Generation of CD1+RelB+ dendritic cells and tartrate-resistant acid phosphatase-positive osteoclast-like multinucleated giant cells from human monocytes. We previously showed that granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) stimulate the differentiation of human monocytes into two phenotypically distinct types of macrophages. However, in vivo, not only CSF but also many other cytokines are produced under various conditions. Those cytokines may modulate the differentiation of monocytes by CSFs. In the present study, we showed that CD14+ adherent human monocytes can differentiate into CD1+relB+ dendritic cells (DC) by the combination of GM-CSF plus interleukin-4 (IL-4) and that they differentiate into tartrate-resistant acid phosphatase (TRAP)-positive osteoclast-like multinucleated giant cells (MGC) by the combination of M-CSF plus IL-4. However, the monocyte-derived DC were not terminally differentiated cells; they could still convert to macrophages in response to M-CSF. Tumor necrosis factor-alpha (TNF-alpha) stimulated the terminal differentiation of the DC by downregulating the expression of the M-CSF receptor, cfms mRNA, and aborting the potential to convert to macrophages. In contrast to IL-4, interferon-gamma (IFN-gamma) had no demonstrable effect on the differentiation of monocytes. Rather, IFN-gamma antagonized the effect of IL-4 and suppressed the DC and MGC formation induced by GM-CSF + IL-4 and M-CSF + IL-4, respectively. Taken together, these results provide a new aspect to our knowledge of monocyte differentiation and provide evidence that human monocytes are flexible in their differentiation potential and are precursors not only of macrophages but also of CD1+relB+DC and TRAP-positive MGC. Such a diverse pathway of monocyte differentiation may constitute one of the basic mechanisms of immune regulation. 

Regulation of [Ca2+]i rise activated by doxepin-sensitive H1-histamine receptors in Jurkat cells, cloned human T lymphocytes. To clarify the presence of histamine receptor and its transmembrane mechanism in human T lymphocytes, we investigated the effects of agonists or antagonists of histamine receptor subtypes and bacterial toxins on intracellular concentration of Ca2+ [Ca2+]i), [3H]pyrilamine binding and c-fos mRNA expression in Jurkat cells, cloned human T lymphocytes. H1-agonists (histamine and 2-methylhistamine) caused a transient rise of [Ca2+], and H1-antagonists (pyrilamine and doxepin) inhibited the histamine-induced [Ca2+]i rise more potently than the H2-antagonist (cimetidine) on the H3-antagonist (impromidine). Binding parameters of [3H]pyrilamine binding were Kd = 5.53 nM and Bmax = 2,647 sites/cell. Pretreatment with B.pertussis, V.cholera. or C.botulinum toxin did not influence histamine-induced [Ca2+]i rise. Western Blot analysis using antibodies against subunits of GTP-binding proteins indicated that Gq/G11 richly existed in Jurkat cells. Histamine induced mRNA expression of an immediate early gene c-fos. Pretreatment with a protein kinase C activator, phorbol 12-myristate 13-acetate, caused almost complete inhibition of histamine-induced [Ca2+]i rise, but did not do so by activators of cAMP- and cGMP-dependent protein kinases. 

T cell response to Epstein-Barr virus transactivators in chronic rheumatoid arthritis. Rheumatoid arthritis is a multistep disorder associated with autoimmune features of yet unknown etiology. Implication of viruses such as Epstein-Barr virus (EBV) in rheumatoid arthritis pathogenesis has been suspected on the basis of several indirect observations, but thus far, a direct link between EBV and rheumatoid arthritis has not been provided. Here we show that a large fraction of T cells infiltrating affected joints from a patient with chronic rheumatoid arthritis recognizes two EBV transactivators (BZLF1 and BMLF1) in a major histocompatibility complex-restricted fashion. Responses to these EBV antigens by synovial lymphocytes from several other chronic rheumatoid arthritis patients were readily detectable. Thus these results suggest a direct contribution of EBV to chronic rheumatoid arthritis pathogenesis. They also demonstrate for the first time the occurrence of T cell responses against EBV transactivating factors, which might be central in the control of virus reactivation. 

Differential nuclear localization of p50, p52, and RelB proteins in human accessory cells of the immune response in situ. The Rel/NF-kappa B proteins, p50, p52, p65, c-Rel, and RelB, constitute a family of transcription factors involved in the positive regulation of a variety of genes during the immune response. Recently, it has been shown that RelB knockout mice have no dendritic cells (DC). An overexpression of p50 has been described in follicular dendritic cells (FDC). A constitutive NF-kappa B activity has been reported in mature macrophages. This led to the hypothesis that some of the Rel/NF-kappa B proteins were key nuclear factors in functions of accessory cells of the immune response. Therefore, we investigated in situ the nuclear localization of Rel/NF-kappa B proteins in accessory cells of the immune system by immunohistochemistry and double labeling by immunofluorescence from five normal human tonsils and five lymph nodes with follicular hyperplasia. Nuclear p65 and c-Rel proteins were found in all cell types including lymphocytes. In germinal centers GC, p50, p52, and RelB were found in the nuclei of FDC only and were not detected in the nuclei of CD68+ cells. In T cell areas, p50, p52, and RelB were found in the nuclei of HLA-DR+ cells with an antigen-presenting cell (APC) morphology. p52 and RelB were detected in the nuclei in both CD1a+ and CD68+ cells from the T cell area, whereas p50 was found only in CD68- and CD1a- cells. Cells with nuclear p50 were negative for the CD38, CD20 and CD2 markers. These results show that, physiologically, high levels of nuclear of p50, p52 and RelB are restricted to accessory cells of the immune system, which include FDC in GC, and DC and macrophages in the T cell zone, that specialized scavenger macrophages from GC do not have detectable levels of p52 and RelB, whereas macrophages from the T cell area, known to present the antigen to T cells, do have both nuclear p52 and RelB, and that in the T cell zone, p52 and RelB are located in nuclei of both CD1a+, CD68+ or both, cells APC, whereas p50 is restricted to CD1a- and CD68- APC. The different patterns of p50, p52 and RelB protein nuclear localization may provide insight into their different roles during the immune response in vivo. 

Signal transduction by DR3, a death domain-containing receptor related to TNFR-1 and CD95. Tumor necrosis factor receptor-1 (TNFR-1) and CD95 (also called Fas or APO-1) are cytokine receptors that engage the apoptosis pathway through a region of intracellular homology, designated the ``death domain. Another death domain-containing member of the TNFR family, death receptor 3 (DR3), was identified and was shown to induce both apoptosis and activation of nuclear factor kappaB. Expression of DR3 appears to be restricted to tissues enriched in lymphocytes. DR3 signal transduction is mediated by a complex of intracellular signaling molecules including TRADD, TRAF2, FADD, and FLICE. Thus, DR3 likely plays a role in regulating lymphocyte homeostasis. 

Cloning and expression of the Epstein-Barr virus-encoded dUTPase: patients with acute, reactivated or chronic virus infection develop antibodies against the enzyme. The gene encoding the Epstein-Barr virus (EBV)-specific dUTPase was amplified from virus DNA by PCR. The active enzyme was expressed in Escherichia coli and in insect cells as a non-fusion protein. The protein from E. coli specifically converted dUTP to dUMP and did not react with other dNTPs or NTPs. Preliminary experiments yielded a Km value of about 0.8 microM for dUTP. MAbs against the dUTPase reacted with a protein of approximately 31 kDa in 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-stimulated B cells harbouring either type 1 or type 2 EBV. The protein was found in untreated cells at low levels, whereas induction of the lytic replication cycle by TPA treatment or by providing the immediate early transactivator BZLF1 in trans resulted in increased expression. We demonstrated that the virus dUTPase isolated from EBV-infected cells is a phosphoprotein. The protein expressed in insect cells was used to test for the presence of specific antibodies in sera from normal, healthy carriers and from patients with various diseases. While the sera of EBV-negative individuals (0/3) or healthy carriers (0/33) did not contain detectable levels of antibodies, patients with mononucleosis (5/18), chronic EBV infection (2/7), EBV reactivation (7/20) and human immunodeficiency virus infection (5/24) showed elevated antibody titres against the enzyme. This indicated that the dUTPase is expressed during EBV replication and reactivation. The enzyme might therefore be a potential target for drug therapy under conditions of active DNA replication. 

Prostaglandin E2 induction of binding activity to CRE and AP-2 elements in human T lymphocytes. Prostaglandins of the E series are immunomodulatory agents which exert inhibitory as well as stimulatory effects on a variety of immune responses. Since it is known that PGE2 is able to increase cAMP levels, we investigated whether it can affect gene expression through the activation of the transcription factors which bind enhancer elements in the promoter regions of cAMP-regulated genes. Using electrophoretic mobility shift assay, we demonstrated that a short treatment of human T lymphocytes with PGE2 induces specific binding activity to CRE and AP-2, but not AP-1, DNA elements. Since the okadaic acid, a potent protein phosphatase inhibitor, prolongs the induction of the binding activity, phosphorylation events are likely to occur. This activity seems to be due to increased cAMP levels because forskolin and IBMX mimic the effects of PGE2. More interestingly, transfection experiments with CRE-CAT plasmide show that PGE2 activates the transcription of a CRE-containing promoter. These data support the positive role for PGE2 on some immune functions. 

Regulation of cytokine and cytokine receptor expression by glucocorticoids. Glucocorticoids (GCS) profoundly inhibit several aspects of T cell immunity largely through inhibition of cytokine expression at the transcriptional and posttranscriptional levels. GCS were also reported to act indirectly by inducing transforming growth factor-beta expression, which in turn blocks T cell immunity. In exerting their antiproliferative effects, GCS diffuse into target cells where they bind their cytoplasmic receptor, which in turn translocates to the nucleus where it inhibits transcription of cytokine genes through direct binding to the glucocorticoid response elements (GRE), which are located in the promoter region of cytokine genes or, alternatively, through antagonism of the action of transcription factors required for optimal transcriptional activation. In contrast to their inhibitory effects on cytokine expression, GCS up-regulate cytokine receptor expression that correlates with enhanced cytokine effects on target cells. In this review, we summarize the current state of knowledge of the mechanism of action of GCS, including the phenomenon of steroid-induced rebound, which ensues upon GCS withdrawal. 

The Oct-2 transcription factor. The Oct-2 transcription factor is a member of the POU (Pit-Oct-Unc) family of transcription factors and is expressed only in B lymphocytes and in neuronal cells but not in other cell types. The primary RNA transcript of the gene is subject to alternative splicing to yield different variants which can either activate or repress gene expression. The forms produced in B lymphocytes have a predominantly activating effect on gene expression whereas those produced in neuronal cells have a predominantly inhibitory effect and can repress the expression of both the herpes simplex virus immediate-early genes and the cellular tyrosine hydroxylase gene. Thus Oct-2 plays an important role in the regulation of cellular gene expression in both B cells and neuronal cells as well as in the control of viral latency. 

Tissue and cell-type specific expression of the tuberous sclerosis gene, TSC2, in human tissues. TSC2 is a gene on chromosome 16p13.3 associated with the autosomal dominant neurocutaneous disorder, tuberous sclerosis complex (TSC). By using a partial nucleotide sequence from the cloned TSC2 and polymerase chain reaction methodology, we constructed a digoxigenin-labeled complementary DNA probe to examine TSC2 gene expression in autopsy- or biopsy-derived human tissues by in situ hybridization. TSC2 messenger RNA was widely expressed in various cell types throughout the body, including epithelia, lymphocytes, and cells with endocrine functions, e.g., adrenal cortex and anterior pituitary. It was prominently and selectively (within the central nervous system) expressed in pyramidal cells of the cerebral cortex and other motor neurons, e.g., in spinal cord and brainstem nuclei. Visceral TSC2 expression was comparable in autopsy tissues from patients with and without TSC; TSC2 messenger RNA expression was most prominent in cells with a rapid mitotic rate and turnover, e.g., epithelia and lymphocytes, with central nervous system pyramidal cells and other neurons being an obvious exception, and/or in cells with important secretory/transport functions. This widespread expression of the TSC2 gene supports the view that it encodes a protein vital to cell growth and metabolism or one that functions as a tumor/growth suppressor. 

Cell specific expression of human Bruton's agammaglobulinemia tyrosine kinase gene (Btk) is regulated by Sp1- and Spi-1/PU.1-family members. Bruton's agammaglobulinemia tyrosine kinase (Btk) is a cytoplasmic tyrosine kinase involved in the human disease X-linked agammaglobulinemia (XLA). The gene is expressed in all hematopoietic cells with the exception of T-cells and plasma cells. For this expression pattern the first 280 bp upstream of the major transcriptional start site seems to be sufficient. In vitro footprinting analysis within this part of the promoter revealed two Sp1 binding sites as well as a PU-box. The transcription factor Spi-1/PU.1 as well as the closely related factor Spi-B bound to the PU-box in B-cells. In the erythroleukemia cell line K562, due to the absence of Spi-B, only PU.1 bound to the Btk promoter. Mutation of either site reduced the expression in transient transfection experiments. However, mutation of the PU box had no effect in the T-cell line Jurkat, where none of the Spi-1 family members is expressed. In addition Spi-B as well as PU.1 were able to transactivate Btk expression. In fetal liver of PU.1-/- mice, which lack lymphoid and myeloid cells, expression of Btk was reduced two- to threefold but not abolished. Collectively this study shows that expression of the Btk gene is regulated by the combined action of Sp1- and PU.1-family members. 

Elf-1 and Stat5 bind to a critical element in a new enhancer of the human interleukin-2 receptor alpha gene [published erratum appears in Mol Cell Biol 1997 Apr;17(4):2351] The interleukin 2 receptor alpha-chain (IL-2R alpha) gene is a key regulator of lymphocyte proliferation. IL-2R alpha is rapidly and potently induced in T cells in response to mitogenic stimuli. Interleukin 2 (IL-2) stimulates IL-2R alpha transcription, thereby amplifying expression of its own high-affinity receptor. IL-2R alpha transcription is at least in part controlled by two positive regulatory regions, PRRI and PRRII. PRRI is an inducible proximal enhancer, located between nucleotides -276 and -244, which contains NF-kappaB and SRE/CArG motifs. PRRII is a T-cell-specific enhancer, located between nucleotides -137 and -64, which binds the T-cell-specific Ets protein Elf-1 and HMG-I(Y) proteins. However, none of these proximal regions account for the induction of IL-2R alpha transcription by IL-2. To find new regulatory regions of the IL-2R alpha gene, 8.5 kb of the 5' end noncoding sequence of the IL-2R alpha gene have been sequenced. We identified an 86-nucleotide fragment that is 90% identical to the recently characterized murine IL-2-responsive element (mIL-2rE). This putative human IL-2rE, designated PRRIII, confers IL-2 responsiveness on a heterologous promoter. PRRIII contains a Stat protein binding site that overlaps with an EBS motif (GASd/EBSd). These are essential for IL-2 inducibility of PRRIII/CAT reporter constructs. IL-2 induced the binding of Stat5a and b proteins to the human GASd element. To confirm the physiological relevance of these findings, we carried out in vivo footprinting experiments which showed that stimulation of IL-2R alpha expression correlated with occupancy of the GASd element. Our data demonstrate a major role of the GASd/EBSd element in IL-2R alpha regulation and suggest that the T-cell-specific Elf-1 factor can serve as a transcriptional repressor. 

Association of TRAF1, TRAF2, and TRAF3 with an Epstein-Barr virus LMP1 domain important for B-lymphocyte transformation: role in NF-kappaB activation. The Epstein-Barr virus (EBV) transforming protein LMP1 appears to be a constitutively activated tumor necrosis factor receptor (TNFR) on the basis of an intrinsic ability to aggregate in the plasma membrane and an association of its cytoplasmic carboxyl terminus (CT) with TNFR-associated factors (TRAFs). We now show that in EBV-transformed B lymphocytes most of TRAF1 or TRAF3 and 5% of TRAF2 are associated with LMP1 and that most of LMP1 is associated with TRAF1 or TRAF3. TRAF1, TRAF2, and TRAF3 bind to a single site in the LMP1 CT corresponding to amino acids (aa) 199 to 214, within a domain which is important for B-lymphocyte growth transformation (aa 187 to 231). Further deletional and alanine mutagenesis analyses and comparison with TRAF binding sequences in CD40, in CD30, and in the LMP1 of other lymphycryptoviruses provide the first evidence that PXQXT/S is a core TRAF binding motif. The negative effects of point mutations in the LMP1(1-231) core TRAF binding motif on TRAF binding and NF-kappaB activation genetically link the TRAFs to LMP1(1-231)-mediated NF-kappaB activation. NF-kappaB activation by LMP1(1-231) is likely to be mediated by TRAF1/TRAF2 heteroaggregates since TRAF1 is unique among the TRAFs in coactivating NF-kappaB with LMP1(1-231), a TRAF2 dominant-negative mutant can block LMP1(1-231)-mediated NF-kappaB activation as well as TRAF1 coactivation, and 30% of TRAF2 is associated with TRAF1 in EBV-transformed B cells. TRAF3 is a negative modulator of LMP1(1-231)-mediated NF-kappaB activation. Surprisingly, TRAF1, -2, or -3 does not interact with the terminal LMP1 CT aa 333 to 386 which can independently mediate NF-kappaB activation. The constitutive association of TRAFs with LMP1 through the aa 187 to 231 domain which is important in NF-kappaB activation and primary B-lymphocyte growth transformation implicates TRAF aggregation in LMP1 signaling. 

CD40, but not lipopolysaccharide and anti-IgM stimulation of primary B lymphocytes, leads to a persistent nuclear accumulation of RelB. In this study we analyzed the effect of CD40 stimulation on the activity and nuclear appearance of Rel/nuclear factor kappaB (NF-kappaB) factors in primary murine B lymphocytes. We show that triggering of CD40 signaling pathway(s) by CD40 ligands expressed on L cells led to strong activation of an NF-kappaB-controlled beta-globin reporter gene in primary B lymphocytes from transgenic mice. Analyses of nuclear translocation of individual members of Rel proteins after CD40 induction of primary B cells showed a strong and long-lasting accumulation of RelB and, less pronounced, of c-Rel. LPS stimulation did not give rise to a persistent nuclear accumulation of RelB and c-Rel, whereas nuclear c-Rel, but not RelB, accumulated after B cell receptor stimulation. CD40 induced not only nuclear translocation but also de novo synthesis of RelB RNA and protein. S107 plasmacytoma cells, which express CD40 but are defective for the nuclear appearance of p50/p65-NF-kappaB, do not express RelB after CD40 stimulation. In S107 cells stably transfected with relB genes, stimulation of nuclear RelB translocation by CD40 was observed. These results indicate that stimulation of CD40 signaling pathways exerts a long-lasting stimulatory effect on both the transcription and nuclear translocation of RelB. Since LPS and anti-IgM were unable to activate RelB, CD40 appears to trigger a special program of gene expression involved in the proliferation and/or differentiation of B lymphocytes. 

Sterol dependent LDL-receptor gene transcription in lymphocytes from normal and CML patients. Sterol regulatory element (SRE) has been recognized to regulate various key genes coding for especially low density lipoprotein (LDL)-receptor, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and HMG-CoA synthase known to play a crucial role in the cholesterol feedback mechanism. The deranged cholesterol feedback mechanism has been widely recognised in initiation as well as progression of various types of cancers including chronic myeloid leukaemia (CML). Consequently, the present study was addressed to understand this phenomenon and revealed the existence of a unique 47 kDa protein factor having affinity for this SRE sequence in lymphocytes from normal subjects as well as its absence in lymphocytes from untreated CML patients. However, this factor appeared when the CML patients achieved complete haematological remission (CHR) through alpha-interferon therapy. Further, an inverse relationship was also observed between sterol modulated LDL-receptor gene transcription and the binding affinity of this 47 kDa factor to the SRE sequence. Based upon these results we propose that alpha-interferon through its receptor initiates phosphatidic acid dependent signalling which in turn regulates the affinity of 47 kDa sterol regulatory element binding factor as well as LDL-receptor gene transcription in lymphocytes from CML patients. 

Effects of Ara-C on neutral sphingomyelinase and mitogen- and stress- activated protein kinases in T-lymphocyte cell lines. Neutral sphingomyelinase (SMase) can be activated by extracellular signals to produce ceramide, which may affect mitogen-activated protein kinase (MAPK) activities. Neutral SMase activity was assessed in membranes from Jurkat, a human T-cell line, and EL4, a murine T-cell line. Ara-C activated SMase with 10 minutes in both Jurkat and EL4 cells, while phorbol ester (PMA) had no effect. PMA, but not Ara-C or ceramides, activated ERK MAPKS, in Jurkat and EL4. PMA acted synergistically with ionomycin to activate JNK MAPKs in Jurkat and EL4 within 10 minutes. Ara-C activated JNKs only after prolonged incubation (90-120 minutes). Thus, ceramide is not a positive signal for ERK activation in T-cell lines. The effects of Ara-C on JNK activity may be mediated through secondary response pathways. 

[Cortisone-resistant bronchial asthma] There is general agreement on the inflammatory pathogenesis of bronchial asthma: an accumulation of activated eosinophils, degranulated mast cells, T lymphocytes and in very severe forms, granulocytes has constantly been found in the bronchial mucosa. In allergic bronchial asthma, inflammation seems to be orchestrated predominantly by a subset of T lymphocytes, with a phenotype similar to the Th2 subset able to produce IL-4 and IL-5. Although corticosteroids are the most potent therapeutic agents used for this disease, their anti-inflammatory effect differs from patient to patient. Some criteria which can be used to define steroid-resistant bronchial asthma are listed here. This review analyzes various molecular alterations responsible for the deficient response to corticosteroid treatment observed in steroid-resistant bronchial asthmatic subjects. New knowledge on the mechanism of steroid resistance may have important implications for the treatment of chronic asthma and other diseases. 

Expression of Egr-1 correlates with the transformed phenotype and the type of viral latency in EBV genome positive lymphoid cell lines. In this paper we have investigated the role of Egr-1 in B cell growth regulation by examining the gene expression in a panel of B cell lines, including both EBV genome negative and EBV carrying cell lines. Egr-1 expression correlates with the cellular phenotype and the specific pattern of viral latency established within the individual cell lines. Thus, constitutive activation of Egr-1 gene is invariably associated with unrestricted expression of viral latent genes in all group III EBV genome carrying cell lines. In contrast, Egr-1 expression is abrogated in group I Burkitt tumor cells, irrespective of the EBV genome carrying status. Activated viral gene expression associated with phenotypic conversion of group I cell lines in to group II or III restores the Egr-1 gene expression. Several forms of EGR-1 protein are found within the different groups of cell lines, and the binding activity to DNA consensus sequences was investigated. Finally, time course analysis of Egr-1 expression during the early steps of EBV infection in vitro demonstrated that Egr-1 is upregulated within minutes from the initial interaction with the B lymphocyte. 

The proximal regulatory element of the interferon-gamma promoter mediates selective expression in T cells. Interferon-gamma (IFN-gamma) is produced by natural killer cells and certain subsets of T cells, but the basis for its selective expression is unknown. Within the region between -108 and -40 base pairs of the IFN-gamma promoter are two conserved and essential regulatory elements, which confer activation-specific expression in T cells. This report describes studies indicating that the most proximal of these two regulatory elements is an important determinant of its restricted expression. The proximal element is a composite site that binds members of the CREB/ATF, AP-1, and octamer families of transcription factors. Jun is essential for activation-induced transcription and binds preferably as a heterodimer with ATF-2. In contrast, CREB appears to dampen transcription from this element. The CpG dinucleotide in this element is selectively methylated in Th2 T cells and other cells that do not express IFN-gamma, and methylation markedly reduces transcription factor binding. As a target for DNA methylation and for binding of transcription factors that mediate or impede transcription, this element appears to play a central role in controlling IFN-gamma expression. 

