Endocytosis of interleukin 2 receptors in human T lymphocytes: distinct intracellular localization and fate of the receptor alpha, beta, and gamma chains.

Members of the cytokine receptor family are composed of several noncovalently linked chains with sequence and structure homologies in their extracellular domain. Receptor subfamily members share at least one component: thus the receptors for interleukin (IL) 2 and IL15 have common beta and gamma chains, while those for IL2, 4, 7, and 9 have a common gamma chain. The intracellular pathway followed by IL2 receptors after ligand binding and endocytosis was analyzed by immunofluorescence and confocal microscopy in a human T lymphocytic cell line. Surprisingly, the alpha, beta, and gamma chains had different intracellular localizations after being endocytosed together. The alpha chain was always in transferrin-positive compartments (early/recycling endosomes), both at early and late internalization times, but was never detected in rab7-positive compartments (late endosomes). On the other hand, at late internalization times, the beta and gamma chains were excluded from transferrin-positive organelles and did not colocalize with alpha. Furthermore, beta could be found in rab7-positive vesicles. These differences suggest that the alpha chain recycles to the plasma membrane, while the beta and gamma chains are sorted towards the degradation pathway. The half-lives of these three chains on the cell surface also reflect their different intracellular fates after endocytosis. The beta and gamma chains are very short-lived polypeptides since their half-life on the surface is only approximately 1 h, whereas alpha is a much more stable surface protein. This shows for the first time that components of a multimeric receptor can be sorted separately along the endocytic pathway.

Both the alpha and beta chains of high-affinity interleukin 2 receptors are located in intracellular vesicles when their ligand is endocytosed.

The growth factor interleukin 2 (IL2) binds to high and low-affinity receptors (Kd approximately 10-100 pM and 10 nM, respectively) present on activated T lymphocytes. High-affinity receptors are composed of two non-convalently linked polypeptides, alpha and beta of 55 and 70 kDa. These two polypeptides do not share any sequence homology, but each of them, in the absence of the other, binds IL2: alpha with a Kd approximately 10 nM and beta with a Kd approximately 1 nM. When these two chains are associated in lymphocytes, they form high-affinity receptors that mediate IL2 endocytosis and degradation, and transduce IL2 signaling. On cells that physiologically express IL2 receptors, such as activated T lymphocytes, both high and low affinity-receptors are present simultaneously on the cell surface, and low-affinity receptors (alpha without beta) are, in most instances, more abundant by a factor 5 to 10 than high-affinity receptors (alpha associated to beta). Low-affinity receptors bind IL2 but do not induce its internalization and signaling. The physiological role of the complexity of this receptor system is not fully understood. In the present study, we have investigated directly the fate of the high-affinity receptors when the ligand is endocytosed. By confocal microscopy, using two monoclonal antibodies specific for alpha and for beta, respectively, we show that each of these two polypeptides is located in intracellular endocytic compartments. Therefore, when the alpha chain is part of high-affinity receptors, it is endocytosed, as opposed to when it is part of low-affinity receptors and is not endocytosed.(ABSTRACT TRUNCATED AT 250 WORDS)

The cytosolic domain of APP induces the relocalization of dynamin 3 in hippocampal neurons.

Amyloid precursor protein (APP) has been the subject of intense research to uncover its implication in Alzheimer's disease. Its physiological function is, however, still poorly understood. Herein, we investigated its possible influence on the development of cultured hippocampal neurons. A peptide corresponding to the APP intracellular domain linked to a cell-penetrating peptide was used to alter the interactions of APP with its cytosolic partners. This treatment promoted the concentration of the cytosolic GTPase dynamin 3 (Dyn3) in neurite segments when most untreated cells displayed a homogenous punctate distribution of Dyn3. The Dyn3-labelled segments were excluded from those revealed by APP staining after aldehyde fixation. Interestingly, after aldehyde fixation MAP2 also labelled segments excluded from APP-stained segments. Thus APP is also a marker for the spacing pattern of neurites demonstrated by Taylor & Fallon (2006)J. Neurosci., 26, 1154-4463.

Cyclosporin A inhibits the interleukin 2 receptor alpha chain gene transcription but not its cell surface expression: the alpha chain stability can explain this discrepancy.

