Lymphokine Genes Research Articles

New insights into IL-7 signaling pathways during early and late T cell development

New insights into IL-7 signaling pathways during early and late T cell development

Current biochemistry, molecular biology, and clinical relevance of natriuretic peptides

The mammalian natriuretic peptide family consists of atrial (ANP), brain [B-type; BNP] and C-type natriuretic peptide (CNP) and three receptors, natriuretic receptors-A (NPR-A), -B (NPR-B) and -C (NPR-C). Both ANP and BNP are abundantly expressed in the heart and are secreted mainly from the atria and ventricles, respectively. By contrast, CNP is mainly expressed in the central nervous system, bone and vasculature. Plasma concentrations of both ANP and BNP are elevated in patients with cardiovascular disease, though the magnitude of the increase in BNP is usually greater than the increase in ANP. This makes BNP is a clinically useful diagnostic marker for several pathophysiological conditions, including heart failure, ventricular remodeling and pulmonary hypertension, among others. Recent studies have shown that in addition to BNP-32, proBNP-108 also circulates in human plasma and that levels of both forms are increased in heart failure. Furthermore, proBNP-108 is O-glycosylated and circulates at higher levels in patients with severe heart failure. In this review we discuss recent progress in our understanding of the biochemistry, molecular biology and clinical relevance of the natriuretic peptide system.

Runx proteins regulate Foxp3 expression

Runx proteins are essential for hematopoiesis and play an important role in T cell development by regulating key target genes, such as CD4 and CD8 as well as lymphokine genes, during the specialization of naive CD4 T cells into distinct T helper subsets. In regulatory T (T reg) cells, the signature transcription factor Foxp3 interacts with and modulates the function of several other DNA binding proteins, including Runx family members, at the protein level. We show that Runx proteins also regulate the initiation and the maintenance of Foxp3 gene expression in CD4 T cells. Full-length Runx promoted the de novo expression of Foxp3 during inducible T reg cell differentiation, whereas the isolated dominant-negative Runt DNA binding domain antagonized de novo Foxp3 expression. Foxp3 expression in natural T reg cells remained dependent on Runx proteins and correlated with the binding of Runx/core-binding factor β to regulatory elements within the Foxp3 locus. Our data show that Runx and Foxp3 are components of a feed-forward loop in which Runx proteins contribute to the expression of Foxp3 and cooperate with Foxp3 proteins to regulate the expression of downstream target genes.

PD-1+memory phenotype CD4+T cells expressing C/EBPα underlie T cell immunodepression in senescence and leukemia

Although altered T cell function plays a part in immunosenescence, the mechanisms remain uncertain. Here we identify a bona fide age-dependent PD-1(+) memory phenotype (MP) CD4(+) T cell subpopulation that hardly proliferates in response to T cell receptor (TCR) stimulation and produces abundant osteopontin at the cost of typical T cell lymphokines. These T cells demonstrate impaired repopulation in Rag2(-/-) mice, but a homeostatic proliferation in gamma-ray-irradiated mice. These T cells also reveal a unique molecular signature, including a strong expression of C/EBPalpha normally expressed in myeloid-lineage cells, with diminished c-Myc and cyclin D1. Transduction of Cebpa in regular CD4(+) T cells inhibited the TCR-mediated proliferation with c-Myc and cyclin D1 repression and caused a striking activation of Spp1 encoding osteopontin along with concomitant repression of T cell lymphokine genes. Although these T cells gradually increase in number with age and become predominant at the senescent stage in normal mice, the generation is robustly accelerated during leukemia. In both conditions, their predominance is associated with the diminution of specific CD4(+) T cell response. The results suggest that global T cell immunodepression in senescence and leukemia is attributable to the increase in PD-1(+) MP CD4(+) T cells expressing C/EBPalpha.

Aging and lymphokine gene expression by T cell subsets.

Aging and lymphokine gene expression by T cell subsets.

Analysis of proteins that interact with the IL-2 regulatory region in patients with rheumatic diseases.

