The impairment of immunoglobulin transcription with a down-regulated synthesis of Oct2 and BOB.1/OBF.1 is the first established general recurrent defect found in HRS cells .


Constitutive expression of NF-kappa B is a characteristic feature of mycosis fungoides : implications for apoptosis resistance and pathogenesis .


The NF-kappa B family of transcription factors is an important regulator of genes expressed during inflammatory responses , immunoglobulin ( Ig ) class switching , cellular differentiation , and apoptosis .


Recently , members of the NF-kappaB family , including p65 ( Rel A ) , have been implicated in promoting survival of various hematopoeitic neoplasms , including T cell malignancies such as adult T cell leukemia-lymphoma .


We investigated the expression of active NF-kappa B p65 ( Rel A ) in cases of mycosis fungoides ( MF ) and the effect of chemical inhibitors of NF-kappa B on apoptosis in cutaneous T cell lymphoma ( CTCL ) cell lines .


Paraffin-embedded tissues from 23 cutaneous lesions and a single lymph node biopsy from patients diagnosed with MF were evaluated for p65 ( Rel A ) expression by using a monoclonal mouse antibody that detects the activated form of p65 ( Rel A ) .


Apoptosis after treatment with the NF-kappa B inhibitors gliotoxin , MG132 , BAY 11-7082 , and BAY 11-7085 was quantitatively measured in the CTCL cell lines HuT-78 and HH by propidium iodide ( PI ) /cell cycle analysis for detection of a hypodiploid ( sub-G ( 0 ) ) population and by determination of increased Annexin V/7-amino-actinomycin D ( 7-AAD ) expression .


Nuclear extracts from CTCL cells before and after chemical inhibition were analyzed for NF-kappa B nuclear DNA-binding activity by electrophoretic mobility shift assay ( EMSA ) with quantitative densitometry .


Nuclear expression of p65 ( Rel A ) before and after treatment with the various inhibitory compounds was measured by immunofluorescence staining in each CTCL cell line .


Neoplastic T lymphocytes from 22 of 24 cases of MF showed strong nuclear and cytoplasmic expression of active p65 ( Rel A ) .


Compared with untreated control cells , a marked increase in apoptosis , a significant decrease in NF-kappa B DNA-binding activity , and a marked decrease in nuclear p65 ( Rel A ) expression were seen in cells from both CTCL cell lines after chemical NF-kappa B inhibition .


These data show that the active form of NF-kappa B p65 ( Rel A ) is commonly expressed in neoplastic T lymphocytes in patients with MF .


In CTCL cell lines , the significant decrease in nuclear NF-kappa B expression and the marked increase in spontaneous apoptosis caused by chemical NF-kappa B inhibition suggest a critical role for NF-kappa B in the pathogenesis and tumor cell maintenance of CTCLs .


Human T-cell leukemia virus type 1 tax protein activates transcription through AP-1 site by inducing DNA binding activity in T cells .


Human T-cell leukemia virus type 1 ( HTLV-1 ) Tax protein induces the expression of various family members of the transcription factor AP-1 , such as c-Jun , JunD , c-Fos , and Fra-1 , at the level of RNA expression in T cells .


We examined the activity of Tax in transcription through AP-1-binding sites ( AP-1 site ) in T cells .


Transient transfection studies showed that Tax activated the expression of a luciferase gene regulated by two copies of an AP-1 site in the human Jurkat T-cell line .


Tax activates the expression of viral and cellular genes through two different enhancers : a cAMP-responsive ( CRE ) -like element and a kappaB element .


Two Tax mutants differentially activated expression of these two elements .


Tax703 preferentially activated the kappaB element but not the CRE-like one , whereas TaxM22 showed the reverse .


In addition , Tax703 and Tax , but not TaxM22 , converted cell growth of a mouse T-cell line from being interleukin ( IL ) -2-dependent to being IL-2-independent .


Unlike the wild-type Tax , Tax703 and TaxM22 only weakly activated the AP-1 site in the T-cell line .


Thus , Tax seems to activate the AP-1 site via mechanisms distinct from those of kappaB or CRE-like elements , and the activation of the AP-1 site is dispensable for IL-2-independent growth of CTLL-2 .


Electrophoretic mobility shift assays showed that Tax induced strong binding activity to an AP-1 site in CTLL-2 , whereas Tax703 did not , indicating that the induction of binding activity to the AP-1 site is essential for the transcriptional activation by Tax .


The binding complex induced by Tax in CTLL-2 contained JunD and Fra-2 .


Activation of transcription through the AP-1 site in Jurkat cells by JunD and/or Fra-2 was weak .


c-Jun , JunB , and c-Fos activation was greater , although the level was still less than that with Tax .


Thus , the induction of AP-1 mRNA by Tax may not be sufficient for a complete activation of AP-1 site by Tax .


Our results suggest that Tax activates the transcription of cellular genes with AP-1 sites by inducing the DNA-binding activity of AP-1 proteins in T cells , a mechanism distinct from those of CRE-like and kappaB elements .


Strict control of human immunodeficiency virus type 1 replication by a genetic switch : Tet for Tat .


Live-attenuated human immunodeficiency virus type 1 ( HIV-1 ) variants have shown great promise as AIDS vaccines , but continued replication can lead to the selection of faster-replicating variants that are pathogenic .


We therefore designed HIV-1 genomes that replicate exclusively upon addition of the nontoxic effector doxycycline ( dox ) .


This was achieved by replacement of the viral TAR-Tat system for transcriptional activation by the Escherichia coli-derived Tet system for inducible gene expression .


These designer `` HIV-rtTA '' viruses replicate in a strictly dox-dependent manner both in a T-cell line and in primary blood cells , and the rate of replication can be fine-tuned by simple variation of the dox concentration .


These HIV-rtTA viruses provide a tool to perform genetics , e.g. , selection and optimization experiments , with the E. coli-derived Tet reagents in a eukaryotic background .


Furthermore , such viruses may represent improved vaccine candidates because their replication can be turned on and off at will .


Synovial fluid induced nuclear factor-kappaB DNA binding in a monocytic cell line .


OBJECTIVE : To determine the effects of synovial fluids ( SF ) on DNA binding activity of transcription factor nuclear factor-kappaB ( NF-kappaB ) in the Mono Mac 6 monocytic/macrophage cell line as a model for the interaction between SF and synovial tissue macrophages in arthritis .


METHODS : Mono Mac 6 cells were incubated with SF from the knee joints of human subjects with rheumatoid arthritis ( RA ) , undifferentiated seronegative oligoarthritis , and osteoarthritis ( OA ) .


Nuclear extracts prepared from the Mono Mac 6 cells and RA synovial tissue were analyzed by electrophoretic mobility shift analysis ( EMSA ) for NF-kappaB DNA binding proteins .


RESULTS : Induction of NF-kappaB DNA binding by the p65 ( RelA ) /p50 heterodimer was observed in response to incubation of Mono Mac 6 cells with SF ( 20 % in culture medium ) from 5 of 8 subjects with RA , 4 of 5 with OA , and none of 3 with undifferentiated seronegative oligoarthritis .


Incubation of SF with neutralizing antibodies against tumor necrosis factor-alpha ( TNF-alpha ) , but not antibodies against interleukin 6 ( IL-6 ) , significantly reduced the induction of p65/p50 binding activity in SF from subjects with RA and OA .


Unexpectedly , a slowly migrating SF inducible NF-kappaB -binding complex was observed in EMSA of Mono Mac 6 cells after incubation with SF from 5 of 8 RA and 2 of 5 OA subjects .


The induction of this complex by SF was not affected by neutralization of TNF-alpha or IL-6 in SF , and the complex was not inducible by TNF-alpha , IL-1beta , TNF-alpha / IL-1beta , IL-6 , platelet derived growth factor , lipopolysaccharide , or tetradecanoyl phorbol acetate .


The slowly migrating complex could not be supershifted with antibodies against NF-kappaB , Jun , or the transcriptional coactivators p300 or CBP .


A NF-kappaB-binding complex with similar slow mobility was observed in nuclear extracts prepared from fresh human RA synovial tissue .


CONCLUSION : Biological activity of TNF-alpha in SF from RA and OA subjects is capable of inducing p65/p50 NF-kappaB DNA binding activity in macrophages .


A property of SF that is independent of TNF-alpha and other cytokines is responsible for the induction of a novel slowly migrating NF-kappaB-binding complex .


Soluble mediators in SF of subjects with RA and OA can therefore modulate binding of nuclear proteins to the NF-kappaB binding site in macrophages and may play a role in inflammatory gene expression in arthritis .


Peroxisome proliferator activator receptor-gamma agonists and 15-deoxy-Delta ( 12 , 14 ) ( 12 , 14 ) -PGJ ( 2 ) induce apoptosis in normal and malignant B-lineage cells .


The research described herein evaluates the expression and functional significance of peroxisome proliferator activator receptor-gamma ( PPAR-gamma ) on B-lineage cells .


Normal mouse B cells and a variety of B lymphoma cells reflective of stages of B cell differentiation ( e.g. , 70Z/3 , CH31 , WEHI-231 , CH12 , and J558 ) express PPAR-gamma mRNA and , by Western blot analysis , the 67-kDa PPAR-gamma protein .


15-Deoxy-Delta ( 12 , 14 ) -PGJ ( 2 ) ( 15d-PGJ ( 2 ) ) , a PPAR-gamma agonist , has a dose-dependent antiproliferative and cytotoxic effect on normal and malignant B cells as shown by [ ( 3 ) H ] thymidine and 3- [ 4 , 5-dimethylthiazol-2-yl ] -2 , 5-diphenyltetrazolium bromide assays .


Only PPAR-gamma agonists ( thiazolidinediones ) , and not PPAR-alpha agonists , mimicked the effect of 15d-PGJ ( 2 ) on B-lineage cells , indicating that the mechanism by which 15d-PGJ ( 2 ) negatively affects B-lineage cells involves in part PPAR-gamma .


The mechanism by which PPAR-gamma agonists induce cytotoxicity is via apoptosis , as shown by annexin V staining and as confirmed by DNA fragmentation detected using the TUNEL assay .


Interestingly , addition of PGF ( 2alpha ) , which was not known to affect lymphocytes , dramatically attenuated the deleterious effects of PPAR-gamma agonists on B lymphomas .


Surprisingly , 15d-PGJ ( 2 ) induced a massive increase in nuclear mitogen-activated protein kinase activation , and pretreatment with PGF ( 2alpha ) blunted the mitogen-activated protein kinase activation .


This is the first study evaluating PPAR-gamma expression and its significance on B lymphocytes .


PPAR-gamma agonists may serve as a counterbalance to the stimulating effects of other PGs , namely PGE ( 2 ) , which promotes B cell differentiation .


Finally , the use of PGs , such as 15d-PGJ ( 2 ) , and synthetic PPAR-gamma agonists to induce apoptosis in B-lineage cells may lead to the development of novel therapies for fatal B lymphomas


Inhibition of STAT3 signaling leads to apoptosis of leukemic large granular lymphocytes and decreased Mcl-1 expression .


Large granular lymphocyte ( LGL ) leukemia is characterized by the expansion of antigen-activated cytotoxic T lymphocytes .


These leukemic cells are resistant to Fas-mediated apoptosis despite expressing high levels of Fas .


We found that leukemic LGL from 19 patients displayed high levels of activated STAT3 .


Treatment of leukemic LGL with the JAK-selective tyrosine kinase inhibitor AG-490 induced apoptosis with a corresponding decrease in STAT -DNA binding activity .


Moreover , using an antisense oligonucleotide approach to diminish STAT3 expression , we found that Fas sensitivity was restored in leukemic LGL .