The effect of the immunosuppressor cyclosporin A (CsA) on the expression of interleukin (IL) 2 receptors was investigated in a human T cell line IARC301 which constitutively expresses such receptors. This cell line also spontaneously secretes IL2 which supports its autocrine growth. We have previously shown that CsA prevents the constitutive transcription of the IL2 gene in these cells. Here we show that as soon as 4 h after CsA addition, the transcription of the gene encoding the alpha chain (p55) of IL2R was inhibited. IL2 can transiently increase the expression of this gene. CsA did not prevent this transient IL2-dependent induction of IL2R alpha, but could still partially inhibit it. Once IL2 induction was over, CsA exerted its full inhibition. Thus, CsA does not seem to inhibit IL 2R alpha gene transcription simply by inhibition of IL2 synthesis. However, no modification of IL2R alpha expression on the cell surface could be detected after 48 h in the presence of CsA. This discrepancy between the effect of CsA on IL2R alpha expression as probed at the mRNA or the protein level can be accounted for by the stability of the IL2R alpha protein after synthesis. Indeed, the half-life of IL2R alpha chain is longer than 40 h. This suggests that the alpha chain, after it is endocytosed together with the beta chain as a component of high-affinity IL2R, might recycle back to the cell surface.

Hairpin, dumbbell, and single-stranded phosphodiester oligonucleotides exhibit identical uptake in T lymphocyte cell lines.

The cellular binding, uptake, and intracellular distribution of structured double-stranded phosphodiester oligonucleotides (decoys) have been examined in T lymphocytes using fluorescein-labeled molecules. Intracellular localization of hairpin and dumbbell decoys was similar to that of single-stranded oligonucleotides. At short incubation times, oligonucleotides were localized only in cytoplasmic vesicles, whereas at longer times, they were also found in the nucleus. Cellular uptake was dependent on temperature, time, and extracellular concentration. Oligonucleotide efflux was similar for all types of molecules and was very rapid (t1/2 = 10-15 minutes). These results imply that phosphodiester double-stranded oligonucleotides can enter cellular compartments of interest for their potential biologic function and, thus, provide a potential tool to control gene transcription.

Rapid endocytosis of interleukin 2 receptors when clathrin-coated pit endocytosis is inhibited.

The cytokine interleukin 2 (IL2) is produced by activated helper T lymphocytes and modulates the growth and activity of cells expressing high-affinity surface IL2 receptors that transduce its signaling. After ligand binding to receptors on the plasma membrane, receptor-ligand complexes are rapidly endocytosed and IL2 is degraded in acidic compartments. The best known receptor-mediated endocytosis pathway involves clathrin-coated pits. Receptors that carry an internalization signal recognized by adaptors on the cytosolic side of the plasma membrane are clustered into the coated pits and enter cells very efficiently. Many receptors use this pathway, but other endocytic pathways have also been reported, for ricin, EGF and insulin, for instance, which seem to be less efficient than the coated one. We compared the endocytosis of IL2 and its receptors to that of transferrin, a marker of the coated pit pathway. Under normal conditions, the kinetics of entry of IL2 was two times slower than that of transferrin. When internalization via coated pits was inhibited by two different methods, potassium depletion and cytosol acidification, endocytosis of IL2 and its receptors was only partly inhibited, while transferrin entry was strongly affected. Treatment with the cationic amphiphilic drug chlorpromazine, which induces a redistribution of a clathrin-coated pit component, AP-2, to endosomes, reduced transferrin, but not IL2 internalization. Thus, unexpectedly, this cytokine and its receptors can still be rapidly endocytosed in the absence of functional clathrin-coated structures. We propose a model for receptor-mediated endocytosis that may account for these results and published data on other receptors.

Autocrine growth of a human T-cell line is inhibited by cyclosporin A.

The effect of cyclosporin A (CsA), a potent immunosuppressive agent, on a human T-cell line, IARC 301, which constitutively secretes interleukin-2 (IL-2) and expresses high-affinity IL-2 receptors, was investigated. We show that CsA inhibits IARC 301 cell growth. CsA also prevents the constitutive secretion of IL-2 in this T-cell line by blocking transcription of the IL-2 gene. If exogenous IL-2 is added together with CsA for 3 days, the cells grow as well as untreated controls. Thus, under such conditions, CsA inhibits IARC 301 growth by preventing its endogenous constitutive IL-2 synthesis. This demonstrates that IL-2 stimulates the proliferation of this cell line by an autocrine pathway, in agreement with our previous data. We also show for the first time, that CsA not only can inhibit IL-2 production of T cells upon activation, but that it can also prevent ongoing constitutive IL-2 synthesis of a T-cell line. Autocrine growth stimulation of tumor cells by cytokines has been demonstrated in a few cases. CsA inhibits synthesis of several cytokines. Probing for the autocrine growth of tumor cells by studying the effect of CsA and its reversibility by cytokines on their proliferation may be simple and useful.

