Superantigen antagonist blocks Th1 cytokine gene induction and lethal shock.

Bacterial superantigens trigger an excessive, Th1-cytokine response leading to toxic shock. We designed a peptide antagonist that inhibits SEB-induced expression of human genes for IL-2, IFN-gamma, and TNF-beta, cytokines that mediate shock. The peptide antagonist shows homology to a beta-strand-hinge-alpha-helix domain that is conserved structurally in superantigens produced by Staphylococcus aureus and Streptococcus pyogenes yet remote from known binding sites for the major histocompatibility class II molecule and T-cell receptor. For Th1-cell activation, superantigens depend on this domain. The peptide protected mice against lethal challenge with SEB or SEA. Moreover, it rescued mice undergoing toxic shock. Surviving mice rapidly developed broad-spectrum, protective immunity, which rendered them resistant to further lethal challenges with different staphylococcal and streptococcal superantigens. Thus, the lethal effect of superantigens, mediated by Th1 cytokines, can be blocked with a peptide antagonist that inhibits their action at the top of the toxicity cascade, before activation of T cells takes place.

Cytokines revisited at Hilton head.

The Seventh International Cytokine Society Meeting, held in December 1999 at Hilton Head, provided once more an ideal setting for an intense discussion and remodeling of concepts regarding the biology and molecular biology of cytokines and chemokines. Approximately 400 scientists gathered at the beach-side resort, forgoing the beauty of the ocean to listen to their colleagues in the field, in a program ably organized by Jan Vilcek, Scott Durum, Bruce Beutler and Ann Richmond. Here, we attempt to cover some of the highlights on gene regulation, T helper cell differentiation, signal transduction, receptor trafficking, chemokines, apoptosis, disease models, and new cytokines.

Superantigen antagonist protects against lethal shock and defines a new domain for T-cell activation.

Superantigens trigger an excessive cellular immune response, leading to toxic shock. We have designed a peptide antagonist that inhibits superantigen-induced expression of human genes for interleukin-2, gamma interferon and tumor necrosis factor-b, which are cytokines that mediate shock. The peptide shows homology to a b-strand-hinge-a-helix domain that is structurally conserved in superantigens, yet is remote from known binding sites for the major histocompatibility class II molecule and T-cell receptor. Superantigens depend on this domain for T-cell activation. The peptide protected mice against lethal challenge with staphylococcal and streptococcal superantigens. Moreover, it rescued mice undergoing toxic shock. Surviving mice rapidly developed protective antibodies against superantigen that rendered them resistant to further lethal challenges, even with different superantigens. Thus, the lethal effect of superantigens can be blocked with a peptide antagonist that inhibits their action at the beginning of the toxicity cascade, before activation of T cells takes place.

Recognition of 5'-terminal TAR structure in human immunodeficiency virus-1 mRNA by eukaryotic translation initiation factor 2.

TAR, a 59 nt 5'-terminal hairpin in human immuno-deficiency virus 1 (HIV-1) mRNA, binds viral Tat and several cellular proteins. We report that eukaryotic translation initiation factor 2 (eIF2) recognizes TAR. TAR and the AUG initiation codon domain, located well downstream from TAR, both contribute to the affinity of HIV-1 mRNA for eIF2. The affinity of TAR for eIF2 was insensitive to lower stem mutations that modify sequence and structure or to sequence changes throughout the remainder that leave the TAR secondary structure intact. Hence, eIF2 recognizes structure rather than sequence in TAR. The affinity for eIF2 was severely reduced by a 3 nt change that converts the single A bulge into a 7 nt internal loop. T1 footprinting showed that eIF2 protects nucleotides in the loop as well as in the strand opposite the A bulge. Thus, eIF2 recognizes the TAR loop and lower part of the sub-apical stem. Though not contiguous, these regions are brought into proximity in TAR by a bend in the helical structure induced by the UCU bulge; binding of eIF2 opens up the bulge context and apical stem. The ability to bind eIF2 suggests a function for TAR in HIV-1 mRNA translation. Indeed, the 3 nt change that reduces the affinity of TAR for eIF2 impairs the ability of reporter mRNA to compete in translation. Interaction of TAR with eIF2 thus allows HIV-1 mRNA to compete more effectively during protein synthesis.

Cell-mediated suppression of human interleukin-2 gene expression at splicing of mRNA.