In order to investigate transcriptional regulation of lymphokine genes in rheumatic diseases, peripheral blood mononuclear cells from patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and systemic sclerosis (SSc) were analysed for expression of DNA-binding proteins. Nuclear extracts prepared from unstimulated and mitogen-activated cells were studied for their ability to bind to 32P-labelled oligonucleotides containing the AP-1, NF-AT, NF-B and CD28RC sites of the IL-2 promoter. Using gel mobility-shift assay, detection of protein binding to the AP-1 site was reduced in SLE compared with controls. NF-AT binding activity was enhanced in all groups of patients, and was associated with measures of disease activity in RA. In addition, SSc patients showed increased NF-kappa B binding activity. Altered patterns of DNA-binding proteins suggest disturbed intracellular signalling which may contribute to abnormal lymphokine production in rheumatic diseases.

Predominance of a type 2 intratumoural immune response in fresh tumour-infiltrating lymphocytes from human gliomas.

Increasing evidence suggests the existence of polarized human T cell responses described as Th1-type (promoting cell-mediated immunity) and Th2-type (promoting humoral immunity), characterized by a dominant production of either interferon-gamma (IFN-gamma) or IL-4, respectively. Little is known about the intratumoural activation of infiltrating lymphocytes (TIL) in human gliomas. Therefore, we assessed fresh TIL at cellular and molecular levels to find out if they were activated and polarized into a type 1 or 2 immune response. Flow cytometry analysis of TIL revealed that the major subset was made of T lymphocytes. Double labelling with alpha-CD3 and adhesion/ activation markers revealed T cell subsets expressing CD49a, CD49b, CD54, and CD15, some of which were almost absent in autologous T peripheral blood lymphocytes (T-PBL). Furthermore, the proportions of T-TIL expressing CD56, CD65, or CD25 were several-fold higher than in T-PBL. Intratumoural functional activation of TIL was tested by semiquantitative assessment in relative units (RU) of lymphokine gene activation with mRNA reverse transcriptase-polymerase chain reaction (RT-PCR). All TIL populations except one significantly expressed IL-4 1 to 2 logs of RU above healthy PBL baseline. Similarly, all patients expressed granulocyte-macrophage colony-stimulating factor (GM-CSF) in a range comparable to IL-4. However, most TIL populations did not express IFN-gamma, IL-2, and tumour necrosis factor-beta (TNF-beta) at higher levels than healthy normal PBL. The increase proportion of T cells expressing activation markers and the consistent detection of significant IL-4 and GM-CSF lymphokine gene activation in TIL populations suggested a predominant type 2 intratumoural immune response that does not promote cell-mediated tumouricidal activity and may contribute to the inefficiency of the antiglioma immune response.

Cytokine profile of the rheumatoid nodule suggests that it is a Th1 granuloma.

To define the cytokine profile within rheumatoid subcutaneous nodules, and to determine whether the destructive inflammatory process in this lesion displays features of a lymphocyte-driven Th1 or Th2 granuloma. Subcutaneous nodules excised from 10 patients with rheumatoid arthritis were examined. Transcripts for interleukin 1beta (IL-1beta) IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, IL-15, IL-18, and for tumor necrosis factor alpha (TNFalpha) and interferon-gamma (IFNgamma) were detected by reverse transcription-polymerase chain reaction of extracted RNA. Nine of 10 nodules contained transcripts for IFNgamma. We observed no evidence for the expression of IL-2, IL-4, or IL-5 among the lymphokine genes analyzed. Transcripts for TNFalpha, IL-1beta, IL-10, IL-15, and IL-18 were present in all 10 nodules. Transcripts for IL-12 were present in all but one nodule. Expression of IL-13 messenger RNA was observed in only 5 nodules. The cytokine profile within the rheumatoid nodule (i.e., presence of IFNgamma but not IL-2, and prominent expression of IL-1beta and TNFalpha together with IL-12, IL-18, IL-15, and IL-10) is similar to the profile of cytokines in the synovial lesion of rheumatoid arthritis, which is generally accepted as being attributable to a Th1-mediated inflammatory mechanism. Our results suggest that damage to affected synovial membrane or subcutaneous tissue is caused by the same inflammatory mechanisms, and that the nodule is a Th1 granuloma.