Immunoregulatory functions of paf-acether. III. Down-regulation of CD4+ T cells high-affinity IL-2 receptor expression.

In the present report, we further explored the mechanisms by which 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (paf-acether), a phospholipid mediator of inflammation inhibited PHA-induced CD4+ cell proliferation. Evidence was obtained that CD4+ cells stimulated with either PHA or immobilized OKT3 in the presence of paf at concentrations that block CD4+ cell proliferation, exhibited a marked decrease in high affinity IL-2R expression. Importantly, paf did not prevent the binding of IL-2 to its receptor. Scatchard analysis of the binding data indicated that paf caused more than 50% decrease in the number of IL-2 high affinity sites per cell, whereas the receptor ligand affinity remained essentially constant. Moreover, the down-regulation of high affinity IL-2R was also accompanied by a loss of IL-2-dependent proliferative capacity. Together these data suggest that decreased expression of high affinity IL-2R may contribute to the diminished proliferative activity observed in CD4+ cells stimulated with PHA or immobilized OKT3 in the presence of paf. They further emphasize the potential role of lipid proinflammatory mediators such as paf in the regulation of T cell activation.

Activity of the kappa B enhancer of the interleukin-2 receptor alpha chain in somatic cell hybrids is accompanied by the nuclear localization of NF-kappa B.

The two nuclear proteins NF-kappa B (consisting of subunits p50 an dp65) and the DNA-binding subunit of NF-kappa B (p50) by itself, also called KBF1, are constitutively expressed and localized in the nucleus of the human T-cell line IARC 301.5. In order to define the roles of these two factors, which bind to the same kappa B enhancers, in transcription activation we have prepared somatic cell hybrids between IARC 301.5 and a murine myeloma. Most hybrids express both KBF1 and NF-kappa B in their nuclei, but one hybrid expresses only KBF1. The kappa B enhancer of the gene encoding the interleukin-2 (IL-2) receptor alpha chain (IL-2R alpha) is functional only in the hybrids expressing nuclear NF-kappa B. These findings show that nuclear NF-kappa B is necessary to activate the kappa B enhancer, while KBF1 by itself is not sufficient. We propose that KBF1 is a competitive inhibitor of NF-kappa B and discuss how these factors may be involved in the transient expression of IL-2 and IL-2R alpha genes during the immune response.

Endocytosis of the beta chain of interleukin-2 receptor requires neither interleukin-2 nor the gamma chain.

Interleukin-2 (IL-2) and IL-2 receptors (IL-2R) critically regulate the magnitude and duration of T cell expansion required in an immune response. Modulation occurs at the level of receptor number and affinity. IL-2R is a multisubunit receptor which contains at least three chains, IL-2R alpha (p55), IL-2R beta (p70) and IL-2R gamma (p64). Some components of high-affinity receptors (alpha beta gamma) are continuously internalized in the absence as well as in the presence of IL-2. From studies on other receptors, it is known that endocytosis of ligand-receptor complexes is due to an intrinsic property of the receptor. However, the specific chains responsible for endocytosis of high-affinity IL-2 receptors have not been fully elucidated. IL-2R gamma has been reported to be necessary for IL-2 internalization, based on the fact that fibroblasts transfected with IL-2R alpha and -beta do not internalize IL-2. However, IL-2 dissociates too rapidly from IL-2R alpha beta receptors to allow for its internalization. From the reported results on IL-2 internalization in transfected fibroblasts, it cannot be concluded as to the respective roles of IL-2R beta and/or IL-2R gamma in endocytosis. As modulation of receptor number is important for biological activity, we have attempted to define the chains responsible for receptor internalization. In this work, we have studied the endocytic properties of IL-2R beta. We demonstrate that IL-2R beta is constitutively endocytosed in a B cell line, derived from a X-linked severe combined immunodeficiency patient, which lacks expression of IL-2R gamma. IL-2R beta was also constitutively internalized in T and natural killer cell lines independently of IL-2R gamma. These results suggest that IL-2R beta is endowed with endocytic capacity and carries internalization signals.

Dendroaxonal transcytosis of transferrin in cultured hippocampal and sympathetic neurons.