AG-490-induced apoptosis in leukemic LGL was independent of Bcl-xL or Bcl-2 expression .


However , we found that the Bcl-2-family protein Mcl-1 was significantly reduced by AG-490 treatment .


Activated STAT3 was shown to bind an SIE-related element in the murine mcl-1 promoter .


Using a luciferase reporter assay , we demonstrated that v-src overexpression in NIH3T3 induced STAT3 -dependent transcriptional activity from the mcl-1 promoter and increased endogenous Mcl-1 protein levels .


We conclude that STAT3 activation contributed to accumulation of the leukemic LGL clones .


These findings suggest that investigation should focus on novel strategies targeting STAT3 in the treatment of LGL leukemia .


Decreased immediate inflammatory gene induction in activating transcription factor-2 mutant mice .


Transcription factor activating transcription factor ( ATF ) -2 is activated by inflammatory signals transduced by the JNK and p38 MAP kinase pathways .


To better define the role of ATF-2 in inflammation , adult mice expressing small amounts of a mutant ATF-2 protein were challenged with lipopolysaccharide ( LPS ) , anti-CD3 antibody or virus .


Within 3 h of challenge by LPS , ATF-2 mutant mice had decreased induction of the adhesion molecules E-selectin , P-selectin and VCAM-1 as well as the cytokines tumor necrosis factor-alpha , IL-1beta and IL-6 compared with control mice .


Stimulation of T lymphocytes by anti-CD3 antibody also showed less induction of IL-1 and IL-6 in ATF-2 mutant tissues .


ATF-2 mutant thymocytes treated with anti-CD3 antibody in vitro demonstrated reduced induction of c-Jun , JunB , JunD and Fra-2 .


However , similar to what was observed after p38 kinase inhibition in normal mice , relative ATF-2 deficiency did not prevent the development of a mononuclear cell infiltrate in the week following an inflammatory stimulus .


ATF-2 mutant mice proved more susceptible to death than control mice from LPS plus D-galactosamine injection or Coxsackievirus B3 infection and had a higher incidence of mononuclear pulmonary infiltrates after exposure to Herpes simplex virus-1 .


ATF-2 is essential for maximal immediate induction of adhesion molecules and cytokine genes , but at later time points may even protect against overactive immune responses .


LIGHT , a TNF-like molecule , costimulates T cell proliferation and is required for dendritic cell -mediated allogeneic T cell response .


LIGHT is a recently identified member of the TNF superfamily and its receptors , herpesvirus entry mediator and lymphotoxin beta receptor , are found in T cells and stromal cells .


In this study , we demonstrate that LIGHT is selectively expressed on immature dendritic cells ( DCs ) generated from human PBMCs .


In contrast , LIGHT is not detectable in DCs either freshly isolated from PBMCs or rendered mature in vitro by LPS treatment .


Blockade of LIGHT by its soluble receptors , lymphotoxin beta receptor-Ig or HVEM-Ig , inhibits the induction of DC -mediated primary allogeneic T cell response .


Furthermore , engagement of LIGHT costimulates human T cell proliferation , amplifies the NF-kappaB signaling pathway , and preferentially induces the production of IFN-gamma , but not IL-4 , in the presence of an antigenic signal .


Our results suggest that LIGHT is a costimulatory molecule involved in DC -mediated cellular immune responses .


Suppression of HIV type 1 replication by a dominant-negative Ets-1 mutant .


Activity of the distal region of the human immunodeficiency virus ( HIV-1 ) long terminal repeat ( LTR ) , which contains binding sites for the Ets-1 and USF-1 proteins , is integral for HIV-1 replication .


The Ets-1 and USF-1 proteins play a critical role in the activity of the HIV-1 LTR distal enhancer region , as indicated by the potent dominant negative effect of a mutant Ets-1 lacking trans-activation domains on the transcriptional activity of the LTR .


To determine the biological relevance of the Ets-1 and USF-1 proteins in HIV-1 replication , we examined the effect of expression of the dominant-negative mutant of Ets-1 ( dnEts-1 ) on HIV-1 infection of T cells .


We demonstrated that expression of dnEts markedly suppressed HIV-1 infection of a T cell line .


This finding indicates that formation of a transcriptionaly active USF-1/Ets-1 complex is important in the productive infection of cells by HIV-1 , and suggests that inhibition of the interaction between USF-1 and Ets-1 with the HIV-1 LTR may provide a new target for anti-HIV-1 gene therapy .


Human eosinophils constitutively express nuclear factor of activated T cells p and c .


BACKGROUND : Eosinophils are now known to produce a variety of proinflammatory cytokines , although the molecular factors that regulate their production are poorly understood .


The expression of almost all of the cytokines produced by eosinophils , including the proallergic cytokine IL-4 , is now known to be regulated at the level of transcription by members of the nuclear factor of activated T cells ( NFAT ) family of transcription factors .


OBJECTIVE : We sought to characterize the expression of different NFAT proteins in resting and activated eosinophils .


METHODS : Nuclear and whole cell extracts were obtained from both peripheral blood eosinophils and those obtained from bronchoalveolar lavage fluid of asthmatic subjects after endobronchial allergen challenge .


NFAT expression was determined by using immunoprecipitation and Western blot analysis , DNA-binding assays , and RT-PCR analysis of eosinophil mRNA .


RESULTS : Both peripheral blood and bronchoalveolar lavage fluid eosinophils expressed NFATp and NFATc protein .


Unlike activated T cells , which express multiple NFATc isoforms , eosinophils preferentially express the approximately 85-kd isoform .


In addition , eosinophils were found to constitutively express NFATc mRNA .


A brief incubation with the T ( H ) 2 cytokines IL-4 and IL-5 was sufficient to induce the nuclear translocation of NFATc .


Eosinophil nuclear extracts contain multiple factors that can specifically recognize the IL-4 promoter P1 NFAT site in DNA-binding assays , including NFATp .


CONCLUSION : NFATp and NFATc can regulate the expression of cytokines and other genes in eosinophils but appear to be regulated by a novel signal transduction mechanism in these cells .


BLyS BINDS TO B CELLS WITH HIGH AFFINITY AND INDUCES ACTIVATION OF THE TRANSCRIPTION FACTORS NF-kappaB AND ELF-1 .


B lymphocyte stimulator ( BLyS ) is a novel member of the TNF family of proteins expressed by myeloid cells as membrane-bound and soluble forms .


BLyS was shown to act specifically on B cells , inducing proliferation and immunoglobulin production both in vitro and in vivo .


The present study was undertaken to characterize binding of radiolabeled BLyS to its cognate receptor on human B lymphocytes and examine intracellular events initiated by BLyS binding .


Similar to other TNF family members , BLyS is present in solution as a homotrimer as determined by gel filtration chromatography and light scattering analysis .


BLyS binding to B cells is specific as other TNF family members tested did not compete for ( 125 ) I- BLyS binding .


Analysis of equilibrium binding of ( 125 ) I-labeled BLyS to purified human tonsillar B cells demonstrated saturable binding .


Scatchard analysis of the binding data revealed a single class of high-affinity binding on human B cells with approximately 2600 binding sites per cell and an apparent dissociation constant ( K ( D ) ) of about 0.1 nM .


In addition we report that BLyS binding to B cells results in the activation of NF-kappaB and the Ets family transcription factor , ELF-1 , and in the induction of mRNA for Polo-like kinase ( PLK ) .


Design and use of an inducibly activated human immunodeficiency virus type 1 Nef to study immune modulation .


The Nef protein of the human immunodeficiency virus type 1 ( HIV-1 ) has been shown to enhance the infectivity of virus particles , downmodulate cell surface proteins , and associate with many intracellular proteins that are thought to facilitate HIV infection .


One of the challenges in defining the molecular events regulated by Nef has been obtaining good expression of Nef protein in T cells .


This has been attributed to effects of Nef on cell proliferation and apoptosis .


We have designed a Nef protein that is readily expressed in T-cell lines and whose function is inducibly activated .


It is composed of a fusion between full-length Nef and the estrogen receptor hormone-binding domain ( Nef-ER ) .


The Nef-ER is kept in an inactive state due to steric hindrance , and addition of the membrane-permeable drug 4-hydroxytamoxifen ( 4-HT ) , which binds to the ER domain , leads to inducible activation of Nef-ER within cells .


We demonstrate that Nef-ER inducibly associates with the 62-kDa Ser/Thr kinase and is localized to specific membrane microdomains ( lipid rafts ) only after activation .


Using this inducible Nef , we also compared the specific requirements for CD4 and HLA-A2 downmodulation in a SupT1 T-cell line .


Half-maximal downmodulation of cell surface CD4 required very little active Nef-ER and occurred as early as 4 h after addition of 4-HT .


In contrast , 50 % downmodulation of HLA-A2 by Nef required 16 to 24 h and about 50- to 100-fold-greater concentrations of 4-HT .


These data suggest that HLA-A2 downmodulation may require certain threshold levels of active Nef .


The differential timing of CD4 and HLA-A2 downmodulation may have implications for HIV pathogenesis and immune evasion .


Regulation of chemokine mRNA expression in a rat model of vanadium-induced pulmonary inflammation .


Environmental and occupational exposure to vanadium dusts results in toxic effects mainly confined to the respiratory system .


Using a rat model of acute lung inflammation induced by intratracheal instillation of sodium metavanadate ( NaVO3 ) at the dose of 200 microg V/kg , we investigated the relationship between the cytologic characterization of pulmonary inflammation and the expression of chemokine mRNA .


Significant polymorphonuclear leukocyte ( PMN ) influx ( P < 0.01 ) into the lung was noted 4 h after NaVO3 instillation , whereas alveolar macrophages ( AMs ) in bronchoalveolar lavage ( BAL ) cells appeared to decrease significantly .


In contrast , neither PMNs nor AMs changed substantially 1 h after NaVO3 instillation .


By Northern analysis , macrophage inflammatory protein ( MIP ) -2 mRNA in BAL cells increased markedly 1 h after NaVO3 instillation and reduced a little bit at 4 h , whereas MIP-1alpha mRNA in BAL cells was expressed relatively high 1 h after NaVO3 instillation , although a basal expression was detected in control group , and returned rapidly nearly to control level at 4 h .


Since MIP-2 is a potent PMN chemoattractant and MIP-1alpha is a potent macrophage/monocyte chemoattractant has been well known .


The facts that PMN influx was preceded by increased MIP-2 mRNA expression , suggesting that MIP-2 is involved in the development of NaVO3-induced pulmonary inflammation , whereas increased MIP-1alpha mRNA expression was followed by decreased AMs in BAL cells , suggesting AMs might be activated by MIP-1alpha , adherent to the lining surface of the airways and then resistant to be washed out .


To delineate the mechanisms of transcriptional activation , we recently cloned the 5'-flanking region of the MIP-2 gene .


The promotor region contains consensus binding sites for transcription factor nuclear factor kappaB ( NF-kappaB ) and activator protein-1 ( AP-1 ) .


Using electrophoretic mobility shift assay , increased nuclear NF-kappaB , not AP-1 , binding activity was detected 1 h after NaVO3 instillation , which correlated with the induction of MIP-2 mRNA .


p65 ( Rel A ) and p50 protein appears to be involved in MIP-2 NF-kappaB binding .


Taken together , our studies suggest that MIP-2 is an important mediator of NaVO3-induced pulmonary inflammation in the rat model .


In addition , elevated MIP-2 mRNA levels are accompanied by increased NF-kappaB binding activity in BAL cells , suggesting possible MIP-2 transcriptional regulation through NF-kappaB .