Human IL-2 gene expression is regulated by cell-mediated suppression. Mitogenic stimulation of PBMC induces transient activation of CD8 cells that inhibit expression of this gene. Depletion of CD8 cells elicits marked superinduction of IL-2 mRNA; reintroduction of CD8 cells causes severe inhibition. Moreover, during IL-2 gene induction, splicing of IL-2 precursor transcripts becomes inhibited, resulting in a transient mRNA wave. This block in IL-2 mRNA splicing is relieved by the translation inhibitor, cycloheximide (CHX), which does not stimulate transcription [Gerez et al., J. Biol. Chem. 270, 15569 (1995)]. We show that suppression of IL-2 mRNA expression, whether by CD8 cells, soluble mediators derived from them, or IL-10, is relieved completely by CHX. Hence, suppression involves a CHX-sensitive step. Response to CHX, manifested in superinduction of IL-2 mRNA, is enhanced 10-fold during suppression. Suppression by CD8 cells or soluble mediators leads to rapid degradation of precursor transcripts while relief from suppression leads to a significant rise in precursor RNA. These changes precede effects at the mRNA level. We conclude that suppression induces a block in mRNA splicing and degradation of blocked precursor transcripts. The near-complete absence of IL-2 mRNA superinduction by CHX in Jurkat Th cells, lacking cells with suppressive capacity, supports this interpretation.

A cis-acting element in the 3'-untranslated region of human TNF-alpha mRNA renders splicing dependent on the activation of protein kinase PKR.

We report a role for the 3'-untranslated region in control of mRNA splicing and show that human TNF-alpha 3' UTR harbors a cis-acting element that renders splicing of precursor transcripts dependent on activation of PKR, the RNA-activated protein kinase that phosphorylates eukaryotic initiation factor 2 (eIF2). When this element, designated 2-APRE, is present, splicing becomes sensitive to inhibition by the PKR inhibitor, 2-aminopurine, or by coexpression of transdominant-negative mutant PKR. Our results reveal that activation of PKR is required for splicing of mRNA when precursor transcripts contain the 2-APRE and that increased expression of wild-type PKR enhances their splicing efficiency. Thus, PKR responds as trans-acting factor to the 2-APRE. 2-APRE RNA forms a stable, 17-bp stem-loop structure and strongly activates PKR in vitro, inducing eIF2alpha phosphorylation. Despite its ability to activate PKR during splicing, the 2-APRE within the 3' UTR does not affect translation efficiency of the resulting TNF-alpha mRNA in transfected cells. PKR and the 3' UTR thus interact during mRNA splicing to confer a novel type of regulation on expression of the TNF-alpha gene.

Cytokine and interferon research in Israel.

From its inception, the field of interferons and cytokines has occupied an important position in Israeli biological science. With the Second Joint Meeting of the International Society for Interferon and Cytokine Research and the International Cytokine Society taking place in Jerusalem in 1998, it is timely to review here current Israeli research on the biology, gene regulation, receptors, signal transduction, mode of action and clinical aspects of interferons and cytokines.

Hyperinducible expression of the interferon-gamma (IFN-gamma) gene and its suppression in systemic lupus erythematosus (SLE).

Transient expression of IFN-gamma and IL-2 mRNA and its control by post-transcriptional and suppressive mechanisms were analysed in phytohaemagglutinin-induced peripheral blood mononuclear cells (PBMC) from 47 patients with SLE and 31 age-matched normal donors, using quantitative hybridization with antisense RNA probes. In SLE, basal levels of gene expression did not deviate from those of normal donors, but strongly aberrant patterns were obtained upon induction. The ratio of subjects exhibiting highly inducible IFN-gamma gene expression in their PBMC to those showing moderate or low inducibility was increased five-fold in SLE (P = 0.003). High inducibility was observed for 43% of SLE patients and was equally pronounced in partial remission, mild or active disease. Inducibility of IL-2 mRNA, by contrast, remained similar to that for normal donors. However, regulation of IFN-gamma gene expression differed for mild SLE. Patients with mild disease showing high inducibility of IFN-gamma mRNA in their PBMC not only had the highest frequency of responders, but also the highest extent of an individual response, defined by superinduction of mRNA, to agents that relieve suppression (gamma-irradiation) or post-transcriptional down-regulation (cycloheximide). By contrast, patients with active SLE showing high IFN-gamma mRNA inducibility had normal suppressive capacity as well as post-transcriptional control. Hence, both high inducibility of the IFN-gamma gene and its suppression are relevant to disease. Hyperactivation of the IFN-gamma gene may be alleviated in mild SLE by a vigorous, concomitant activation of post-transcriptional control and of cell-mediated suppression.