The role of calcineurin in lymphocyte activation

Lymphokine gene transcription involves numerous signal transduction molecules and second messengers. The serine/threonine phosphatase calcineurin has been demonstrated to play a central role in the immediate, early activation of numerous lymphokines (such as interleukin [IL]-2) and in the regulation of cell surface receptors such as CD40L, CD95, and recently CD25 α (the α chain of the IL-2 receptor). In addition to lymphocyte activation, calcineurin functions include control of neuronal signaling, muscle contraction, muscle hypertrophy and cellular death. Therefore, calcineurin not only plays a vital role in the regulation of T lymphocyte function, but also functions in cellular environments outside the immune system.

FK506, an immunosuppressant targeting calcineurin function.

The macrolactam natural product, FK506 (Tacrolimus), acts as a powerful and clinically useful immnosuppressant through disruption of signaling events mediated by the calcium-dependent serine/threonine protein phosphatase, calcineurin (CaN), in T lymphocytes. Its mechanism of action involves the formation of a molecular complex with the intracellular FK506-binding protein-12 (FKBP12), thereby acquiring the ability to interact with CaN and to interfere with its access to and dephosphorylation of various substrates. Among the CaN substrates whose activity is altered by FK506, the nuclear factors of activated T cells (NFAT), a family of transcription factors regulating lymphokine gene expression, have been shown to play a prominent role in FK506-induced immunosuppression. Over the past few years, additional members of the FKBP and NFAT families of proteins have been identified, providing further insights into the complexity of FK506 biological effects. Furthermore, it has become clear that, predominantly as a result of CaN inhibition, FK506 alters multiple biochemical processes in a variety of cells besides lymphocytes. This may account for the adverse side effects of the drug, including neurotoxicity and nephrotoxicity. Extensive medicinal chemistry efforts have been devoted to the generation of analogs of FK506 with the hope of identifying compounds with an improved therapeutic index, that could have broader therapeutic utility than the parent drug. These efforts yielded several compounds with unique biochemical attributes, showing evidence for a dissociation between immunosuppressive and toxic properties, which may pave the way towards designing safer FK506-related immunosuppressants.

Synergism between the Calmodulin-binding and Autoinhibitory Domains on Calcineurin Is Essential for the Induction of Their Phosphatase Activity

Elevation of the intracellular calcium concentration is necessary for cell growth and the activation of several lymphokine genes. The immunosuppressive drugs cyclosporin A and FK506 profoundly inhibit the calcium-dependent signaling pathway in T lymphocytes by interfering with the activity of the calcium/calmodulin (CaM)-dependent serine/threonine phosphatase, calcineurin (CN). Little is known, however, about how activation of CN enzyme activity or interaction with its substrate, nuclear factor of activated T cell (NF-AT), is regulated. We show here that the binding of CaM to CN may affect the conformation of CN at both the CaM-binding and the autoinhibitory (AI) domains and that this is critical for activation of CN to dephosphorylate NF-AT. Dissociation of the AI domain from the enzyme active site on CN leads to expose a binding site for NF-AT at the N terminus of CN and allows the CN binding to NF-AT. Since the cytoplasmic form of NF-AT can interact with CN mutants lacking enzyme activity, the interaction of the two molecules is independent of CN enzyme activity and occurs prior to the dephosphorylation of NF-AT. Dephosphorylation converts NF-AT from the cytoplasmic to the nuclear form by exposing the DNA-binding domain on NF-AT, and the nuclear form of NF-AT brings CN together into the nuclei. We therefore propose that the activation of CN by the CaM binding independently regulates the interaction with NF-AT and the dephosphorylation of NF-AT.