Activation of signal transduction and apoptosis in healthy lymphomonocytes exposed to bystander HIV-1-infected cells .


Persistent activation of the immune system is one of the hallmarks of HIV-1 infection .


In this study we analysed the induction of factors involved in cytokine signal transduction , such as STAT 1 proteins and IRF-1 mRNA , in normal peripheral blood mononuclear cells ( PBMC ) exposed to HIV-infected cells , and the induction of apoptosis .


Western blot analyses and reverse transcriptase-polymerase chain reaction results indicate that both cells infected with a X4 strain and cells infected with a R5 strain are able to increase intracellular levels of STAT 1alpha and beta proteins as well as IRF-1 mRNA .


This effect was prevented by neutralizing antibodies against interferon-alpha ( IFN-alpha ) .


HIV-1-infected cells dose-dependently induced apoptotic commitment in normal PBMC , as revealed by DNA fragmentation analysis , but this was not accompanied by an increase of caspase-3 activity , even if a slight up-regulation of IL-1beta-converting enzyme mRNA was detected .


Apoptosis induction could be abrogated mainly by antibodies against tumour necrosis factor-alpha ( TNF-alpha ) and , to a lesser extent , by antibodies against IFN-gamma .


All these findings suggest that uninfected PBMC can undergo activation of signal transduction and apoptosis after exposure to bystander HIV-infected cells , subsequent to the induction of cytokines such as IFNs and TNF-alpha .


The physical association of protein kinase C theta with a lipid raft-associated inhibitor of kappa B factor kinase ( IKK ) complex plays a role in the activation of the NF-kappa B cascade by TCR and CD28 .


We investigated the role of protein kinase C theta ( PKCtheta ) in the activation of the NF-kappaB cascade in primary human CD4 ( + ) lymphocytes .


Among six or so PKC isoforms expressed in T cells , only PKCtheta participates in the assembly of the supramolecular activation clusters at the contact site of the TCR with Ag .


Signaling via both the TCR and CD28 is required for optimal activation of the multisubunit IkappaB kinase ( IKK ) complex in primary human T lymphocytes ; this activation could be inhibited by a Ca ( 2+ ) -independent PKC isoform inhibitor , rottlerin .


Moreover , endogenous PKCtheta physically associates with activated IKK complexes in CD3/CD28-costimulated primary CD4 ( + ) T cells .


The same set of stimuli also induced relocation of endogenous PKCtheta and IKKs to a GM1 ganglioside-enriched , detergent-insoluble membrane compartment in primary T cells .


IKKs recruited to these lipid rafts were capable of phosphorylating a recombinant IkappaBalpha sustrate .


Confocal microscopy further demonstrated that exogenously expressed PKCtheta and IKKss colocalize in the membrane of CD3/CD28-costimulated Jurkat T cells .


Constitutively active but not kinase-inactive PKCtheta activated IKKbeta in Jurkat T cells .


Expression of dominant-active PKCtheta also had stimulatory effects on the CD28 response element of the IL-2 promoter .


Taken together , these data show that the activation of PKCtheta by the TCR and CD28 plays an important role in the assembly and activation of IKK complexes in the T cell membrane


T-cell -mediated regulation of osteoclastogenesis by signalling cross-talk between RANKL and IFN-gamma .


Bone resorption is regulated by the immune system , where T-cell expression of RANKL ( receptor activator of nuclear factor ( NF ) -kappaB ligand ) , a member of the tumour-necrosis factor family that is essential for osteoclastogenesis , may contribute to pathological conditions , such as autoimmune arthritis .


However , whether activated T cells maintain bone homeostasis by counterbalancing the action of RANKL remains unknown .


Here we show that T-cell production of interferon ( IFN ) -gamma strongly suppresses osteoclastogenesis by interfering with the RANKL -RANK signalling pathway .


IFN-gamma induces rapid degradation of the RANK adapter protein , TRAF6 ( tumour necrosis factor receptor-associated factor 6 ) , which results in strong inhibition of the RANKL -induced activation of the transcription factor NF-kappaB and JNK .


This inhibition of osteoclastogenesis is rescued by overexpressing TRAF6 in precursor cells , which indicates that TRAF6 is the target critical for the IFN-gamma action .


Furthermore , we provide evidence that the accelerated degradation of TRAF6 requires both its ubiquitination , which is initiated by RANKL , and IFN-gamma -induced activation of the ubiquitin-proteasome system .


Our study shows that there is cross-talk between the tumour necrosis factor and IFN families of cytokines , through which IFN-gamma provides a negative link between T-cell activation and bone resorption .


Our results may offer a therapeutic approach to treat the inflammation-induced tissue breakdown .


Stromal-derived factor 1 and thrombopoietin regulate distinct aspects of human megakaryopoiesis .


The role of the chemokine binding stromal-derived factor 1 ( SDF-1 ) in normal human megakaryopoiesis at the cellular and molecular levels and its comparison with that of thrombopoietin ( TPO ) have not been determined .


In this study it was found that SDF-1 , unlike TPO , does not stimulate alpha ( IIb ) beta ( 3 ) ( + ) cell proliferation or differentiation or have an antiapoptotic effect .


However , it does induce chemotaxis , trans-Matrigel migration , and secretion of matrix metalloproteinase 9 ( MMP-9 ) and vascular endothelial growth factor ( VEGF ) by these cells , and both SDF-1 and TPO increase the adhesion of alpha ( IIb ) beta ( 3 ) ( + ) cells to fibrinogen and vitronectin .


Investigating the intracellular signaling pathways induced by SDF-1 and TPO revealed some overlapping patterns of protein phosphorylation/activation ( mitogen-activated protein kinase [ MAPK ] p42/44 , MAPK p38 , and AKT [ protein kinase B ] ) and some that were distinct for TPO ( eg , JAK-STAT ) and for SDF-1 ( eg , NF-kappa B ) .


It was also found that though inhibition of phosphatidyl-inositol 3-kinase ( PI-3K ) by LY294002 in alpha ( IIb ) beta ( 3 ) ( + ) cells induced apoptosis and inhibited chemotaxis adhesion and the secretion of MMP-9 and VEGF , the inhibition of MAPK p42/44 ( by the MEK inhibitor U0126 ) had no effect on the survival , proliferation , and migration of these cells .


Hence , it is suggested that the proliferative effect of TPO is more related to activation of the JAK-STAT pathway ( unique to TPO ) , and the PI-3K-AKT axis is differentially involved in TPO- and SDF-1-dependent signaling .


Accordingly , PI-3K is involved in TPO -mediated inhibition of apoptosis , TPO- and SDF-1-regulated adhesion to fibrinogen and vitronectin , and SDF-1 -mediated migration .


This study expands the understanding of the role of SDF-1 and TPO in normal human megakaryopoiesis and indicates the molecular basis of the observed differences in cellular responses .


Adhesion of immature and mature T cells induces in human thymic epithelial cells ( TEC ) activation of IL-6 gene trascription factors ( NF-kappaB and NF-IL6 ) and IL-6 gene expression : role of alpha3beta1 and alpha6beta4 integrins .


T cell precursors homed to thymus develop in close contact with stromal cells .


Among them , thymic epithelial cells ( TEC ) are known to exert dominant roles in their survival and functional shaping .


Key molecules mediating TEC / thymocytes interactions include cytokines and growth factors secreted by the two cell types and adhesion receptors mediating cell contact .


Signaling events triggered in thymocytes by adhesion to epithelial cells have been extensively investigated , whereas little is known on the opposite phenomenon .


We have previously investigated this issue in a co-culture system composed of TEC cultures derived from human normal thymus and heterologous thymocytes .


We demonstrated that thymocytes adhere to TEC involving beta1 and beta4 integrins and induce the clustering of alpha3beta1 and alpha6beta4 heterodimers at the TEC surface .


In addition thymocyte adhesion was followed by activation of NF-kappaB and NF-IL6 gene transcription factors and enhanced IL-6 production .


The two latter phenomena were reproduced by the cross-linking of the alpha3 , alpha6 , beta1 and beta4 integrins , thus implying that the alpha3beta1 and alpha6beta4 heterodimers can signal during thymocyte adhesion .


We have extended our previous work investigating in the same experimental setting the inducing activity of non stimulated or activated policlonal or clonal mature T cells as representative of the more mature thymocyte subset .


We found that adhesion of unstimulated T cell i ) involved beta1 , but not beta4 integrin functions at the surface ii ) induced the clustering of alpha3beta1 , but not alpha2beta1 heterodimers at the TEC surface and iii ) up-regulated the nuclear binding activity of NF-kappaB transcription factor and the IL-6 secretion .


We propose that alpha3beta1 and alpha6beta4 heterodimers are induced to cluster at the TEC surface recognizing yet unknown cellular ligands differentially expressed during T cell development .


Identification and characterization of SKAT-2 , a novel Th2-specific zinc finger gene .


We have identified a novel Kruppel-type zinc finger ( ZF ) gene , SKAT-2 , which is selectively expressed by murine Th2 cells .


The protein encoded by this gene has 14 C2H2-type ZF tandemly arrayed at its C terminus and N-terminal SCAN box and KRAB domains .


SKAT-2 is tissue restricted in expression at the RNA level , detectable only in brain and at low levels in kidney and spleen and few hematopoietic cell lines .


By in situ hybridization , SKAT-2 expression was found to peak in antigen-stimulated CD4 ( + ) T cells after 2-3 days of culture under Th2 but not Th1 biasing conditions .


This pattern of expression closely mirrored that of GATA-3 in the same cells .


In transient transfection experiments in phorbol 12-myristate 13-acetate/ionomycin-stimulated EL4 cells , SKAT-2 was found to up-regulate the activity of the IL-4 but not the IL-5 promoter , contrasting with the ability of GATA-3 to activate both promoters .


This result was confirmed using clones of EL4 cells stably expressing an inducible form of SKAT-2 , thus SKAT-2 is a novel Th2-specific gene that may play a role in selective regulation of cytokine genes in T cells .


hsp70 interacting protein Hip does not affect glucocorticoid receptor folding by the hsp90-based chaperone machinery except to oppose the effect of BAG-1 .


Reticulocyte lysate contains a chaperone system that assembles glucocorticoid receptor ( GR ) .hsp90 heterocomplexes .


Using purified proteins , we have prepared a five-protein heterocomplex assembly system consisting of two proteins essential for heterocomplex assembly- hsp90 and hsp70 -and three proteins that act as co-chaperones to enhance assembly- Hop , hsp40 , p23 [ Morishima , Y. , Kanelakis , K. C. , Silverstein , A. M. , Dittmar , K. D. , Estrada , L. , and Pratt , W. B. ( 2000 ) J. Biol. Chem. 275 , 6894-6900 ] .


The hsp70 co-chaperone Hip has been recovered in receptor.hsp90 heterocomplexes at an intermediate stage of assembly in reticulocyte lysate , and Hip is also thought to be an intrinsic component of the assembly machinery .


Here we show that immunodepletion of Hip from reticulocyte lysate or addition of high levels of Hip to the purified five-protein system does not affect GR.hsp90 heterocomplex assembly or the activation of steroid binding activity that occurs with assembly .


Despite the fact that Hip does not affect assembly , it is recovered in GR.hsp90 heterocomplexes assembled by both systems .


In the five-protein system , Hip prevents inhibition of assembly by the hsp70 co-chaperone BAG-1 , and cotransfection of Hip with BAG-1 opposes BAG-1 reduction of steroid binding activity in COS cells .


We conclude that Hip is not a component of the assembly machinery but that it could play a regulatory role in opposition to BAG-1 .


Cutting edge : STAT6-deficient mice have enhanced tumor immunity to primary and metastatic mammary carcinoma .