Previous studies using overexpressed polymeric immunoglobulin receptor in cultured neurons have suggested that these cells may use a dendroaxonal transcytotic pathway (Ikonen et al., 1993; de Hoop et al., 1995). By using a combination of semiquantitative light microscopy, video microscopy, and a biochemical assay, we show that this pathway is used by the endogenous ligand transferrin (Tf) and its receptor. Labeled Tf added to fully mature hippocampal neurons changes the intracellular distribution of its receptor from preferentially dendritic shortly after addition to dendritic and axonal at longer times. Incubation of living neurons with (caged)FITC-Tf followed by uncaging in the dendrites results in the later appearance of fluorescence in the axon of the same cell. In "chambered" sympathetic neurons in culture, 125I-Tf or iron as 55Fe-Tf added to the cell body/dendrite chamber is recovered in the axonal chamber, showing that internalized ligand from the cell body-dendrite area is released at the axonal end. Finally, we show that excitatory neurotransmitters increase Tf receptor transcytosis, whereas inhibitory neurotransmitters reduce it. The dendritic uptake, transcytotic transport, and axonal release of physiologically active Tf demonstrated here could be envisioned for other trophic factors and therefore have important consequences for neuronal anterograde target maturation. Moreover, the changes in transcytosis after neurotransmitter addition may be important in the cellular responses that follow electrical activation.

Kappa B binding proteins are constitutively expressed in an IL-2 autocrine human T cell line.

The IL-2 and the IL-2-R alpha genes are both expressed transiently in normal T lymphocytes after Ag or mitogen activation. In contrast, the human T cell line, IARC 301, expresses these two genes constitutively and we have previously demonstrated that its growth depends on the autocrine production of this T cell growth factor and high affinity IL-2R. To dissect the molecular basis for the unusual persistent expression of the IL-2 and IL-2-R alpha genes in these IARC 301 T cells, we have analyzed the interactions of constitutively expressed nuclear proteins with the 5' flanking regions of the IL-2 and IL-2-R alpha genes using both DNase I footprinting and gel retardation techniques. We have found that a region in both genes (-276 to -250 for IL-2-R alpha and -203 to -183 for IL-2), which corresponds to a kappa B enhancer element, is specifically protected by nuclear proteins from IARC 301. In agreement with this finding, both the IL-2 and IL-2-R alpha promoters are active in transient transfection assays in IARC 301 cells. In contrast, mutation of the kappa B enhancer results in markedly attenuated activities of both promoters. Two proteins binding the kappa B sequence, NF-kappa B and KBF1, are constitutively expressed in IARC 301 nuclei and induced by PMA and PHA in Jurkat. They bind to the kappa B motifs with different relative affinities that may reflect their different contribution in the expression of various promoters.

The product of the c-ets-1 proto-oncogene and the related Ets2 protein act as transcriptional activators of the long terminal repeat of human T cell leukemia virus HTLV-1.

The c-ets-1 proto-oncogene and the related c-ets-2 gene encode related nuclear chromatin-associated proteins which bind DNA in vitro. To investigate the possibility that Ets1 and Ets2 are transcriptional activators, we analyzed the ability of these proteins to trans-activate promoter/enhancer sequences in transient co-transfection experiments. A CAT construct driven by the long terminal repeat of the human T cell leukemia virus, HTLV-1 was found to be trans-activated by both Ets1 and Ets2 in NIH3T3 and HeLa cells. The increased levels of CAT activity were paralleled by increased levels of correctly initiated CAT mRNA. Mutant Ets1 proteins unable to accumulate in the nucleus were found to be inactive. An ets-responsive sequence between positions -117 and -160 of the LTR was identified by analyses of a series of 5' deletion mutants of the HTLV-1 LTR and of dimerized versions of specific motifs of the LTR enhancer region. Using a gel shift binding assay, Ets1 was found to bind specifically to an oligonucleotide corresponding to region -117 to -160. This sequence, which also contributes to Tax1 responsiveness of the HTLV-1 LTR, is characterized by the presence of four repeats of a pentanucleotide sequence of the type CC(T/A)CC. Competition experiments show that integrity of repeats 1 and 4 is important for Ets1 binding. These results show that Ets1 and Ets2 are sequence-specific transcriptional activators. In view of the high level expression of Ets1 in lymphoid cells, Ets1 could be part of the transcription complex which mediates the response to Tax1 and the control of HTLV-1 replication. More generally, Ets1 and Ets2 could regulate transcription of cellular genes.

The Staphylococcus aureus enterotoxin B superantigen induces specific T cell receptor down-regulation by increasing its internalization.