Activation of nuclear factor-kappaB via T cell receptor requires a Raf kinase and Ca2+ influx. Functional synergy between Raf and calcineurin. Signals transduced via the TCR activate the transcription factor nuclear factor-kappaB (NF-kappaB), which, in turn, is critical to the transcriptional induction of many genes important for the proliferation and expression of a differentiated phenotype. Treatment of T cells with the protein kinase C activator PMA in combination with Ca2+ ionophores mimics this process, and the two agents are often substituted for TCR stimulation, bypassing the TCR. Here we identify intracellular signaling components involved in activation of NF-kappaB following TCR stimulation. TCR signaling was triggered by treating Jurkat T cells with PHA or anti-CD3 Abs, and NF-kappaB activation was monitored by electrophoretic mobility shift assays and/or by kappaB-dependent reporter assays. Contrary to the idea that protein kinase C is involved in TCR-mediated activation of NF-kappaB, high doses of staurosporine did not interfere with activation of NF-kappaB by PHA, while the same dose of staurosporine completely blocked activation by PMA. PHA-induced kappaB-dependent reporter activity was, however, effectively blocked by a dominant negative form of Raf-1, suggesting a critical role for a Raf kinase. The TCR-mediated activation of NF-kappaB was also dependent on a Ca2+ influx, because the Ca2+ channel blocker, SK&F 96365, as well as other agents that prevented the Ca2+ influx, inhibited NF-kappaB activation. Cotransfection of a constitutively active form of calcineurin largely substituted for the Ca2+ requirement and reversed the blockade by SK&F 96365. Consistent with these observations, coexpression of constitutively active forms of Raf-1 and calcineurin synergistically induced kappaB-dependent reporter activity, suggesting a physiologically relevant functional interaction between the kinase and the phosphatase. 

Induction of the CD11b gene during activation of the monocytic cell line U937 requires a novel nuclear factor MS-2 [published erratum appears in J Immunol 1999 Jul 15;163(2):1091] The differentiation of myeloid precursors into mature myelomonocytic cells is characterized by the induction of the gene encoding the beta2 integrin CD11b. The transcription factors Sp1 and PU.1 prime the CD11b promoter, but the nature of the factors responsible for its inducible expression are unknown. In addition to the CD11b gene, the homologous genes encoding CD11a and CD11c also exhibit inducible expression during myeloid differentiation. Therefore, we compared the nucleotide sequences of the CD11a, CD11b, and CD11c gene promoters to identify common elements that might contribute to inducible expression. This analysis identified one such element repeated four times within the CD11b promoter. Mutation of these elements indicated that two, MS-2beta and MS-2gamma, are critical to the induction of the CD11b gene during differentiation of the pro-monocytic cell line U937. Electrophoretic mobility shift assays indicate that MS-2beta and MS-2gamma interact with nuclear factors that are induced during U937 differentiation. These factors are detected at the time the CD11b promoter is activated. The molecular mass of these factors is approximately 28 kDa, and their DNA binding characteristics are indistinguishable from those of the novel nuclear factor MS-2. Taken together, our data indicate that MS-2 mediates induction of the CD11b gene as cells of the monocytic lineage mature. The presence of multiple potential binding sites for MS-2 in the promoter regions of a wide range of genes expressed in mature myeloid cells suggests this factor plays a general role in myeloid differentiation. 

HIV-1 LTR activity in human CD40-activated B lymphocytes is dependent on NF-kappaB. CD40-stimulated human B lymphocytes are highly permissive to a productive infection by the human immunodeficiency virus type 1. In these cells, nuclear factors involved in activation of the HIV-1 LTR, which contains the transcriptional control elements of the virus, are unknown. Transient expression assays with plasmids containing deleted parts of the LTR region linked to a reporter gene showed that the NF-kappaB binding site was essential for HIV-1 LTR activity in CD40-stimulated B lymphocytes. In addition, electrophoretic mobility shift and supershift assays revealed that important NF-kappaB binding activity composed of at least p50, p65, and c-Rel NF-kappaB subunits was present in nuclei of CD40-stimulated B cells. These results confirm at a molecular level the ability of HIV-1 to replicate in B cells and that this activity is strongly associated with NF-kappaB. 

Mutation of tyrosines 492/493 in the kinase domain of ZAP-70 affects multiple T-cell receptor signaling pathways. The protein-tyrosine kinase ZAP-70 is implicated, together with the Src kinase p56(lck), in controlling the early steps of the T-cell antigen receptor (TCR) signaling cascade. To help elucidate further the mechanism by which ZAP-70 regulates these initial events, we used a dominant-negative mutant approach. We overexpressed in the Jurkat T-cell line ZAP-70 mutated on Tyr-492 and Tyr-493 in the putative regulatory loop of its kinase domain. This mutant inhibited TCR-induced activation of nuclear factor of activated T cells by interfering with both intracellular calcium increase and Ras-regulated activation of extracellular signal-regulated kinases. Moreover, TCR-induced phosphorylation of pp36-38, thought to play a role upstream of these pathways, was found to be reduced. In contrast, overexpression of wild-type ZAP-70 induced constitutive activation of nuclear factor of activated T cells. The ZAP-70 mutant studied here could be phosphorylated on tyrosine when associated to the TCR zeta chain and was able to bind p56(lck). This result demonstrates that Tyr-492 and Tyr-493 are not responsible for the Src homology domain 2-mediated association of p56(lck) with ZAP-70. Our data are most consistent with a model in which recruitment to the TCR allows ZAP-70 autophosphorylation and binding to p56(lck), which in turn phosphorylates Tyr-492 and/or Tyr-493 with consequent up-regulation of the ZAP-70 kinase activity. ZAP-70 will then be able to effectively control phosphorylation of its substrates and lead to gene activation. 

Dexamethasone suppression test: corticosteroid receptors regulation in mononuclear leukocytes of young and aged subjects. The dexamethasone suppression test (DST) is considered an indicator of the function of the adrenal pituitary axis. The effect of the steroid is mediated by its binding to corticosteroid receptors. We previously suggested that the measurement of corticosteroid receptors in lymphocytes is an index of an analogous pattern in brain. In the present study, corticosteroid Type I and Type II receptors in mononuclear leukocytes were measured in 10 elderly subjects and in 9 young adults, before and after overnight DST (1 mg). Receptors were measured by radioreceptor assay. In all the subjects, dexamethasone was able to suppress plasma cortisol. The number of Type I and Type II receptors before the test was lower in elderly subjects than in adults. In the control group, dexamethasone produced a significant depression of Type I receptors (from 267 +/- 72 to 169 +/- 71 receptors per cell), which can be interpreted as a primary involvement of Type I receptors in the response to dexamethasone; Type II receptors decreased in half the subjects (from 2849 +/- 703 to 2345 +/- 569 receptors per cell). In elderly healthy subjects, Type II receptors were also significantly decreased (from 1796 +/- 671 to 720 +/- 345). We suggest that in young subjects Type II receptors are initially up-regulated by dexamethasone, and then down-regulated, while in aged subjects an up-regulation cannot be achieved, as suggested by the higher values of plasma cortisol usually found in aging subjects. 

Lymphocytes from CML patients lack a 47 kDa factor having affinity for a genomic sterol regulatory sequence. Deranged cellular cholesterol homeostasis has been widely recognized in the initiation as well as progression of various types of cancers including chronic myeloid leukaemia (CML). Since the human genomic sterol regulatory element (SRE) has been shown to regulate various key genes involved in this phenomenon, the present study revealed the existence of a unique 47 kDa protein factor having affinity for this SRE sequence in lymphocytes from normal subjects, as well as its absence in lymphocytes from untreated CML patients. However, this factor appeared when these CML patients achieved complete haematological remission (CHR) through alpha-interferon therapy. Furthermore, an inverse relationship was also observed between the LDL receptor gene expression at the transcriptional level and the binding affinity of this 47 kDa protein factor to the SRE sequence. Based upon these results we propose that this factor may have a role in pathophysiology of chronic myeloid leukaemia. 

A novel immunosuppressive factor in bovine colostrum blocks activation of the interleukin 2 gene enhancer at the NFAT site. A factor in bovine colostrum (colostrum inhibitory factor, CIF) inhibits interleukin 2 (IL2) production in activated T helper cells by blocking the accumulation of IL2 mRNA. To determine whether CIF blocks at the level of IL2 transcription, we introduced reporter plasmids into the human T leukemia cell line Jurkat by transient transfection. These contained the luciferase gene under the control of either the human IL2 upstream enhancer region (segments -326 to +45) or three repeats of the NFAT element contained within it (segments -255 to -285). Expression of luciferase in these cells was induced by phorbol myristate acetate plus a calcium ionophore. CIF inhibited induction of either construct as did cyclosporine, which is known to block activation of the NFAT element. CIF failed to inhibit several other enhancer elements. The NFAT-controlled luciferase gene system distinguishes CIF from other T cell inhibitory activities present in colostrum, in particular, TGF beta 1 and TGF beta 2 and the glucocorticoids. Stably transfected Jurkat cells behaved similarly to the transiently transfected ones with respect to inhibition by CIF and cyclosporine. The NFAT-luc assay is a useful technique for the rapid, sensitive measurement of CIF or other immunosuppressants with a similar mode of action. 

Synergistic interactions between overlapping binding sites for the serum response factor and ELK-1 proteins mediate both basal enhancement and phorbol ester responsiveness of primate cytomegalovirus major immediate-early promoters in monocyte and T-lymphocyte cell types. Cytomegalovirus (CMV) infection is nonpermissive or persistent in many lymphoid and myeloid cell types but can be activated in differentiated macrophages. We have shown elsewhere that both the major immediate-early gene (MIE) and lytic cycle infectious progeny virus expression can be induced in otherwise nonpermissive monocyte-like U-937 cell cultures infected with either human CMV (HCMV) or simian CMV (SCMV) by treatment with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Two multicopy basal enhancer motifs within the SCMV MIE enhancer, namely, 11 copies of the 16-bp cyclic AMP response element (CRE) and 3 copies of novel 17-bp serum response factor (SRF) binding sites referred to as the SNE (SRF/NFkappaB-like element), as well as four classical NFkappaB sites within the HCMV version, contribute to TPA responsiveness in transient assays in monocyte and T-cell types. The SCMV SNE sites contain potential overlapping core recognition binding motifs for SRF, Rel/NFkappaB, ETS, and YY1 class transcription factors but fail to respond to either serum or tumor necrosis factor alpha. Therefore, to evaluate the mechanism of TPA responsiveness of the SNE motifs and of a related 16-bp SEE (SRF/ETS element) motif found in the HCMV and chimpanzee CMV MIE enhancers, we have examined the functional responses and protein binding properties of multimerized wild-type and mutant elements added upstream to the SCMV MIE or simian virus 40 minimal promoter regions in the U-937, K-562, HL-60, THP-1, and Jurkat cell lines. Unlike classical NFkappaB sites, neither the SNE nor the SEE motif responded to phosphatase inhibition by okadaic acid. However, the TPA responsiveness of both CMV elements proved to involve synergistic interactions between the core SRF binding site (CCATATATGG) and the adjacent inverted ETS binding motifs (TTCC), which correlated directly with formation of a bound tripartite complex containing both the cellular SRF and ELK-1 proteins. This protein complex was more abundant in U-937, K-562, and HeLa cell extracts than in Raji, HF, BALB/c 3T3, or HL-60 cells, but the binding activity was altered only twofold after TPA treatment. A 40-fold stimulation of chloramphenicol acetyltransferase activity mediated by four tandem repeats of the SNE could be induced within 2 h (and up to 250-fold within 6 h) after addition of TPA in DNA-transfected U-937 cells, indicating that the stimulation appeared likely to be a true protein kinase C-mediated signal transduction event rather than a differentiation response. Slight differences in the sequence of the core SRF binding site compared with that of the classical c-Fos promoter serum response element, together with differences in the spacing between the SRF and ETS motifs, appear to account for the inability of the SCMV SNEs to respond to serum induction. 

Energy substrates, hormone responses and glucocorticoid binding in lymphocytes during intense physical exercise in humans following phosphocreatine administration. Eight healthy untrained male volunteers pedalled a cycle ergometer according to two exercise protocols: the first involved step-wise increasing physical exercise to maximal (MPE); the second involved prolonged (35 min) submaximal physical exercise (PPE) at 70% of the individual's maximal oxygen uptake. Each volunteer performed these exercise twice, following either an intravenous injection of phosphocreatine (PCr) or a placebo of an isotonic NaCl solution. Anaerobic threshold (AT) was determined from the point of departure of the ventilatory response from linearity and from the sudden increase in venous blood lactate concentrations during MPE. After exercise following placebo administration we observed increases in concentrations of blood substrates, plasma adrenocorticotropin (ACTH), growth hormone and cortisol and in the number of glucocorticoid receptors in lymphocytes without changes in the dissociation constant. Intravenous administration of PCr (starting 1 day before exercise) led to an increase in the total workload (on average by 5.8%) and in AT (on average by 6.8%) during MPE and to a better tolerance of exercise during PPE. Following PCr administration we observed lower blood lactate concentrations and different patterns of some enzyme activities, less pronounced changes in plasma ACTH and cortisol concentrations and in glucocorticoid binding in lymphocytes, but no changes in plasma growth hormone concentrations compared to the placebo. The results showed that intense physical exercise led not only to increases in blood hormone concentrations but also to an increase in the density of glucocorticoid receptors in lymphocytes. Intravenous PCr injection led to smaller changes in ACTH and cortisol concentrations as well as to a lower activation of glucocorticoid binding in lymphocytes. 

Acetylsalicylic acid and sodium salicylate inhibit LPS-induced NF-kappa B/c-Rel nuclear translocation, and synthesis of tissue factor (TF) and tumor necrosis factor alfa (TNF-alpha) in human monocytes. We have investigated the effects of acetylsalicylic acid and sodium salicylate on the LPS-induced synthesis of the pro-coagulant protein tissue factor (TF) and the pro-inflammatory protein tumor necrosis factor-alpha (TNF-alpha), as well as the prostaglandin PGE2 in human monocytes. Both drugs dose-dependently inhibited LPS-induced TF and TNF-alpha synthesis at the mRNA and the protein level, and reduced PGE2 production. As evidenced by electro mobility shift assay (EMSA) and the use of a NF-kappa B prototypic probe, these drugs probably exert their inhibitory effects by interference with the nuclear translocation of NF-kappa B/c-Rel proteins. These data may expand the understanding of the anti-thrombotic and anti-inflammatory effects of these drugs when activation of monocytes occurs. 

Octamer independent activation of transcription from the kappa immunoglobulin germline promoter. Previous analyses of immunoglobulin V region promoters has led to the discovery of a common octamer motif which is functionally important in the tissue-specific and developmentally regulated transcriptional activation of immunoglobulin genes. The germline promoters (Ko) located upstream of the J region gene segments of the kappa locus also contain an octamer motif (containing a single base pair mutation and referred to as the variant octamer) which has been shown previously to bind Oct-1 and Oct-2 transcription factors in vitro. To further elucidate the role of this variant octamer motif in the regulation of germline transcription from the unrearranged kappa locus, we have quantitated the relative binding affinity of Oct-1 and Oct-2 for the variant octamer motif and determined the functional role of this octamer motif in transcriptional activation. We find that, although the variant octamer motif binds Oct-1 and Oct-2 in vitro with 5-fold lower affinity than the consensus octamer motif, mutation of the variant octamer motif to either a consensus octamer or non-octamer motif has no effect on transcriptional activation from the germline promoter. We also find significant differences in activation of germline and V region promoters by kappa enhancers. Our results suggest that the germline promoters and V region promoters differ in their dependence on octamer for activation and respond differently to enhancer activation. These findings have important implications in regulation of germline transcription as well as concomitant activation of the V-J recombination of the kappa light chain locus. 

Characterization of the murine cyclin-dependent kinase inhibitor gene p27Kip1. The cyclin-dependent kinase inhibitor p27Kip1 plays an important role in regulating cell-cycle progression. p27Kip1 directly inhibits the catalytic activity of cyclin/cdks (cyclin-dependent kinase) complexes and/or interferes physically with cyclin/cdks activation by CAK. Interestingly, the expression level of p27Kip1 mRNA was maximal in resting Go T-cells and rapidly declined following anti-CD3 activation. We report here the cloning of p27Kip1 gene from murine genomic DNA and the functional analysis of the promoter of the p27Kip1 gene. The gene consists of at least three exons and spans more than 5.6 kb of DNA. Primer extension and nuclease S1 protection analysis revealed two major transcription initiation sites. The promoter region lacked a TATA box but contained potential binding sites for the transcriptional factors including two Sp1, CRE, Myb and NFkB located at positions -153, -178, -286, -875, and -1011, respectively. To analyze the regulatory mechanisms controlling p27Kip1 gene expression, we characterized the 5'-flanking region from nt -1609 to +178. The -326 to -615 region contained positive regulatory elements. 

Glucocorticoid-mediated inhibition of RANTES expression in human T lymphocytes. The chemokine RANTES has been implicated in the pathogenesis of allergic inflammatory diseases including asthma and rhinitis which are frequently treated with glucocorticoids. We observed that dexamethasone dramatically inhibited RANTES mRNA expression dose dependently in anti-CD3 activated Hut-78 T cells and human PBMCs. Inhibition of RANTES expression did not appear to be secondary to IL-2 inhibition and required binding to the intracellular glucocorticoid receptor. The down-regulation of RANTES expression by glucocorticoids in T cells may directly contribute to the efficacy of these agents in suppressing cellular infiltration and to their anti-inflammatory properties. 

The state of maturation of monocytes into macrophages determines the effects of IL-4 and IL-13 on HIV replication. The molecular mechanisms of the effects of IL-4 and IL-13 on HIV infection in human monocytes as they matured into monocyte-derived macrophages over 7 days were investigated using HIV-1(BaL), and low passage clinical strains. IL-4 and IL-13 up-regulated the expression of both genomic and spliced HIV mRNA in monocytes cultured on Teflon, as determined by Northern analysis and p24 Ag assay. Using a nuclear run-on assay, IL-4 stimulation was shown to enhance transcription by two- to threefold. IL-4 stimulated nuclear factor-kappaB nuclear translocation and binding before enhancement of HIV RNA expression. Conversely, IL-4 and IL-13 markedly and significantly inhibited HIV replication at the transcriptional level in monocyte-derived macrophages, and this occurred whether these cytokines were added before or after HIV infection. The reversal from stimulation to inhibition occurred after 3 to 5 days of adherence to plastic. IL-4 had no significant effect on HIV reverse transcription. The effect of both cytokines on the monocyte maturation/differentiation (CD11b, CD13, and CD26) and other macrophage markers (CD14 and CD68) was examined. IL-4 enhanced CD11b, but inhibited CD26 expression and delayed CD13 loss. IL-13 had similar effects on CD11b and CD13, but no effect on CD26. Hence, these cytokines do not simply enhance monocyte differentiation, but have complex and slightly divergent effects that impact on HIV replication probably through cell signaling pathways and nuclear factor-kappaB translocation. 

A novel SP-1 site in the human interleukin-1 beta promoter confers preferential transcriptional activity in keratinocytes. To investigate the mechanisms of transcriptional activation of interleukin-1beta (IL-1beta) in non-monocytic cells, we constructed a series of reporter plasmids with the bacterial chloramphenicol acetyltransferase gene linked to various parts of the human IL-1beta promoter and performed transient transfection experiments. We identified a promoter segment that activates transcription most efficiently in keratinocytes. Electrophoretic mobility shift assays (EMSA) with a 43-mer oligonucleotide derived from the functionally identified cis-acting element revealed specific complexes. By competition analysis with transcription factor consensus sequence oligonucleotides and by immunosupershift, transcription factor SP-1 or a closely related protein was shown to bind to this regulatory element. The closest match to the known SP-1 consensus sequence within the respective region is a TCCCCTCCCCT motif. Mutation of this motif almost completely, and specifically, abolished the binding of two low-mobility complexes and led to a 95% decrease of constitutive transcriptional activation of a reporter construct IL-1beta (-170/+108). Likewise, activation of this reporter construct by tumor necrosis factor-alpha depended on the SP-1 site. These observations suggest that a so-far-unrecognized SP-1 site in the human IL-1beta promoter may participate in the transcriptional regulation of this gene in keratinocytes. 

Effects of glucocorticoids on lymphocyte activation in patients with steroid-sensitive and steroid-resistant asthma. BACKGROUND: Glucocorticoids are important medications used to control the airway inflammation associated with asthma. Synthetic glucocorticoids vary in their binding affinity for the glucocorticoid receptor (GCR). METHODS: We compared hydrocortisone, beclomethasone dipropionate, triamcinolone acetonide, flunisolide, and budesonide with regard to their capacity to inhibit phytohemagglutinin-induced peripheral blood mononuclear cell proliferation from six patients with steroid-sensitive asthma and seven patients with steroid-resistant asthma. Peripheral blood mononuclear cell GCR binding affinities for dexamethasone and budesonide were also determined for both patient groups by using a radioligand binding assay and Scatchard analysis. RESULTS: Dose-dependent inhibition was demonstrated for all glucocorticoids in both patient groups, with the steroid-resistant group requiring approximately 2 log-fold more glucocorticoids for an equivalent degree of inhibition. The mean concentrations necessary to cause 50% inhibition of lymphocyte proliferation (IC50s) for the steroid-sensitive group ranged from 2 x 10(-10) mol/L for budesonide to 7 x 10(-8) mol/L for hydrocortisone, whereas the mean IC50s for the steroid-resistant group ranged from approximately 2 x 10(-8) mol/L for budesonide to greater than 10(-6) mol/L for hydrocortisone. In addition, a significant correlation was noted between the degree of inhibition of lymphocyte proliferation (IC50) and the binding affinity of dexamethasone to the GCR. Patients with steroid-resistant asthma have been shown to have a reduced GCR binding affinity. The GCR binding affinity for budesonide was significantly higher in both groups (i.e., lower dissociation constant) than that obtained for dexamethasone. CONCLUSION: These data suggest that glucocorticoids such as budesonide, by virtue of their high GCR binding affinities and greater ability to suppress lymphocyte proliferation, may therefore be beneficial in the management of difficult-to-control asthma. 

Involvement of nuclear factor-kappa B activation in IgE synthesis in human B cells. Nuclear factor-kappa B (NF-kappa B) is a transcription factor that binds to the consensus DNA sequence in the cis-acting elements of various genes. Although NF-kappa B activates the expression of many genes involved in immune and inflammatory responses, little is known about the role of NF-kappa B activation in the induction of IgE synthesis in human B cells. Therefore we first examined the participation of NF-kappa B in germline C epsilon transcription in a human Burkitt lymphoma B cell line, DND39. Stimulation of DND39 cells with IL-4 or anti-CD40 monoclonal antibody (mAb) activated phosphatidylinositol 3-kinase and subsequently induced nuclear expression of NF-kappa B, which was identified by electrophoretic mobility shift assays. n-Acetyl-L-cysteine (NAC), a potent antioxidant, blocked NF-kappa B activation caused by IL-4 and by anti-CD40 mAb. Although inhibition of IL-4-driven germline C epsilon transcription by NAC was not sufficient, the agent remarkably diminished anti-CD40 mAb-mediated up-regulation of germline C epsilon transcription. Second, we studied the effect of NAC on IgE synthesis in human normal B cells costimulated with IL-4 and anti-CD40 mAb. NAC was effective in inhibiting mature C epsilon transcription and IgE synthesis in the T cell-independent culture system. However, NAC did not significantly affect the spontaneous production of IgE by atopic B cells. These results indicate that NF-kappa B activity is commonly inducible in DND39 cells by IL-4 and anti-CD40 mAb and suggest that NF-kappa B sensitive to NAC may play a role in regulating IgE synthesis in B cells. 

[Molecular mechanisms of age-related lymphocyte dysfunction] Aging is classically accompanied by a dysregulation of the immunologic machinery. As a consequence, the immune response developed in senescent organisms is usually inappropriate, often inefficient, sometimes aberrant, and potentially detrimental. The age-associated immune dysfunction may be implicated to some degree in the extreme susceptibility of the elderly to infection and neoplasia and may even participate in various aspects of senescence. The current understanding of the molecular mechanisms underlying immunosenescence is still fragmentary. The most extensively studied phenomenon is the progressive decline in the proliferative capacities of T lymphocytes with aging. The loss of proliferative potential in response to antigenic challenge is a characteristic feature of immune senescence. It is directly implicated in the emergence of the age-related immune deficiency. The purpose of this review is to show how the accumulation of various biochemical lesions with advancing age leads to the failure of a critical cell function, namely the activation-induced lymphocyte proliferation. The biochemical modifications responsible for the defect in transduction and execution of the proliferative signal are analyzed as a function of age. The multiple alterations observed on the various biochemical pathways may appear as a consequence of a unique deleterious mechanism more fundamentally related to the process of senescence such as the inability to cope with oxidative stress. 