STAT4 and STAT6 are essential for the development of CD4 ( + ) Th1 and Th2 development , respectively .


Tumor immunologists have hypothesized that Th1 cells are critical in tumor immunity because they facilitate differentiation of CD8 ( + ) T cells , which are potent anti-tumor effectors .


We have used STAT4 ( -/- ) and STAT6 ( -/- ) mice to test this hypothesis .


BALB/c and knockout mice were challenged in the mammary gland with the highly malignant and spontaneously metastatic BALB/c-derived 4T1 mammary carcinoma .


Primary tumor growth and metastatic disease are reduced in STAT6 ( -/- ) mice relative to BALB/c and STAT4 ( -/- ) mice .


Ab depletions demonstrate that the effect is mediated by CD8 ( + ) T cells , and immunized STAT6 ( -/- ) mice have higher levels of 4T1-specific CTL than BALB/c or STAT4 ( -/- ) mice .


Surprisingly , Th1 or Th2 cells are not involved , because CD4 depletion does not diminish the anti-tumor effect .


Therefore , deletion of the STAT6 gene facilitates development of potent anti-tumor immunity via a CD4 ( + ) -independent pathway .


Activation of oncogenic transcription factor AP-1 in T cells infected with human T cell leukemia virus type 1 .


Human T cell leukemia virus type 1 ( HTLV-1 ) Tax protein transforms primary human T cells in vitro .


We previously showed that Tax induces the expression of various family members of the transcription factor AP-1 such as c-Jun , JunD , c-Fos , and Fra-1 at the mRNA level in T cells .


In this study , we have examined the ability of Tax to activate transcription through the AP-1-binding site ( AP-1 site ) .


A transient transfection study showed that Tax can activate transcription through the AP-1-binding site in a human T cell line , whereas any combination of AP-1 proteins did so much less than Tax , indicating that the activation of the AP-1 site by Tax may require a mechanism other than the induction of AP-1 mRNA .


Fresh peripheral blood leukemia cells of all surveyed ATL patients displayed constitutive AP-1 DNA-binding activity , whereas no normal individuals did .


However , the HTLV-1 genes , including tax , are not significantly expressed in fresh leukemia cells from ATL patients .


Our present results suggest that activation of AP-1 occurs through Tax-dependent and -independent mechanisms in HTLV-1-infected T cells , which may play some roles in dysregulated phenotypes of HTLV-1-infected cells .


2 , 2 ' , 4 , 6 , 6'-pentachlorobiphenyl induces apoptosis in human monocytic cells .


Polychlorinatedbiphenyls ( PCBs ) are a group of persistent and widely dispersed environmental pollutants , some of which may be immunotoxic .


In the present study , we investigated the effect of PCBs on immune system by assessing apoptotic cell death in human monocytic U937 cells .


Among the various congeners tested , 2 , 2 ' , 4 , 6 , 6'-pentachlorobiphenyl ( PeCB ) , a highly ortho-substituted congener , specifically induced DNA fragmentation , a hallmark of apoptosis , while the other examined di- , tri- , tetra- , and pentachlorobiphenyls did not .


To further study the 2 , 2 ' , 4 , 6 , 6'-PeCB-induced cell death , various features of apoptosis were examined .


2 , 2 ' , 4 , 6 , 6'-PeCB caused a decrease in cell viability and induced cellular morphologic features characteristic of apoptosis such as chromatin aggregation and apoptotic bodies .


In addition , caspase-3 , an executioner of apoptosis , was activated and its substrate , poly ( ADP-ribose ) polymerase ( PARP ) , was cleaved during 2 , 2 ' , 4 , 6 , 6'-PeCB-induced apoptosis .


In contrast , 3 , 3 ' , 4 , 4 ' , 5-PeCB , a congener of coplanar structure , as well as 2 , 3 , 7 , 8-TCDD did not induce apoptosis in these human monocytic cells , although they potently induced CYP 1A1 in human hepatoma Hep G2 cells .


Taken together , the data indicate that 2 , 2 ' , 4 , 6 , 6'-PeCB induces apoptosis in human monocytic cells through a mechanism that is independent of the arylhydrocarbon receptor .


This suggests a possibly separate mechanism by which PCBs cause immunosuppression .


Effects of deregulated Raf activation on integrin , cytokine-receptor expression and the induction of apoptosis in hematopoietic cells .


The effects of deregulated Raf activation on the growth and differentiation of hematopoietic cells were investigated .


The cytokine-dependent murine myeloid FDC-P1 and human erythroleukemic TF-1 cell lines were transformed to grow in response to deregulated Raf expression in the absence of exogenous cytokines .


The conditionally active Raf proteins were regulated by beta-estradiol as cDNAs containing the Raf catalytic , but lacking negative-regulatory domains , were ligated to the hormone binding domain of the estrogen receptor ( deltaRaf : ER ) .


Continuous deltaRaf expression prevented apoptosis in the absence of exogenous cytokines and altered the morphology of the FD/deltaRaf : ER cells as they grew in large aggregated masses ( > 100 cells ) whereas the parental cytokine-dependent FDC-P1 cells grew in smaller grape-like clusters ( < 10 cells ) .


FD/deltaRaf-1 : ER cells growing in response to Raf activation displayed decreased levels of the Mac-2 and Mac-3 molecules on their cell surface .


In contrast , when these cells were cultured in IL-3 , higher levels of these adhesion molecules were detected .


Expression of activated Raf oncoproteins also abrogated cytokine dependency and prevented apoptosis of TF-1 cells .


Moreover , the differentiation status of these Raf-responsive cells was more immature upon Raf activation as culture with the differentiation-inducing agent phorbol 12 myristate 13-acetate ( PMA ) and beta-estradiol resulted in decreased levels of the CD11b and CD18 integrin molecules on the cell surface .


In contrast when the Raf-responsive cells were induced to differentiate with PMA and GM-CSF , in the absence of deltaRaf : ER activation , increased levels of the CD11b and CD18 molecules were detected .


Retinoic acid ( RA ) inhibited 3H-thymidine incorporation in response to GM-CSF .


Interestingly , Raf activation counterbalanced the inhibition of DNA synthesis caused by RA but not PMA .


Thus deregulated Raf expression can alter cytokine dependency , integrin expression and the stage of differentiation .


These Raf-responsive cell lines will be useful in elucidating the roles of the MAP kinase cascade on hematopoietic cell differentiation and malignant transformation


Cyclic AMP activates p38 mitogen-activated protein kinase in Th2 cells : phosphorylation of GATA-3 and stimulation of Th2 cytokine gene expression .


cAMP is an important second messenger with immunomodulatory properties .


Elevation of intracellular cAMP in T cells , induced by agents such as IL-1alpha or PGs , inhibits T cell activation .


In effector T cells , an increase in the level of intracellular cAMP inhibits cytokine production in Th1 cells but stimulates cytokine production in Th2 cells .


Here we report that cAMP-induced effects in Th2 cells occur independently of the protein kinase A pathway , which is the major mediator of cAMP-induced signaling events in most cell types .


Instead , cAMP stimulates activation of p38 mitogen-activated protein kinase in Th2 cells .


This appears to be a Th2 -selective event because cAMP barely increased p38 phosphorylation in Th1 cells .


We show that in Th2 cells , cAMP promotes the production of both IL-5 and IL-13 , which play distinct but critical roles in asthma pathogenesis .


Our data also show that cAMP causes increased phosphorylation of the transcription factor GATA-3 , which we have shown is a critical regulator of Th2 cytokine gene expression and , in turn , of airway inflammation in mice .


Thus , Th2 -specific GATA-3 expression and p38 mitogen-activated protein kinase activation together provide a molecular basis for the differential effects of cAMP in the two T helper cell subsets .


Characterization of IL-4 and IL-13 signals dependent on the human IL-13 receptor alpha chain 1 : redundancy of requirement of tyrosine residue for STAT3 activation .


IL-4 and IL-13 are pleiotropic cytokines whose biological activities overlap with each other .


IL-13 receptor alpha chain 1 ( IL-13R alpha 1 ) is necessary for binding to IL-13 , and the heterodimer composed of IL-13R alpha 1 and IL-4R alpha chain transduces IL-13 and IL-4 signals ; however , the functional mapping of the intracellular domain of IL-13R alpha 1 is not fully understood .


In this study , we constructed wild and mutated types of human IL-13R alpha 1 , and analyzed IL-4 and IL-13 signals using an IL-13R alpha 1 -transfected human B cell line .


Expression of IL-13R alpha 1 evoked STAT3 activation by IL-4 and IL-13 , and in stimulated human B cells , on which IL-13R alpha 1 was highly expressed , IL-4 and IL-13 induced STAT3 activation .


Replacement of the two tyrosine residues completely abolished STAT3 activation , although replacing either tyrosine residue alone retained it .


Furthermore , we found that the Box1 region and the C-terminal tail of IL-13R alpha 1 were critical for binding to Tyk2 , and activation of Jak1 , Tyk2 , the insulin receptor substrate-1 and STAT6 respectively .


These results suggest that STAT3 activation is involved with IL-4 and IL-13 signals in human B cells along with the activation of STAT6 , and that there is a unique sequence in IL-13R alpha 1 to activate STAT3 .


Functional uncoupling of the Janus kinase 3-Stat5 pathway in malignant growth of human T cell leukemia virus type 1-transformed human T cells .


Human T cell leukemia virus type 1 ( HTLV-1 ) transforms cytokine -dependent T lymphocytes and causes adult T cell leukemia .


Janus tyrosine kinase ( Jak ) 3 and transcription factors Stat5a and Stat5b are essential for the proliferation of normal T cells and are constitutively hyperactivated in both HTLV-1-transformed human T cell lines and lymphocytes isolated from HTLV-1-infected patients ; therefore , a critical role for the Jak3-Stat5 pathway in the progression of this disease has been postulated .


We recently reported that tyrphostin AG-490 selectively blocked IL-2 activation of Jak3/Stat5 and growth of murine T cell lines .


Here we demonstrate that disruption of Jak3/Stat5a/b signaling with AG-490 ( 50 & mgr ; M ) blocked the proliferation of primary human T lymphocytes , but paradoxically failed to inhibit the proliferation of HTLV-1-transformed human T cell lines , HuT-102 and MT-2 .


Structural homologues of AG-490 also inhibited the proliferation of primary human T cells , but not HTLV-1-infected cells .


Disruption of constitutive Jak3/Stat5 activation by AG-490 was demonstrated by inhibition of 1 ) tyrosine phosphorylation of Jak3 , Stat5a ( Tyr ( 694 ) ) , and Stat5b ( Tyr ( 699 ) ) ; 2 ) serine phosphorylation of Stat5a ( Ser ( 726 ) ) as determined by a novel phosphospecific Ab ; and 3 ) Stat5a/b DNA binding to the Stat5-responsive beta-casein promoter .


In contrast , AG-490 had no effect on DNA binding by p50/p65 components of NF-kappaB , a transcription factor activated by the HTLV-1-encoded phosphoprotein , Tax .


Collectively , these data suggest that the Jak3-Stat5 pathway in HTLV-1-transformed T cells has become functionally redundant for proliferation .


Reversal of this functional uncoupling may be required before Jak3/Stat5 inhibitors will be useful in the treatment of this malignancy .


The Epstein-Barr virus promoter initiating B-cell transformation is activated by RFX proteins and the B-cell-specific activator protein BSAP/Pax5 .


Epstein-Barr virus ( EBV ) -induced B-cell growth transformation , a central feature of the virus ' strategy for colonizing the human B-cell system , requires full virus latent gene expression and is initiated by transcription from the viral promoter Wp .


