Requirements for the induction of the interleukin-2 receptor complex in a human pre-T-cell line.

Cell line PER-117 is a T-cell receptor negative human T-cell line that can be induced to express a functional interleukin-2 receptor (IL-2R). Recombinant interleukin-1 (IL-1) as well as certain combinations of inducer substances could be shown to stimulate the expression of the p55 (alpha)-chain of the IL-2R in PER-117 cells. The synergistic increases in IL-2R alpha expression were demonstrated at the cell surface as well as at the mRNA level. The results suggested that in PER-117 cells IL-1 appears to induce expression of the alpha-chain by pathways that are different to activation via protein kinase C (PKC), and that drug-induced cyclic AMP (cAMP) activation did not substitute for IL-1. We found that the regulation of mRNA for IL-2R beta (p75) differed significantly from that seen for IL-2R alpha. Moreover, the requirements for IL-2R alpha induction determined for this cell line differ from other human cell lines, which may reflect that there are distinct requirements for activation depending on the stage of differentiation and/or lineage of the cells. The PER-117 cell line provides a unique model to examine further the mechanism leading to induction of a functional IL-2R at an early stage of human T-cell differentiation.     

IL-13 signal transduction in human monocytes: phosphorylation of receptor components,association with Jaks,and phosphorylation/activation of Stats

Interleukin (IL)-13 regulates monocyte function and is a potent stimulator of 15-lipoxygenase expression. In different cell types, the functional IL-13 receptor complex can be comprised of variable protein components and has not been thoroughly examined in human monocytes. Here, we identify the receptor components and upstream signaling events initiated by IL-13 in primary human blood monocytes. The expression, phosphorylation and associated Jak kinases of the known, variable receptor components, IL-4R(alpha), IL-2Rgammac, IL-13R(alpha)1 and IL-13R(alpha)2, were examined. We determined that IL-4R(alpha) and IL13R(alpha)1 are phosphorylated upon exposure to IL-13. Although IL-2Rgammac is also expressed, it is not phosphorylated upon exposure to IL-13. Evaluation of the presence of IL-13R(alpha)2 failed to reveal significant mRNA or protein expression. Earlier, our laboratory showed that IL-13 induced the phosphorylation of Jak2 and Tyk2 in monocytes and that expression of both Jaks was essential for downstream signaling by IL-13. Here, we report that Jak2 is associated with IL-4R(alpha), and Tyk2 is associated with the IL-13R(alpha)1 component of the IL-13 receptor complex. Additionally, Stat proteins 1alpha, 3, 5A, 5B, and 6 are phosphorylated in response to IL-13. Further, the nuclear translocation and DNA binding of each of these Stats were induced by IL-13. These data represent the first complete report of the functional IL-13 receptor complex and early signaling events in human monocytes. This information is critical for understanding the IL-13 response of monocytes in inflammation.     

Molecular cloning, expression and characterization of the ovine IL-2R alpha chain.

Interleukin-2 (IL-2) stimulates the proliferation of activated antigen-specific T cells through its interaction with high affinity receptors. This event is largely regulated by the inducible expression of the alpha-chain (CD25) which, in combination with the beta-chain and possibly additional chains, forms the high affinity IL-2 receptor (IL-2R) complex. From a concanavalin A (Con A)-activated ovine T-cell complementary DNA (cDNA) library we have isolated two cDNA clones which together constitute a 2650 base pair (bp) messenger RNA (mRNA) species encoding the ovine IL-2R alpha chain. The nucleotide sequence has high homology with analogous cDNA from other species and predicts a mature protein of 254 amino acids. In addition to the predominate 2.6 kilobase (kb) ovine IL-2R alpha chain mRNA species. Northern blot analysis of activated T-cell RNA revealed two larger mRNA species. The ovine IL-2R alpha chain cDNA was transfected into CHO cells and low affinity binding of human recombinant IL-2 demonstrated. Polyclonal antisera generated against the transfected cells cross-reacted with Con A-activated ovine lymphocytes. In addition these antisera were used to immunoprecipitate a unique 50,000 MW protein from the transfected cells. It is likely that this protein represents the expressed ovine IL-2R alpha chain cDNA which is heavily glycosylated as distinct from the 30,869 MW primary translation product. Southern blot analysis of ovine genomic DNA suggests that the ovine IL-2R alpha chain is encoded by a single copy gene.     