Engagement of the Lewis X antigen (CD15) results in monocyte activation. We previously reported that monocyte adhesion to tumor necrosis factor-alpha (TNF-alpha)-treated endothelial cells increased expression of tissue factor and CD36 on monocytes. Using immunological cross-linking to mimic receptor engagement by natural ligands, we now show that CD15 (Lewis X), a monocyte counter-receptor for endothelial selectins may participate in this response. We used cytokine production as a readout for monocyte activation and found that CD15 cross-linking induced TNF-alpha release from peripheral blood monocytes and cells from the monocytic cell line MM6. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) showed an increase in steady-state TNF-alpha mRNA after 3 to 4 hours of cross-linking. CD15 cross-linking also concomitantly increased interleukin-1 beta (IL-1 beta) mRNA, while no apparent change was observed in the levels of beta-actin mRNA, indicating specificity. To examine transcriptional regulation of cytokine genes by CD15 engagement, a CAT plasmid reporter construct containing IL-1 beta promoter/enhancer sequences was introduced into MM6. Subsequent cross-linking of CD15 increased CAT activity. CD15 engagement by monoclonal antibody also attenuated IL-1 beta transcript degradation, demonstrating that signaling via CD15 also had posttranscriptional effects. Nuclear extracts of anti-CD15 cross-linked cells demonstrated enhanced levels of the transcriptional factor activator protein-1, minimally changed nuclear factor-kappa B, and did not affect SV40 promoter specific protein-1. We conclude that engagement of CD15 on monocytes results in monocyte activation. In addition to its well-recognized adhesive role, CD15 may function as an important signaling molecule capable of initiating proinflammatory events in monocytes that come into contact with activated endothelium. 

Activation of signaling pathways and prevention of apoptosis by cytokines in eosinophils. Inhibition of apoptosis in eosinophils by cytokines such as IL-5 and GM-CSF may play an important role in the pathogenesis of allergic and parasitic disorders. Recently, there has been some progress in the understanding of the signal transduction pathways activated by these cytokines in eosinophils. The IL-3, IL-5 and GM-CSF receptors share a common signal transducer that possesses no intrinsic kinase domain. It has been shown that eosinophil stimulation by these cytokines is associated with increases in tyrosine phosphorylation of several cellular substrates. In the past few years, there has been some progress in defining the tyrosine kinases that are activated by the IL-3/IL-5/GM-CSF receptor beta-subunit in eosinophils. This review will concentrate on this topic and on its role for the regulation of eosinophil apoptosis. 

Stimulation of human peripheral blood mononuclear cells by zinc and related cations. Zinc is an important trace element for immune function. Here, we show that zinc addition in a serum- and lipopolysaccharide-free cell culture system leads to significantly enhanced levels of interleukin 1 beta (IL-1 beta) and tumour necrosis factor alpha (TNF-alpha) and to expression of the corresponding mRNA in human peripheral blood mononuclear cells (PBMC). Structurally related divalent cations like cobalt, nickel, and mercury also partially increase monokine secretion but to a much lower and thus insignificant extent. They fail to induce mRNA of TNF-alpha after 3 h of culture. Therefore, monokine induction is a zinc-specific effect influenced by the physicochemical properties of the ion. Confirmation of the unique significance of zinc for immune function provides a better understanding of the mechanisms of specific zinc-mediated immune modulation. 

Sequence analysis and expression in cultured lymphocytes of the human FOSB gene (G0S3). G0S3 is a member of a set of putative G0/G1 switch regulatory genes (G0S genes) selected by screening cDNA libraries prepared from human blood mononuclear cells cultured for 2 hr with lectin and cycloheximide. The sequence shows high homology with the murine FOSB gene, which encodes a component of the AP1 transcriptional regulator. Comparison of cDNA and genomic sequences reveals a 4-exon structure characteristic of the FOS family of genes. Freshly isolated cells show high levels of FOSB/G0S3 and FOS/G0S7 mRNAs, which decline rapidly during incubation in culture medium. The kinetics of expression suggest that the high initial levels are caused by the isolation procedure, and do not reflect constitutive expression. In cells preincubated for a day, levels of FOS mRNA reach a maximum 20 min after the addition of lectin and decline to control levels over the next 3 hr. Levels of FOSB mRNA reach a maximum 40 min after the addition of lectin and decline to control levels over the next 6 hr. In freshly isolated cells, both FOS and FOSB mRNAs increase dramatically in response to the protein synthesis inhibitor cycloheximide. In preincubated cells, the cycloheximide response is decreased, especially in the case of FOSB. These differences in expression of FOS and FOSB suggest different roles and regulation. Regions of low base order-dependent stem-loop potential in the region of the gene are defined. These indicate where base order has been adapted for purposes other than stem-loop stability (e.g., encoding proteins or gene regulation). Regions of low potential in a 68.5-kb genomic segment containing the FOSB gene suggest that the potential may help locate genes in uncharted DNA sequences. 

STAT1 pathway is involved in activation of caprine arthritis-encephalitis virus long terminal repeat in monocytes. The caprine arthritis-encephalitis virus (CAEV) long terminal repeat (LTR) is activated by gamma interferon (IFN-gamma) in promonocytic cells. We have previously shown that a 70-bp element is necessary and sufficient for the response of the CAEV LTR to this cytokine. At the 5' end, this 70-bp IFN-gamma response element contains sequence similarity to the gamma activated site (GAS). Here we demonstrate that the putative GAS element in the CAEV LTR binds specifically to a cellular factor induced by IFN-gamma in promonocytic cells. Substitution mutations in this consensus sequence eliminate binding of the inducible factor. The GAS element from the 70-bp motif is sufficient to confer responsiveness to IFN-gamma using a heterologous minimal promoter. Consistent with the binding data, the same mutations in the GAS element eliminate responsiveness to IFN-gamma in the context of both a functional CAEV LTR and a heterologous promoter. The cellular factor that binds to the GAS element is present from 5 min to 14 h after stimulation with IFN-gamma. Binding of the nuclear factor to the GAS element in the CAEV LTR is inhibited by antibody directed against STAT1 (p91/84). Thus, the GAS sequence in the CAEV LTR is essential for the response to IFN-gamma and a STAT1-like factor binds to this site. The STAT-1 signaling pathway provides at least one mechanism for activation of the CAEV LTR by IFN-gamma in monocytes. These data are the first demonstration of a role for a STAT family member in the regulation of a viral promoter. 

Signaling via IL-2 and IL-4 in JAK3-deficient severe combined immunodeficiency lymphocytes: JAK3-dependent and independent pathways. Both IL-2 and IL-4 bind to receptors containing the common gamma chain and JAK3. Although JAK3 is required for proper lymphoid development, the precise roles of this kinase in IL-2 and IL-4 signaling in lymphocytes have not been defined. Here, we have studied IL-2 and IL-4 signaling in B cell lines lacking JAK3. Although IL-2-induced phosphorylation of IL-2R beta, JAK1, and STAT5 all required the presence of JAK3, IL-4-mediated phosphorylation of JAK1, STAT6, and insulin receptor substrates 1 and 2 did not. However, IL-4-induced effects were clearly improved following JAK3 expression. These data indicate that IL-4 signaling occurs in the absence of of JAK3, but is comparatively inefficient. These findings may help in understanding the pathogenesis of the immunodeficiency that occurs with mutations of JAK3 and may suggest a mechanism for the pleiotropic effects of IL-4. 

Differentiation-dependent expression of a human carboxylesterase in monocytic cells and transcription factor binding to the promoter. Carboxylesterases play an important role in defense and clearance mechanisms of the monocyte/macrophage system. During the differentiation process of cells from the monocytic cell line THP-1 we observed a transient transcriptional upregulation of a human carboxylesterase analyzed by means of Northern blots. In PMA-treated THP-1 cells we could detect three major transcription initiation sites as revealed by Nuclease Protection Assay carried out with two overlapping antisense RNA probes. We have recently cloned the carboxylesterase upstream sequence and showed its basal promoter activity in CHO cells. Using electrophoretic mobility shift analysis we demonstrated that the promoter region spanning base pairs -1 to -275, which contains several putative binding sites for transcription factors, is bound by nuclear factors Sp1 and IRBP but not by C/EBPs. Taken together these data indicate that carboxylesterase gene transcription in THP-1 cells starts at multiple initiation sites and that Sp1 and IRBP may be critical factors for modulating the differentiation-dependent transcription of this human carboxylesterase gene. 

Glycation-dependent, reactive oxygen species-mediated suppression of the insulin gene promoter activity in HIT cells. Prolonged poor glycemic control in non-insulin-dependent diabetes mellitus patients often leads to a decline in insulin secretion from pancreatic beta cells, accompanied by a decrease in the insulin content of the cells. As a step toward elucidating the pathophysiological background of the so-called glucose toxicity to pancreatic beta cells, we induced glycation in HIT-T15 cells using a sugar with strong deoxidizing activity, D-ribose, and examined the effects on insulin gene transcription. The results of reporter gene analyses revealed that the insulin gene promoter is more sensitive to glycation than the control beta-actin gene promoter; approximately 50 and 80% of the insulin gene promoter activity was lost when the cells were kept for 3 d in the presence of 40 and 60 mM D-ribose, respectively. In agreement with this, decrease in the insulin mRNA and insulin content was observed in the glycation-induced cells. Also, gel mobility shift analyses using specific antiserum revealed decrease in the DNA-binding activity of an insulin gene transcription factor, PDX-1/IPF1/STF-1. These effects of D-ribose seemed almost irreversible but could be prevented by addition of 1 mM aminoguanidine or 10 mM N-acetylcysteine, thus suggesting that glycation and reactive oxygen species, generated through the glycation reaction, serve as mediators of the phenomena. These observations suggest that protein glycation in pancreatic beta cells, which occurs in vivo under chronic hyperglycemia, suppresses insulin gene transcription and thus can explain part of the beta cell glucose toxicity. 

A novel transcription factor regulates expression of the vacuolar H+-ATPase B2 subunit through AP-2 sites during monocytic differentiation. During monocyte-to-macrophage differentiation, the cellular content of vacuolar H+-ATPase (V-ATPase) increases more than 4-fold. We have shown previously that amplified expression of the B2 subunit of the V-ATPase occurs solely by increased transcription, and that the 5'-untranslated region of the B2 gene, containing multiple consensus binding sites for the transcription factors AP-2 and Sp1, is required for this expression. The present study demonstrates that AP-2 binding sequences are essential for increased transcription from the B2 promoter during monocyte-macrophage differentiation and that AP-2, expressed exogenously in THP-1 and other cells, activates transcription from the B2 promoter. In mobility shift assays, a nuclear factor from THP-1 and U-937 cells was identified that binds to several AP-2 response elements within the B2 promoter, but does not react with AP-2 antibodies, and has a DNA sequence binding affinity profile that differs from AP-2. These findings suggest that a novel AP-2-like transcription factor is responsible for V-ATPase B subunit amplification during monocyte differentiation. 

Identification of Bcd, a novel proto-oncogene expressed in B-cells. A novel B-cell derived (Bcd) oncogene has been isolated from the peripheral blood lymphocytes of one B-cell chronic lymphocytic leukemia (B-CLL) patient using DNA transfer and a mouse tumorigenicity assay. The Bcd proto-oncogene was activated by a truncation in the 5' UTR. It predicts for two open reading frames (ORFs). ORF1 consists of 240 bp that would encode 80 amino acids, while the major ORF2 consists of 648 bp capable of coding for 216 amino acids. Predicted peptide sequence of ORF2 contained a zinc finger domain which showed significant homology to GC box binding proteins BTEB2 and SP1. Transfection of an expression vector containing ORF2 but not full length cDNA was able to transform NIH3T3 cells and induce tumors in nude mice. Bcd mRNA transcripts of < or = 2.6 kb were selectively expressed in PBL and testis of healthy individuals. Within the PBL, Bcd gene expression was restricted to CD19+ B-cells and absent from CD14+ monocytes and T-cells. Bcd transcripts were detected in all normal PBL samples tested but not in several malignant human B-cell lines and not in 50% of B-cells from B-CLL patients. However, stimulation of B-cells from B-CLL patients under conditions which induced differentiation into plasma cells was associated with induction of Bcd gene expression. The Bcd gene may therefore play an important role in B-cell growth and development. 

GATA-1 DNA binding activity is down-regulated in late S phase in erythroid cells. We have set out to test a model for tissue-specific gene expression that relies on the early replication of expressed genes to sequester limiting activating transcription factors. Using an erythroid cell line, we have tested the changes in the DNA binding activity of the lineage-restricted transcription factor GATA-1 through the cell cycle. We find that GATA-1 activity is low in G1, peaks in mid-S phase, and then decreases in G2/M. In contrast, the binding activities of two ubiquitous transcription factors, Oct1 and Sp1, remain high in G2/M. GATA-1 protein and mRNA vary in a similar manner through the cell cycle, suggesting that the expression of the gene or the stability of its message is regulated. Although a number of transcription factors involved in the control of the cell cycle or DNA replication have been shown to peak in S phase, this is the first example of a lineage-restricted transcription factor displaying S phase-specific DNA binding activity. One interpretation of these data leads to a model in which the peak in GATA-1 DNA binding amplifies the effect of early replication on the activation of erythroid-specific genes at the same time as preventing activation of non-erythroid genes containing GATA-responsive elements. These results may also relate to recent data implicating GATA-1 function in apoptosis and cell cycle progression. 

Constitutive dephosphorylation and activation of a member of the nuclear factor of activated T cells, NF-AT1, in Tax-expressing and type I human T-cell leukemia virus-infected human T cells. The tax gene product of the type I human T-cell leukemia virus (HTLV-I) transactivates interleukin-2 (IL-2) gene through activation of an enhancer termed CD28 responsive element (CD28RE). Tax activation of the CD28RE is partially mediated by a member of the nuclear factor of activated T cells, NF-AT1. We have previously shown that NF-AT1 is constitutively active in Jurkat T cells stably transfected with the Tax cDNA, although the underlying molecular mechanism and physiological relevance of this finding remain unclear. In this report, we demonstrate that the active form of NF-AT1 is also present in the nuclei of HTLV-I-transformed T cells that express the Tax protein. Interestingly, the constitutive activation of NF-AT1 in these T cells is associated with its dephosphorylation. Furthermore, the dephosphorylated NF-AT1 can be rapidly rephosphorylated when the cells are incubated with cyclosporin A, an immunosuppressant inhibiting the serine/threonine phosphatase calcineurin. These results suggest that activation of NF-AT1 in Tax-expressing and HTLV-I-transformed T cells results from its dephosphorylation, which in turn may be due to deregulation of calcineurin. 

Elf-2, a rhombotin-2 binding ets transcription factor: discovery and potential role in T cell leukemia. Rhombotin-2 (RBTN-2) is a proto-oncogene only in the context of T lymphocytes. We postulated that the oncogenic effect of RBTN-2 in T cells is likely mediated by binding protein(s) with T cell-specific expression. By screening a T cell cDNA library, we identified a novel ets transcription factor that binds RBTN-2. This protein was named elf-2 because its DNA-binding domain is virtually identical to that of ets family member elf-1. Northern analyses showed similar levels of two elf-2 transcripts (3.5 kb and 3.8 kb) in all tissues except thymus. Thymocytes expressed four- to 10-fold greater amounts of the 3.5 kb transcript than other tissues. Sequence analyses of cDNA clones indicated that these transcripts encode proteins differing only at their amino termini, and likely represent alternatively spliced isoforms. These isoforms (elf-2a and elf-2b) contain identical RBTN-2 binding regions and DNA-binding domains. Elf-2b lacks a putative transactivation domain. The expression patterns suggest that RBTN-2 normally interacts equally with elf-2a and elf-2b. In contrast, when RBTN-2 is inappropriately expressed in T cells, RBTN-2 would interact predominantly with elf-2b; this interaction may lead to T cell proliferation. 

Isolation of a B-cell-specific promoter for the human class II transactivator. The class II transactivator (CIITA) is essential for the expression of major histocompatibility complex (MHC) class II antigens. The tissular patterns of CIITA and MHC class II gene expression are tightly correlated: CIITA mRNA is highly expressed in B cells, and is induced by interferon gamma (IFN-gamma) in macrophage and epithelial cell lines. We first isolated two overlapping cosmids encoding human CIITA which, when co-transfected, are able to restore MHC class II expression in a B-lymphoblastoid cell line (B-LCL) defective for CIITA. Subsequently, a 1.8 kilobase (kb) fragment of the CIITA promoter was isolated and sequenced. A motif presenting a strong similarity to an initiator was detected, as well as putative binding sites for Sp1, GATA-2, LyF-1, ets-1, AP1, and MZF1 transcription factors, and two GAS motifs. When introduced in front of a luciferase reporter gene, this promoter is able to direct a high luciferase activity in a human B-LCL. In contrast, luciferase expression was not stimulated after IFN-gamma treatment when the construct was transfected in macrophage or in epithelial cell lines. However, an induction of the human CIITA gene was observed in mouse macrophage and fibrosarcoma cell lines, when the cells were transfected with a cosmid containing the human CIITA gene, but lacking the 1.8 kb promoter described above. Taken together, these data suggest the existence of an intragenic promoter driving an IFN-gamma-inducible expression of CIITA. 

Shared gamma(c) subunit within the human interleukin-7 receptor complex. A molecular basis for the pathogenesis of X-linked severe combined immunodeficiency. Genetic evidence suggests that mutations in the gamma(c) receptor subunit cause X-linked severe combined immunodeficiency (X-SCID). The gamma(c) subunit can be employed in receptor complexes for IL-2, -4, -7, -9, and -15, and the multiple signaling defects that would result from a defective gamma(c) chain in these receptors are proposed to cause the severe phenotype of X-SCID patients. Interestingly, gene disruption of either IL-7 or the IL-7 receptor (IL-7R) alpha subunit in mice leads to immunological defects that are similar to human X-SCID. These observations suggest the functional importance of gamma(c) in the IL-7R complex. In the present study, structure/function analyses of the IL-7R complex using a chimeric receptor system demonstrated that gamma(c) is indeed critical for IL-7R function. Nonetheless, only a limited portion of the cytoplasmic domain of gamma(c) is necessary for IL-7R signal transduction. Furthermore, replacement of the gamma(c) cytoplasmic domain by a severely truncated erythropoeitin receptor does not affect measured IL-7R signaling events. These findings support a model in which gamma(c) serves primarily to activate signal transduction by the IL-7R complex, while IL-7R alpha determines specific signaling events through its association with cytoplasmic signaling molecules. Finally, these studies are consistent with the hypothesis that the molecular pathogenesis of X-SCID is due primarily to gamma(c)-mediated defects in the IL-7/IL-7R system. 

V3 loop of human immunodeficiency virus type 1 suppresses interleukin 2-induced T cell growth [published erratum appears in AIDS Res Hum Retroviruses 1997 May 1;13(7):633] We tested the effect of three linear or two loop peptides derived from the V3 region of the HTLV-III BH10 clone or the SF2 strain of human immunodeficiency virus type 1 on IL-2-driven T cell proliferation. V3-BH10, which consists of 42 amino acids and has a loop structure, suppressed IL-2-driven proliferation of all IL-2-dependent cells [Kit225, ED-40515(+), KT-3, 7-day PHA-blasts, and fresh peripheral blood mononuclear cells] tested, whereas it did not suppress the cell growth of IL-2-independent cell lines (Hut102, Molt-4, and Jurkat). This suppressive effect was also seen in IL-2-driven cell growth of CD8-positive lymphocytes purified from 7-day PHA-blasts, indicating that CD4 molecules were not required for the suppression. The treatment with anti-V3 loop monoclonal antibody (902 antibody) completely abolished the suppressive effect of V3-BH10. In addition, V3-BH10 generated the arrest of Kit225 cells and also purified CD8-positive lymphocytes in G1 phase in the presence of IL-2. Neither chromatin condensation nor DNA fragmentation was detected in Kit225 cells cultured with V3-BH10 and IL-2. V3-BH10 neither blocked radiolabeled IL-2 binding to IL-2 receptors nor affected tyrosyl phosphorylation of several cellular proteins (p120, p98, p96, p54, and p38), which is immediately induced by IL-2 stimulation. However, V3-BH10 enhanced IL-2-induced mRNA expression of c-fos but not c-myc or junB. Thus, the binding of V3 loop of gp120 to the cell surface molecule(s) appears to affect intracellular IL-2 signaling, which leads to the suppression of IL-2-induced T cell growth. 

Nuclear factor-kappaB activation in human monocytes stimulated with lipopolysaccharide is inhibited by fibroblast conditioned medium and exogenous PGE2. The nuclear factor kappaB (NF-kappaB) is thought to be crucially involved in the gene activation of several cytokines, including tumor necrosis factor alpha (TNF). Previously, we showed that fibroblast conditioned medium (FCM) is able to inhibit both TNF mRNA accumulation and protein release in peripheral blood-derived human monocytes (PBM) stimulated with lipopolysaccharide (LPS). In this study we have investigated the effect of FCM on the LPS-induced DNA-binding activity of NF-kappaB, by means of electrophoretic shift assay (EMSA). We provide evidence that FCM strongly inhibits the LPS-induced NF-kappaB activation in PBM. Furthermore, we show that exogenous PGE2 mimics the NF-kappaB inhibitory effect of FCM. On the other hand, FCM produced in the presence of indomethacin does not inhibit NF-kappaB activation by LPS. Our results lend further support to the hypothesis that inflammatory and immune responses of monocytes/macrophages may be modulated at the molecular level by signals originating from tissue structural cells such as fibroblasts. 

Differential interaction of nuclear factors with the PRE-I enhancer element of the human IL-4 promoter in different T cell subsets. The immunomodulatory cytokine IL-4 affects cells of most hemopoietic lineages. IL-4 is secreted by activated Th2 but not Th1 cells and plays a major role in the immune response by modulating the differentiation of naive Th cells toward the Th2 phenotype. We have previously identified an enhancer element, PRE-I, that is essential for the function of the human IL-4 promoter. To investigate the mechanisms responsible for tissue-specific expression of the IL-4 gene, we analyzed nuclear factors binding to the PRE-I site and compared the binding activities of these factors to the IL-4 promoter of Th1 and Th2 cells. We show that PRE-I interacts with PMA- and PMA/ionomycin-inducible, cyclosporin A-sensitive nuclear factors. Using anti-C/EBPbeta (NF-IL6), anti-C/EBPdelta (NF-IL6beta), anti-NF-ATc, anti-NF-ATp, anti-Fos, and anti-Jun Abs we demonstrate that the previously identified PRE-I binding factor POS-1 is composed of different transcription factors in different Th cell subsets. In the IL-4-producing Th0-like human Jurkat and mouse EL-4 cells, POS-1 (designated POS-1a) contains NF-IL6beta and Jun. In the mouse Th2 D10 cells and in the human Th2 clones, POS-1 (designated POS-1b) contains NF-IL6beta, Jun, and NF-ATc/p. In contrast, POS-1 was not found in nuclear extracts of human Th1 clones. These findings suggest that PRE-I may play a role in the differential regulation of IL-4 gene expression levels. 

Inhibitory effect of growth hormone on TNF-alpha secretion and nuclear factor-kappaB translocation in lipopolysaccharide-stimulated human monocytes. Several studies have pointed to a link between immune and endocrine systems, including a regulatory function of GH on monocyte activation. The present study demonstrates that human THP-1 promonocytic cells, engineered by gene transfer to constitutively produce human growth hormone (hGH), secreted depressed amounts of TNF-alpha in response to challenge by LPS. The effect of GH appears to occur in an autocrine fashion, since the inhibitory effect on TNF-alpha secretion by constitutive GH production could be abolished in the presence of anti-hGH mAb. The GH-induced inhibitory effect was also observed using normal human monocytes and monocyte-derived macrophages. Inhibition of TNF-alpha production by THP-1-hGH-transfected cells cultured in the presence of LPS is dependent on a selective pathway, since no inhibition of TNF-alpha production was observed when cells were cultured in the presence of PMA. Inhibition of TNF-alpha secretion by LPS-stimulated THP-1-hGH cells was associated with a decrease in nuclear translocation of nuclear factor-kappaB. The capacity of GH to inhibit LPS-induced TNF-alpha production by monocytes without altering other pathways leading to TNF-alpha production may be of potential relevance in septic shock, since GH is available for clinical use. 