Superantigens are able to stimulate T lymphocyte populations expressing T cell antigen receptors (TCR) belonging to particular V beta families. Moreover, the presence of these superantigens may induce long term unresponsiveness (anergy) of these sensitive cells. Some bacterial toxins are potent superantigens. We have analyzed in vitro the capacity of some Staphylococcus aureus enterotoxin superantigens to modulate T cell antigen receptor expression and the cellular mechanisms involved. Staphylococcus enterotoxin B (SEB) induced rapid down-regulation of surface T cell antigen receptors in V beta 3-expressing T lymphocytes, as assessed by flow cytometry. This phenomenon was a consequence of the direct interaction between the toxin and the TCR since it was observed in the absence of cells expressing major histocompatibility complex class II molecules. The cellular mechanism involved in SEB-induced down-regulation of TCR was further investigated. Immunofluorescence and confocal microscopy experiments showed that toxin B induced intracellular accumulation of TCR.CD3 in endocytic vesicles. Moreover, SEB induced an increase in T cell receptor endocytosis as measured using radiolabeled Fab fragments of an anti-CD3 monoclonal antibody. Taken together, our observations indicate that Staphylococcus enterotoxin B superantigen induced changes in the dynamics of surface T cell receptors, which resulted in the fast reduction of membrane receptor numbers.

Transcriptional and post-transcriptional mechanisms are involved in the absence of CD4 surface expression in two HIV-1 chronically infected T cell lines.

In vivo infection of human T cell lymphocytes by HIV-1 is mediated by the specific binding of the HIV-1 envelope glycoprotein gp120 to the T cell CD4 receptor. One of the post-infection events observed in vivo is the progressive loss of CD4+ T cells. One possible mechanism is the production of infected T cells which are lacking in surface expression of the CD4 receptor protein. We have analysed this possibility utilizing the two HIV-1 chronically-infected CD4- cell lines, 8E5 and ACH-2, both of which are derived from a CD4+ parental strain (A3.01) after HIV-1 infection. In each cell (8E5 and ACH-2) the loss of CD4 surface expression was found to occur by different mechanisms. In ACH-2 cells, neither CD4 protein nor the 3 kb CD4 RNA transcript could be detected. However, treatment of ACH-2 cells with cycloheximide elicited production of the 3 kb transcript suggesting the possibility for a repressor protein(s) to act at the level of transcription and/or stability of the 3 kb mRNA. In contrast, in 8E5 cells the level of the 3 kb CD4 RNA was comparable with that found in the CD4+ A3.01 parental strain. Analysis of the 8E5 strain revealed the presence of a CD4- gp160 bimolecular protein complex sequestered internally in the rough endoplasmic reticulum (RER). Finally, the protein tyrosine kinase p56lck, normally associated with the cellular membrane, appeared to be linked to the RER and bound to the CD4- gp160 proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

A triple helix-forming oligonucleotide-intercalator conjugate acts as a transcriptional repressor via inhibition of NF kappa B binding to interleukin-2 receptor alpha-regulatory sequence.

Oligonucleotide-directed triplex formation within upstream regulatory sequences is envisioned as a potential tool for gene inhibition. However, this approach requires that triple helix-forming oligonucleotides are chemically modified, so that the triplex is stable under physiological conditions. Here, we have compared several chemical modifications of an oligonucleotide, targeted to a natural 15-base pair homopyrimidine.homopurine sequence located in the upstream regulatory region of the gene encoding the interleukin-2 receptor alpha chain (p55, IL-2 R alpha). Methylation of the cytosines strongly stabilized the triplex. Further attachment of an intercalating agent (acridine) dramatically increased the stability of the triplex, as assessed by Tm measurements or by band shift assays. Furthermore, the acridine-derivatized oligonucleotide was more efficient in competing away high affinity DNA-binding proteins, as assessed by restriction enzyme inhibition assays. Using a novel footprinting assay, we have further shown that the interaction of the methylcytosine-substituted, acridine-derivatized oligonucleotide with a plasmidic target, harboring the IL-2 R alpha regulatory region, remains highly sequence specific, occurs at physiological pH and is independent of the superhelicity of the plasmid. Acridine derivatization did not impair the exquisite target specificity of triplex formation, since the derivatized oligonucleotide inhibited the binding of nuclear proteins to the overlapping NF kappa B enhancer sequence on an IL-2 R alpha target and not on the related human immunodeficiency virus long terminal repeat target. Finally, the oligonucleotide inhibited the NF kappa B-dependent tax-induced transcriptional activation of the IL-2 R alpha chloramphenicol acetyltransferase construct in live cells, whereas it did not have any effect on a human immunodeficiency virus long terminal repeat chloramphenicol acetyltransferase construct. We conclude that this modified oligonucleotide acts as a transcriptional repressor for the IL-2 R alpha gene via triple helix formation with regulatory sequences.