Down-regulation of human tumor necrosis factor-beta gene expression by cells with suppressive activity.

Human TNF-beta (lymphotoxin) gene expression is down-regulated by immunosuppression. Induction of TNF-beta mRNA in lymphoid cells is greatly enhanced by gamma-irradiation, cyclophosphamide and cimetidine, agents that each inhibit activation of suppressive cells. The level of TNF-beta mRNA expressed in response to stimulation, whether by mitogen or antigen, is reduced strongly by concomitant activation of suppressive cell subsets. Removal of CD8 or CD11b cells leads to a pronounced superinduction of TNF-beta mRNA in the depleted cell population. Induction of TNF-beta mRNA precedes appearance of suppressive cell activity, allowing for temporary expression. The TNF-beta gene is as sensitive as IFN-gamma and IL-2 genes to suppression. Hence, three genes characteristically expressed in Th1 cells, encoding IL-2, IFN-gamma, and TNF-beta, are similarly regulated by cell-mediated suppression. Actual levels of TNF-beta during an immune response are determined by the balance between activities of expressing and suppressing cell subsets, both transiently manifested.

Linomide, an immunomodulator that inhibits Th1 cytokine gene expression.

Linomide (LS-2616, quinoline-3-carboxamide) has strong immunomodulating effects in animal models, inhibiting toxic shock, progressive autoimmune disease and cancer. In humans, linomide strongly reduced the appearance of new lesions in multiple sclerosis yet enhanced immune responses after bone marrow transplantation. In contrast to these clear effects in vivo, attempts to show an effect of linomide in vitro have not been successful and its mode of action remains to be elucidated. Here we show that at concentrations effective in vivo, linomide is active on human peripheral blood mononuclear cells (PBMC), severely inhibiting the induction by Staphylococcus aureus enterotoxin B of mRNA of three cytokine genes expressed in Th1 cells, those for IFN-gamma, IL-2, and tumor necrosis factor-beta. Yet, cell viability was not affected by linomide. The extent of inhibition is dose-dependent on linomide. Linomide also blocked induction of IL-2 and IFN-gamma mRNA by phytohemagglutinin. The inhibitory effect is expressed immediately but can be enhanced significantly by a prolonged exposure of PBMC to linomide, reaching 10-fold. These results support the concept that linomide antagonizes the activation of Th1 cells during a cellular immune response.

2-Aminopurine selectively inhibits splicing of tumor necrosis factor alpha mRNA.

2-Aminopurine (2-AP) inhibits specific kinases that phosphorylate the alpha subunit of eukaryotic translation initiation factor 2. One of these, PKR, is also involved in signal transduction. We show here that 2-AP selectively inhibits expression of tumor necrosis factor alpha (TNF-alpha) mRNA in primary human lymphoid cells. 2-AP does not inhibit transcription of the human TNF-alpha gene, nor does it affect mRNA stability. Instead, the flow of short-lived precursor transcripts into mature TNF-alpha mRNA is blocked. When 2-AP is present during induction, unspliced TNF-alpha precursor transcripts accumulate at the expense of mRNA. Using RNase protection analysis with genomic probes for different exon-intron junctions, we show that 2-AP blocks splicing of TNF-alpha mRNA. Neither the TNF-beta nor the interleukin-1 beta gene shows such regulation. 2-AP also inhibits splicing of precursor RNA transcribed from an exogenous human TNF-alpha gene. Sequences within this gene thus confer sensitivity to 2-AP. Yet, control is not exerted at a specific splice site. Our results reveal the involvement of a 2-AP-sensitive component, expressed in functional form before induction, in the splicing of TNF-alpha mRNA.

Prediction of response to treatment in superficial bladder carcinoma through pattern of interleukin-2 gene expression.