Impaired induction of c-fos/c-jun genes and of transcriptional regulatory proteins binding distinct c-fos/c-jun promoter elements in activated human T cells during aging. The activation of transcriptional factor c-Fos/c-Jun AP-1 is essential for normal T cell responsiveness and is often impaired in T cells during aging. In the present study, we investigated whether aberrancies in the regulation of c-fos/c-jun at the mRNA or protein level might underlie the age-associated impairments of AP-1 in human T cells. Whereas T cells from young subjects stimulated with cross-linked anti-CD3epsilon mAb OKT3 plus PMA or with the lectin PHA plus PMA demonstrated considerable increases in c-Fos protein expression, the expression of c-Fos but not c-Jun was markedly reduced in stimulated T cells from certain elderly subjects. In addition, RNase protection assays revealed that anti-CD3/PMA-stimulated T cells from a substantial proportion of elderly subjects exhibited decreased levels of c-fos and/or c-jun mRNA compared to T cells from young subjects. Using electrophoretic mobility shift assays, the levels of nuclear regulatory proteins recognizing the AP-1 consensus TRE motif, the proximal c-jun TRE-like promoter element, and the c-fos serum response element (SRE) were determined in resting and stimulated T cells. Although the stimulation of T cells from young subjects resulted in coordinated increases of nuclear protein complexes binding the AP-1 TRE, c-jun TRE, and c-fos SRE DNA sequence motifs, age-related reductions in the activation of AP-1 were accompanied by decreased levels of c-jun TRE and c-fos SRE binding complexes. Furthermore, the nuclear protein complexes binding the SRE motif induced in activated T cells of young and elderly subjects contained serum response factor and Elk-1 pointing toward age-related defects in the activation of transcriptional regulatory proteins distinct from c-jun/AP-1. These results suggest that underlying aberrancies in the induction of c-fos/c-jun as well as their nuclear regulatory proteins may contribute to the age-related impairments of AP-1 activation in human T cells. 

Dissociation of the Jak kinase pathway from G-CSF receptor signaling in neutrophils. Activation of the granulocyte colony-stimulating factor receptor (G-CSFR) induces rapid tyrosine phosphorylation of multiple intracellular substrates in proliferating cells and nonproliferating, terminally differentiated neutrophils. The kinases that couple ligand binding to tyrosine phosphorylation of cellular substrates by the G-CSFR with activation of specific functional programs are largely unknown. In this study, we examined early signaling events in proliferating and terminally differentiated cells following G-CSF stimulation to determine whether identical signaling cascades are activated. In murine Ba/F3 cells transfected with the human G-CSFR and NFS-60 cells constitutively expressing the murine G-CSFR, G-CSF induced tyrosine phosphorylation and activation of Jak1, Jak2, and Tyk2. Tyrosine phosphorylation of Stat3 and, to a lesser extent, Stat1 was also detected following G-CSF stimulation. Using a mitogenically incompetent human G-CSFR mutant in which Pro639 and Pro641 were substituted by alanine, the box 1 PDP motif was found to be required for activation of Jak kinases, tyrosine phosphorylation of the G-CSFR, and recruitment of Stat proteins. Notably, no activation of Jak1, Jak2, Tyk2, Stat1, or Stat3 was observed in neutrophils following G-CSF stimulation. In addition, there was no detectable activation in neutrophils of the recently cloned Jak3 kinase, which has been reported to be expressed at high levels as myeloid cells undergo terminal neutrophilic maturation. These results indicate a lack of involvement of Jak kinases in signaling by the G-CSFR in neutrophils, and suggest utilization of alternative signal transduction pathways distinct from those in proliferating cells. Activation of the Jak-Stat pathway correlates with proliferative signaling by the G-CSFR and requires the membrane-proximal box 1 PXP motif, which is conserved in members of the cytokine receptor superfamily. 

Adenovirus E1B 19K protein is required for efficient DNA replication in U937 cells. The adenovirus E1B 19K gene plays an essential role in transformation of primary rodent cells in cooperation with E1A and in the inhibition of apoptosis during lytic infection. It has been shown that this E1B 19K protein is not necessary for viral DNA replication in human cell lines, such as HeLa and KB. We reported here that the E1B 19K mutant viruses were unable to replicate efficiently in a monocyte cell line, U937. Viral DNA synthesis and late gene expression were found to be defective in U937 cells infected with E1B 19K mutants compared with wild-type virus. Early viral RNA splicing patterns also differ between wild-type and dl337-infected cells. Furthermore, the defect in viral replication could be complemented by dl312 virus defective in E1A expression 4 days after infection with E1B mutants, suggesting persistence of the E1B mutant genome in the infected cells despite defective onset of the late phase of replication. These results imply that E1B 19K is required for efficient viral DNA replication in U937 cells. Inefficient DNA replication is also found in another monocyte cell line, THP-1. 

Interaction of transcription factors RFX1 and MIBP1 with the gamma motif of the negative regulatory element of the hepatitis B virus core promoter. The negative regulatory element (NRE) of the hepatitis B virus (HBV) core promoter contains three subregions which act synergistically to suppress core promoter activity. One of these subregions, NRE gamma, is active in both HeLa cervical carcinoma cells and Huh7 hepatoma cells and was found to be bound by a protein factor present in both cell types. Here we show that the transcription factor RFX1 can bind to NRE gamma and transactivate the core promoter through this site. Mutations which abrogated the gene-suppressive activity of NRE gamma prevented RFX1 from binding to NRE gamma. In addition, RFX1 can bind simultaneously, most likely as a heterodimer, with the transcription factor MIBP1 to NRE gamma. In the absence of a cloned MIBP1 gene for further studies, we hypothesize that RFX1 acts with MIBP1 to negatively regulate the core promoter activity through the NRE gamma site. The ability of RFX1 to transactivate the core promoter raises the possibility that RFX1 may play a dual role in regulating HBV gene expression. 

Evaluation of monoclonal anti-D reagents using D variant cells. Monoclonal anti-D antibodies submitted to the Third Monoclonal International Workshop were evaluated against a number of D variant cells using standard serological techniques. The monoclonal antibodies were able to discriminate between the cells of Categories Va, VI and DFR but not Category III cells. Cells within each category did not give any aberrant results. The Rh:33 cells behaved as normal Rh(D) positive cells. 

Pancreatic development and maturation of the islet B cell. Studies of pluripotent islet cultures. Pancreas organogenesis is a highly regulated process, in which two anlage evaginate from the primitive gut. They later fuse, and, under the influence of the surrounding mesenchyme, the mature organ develops, being mainly composed of ductal, exocrine and endocrine compartments. Early buds are characterized by a branching morphogenesis of the ductal epithelium from which endocrine and exocrine precursor cells bud to eventually form the two other compartments. The three compartments are thought to be of common endodermal origin; in contrast to earlier hypotheses, which suggested that the endocrine compartment was of neuroectodermal origin. It is thus generally believed that the pancreatic endocrine-lineage possesses the ability to mature along a differentiation pathway that shares many characteristics with those of neuronal differentiation. During recent years, studies of insulin-gene regulation and, in particular, the tissue-specific transcriptional control of insulin-gene activity have provided information on pancreas development in general. The present review summarizes these findings, with a special focus on our own studies on pluripotent endocrine cultures of rat pancreas. 

Induction of vascular cell adhesion molecule-1 by low-density lipoprotein. Low-density lipoprotein (LDL) is a well-established risk factor for atherosclerosis. When endothelial cells are incubated with this lipoprotein in pathophysiologic amounts, the cells are activated. Among the documented cellular responses to LDL is increased recruitment of monocytes, which are believed to play a major role in promoting intimal plaque formation. The findings presented here link an atheogenic lipoprotein, LDL, with the induction of an adhesion molecule important in atherogenesis Human LDL induces the vascular cell adhesion molecule-1 (VCAM-1) transcriptionally with an increase in mRNA levels through activation of the VCAM promoter. This effect is blocked by anti-VCAM antibodies. After a 2-day incubation in LDL, the binding of NF-kappa B, which is believed to be a key oxidative-stress sensor for VCAM regulation, remains at basal level. In contrast, the binding activities of AP-1 and GATA, on the other hand, are increased by LDL. Thus, a component of LDL-enhanced endothelial recruitment of monocytes is attributed to VCAM-1 expression, which appears to be mediated through AP-1 and GATA. These data identify LDL as a VCAM-inducer possibly distinct from cytokines and endotoxin. 

Cloning and expression of the glucocorticoid receptor from the squirrel monkey (Saimiri boliviensis boliviensis), a glucocorticoid-resistant primate. New World primates such as the squirrel monkey have elevated cortisol levels and glucocorticoid resistance. We have shown that the apparent binding affinity of the glucocorticoid receptor in squirrel monkey lymphocytes is 5-fold lower than that in human lymphocytes (apparent Kd, 20.9 +/- 1.8 and 4.3 +/- 0.2 nmol/L, respectively; n = 3), consistent with previous studies in mononuclear leukocytes isolated from the two species. As a first step in understanding the mechanism of decreased binding affinity in New World primates, we used reverse transcription-PCR to clone the glucocorticoid receptor from squirrel monkey liver and have compared the sequence to receptor sequences obtained from owl monkey liver, cotton-top tamarin B95-8 cells, and human lymphocytes. The squirrel monkey glucocorticoid receptor is approximately 97% identical in nucleotide and amino acid sequence to the human receptor. The ligand-binding domain (amino acids 528-777) of the squirrel monkey glucocorticoid receptor contains four amino acid differences (Ser551 to Thr, Ser616 to Ala, Ala618 to Ser, and Ile761 to Leu), all of which are present in owl monkey and cotton-top tamarin receptors. The DNA-binding domain (amino acids 421-486) is completely conserved among human, squirrel monkey, owl monkey, and cotton-top tamarin receptors. Twenty-two differences from the human sequence were found in the N-terminal region (amino acids 1-421) of the squirrel monkey receptor. None of the substitutions in the ligand-binding domain matched mutations known to influence binding affinity in other species. To determine whether the substitutions per se were responsible for decreased affinity, squirrel monkey and human glucocorticoid receptors were expressed in the TNT Coupled Reticulocyte Lysate System. Expressions of human and squirrel monkey glucocorticoid receptors and a squirrel monkey receptor in which Phe774 was mutated to Leu (F774L) were similar. When expressed in the TNT System, squirrel monkey and human glucocorticoid receptors had similar, high affinity binding for dexamethasone (apparent Kd, 5.9 +/- 1.2 and 4.3 +/- 0.5 nmol/L, respectively; n = 3), whereas the squirrel monkey F774L receptor had lower affinity binding (apparent Kd, 20.4 +/- 2.0 nmol/L; n = 3). Thus, substitutions within the ligand-binding domain of the squirrel monkey glucocorticoid receptor cannot account for the decreased binding affinity of these receptors in squirrel monkey cells. Rather, the binding affinity is probably influenced by the expression of cytosolic factors that affect glucocorticoid receptor function. 

The NF-kappa B inhibitor, tepoxalin, suppresses surface expression of the cell adhesion molecules CD62E, CD11b/CD18 and CD106. Tepoxalin, a dual enzyme inhibitor of cyclooxygenase and 5-lipoxygenase has been shown to inhibit T-cell activation. Its immunosuppressive property is distinct from cyclosporin because only tepoxalin, but not cyclosporin, suppresses NF-kappa B activation. Here we report that tepoxalin selectively inhibits intercellular adhesion molecule-1 (ICAM-1, CD54)/MAC-1 (CD11b/CD18) dependent adhesion of polymorphonuclear cells to IL-1 activated human umbilical vein endothelial cells. The mechanism of inhibition is related to the surface expression of several cell adhesion molecules. Flow cytometry analyses on cultured cells that were treated with tepoxalin or antisense oligonucleotides to the P65/p50 subunit of NF-kappa B, and then stimulated with PMA, revealed a reduced expression of CD11b/CD18 on monocytic HL60 cells, and endothelial adhesion molecule-1 (CD62E) and vascular adhesion molecule-1 (CD106) on human umbilical vein endothelial cells. Expression of other adhesion molecules such as lymphocyte function associated-antigen-1 (CD11a/CD18) and CD54 were unaffected. Tepoxalin also inhibited the secretion of a NF-kappa B regulated chemokine, IL-8, a known inducer of CD11b/CD18 expression. Thus the suppression of CD11b/CD18 expression by tepoxalin may involve IL-8. Our results suggest that by inhibiting NF-kappa B activation, surface expression of several adhesion molecules can be modulated and that tepoxalin may be useful in treating selected adhesion mediated events such as leukocyte migration or atherosclerotic plaque formation. 

Characterization of the human platelet/endothelial cell adhesion molecule-1 promoter: identification of a GATA-2 binding element required for optimal transcriptional activity. Platelet/endothelial cell adhesion molecule-1 (PECAM-1) is a 130-kD member of the Ig gene superfamily that is expressed on platelets, endothelial cells, and certain leukocyte subsets. To examine the factors controlling vascular-specific expression of PECAM-1, we cloned the 5'-flanking region of the PECAM-1 gene and analyzed its transcriptional activity. 5'-Rapid amplification of cDNA ends (5'-RACE) analysis showed that transcription initiation occurred at several closely spaced nearby sites originating approximately 204 bp upstream from the translation start site. Analysis of the sequence immediately upstream from the transcription initiation site (TIS) showed no canonical TATA or CAAT elements, however an initiator element commonly found in TATA-less promoters encompassed the TIS. 5'-serially truncated PECAM-1 promoter segments cloned in front of a luciferase reporter drove transcription in both a lineage- and orientation-specific manner. Putative cis-acting control elements present within a 300-bp core promoter included two ets sites, an Sp1 site, tandem E-box domains, two GATA-associated sites (CACCC), an AP-2 binding site, and a GATA element at -24. Mutational analysis showed that optimal transcriptional activity required the GATA sequence at position -24, and gel-shift assays further showed that the GATA-2 transcription factor, but not GATA-1, bound to this region of the PECAM-1 promoter. Understanding the cis- and transacting factors that regulate the tissue-specific expression of PECAM-1 should increase our understanding of the mechanisms by which vascular-specific gene expression is achieved. 

Rapid Ca2+-mediated activation of Rap1 in human platelets. Rap1 is a small, Ras-like GTPase whose function and regulation are still largely unknown. We have developed a novel assay to monitor the active, GTP-bound form of Rap1 based on the differential affinity of Rap1GTP and Rap1GDP for the Rap binding domain of RalGDS (RBD). Stimulation of blood platelets with alpha-thrombin or other platelet activators caused a rapid and strong induction of Rap1 that associated with RBD in vitro. Binding to RBD increased from undetectable levels in resting platelets to >50% of total Rap1 within 30 s after stimulation. An increase in the intracellular Ca2+ concentration is both necessary and sufficient for Rap1 activation since it was induced by agents that increase intracellular Ca2+ and inhibited by a Ca2+-chelating agent. Neither inhibition of translocation of Rap1 to the cytoskeleton nor inhibition of platelet aggregation affected thrombin-induced activation of Rap1. In contrast, prostaglandin I2 (PGI2), a strong negative regulator of platelet function, inhibited agonist-induced as well as Ca2+-induced activation of Rap1. From our results, we conclude that Rap1 activation in platelets is an important common event in early agonist-induced signalling, and that this activation is mediated by an increased intracellular Ca2+ concentration. 

Expression of erythroid-specific genes in megakaryoblastic disorders. Currently available data indicate that erythroid and megakaryocytic differentiation pathways are closely related to each other, and there may exist progenitor cells common to those two lineages may exist. Acute megakaryoblastic leukemia (AML-M7) and transient myeloproliferative disorder in Down's syndrome (TMD) are characterized by rapid growth of abnormal blast cells which express megakaryocytic markers. These blast cells express lineage-specific transcription factors such as GATA-1 common to these lineages and frequently express erythroid-specific mRNAs such as gamma-globin and erythroid delta-aminolevulinate synthase (ALAS-E), indicating that most of the blasts in M7 and TMD cases have erythroid and megakaryocytic phenotypes. These results suggest that blasts in M7 and TMD may correspond to progenitors of both erythroid and megakaryocytic lineages. 

Nuclear NF-ATp is a hallmark of unstimulated B cells from B-CLL patients. B lymphocytes from the peripheral blood of patients with chronic lymphocytic leukaemia (CLL) were analysed for the nuclear presence and DNA binding of a panel of transcription factors which are involved in the gene control of lymphoid cells. The following transcription factors were studied: the Octamer factors Oct-1 and Oct-2, members of the AP-1 factor family, NF-AT factors, in particular NF-ATp, and members of the Rel/NF-kB family. We show that the constitutive nuclear translocation of NF-ATp, a member of the growing family of NF-AT factors, is a hallmark of nonstimulated B cells from CLL patients that distinguishes B-CLL cells from 'normal' B lymphocytes. Constitutive nuclear appearance was also observed for NF-kB2/p52. Constitutive binding of further factor proteins to DNA, such as JunD, c-Fos and FosB, was detected in several patients whereas the localisation and DNA binding of other factors such as c-Jun, RelA/p65 and c-Rel was unaltered. It is remarkable that in B-CLL cells the nuclear appearance and DNA binding of specific transcription factors is dramatically affected whereas other members of the same factor family remained unaltered in these leukemic cells. It remains to be shown which molecular events lead to the specific 'pre-activation', i.e. constitutive nuclear translocation and DNA binding, of these members of NF-AT, NF-kB and AP-1 factor families. 

Physical and functional interaction between the human T-cell lymphotropic virus type 1 Tax1 protein and the CCAAT binding protein NF-Y. Tax1, a potent activator of human T-cell lymphotropic virus type 1 (HTLV-1) transcription, has been shown to modulate expression of many cellular genes. Tax1 does not bind DNA directly but regulates transcription through protein-protein interactions with sequence-specific transcription factors. Using the yeast two-hybrid system to screen for proteins which interact with Tax1, we isolated the B subunit of the CCAAT binding protein NF-Y from a HeLa cDNA library. The interaction of Tax1 with NF-YB was specific in that NF-YB did not interact with a variety of other transcription factors, including human immunodeficiency virus Tat, human papillomavirus E6, and Bicoid, or with the M7 (amino acids 29CP-AS) Tax1 mutant. However, NF-YB did interact with the C-terminal Tax1 mutants M22 (130TL-AS) and M47 (319LL-RS). We also show that in vitro-translated NF-YB specifically bound to a glutathione S-transferase-Tax1 fusion protein. Further, Tax1 coimmunoprecipitated with NF-Y from nuclear extracts of HTLV-1-transformed cells, providing evidence for in vivo interaction of Tax1 and NF-YB. We further demonstrate that Tax1 specifically activated the NF-Y-responsive DQbeta promoter, as well as a minimal promoter which contains only the Y-box element. In addition, mutation of the Y-box element alone abrogated Tax1-mediated activation. Taken together, these data indicate that Tax1 interacts with NF-Y through the B subunit and that this interaction results in activation of the major histocompatibility complex class II promoter. Through activation of this and other NF-Y driven promoters, the Tax1-NF-Y interaction may play a critical role in causing cellular transformation and HTLV-1 pathogenesis. 

Cloning of the novel human myeloid-cell-specific C/EBP-epsilon transcription factor. Chicken NF-M transcription factor, in cooperation with either c-Myb or v-Myb, is active in the combinatorial activation of myeloid-cell-specific genes in heterologous cell types, such as embryonic fibroblasts. In humans, similar effects were observed with homologous members of the CCAAT/enhancer-binding protein (C/EBP) family of transcriptional regulators, especially the human homolog of chicken NF-M, C/EBP-beta (NF-IL6). However, the NF-IL6 gene is expressed in a variety of nonmyeloid cell types and is strongly inducible in response to inflammatory stimuli, making it an unlikely candidate to have an exclusive role as a combinatorial differentiation switch during myelopoiesis in human cells. By using a reverse transcription-PCR-based approach and a set of primers specific for the DNA-binding domains of highly homologous members of the C/EBP family of transcriptional regulators, we have cloned a novel human gene encoding a member of the C/EBP gene family, identified as the human homolog of CRP1, C/EBP-epsilon. A 1.2-kb cDNA encoding full-length human C/EBP-epsilon was cloned from a promyelocyte-late myeloblast-derived lambda gt11 library. Molecular analysis of the cDNA and genomic clones indicated the presence of two exons encoding a protein with an apparent molecular mass of 32 kDa and a pI of 9.5. Primer extension analysis of C/EBP-epsilon mRNA detected a single major transcription start site approximately 200 bp upstream of the start codon. The putative promoter area is similar to those of several other myeloid-cell-specific genes in that it contains no TATAAA box but has a number of purine-rich stretches with multiple sites for the factors of the Ets family of transcriptional regulators. Northern blot analyses indicated a highly restricted mRNA expression pattern, with the strongest expression occurring in promyelocyte and late-myeloblast-like cell lines. Western blot and immunoprecipitation studies using rabbit anti-C/EBP-epsilon antibodies raised against the N-terminal portion of C/EBP-epsilon (amino acids 1 to 115) showed that C/EBP-epsilon is a 32-kDa nuclear phosphoprotein. The human C/EBP-epsilon protein exhibited strong and specific binding to double-stranded DNA containing consensus C/EBP sites. Cotransfection of the C/EBP-epsilon sense and antisense expression constructs together with chloramphenicol acetyltransferase reporter vectors containing myeloid-cell-specific c-mim and human myeloperoxidase promoters suggested a role for C/EBP-epsilon transcription factor in the regulation of a subset of myeloid-cell-specific genes. Transient tranfection of a promyelocyte cell line (NB4) with a C/EBP-epsilon expression plasmid increased cell growth by sevenfold, while antisense C/EBP-epsilon caused a fivefold decrease in clonal growth of these cells. 

Transcriptional regulation of the ferritin heavy-chain gene: the activity of the CCAAT binding factor NF-Y is modulated in heme-treated Friend leukemia cells and during monocyte-to-macrophage differentiation. The ferritin H-chain gene promoter regulation was analyzed in heme-treated Friend leukemia cells (FLCs) and during monocyte-to-macrophage differentiation. In the majority of cell lines studied, the regulation of ferritin expression was exerted mostly at the translational level. However, in differentiating erythroid cells, which must incorporate high levels of iron to sustain hemoglobin synthesis, and in macrophages, which are involved in iron storage, transcriptional regulation seemed to be a relevant mechanism. We show here that the minimum region of the ferritin H-gene promoter that is able to confer transcriptional regulation by heme in FLCs to a reporter gene is 77 nucleotides upstream of the TATA box. This cis element binds a protein complex referred to as HRF (heme-responsive factor), which is greatly enhanced both in heme-treated FLCs and during monocyte-to-macrophage differentiation. The CCAAT element present in reverse orientation in this promoter region of the ferritin H-chain gene is necessary for binding and for gene activity, since a single point mutation is able to abolish the binding of HRF and the transcriptional activity in transfected cells. By competition experiments and supershift assays, we identified the induced HRF as containing at least the ubiquitous transcription factor NF-Y. NF-Y is formed by three subunits, A, B, and C, all of which are necessary for DNA binding. Cotransfection with a transdominant negative mutant of the NF-YA subunit abolishes the transcriptional activation by heme, indicating that NF-Y plays an essential role in this activation. We have also observed a differential expression of the NF-YA subunit in heme-treated and control FLCs and during monocyte-to-macrophage differentiation. 