Nucleolin and YB-1 are required for JNK-mediated interleukin-2 mRNA stabilization during T-cell activation

Regulated mRNA turnover is a highly important process, but its mechanism is poorly understood. Using interleukin-2 (IL-2) mRNA as a model, we described a role for the JNK-signaling pathway in stabilization of IL-2 mRNA during T-cell activation, acting via a JNK response element (JRE) in the 5' untranslated region (UTR). We have now identified two major RNA-binding proteins, nucleolin and YB-1, that specifically bind to the JRE. Binding of both proteins is required for IL-2 mRNA stabilization induced by T-cell activation signals and for JNK-induced stabilization in a cell-free system that duplicates essential features of regulated mRNA decay. Nucleolin and YB-1 are required for formation of an IL-2 mRNP complex that responds to specific mRNA stabilizing signals.     

Expression of the IL-2 receptor gamma subunit in resting human CD4 T lymphocytes: mRNA is constitutively transcribed and the protein stored as an intracellular component

IL-2 receptor (IL-2R) is composed of three subunits named IL-2R alpha, IL-2R gamma. Here, we study the expression of the IL-2R gamma in highly purified, resting peripheral human CD4 T lymphocytes. We show by FACS analysis that the IL-2R gamma subunit is not detectable at the cell surface of peripheral CD4 T lymphocytes. This result has been verified after acid treatment of the cell surface and analysis with three specific anti-IL-2R gamma mAb. Using RT-PCR and intracellular FACS analysis, we demonstrate that IL-2R gamma is constitutively expressed at the mRNA level and the protein is stored as an intracellular component in resting CD4 T lymphocytes. IL-2R alpha and beta subunits are not detectable by these methods. In addition, we show that CD4 T cell remain insensitive to a variety of cytokines that share IL-2R gamma as a common subunit of their receptors (e.g. IL-2, IL-4, IL-7 and IL-15). The kinetics of cell surface expression of IL-2R gamma have been studied after activation of CD4 T lymphocytes and compared with induction of IL-2R alpha and IL-2R beta. Maximum expression of IL-2R gamma is observed after 2 days of stimulation, and remains constant and comparable to IL-2R beta up to day 5. We conclude from these studies that IL-2R gamma is translocated to the membrane only after T cell activation and induction of the IL-2R alpha and IL-2R beta genes. We hypothesize that in CD4 T cells a large intracellular pool of IL-2R gamma is present but that its cell surface translocation depends on the expression of alpha and/or beta chains specific for IL-2, IL-4, IL-7, IL-9 or IL-15.      

Regulation of IL-2 beta receptor expression and beta-chain mRNA by human thymocytes.

The high affinity form of the human IL-2 receptor (IL-2R) has two known components, the IL-2R alpha (p55) and the IL-2R beta chain (p75). We have previously shown that recombinant IL-2 (rIL-2) could induce the expression of the alpha-chain (p55) on T cells and thymocytes, and increase this expression following suboptimal activation with concanavalin A (Con A) in combination with IL-2. An increase in the accumulation of IL-2R alpha-specific mRNA induced by rIL-2 in T cells and thymocytes had also been documented. We report here that the expression of IL-2R beta on the cell surface can be demonstrated on human thymocytes by the binding of Mik beta1, a MoAb directed against an epitope of the beta-chain. The IL-2R beta chain is constitutively expressed on freshly isolated thymocytes; this expression can be increased in thymocytes activated with Con A in combination with IL-2 or tetradecanoylphorbol 13-acetate (TPA). Blocking the formation of high affinity receptors with a MoAb directed against the alpha-chain of the receptor results in an increase in the display of IL-2R beta as evidenced by binding of MoAb Mik beta1. The accumulation of IL-2R-beta-specific mRNA is observed in freshly isolated thymocytes and it is increased in thymocytes cultured with rIL-2 alone, with Con A, and further enhanced by the addition of rIL-2 in combination with Con A or with TPA. Cyclosporine (CsA), which inhibits the accumulation of lymphokine-specific mRNA of thymocytes, does not inhibit the induction of the accumulation of IL-2R beta-specific mRNA. This is analogous to its effect on the expression of the alpha-chain (p55), and the accumulation of alpha-chain-specific mRNA.     