Interestingly , when EBV accesses other cell types , this growth-transforming program is not activated .


The present work focuses on a region of Wp which in reporter assays confers B-cell -specific activity .


Bandshift studies indicate that this region contains three factor binding sites , termed sites B , C , and D , in addition to a previously characterized CREB site .


Here we show that site C binds members of the ubiquitously expressed RFX family of proteins , notably RFX1 , RFX3 , and the associated factor MIBP1 , whereas sites B and D both bind the B-cell-specific activator protein BSAP/Pax5 .


In reporter assays with mutant Wp constructs , the loss of factor binding to any one of these sites severely impaired promoter activity in B cells , while the wild-type promoter could be activated in non-B cells by ectopic BSAP expression .


We suggest that Wp regulation by BSAP helps to ensure the B-cell specificity of EBV 's growth-transforming function .


Activation of the Lck tyrosine protein kinase by the Herpesvirus saimiri tip protein involves two binding interactions .


The Tip protein of Herpesvirus saimiri strain 484C binds to and activates the Lck tyrosine protein kinase .


Two sequences in the Tip protein were previously shown to be involved in binding to Lck .


A proline-rich region , residues 132-141 , binds to the SH3 domain of the Lck protein .


We show here that the other Lck-binding domain , residues 104-113 , binds to the carboxyl-terminal half of Lck and that this binding does not require the Lck SH3 domain .


Mutated Tip containing only one functional Lck-binding domain can bind stably to Lck , although not as strongly as wild-type Tip .


Interaction of Tip with Lck through either Lck-binding domain increases the activity of Lck in vivo .


Simultaneous binding of both domains is required for maximal activation of Lck .


The transient expression of Tip in T cells was found to stimulate both Stat3 -dependent and NF-AT -dependent transcription .


Mutant forms of Tip lacking one or the other of the two Lck-binding domains retained the ability to stimulate Stat3 -dependent transcription .


Tip lacking the proline-rich Lck-binding domain exhibited almost wild-type activity in this assay .


In contrast , ablation of either Lck-binding domain abolished the ability of Tip to stimulate NF-AT -dependent transcription .


Full biological activity of Tip , therefore , appears to require both Lck-binding domains .


Accumulation of RXR alpha during activation of cycling human T lymphocytes : modulation of RXRE transactivation function by mitogen-activated protein kinase pathways .


We have previously reported that the activation of resting human immature peripheral blood T ( PBT ) lymphocytes is associated with the loss of retinoid X receptor alpha ( RXRalpha ) expression .


In the present study , we have demonstrated that , unlike resting cells , activation of cycling human mature PBT lymphocytes , and T lymphocyte leukemia cell lines is accompanied by the accumulation of RXRalpha mRNA and protein .


Interestingly , cyclosporin A further augmented RXRalpha expression , indicating the involvement of calcineurin pathways in the process .


9-cis retinoic acid inhibited the accumulation , suggesting that retinoids can regulate the synthesis of their own receptors during T cell activation .


Transfection analysis in Jurkat cells , using RXRE-dependent reporter assays , showed that RXRalpha accumulated during T cell activation was transcriptionally inactive .


To investigate the mechanism of such inhibition , the role of two mitogen-activated protein kinase pathways , c-Jun N-terminal kinase ( JNK ) and extracellular signal-regulated kinase ( ERK ) , in modulating RXRE-dependent transcription , was explored .


The expression of constitutively active MAP/ERK kinase kinase 1 ( MEKK1 ) inhibited RXRE-dependent transcription , whereas dominant negative MEKK1 increased the transcription , indicating the involvement of JNK signaling pathways in the process .


In contrast , expression of constitutively active MEK1 , which activates ERK pathway , enhanced RXRE-dependent activation .


When both were activated simultaneously , JNK pathway was dominant over ERK pathway and resulted in inhibition of RXRE-mediated transcription .


These data demonstrate a dual regulatory control of RXRalpha expression during the activation of resting and cycling T lymphocytes and indicate a dynamic balance between JNK and ERK pathways in modulating RXRE-mediated transactivation .


The proteasome regulates receptor-mediated endocytosis of interleukin-2 .


Recent studies have increasingly implicated the proteasome in the regulation of cell surface receptors .


In the present study , we investigated the role of the proteasome for ligand-dependent endocytosis and degradation of the interleukin-2 ( IL-2 ) -interleukin-2 receptor ( IL-2R ) complex .


Proteasome inhibitors impaired internalization of IL-2.IL-2R and prevented the lysosomal degradation of this cytokine .


Based on time-course studies , proteasome activity is primarily required after initial endocytosis of the IL-2.IL-2R .


Proteasome function was also necessary for the lysosomal degradation of IL-2 internalized by IL-2R that were comprised of cytoplasmic tailless beta- or gamma c-subunits , suggesting that the target protein for the proteasome is independent of either the cytoplasmic tail of the IL-2R beta- or gamma c-subunits and their associated signaling components .


Therefore , a functional proteasome is required for optimal endocytosis of the IL-2R/ligand complex and is essential for the subsequent lysosomal degradation of IL-2 , possibly by regulating trafficking to the lysosome .


Functional characterization of the two alternative promoters of human p45 NF-E2 gene .


OBJECTIVE : The transcription factor NF-E2 , a heterodimeric protein complex composed of p45 and small Maf family proteins , is considered crucial for the proper differentiation of erythrocytes and megakaryocytes in vivo .


We report the results of studies aimed at understanding the regulatory mechanisms controlling p45 gene expression in erythroid cells .


MATERIALS AND METHODS : Human p45 mRNAs have two alternative isoforms , aNF-E2 and fNF-E2 , and these isoforms are transcribed from the alternative promoters .


We investigated lineage-specific expression of both isomers in human erythroid and megakaryocytic cells by reverse transcriptase polymerase chain reaction or Northern blot analysis .


For functional characterization of both promoters , plasmids in which reporter genes were placed under the control of a series of truncated or mutated promoter fragments were transfected to human hematopoietic cell lines .


RESULTS : When CD34 ( + ) cells isolated from human cord blood were induced to unilineage erythroid or megakaryocytic differentiation in liquid suspension culture , both transcripts , although barely detected at day 0 , were induced in both erythroid and megakaryocytic cultures .


fNF-E2 mRNA was found to be more abundant in erythroid cells than megakaryocytic cells at day 7 of culture .


Although both isomers were expressed in human erythroid-megakaryocytic cell lines , megakaryocytic maturation with loss of erythroid phenotype induced by phorbol 12-myristate 13-acetate ( PMA ) resulted in exclusive downregulation of fNF-E2 , suggesting that fNF-E2 promoter is more erythroid specific .


Functional analysis of fNF-E2 promoter showed that the promoter is active only in erythroid-megakaryocytic cells and that the double GATA sit e in the proximal region is necessary for its efficient activity .


CONCLUSION : These results suggest that GATA proteins , which govern the differentiation of erythroid lineage cells , are required for full promoter activity of the p45 gene .


Transcriptional activation of heme oxygenase-1 and its functional significance in acetaminophen-induced hepatitis and hepatocellular injury in the rat .


BACKGROUND/AIM : Glutathione depletion contributes to acetaminophen hepatotoxicity and is known to induce the oxidative stress reactant heme oxygenase-1 .


The metabolites of the heme oxygenase pathway , biliverdin , carbon monoxide , and iron may modulate acetaminophen toxicity .


The aim of this study was to assess cell-type specific expression of heme oxygenase-1 and its impact on liver injury and microcirculatory disturbances in a model of acetaminophen-induced hepatitis .


METHODS : Gene expression of heme oxygenase-1 was studied by Northern- and Western analysis as well as immunohistochemistry .


The time course of heme oxygenase-1 and -2 , cytokine-induced neutrophil chemoattractant-1 , and intercellular adhesion molecule-1 was studied by Northern analysis .


DNA-binding activity of nuclear factor-kappaB was determined by electrophoretic mobility shift assay .


Sinusoidal perfusion and leukocyte-endothelial interactions were assessed by intravital microscopy .


RESULTS : Acetaminophen caused a moderate sinusoidal perfusion failure ( -15 % ) and infiltration of neutrophils along with activation of nuclear factor-kappaB and intercellular adhesion molecule-1 and cytokine-induced neutrophil chemoattractant-1 mRNAs .


Induction of heme oxygenase-1 mRNA and protein ( approximately 30-fold ) in hepatocytes and non-parenchymal cells paralleled the inflammatory response .


Blockade of heme oxygenase activity with tin-protoporphyrin-IX abrogated acetaminophen-induced hepatic neutrophil accumulation and nuclear factor-kappaB activation , but failed to affect sinusoidal perfusion and liver injury .


CONCLUSIONS : The inflammatory response associated with acetaminophen hepatotoxicity is modulated by the parallel induction of the heme oxygenase-1 gene .


However , heme oxygenase-1 has no permissive effect on sinusoidal perfusion and does not affect liver injury in this model .


These data argue against a central role of nuclear factor-kappaB activation and neutrophil infiltration as perpetuating factors of liver injury in acetaminophen toxicity .


Tumor necrosis factor-alpha -induced proliferation requires synthesis of granulocyte-macrophage colony-stimulating factor .


OBJECTIVE : Tumor necrosis factor- alpha ( TNF-alpha ) induces a variety of cellular responses , some of them being at least seemingly contradictory .


Thus , we set out to find differences in the modes of proliferative and apoptotic responses to TNF- alpha .


MATERIALS AND METHODS : We screened a panel of acute myeloid leukemia-derived cell lines for TNF- alpha -responsiveness .


In two lines ( OCI-AML-1 , OCI-AML-11 ) , TNF- alpha acted as an apoptotic agent ; in others ( HU-3 , M-07e , TF-1 ) , it had the opposite effect , preventing apoptosis and inducing proliferation .


Direct and indirect signaling mechanisms , including NF-kappaB activation and cytokine synthesis , were analyzed .


RESULTS : All cell lines tested expressed TNF- alpha receptors I and II and responded to TNF- alpha by upregulation of intercellular adhesion molecule-1 .


In contrast to granulocyte-macrophage colony-stimulating factor ( GM-CSF ) , TNF- alpha did not activate the MAP kinase and p70S6 kinase pathways .


Nevertheless , inhibitors of these pathways clearly reduced the TNF-alpha -induced cell growth , indicating that TNF- alpha-proliferative cells produced a growth factor that induced proliferation upon stimulation of the above pathways .


Anti-GM-CSF antibodies inhibited the TNF-alpha -induced growth , suggesting the presence of an autocrine loop for cell proliferation mediated by GM-CSF .


Supporting this notion , TNF-alpha -induced upregulation of GM-CSF mRNA levels and protein secretion in the TNF-alpha -proliferative , but not in the TNF-alpha-apoptotic cell lines .


CONCLUSION : These data identify GM-CSF synthesis as an early and essential step in TNF- alpha-induced proliferation .


We show for the first time that TNF-alpha-treated cell lines producing no or only minimal amounts of GM-CSF demonstrate an apoptotic phenotype , while cell lines with high GM-CSF expression rates can escape from growth arrest or even apoptosis .


In this context , we discuss arguments pointing at NF-kappaB as regulator of GM-CSF synthesis and thus indirectly as regulator for the escape of TNF-alpha -induced apoptosis


Glucocorticoid receptor content of T lymphocytes : evidence for heterogeneity .


Glucocorticoid receptors were measured in T lymphocytes that were isolated from peripheral blood by either nylon wool filtration or E-rosette sedimentation .