Characterization of a mutant cell line that does not activate NF-kappaB in response to multiple stimuli. Numerous genes required during the immune or inflammation response as well as the adhesion process are regulated by nuclear factor kappaB (NF-kappaB). Associated with its inhibitor, I kappaB, NF-kappaB resides as an inactive form in the cytoplasm. Upon stimulation by various agents, I kappaB is proteolyzed and NF-kappaB translocates to the nucleus, where it activates its target genes. The transduction pathways that lead to I kappaB inactivation remain poorly understood. In this study, we have characterized a cellular mutant, the 70/Z3-derived 1.3E2 murine pre-B cell line, that does not activate NF-kappaB in response to several stimuli. We demonstrate that upon stimulation by lipopolysaccharide, Taxol, phorbol myristate acetate, interleukin-1, or double-stranded RNA, I kappaB alpha is not degraded, as a result of an absence of induced phosphorylation on serines 32 and 36. Neither a mutation in I kappaB alpha nor a mutation in p50 or relA, the two major subunits of NF-kappaB in this cell line, accounts for this phosphorylation defect. As well as culminating in the inducible phosphorylation of I kappaB alpha on serines 32 and 36, all the stimuli that are inactive on 1.3E2 cells exhibit a sensitivity to the antioxidant pyrrolidine dithiocarbamate (PDTC). In contrast, stimuli such as hyperosmotic shock or phosphatase inhibitors, which use PDTC-insensitive pathways, induce I kappaB alpha degradation in 1.3E2. Analysis of the redox status of 1.3E2 does not reveal any difference from wild-type 70Z/3. We also report that the human T-cell leukemia virus type 1 (HTLV-1)-derived Tax trans-activator induces NF-kappaB activity in 1.3E2, suggesting that this viral protein does not operate via the defective pathway. Finally, we show that two other I kappaB molecules, I kappaB beta and the recently identified I kappaB epsilon, are not degraded in the 1.3E2 cell line following stimulation. Our results demonstrate that 1.3E2 is a cellular transduction mutant exhibiting a defect in a step that is required by several different stimuli to activate NF-kappaB. In addition, this analysis suggests a common step in the signaling pathways that trigger I kappaB alpha, I kappaB beta, and I kappaB epsilon degradation. 

Transcription mediated by NFAT is highly inducible in effector CD4+ T helper 2 (Th2) cells but not in Th1 cells. Transcriptional factors of the NFAT family play an important role in regulating the expression of several cytokine genes during the immune response, such as the genes for interleukin 2 (IL-2) and IL-4, among others. Upon antigen stimulation, precursor CD4+ T helper (pTh) cells proliferate and differentiate into two populations of effector cells (eTh1 and eTh2), each one expressing a specific pattern of cytokines that distinguishes them from their precursors. eTh2 cells are the major source of IL-4, while gamma interferon is produced by eTh1 cells. Here we have used reporter transgenic mice to show that DNA binding and transcriptional activities of NFAT are transiently induced during the differentiation of pTh cells into either eTh1 or eTh2 cells to mediate the expression of IL-2 as a common growth factor in both pathways. However, although NFAT DNA binding is similarly induced in both eTh1 and eTh2 cells upon antigen stimulation, only the NFAT complexes present in eTh2 cells are able to mediate high-level transcription, and relatively little NFAT transcriptional activity was induced in eTh1 cells. In contrast to activated pTh cells, neither eTh1 nor eTh2 cells produced significant IL-2 upon stimulation, but the high levels of NFAT transcriptional activities directly correlate with the IL-4 production induced in response to antigen stimulation in eTh2 cells. These data suggest that activated NFAT is involved in the effector function of eTh2 cells and that the failure of eTh1 cells to produce IL-4 in response to an antigen is due, at least partially, to a failure to induce high-level transcription of the IL-4 gene by NFAT. Regulation of NFAT could be therefore a critical element in the polarization to eTh1 or eTh2. 

Constitutive expression of p50 homodimer in freshly isolated human monocytes decreases with in vitro and in vivo differentiation: a possible mechanism influencing human immunodeficiency virus replication in monocytes and mature macrophages. Human immunodeficiency virus type 1 (HIV-1) replicates more efficiently in vitro in differentiated macrophages than in freshly isolated monocytes. We investigated whether this may be partly explained by changes in expression of NF-kappaB with monocyte differentiation. We demonstrated that constitutive expression of NF-kappaB in primary human monocytes changed significantly with differentiation in vitro to monocyte-derived macrophages (MDMs) and differentiation in vivo to alveolar macrophages (AMs). Freshly isolated monocytes constitutively expressed high levels of transcriptionally inactive p50 homodimer which decreased with time in culture in favor of the transcriptionally active p50/p65 and p50/RelB heterodimers. As in MDMs, AMs constitutively expressed p50/p65 and p50/RelB although at lower levels. HIV infection of fresh monocytes failed to induce p50/p65 as seen in MDMs. The replacement of p50 homodimers with transcriptionally active heterodimers following time in culture may partially explain the progressive increase in susceptibility of monocytes to HIV infection during in vitro culture. The change in NF-kappaB components with monocyte differentiation in vivo may also explain the different transcriptional activities of these cell populations in HIV-infected individuals. 

Identification of nucleotide sequences that regulate transcription of the MCF13 murine leukemia virus long terminal repeat in activated T cells. The region downstream of the enhancer (DEN) of the long terminal repeat of the mink cell focus-forming murine leukemia virus is important for viral pathogenicity. Another important activity of DEN is its control of transcription in activated T cells, and we have determined that an NF-kappaB site is critical for this activity. 

Activation of the NF-kappaB transcription factor in a T-lymphocytic cell line by hypochlorous acid. Reactive oxygen species (ROS) such as hydrogen peroxide serve as second messengers in the induction of the transcription factor NF-kappaB, and hence in the activation and replication of human immunodeficiency virus type 1 (HIV-1) in human cells. During inflammatory reactions, many oxidative species are produced, one of which is hypochlorous acid (HOCl), which is responsible for the microbicidal effects of activated human polymorphonuclear leukocytes. Treatment of a T-lymphocytic cell line with micromolar concentrations of HOCl promoted the appearance of transcription factor NF-kappaB (the heterodimer p50/p65) in the nucleus of the cells, even in the absence of de novo protein synthesis. Western blot analysis of the NF-kappaB inhibitory subunits (IkappaB) demonstrated that both IkappaB-alpha proteolysis and p105 processing were induced by the treatment. NF-kappaB activation was very effective when cells were subjected to hyperthermia before being treated with HOCl. Various antioxidants, such as pyrrolidine dithiocarbamate, p-bromophenacyl-bromide and nordihydroguaiaretic acid could strongly reduce NF-kappaB translocation, demonstrating the importance of oxidative species in the transduction mechanism. Moreover, ACH-2 cells treated with HOCl or H2O2 released tumour necrosis factor-alpha (TNF-alpha) in the supernatants. The importance of TNF-alpha release in NF-kappaB induction by HOCl or H2O2 was demonstrated by the fact that: (1) the nuclear appearance of NF-kappaB was promoted in untreated cells; and (2) synergism between TNF-alpha and HOCl was detected. Collectively, these results suggest that HOCl should be considered as an oxidative species capable of inducing NF-kappaB in a T-lymphocytic cell line through a transduction mechanism involving ROS, and having a long-distance effect through subsequent TNF-alpha release. 

NF-kappa B-independent suppression of HIV expression by ascorbic acid. Ascorbic acid (ascorbate or vitamin C) has been shown to suppress the induction of HIV in latently infected T lymphocytic cells following stimulation with a tumor promoter (PMA) and inflammatory cytokine (TNF-alpha). To assess whether this inhibition was mediated via modulation of the cellular transcription factor, NF-kappa B, we carried out gel shift analysis on nuclear extracts prepared under different conditions of cell stimulation in the presence or absence of ascorbate, N-acetylcysteine (NAC), or zidovudine (AZT). Pretreatment of ACH-2 T cells by NAC followed by stimulation with PMA, TNF-alpha, or hydrogen peroxide (H2O2) resulted in strong suppression of NF-kappa B activation. In contrast, neither ascorbate nor AZT affected NF-kappa B activity under all three induction conditions in the ACH-2 cell line. Ascorbate and AZT also had no effect on NF-kappa B activation following TNF-alpha- or PMA-induced stimulation of U1 promonocytic cells. These results suggest that the molecular mechanism of HIV inhibition by ascorbate is not mediated via NF-kappa B inhibition, unlike that seen with other antioxidants. 

Involvement of Egr-1/RelA synergy in distinguishing T cell activation from tumor necrosis factor-alpha-induced NF-kappa B1 transcription. NF-kappa B is an important transcription factor required for T cell proliferation and other immunological functions. The NF-kappa B1 gene encodes a 105-kD protein that is the precursor of the p50 component of NF-kappa B. Previously, we and others have demonstrated that NF-kappa B regulates the NF-kappa B1 gene. In this manuscript we have investigated the molecular mechanisms by which T cell lines stimulated with phorbol 12-myristate 13-acetate (PMA) and phytohemagglutin (PHA) display significantly higher levels of NF-kappa B1 encoding transcripts than cells stimulated with tumor necrosis factor-alpha, despite the fact that both stimuli activate NF-kappa B. Characterization of the NF-kappa B1 promoter identified an Egr-1 site which was found to be essential for both the PMA/PHA-mediated induction as well as the synergistic activation observed after the expression of the RelA subunit of NF-kappa B and Egr-1. Furthermore, Egr-1 induction was required for endogenous NF-kappa B1 gene expression, since PMA/PHA-stimulated T cell lines expressing antisense Egr-1 RNA were inhibited in their ability to upregulate NF-kappa B1 transcription. Our studies indicate that transcriptional synergy mediated by activation of both Egr-1 and NF-kappa B may have important ramifications in T cell development by upregulating NF-kappa B1 gene expression. 

The synthesis and immunogenicity of varicella-zoster virus glycoprotein E and immediate-early protein (IE62) expressed in recombinant herpes simplex virus-1. In order to evaluate the conditions for optimal expression and immunogenicity of varicella-zoster virus (VZV) proteins in a herpes simplex virus-1 (HSV-1) vector, we selected the VZV glycoprotein E (gE), encoded by ORF 68 and the VZV product of ORF 62, an immediate-early major tegument protein (IE62). Three HSV/VZV recombinants were generated: (1) VZV gE protein coding sequences along with the promoter region were inserted into the thymidine kinase (TK) gene of HSV-1 strain KOS; (2) VZV gE expressed from the HSV-1 ICP4 promoter was inserted into the glycoprotein C (gC) gene of HSV-1 strain F; and (3) VZV IE62 protein coding sequences under the control of the HSV-1 ICP4 promoter were inserted into the gC gene of HSV-1 strain F. Immunoblot analysis and immunoperoxidase staining of infected cell monolayers demonstrated vector expression of VZV proteins. Following intracranial inoculation in mice, both VZV gE-HSV (TK) and VZV IE62-HSV (gC) induced an IgG response against VZV gE or VZV IE62. When tested in cytotoxicity assays using T-lymphocytes from VZV immune human donors, the range of precursor frequencies for T-lymphocytes that recognized VZV gE or VZV IE62 was similar whether these proteins were expressed by HSV-1 or a vaccinia vector. These experiments demonstrate that HSV-1 is a competent vector for expression of these VZV proteins and support the feasibility of engineering a combined vaccine for these closely related alpha-herpesviruses. 

[Correlation of lymphocytic infiltration of tumor tissue with the hormonal and metabolic state in patients with breast cancer] Lymphocyte infiltration of tumor was studied vis-a-vis hormone metabolic status, tumor tissue hormone sensitivity and tobacco smoking, in 113 breast cancer patients, aged 25-77. On the average, no correlation was established between degree of lymphocyte infiltration in breast tumor and age and menopause onset. In smoking menopausal patients, lymphocyte infiltration was found to be higher than in non-smokers (p < 0.05). There was a direct correlation between the rate of lymphocyte infiltration and the level of progesterone receptors in tumor. Some subgroups displayed a direct correlation between infiltration and sex-binding globulin, cholesterol, luteinizing hormone in blood, and lean body mass. It was matched by an inverse correlation between lymphocyte infiltration and blood-thyroid hormone concentration, urine catecholamines and free cortisol excretion and fat/lean body mass ratio. Considering the abovesaid as well as the lymphocyte ability to perform the dual function of immunocytes and hormonocytes, it is suggested that the results may be used in both the study of lymphocyte infiltration and research in means of its control. 

Thymocytes control the CD4 gene differently from mature T lymphocytes. We analyzed the activity of the enhancer, the promoter and the silencer of the human CD4 gene during T cell development using transgenic mice. Immunofluorescence studies on thymic populations of mice carrying transgenes in various combinations of these regulatory DNA elements revealed that thymocytes control the CD4 gene in a different manner than mature peripheral T lymphocytes. The 5'-positive regulatory unit, consisting of the promoter and the 5' enhancer, is already active at the CD4-CD8-double-negative (DN) stage of development. However, its activity becomes lower in the double-positive and a fraction of the CD4+ CD8int/- cell population, indicating that an additional enhancer, located in either the first or the third intron of the CD4 gene, is required for CD4 gene expression in this population. The other studied regulatory element is the minimal CD4 silencer which inhibits CD4 gene expression in peripheral CD8 T lymphocytes. This silencer is inactive in the most immature DN thymocytes, which probably use a distinct silencer mechanism to down-regulate CD4 gene expression. Unexpectedly, the CD4 silencer is also active in CD4+ CD8int/- cells of the thymus, implying that an anti-silencer may be required to resume CD4 expression in this cell population. Altogether, the CD4 gene is regulated by several positive and negative regulatory mechanisms which come into play in a developmentally coordinated manner. 

Heat-shock and cadmium chloride increase the vimentin mRNA and protein levels in U-937 human promonocytic cells. Heat-shock for 2 hours at 42 degrees C, or the administration for 3 hours of 100 or 150 microM cadmium chloride, inhibited the subsequent proliferation activity, induced the expression of functional differentiation markers, and caused an increase in the amount of the stress-responsive HSP70 protein in U-937 human promonocytic cells. In addition, both heat and cadmium produced an increase in the amount of the intermediate filament protein vimentin, as determined by immunoblot and immunofluorescence assays. By contrast, the amounts of actin and beta-tubulin were not significantly altered. The amount of vimentin mRNA was also increased during recovery from stress, indicating that vimentin expression was not exclusively regulated at the protein level. Although cadmium caused an early, transient stimulation of c-jun and c-fos expression and AP-1 binding activity, heat-shock failed to alter both protooncogene expression and transcription factor binding, indicating that the stress-induced vimentin increase was not the result of AP-1-mediated transcriptional activation. Finally, it was observed that the rate of decay of vimentin mRNA upon actinomycin D administration was decreased in heat- and cadmium-pretreated cells in comparison to untreated cells. These results indicate that stress treatments cause an increase in vimentin levels in promonocytic cells, which may be explained at least in part by transcript stabilization. 

Jak1 expression is required for mediating interleukin-4-induced tyrosine phosphorylation of insulin receptor substrate and Stat6 signaling molecules. The Jak1, Jak2, Jak3, and Fes tyrosine kinases have been demonstrated to undergo tyrosine phosphorylation in response to interleukin (IL)-4 stimulation in different cell systems. However, it is not clear which, if any, of these kinases are responsible for initiating IL-4-induced tyrosine phosphorylation of intracellular substrates in vivo. In the present study, we have utilized a mutant Jak1-deficient HeLa cell line, E1C3, and its parental Jak1-expressing counterpart, 1D4, to analyze the role of Jak1 in mediating IL-4-induced tyrosine phosphorylation events. IL-4 treatment rapidly induced tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and IRS-2 in 1D4 but not in E1C3 cells. IL-4-mediated tyrosine phosphorylation of Stat6 was pronounced in 1D4 cells, while no IL-4-induced Stat6 phosphorylation was detected in E1C3 cells. IL-4 also induced Stat6 DNA binding activity from lysates of 1D4 but not E1C3 cells utilizing a radiolabeled immunoglobulin heavy chain germline epsilon promotor sequence (Iepsilon) in an electrophoretic mobility shift assay. Reconstitution of Jak1 expression in E1C3 cells restored the ability of IL-4 to induce IRS and Stat6 tyrosine phosphorylation. These results provide evidence that Jak1 expression is required for mediating tyrosine phosphorylation and activation of crucial molecules involved in IL-4 signal transduction. 

Immune hyperactivation of HIV-1-infected T cells mediated by Tat and the CD28 pathway. Human immunodeficiency virus-type 1 (HIV-1) infection is characterized by a chronic state of immune hyperactivation in patients. Infection of human peripheral blood lymphocytes with HIV-1 in vitro resulted in increased interleukin-2 (IL-2) secretion in response to T cell activation via the CD3 and CD28 receptors. Expression of the HIV-1 transactivator Tat recapitulated this phenotype and was associated with increased IL-2 secretion in response to costimulation with CD3 plus CD28. IL-2 superinduction by Tat occurred at the transcriptional level, was mediated by the CD28-responsive element in the IL-2 promoter, and was exclusively dependent on the 29 amino acids encoded by the second exon of Tat. 

c-Rel is a target of pentoxifylline-mediated inhibition of T lymphocyte activation. The possible clinical use of the methyl xanthine derivative, pentoxifylline (PF), for the treatment of T cell-dependent diseases is being noted with increasing interest. In this paper, we studied the molecular consequences of PF treatment during lymphocyte activation. We found that in T cells, anti-CD3-induced c-Rel expression was blocked by PF, whereas the induction of other NF-kappaB family members was not significantly affected. However, induction of NF-AT, which has the same signaling requirements as c-Rel induction, was not inhibited by PF. Among genes that respond to these transcription factors, IL-2 mRNA induction was suppressed by PF, whereas IL-2R(alpha) chain mRNA induction was not affected. These observations implicated c-Rel as an IL-2 promoter factor, for which experimental support was obtained from transient transfection experiments. In contrast with the observation in T cells, c-Rel induction was not blocked by PF in B cells. The greater selectivity of PF, compared with FK506, at both the molecular and cellular levels may prove advantageous in manipulating T cell responses in vivo. 

A T cell-specific enhancer in the interleukin-3 locus is activated cooperatively by Oct and NFAT elements within a DNase I-hypersensitive site. Interleukin-3 (IL-3) is a cytokine that is expressed primarily in activated T cells. Here we identified an inducible T cell-specific enhancer 14 kb upstream of the IL-3 gene that responded to activation of T cell receptor signaling pathways. The IL-3 enhancer spanned an inducible cyclosporin A-sensitive DNase I-hypersensitive site found only in T cells. Four NFAT-like elements exist within the enhancer. The two most active NFAT-like elements were located at the center of the DNase I-hypersensitive site. One of these NFAT-like elements encompassed overlapping Oct- and NFATp/c-binding sites, which functioned in a highly synergistic manner. We suggest that the T cell-specific expression of the IL-3 gene is partly controlled through the enhancer by cooperation between Oct and NFAT family proteins. 

TRAMP, a novel apoptosis-mediating receptor with sequence homology to tumor necrosis factor receptor 1 and Fas(Apo-1/CD95). A novel member of the tumor necrosis factor (TNF) receptor family, designated TRAMP, has been identified. The structural organization of the 393 amino acid long human TRAMP is most homologous to TNF receptor 1. TRAMP is abundantly expressed on thymocytes and lymphocytes. Its extracellular domain is composed of four cysteine-rich domains, and the cytoplasmic region contains a death domain known to signal apoptosis. Overexpression of TRAMP leads to two major responses, NF-kappaB activation and apoptosis. TRAMP-induced cell death is inhibited by an inhibitor of ICE-like proteases, but not by Bcl-2. In addition, TRAMP does not appear to interact with any of the known apoptosis-inducing ligands of the TNF family. 

Regulation of the tissue factor gene in human monocytic cells. Role of AP-1, NF-kappa B/Rel, and Sp1 proteins in uninduced and lipopolysaccharide-induced expression. Tissue factor (TF) expression by peripheral blood monocytes during sepsis initiates intravascular thrombosis. Bacterial lipopolysaccharide (LPS) rapidly induces TF gene transcription in monocytes. The human TF promoter contains binding sites for the transcription factors AP-1, c-Rel/p65, Egr-1, and Sp1. NF-kappa B/Rel proteins have been shown to physically interact with both AP-1 and Sp1 proteins. In this study, we investigated the role of these transcription factors in uninduced and LPS-induced TF gene expression in human monocytic THP-1 cells. Deletional analysis indicated that five Sp1 sites mediated basal expression in uninduced cells. The two AP-1 sites bound c-Fos/c-Jun heterodimers in both unstimulated and LPS-stimulated cells. Maximal LPS induction of the TF promoter required the two AP-1 sites and the kappa B site within the LPS response element. Disruption of the conserved spacing between the proximal AP-1 site and the kappa B site abolished LPS induction. Replacement of the two AP-1 sites with intrinsically bent DNA partially restored LPS induction, suggesting an additional structural role for the AP-1 sites. Synergistic transactivation of the LPS response element in Drosophila Schneider cells by coexpression of c-Fos, c-Jun, c-Rel, and p65 or c-Jun and p65 required the transactivation domains of c-Jun and p65. These data indicated that c-Fos/c-Jun, c-Rel/p65, and Sp1 regulate TF gene expression in human monocytic cells. 

AML1a but not AML1b inhibits erythroid differentiation induced by sodium butyrate and enhances the megakaryocytic differentiation of K562 leukemia cells. AML1 may play a role in growth and differentiation of cells along erythroid and/or megakaryocytic lineages, because a significant level of the AML1 gene is expressed in these cells. We overexpressed AML1a (without the transcription-activating domain) and AML1b (with the domain) proteins in K562 leukemia cells, which can be induced to differentiate into hemoglobin-producing cells and megakaryocytes. The AML1a-transfected K562 cells had a reduced capacity to differentiate in the presence of sodium n-butyrate but not in the presence of other inducers, such as hemin, 1-beta-D-arabinofuranosylcytosine, and herbimycin A. The AML1 antisense oligodeoxynucleotide but not the sense oligomer recovered its differentiation-inducing capacity in the presence of butyrate. On the other hand, AML1b conferred a similar differentiation-inducing capacity upon K562 cells transfected with vector alone. AML1a expression was associated with enhanced sensitivity to megakaryocytic differentiation induced by phorbol ester. These results provide evidence that AML1 proteins play a role in erythroid and megakaryocytic differentiation. 

Differentiation of U-937 promonocytic cells by etoposide and ICRF-193, two antitumour DNA topoisomerase II inhibitors with different mechanisms of action. We have compared the action on U-937 human promonocytic leukemia cells of two DNA topoisomerase II inhibitors, namely the epipodophyllotoxin etoposide and the bisdioxopiperazine ICRF-193. One hour pulse-treatment with 3 microM etoposide caused topoisomerase associated, primary DNA breakage, which was rapidly followed by apoptosis. By contrast, these effects were not observed upon pulse-treatment with 6 microM ICRF-193. However, continuous treatments with subcytotoxic concentrations of etoposide (0.15 microM) and ICRF-193 (0.3 microM) produced several similar effects, namely decreased cell proliferation, accumulation of cells at G2, increase in cell mass, and induction of differentiation. Under these conditions, etoposide produced a biphasic activation of protein kinase C, which consisted in an early transient activation (from hours 1 to 6) of the membrane-bound enzyme followed by a later activation (hour 48) of the total, membrane-bound and cytosolic enzyme. By contrast, ICRF-193 only provoked a late activation (from hours 72 to 96) of the total enzyme. When used at differentiation-inducing concentrations, both topoisomerase inhibitors caused a great stimulation of AP-1 binding activity, with maximum value at hour 12 in etoposide-treated cells and at hour 48 in ICRF-193-treated cells. By contrast, the binding activity of the NF-kappa(B) and EGR-1 transcription factors was little affected. It is concluded that topoisomerase II inhibitors may induce the differentiation of promonocytic cells, independently of their capacity to cause DNA strand breaks. However, there are other effects, such as the early activation of protein kinase C, which are probably derived from the production of primary DNA breakage by some anti-topoisomerase drugs. 