Tacrolimus in refractory poly myositis with inter stitial lung disease

Anti-Jo-1 is the most frequently identified autoantibody in the idiopathic inflammatory myopathies (IIM) and patients with Jo-1 may also develop interstitial pneumonitis, arthritis, and “mechanic’s hands”. Anti-signal recognition particle (SRP) identifies a second subgroup of IIM with severe muscle weakness. Patients with either antibody have a worse outlook.1 We assessed the effectiveness of tacrolimus in treating such patients. Patients were eligible if they had muscle weakness, raised serum creatine kinase (CK), electromyographic and biopsy evidence of active disease, and if treatment with prednisone and at least one other concomitantly adminstered immunosuppressive agent had failed. Patients received oral tacrolimus (0·075 mg/kg daily) in twice daily doses to achieve a 12-h plasma trough concentration of 5–20 ng/mL. Their manual muscle strength, serum CK, functional status as determined by the disability index (DI) of the Health Assessment Questionnaire (HAQ), and daily prednisone dose were recorded as outcome measures. Eight patients (five women, age range 30–70 years), were treated. Time from disease onset to treatment with tacrolimus ranged from 9 months to almost 9 years. Six had anti-Jo-1 and two had anti-SRP autoantibodies. All had corticosteroid and methotrexate-resistant disease; four had failed azathioprine, two cyclophosphamide, one intravenous immune globulin, and one plasmapheresis. All patients had improved muscle strength after tacrolimus and five of the six anti-Jo-1 patients regained normal strength (three within 3 months, two within 6 months). Mean CK of the six anti-Jo-1 patients decreased from 3114 IU/mL to 87 IU/mL (p<0·01) and three returned to normal within 1 month. The two anti-SRP patients had a decrease in their serum CK and strength improved. Patients with the anti-Jo-1 antibody had an improved functional status (mean DI of the HAQ 0·98 to 0·48, p<0·005). Mean daily prednisone dose decreased by 80% in the six anti-Jo-1-positive patients (25 mg to 5 mg, p<0·005, and one patient is off prednisone). Patients have received 368 patient months of tacrolimus treatment (range, 17–74 months; mean, 46 months) and none have had a flare-up of myositis flare when their tacrolimus serum concentrations were within the therapeutic range. Suboptimum concentrations (one patient) and drug discontinuation (two patients, figure), led to flares that responded favourably to restarting tacrolimus. Extramuscular manifestations such as fever, polyarthritis, and “mechanics hands” improved with tacrolimus. Five of the six Jo-1-positive patients had radiographic evidence of diffuse parenchymal interstitial infiltrates and two required treatment with oxygen. One had stable pulmonary function tests, one worsened slightly, and three responded favourably. The mean improvement in the forced vital capacity after 12, 21, and 37 months of tacrolimus in these three patients was 0·62 L (a 22% increase). Carbon monoxide diffusion improved in two patients and total lung capacity increased an average of 15%. Figure 1 Response of serum CK to administration of several immunosuppressives in an anti-Jo-1 antibody-positive patient Tacrolimus was well tolerated. The serum creatinine increased in all patients but dosage modification led to improvement in all but one patient who developed accelerated hypertension and worsening renal function with a creatinine of 4·1 mg/dL. Tacrolimus was stopped and muscle strength and serum CK remain normal 12 months later. Two patients developed mild anaemia and hypertension and one experienced gynaecomastia which resolved. No patients had an opportunistic infection. Another SRP-positive patient stopped tacrolimus due to concerns over side-effects. Only 9% (3/34) of previously reported antisynthetase antibody-positive patients responded completely to treatment.2 Our six Jo-1-positive patients had a good response to tacrolimus and in five it was sustained. Despite a mean tacrolimus treatment duration of 46 months and decreasing corticosteroid doses, no patient flared during treatment. There were three disease exacerbations when tacrolimus was tapered or discontinued. The prevalence of interstitial lung disease is increased in myositis patients with antisynthetase autoantibodies, is associated with reduced survival, and was the immediate cause of mortality in 58% of all deaths related to IIM.3 Four of our five Jo-1-positive patients with interstitial lung disease are stable or have improved. Tacrolimus has been effective in treating pyoderma gangrenosum, psoriasis, uveitis, Behcets disease, nephritic syndrome, and primary sclerosing cholangitis.4 It inhibits activation of CD4+ T helper cells through several calcium-associated events involved in lymphokine gene transcription and interleukin-2 and other cytokines are also inhibitied.5 Because polymyositis is predominantly a T-cell-mediated disease, one might expect IIM patients to respond more favourably to tacrolimus than those with dermatomyositis (a B-cell mediated disease). We suggest that tacrolimus be considered and studied in the treatment of patients with severe myositis with poor prognostic autoantibodies; and patients with immunological (T-cell mediated) interstitial lung disease before they develop pulmonary fibrosis.