Role of neuronal interferon-gamma in the development of myelopathy in rats infected with human T-cell leukemia virus type 1

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of not only adult T-cell leukemia but also HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Among the rat strains infected with HTLV-1, chronic progressive myelopathy, named HAM rat disease, occurs exclusively in WKAH rats. In the present study, we found that HTLV-1 infection induces interferon (IFN)-gamma production in the spinal cords of HAM-resistant strains but not in those of WKAH rats. Neurons were the major cells that produced IFN-gamma in HTLV-1-infected, HAM-resistant strains. Administration of IFN-gamma suppressed expression of pX, the gene critically involved in the onset of HAM rat disease, in an HTLV-1-immortalized rat T-cell line, indicating that IFN-gamma protects against the development of HAM rat disease. The inability of WKAH spinal cord neurons to produce IFN-gamma after infection appeared to stem from defects in signaling through the interleukin (IL)-12 receptor. Specifically, WKAH-derived spinal cord cells were unable to up-regulate the IL-12 receptor beta2 gene in response to IL-12 stimulation. We suggest that the failure of spinal cord neurons to produce IFN-gamma through the IL-12 pathway is involved in the development of HAM rat disease.     

Altered interleukin-2 receptor alpha-chain is expressed in human T-cell leukaemia virus type-I-infected T-cell lines and human peripheral blood mononuclear cells of adult T-cell leukaemia patients through an alternative splicing mechanism.

A polymerase chain reaction (PCR) method was used to detect the interleukin-2 receptor alpha-chain (IL-2R alpha) chain which lacks the conventional transmembrane (TM) domain in mRNA from human T-cell leukaemia virus type-I (HTLV-I)-infected cell lines or peripheral blood mononuclear cells (PBMC) isolated from adult T-cell leukaemia (ATL) patients. Primer pairs encompassing the TM domain were selected to generate a 357-base pair (bp) fragment. A 146-bp PCR product was observed consistently in addition to the target 357-bp PCR product in mRNA from HTLV-I-infected cell lines, such as MT-1, MT-2, MT-4 and in PBMC isolated from ATL patients. However, this 146-bp PCR product was undetectable in HTLV-I-negative cell lines. The product was also detected in PBMC from normal individuals if activated in vitro with phytohaemagglutinin but not without stimulation. DNA sequence analyses revealed that exons from 5 to 7, which define a 211-bp region containing the conventional TM domain, were deleted in the 146-bp PCR product. The C-terminal amino acid sequence starting from Gly174 of the 211-bp-deleted molecule was distinct from that of conventional IL-2R alpha as a result of an altered reading frame. We identified a 45000 MW peptide generated from IL-2R alpha mRNA through this exon skip in cell lysate of MT-1 and MT-2 by Western blot analyses using an antibody raised against the peptides specific to an altered IL-2R alpha. Our results indicate that an altered IL-2R alpha chain is expressed in HTLV-I-infected T lymphocytic cell lines and in ATL patients.     

Interleukin-1 down-regulates gene and surface expression of interleukin-1 receptor type I by destabilizing its mRNA whereas interleukin-2 increases its expression.