Nuclear Rel-A and c-Rel protein complexes are differentially distributed within human thymocytes. Nuclear factor-kappa B (NF-kappa B)/Rel proteins are inducible transcriptional regulators of numerous cellular genes. They are particularly abundant in lymphoid tissues and are thought to be critical for the transcription of genes involved in immune and inflammatory responses. We have reported previously that a nuclear NF-kappa B activity was present in freshly extracted human thymocytes in the absence of in vitro treatment of these cells. In the present report, we identified NF-kappa B proteins extracted from human thymocyte nuclei as being p50/p65 and p50/c-Rel complexes. Immunochemical and immunofluorescent staining of thymus sections using specific Abs allowed visualization of nuclear NF-kappa B proteins in both thymocytes and nonthymocyte cells. This detection suggested a preferential activation of p50/c-Rel in medullary thymocytes, whereas p50/p65 was present in both cortical and medullary regions of human thymus lobules. However, the intensity of p65 labeling was much higher in several thymocytes from the medulla. p65, p50, and c-Rel activities were found in both CD4- and CD8-positive thymocytes. These observations suggest that p65 and c-Rel complexes play distinct roles in gene expression and that both forms of NF-kappa B play critical roles during late stages of the intrathymic maturation of T cells. 

Alteration of a single serine in the basic domain of the Epstein-Barr virus ZEBRA protein separates its functions of transcriptional activation and disruption of latency. The ZEBRA protein from Epstein-Barr virus (EBV) activates a switch from the latent to the lytic expression program of the virus. ZEBRA, a member of the bZIP family of DNA-binding proteins, is a transcriptional activator capable of inducing expression from viral lytic cycle promoters. It had previously been thought that ZEBRA's capacity to disrupt EBV latency resided primarily in its ability to activate transcription of genes that encode products required for lytic replication. We generated a point mutant of ZEBRA, Z(S186A), that was not impaired in its ability to activate transcription; however, this mutation abolished its ability to initiate the viral lytic cascade. The mutant, containing a serine-to-alanine substitution in the DNA-binding domain of the protein, bound to several known ZEBRA-binding sites and activated transcription from reporters bearing known ZEBRA-responsive promoters but did not disrupt latency in EBV-infected cell lines. Therefore, initiation of the EBV lytic cycle by the ZEBRA protein requires a function in addition to transcriptional activation; a change of serine 186 to alanine in the DNA-binding domain of ZEBRA abolished this additional function and uncovered a new role for the ZEBRA protein in disruption of EBV latency. The additional function that is required for initiation of the lytic viral life cycle is likely to require phosphorylation of serine 186 of the ZEBRA protein, which may influence either DNA recognition or transcriptional activation of lytic viral promoters in a chromatinized viral episome. 

Redox regulation of the mitogen-activated protein kinase pathway during lymphocyte activation. We have previously demonstrated an obligatory requirement for intracellular reactive oxygen species generation during T lymphocyte activation, and have proposed that intracellular reactive oxygen species may act as signalling agents in the regulation of certain cellular processes, for example, during cell cycle entry. To test this hypothesis, we have been interested to determine which, if any, cell cycle entry events are affected by oxidative signalling. In earlier studies, we have identified the transcription factors NF-kappa B and AP-1 as molecular targets for oxidative signalling processes during cell cycle entry, and have shown that oxidative signalling is involved in the regulation of early changes in gene expression during the G0 to G1 phase transition. To extend these initial observations, we have examined the effect of antioxidant treatment on the activity of the mitogen-activated protein kinases erk1 and erk2, as members of a signal transduction pathway known to directly regulate transcription factor function. Using as a probe cysteamine, an aminothiol compound with both antioxidant and antiproliferative activity, we have identified erk2, a key element of the MAP kinase pathway, as being responsive to oxidative signalling during lymphocyte activation. These observations provide further evidence to suggest a role for intracellular oxidant generation as a regulatory mechanism during cell cycle entry, and establish a link between oxidative signalling and other aspects of the intracellular signalling network that is activated in response to mitogenic stimulation. 

Common and distinct intracellular signaling pathways in human neutrophils utilized by platelet activating factor and FMLP. Stimulation of human neutrophils with chemoattractants FMLP or platelet activating factor (PAF) results in different but overlapping functional responses. We questioned whether these differences might reflect patterns of intracellular signal transduction. Stimulation with either PAF or FMLP resulted in equivalent phosphorylation and activation of the mitogen-activated protein kinase (MAPk) homologue 38-kD murine MAP kinase homologous to HOG-1 (p38) MAPk. Neither FMLP nor PAF activated c-jun NH2-terminal MAPk (JNKs). Under identical conditions, FMLP but not PAF, resulted in significant p42/44 (ERK) MAPk activation. Both FMLP and PAF activated MAP kinase kinase-3 (MKK3), a known activator of p38 MAPk. Both MAP ERK kinase kinase-1 (MEKK1) and Raf are activated strongly by FMLP, but minimally by PAF. Pertussis toxin blocked FMLP-induced activation of the p42/44 (ERK) MAPk cascade, but not that of p38 MAPk. A specific p38 MAPk inhibitor (SK&F 86002) blocked superoxide anion production in response to FMLP and reduced adhesion and chemotaxis in response to PAF or FMLP. These results demonstrate distinct patterns of intracellular signaling for two chemoattractants and suggest that selective activation of intracellular signaling cascades may underlie different patterns of functional responses. 

Selective expression of an interleukin-12 receptor component by human T helper 1 cells. Interleukin-12 (IL-12), a heterodimeric cytokine produced by activated monocytes and dendritic cells, plays a crucial role in regulating interferon (IFN)-gamma production and in the generation of IFN-gamma-producing T helper 1 (Th1) cells. Here we show that the IL-12 receptor (IL-12R) beta 2 subunit, a recently cloned binding and signal transducing component of the IL-12R, is expressed on human Th1 but not Th2 clones and is induced during differentiation of human naive cells along the Th1 but not the Th2 pathway. IL-12 and type I but not type II interferons induce expression of the IL-12R beta 2 chain during in vitro T cell differentiation after antigen receptor triggering. The selective expression and regulation of the IL-12R beta 2 subunit may help to understand the basis of Th1/Th2 differentiation and may provide therapeutic options for altering the Th1/Th2 balance in several immuno-pathological conditions such as autoimmune diseases and allergies. 

Activation of the transcription factor NF-kappaB in lipopolysaccharide-stimulated U937 cells. During the course of serious bacterial infections, lipopolysaccharide (LPS) interacts with monocyte/macrophage receptors, resulting in the generation of inflammatory cytokines. Transcription factor NF-kappaB is crucial in activating the transcription of genes encoding proinflammatory cytokines. In this paper, we demonstrate that the activation of NF-kappaB by LPS in a promonocytic cell line (U937) followed a rather slow kinetics, depending on the rate of IkappaB-alpha inhibitor hydrolysis. No degradation of p105 and p100 inhibitors was observed under these conditions. The transduction pathway leading to NF-kappaB activation in U937 cells involved the intracellular generation of reactive oxygen species (ROS), as demonstrated by the concomitant inhibitory effects of antioxidants on NF-kappaB activation and the emission of a fluorescent probe reacting intracellularly with hydrogen peroxide. This ROS pathway was also characterized by the use of other inhibitors. This finding indicates that phospholipase A2 and 5-lipoxygenase are also involved. However, the NF-kappaB activation pathway involving the acidic sphingomyelinase of the endolysosomial membrane did not seem to participate in the LPS-induced NF-kappaB activation in U937 cells. 

Detection of adenovirus DNA in peripheral blood mononuclear cells by polymerase chain reaction assay. Adenovirus can establish persistent infections which may reactivate and cause disease in immunocompromised hosts. Lymphocytes have been postulated to serve as a site of adenoviral persistence based upon the ability to isolate adenovirus from tonsils and to detect adenovirus DNA by Southern blot hybridization in peripheral blood mononuclear cells (PBMC). To test this hypothesis, a more sensitive and specific polymerase chain reaction (PCR) assay was developed to detect adenovirus DNA. Two sets of nested primers were designed to conserved sequences in the adenovirus E1A and hexon genes. The E1A and hexon primers amplified DNA from representative adenoviral serotypes in all six adenoviral groups (A-F). Both primers detected a single copy of the adenovirus type 2 genome but were less sensitive for the group B type 35. None of 33 PBMC specimens from healthy adults and only one of 40 pediatric samples was positive (at a low level) for adenovirus DNA by nested PCR assay. In comparison, PBMC from two children with fatal adenoviral infection were both strongly positive for adenovirus DNA. It is concluded that, in contrast to a previous study, PBMC are not a common site of persistent group C adenoviral infection. In addition, assay of PBMC by the adenovirus-specific PCR may help detect early invasive disease and warrants further evaluation. 

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

A PMLRARalpha transgene initiates murine acute promyelocytic leukemia. The malignant cells of acute promyelocytic leukemia (APL) contain a reciprocal chromosomal translocation that fuses the promyelocytic leukemia gene (PML) with the retinoic acid receptor alpha gene (RAR alpha). To test the hypothesis that the chimera PMLRAR alpha plays a role in leukemogenesis, we expressed a PMLRAR alpha cDNA in myeloid cells of transgenic mice. PMLRAR alpha transgenic mice exhibited impaired neutrophil maturation early in life, which progressed at a low frequency over the course of several months to overt APL. Both the preleukemic state and the leukemia could be transplanted to nontransgenic mice, and the transplanted preleukemia could progress to APL. The APL recapitulated features of the human disease, including a response to retinoic acid. Retinoic acid caused the leukemic cells to differentiate in vitro and in vivo, eliciting remissions of both the preleukemic state and APL in mice. Our results demonstrate that PMLRAR alpha impairs neutrophil differentiation and initiates the development of APL. The transgenic mice described here provide an apparently accurate model for human APL that includes clear evidence of tumor progression. The model should be useful for exploring the molecular pathogenesis of APL and the mechanisms of the therapeutic response to retinoic acid, as well as for preclinical studies of therapeutic regimens. 

Signal transducer and activator of transcription-3 (STAT3) is constitutively activated in normal, self-renewing B-1 cells but only inducibly expressed in conventional B lymphocytes [see comments] Cytokine and growth factor receptor engagement leads to the rapid phosphorylation and activation of latent, cytosolic signal transducers and activators of transcription (STAT) proteins, which then translocate to the nucleus where they regulate transcriptional events from specific promoter sequences. STAT3 expression in particular has been associated with Abl, Src, and HTLV-1 transformation of normal cells. B-1 lymphocytes are self-renewing, CD5+ B cells that display a propensity for malignant transformation and are the normal counterpart to human chronic lymphocytic leukemias. Further, B-1 cells are characterized by aberrant intracellular signaling, including hyperresponsiveness to phorbol ester PKC agonists. Here we demonstrate that B-1 lymphocytes constitutively express nuclear activated STAT3, which is not expressed by unmanipulated conventional (B-2) lymphocytes. In contrast, STAT3 activation is induced in B-2 cells after antigen receptor engagement in a delayed fashion (after 3 h). Induction of STAT3 is inhibited by both the serine/threonine protein kinase inhibitor H-7 and the immunosuppressive drug rapamycin and requires de novo protein synthesis, demonstrating novel coupling between sIg and STAT proteins that differs from the classical paradigm for STAT induction by cytokine receptors. The inability of prolonged stimulation of conventional B-2 cells with anti-Ig, a treatment sufficient to induce CD5 expression, to result in sustained STAT3 activation suggests that STAT3 is a specific nuclear marker for B-1 cells. Thus, STAT3 may play a role in B cell antigen-specific signaling responses, and its constitutive activation is associated with a normal cell population exhibiting intrinsic proliferative behavior. 

The T cell activation factor NF-ATc positively regulates HIV-1 replication and gene expression in T cells. Clinical deterioration in human immunodeficiency virus type 1 (HIV-1) infection is associated with increased levels of viral replication and burden in the peripheral blood and lymphoid organs. T cell activation and ensuing cellular gene activation can be critical for HIV-1 replication. The hypothesis that the nuclear factor of activated T cells (NF-AT) may influence HIV-1 replication is therefore compelling given the tight correlation of HIV-1 transcriptional induction to T cell activation. We report that certain NF-AT(Rel) family members productively bind the kappaB regulatory elements, synergize with NF-kappaB and Tat in transcriptional activation of HIV-1, and enhance HIV-1 replication in T cells. These results link regulatory factors critical to T cell commitment directly to HIV-1 replication. 

ALY, a context-dependent coactivator of LEF-1 and AML-1, is required for TCRalpha enhancer function. LEF-1 is a transcription factor that participates in the regulation of the T-cell receptor alpha (TCR alpha) enhancer by facilitating the assembly of multiple proteins into a higher order nucleoprotein complex. The function of LEF-1 is dependent, in part, on the HMG domain that induces a sharp bend in the DNA helix, and on an activation domain that stimulates transcription only in a specific context of other enhancer-binding proteins. With the aim of gaining insight into the function of context-dependent activation domains, we cloned ALY, a novel LEF-1-interacting protein. ALY is a ubiquitously expressed, nuclear protein that specifically associates with the activation domains of LEF-1 and AML-1 (CBF alpha2, PEBP2 alpha(B), which is another protein component of the TCR alpha enhancer complex. In addition, ALY can increase DNA binding by both LEF-1 and AML proteins. Overexpression of ALY stimulates the activity of the TCR alpha enhancer complex reconstituted in transfected nonlymphoid HeLa cells, whereas down-regulation of ALY by anti-sense oligonucleotides virtually eliminates TCR alpha enhancer activity in T cells. Similar to LEF-1, ALY can stimulate transcription in the context of the TCR alpha enhancer but apparently not when tethered to DNA through an heterologous DNA-binding domain. We propose that ALY mediates context-dependent transcriptional activation by facilitating the functional collaboration of multiple proteins in the TCR alpha enhancer complex. 

Differentiation of T-helper lymphocytes: selective regulation by members of the STAT family of transcription factors. Interleukin-4 (IL-4) and interleukin-12 (IL-12) control the differentiation of T-helper cells. Here we summarize studies which investigate the mechanism by which these cytokines selectively reprogramme gene expression in T-lymphocytes. Cytokine stimulation leads to the phosphorylation of specific tyrosine residues within the intracellular domain of the corresponding cytokine receptor. These phosphotyrosines serve as docking sites for latent, cytoplasmic transcription factors known as signal transducers and activators of transcription (Stat) proteins. Receptor/Stat interaction is mediated by the src homology 2 (SH2) domain of the corresponding Stat protein. Although Stat binding to the intracellular domain of the cytokine receptor strongly depends on the phosphotyrosine residue, the recruitment of a specific Stat protein is dictated by amino acid residues C-terminal to the phosphotyrosine. Specific docking sites within individual cytokine receptors have been identified for almost all Stat proteins. The direct coupling between cytokine receptor and transcription factor helps to explain how different cytokines elicit distinct patterns of gene expression. 

Cell-to-cell contact activates the long terminal repeat of human immunodeficiency virus 1 through its kappaB motif. Cell-to-cell contact between peripheral blood lymphocytes and transfected human colonic carcinoma cell line HT29 activates transcription of the long terminal repeats (LTR) of human immunodeficiency virus. HIV-1 LTR transcription is controlled by a complex array of virus-encoded and cellular proteins. Using various constructs expressing a lacZ reporter gene under the control of the intact or three deleted forms of HIV-1 LTR, we obtained evidence that the kappaB regulatory elements located in the U3 region are involved in cell-to-cell activation of HIV-1 LTR. Cell-to-cell contact activates in vitro binding of the nuclear factor kappaB (NF-kappaB) p50/p65 heterodimer to an HIV-1 kappaB oligonucleotide. Cell-to-cell contact activation of NF-kappaB was only partially inhibited by 100 microM pyrrolidine dithiocarbamate and was not correlated with a significant decrease of cellular inhibitor kappaB alpha. NF-kappaB nuclear activation was not detectable before 1 h after cell contact and was dependent on protein synthesis. 

Lineage- and stage-specific expression of runt box polypeptides in primitive and definitive hematopoiesis. Translocations involving the human CBFA2 locus have been associated with leukemia. This gene, originally named AML1, is a human homologue of the Drosophila gene runt that controls early events in fly embryogenesis. To clarify the role of mammalian runt products in normal and leukemic hematopoiesis, we have studied their pattern of expression in mouse hematopoietic tissues in the adult and during ontogeny using an anti-runt box antiserum. In the adult bone marrow, we found expression of runt polypeptides in differentiating myeloid cells and in B lymphocytes. Within the erythroid lineage, runt expression is biphasic, clearly present in the erythroblasts of early blood islands and of the fetal liver, but absent in the adult. Biochemical analysis by Western blotting of fetal and adult hematopoietic populations shows several runt isoforms. At least one of them appears to be myeloid specific. 

Cytokine signal networks and a new era in biomedical research. Elucidation of the biochemical nature of the signal transduction pathway that regulate transcription and replication is the focus of attention in molecular biology. This research may make feasible manipulation of growth and differentiation of mammalian cells, which in turn would have profound implication in biomedical research on cell and gene therapy, and development of pharmaceutical products. Cytokines control growth, differentiation, death, and function of cells of lymphocytic, hemopoietic systems, and together with nerve cells provide a pertinent model to study intercellular communications and intercellular signal networks. This review outlines general features of signal transduction and several aspects of cytokine networks are discussed with emphasis on: transcriptional regulation of Th1 and Th2-specific cytokine genes in T cells, the roles of cytokines and their receptors in growth and differentiation of hemopoietic cells, and the manipulation of cytokine networks. 

Nuclear factor-kappa B potently up-regulates the promoter activity of RANTES, a chemokine that blocks HIV infection. The complex network of cytokines that are involved in inflammatory and immunoregulatory responses plays a critical role in the pathogenesis of HIV infection. RANTES (regulated upon activation, normal T cell expressed and secreted) is a cytokine that belongs to the beta-chemokine family; it is chemoattractant for CD4+/CD45RO T cells, it is produced by various cell types including CD8+ and CD4+ T cells as well as monocytes/macrophages, and has recently been shown to suppress replication of macrophage-tropic strains of HIV in CD4+ T cells. To investigate the molecular mechanisms of RANTES expression, the RANTES promoter region was analyzed by transient expression and gel-mobility shift assays. We demonstrate that: 1) RANTES promoter activity is up-regulated by PMA plus ionomycin, coexpression of the p65 subunit of nuclear factor (NF)-kappa B, the proinflammatory cytokines TNF-alpha and IL-1 beta, and the CD28 costimulatory pathway; 2) the RANTES promoter region contains four NF-kappa B binding sites at positions -30, -44, -213, and -579 relative to the transcription start site; 3) one site (-213) is an NF-AT (nuclear factor of activated T cells) binding site that also has weak affinity to NF-kappa B, and the most distal site (-579) also serves as a CD28-responsive element; and 4) mutation on any of those NF-kappa B sites or coexpression of I kappa B alpha (cytoplasmic inhibitor of NF-kappa B) markedly reduced the promoter activity. Thus, NF-kappa B, a potent transcriptional activator of HIV expression, is also involved in the expression of RANTES, a chemokine that blocks infection by macrophage-tropic strains of HIV. 

Response to intranasal fluticasone propionate in perennial allergic rhinitis not associated with glucocorticoid receptor characteristics. BACKGROUND: The reduction of symptoms due to treatment with corticosteroids varies among patients with perennial rhinitis. Most patients will respond but a few patients respond less to these drugs. OBJECTIVE: To investigate the association in reduction of symptoms due to glucocorticoids and glucocorticoid receptor characteristics in patients with perennial allergic rhinitis, in vitro glucocorticoid receptor binding studies were performed with peripheral blood mononuclear cells using dexamethasone and in vitro production of mediators were measured. METHODS: During a double-blind placebo-controlled crossover study, 200 micrograms fluticasone propionate aqueous nasal spray (in the active treatment period) and placebo (in the placebo treatment period) were administered twice daily for 2 weeks to 22 patients allergic to house dust mite. At the end of both treatment periods symptoms were scored after allergen provocation (100, 1000, 10000 BU/mL) and during the 9.5 hours after this challenge. Receptor binding studies with dexamethasone were performed with peripheral blood mononuclear cells. Leukotriene B4 produced by monocytes in vitro and soluble interleukin-2 receptor released by lymphocytes in vitro and cortisol levels in plasma were determined. RESULTS: No significant partial correlations of the number of the peripheral blood mononuclear cell glucocorticoid receptors (6821 +/- 5669 binding sites per cell) and the affinity (Kd: 16.5 +/- 13.51 nmol/L) for the glucocorticoid receptors with the symptom score (placebo: 4.3 +/- 2.45 pts; fluticasone: 2.4 +/- 1.55 pts) after active treatment were found. Also no significant partial correlations of the levels of leukotriene B4 (45.6 +/- 105.3 ng/10(6) cells) produced by monocytes in vitro, soluble interleukin-2 receptor (734 +/- 237 ng/10(6) cells) released by lymphocytes in vitro and cortisol levels (571 +/- 236 ng/mL) in plasma with the symptom score after active treatment were found. CONCLUSIONS: The reduction of symptoms due to topical fluticasone propionate in patients with rhinitis and allergy to house dust mite is not correlated with the characteristics of the glucocorticoid receptor. 

HIV does not replicate in naive CD4 T cells stimulated with CD3/CD28. In this report, we demonstrate that the T cell tropic strain of HIV, LAI, does not replicate in naive CD4 T cells stimulated by cross-linking CD3 and CD28. In contrast, LAI replicates well in memory CD4 T cells stimulated in the same way. Unlike this physiologically relevant stimulation, PHA stimulates productive LAI replication in both naive and memory T cells. These studies were conducted with highly purified (FACS-isolated) subsets of CD4 T cells identified by expression of both CD45RA and CD62L. Remixing of purified T cells showed that naive T cells do not suppress LAI replication in memory T cells and that memory T cells do not restore LAI expression in naive T cells. The suppression of productive LAI replication in naive T cells is not due to differential expression of viral coreceptors, nor is it due to inhibition of activation of the important HIV transcription factors, nuclear factor-kappaB and activator protein-1. The inherent resistance of naive T cells to productive HIV infection, coupled with their proliferative advantage as demonstrated here, provides a sound basis for proposed clinical therapies using ex vivo expansion and reinfusion of CD4 T cells from HIV-infected adults. 

The Pax-5 gene is alternatively spliced during B-cell development. The transcription factor Pax-5 is expressed during the early stages of B-cell differentiation and influences the expression of several B-cell-specific genes. In addition to the existing isoform (Pax-5, which we have named Pax-5a), we have isolated three new isoforms, Pax-5b, Pax-5d, and Pax-5e, from murine spleen and B-lymphoid cell lines using library screenings and polymerase chain reaction amplification. Isoforms Pax-5b and Pax-5e have spliced out their second exon, resulting in proteins with only a partial DNA-binding domain. Isoforms Pax-5d and Pax-5e have deleted the 3'-region, which encodes the transactivating domain, and replaced it with a novel sequence. The existence of alternative Pax-5 transcripts was confirmed using RNase protection assays. Furthermore, Pax-5a and Pax-5b proteins were detected using Western blot analysis. Pax-5a was detectable in pro-, pre-, and mature B-cell lines, but not in two plasmacytomas; Pax-5b was shown to be present at low levels in mature B-cell lines and, unexpectedly, in one plasma cell line, but not in pro-B-cell or T-cell lines. Mobility shift assays showed that in vitro translated Pax-5a and Pax-5d, but not Pax-5b or Pax-5e, could interact with a B-cell-specific activator protein-binding site on the blk promoter. Using this assay, we also showed that Pax-5d was present in nuclear extracts of some (but not all) B-lymphoid lines and interacts with the B-cell-specific activator protein-binding site. The pattern of differential expression of alternatively spliced Pax-5 isoforms suggests that they may be important regulators of transcription during B-cell maturation. 

Oxidant-regulation of gene expression in the chronically inflamed intestine. It is becoming increasingly apparent that the chronic gut inflammation observed in the idiopathic inflammatory bowel diseases (e.g. ulcerative colitis, Crohn's disease) is associated with enhanced production of leukocyte-derived oxidants. Oxidants such as hydrogen peroxide are known to activate certain transcription factors such as nuclear transcription factor kappa beta. Nuclear transcription factor kB (NF-kappa B) is a ubiquitous transcription factor and pleiotropic regulator of numerous genes involved in the immune and inflammatory responses. This transcription factor is activated via the selective phosphorylation, ubiquination and degradation of its inhibitor protein I-kB thereby allowing translocation of NF-kappa B into the nucleus where it upregulates the transcription of a variety of adhesion molecules (e.g. ICAM-1, VCAM-1), cytokines (TNF, IL-1, IL-6) and enzymes (iNOS). The proteolytic degradation of the post-translationally modified I-kappa B is known to be mediated by the 26S proteasome complex. Based upon work from our laboratory, we propose that inhibition of NF-kappa B activation produces significant anti inflammatory activity which may be mediated by the inhibition of transcription of certain pro-inflammatory mediators and adhesion molecules. 