T cells isolated by nylon wool filtration specifically bind 6.7 +/- 0.2 fmol of dexamethasone per million cells ( equivalent to 4000 +/- 200 receptors per cell ) , whereas T cells isolated by E-rosette sedimentation bind 12.0 +/- 0.7 fmol of dexamethasone per million cells ( equivalent to 7200 +/- 400 receptors per cell ) .


This difference in the amount of dexamethasone bound by the two T cell preparations was significant ( p less than .001 ) and was present immediately after cell isolation .


The binding affinities of the different T cell preparations for dexamethasone were similar .


T cells that are isolated by a combination of nylon wool filtration followed by E-rosette sedimentation bind the same amount of dexamethasone as T cells isolated by nylon wool filtration alone .


T cells isolated by a combination of E-rosette sedimentation following by nylon wool filtration bind less dexamethasone than do T cells isolated by E-rosette sedimentation alone .


These findings suggest that T cells are heterogeneous with respect to their quantity of glucocorticoid receptors .


Isolation of T cells by E-rosette sedimentation enriches for T cells that have a greater number of glucocorticoid receptors , and isolation of T cells by nylon wool filtration enriches for T cells that have a lesser number of glucocorticoid receptors .


Glucocorticoid receptors and glucocorticoid sensitivity of human leukemic cells .


We have established optimal conditions for the measurement of glucocorticoid receptors ( GR ) in human white cells using a whole-cell binding assay with [ 3H ] dexamethasone as the ligand , and the subsequent determination of the GR content in normal human lymphocytes and in leukemic cells of patients with various forms of acute and chronic leukemia .


A number of leukemia cell lines in continuous culture were also subjected to the GR assay , and the results were correlated with the sensitivity of these cell lines to glucocorticoid steroids in vitro .


The GR content of normal human lymphocytes amounted to 4 , 850 +/- 1 , 340 ( mean +/- SD ) receptors/cell .


The mean equilibrium dissociation constant ( KD ) of the interaction of [ 3H ] dexamethasone with the GR was 1.2 x 10 ( -8 ) M .


Steroidal compounds with a known glucocorticoid potency effectively competed for the binding , whereas steroids devoid of glucocorticoid activity ( e.g. estradiol-17 beta and testosterone ) were ineffective .


The GR content of the blast cells obtained from eight patients suffering from acute leukemia and four patients with a blast crisis of chronic myelocytic leukemia was found to be highly variable ( 3 , 230-29 , 900 receptors/cell ) , while the lymphocytes of six patients with chronic lymphatic leukemia contained a rather stable GR content ( 2 , 930-5 , 120 receptors/cell ) , which was comparable with that of normal lymphocytes .


GR was identified in all the 12 malignant continuous white cell lines studied .


Large cells contained more GR than the smaller ones .


There was no apparent correlation between the GR concentration and the sensitivity of the cells in vitro to glucocorticoids as judged by [ 3H ] thymidine incorporation studies .


Distribution of the surface markers in the leukemic cell lines did not relate to the GR concentration .


We conclude that the presence of GR is probably a universal feature of the leukemic cells , and , from a clinical standpoint , probably does not alone imply steroid responsiveness .


Evidence for a steroid receptor in rheumatoid synovial tissue cells .


One mechanism by which glucocorticoids could exert their anti-inflammatory action is via rapidly saturable , stereo-specific cytoplasmic protein receptors .


This report is of an investigation into such a possibility in synovial cells .


Synovium , obtained from knee joints of rheumatoid patients undergoing surgery , was incubated with clostridiopeptidase A and trypsin-EDTA to obtain cell suspensions .


These , together with cells obtained from synovial fluid aspirated from patients with rheumatoid arthritis , were identified by electron microscopy .


Duplicate samples of these cell suspensions were incubated with increasing concentrations of H3Dexamethasone ( 1 x 10 ( -10 ) M-1 x 10 ( -9 ) M ) for 30 minutes at 37 degrees C .


Analysis of the proportion of steroid bound to whole cells showed evidence for specific , rapidly saturable , receptors in the cells obtained from synovial tissue , but this was not found in synovial fluid cells .


Electron micrographs showed that cells obtained from synovial tissue consisted of synovial fibroblast - and macrophage-types , lymphocytes , monocytes and macrophages .


Polymorphonuclear leucocytes appeared to be absent .


However , in synovial fluid cell type polymorphonuclear leucocytes were the predominant cell type .


We concluded from this , that one or more of the cell types present in synovial tissue contain a specific steroid receptor , but that this is lacking in synovial fluid polymorphonuclear leucocytes .


Clinical implications of glucocorticoid receptors in human leukemia .


Glucorticoid receptors were studied in various populations of normal human peripheral blood lymphocytes and leukemic lymphoblasts .


Normal lymphocytes contain low levels of glucocorticoid receptor ( approximately 2 , 500 sites/cell ) which are identical in T- and non-T-fractions .


Phytohemagglutinin treatment increases levels about 3-fold .


Leukemic lymphoblasts contain larger numbers of receptor sites .


Presence of receptor is correlated with in vitro sensitivitiy to glucocorticoids and in vivo response to therapy .


Quantity of receptor is also correlated with complete remission duration independently of leukemic cell type ( T or null ) , initial WBC , or age of patient .


Quantitative determination of glucocorticoid receptor levels in acute lymphoblastic leukemia may be of value both as an independent prognostic variable and in suggesting which patients should receive glucocorticoid therapy .


Functional and physical interaction of protein-tyrosine kinases Fyn and Csk in the T-cell signaling system .


The Src-like protein-tyrosine kinase Fyn is associated with T-cell antigen receptor .


Transient expression of actively mutated Fyn , having Phe-528 instead of Tyr-528 or Thr-338 instead of Ile-338 , in Jurkat T-cells stimulated the serum response element ( SRE ) , 12-O-tetradecanoyl-phorbol-13-acetate response element , cyclic AMP response element , and c-fos promoter .


The stimulation of SRE was particularly prominent not only with active Fyn but also with normal ( wild-type ) Fyn .


SRE was also stimulated by both normal and active Lck .


Furthermore , normal and active Fyn stimulated transcription from the IL-2 gene promoter when transfected cells were stimulated by concanavalin A plus 12-O-tetradecanoylphorbol-13-acetate .


Under the same conditions , Lck did not stimulate IL-2 promoter unless it was activated by mutation .


Interestingly , a mutant Fyn , which has deletions within the SH2 region and so is able to transform chicken embryo fibroblasts , did not stimulate either the c-fos or IL-2 promoter , suggesting the importance of this region in T-cell signaling .


Csk , which phosphorylates tyrosine residues in the negative regulatory sites of Src family kinases , down-regulated Fyn- and Lck-mediated stimulation of the serum response element and Fyn-mediated enhancement of IL-2 promoter activity .


These data suggest that Fyn and Lck , whose activities are regulated by Csk , are involved in different phases of T-cell activation .


A novel NF-kappa B complex containing p65 homodimers : implications for transcriptional control at the level of subunit dimerization .


The predominant inducible form of the NF-kappa B transcription factor is a heteromeric complex containing two Rel-related DNA-binding subunits , termed p65 and p50 .


Prior transfection studies have shown that when these p65 and p50 subunits are expressed independently as stable homodimers , p65 stimulates kappa B-directed transcription , whereas p50 functions as a kappa B-specific repressor .


While authentic p50 homodimers ( previously termed KBF1 ) have been detected in nuclear extracts from nontransfected cells , experimental evidence supporting the existence of p65 homodimers in vivo was lacking .


We now provide direct biochemical evidence for the presence of an endogenous pool of inducible p65 homodimers in intact human T cells .


As with the prototypical NF-kappa B p50-p65 heterodimer , this novel p65 homodimeric form of NF-kappa B is functionally sequestered in the cytoplasm but rapidly appears in the nuclear compartment following cellular stimulation .


Site-directed mutagenesis studies indicate that the homodimerization function of p65 is dependent upon the presence of cysteine 216 and a conserved recognition motif for protein kinase A ( RRPS ; amino acids 273 to 276 ) , both of which reside within a 91-amino-acid segment of the Rel homology domain that mediates self-association .


In contrast , mutations at these two sites do not affect heterodimerization of p65 with p50 or its functional interaction with I kappa B alpha .


These later findings indicate that neither homo- nor heterodimer formation is an absolute prerequisite for I kappa B alpha recognition of p65 .


Taken together with prior in vivo transcription studies , these results suggest that the biological activities of p65 and p50 homodimers are independently regulated , thereby providing an integrated and flexible control mechanism for the rapid activation and repression of NF-kappa B / Rel -directed gene expression .


Carrier determination for X-linked agammaglobulinemia using X inactivation analysis of purified B cells .


We report the development of a relatively quick and simple method for the assessment of X inactivation status for carrier determination in families affected by X-linked agammaglobulinemia ( XLA ) .


This method utilises an immunomagnetic separation technique for B cell purification and a polymerase chain reaction ( PCR ) based assay for the determination of methylation status at the androgen receptor ( AR ) gene locus to assess whether X inactivation is random or non-random at this locus .


We report the results we have obtained using this assay to investigate females known to be carriers of various X-linked immunodeficiency disorders .


In addition , we investigated four females from different families affected by XLA , two of whom were of unknown carrier status , and we discuss the results obtained with this and other X-inactivation assays .


A similar assay has recently been described by Allen et al. ( 1992 ) and applied to members of one family affected by XLA .


Effects of IL-4 and Fc gamma receptor II engagement on Egr-1 expression during stimulation of B lymphocytes by membrane immunoglobulin crosslinking .


Egr-1 is an immediate early gene that is rapidly upregulated in response to mitogenic signals induced by antigen receptor crosslinking on murine B lymphocytes .


It has been shown that levels of Egr-1 expression are closely correlated with B cell proliferation in several models of B cell activation and tolerance .


We compared the expression of Egr-1 during B cell stimulation with Fab'2 and IgG anti-immunoglobulin ( anti-Ig ) , since it is known that Fab'2 anti-Ig is mitogenic while IgG anti-Ig is not , owing to a dominant inhibitory effect of crosslinking the B cell Fc gamma RII to membrane Ig .


While mitogenic doses of Fab'2 anti-Ig induce large and rapid increases in Egr-1 expression , IgG anti-Ig results in smaller increases in Egr-1 mRNA , comparable to that seen with submitogenic concentrations of Fab'2 anti-Ig .


However , the correlation between Egr-1 expression and B cell proliferation breaks down when IL-4 is added as a co-mitogen to induce B cell proliferation with IgG anti-Ig or submitogenic concentrations of Fab'2 anti-Ig .


No corresponding increases in Egr-1 mRNA levels are observed when IL-4 is added .


Therefore , IL-4 overcomes Fc receptor-mediated inhibition of B cell proliferation without affecting inhibition of Egr-1 mRNA induction , as demonstrated earlier for c-myc mRNA in this system .


Identification of a novel cyclosporin-sensitive element in the human tumor necrosis factor alpha gene promoter .


Tumor necrosis factor alpha ( TNF-alpha ) , a cytokine with pleiotropic biological effects , is produced by a variety of cell types in response to induction by diverse stimuli .


In this paper , TNF-alpha mRNA is shown to be highly induced in a murine T cell clone by stimulation with T cell receptor ( TCR ) ligands or by calcium ionophores alone .


Induction is rapid , does not require de novo protein synthesis , and is completely blocked by the immunosuppressant cyclosporin A ( CsA ) .


We have identified a human TNF-alpha promoter element , kappa 3 , which plays a key role in the calcium-mediated inducibility and CsA sensitivity of the gene .


In electrophoretic mobility shift assays , an oligonucleotide containing kappa 3 forms two DNA protein complexes with proteins that are present in extracts from unstimulated T cells .