PURPOSE: Superficial bladder carcinoma, treated by resection and intravesical administration of bacillus Calmette-Guérin (BCG), yields a remission rate that approaches 70%. We examined whether expression of interleukin-2 (IL-2) or interferon-gamma (IFN-gamma) genes can serve to predict response. PATIENTS AND METHODS: During BCG treatment, we analyzed induction of IL-2 and IFN-gamma mRNA in peripheral-blood mononuclear cells (PBMC) from 73 patients: 51 with papillary tumors and 22 with carcinoma-in-situ (CIS). Results were correlated with remission, relapse, or tumor persistence over a 4-year follow-up period. RESULTS: Independent of tumor type, induction of IL-2 mRNA was observed for patients who responded with remission, but not for those who relapsed (P = .0001). Multivariate logistic analysis showed that inducibility of IL-2 mRNA is the discriminating parameter, which yields a predictive value of 97% for remission. Of 23 patients with relapse/persistence, 22 lacked inducibility of IL-2 mRNA (sensitivity, 95.6%), while 35 of 50 patients in remission exhibited inducibility (specificity, 70%). For patients with carcinoma-in-situ, in which remission or failure depends solely on response to BCG, sensitivity and specificity were 88% and 86%, respectively; for patients with papillary tumors, they were 100% and 64%. IFN-gamma mRNA, by contrast, was clearly inducible in PBMC from all patients (P = .51). The disease-free interval increased progressively with inducibility of IL-2 mRNA; this trend was highly significant (P = .0001). CONCLUSION: IL-2 gene expression is essential for mounting an antitumor response in superficial bladder carcinoma. Inducibility of IL-2 mRNA is an independent prognostic parameter and useful predictive indicator of remission versus relapse.

Dual control of human interleukin-2 and interferon-gamma gene expression by histamine: activation and suppression.

Histamine is considered to be an activator of cells with suppressive capacity. In agreement with this concept, we show that histamine elicits a strong inhibition of the induced expression of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) genes. However, our experiments reveal a novel property of histamine: early in the induction process, it strongly stimulates expression of these two genes in cultured human peripheral blood mononuclear cells (PBMC). The histamine-mediated superinduction of IL-2 mRNA is seen also in a Th cell line, showing that such cells respond directly to histamine. In the course of mitogenic induction, a 20-fold stimulation by histamine is converted into an equally strong inhibition. The response of a PBMC population to histamine thus undergoes a remarkable change following T cell activation. The dual effect of histamine can be blocked by the H2 histamine receptor antagonist cimetidine, while the early activation by histamine is mimicked by the H2 agonist impromidine, showing that both activation and inhibition of IL-2 and IFN-gamma gene expression by histamine are exerted via this receptor. These results support the concept that histamine, released during an immune response, exerts opposite regulatory effects by first activating cells able to express the IL-2 and IFN-gamma genes and only then suppressive cells that become responsive to histamine more slowly, but once activated shut off the expression of these genes.

Post-transcriptional regulation of human interleukin-2 gene expression at processing of precursor transcripts.

Interleukin-2 (IL-2) regulates the clonal expansion of activated T cells and is produced in limited amounts during an immune response. Mitogenic induction of human IL-2 gene expression elicits a transient wave of unstable mRNA. We show here that transcription continues unabated during and well beyond the time when the wave is subsiding, yet few, if any, new mRNA molecules are generated once the wave has reached its maximum. Instead, IL-2 precursor transcripts accumulate, becoming the majority of expressed IL-2 RNA molecules. The flow of precursor transcripts into mature mRNA becomes inhibited in the course of induction. When translation is blocked (e.g. by cycloheximide), expression of IL-2 mRNA can be superinduced by 2 orders of magnitude. This superinduction is completely dependent upon transcription, yet is not accompanied by any significant increase in the rate of primary transcription or in mRNA stability. Instead, the processing of nuclear IL-2 precursor transcripts is greatly facilitated, resulting in pronounced superinduction of cytoplasmic mRNA. Once its transcription has been induced, therefore, expression of the IL-2 gene is down-regulated extensively at the level of precursor RNA processing.

Transient expression of human interleukin-2 and interferon-gamma genes is regulated by interaction between distinct cell subsets.

The level of transient expression of human IL-2 and IFN-gamma genes, we show, is regulated by dynamic interaction between two functionally distinct cell populations. One is able to express these genes, while the other, bearing one of several specific surface markers, actively inhibits their expression. Defined cell subsets were isolated from PBMC and tonsil cells using immunomagnetic beads coated with monoclonal antibodies directed against surface markers. Depletion of CD8, CD11a (Leu15), or Leu8 subsets led to a pronounced superinduction of IL-2 and IFN-gamma gene expression when the remaining cell population was stimulated with mitogen (PHA) or antigen (SEB). Thus, a 10-fold increase in production of IFN-gamma was observed after removal of CD11a (Leu15) cells constituting only a small percentage of the total cell population. By contrast, depletion of cells expressing CD19, a B cell marker, did not yield any superinduction. Conversely, CD8, CD11a (Leu15), or Leu8 cell subsets, but not CD19 cells, each inhibited the induction of IL-2 and IFN-gamma gene expression almost completely in depleted or total cell populations from which they were derived. Gene expression occurring within one cell subset could be effectively inhibited by cells from a second subset. Introduction of inhibitory cells (Leu8) into a population that actively expressed IL-2 and IFN-gamma mRNA resulted in an immediate cessation of gene expression. This suppression involves a soluble mediator, since the culture medium in which such cells were activated exerted a similarly effective inhibition.