Expression of LAZ3/BCL6 in follicular center (FC) B cells of reactive lymph nodes and FC-derived non-Hodgkin lymphomas. Chromosomal translocation resulting in abnormal expression of the LAZ3/BCL6 gene in B cells has been implicated in the tumorigenesis of non-Hodgkin lymphoma (NHL). Therefore we studied the expression pattern of LAZ3/BCL6 by in situ hybridization with synthetic oligonucleotide probes in frozen tissue sections from five reactive lymph nodes and 38 B cell and non-B NHL. In addition, we investigated the expression of LAZ3/BCL6 by Northern blot analysis on multiple human tissues. The LAZ3/BCL6 transcript was found in a variety of tissues, including skeletal muscle, peripheral blood leukocytes, and weakly in normal lymph nodes. In the tumor samples, expression of LAZ3/BCL6 was observed in 68% of all B cell NHL and none of the non-B lymphomas. All cases of follicular, mixed small and large cell lymphomas showed LAZ3/BCL6 expression confined to the neoplastic follicles. A follicular expression pattern was also found in all non-malignant reactive lymph nodes. Hence, the expression of LAZ3/BCL6 does not correlate to malignancy, but reflects the origin of B cells from the germinal centers. 

Estrogen and progesterone receptors in vernal keratoconjunctivitis. PURPOSE: Sex-related influences have been implicated in the pathogenesis of vernal keratoconjunctivitis (VKC), an allergic eosinophilic disease. METHODS: The authors evaluated tarsal and bulbar conjunctival biopsies from seven patients with severe and symptomatic VKC for the presence of estrogen and progesterone receptors by using monoclonal antibodies with a peroxidase-antiperoxidase technique. RESULTS: Both the epithelium and subepithelium of the tarsal and bulbar conjunctiva of patients with VKC, but not those of four nonatopic control subjects, showed intense positive staining for estrogen and progesterone receptors. Immunofluorescence colocalization of both estrogen and progesterone receptors with eosinophil cationic protein showed that approximately 70% of positive cells were eosinophils. CONCLUSIONS: Sexual hormones, through their receptors, may influence the activity of eosinophils in patients with VKC. 

Use of new biologic markers in the ovulation induction. Biological markers of ovulation, after a great in the past, have been fallen into disuse for the large diffusion of biochemical and biophysical ones. However, the real effect of hormones involved in ovulation is expressed by biological modifications on target tissues. To explore the modifications of not reproductive target tissues as ovulation markers we studied the behaviour of Albuminemia, Platelet Factor IV (as indicator of Platelet Aggregation), Type II estrogenic receptors in 42 ovulation induced women, undergoing our observation. 33 of them had ovulation and 9 developed a LUF syndrome, constituting two biological models of an opposite situation for the three markers observed. All the markers considered were sufficiently sensitive, but among them, Platelet Factor IV was the most reliable to the hormonal ovulatory situation. 

Induction of relA(p65) and I kappa B alpha subunit expression during differentiation of human peripheral blood monocytes to macrophages. We evaluated the expression and DNA binding activity of nuclear factor (NF)-kappa B subunits in human peripheral blood monocytes and in monocyte-derived macrophages (MDMs). Constitutive DNA binding activity consisting of p50 homodimers was detected in nuclear extracts from both cell types. An additional complex composed of p50/RelA(p65) heterodimers appeared only in nuclear extracts from 7-day MDMs. Immunoblot analysis showed that the p50 subunit was constitutively expressed in monocytes and MDMs. In contrast, the RelA(p65) subunit was barely detectable in monocytes, but its level increased markedly in MDMs. Analysis of RelA(p65) mRNA revealed that the stability of RelA(p65) mRNA was significantly higher in MDMs, compared with monocytes. In MDMs, an upregulation of I kappa B alpha synthesis as well as the appearance of a novel M(r) 40,000 form of I kappa B alpha were also observed. These results suggest that macrophage differentiation results in the expression of active p50/RelA(p65) heterodimers with the capacity to activate target gene expression. The parallel induction of I kappa B alpha synthesis may allow for the continuous presence of a cytoplasmic reservoir of p50/RelA(p65) complexes that are readily available for inducer-mediated stimulation. 

Requirement of prestimulated THP-1 monocytic cells for endothelial cell activation. Involvement of TNF alpha. Blood monocytes spontaneously activate endothelial cells in culture, leading to adhesion of monocytic cells onto the endothelial surface and overproduction of endothelial proteins such as von Willebrand factor (vWf) and plasminogen activator inhibitor type 1 (PAI-1). To overcome the difficulty in obtaining quiescent monocytes, we studied the ability of promonocytic THP-1 cells to activate endothelial cells. Lipopolysaccharide (LPS)-prestimulated and untreated THP-1 cells were cocultured with resting human umbilical vein endothelial cells (HUVEC) for 3 and 24 h in the presence of colimycin to neutralize LPS traces. Addition of untreated THP-1 cells had little effect on HUVEC adhesiveness. Addition of prestimulated THP-1 cells was followed by a noticeable adhesion after 3 h which reversed to basal values within 24 h. Under these conditions HUVEC adhesion molecules, E-selectin, VCAM-1 and ICAM-1, were increased at 3 h with only ICAM-1 remaining overexpressed at 24 h. Diffusible endothelial proteins such as soluble E-selectin, PAI-1 and vWf to a minimal extent, increased in supernatants from HUVEC cocultured for 24 h with prestimulated THP-1 cells. In those cocultures, TNF alpha concentrations peaked at 3 h whereas IL-1 beta levels progressively rose until 24 h. Addition of an anti-TNF alpha antibody decreased by 40% E-selectin and ICAM-1 induction and suppressed PAI-1 overproduction with a weak effect on vWf. An anti-IL-1 beta antibody had negligible effects on HUVEC adhesion molecules, PAI-1 or vWf production. These results provide evidence that promonocytic THP-1 cells require prestimulation in order to activate HUVEC and that TNF alpha contributes to this phenomenon. 

c-Jun and GST-pi expression in human plasma cells. Bone marrow samples from 33 patients affected by MM and MGUS, and 8 patients not affected by lymphoproliferative diseases were studied for expression of c-Jun (a component of the transcription factor AP-1) and glutathione-S-transferase pi (GST-pi) using immunocytochemical methods. A high and frequent expression of these two proteins was found both in MM and MGUS patients (31/33 patients positive for c-Jun and 29/33 patients positive for GST-pi) and in controls not affected by monoclonal gammopathy (7/8 patients positive for both c-Jun and GST-pi). No statistically significant correlation was found between c-Jun- and GST-pi-positive plasma cells. The expression of these two proteins was not related to clinical or laboratory data. Our results seem to confirm a possible role of the transcriptional complex AP-1 in activating GST-pi promoter in human plasma cells. 

Two distinct pathways of interleukin-5 synthesis in allergen-specific human T-cell clones are suppressed by glucocorticoids. Glucocorticoids (GC) have long been used as the most effective agents for the treatment of allergic diseases accompanied by eosinophilia such as chronic asthma and atopic dermatitis. The development of chronic eosinophilic inflammation is dependent on interleukin-5 (IL-5), a selective eosinophil-activating factor, produced by helper T cells. To delineate the regulatory mechanisms of human IL-5 synthesis, we established allergen-specific CD4+ T-cell clones from asthmatic patients. GC efficiently suppressed IL-5 synthesis of T-cell clones activated via either T-cell receptor (TCR) or IL-2 receptor (IL-2R). Induction of IL-5 mRNA upon TCR and IL-2R stimulation was totally inhibited by dexamethasone. Human IL-5 promoter/enhancer-luciferase gene construct transfected to T-cell clones was transcribed on either TCR or IL-2R stimulation and was clearly downregulated by dexamethasone, indicating that the approximately 500-bp human IL-5 gene segment located 5' upstream of the coding region contains activation-inducible enhancer elements responsible for the regulation by GC. Electrophoretic mobility shift assay analysis suggested that AP-1 and NF-kappaB are among the possible targets of GC actions on TCR-stimulated T cells. NF-AT and NF-kappaB were not significantly induced by IL-2 stimulation. Our results showing that GC suppressed IL-5 production by human CD4+ T cells activated by two distinct stimuli, TCR and IL-2R stimulation, underscore the efficacy of GC in the treatment of allergic diseases via suppression of T-cell IL-5 synthesis. 

Experimental expression in mice and spontaneous expression in human SLE of polyomavirus T-antigen. A molecular basis for induction of antibodies to DNA and eukaryotic transcription factors. We have previously demonstrated that experimental expression of the polyomavirus transcription factor T-antigen has the potential to induce anti-DNA antibodies in mice. Two sets of independent evidences are presented here that demonstrate a biological relevance for this model. First, we describe results demonstrating that mice inoculated with T-antigen-expressing plasmids produced antibodies, not only to T-antigen and DNA, but also to the DNA-binding eukaryotic transcription factors TATA-binding protein (TBP), and to the cAMP-response-element-binding protein (CREB). Secondly, we investigated whether polyomavirus reactivation occurs in SLE patients, and whether antibodies to T-antigen, DNA, and to TBP and CREB are linked to such events. Both within and among these SLE patients, frequent polyomavirus reactivations were observed that could not be explained by certain rearrangements of the noncoding control regions, nor by corticosteroid treatment. Linked to these events, antibodies to T-antigen, DNA, TBP, and CREB were detected, identical to what we observed in mice. Antibodies recognizing double-stranded DNA were confined to patients with frequent polyomavirus reactivations. The results described here indicate that cognate interaction of B cells recognizing DNA or DNA-associated proteins and T cells recognizing T antigen had taken place as a consequence of complex formation between T ag and DNA in vivo in the context of polyomavirus reactivations. 

Involvement of an SAF-like transcription factor in the activation of serum amyloid A gene in monocyte/macrophage cells by lipopolysaccharide. Serum amyloid A (SAA) has been linked to atherosclerosis because of its ability to remodel high-density lipoprotein by the depletion of apolipoprotein A1, its ability to bind cholesterol, and its presence in the atherosclerotic plaques of coronary and carotid arteries. In the present study, we investigated the induction mechanism of SAA gene in THP-1 monocyte/macrophage cells which play a critical role in the development of atherosclerotic fatty streak and plaque formation. We and others have shown that SAA gene is induced in monocyte/macrophage cells by lipopolysaccharide (LPS). By promoter function analysis, we show that the SAA promoter sequence between -280 and -226 can confer LPS responsiveness. Gel electrophoretic mobility shift assay detected an induced DNA-binding activity in these cells in response to LPS. Characterization of the DNA-binding protein by UV cross-linking, Southwestern blot, and antibody ablation/supershift assays revealed that it is similar to a recently reported nuclear factor designated SAF. These results demonstrated that LPS-mediated SAA gene induction in monocyte/macrophage cells is primarily due to the induction of SAF activity. 

Expression of NFAT-family proteins in normal human T cells. NFAT proteins constitute a family of transcription factors involved in mediating signal transduction. Using a panel of specific antisera in immunoprecipitation assays, we found that NFATp (135 kDa) is constitutively expressed in normal human T cells, while synthesis of NFATc (predominant form of 86 kDa) is induced by ionomycin treatment. NFAT4/x was very weakly expressed in unstimulated cells, and its level did not increase upon treatment with activating agents. NFAT3 protein was not observed under any conditions. Higher-molecular-weight species of NFATc (of 110 and 140 kDa) were also detected. In addition, translation of NFATc mRNA apparently initiates at two different AUG codons, giving rise to proteins that differ in size by 36 amino acids. Additional size heterogeneity of both NFATc and NFATp results from phosphorylation. In contrast to ionomycin treatment, exposure of cells to phorbol myristate acetate (PMA) plus anti-CD28 did not induce NFATc, indicating that under these conditions, interleukin-2 synthesis by these cells is apparently independent of NFATc. In DNA binding assays, both PMA plus anti-CD28 and PMA plus ionomycin resulted in nuclear NFAT. Surprisingly, the PMA-ionomycin-induced synthesis of NFATc that was detected by immunoprecipitation was not mirrored in the DNA binding assays: nearly all of the activity was due to NFATp. This is the first study of expression of all family members at the protein level in normal human T cells. 

High levels of the DNA-binding activity of E2F in adult T-cell leukemia and human T-cell leukemia virus type I-infected cells: possible enhancement of DNA-binding of E2F by the human T-cell leukemia virus I transactivating protein, Tax. Transcription factor E2F binds to cellular promoters of certain growth- and cell cycle-controlling genes and forms distinct heteromeric complexes with other nuclear proteins. It has therefore been proposed that E2F is involved in cellular proliferation control. Human T-cell leukemia virus type I (HTLV-I) is an etiological agent of adult T-cell leukemia (ATL). We show here by mobility-shift assay that E2F-containing DNA-binding complexes were detected in HTLV-I-infected T-cell lines and leukemic cells obtained from ATL patients but not in an uninfected T-cell line, Jurkat, and normal peripheral blood mononuclear cells. The Tax protein, encoded by HTLV-I, is a potent transcription activator of viral and several cellular genes. We demonstrate that expression of Tax can induce the E2F-containing DNA-binding complexes in Jurkat T cells. Thus, Tax, through enhancement of the DNA-binding activity of E2F, may be capable of regulating cellular gene expression implicated in the proliferation and transformation of T cells in ATL. This activity may be relevant to the mechanisms whereby HTLV-I which does not contain oncogenes induces neoplasia. 

Oncogenic forms of NOTCH1 lacking either the primary binding site for RBP-Jkappa or nuclear localization sequences retain the ability to associate with RBP-Jkappa and activate transcription. Truncated forms of the NOTCH1 transmembrane receptor engineered to resemble mutant forms of NOTCH1 found in certain cases of human T cell leukemia/lymphoma (T-ALL) efficiently induce T-ALL when expressed in the bone marrow of mice. Unlike full-sized NOTCH1, two such truncated forms of the protein either lacking a major portion of the extracellular domain (DeltaE) or consisting only of the intracellular domain (ICN) were found to activate transcription in cultured cells, presumably through RBP-Jkappa response elements within DNA. Both truncated forms also bound to the transcription factor RBP-Jkappa in extracts prepared from human and murine T-ALL cell lines. Transcriptional activation required the presence of a weak RBP-Jkappa-binding site within the NOTCH1 ankyrin repeat region of the intracellular domain. Unexpectedly, a second, stronger RBP-Jkappa-binding site, which lies within the intracellular domain close to the transmembrane region and significantly augments association with RBP-Jkappa, was not needed for oncogenesis or for transcriptional activation. While ICN appeared primarily in the nucleus, DeltaE localized to cytoplasmic and nuclear membranes, suggesting that intranuclear localization is not essential for oncogenesis or transcriptional activation. In support of this interpretation, mutation of putative nuclear localization sequences decreased nuclear localization and increased transcriptional activation by membrane-bound DeltaE. Transcriptional activation by this mutant form of membrane-bound DeltaE was approximately equivalent to that produced by intranuclear ICN. These data are most consistent with NOTCH1 oncogenesis and transcriptional activation being independent of association with RBP-Jkappa at promoter sites. 

Activation of the NF-kappaB pathway by inflammatory stimuli in human neutrophils. Activated neutrophils have the ability to upregulate the expression of many genes, in particular those encoding cytokines and chemokines, and to subsequently release the corresponding proteins. Although little is known to date concerning the regulation of gene transcription in neutrophils, it is noteworthy that many of these genes depend on the activation of transcription factors, such as NF-kappaB, for inducible expression. We therefore investigated whether NF-kappaB/Rel proteins are expressed in human neutrophils, as well as their fate on cell activation. We now report that dimers consisting of p50 NFkappaB1, p65 RelA, and/or c-Rel are present in neutrophils and that the greater part of these protein complexes is physically associated with cytoplasmic IkappaB-alpha in resting cells. Following neutrophil stimulation with proinflammatory agonists (such as lipopolysaccharide [LPS], tumor necrosis factor-alpha [TNF-alpha], and fMet-Leu-Phe) that induce the production of cytokines and chemokines in these cells, NF-kappaB/Rel proteins translocated to nuclear fractions, resulting in a transient induction of NF-kappaB DNA binding activity, as determined in gel mobility shift assays. The onset of both processes was found to be closely paralleled by, and dependent on, IkappaB-alpha degradation. Proinflammatory neutrophil stimuli also promoted the accumulation of IkappaB-alpha mRNA transcripts, resulting in the reexpression of the IkappaB-alpha protein. To our knowledge, this constitutes the first indication that NF-kappaB activation may underlie the action of proinflammatory stimuli towards human neutrophil gene expression and, as such, adds a new facet to our understanding of neutrophil biology. 

Glucocorticoid-resistance in peripheral-blood lymphocytes does not correlate with number of affinity of glucocorticoid-receptors in chronic renal failure patients. Glucocorticoid (GC) resistance in patients with chronic renal failure (CRF) seriously impairs successive GC therapy after renal transplantation. We examined the relationship between GC-receptor (GC-R) parameters in peripheral-blood mononuclear cells (PBMC) and PBMC resistance to GC in 21 CRF patients and 18 healthy subjects. Each subject group was divided into two subgroups according to PBMC sensitivity to prednisolone in a mitogen assay procedure; i.e., sensitive (IC50 < 381 ng/mL) and resistant (IC50 > 381 ng/mL) groups. In healthy subjects, the mean GC-R Bmax and Kd in quiescent PBMC of the GC-sensitive group were 2.89 +/- 1.23 fmol/10(6) cells and 4.00 +/- 2.24 nM, respectively. The Bmax in these subjects significantly increased to 6.61 +/- 2.02 (257.7 +/- 107.8%) after 24 h stimulation with concanavalin A (p < 0.01), while the Kd change was not significant. The GC-R Bmax and Kd in quiescent PBMC of the GC-resistant group were 5.33 +/- 1.37 fmol/10(6) cells and 3.20 +/- 1.39 nM, respectively. Both of these parameters, however, did not change significantly after mitogen stimulation. There was a significant negative correlation between IC50S of prednisolone and increase-ratios (post/pre ratio) of Bmax after mitogen stimulation (p < 0.05). In CRF patients, Bmax and Kd in quiescent PBMC of the GC-sensitive group were 6.04 +/- 2.35 fmol/10(6) cells and 3.49 +/- 1.72 nM, respectively, while those in PBMC of the GC-resistant group were 5.13 +/- 2.31 fmol/10(6) cells and 4.04 +/- 1.62 nM, respectively. The Bmax and Kd were not significantly changed after mitogen stimulation in both subgroups of CRF. Moreover, in contrast to healthy subjects, there was no correlation between IC50 and GC-R parameters in CRF. We concluded that, in healthy subjects, decreased PBMC capacity to amplify GC-R numbers in response to mitogen is correlated with GC resistance, whereas in CRF patients the resistant mechanism is not correlated with GC-R parameters. An unknown event might be involved in GC-resistance of CRF. 

Significance of quantitative analysis of AML1/ETO transcripts in peripheral blood stem cells from t(8;21) acute myelogenous leukemia. Autologous peripheral blood stem cell transplantation (PBSCT) is replacing autologous bone marrow transplantation (BMT) in the treatment of leukemia. One of the potential advantages of autologous PBSCT is the possibility that peripheral blood stem cells (PBSC) are less likely to be contaminated by leukemic cells than bone marrow grafts. However, the major problem still remains the high incidence of leukemic relapse following autologous PBSCT, which may be caused by the reinfusion of PBSC contaminated by leukemic cells. Recently, we have developed a quantitative assay using competitive reverse transcriptase polymerase chain reaction that estimates the number of AML1/ETO transcripts in t(8;21) acute myelogenous leukemia (AML), in order to determine the degree of leukemic cell contamination in PBSC harvests, and to monitor minimal residual disease (MRD) quantitatively in patients with t(8;21) AML. Our data indicate that although PBSC harvests collected after consolidation chemotherapy are contaminated by leukemic cells, the degree of leukemic cell contamination decreases with repeated cycles of chemotherapy. Furthermore, the MRD in PBSC harvests is less than in the corresponding bone marrow obtained on the day of the PBSC collection. There appears to be no relationship between the number of AML1/ETO transcripts found in the infused PBSC harvests and the incidence of leukemic relapse following autologous PBSCT in our study. However, a substantial decrease of AML1/ETO transcripts was seen following autologous PBSCT. Thus, the quantitative analysis of AML1/ETO transcripts may be clinically useful in patients with t(8;21) AML. 

DNA methylation changes in hematologic malignancies: biologic and clinical implications. DNA methylation changes are among the most common detectable abnormalities in human neoplasia. Hypermethylation within the promoters of selected genes appears to be especially common in all types of human hematopoietic neoplasms, and is usually associated with inactivation of the involved gene(s). Such hypermethylation-associated silencing of gene expression has been shown for several genes regulating the growth and differentiation of hematopoietic cells, including the estrogen receptor (ER) gene, P15, P16 and others. Hypermethylation within the promoters of some genes appear to be an early event in the pathogenesis of neoplasia (ER, P15), while other genes seem to become methylated during the progression of leukemias (HIC1, c-abl). The high prevalence of promoter methylation suggests that this molecular abnormality can be used to monitor disease activity during therapy. In addition, new technology allows the sensitive identification of gene hypermethylation in a background of normal cells, suggesting possible new strategies for the detection of minimal residual disease. Finally, reactivation of tumor-suppressor gene expression through pharmacologic inhibition of DNA methyltransferase and resultant DNA demethylation appears to be a promising new avenue of therapy in acute leukemia. 

Transcriptional induction of collagenase-1 in differentiated monocyte-like (U937) cells is regulated by AP-1 and an upstream C/EBP-beta site. In this report, we demonstrate that the AP-1 site and a distal promoter element regulate transcriptional induction of collagenase-1 during monocytic differentiation. Chloramphenicol acetyltransferase expression constructs containing regions of the human collagenase-1 promoter were stably or transiently transfected into U937 cells, and reporter activity was assessed at various times after the onset of phorbol 12-myristate 13-acetate (PMA)-mediated differentiation. Rapid and strong induction of promoter activity was lost in constructs with a mutant AP-1 element; however, at 16-96 h post-PMA, the mutant collagenase-1 promoter displayed AP-1 independent PMA-mediated transactivation. The AP-1 mutant constructs also showed delayed transcriptional activation in PMA-treated fibroblasts. Western and supershift analyses indicated that functional Jun and Fos proteins were present in nuclear extracts of PMA-differentiated U937 cells. Promoter deletion constructs demonstrated the potential role of distal promoter sequences in regulating collagenase-1 transcription. In particular, Western, supershift, and promoter deletion analyses suggested a role for CCAAT/enhancer-binding protein-beta (C/EBP-beta) binding site between -2010 and -1954 in regulating transcription of collagenase-1 in monocytic cells. Our findings suggest that distinct regulatory elements, acting somewhat independently of each other, control expression of collagenase-1. In addition, our data suggests that the rapid PMA-mediated induction of collagenase-1 transcription is controlled by a mechanism distinct from that regulating the sustained expression of this proteinase in activated macrophages. 

The tumour associated cell surface antigen A6H is costimulatory for human CD4+ but not CD8+ T cells. The A6H monoclonal antibody (mAb) recognizes a 120,000-140,000 MW antigen that is expressed at similar densities on 85-90% of human CD4+ and CD8+ T cells and on renal cell carcinomas. The binding of the A6H mAb induced a costimulatory signal in anti-CD3 activated T cells. In the present report, we show that A6H costimulated cell proliferation and cytokine production in purified CD4+ T cells. Unexpectedly, the CD8+ T-cell subpopulation failed to respond. CD4+ T cells costimulated with the A6H mAb upregulated CD80, CD86, CD71, interleukin-2 (IL-2)R alpha, IL-2R beta and IL-2R gamma, while no corresponding up-regulation of these cell surface molecules was seen in CD8+ T cells. In order to investigate the nature of the A6H mAb costimulus at the transcriptional level we have examined induction of the transcription factors OCT-1, AP-1 and NF-kappa B which are known to be transcriptional regulators of several cytokine and cytokine receptor genes, including the IL-2 and IL-2R genes. Co-ligation of the A6H antigen and the CD3 complex induced expression of the transcription factor AP-1 in CD4+ T cells, whereas no increase in NF-kappa B and octamer-binding (Oct) proteins was seen compared to T cells stimulated with anti-CD3 alone. Furthermore, no induction of AP-1 was seen in A6H costimulated CD8+ T cells. These results suggests that both proximal steps in CD8+ T-cell activation as well as the later phases are unresponsive to A6H ligation. Molecular differences of the A6H molecule or distinct regulation of the A6H transduced AP-1 activation pathway may exist in CD4+ and CD8+ T cell subpopulations. 