Interaction of AU-rich sequence binding proteins with actin: possible involvement of the actin cytoskeleton in lymphokine mRNA turnover.

In the current study, we report that cytochalasin-induced disruption of microfilaments stabilizes lymphokine mRNAs in activated human peripheral blood lymphocytes. Parallel with this, a dose- and time-dependent increase in AU-rich sequence binding protein (AUPB) activities is apparent in the nonionic detergent-resistant fractions of these cells, suggesting that cytochalasin-induced modulation of lymphokine mRNA stability might be mediated through cytoplasmic AUBPs. We provide evidence that some of the AUBPs can be immunoprecipitated with anti-actin antibodies, implicating the potential of these proteins to associate with the actin-based cytoskeleton in vivo. Moreover, disruption of the microfilament network by cytochalasins produces increased immunoprecipitable actin-AUBP complexes in the detergent-resistant cytoplasmic subfractions of lymphocytes. We show that cytochalasin-induced changes in AUBP activities are parallel with their higher binding affinity to RNA containing AU-rich instability sequence element as judged by in vitro competition and in vivo ultraviolet-crosslinking analysis. Correlation of these findings with changes in mRNA stability indicates that the actin cytoskeleton may play a physiologically important role in posttranscriptional regulation of lymphokine gene expression during early lymphocyte activation.

Heterogeneity of biologic responses of melanoma-specific CTL.

To better understand how Ag density influences the various biologic responses of CTL, we analyzed lysis and, at the single cell level, cytokine production by a panel of melanoma-specific CTL clones. Titration experiments done with peptide-pulsed TAP-deficient T2 cells indicated that: 1) Ag density affects both the fraction of responding cells and the amount of cytokine secreted by each cell. 2) Different responses have a relative Ag requirement that may vary between clones. Lysis and secretion of IFN-gamma, and for most clones' secretion of TNF-alpha, required lower Ag densities, by one or two logs, than IL-2 and granulocyte-macrophage CSF secretion. 3) In a significant fraction of IFN-gamma-secreting cells, IL-2 production is not induced. 4) A large fraction of cloned cells is refractory to lymphokine gene activation for about 2 wk after previous stimulation. Together these data indicate that CTL biologic responses are controlled by variable Ag thresholds and by additional parameters affecting activation requirements of each cell. A similar heterogeneity of cytokine responses was observed to Ag endogenously presented by melanoma cells. As a consequence, most melanoma lines, including those with the highest Ag expression, could trigger only low CTL fractions to secrete IL-2 and, also for most clones, granulocyte-macrophage CSF. This may be an important component of the inefficiency of specific CTL in cancer patients.

Reverse transcriptase-polymerase chain reaction amplification and partial sequence of T helper 1- and T helper 2-type lymphokine genes from the owl monkey (Aotus trivirgatus).