Interleukin-2 induces an early step in the activation of interferon-gamma gene expression.

Establishment of protective immunity depends critically on IFN-gamma. We show that in human peripheral blood mononuclear cells, low doses of IL-2 greatly potentiate the response of the IFN-gamma gene to mitogen, by over 100-fold. By itself, IL-2 is unable to induce IFN-gamma mRNA to a significant extent. Yet, exposure to IL-2 leads to cellular commitment within a few hours, expressed by greatly enhanced accumulation of IFN-gamma mRNA upon subsequent exposure to phytohemagglutinin. Changes induced by IL-2 do not relieve the requirement for de novo protein synthesis during the early phases of induction of IFN-gamma gene expression. IL-2 may induce a component essential for induction of IFN-gamma mRNA that is utilized during subsequent exposure to a mitogenic signal. Our results demonstrate synergy between IL-2 and mitogen in IFN-gamma gene induction.

Induction of human interleukin-1 gene expression by retinoic acid and its regulation at processing of precursor transcripts.

Retinoic acid (RA), we show, induces in peripheral blood mononuclear cells a transient wave of newly transcribed, unstable interleukin-1 alpha (IL-1 alpha) and IL-1 beta mRNA. Tumor necrosis factor-alpha mRNA, by contrast, is expressed in multiple waves. IL-1 genes are primary targets for RA. Most IL-1 beta gene transcription induced by RA fails to yield mature mRNA. Instead, precursor transcripts accumulate, detected by ribonuclease protection analysis. The flow of precursors into IL-1 beta mRNA becomes inhibited during induction. When translation is blocked, e.g. by cycloheximide, expression of IL-1 beta mRNA is superinduced by 2 orders of magnitude. Superinduction is dependent on transcription, yet is unaccompanied by increased primary transcription or mRNA stability. Instead, processing of unstable IL-1 beta precursor transcripts into mature mRNA is greatly facilitated. Control is not narrowly localized within precursors: splicing of distinct exons and intron excision are enhanced by cycloheximide. Pre-mRNA processing thus is a limiting step in RA-induced IL-1 beta gene expression. This regulation is specific for RA: when induced by phorbol ester, IL-1 beta gene expression is also superinduced by cycloheximide but that response is accompanied by enhanced mRNA stability. Thus, IL-1 beta gene transcription is induced by RA, yet, unlike for other primary target genes, mRNA expression is regulated at pre-mRNA processing.

Regulation of the human interleukin-2/interleukin-2 receptor system: a role for immunosuppression.

The strength of the cellular immune response is regulated to a large extent by the amount of interleukin-2 (IL-2) produced in response to a stimulus. The ability of lymphocytes and other cells to respond to IL-2 depends upon the expression of cell surface IL-2 receptors. Formation of a high-affinity IL-2 receptor is regulated primarily through induction of its alpha subunit, IL-2R alpha. Once formed, the IL-2R alpha chain turns over rapidly, rendering expression of high-affinity IL-2 receptors during the immune response dependent upon continuous activity of the IL-2R alpha gene. The induced expression of both human IL-2 and IL-2R alpha chains is sensitive to cell-mediated suppression by CD8 cells; depletion of CD8 cells leads to extensive superinduction. This coupled suppression of IL-2 and IL-2R alpha genes greatly increases the extent of control, and strongly limits the strength, of the signal transduced by this ligand/receptor system during an immune response.

Is cord blood erythropoietin a marker of intrapartum hypoxia?

A sensitive assay was used to compare the biological activity of cord serum erythropoietin in two groups of infants born with or without labor-induced hypoxia. The mean cord serum erythropoietin activity in 161 infants delivered after vaginal labor was 116 +/- 36 mU/mL, and was indistinguishable from that observed in 23 infants delivered by preplanned, elective cesarean section, 114 +/- 12 mU/mL (P = .75). The bioassay measured effective erythropoietin activity, including the contribution of potentiators in serum. These results indicate that duration and intensity of labor are insufficient to cause a significant increase in effective erythropoietin activity.