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

Molecular actions of prolactin in the immune system. The immunoregulatory properties of prolactin, a pituitary peptide hormone, have received renewed attention. The prolactin receptor, a member of the hematopoietin/cytokine receptor superfamily, is ubiquitously expressed by cells in the immune system. Certain subpopulations of lymphocytes synthesize and secrete biologically active prolactin, which suggests that prolactin can act as an autocrine and/or paracrine factor to modulate the activities of cells of the immune system. This review focuses on the molecular actions of prolactin in the immune system. Emphasis is given to recent information about the molecular mechanisms of prolactin receptor signal transduction, and the signaling molecules and prolactin-inducible target genes that participate in these responses. In particular, the prolactin-inducible interferon regulatory factor-1 gene and its roles in mediating diverse immune responses. 

Spontaneous occurrence of early region 1A reiteration mutants of type 5 adenovirus in persistently infected human T-lymphocytes. Mutants of type 5 adenovirus (Ad5) with reiterated DNA sequences in the E1a region appeared in a human T-lymphocyte cell line, Molt-4, persistently infected with H5sub304, a deletion/substitution mutant that has a wild-type phenotype in viral replication. Endonuclease analyses and DNA sequencing revealed DNA reiteration in each mutant. In the four representative mutants investigated, the DNA reiterations all started within a six-base-pair consensus sequence, G(or C)CTGTG, located in the second exon of the E1a region (at nt 1333, 1367, or 1419). There was not any DNA homology between the breakpoints in the second exon and the inserting sequences (starting at nt 532, 710, or 792). Northern analyses suggested that the reiterated splicing sites of the representative mutants were all used in RNA splicing, and the closest donor and recipient joints were used most frequently. These observations imply that during persistent infection Ad5 underwent spontaneous mutations by sequence-specific breakage and nonhomologous end-end joining recombination events. These E1a reiteration mutants could be propagated in HeLa, A549, and KB cells; they were genetically stable; and they killed CREF cells at a strikingly high frequency. Preliminary observations tend to correlate this CREF cell killing with the accumulation of the early viral proteins and/or viral DNA in the infected cells. This degree of cell damage was not observed in Ad5wt or H5sub304 infection of CREF cells. The observed E1a reiterations provide a model to gain insight into understanding the evolutionary events of some, if not all, adenovirus types during many years of symbiotic, persistent relationship in human tonsils and adenoids and possibly other lymphoid organs. 

Quantification of vitamin D receptor mRNA by competitive polymerase chain reaction in PBMC: lack of correspondence with common allelic variants. It has been recently claimed that polymorphism for the vitamin D receptor (VDR) influences several aspects of calcium and bone metabolism. To evaluate the physiologic plausibility of these claims, we compared the abundance of the VDR mRNA in peripheral blood mononuclear cells (PBMCs) between different VDR genotypes using a quantitative reverse transcribed polymerase chain reaction-based method. The method is based on the coamplification of VDR cDNA and an internal standard consisting of known concentrations of a human VDR CDNA mutated at a BglII restriction site; the interassay coefficient of variation is 11%. To validate the method, we made use of earlier receptor binding studies indicating that normal human monocytes and activated, but not resting, lymphocytes expressed the VDR. The concentration of the VDR mRNA was 10(-8) to 10(-7) g/g of total RNA in cell-sorted monocytes and in in vitro activated lymphocytes, but only 10(-12) g/g of total mRNA in resting lymphocytes, establishing that the VDR mRNA determined by our method in PBMCs is due to constitutive expression in monocytes. Following an initial genotype screening of 85 normal volunteers by polymerase chain reaction or restriction fragment length polymorphism analysis, 14 individuals with the Bb genotype, 12 with the bb genotype, and 12 with the BB genotype were selected. The concentration of the VDR mRNA, corrected for the number of monocytes, was similar among the three genotype groups, as were the other variables examined: serum calcitriol, serum osteocalcin, and vertebral and hip bone density. We conclude that VDR polymorphism does not affect the abundance of the VDR mRNA. 

HLA-DMA and HLA-DMB gene expression functions through the conserved S-X-Y region. The MHC class II homologous proteins HLA-DMA and HLA-DMB function in the loading of peptides onto class II molecules. Like the class II genes, the HLA-DM genes contain upstream regulatory sequences similar to the S-X-Y regulatory region as well as additional putative regulatory sites. To determine whether the DM genes are regulated in a similar manner as class II genes, a series of in vivo and in vitro analyses was performed. Deletion analysis showed that expression from the DM promoters is dependent on the conserved S-X-Y region. The class II-specific transcription factors RFX and CIITA are also required for expression, as cell lines deficient in these factors failed to allow transcription from the DM promoters. In addition, in vivo footprint analysis showed the putative X and Y boxes to be occupied by transcription factors in wild-type B cells, but not in RFX-deficient B cells. In astrocytes, IFN-gamma treatment induced increased occupancy of these sites. None of the other putative regulatory sites was occupied in vivo, indicating that they may not be functional. Finally, gel shift analysis showed synergistic complex formation between proteins that bind to the putative X boxes of the DM genes, as is found for the DRA gene. Therefore, the DM genes share a common mechanism of regulation with the class II genes. 

An enhancer-blocking element between alpha and delta gene segments within the human T cell receptor alpha/delta locus. T cell receptor (TCR) alpha and delta gene segments are organized within a single genetic locus but are differentially regulated during T cell development. An enhancer-blocking element (BEAD-1, for blocking element alpha/delta 1) was localized to a 2.0-kb region 3' of TCR delta gene segments and 5' of TCR alpha joining gene segments within this locus. BEAD-1 blocked the ability of the TCR delta enhancer (Edelta) to activate a promoter when located between the two in a chromatin-integrated construct. We propose that BEAD-1 functions as a boundary that separates the TCR alpha/delta locus into distinct regulatory domains controlled by Edelta and the TCR alpha enhancer, and that it prevents Edelta from opening the chromatin of the TCR alpha joining gene segments for VDJ recombination at an early stage of T cell development. 

Plasma sialyltransferase levels in psychiatric disorders as a possible indicator of HPA axis function. A dysfunction in the regulation of the hypothalamic-pituitary-adrenal (HPA) axis, possibly attributed to a change in glucocorticoid receptor (GR) functionality, has been implicated in depression. We have measured both lymphocyte GR receptor binding parameters and plasma sialyltransferase activity, as a biochemical marker of GR function, in two groups of patients suffering from depression or schizophrenia and in a group of age- and sex-matched controls. While there was a significant increase in plasma cortisol levels in the depressed group, there were no changes in the lymphocyte GR binding parameters (K(m) and Bmax). There was, however, a significant decrease in the plasma sialyltransferase: cortisol ratio in the depressed group suggesting an inability of the raised cortisol levels to induce enzyme expression and this ratio may provide a useful biochemical marker of cortisol receptor function. Although there was an increase in the plasma activity of the alpha 2,6 sialyltransferase isozyme in the schizophrenic group, no other changes were determined. Therefore, while the total plasma sialyltransferase:cortisol ratio reflects HPA axis function, alterations in specific isozyme activity may also be associated with other CNS disease states. 

Involvement of the N-terminal region of the human mineralocorticoid receptor hormone-binding domain in agonist and antagonist binding as revealed by a new monoclonal antibody. To gain a better understanding of the mechanism of binding to the human mineralocorticoid receptor (hMR), we developed a new monoclonal antibody (mAb) raised against the hormone-binding domain (HBD). For this purpose, mice were immunized with a fusion protein including the sequence Thr729-Lys984 of hMR. After ELISA screening, mAb 18C7 was selected for its specificity towards the HBD. This antibody recognized both the denatured and native MR forms, as well as the hetero-oligomeric MR form and the transformed MR state. By using several HBD subfragments, the mAb 18C7 epitope was located in the N-terminal region of the HBD from Thr729 to Leu765. We then studied the effect of the antibody on aldosterone and progesterone binding to the hMR. When 18C7 was incubated with liganded MR, it was able to partly displace (20%) the hormone from its binding site. When 18C7 was incubated with MR before aldosterone or progesterone, the antibody inhibited 75-80% of the binding. The effect of 18C7 on the binding was similar with both hormones. A sucrose gradient analysis indicated the simultaneous presence of two kinds of receptor complexes: the steroid-MR complex and the antibody-MR complex. After its associated proteins, especially the heat-shock protein hsp90, had been cross-linked with the hMR by dimethylpimelimidate, 18C7 was still able to react with the receptor. Our results indicated that the epitope recognized by 18C7 was directly implicated in hormone binding. The lack of steroid binding of HBD mutants with the Thr729-Leu765 sequence deleted [Jalaguier, Mesnier, Leger and Auzou (1996) J.Steroid Biochem.Mol.Biol.57, 43-50] supports this hypothesis. Because of the similar behaviours of aldosterone and progesterone, we conclude that the N-terminal Thr729-Leu765 region of the HBD is similarly involved in the binding of both hormones. 

Triggering of HLA-DR antigens differentially modulates tumor necrosis factor alpha release by B cells at distinct stage of maturation. Triggering of HLA class II antigens by the anti-HLA-DR monoclonal antibody (mAb) L243 significantly (P < 0.05) and differentially enhanced the release of tumor necrosis factor alpha (TNF-alpha) by the non-Hodgkin's lymphoma cells Ri-I, Ci-I, and Sc-I, which are at a distinct stage of B-cell differentiation, and by the more mature Burkitt lymphoma cell Raji; in contrast, it did not induce TNF-alpha release by the pre-B leukemia cells Nalm-6 and BV173. TNF-alpha release peaked at 24 h and decreased thereafter, and it was dose dependent and preceded by an increase of TNF-alpha mRNA detectable after 3 h of stimulation with mAb L243. Secreted TNF-alpha mediated the enhancement of nuclear factor kappa B (NF-kappa B) and activator protein-1 (AP-1) binding activity; in fact, the triggering of HLA-DR antigens in the presence of antihuman TNF-alpha-neutralizing antibodies did not upregulate NF-kappa B and AP-1. In contrast, released TNF-alpha was not responsible for the homotypic aggregation of Ri-I, Ci-I, Sc-I, and Raji cells induced by mAb L243, and it did not affect the proliferation of B cells investigated. Altogether, our data demonstrate that: (a) the ability of B cells to release TNF-alpha after triggering of HLA-DR antigens depends on their stage of differentiation; (b) levels of released TNF-alpha seem to correlate with the stage of B-cell maturation but do not correlate with the amounts of cell surface HLA-DR antigens; (c) secreted TNF-alpha regulates the levels of expression of NF-kappa B and AP-1 by an autocrine loop; and (d) intracellular signals mediating TNF-alpha release by B cells are distinct from those regulating homotypic aggregation and proliferation. 

Immediate early and early lytic cycle proteins are frequent targets of the Epstein-Barr virus-induced cytotoxic T cell response. Epstein-Barr virus (EBV), a human gamma-herpesvirus, can establish both nonproductive (latent) and productive (lytic) infections. Although the CD8+ cytotoxic T lymphocyte (CTL) response to latently infected cells is well characterized, very little is known about T cell controls over lytic infection; this imbalance in our understanding belies the importance of virus-replicative lesions in several aspects of EBV disease pathogenesis. The present work shows that the primary CD8+ CTL response to EBV in infectious mononucleosis patients contains multiple lytic antigen-specific reactivities at levels at least as high as those seen against latent antigens; similar reactivities are also detectable in CTL memory. Clonal analysis revealed individual responses to the two immediate early proteins BZLF1 and BRLF1, and to three (BMLF1, BMRF1, and BALF2) of the six early proteins tested. In several cases, the peptide epitope and HLA-restricting determinant recognized by these CTLs has been defined, one unusual feature being the number of responses restricted through HLA-C alleles. The work strongly suggests that EBV-replicative lesions are subject to direct CTL control in vivo and that immediate early and early proteins are frequently the immunodominant targets. This contrasts with findings in alpha- and beta- herpesvirus systems (herpes simplex, cytomegalovirus) where viral interference with the antigen-processing pathway during lytic infection renders immediate early and early proteins much less immunogenic. The unique capacity of gamma-herpesvirus to amplify the viral load in vivo through a latent growth-transforming infection may have rendered these agents less dependent upon viral replication as a means of successfully colonizing their hosts. 

A new mouse gene, SRG3, related to the SWI3 of Saccharomyces cerevisiae, is required for apoptosis induced by glucocorticoids in a thymoma cell line. We isolated a new mouse gene that is highly expressed in thymocytes, testis, and brain. This gene, SRG3, showed a significant sequence homology to SWI3, a yeast transcriptional activator, and its human homolog BAF155. SRG3 encodes 1,100 amino acids and has 33-47% identity with SWI3 protein over three regions. The SRG3 protein contains an acidic NH2 terminus, a myb-like DNA binding domain, a leucine-zipper motif, and a proline- and glutamine-rich region at its COOH terminus. Rabbit antiserum raised against a COOH-terminal polypeptide of the SRG3 recognized a protein with an apparent molecular mass of 155 kD. The serum also detected a 170-kD protein that seems to be a mouse homologue of human BAF170. Immunoprecipitation of cell extract with the antiserum against the mouse SRG3 also brought down a 195-kD protein that could be recognized by an antiserum raised against human SWI2 protein. The results suggest that the SRG3 protein associates with a mouse SWI2. The SRG3 protein is expressed about three times higher in thymocytes than in peripheral lymphocytes. The expression of anti-sense RNA to SRG3 mRNA in a thymoma cell line, S49.1, reduced the expression level of the SRG3 protein, and decreased the apoptotic cell death induced by glucocorticoids. These results suggest that the SRG3 protein is involved in the glucocorticoid-induced apoptosis in the thymoma cell line. This implicates that the SRG3 may play an important regulatory role during T cell development in thymus. 

Cytomegalovirus immediate early genes upregulate interleukin-6 gene expression. BACKGROUND: The immediate early genes (IE) of human cytomegalovirus (CMV) can be expressed in monocytic cells and are known to regulate viral and cellular genes. Interleukin-6 (IL-6) plays a central role in numerous inflammatory and immune processes. Interleukin-6 levels are increased in lung transplant patients clinically diagnosed with CMV pneumonitis. The regulation of IL-6 is dependent on various stimuli that include lipopolysaccharide (LPS), viruses, and other cytokines. These studies examined the ability of CMV IE gene products to modulate IL-6 production. METHODS: THP-1 cells, a monocytic cell line, were transfected with the CMV IE genes. Interleukin-6 protein and IL-6 mRNA were measured in control and CMV immediate early transfected cells. Cotransfection of CMV IE genes and IL-6 chloramphenicol acetyl transferase (CAT) or IL-6 luciferase constructs were used to study IL-6 promoter activity. RESULTS: Interleukin-6 protein and mRNA production were significantly increased in cells transfected with the CMV IE genes and stimulated with LPS compared to LPS-stimulated control cells. Cytomegalovirus IE gene products significantly enhanced LPS stimulation of IL-6 promoter activity in both IL-6 CAT and IL-6 luciferase assays. A deletion construct that contains a NF-kappa B site but is missing the multiple response region demonstrated a continued increase in IL-6 luciferase activity in LPS-stimulated CMV transfected cells. CONCLUSION: Cytomegalovirus immediate early gene products significantly enhanced expression of IL-6 in LPS-stimulated cells. The increase in IL-6 luciferase activity occurs in the absence of the multiple response region, the area of the IL-6 promoter responsive to IL-1, TNF alpha, cyclic amp, and phorbol 12-myristate 13-acetate. The ability of CMV IE gene products to enhance IL-6 production may play an important role in immune inflammatory states associated with CMV infection. 

Cellular redox status influences both cytotoxic and NF-kappa B activation in natural killer cells. The role of cellular redox status in both cytotoxic activity and NF-kappa B activation in natural killer (NK) cells was investigated. The results indicate that stimulation of NK cells, either freshly isolated from peripheral blood lymphocytes (PBL) or long-term cultured NK clones, with specific cell targets results in an increased binding activity of NF-kappa B and AP-1 transcription factors measured by gel retardation. Pretreatment of NK cells with the antioxidant pyrrolidine dithiocarbarmate (PDTC) leads to the inhibition of NF-kappa B activation but the AP-1 binding to DNA was superinduced. The inhibition of NF-kappa B by PDTC paralleled with an inhibition of spontaneous cytotoxicity mediated by NK cells. Moreover, the inhibitors of serine proteases, N-alpha-tosyl-L-lysine chloromethyl ketone and N-alpha-tosyl-L-phenylalanine chloromethyl ketone, also blocked the cytolytic activity of NK cells against the sensitive target K562. In contrast, NK activity was not affected by pretreatment of the effector cells with the proteasome inhibitor N-acetyl-leu-leu-norleucinal which selectively inhibits NF-kappa B activation. Altogether, these results support the hypothesis that the activation of NK cells involved transcriptional and post-transcriptional events, and that reactive intermediates may play an important role in the molecular processes related with the generation of a cytotoxic response by NK cells. 

Abnormal T lymphocyte development induced by targeted overexpression of IkappaB alpha. A role in thymic maturation for factors of the NF-kappaB family has long been suspected, but not yet proven. Transgenic mice with a lymphocyte-specific defect in NF-kappaB activation were produced by targeted expression of human IkappaB alpha. The thymic cellularity of these mice was significantly decreased. The proportion of mature, TCRhigh thymocytes of the alphabeta lineage was reduced, and the remaining TCRhigh population contained an unusually high proportion of double-positive cells. This defect in maturation resulted in a transgene dose-dependent reduction in peripheral T lymphocytes, with the CD8 lineage being more severely affected. These data provide direct evidence for the involvement of NF-kappaB/Rel family proteins in late stages of T lymphocyte development, coincident with positive and negative selection. 

Sequential development of structural and functional alterations in T cells from tumor-bearing mice. The TCR alpha beta or -gamma delta chains bind the peptide ligand, whereas the associated CD3 delta epsilon gamma and TCR zeta subunits couple the TCR to intracellular signal transduction components. Recently, several groups have described marked alterations in signal transduction elements in T cells from cancer patients or in mice bearing tumor for a few weeks (>26 days). The sequence in which these alterations develop is unknown. The aim of this study was to explore the kinetics of the development of alterations in signal transduction molecules (TCR zeta chain, NF kappaB family proteins, and tyrosine kinase p56(lck)) in mice bearing MC38 colon adenocarcinoma. The results demonstrate that alterations in NF kappaB family proteins, specifically the failure of p65 translocation to the nucleus, occur earlier and more frequently than the decrease in zeta-chain. These defects are paralleled by an impaired ability to produce Th1 cytokines (IL-2 and IFN-gamma). These initial changes are followed by the eventual loss of TCR zeta chain and p56(lck) and a marked decrease in cytotoxic function. An increased rate of lysosomal degradation is one of the mechanisms responsible for the loss of zeta-chain. 

Characterization of interleukin-10 receptor expression on B-cell chronic lymphocytic leukemia cells. B-cell chronic lymphocytic leukemia (B-CLL) cells accumulate in vivo in the G0/G1 phase of the cell cycle, suggesting that their malignant expansion is due, at least in part, to a delay in cell death. However, the cellular or molecular factors responsible for a delay in B-CLL cell death are unknown. B-CLL cells do express receptors for interferon-alpha (IFN-alpha) and IFN-gamma, and activation of both has been shown to promote B-CLL survival in vitro by preventing apoptosis. The interleukin-10 (IL-10) receptor is another member of the IFN receptor family, but its ligand, IL-10, has been reported to induce apoptosis in B-CLL cells. In the current study, we undertook a biochemical analysis of IL-10 receptor expression on freshly isolated B-CLL cells and characterized the functional responsiveness of IL-10 binding to its constitutively expressed receptor. We show that B-CLL cells bind IL-10 with significant specificity and express between 47 and 127 IL-10 receptor sites per cell, with a dissociation constant in the range of 168 to 426 x 10(-12) mol/L. Ligand binding and activation of the IL-10 receptor expressed on B-CLL cells results in the phosphorylation of signal transducer and activator of transcription 1 (STAT1) and STAT3 proteins. This pattern of STAT protein phosphorylation is identical to IL-10 receptor activation on normal cells and similar to IFN-alpha (STAT1 and STAT3) and IFN-gamma (STAT1) receptor activation in CLL. Further, in consecutive samples of fresh blood obtained from patients with B-CLL cells, the addition of IL-10 inhibited B-CLL proliferation, enhanced B-CLL differentiation, but did not induce apoptosis. Indeed, IL-10, like IFN-gamma, was able to significantly reduce the amount of B-CLL cell death caused by hydrocortisone-induced apoptosis. We conclude that cytokines, which signal through the interferon family of receptors, have comparable functional effects on B-CLL cells. 

An isotype-specific activator of major histocompatibility complex (MHC) class II genes that is independent of class II transactivator. Patients with one type of major histocompatibility complex class II combined immunodeficiency have mutations in a gene termed class II transactivator (CIITA), which coordinately controls the transcription of the three major human class II genes, HLA-DR, -DQ, and -DP. However, the experimentally derived B-lymphoblastoid cell line, clone 13, expresses high levels of HLADQ in the absence of HLA-DR and HLA-DP, despite its mapping by complementation analysis to this group. It was possible that one of the clone 13 CIITA alleles bore a mutation that allowed HLA-DQ, but not HLA-DR or -DP transcription. Alternatively, another factor, distinct from CIITA, might control HLA-DQ expression. We report here that ectopic expression of CIITA cDNAs derived by reverse transcriptase polymerase chain reaction from clone 13 do not restore expression of HLA-DQ in another CIITA-deficient cell line, RJ2.2.5. In addition, no CIITA protein is detectable in clone 13 nuclear extracts. In contrast, somatic cell fusion between clone 13 and RJ2.2.5 restored expression of the HLA-DQ haplotype encoded by the RJ2.2.5 DQB gene. Taken together, these data demonstrate the existence of an HLA-DQ isotype-specific trans-acting factor, which functions independently of CIITA. 

Uncoupling of cell cycle arrest from the expression of monocytic differentiation markers in HL60 cell variants. Differentiation generally leads to cell cycle arrest. Human leukemia HL60 cells respond to the presence of 1,25-dihydroxyvitamin D3 (1,25D3) by expressing a number of markers of the monocyte/macrophage phenotype and become arrested predominantly in the G1 phase of the cell cycle. We have recently reported a series (A) of 1,25D3-resistant variants of HL60 cells which proliferate in the presence of 1,25D3 and do not express differentiation markers (Exp. Cell Res. 224, 312, 1996). We now describe another series (B) of such variants, which differ from A series cells grown in similar concentrations of 1,25D3 in that they express the CD14 antigen and nonspecific esterase, characteristic of the monocyte, while continuing to proliferate and they develop hypotetraploid DNA (4C) content at higher concentrations of ambient 1,25D3 than the A series cells. Cells in the B series with 4C DNA content (100B and 200B) also differed from the A series 4C cells by the absence of DNA binding by the full-length Sp1 transcription factor. However, B series cells resembled the A series cells in exhibiting faster growth rates than the parental HL60 cells and showed high levels of vitamin D receptor and retinoid receptor X proteins. These results show that the initial steps in the 1,25D3 signaling pathway are intact in B series resistant cells and lead to the appearance of early markers of monocytic differentiation. However, the progression to subsequent events which comprise terminal differentiation and cell cycle arrest is halted during the adaptation to the presence of 1,25D3 in these cells. Thus, the availability of these variant cells should provide a system for studying the link between differentiation and cell cycle arrest. 