The reverse transcriptase-polymerase chain reaction (RT-PCR) was used to amplify selected lymphokine mRNAs from phytohemagglutinin-activated leukocytes of the owl monkey (Aotus trivirgatus). Interleukin-2 (IL-2), IL-4, IL-13, and interferon-gamma were selected as lymphokine mRNAs of interest, since expression of these cytokines helps define the type of T helper lymphocyte response (i.e., TH1 versus TH2). Because sequences for these lymphokine genes were not available for the owl monkey, multiple PCR primers for each lymphokine gene were designed based on published human sequences. Various PCR primer pairs were then used in the RT-PCR to determine the conditions for optimal amplification of each owl monkey cytokine mRNA. In addition, each PCR primer pair was compared for the ability to amplify lymphokine mRNAs from other primate species, including African green (Cercopithecus aethiops), squirrel (Saimiri sciureus), and rhesus (Macaca mulatta) monkeys. The specificity and sensitivity of optimal primer pair was also demonstrated by amplification of as little as 10 fg of each lymphokine gene in a background of 300 ng of irrelevant cDNA. Finally, partial sequences of owl monkey coding regions for IL-2, IL-13, and interferon-gamma were determined and compared for homology with their human counterparts. Together, these studies define specific and sensitive conditions for detection of lymphokine mRNA expression in the owl monkey and provide partial sequence information of the coding region for these lymphokines. This investigation should provide molecular probes to investigate the immune response against malaria and the effectiveness of malaria vaccines in the owl monkey that models this human disease.

Oxidative and chemical stress mimic insulin by selectively inhibiting the expression of phosphoenolpyruvate carboxykinase in hepatoma cells.

Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the rate-limiting step in hepatic gluconeogenesis. Glucagon (via the second messenger cAMP), retinoic acid, and glucocorticoids stimulate transcription of the PEPCK gene, whereas insulin and phorbol esters have a dominant inhibitory effect. We now show that oxidative and chemical stress (hydrogen peroxide and sodium meta-arsenite, respectively) also produce a dominant inhibitory effect, both on the endogenous PEPCK gene and on a stably transfected PEPCK-chloramphenicol acetyl transferase (CAT) fusion gene. Wortmannin, an inhibitor of 1-phosphatidylinositol 3-kinase (PI 3-kinase), blocks the inhibition of glucocorticoid and cAMP-induced PEPCK gene transcription by insulin; however, it has no effect on the inhibition elicited by oxidative or chemical stress. Thus, the mechanism(s) used by hydrogen peroxide and sodium meta-arsenite to regulate PEPCK gene expression are PI 3-kinase independent. This suggests that these agents operate by a pathway distinct from that used by insulin or that the pathways converge at a point downstream of PI 3-kinase. The reactivating kinase (RK, also known as p38 mitogen activated protein kinase) is induced by insulin, hydrogen peroxide, or sodium meta-arsenite in hepatoma cells, and these effects are blocked by SB203580, a selective inhibitor of RK. However, SB203580 has no effect on the ability of any of these agents to regulate PEPCK-CAT fusion gene expression. Thus, although RK has a role in the regulation of lymphokine gene expression in monocytes, it is not required for the regulation of PEPCK expression by either insulin or oxidative and chemical stress in hepatoma cells.

The conserved lymphokine element-0 in the IL5 promoter binds to a high mobility group-1 protein

The conserved lymphokine elements-0 ( CLE0) in the IL5 promoter is essential for the expression of IL-5. Here, we report the cloning and expression of a cDNA encoding a novel CLE0-binding protein, CLEBP-1 from a mouse Th2 clone, D10.G4.1. Interestingly, it was found that the CLEBP1 cDNA sequence was almost identical to the sequences of known high mobility group-1 ( HMG1) cDNAs. When expressed as a recombinant fusion protein in Escherichia coli, CLEBP-1 was shown to bind to the IL5-CLE0 element in electrophoretic mobility-shift assays (EMSA) and southwestern blot analysis. The CLEBP-1 fusion protein cross-reacts with and-HMG- 1 2 in Western blot analysis. It also binds to the CLE0 elements of IL4, GMCSF and GCSF genes. CLEBP-1 and closely related HMG-1 and HMG-2 proteins may play key roles in facilitating the expression of the lymphokine genes that contain CLE0 elements.