The interleukin-1 receptor type I (IL-1RtI) plays an important role in the biological effects of IL-1, but regulation of its surface and gene expression remains unknown. We found that occupancy of 2-15% of the IL-1 surface receptor results in dramatic down-regulation of IL-1RtI both at the mRNA and cell surface level in murine D10S cells, a subline of T-helper type 2 cells. At these low occupancy levels (3 x 10(-12) to 3 x 10(-13) M), the reduction in IL-1RtI surface expression appears at 24 hr and continues to 48 and 72 hr. At the mRNA level, low occupancy of the IL-1R results in decreased IL-1RtI mRNA stability; steady state half-life of the IL-1RtI mRNA is reduced from 6 to 1 hr after exposure to 3 x 10(-12) M IL-1. This down-regulation of IL-1RtI by IL-1 is blocked by cycloheximide, suggesting de novo protein synthesis is necessary for decreased RNA stability. Low concentrations of human IL-1 beta, murine and rabbit IL-1 alpha or beta similarly down-regulated IL-1RtI, whereas low concentrations of human IL-1 alpha failed to reduce the receptor surface expression, despite inducing a full proliferative response. We also observed that the effect of IL-1 on this down-regulation was not through protein kinase C (PKC), since PMA rapidly increased IL-1RtI mRNA levels within 30 min and persisted for 24 hr. IL-2 up-regulated IL-1RtI in D10S cells at both mRNA and protein levels. These results demonstrate that low occupancy of IL-1 receptors induces down-regulation of IL-1RtI surface as well as mRNA expression. The regulation of IL-1RtI gene expression may be one of the mechanisms by which IL-1-mediated events are controlled.     

Phorbol ester synergizes with Ca2+ ionophore in activation of protein kinase C (PKC)alpha and PKC beta isoenzymes in human T cells and in induction of related cellular functions.

Studies described herein were designed to examine the effects of 12-O-tetradecanoyl phorbol-13-acetate (TPA), and a Ca2+ ionophore (ionomycin), singly or in combination, on the activation and expression of the Ca(2+)-dependent protein kinase C (PKC) isoenzymes (alpha, beta and gamma) at the protein and messenger RNA (mRNA) levels in T cells. These two agents induce the activation and proliferation of T lymphocytes by mimicking the action of inositol phospholipid-derived second messengers normally generated by triggering of the antigen-specific T-cell receptor (TcR)/CD3 complex. TPA-induced T-cell proliferation, expression of interleukin-2 receptor-alpha subunit (IL-2R alpha) and transferrin receptor, CD3 down-regulation and, lastly, the cytosol-to-membrane PKC translocation (determined by an enzymatic assay or by immunoblotting with a cross-reactive anti-PKC peptide antibody) were all facilitated by ionomycin. Immunoblots with isoenzyme-specific anti-PKC monoclonal antibodies demonstrated expression of immunoreactive PKC alpha, PKC beta and PKC gamma proteins that were translocated to the membrane upon TPA plus ionomycin stimulation. Resting T cells expressed abundant levels of mRNA for PKC alpha and PKC beta, but very low levels (relative to brain) of PKC gamma. TPA increased by two- to threefold the expression of PKC beta, but not of PKC alpha or PKC gamma, mRNA within 12 hr of stimulation. Ionomycin synergized with TPA in increasing the expression of PKC alpha and PKC beta mRNA. The two agents also synergized in inducing expression of additional activation/growth-associated genes, namely the c-myc protooncogene, ornithine decarboxylase (ODC) and IL-2R alpha. Ionomycin alone was inactive (or marginally active) in all of these assays. The translocation of distinct Ca(2+)-dependent PKC isoenzymes to the membrane and the up-regulation of PKC alpha and beta mRNA suggest that at least these two isoenzymes are involved in discrete steps of the pathway leading to T-cell activation and proliferation. Moreover, the combined effects of TPA and ionomycin on T-cell function and cell-surface antigen expression appear to be due, at least in part, to their synergistic activation of distinct PKC isoenzyme(s).     

Tumour-bearing animals synthesize a decreased level of mRNA for the inducible 55,000 MW interleukin-2 receptor.

Spleen cells from mice bearing progressive growing methylcholanthrene-induced syngenic tumours were deeply hyporeactive in response to T-cell mitogens. This hyporeactivity was associated with decreased ability to synthesize mRNA for the inducible 55,000 MW interleukin-2 receptor (IL-2R). Since the expression of functional IL-2R represents one of the early and pivotal events in lymphoid-cell activation, it is suggested that the defect in effective IL-2R expression may be one of the primary factors responsible for the immunological hyporeactivity of tumour-bearing hosts.