These complexes appear in nuclear extracts only after T cell stimulation .


Induction of the inducible nuclear complexes is rapid , independent of protein synthesis , and blocked by CsA , and thus , exactly parallels the induction of TNF-alpha mRNA by TCR ligands or by calcium ionophore .


Our studies indicate that the kappa 3 binding factor resembles the preexisting component of nuclear factor of activated T cells .


Thus , the TNF-alpha gene is an immediate early gene in activated T cells and provides a new model system in which to study CsA-sensitive gene induction in activated T cells .


Differences in expression of transcription factor AP-1 in human promyelocytic HL-60 cells during differentiation towards macrophages versus granulocytes .


Commitment of HL-60 cells to macrophage or granulocytic differentiation was achieved by incubation with 4 beta-phorbol 12-myristate 13-acetate ( PMA ) for 30-60 min or with dimethyl sulphoxide ( DMSO ) for 24 h respectively .


The commitment stage towards PMA-induced macrophage differentiation was associated with increases in jun B and c-fos mRNA levels , as well as with an increase in the binding activity of transcription factor AP-1 .


Nevertheless , gel retardation analysis indicated that the AP-1 activity detected in untreated cells was drastically reduced during the commitment stage of DMSO-induced HL-60 differentiation towards granulocytes .


When HL-60 cells were treated with sodium butyrate , which induced monocytic differentiation , a remarkable increase in AP-1 binding activity was detected .


Treatment of HL-60 cells with 1 alpha , 25-dihydroxyvitamin D3 , another monocytic differentiation agent , induced a weak , but appreciable , increase in AP-1 activity .


Furthermore , addition of sodium butyrate or 1 alpha , 25-dihydroxyvitamin D3 to HL-60 cells induced the expression of c-fos , c-jun , jun B and jun D proto-oncogenes .


In contrast , when HL-60 cells were treated with retinoic acid , a granulocytic differentiation inducer , no enhanced AP-1 binding activity was observed , and only a weak increase in jun D mRNA level was detected .


These data indicate that formation of AP-1 is not required for the induction of HL-60 differentiation towards granulocytes , whereas induction of monocytic differentiation is correlated with an increase in AP-1 activity .


The differential expression of AP-1 activity may be critical in the differentiation of HL-60 cells towards monocytic or granulocytic lineages


Glucocorticoid receptors and sensitivity in leukemias .


In an attempt to investigate the utility of glucocorticoid receptor determination to predict clinical responsiveness in human leukemias we have studied glucocorticoid receptors in the leukemic cells from 46 patients and in the lymphocytes from 18 normal donors .


In the normal lymphocytes there were 3 , 875 ( Median ) specific binding sites per cell .


The blasts from 17 patients with ANLL had on average higher levels of binding sites per cell ( Median = 7 , 250 , range : 0 to 15 , 295 ) than the other leukemias .


Of the 15 patients with CLL , six had received glucocorticoid treatment for 3 to 5 years .


Their lymphocytes had lower number of receptors ( Median = 2 , 000 ) than the other cases which were newly diagnosed ( Median = 4 , 500 ) .


Four patients had ALL/AUL , three patients had blast crisis as terminal phase of CML , and seven had leukemic Non-Hodgkin lymphomas ( Median = 3 , 500 sites/cell ) .


In 24 patients we have also studied the in vitro sensitivity of the leukemic cells to dexamethasone .


There was no marked correlation between glucocorticoid receptor levels and in vitro sensitivity .


An attempt to correlate receptor levels with clinical responsiveness demonstrated that glucocorticoid receptor determination might be of value in patients with lymphoid malignancies but probably not in patients with other leukemias .


'Activation-labile ' glucocorticoid-receptor complexes of a steroid-resistant variant of CEM-C7 human lymphoid cells .


For cytoplasmic glucocorticoid-receptor complexes to enter and accumulate in the nucleus a temperature-dependent event , 'activation ' is required .


Activation can be achieved in vitro by increased ionic strength , dilution or gel filtration and is manifested by an increased affinity of steroid-receptor complex for DNA and an altered elution profile from ion-exchange resins .


Munck and Foley have shown that activated complexes isolated from thymocytes elute from DEAE-cellulose in a manner identical to complexes activated in vitro .


We report here that DEAE-cellulose chromatography of steroid-receptor complexes from CEM-C7 , a cloned human leukaemic T-cell line sensitive to the cytolytic action of glucocorticoids , and its steroid-resistant subclone 4R4 demonstrated that steroid receptors of clone 4R4 can not form stable activated complexes .


This defines a new defect in receptor action , activation lability ( r+act1 ) , which is unlike either the r- , r+nt- , or r+nti phenotypes previously described for mouse lymphoid variants .


Granulocytes in the endometrium of post-partum women .


Endometrial samples of women at various stages of gonadal activity after parturition were examined for the presence and numbers of endometrial granulocytes .


Although samples at all the stages contained significant numbers of the granulocytes ( i.e. greater than 7/high-power field ) , the 100 % values for late-proliferative and adaptation hyperplasia were significantly higher than the values for the resting ( 81.8 % ) , early ( 82.4 % ) and mid- ( 87.9 % ) proliferative and secretory ( 83.3 % ) phases .


We suggest that this correlates with the suggestion that the granulocytes constitute a receptor system for oestrogens .


Interaction of glucocorticoids with macrophages .


Identification of glucocorticoid receptors in monocytes and macrophages .


Glucocorticoid binding was measured in resident and thioglycollate-elicited mouse peritoneal macrophages , rabbit alveolar macrophages , and human monocytes .


Two assays of binding were used -- an assay with intact cells in suspension or monolayers , and an assay of cytosol and nuclear forms of glucocorticoid receptors .


The mononuclear phagocytes contained approximately equal to 4 -- 10 X 10 ( 3 ) high affinity receptor sites per cell , with dissociation constants of approximately equal to 2 -- 8 nM dexamethasone .


The binding to the saturable sites was specific for steroids with glucocorticoid or antiglucocorticoid activity .


Cortisol , corticosterone , and progesterone competed with dexamethasone for binding , whereas estradiol , dihydrotestosterone , and 11-epicortisol competed very little .


Binding of dexamethasone to cytosol and nuclear forms of the receptor complex and temperature-sensitive translocation of cytosol forms to nuclear forms were shown .


At 37 degrees C the predominant form of the hormone-receptor complex was nuclear .


These results demonstrate that corticosteroids interact with macrophages at physiological concentrations .


Nitric oxide signaling : a possible role for G proteins .


We have previously reported various inductive effects of nitric oxide on human PBMC .


We describe a novel and potentially important mechanism of nitric oxide signaling-through direct activation of guanine nucleotide-binding proteins ( G proteins ) .


We have found that nitric oxide treatment of membranes isolated from fresh human PBMC enhances the ability of these membranes to hydrolyze [ gamma-32P ] GTP and bind [ gamma-35S ] GTP .


In addition , treatment of whole cells with nitric oxide yielded membranes with enhanced GTPase activity .


Furthermore , the GTPase activity of pure , recombinant Gs alpha , Gi alpha 1 , and p21ras was greatly enhanced by nitric oxide .


In support of the existence of this pathway in whole cells , we found that the G protein inhibitor , GDP-beta-S , blocked NF-kappa B translocation induced by nitric oxide or LPS in permeabilized cells .


In addition , nitric oxide greatly reduced the pertussis toxin-mediated ADP-ribosylation of 45- and 41-kDa proteins in membranes of these cells .


Because G proteins play a central role in many diverse signaling systems , activation by an endogenous and inducible oxidant may represent a novel signaling pathway .


The granulocyte-macrophage colony-stimulating factor promoter cis-acting element CLE0 mediates induction signals in T cells and is recognized by factors related to AP1 and NFAT .


Expression of the granulocyte-macrophage colony-stimulating factor ( GM-CSF ) gene in T cells is activated by the combination of phorbol ester ( phorbol myristate acetate ) and calcium ionophore ( A23187 ) , which mimic antigen stimulation through the T-cell receptor .


We have previously shown that a fragment containing bp -95 to +27 of the mouse GM-CSF promoter can confer inducibility to reporter genes in the human Jurkat T-cell line .


Here we use an in vitro transcription system to demonstrate that a cis-acting element ( positions -54 to -40 ) , referred to as CLE0 , is a target for the induction signals .


We observed induction with templates containing intact CLE0 but not with templates with deleted or mutated CLE0 .


We also observed that two distinct signals were required for the stimulation through CLE0 , since only extracts from cells treated with both phorbol myristate acetate and A23187 supported optimal induction .


Stimulation probably was mediated by CLE0-binding proteins because depletion of these proteins specifically reduced GM-CSF transcription .


One of the binding factors possessed biochemical and immunological features identical to those of the transcription factor AP1 .


Another factor resembled the T-cell-specific factor NFAT .


The characteristics of these two factors are consistent with their involvement in GM-CSF induction .


The presence of CLE0-like elements in the promoters of interleukin-3 ( IL-3 ) , IL-4 , IL-5 , GM-CSF , and NFAT sites in the IL-2 promoter suggests that the factors we detected , or related factors that recognize these sites , may account for the coordinate induction of these genes during T-cell activation .


Identification and characterization of an Alu-containing , T-cell-specific enhancer located in the last intron of the human CD8 alpha gene .


Expression of the human CD8 alpha gene is restricted to cells of the lymphoid lineage and developmentally regulated during thymopoiesis .


As an initial step towards understanding the molecular basis for tissue-specific expression of this gene , we surveyed the surrounding chromatin structure for potential cis-acting regulatory regions by DNase I hypersensitivity mapping and found four hypersensitive sites , three of which were T cell restricted .


By using a reporter-based expression approach , a T-cell-specific enhancer was identified by its close association with a prominent T-cell-restricted hypersensitive sites in the last intron of the CD8 alpha gene .


Deletion studies demonstrated that the minimal enhancer is adjacent to a negative regulatory element .


DNA sequence analysis of the minimal enhancer revealed a striking cluster of consensus binding sites for Ets-1 , TCF-1 , CRE , GATA-3 , LyF-1 , and bHLH proteins which were verified by electrophoretic mobility shift assays .


In addition , the 5 ' end of the enhancer was composed of an Alu repeat which contained the GATA-3 , bHLH , and LyF-1 binding sites .


Site-directed mutation of the Ets-1 and GATA-3 sites dramatically reduced enhancer activity .


The functional importance of the other binding sites only became apparent when combinations of mutations were analyzed .


Taken together , these results suggest that the human CD8 alpha gene is regulated by the interaction of multiple T-cell nuclear proteins with a transcriptional enhancer located in the last intron of the gene .


Comparison of the CD8 alpha enhancer with other recently identified T-cell-specific regulatory elements suggests that a common set of transcription factors regulates several T-cell genes .


Molecular regulation of the human IL-3 gene : inducible T cell-restricted expression requires intact AP-1 and Elf-1 nuclear protein binding sites .


Interleukin 3 ( IL-3 ) is a hematopoietic stem-cell growth and differentiation factor that is expressed solely in activated T and NK cells .


Studies to date have identified elements 5 ' to the IL-3 coding sequences that regulate its transcription , but the sequences that confer T cell-specific expression remain to be clearly defined .


We have now identified DNA sequences that are required for T cell-restricted IL-3 gene transcription .


A series of transient transfections performed with human IL-3-chloramphenicol acetyltransferase ( CAT ) reporter plasmids in T and non-T cells revealed that a plasmid containing 319 bp of 5 ' flanking sequences was active exclusively in T cells .