Production of tumor necrosis factor-alpha as a result of glia-T-cell interaction correlates with the pathogenic activity of myelin basic protein-reactive T cells in experimental autoimmune encephalomyelitis.

Tumor necrosis factor-alpha (TNF-alpha) has attracted the greatest attention as a major factor in experimental autoimmune encephalomyelitis (EAE) pathogenesis. We compared rats undergoing EAE with manipulated but healthy animals by examining TNF-alpha gene expression in cells recovered from the brain. We used reverse transcriptase-polymerase chain reaction (RT-PCR) as a sensitive assay for detection and Northern blot hybridization as a reliable quantitative assay of TNF-alpha mRNA. TNF-alpha gene expression was consistently detected in rats immunized with myelin basic protein (MBP) emulsified in complete Freund adjuvant (CFA), but not in rats immunized with MBP emulsified in incomplete Freund adjuvant (IFA), which does not induce EAE. Similarly, brain-derived cells from rats injected with cloned encephalitogenic T cells contained increased amounts of TNF-alpha mRNA compared with rats injected with nonencephalitogenic T cell clones similar in antigen specificity and in vitro lymphokine-producing capacity. Considering that the differing pathogenic capacity of MBP-reactive T cells might result from differing patterns of interaction with glia, we examined the impact of T-cell-glia interaction in vitro on cytokine gene expression in both cell types. Glial components were efficient in inducing TNF-alpha expression in T cells; T cells and T-cell-derived cytokines could elicit expression of several lymphokine genes in glial cells. Comparison of RT-PCR and blot hybridization assays, however, suggested that cytokine expression was much more efficient, on a per cell basis, in T cells than in glia. TNF-alpha was shown to have direct cytotoxic effect on glial cells, which was greatly enhanced by small amounts of interferon-gamma (IFN-gamma).

Interactions of a Transcriptional Activator in the env Gene of the Mouse Mammary Tumor Virus with Activation-dependent, T Cell-specific Transacting Factors

The mouse mammary tumor virus env gene contains a transcriptional activator (META) that can control transcription of the adjacent long terminal repeat region. Transcriptional control by META parallels that of several lymphokine genes, being specific to T cells, dependent on their activation, and inhibited by the immunosuppressive drug cyclosporine (CsA). DNase I footprinting indicated that nuclear factors from activated T lymphocytes bound a promoter-proximal site, META(P), and a promoter-distal site, META(D+), within the 400-base pair META region. Nuclear factors from unstimulated, but not from activated cells, bound a site, META(D-), adjacent to META(D+). META(D+) directed transcription of a linked luciferase gene, and gel shift analysis revealed binding of inducible, CsA-sensitive T cell factors, in parallel with transfection results. Authentic NFAT and NF-kappaB targets did not compete for the META(D+) binding factor(s). The SV40 core sequence competed for META(D+) binding factors, but META(D+) failed to compete for the complexes obtained with the SV40 probe. Our results, taken together, indicate that META(D+) is a novel transcriptional enhancer element that is similar in its cell-type specificity, activation dependence, and CsA sensitivity to the NFAT element. It may be relevant to the role of MMTV in expression of Mls antigens or the induction of T cell lymphomas.

How is expression of the interleukin-5 gene regulated?

Eosinophilia is a uniquely specific phenomenon regulated by interleukin-5 (IL-5), suggesting specific control for IL-5 expression. However in eosinophilia IL-5 is often co-expressed with other lymphokines such as IL-4, indicating that common, as well as independent, control mechanisms must exist. IL-5 gene expression is regulated at the transcriptional level. The molecular analysis of the IL-5 promoter region reveals the presence of positive regulatory sites that are common to many lymphokine genes. Results from immunosuppression studies suggest that the key control mechanism of IL-5 regulation may not depend on specific regulatory factors but on how gene expression is activated.