory agents which exert inhibitory as well as stimulatory effects on a variety of immune responses. Since it is known that PGE2 is able to increase cAMP levels, we investigated Using electrophoretic mobility shift assay, we demonstrated that a short treatment of human T lymphocytes with PGE2 induces specific binding activity to CRE and AP-2, but Since the okadaic acid, a potent protein phosphatase inhibitor, prolongs the induction of the binding activity, More interestingly, transfection experiments with CRE-CAT plasmide show that PGE2 activates the transcription of a CRE-containing promoter. These data support the positive role for PGE2 on some immune functions.

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BACKGROUND . Increased expression of the HER-2/neu oncogene in breast cancer correlates with decreased estrogen receptor concentration and The authors investigated METHOD . A Western blot analysis was used to investigate HER-2/neu expression, whereas a chromium-release assay using the K562 cell line as target was used to measure natural killer (NK) cell activity. RESULTS . In patients with breast cancer, NK cell activity was significantly higher compared with patients with benign tumors (P = 0.006) or healthy control subjects (P = 0.002). Moreover, 23.3 % of patients with breast cancer showed an overexpression of HER-2/neu protein. Within this group of patients, NK cell activity was significantly lower (45.6 ± 16.1%) compared with the group with NK cell activity did Thus, there was a statistically significant correlation of cytolytic effector cell function with HER-2/neu expression of the tumor (P = 0.003), and HER-2/neu overexpression correlated with a negative estrogen receptor status (P = 0.005). CONCLUSION . These data add further evidence to previous observations from the authors' laboratory that

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The leukocyte-specific, cytoskeleton-binding pp52 (LSP-1, WP-34) protein is widely expressed in multiple leukocyte lineages, including B and T lymphocytes, granulocytes, and macrophages. We previously detected a tissue-specific promoter preceding the exon encoding the N terminus of the pp52 leukocyte protein. Here we describe the functional characterization of this promoter and identification of the factors in B and T cells that regulate its activity. The pp52 promoter contains an initiator specifying the unique 5' terminus of pp52 mRNA, tandem pairs of Ets and SP1 motifs, and a lone C/EBP motif. All these motifs are essential and collectively control transcriptional activity. DNA binding studies and Ab supershift assays revealed that different combinations of factors interact with these motifs in B cells vs T cells. The Ets motifs are preferentially bound by PU-1 in B cell extracts from all stages of development, whereas a different Ets family member reacts with these motifs in T cell extracts. The C/EBP motif is bound by Ig/EBP-1 in pre-B cell and T cell extracts, but is replaced by nuclear factor-IL-6beta or a nuclear factor-IL-6beta-Ig/EBP-1 heterodimer in plasmacytoma cell extracts. Despite its reported role as a negative regulator of transcription, These findings reveal the features controlling the pp52 promoter in B and T cells and provide the foundation for determining the regulation of this promoter in other leukocyte lineages.

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The multiple biological activities of tumor necrosis factor (TNF) are mediated by two distinct cell surface receptors of 55 kd (TNFRp55) and 75 kd (TNFRp75). Using gene targeting, we generated a TNFRp55-deficient mouse strain. Cells from TNFRp55-/-mutant mice Thymocyte development and lymphocyte populations are unaltered, and However, TNF signaling is largely abolished, as judged by the failure of TNF to induce NF-kappa B in T lymphocytes from TNFRp55-deficient mice. The loss of TNFRp55 function renders mice resistant to lethal dosages of either lipopolysaccharides or S. aureus enterotoxin B. In contrast, TNFRp55-deficient mice are severely impaired to clear L. monocytogenes and readily succumb to infection. Thus, the 55 kd TNFR plays a decisive role in the host's defense against microorganisms and their pathogenic factors.

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The immunosuppressant drug cyclosporin A blocks a calcium-dependent signal from the T-cell receptor (TCR) that normally leads to T-cell activation. When bound to cyclophilin, cyclosporin A binds and inactivates the key signalling intermediate calcineurin. To identify One gene product, when overexpressed in Jurkat T cells, specifically induced transcription from the interleukin-2 enhancer, by activating the T-cell-specific transcription factors NF-AT and NF-IL2A. This protein, termed calcium-signal modulating cyclophilin ligand (CAML), acts downstream of the TCR and upstream of calcineurin by causing an influx of calcium.

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Monocyte/macrophages The effect of LPS and TNF-alpha is mediated by their ability to induce nuclear translocation of the DNA-binding heterodimer NF-kappa B (p50/p65), which binds to a specific sequence in the HIV-long terminal repeat. The present study demonstrates that triggering of complement receptors CR1 (CD35) and CR3 (CD11b/CD18) enhances viral replication in HIV-infected human monocytic cells. Monocytic cell lines and normal peripheral blood monocytes were infected with HIV-1 in vitro and cultured in the presence or Stimulation of CR1 or CR3 induces a two- to fourfold increase in the amount of cell-associated and released p24 Ag in cell cultures that was equivalent to that observed in control cultures triggered with LPS. We further observed that stimulation of CR1 or CR3 induces the nuclear translocation of NF-kappa B p50/p65 in infected cells. Translocation of NF-kappa B p50/p65 was also observed following stimulation of CR1 or CR3 of uninfected peripheral blood monocytes from HIV-seronegative donors. The amount of protein translocated was similar to that observed when cells were stimulated with rhTNF-alpha. TNF-alpha did Taken together, these observations

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Present evidence indicates a pathway of signal transmission in T cells that is outlined in figure 1. The elevation in intracellular calcium that is induced by interactions at the antigen receptor leads to the activation of the calcium-dependent phosphatase calcineurin. This in turn leads to the nuclear association of the cytosolic component of NF-ATc. The activation of calcineurin and the nuclear import of NF-ATc can both be blocked by cyclosporin A or FK506 in complex with their respective immunophilins. Once in the nucleus, NF-ATc interacts with NF-ATn to form an active transcriptional complex. NF-ATn is a ubiquitous protein, can be synthesized in response to PMA, and has many similarities to AP-1. The mechanism by which NF-ATc enters the nucleus is unknown, and although Alternative mechanisms include the Although NF-ATp copurifies with NF-ATc, there is as yet Now that these proteins are purified and cloned, the major goals will be to understand their role and the roles of other family members in thymic development.

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