Deletion analysis revealed that T cell specificity was conferred by a 49-bp fragment ( bp -319 to -270 ) that included a potential binding site for AP-1 transcription factors 6 bp upstream of a binding site for Elf-1 , a member of the Ets family of transcription factors .


DNaseI footprint and electrophoretic mobility shift assay analyses performed with MLA-144 T cell nuclear extracts demonstrated that this 49-bp region contains a nuclear protein binding region that includes consensus AP-1 and Elf-1 binding sites .


In addition , extracts prepared from purified human T cells contained proteins that bound to synthetic oligonucleotides corresponding to the AP-1 and Elf-1 binding sites .


In vitro-transcribed and -translated Elf-1 protein bound specifically to the Elf-1 site , and Elf-1 antisera competed and super shifted nuclear protein complexes present in MLA-144 nuclear extracts .


Moreover , addition of anti-Jun family antiserum in electrophoretic mobility shift assay reactions completely blocked formation of the AP-1-related complexes .


Transient transfection studies in MLA-144 T cells revealed that constructs containing mutations in the AP-1 site almost completely abolished CAT activity while mutation of the Elf-1 site or the NF-IL-3 site , a previously described nuclear protein binding site ( bp. -155 to -148 ) in the IL-3 promoter , reduced CAT activity to < 25 % of the activity given by wild-type constructs .


We conclude that expression of the human IL-3 gene requires the AP-1 and Elf-1 binding sites ; however , unlike other previously characterized cytokine genes such as IL-2 , the AP-1 and Elf-1 factors can bind independently in the IL-3 gene .


Combination IL-2 and IL-4 reduces glucocorticoid receptor-binding affinity and T cell response to glucocorticoids .


The mechanisms contributing to persistent T cell activation and poor response to glucocorticoids in chronic inflammatory illnesses such as steroid resistant ( SR ) asthma are poorly defined .


We examined the possibility that certain cytokines , specifically IL-2 and IL-4 , could affect T cell response to glucocorticoids .


A [ 3H ] dexamethasone radioligand-binding assay was used to measure the number of glucocorticoid receptors ( GR ) and dissociation constant ( Kd ) in PBMC from normal donors and patients with SR asthma , cultured in the absence and presence of these cytokines .


PBMC from normal donors incubated for 48 h in the presence of combination IL-2 + IL-4 had nuclear GR with significantly reduced binding affinity ( GR Kd = 36.1 +/- 1.63 nM , mean +/- SEM ; p = 0.0001 ) as compared with PBMC incubated with medium alone ( GR Kd = 6.74 +/- 0.46 nM ) .


The cytosolic GR Kd remained unchanged .


However , when PBMC were incubated with IL-2 alone or IL-4 alone , no change in GR-binding affinity was observed .


Furthermore , when T cells and non-T cells were individually stimulated with combination IL-2 + IL-4 , a significant reduction in GR-binding affinity was observed only in the T cell population ( p = 0.0001 ) .


The IL-2 + IL-4-induced alteration in PBMC GR Kd was associated with an increase in GR number ( 8348 +/- 964 vs 1710 +/- 228 sites/cell ; p = 0.0003 ) .


More importantly , the alteration in PBMC GR-binding affinity with IL-2 + IL-4 was associated with a functional change in T cell response to methylprednisolone MPN , i.e. , a reduced inhibitory effect of MPN on PMA/ionomycin-induced T cell proliferation .


These effects of IL-2 + IL-4 on PBMC GR affinity and response to MPN were blocked by co-incubation with IFN-gamma .


Freshly isolated PBMC from four patients with SR asthma had a significantly reduced GR-binding affinity ( Kd = 40.0 +/- 2.68 nM ; p = 0.0001 ) when compared with seven normal subjects ( 7.15 +/- 0.41 nM ) .


The altered PBMC GR binding from patients with SR asthma reversed to normal when incubated with medium alone , but was sustained with IL-2 + IL-4 .


These observations suggest that with persistent inflammation certain cytokines may contribute to an impaired response to glucocorticoids .


Furthermore , the effects of IL-2 and IL-4 were blocked by IFN-gamma .


Characterization of the human CD4 gene promoter : transcription from the CD4 gene core promoter is tissue-specific and is activated by Ets proteins .


We analyzed the 5 ' transcription control sequences of the human CD4 gene .


We located the transcription initiation site and showed that the CD4 core promoter ( positions -40 to +16 ) lacks a classical `` TATA '' or initiator positioning consensus sequence but directs precise and efficient transcription when coupled to the ubiquitously active simian virus 40 enhancer .


The transcriptional activity of the CD4 gene promoter correlated with CD4 expression in various cell types .


Interestingly , the CD4 core promoter also displayed a tissue-specific transcriptional activity .


Within this fragment , three nucleic acid sequences are completely conserved in the murine CD4 gene .


One of these sequences contains a perfect ETS consensus sequence .


Another ETS consensus sequence is located 1060 nt upstream .


Electrophoretic-mobility-shift assays showed that the core promoter ETS motif binds an Ets-related protein specifically expressed at high levels in CD4+ cells .


Moreover , in CD4- cells , overexpression of Ets-1 or Ets-2 efficiently and specifically activated transcription from the CD4 promoter and core promoter .


These data indicate that Ets transcription factors play a central role in controlling CD4 gene expression , by binding to both a classical remote site and an unusual proximal activator sequence


Glucocorticoid receptor activation and inactivation in c ultured human lymphocytes .


Although glucocorticoids are not cytolytic for and do not inhibit the growth of the IM-9 line of cultured human lymphoblasts , these cells have a high steroid-binding capacity .


We have used IM-9 cells in order to examine whether unoccupied glucocorticoid receptors are inactivated and activated in intact cells .


when IM-9 cells are incubated in glucose-free medium in a nitrogen atmosphere , both their ability to bind triamcinolone acetonide and their ATP levels decline and , when glucose and oxygen are reintroduced , ATP levels and receptor activity return .


The specific glucocorticoid-binding activity of cytosol prepared from cells exposed to various degrees of energy limitation is directly correlated with the ATP content .


Receptor activation in intact cells is rapid and independent of protein synthesis .


Cytosol prepared from inactivated cells can not be activated by addition of ATP .


The inactivation of glucocorticoid receptors that occurs when cytosol from normal IM-9 cells is incubated at 25 degrees C is inhibited by molybdate , vanadate , fluoride , ATP , and several other nucleotides .


The experiments with intact human lymphoblasts suggest that assays of specific glucocorticoid-binding capacity do not necessarily reflect the cellular content of receptor protein .


Cell cycle-dependent changes in glucocorticoid receptor content .


To study variations in glucocorticoid receptor levels during the cell cycle , we have separated mitogen-stimulated human peripheral lymphocytes and rat lymph node cells by unit gravity sedimentation and measured glucocorticoid binding in the resultant fractions .


By morphologic criteria and thymidine incorporation , the fractions were separated into populations of G0 and G1 phase and S and post-S phase cells .


A 2- to 3-fold increase in glucocorticoid receptor sites per cell , for cells in the S and post-S phase over those in G0 and G1 , was observed with both nonstimulated rat lymph node cell suspensions and concanavalin A-stimulated human peripheral lymphocytes .


These observations together with those from other studies indicate that formation of new glucocorticoid receptors near the S phase may be a general phenomenon in proliferating cells .


We propose that this increase in glucocorticoid receptors during the cell cycle may explain the increase in glucocorticoid receptors in mitogen-stimulated lymphocytes .


The leukocyte migration inhibition response to certain breast cancer-related antigens ( MCF-7 and MuMTV ) : their potential as discriminants .


Certain oncogenic viruses have been implicated in human breast cancer , including the murine mammary tumor virus ( MuMTV ) and the Mason-Pfizer monkey virus ( MPMV ) .


We have used the leukocyte migration inhibition ( LMI ) response to assay the response to several potential breast cancer-related antigens , including MuMTV , MPMV , and a breast cancer cultured cell line , MCF-7 , in 96 breast cancer patients , in 32 women with benign breast disease , and in 67 normal women .


The lowest tenth percentile of control ( LMI ) responses was used as the cutoff point to designate responders .


Breast cancer patients showed significant responses to MuMTV ( 49 % and to MCF-7 ( 50 % ) , but not to MPMV ( 29 % ) .


In a paired-antigen study using MuMTV and MCF-7 , 75 % of the breast cancer patients responded , versus 18 % of the normal women ( P less than 0.0050 ) .


The potential for this assay to distinguish `` normal '' from `` breast cancer '' was analyzed using a migration index derived from discriminant analysis .


The ability of the assay to discriminate `` normal '' from `` cancer '' was significant ( P less than 0.001 ) and showed a sensitivity of detecting `` cancer '' of 75 % .


The overall responses to MuMTV and MCF-7 were analyzed with reference to certain prognostic factors , but showed no relation to age , menstrual status , estrogen receptor status , or stage of disease .


The above reactions suggest that a large proportion of breast cancer patients exhibit presensitization to antigenfs found in MuMTV and MCF-7 , which may be cross-reactive with antigens in the primary cancer .


These responses appear to be independent of major prognostic variables .


Further refinement of this assay may yield one which is more highly discriminating for breast cancer .


Chronic lymphatic leukaemia : cellular effects of glucocorticoids in vitro .


Glucocorticoid receptor levels and steroid induced inhibition of nucleic acid precursors have been examined in lymphocytes from 27 patients at different stages of chronic lymphatic leukaemia .


No correlation can be found between the level of glucocorticoid receptors and the stage of the disease .


On the other hand , a significant difference ( P less than 0.02 ) was found between stage O and stage III/IV patients , in terms of the in vitro effect of dexamethasone on [ 3H ] uridine incorporation .


Pyrrolidine dithiocarbamate inhibits NF-kappa B mobilization and TNF production in human monocytes .


The human TNF promoter contains four potential nuclear factor-kappa B ( NF-kappa B ) -binding sites , with the strongest binding seen for the -605 motif .


Nuclear extracts from unstimulated cells of the human monocytic cell line , Mono Mac 6 , contain one specific binding protein ( complex II ) , consistent with a constitutive p50 homodimer .


Stimulation of Mono Mac 6 cells with LPS will increase complex II and will strongly induce a second specific complex ( complex I ) , which represents the p50/65 heterodimer .


Treatment of Mono Mac 6 cells with pyrrolidine-dithiocarbamate ( PDTC ) at 300 microM will block the LPS-induced complex I almost completely and will reduce complex II to the constitutive level .


Binding activity of other nuclear factors that recognize the SP-1 and c/EBP motifs of the human TNF promoter is not affected by such treatment .


Northern blot analysis demonstrates that PDTC treatment will strongly reduce LPS-induced TNF transcripts .


Secreted TNF protein as detected in the Wehi 164S/ActD bioassay and in a sandwich immunoassay was similarly reduced by PDTC .


Kinetic analyses show that after LPS stimulation , NF-kappa B will peak at 1 h , TNF transcript prevalence at 2 h , and TNF protein at 4 h .


PDTC did not shift this response to LPS to a later time , but suppressed NF-kappa B mobilization , TNF transcripts , and TNF protein over the entire 8-h observation period .


Analysis of freshly isolated , LPS-stimulated blood monocytes showed a similar blockade of NF-kappa B .


Furthermore , in these primary cells , induction of TNF transcripts , as determined by Northern blot analysis and by quantitative polymerase chain reaction , was prevented by PDTC as was TNF protein production .


These data show that dithiocarbamates can profoundly affect cytokine expression and suggest that NF-kappa B is involved in LPS-induced TNF gene expression in human monocytes .


Comparing regions of the Epstein-Barr virus ZEBRA protein which function as transcriptional activating sequences in Saccharomyces cerevisiae and in B cells .