Interferon-gamma and interleukin-4 regulate T cell interleukin-12 responsiveness through the differential modulation of high-affinity interleukin-12 receptor expression

Interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) are mutually antagonistic cytokines that stimulate CD4+ T cells to develop into either Th1 or Th2 cells. One feature of Th2 differentiation in mice is the loss of IL-12-induced Jak2 and Stat4 activation, which is accompanied by the inability to produce IFN-gamma in response to IL-12. In this report, we show that freshly isolated human T cells activated with phytohemagglutinin (PHA) in the presence of IL-4 exhibit a greatly diminished response to IL-12, whereas the IL-12 response of T cells activated with PHA plus IFN-gamma is enhanced. Radiolabeled IL-12 binding studies demonstrate that the impairment of T cell IL-12 responsiveness by IL-4 is associated with the down-regulation of high-affinity IL-12 receptor expression. In contrast, the enhancement of IL-12 responsiveness by IFN-gamma is associated with the upregulation of high-affinity IL-12 receptor expression. Through the use of a newly synthesized neutralizing antibody to the low-affinity IL-12 receptor beta subunit (IL-12Rbeta), we show that neither IL-4 nor IFN-gamma affect the expression of IL-12Rbeta, which we determine to be one of at least two low-affinity subunits required for high-affinity IL-12 binding. These findings suggest that IL-4 and IFN-gamma exert opposite effects on T cell IL-12 responsiveness by differentially modulating the expression of low-affinity IL-12 receptor subunits that are distinct from IL-12Rbeta and required, together with IL-12Rbeta, for high-affinity IL-12 binding and IL-12 responsiveness. This provides a basis for understanding the interplay between different cytokines at the level of cytokine receptor expression, and offers insight into one of the mechanisms governing Th1 and Th2 development.     

Suppression of interleukin-2 receptor expression on mouse CD4+ T cells by bovine kappa-caseinoglycopeptide

Bovine kappa-caseinoglycopeptide (residues 106-169, CGP) completely inhibited the PHA-induced proliferation of mouse splenocytes when CGP was added simultaneously with PHA. The inhibitory effect, however, was reduced to about one-half when CGP was added after 24 h of cultivating the splenocytes with PHA or when anti-IL-1ra antibody was added simultaneously with PHA and CGP. On the other hand, CGP bound to mouse CD4+ T cells but not to CD8+ T cells. CGP suppressed IL-2 receptor expression of the PHA-stimulated mouse CD4+ T cells.     

Type D SRV-2 virus-specific CD8+ and CD4- CD8- T cells that regulate virus-induced T cell proliferation in Celebes macaques

These studies defined SRV-2 envelope peptides 96-102, 127-152, and 233-249 as T cell epitopes that induce significant T cell proliferation. Peripheral blood lymphocytes of Celebes macaques (Macaca nigra) exposed to SRV-2 and currently virus- antibody+, cultured with SRV-2 virus show strongly suppressed T cell responses and have two immunoregulatory T cell populations.     

Retinoic acid receptors regulate expression of retinoic acid 4-hydroxylase that specifically inactivates all-trans retinoic acid in human keratinocyte HaCaT cells

Among oxysterols oxidized at C7 (7alpha-, 7beta-hydroxycholesterol, and 7-ketocholesterol), 7beta-hydroxycholesterol and 7-ketocholesterol involved in the cytotoxicity of oxidized low density lipoproteins (LDL) are potent inducers of apoptosis. Here, we asked whether all oxysterols oxidized at C7 were able to trigger apoptosis, to stimulate interleukin (IL)-Ibeta and/or tumor necrosis factor (TNF)-alpha secretion, and to enhance adhesion molecule expression (intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin) on human umbilical venous endothelial cells (HUVECs). Only 7beta-hydroxycholesterol and 7-ketocholesterol were potent inducers of apoptosis and of IL-1beta secretion. TNF-alpha secretion was never detected. Depending on the oxysterol considered, various levels of ICAM-1, VCAM-1 and E-selectin expression were observed. So, oxysterols oxidized at C7 differently injure and activate HUVECs, and the alpha- or beta-hydroxyl radical position plays a key role in apoptosis and IL-1beta secretion.     

Multiple developmental stage-specific enhancers regulate CD8 expression in developing thymocytes and in thymus-independent T cells

We and others have recently identified a CD8 locus enhancer (E8) that directs expression in mature CD8 single-positive thymocytes and peripheral CD8+ T cells and in extrathymically derived intestinal intraepithelial lymphocytes (IEL). In this study, we show that deletion of E8, by homologous recombination results in reduced CD8alphaalpha homodimer expression on IEL. Since CD8 expression on thymus-derived T cells was normal, other enhancers regulate CD8 expression in these cells. By exploiting a transgenic reporter expression assay, we identified three additional enhancers that directed expression in diverse thymocyte subsets and mature T cells but not in CD8alphaalpha+ IEL. The results suggest that CD8alpha expression is primarily regulated by E8, in IEL and by the novel enhancers in the thymus-dependent lineages.     

T cell-derived IL-4 and dendritic cell-derived IL-12 regulate the lymphokine-producing phenotype of alloantigen-primed naive human CD4 T cells

Previous studies on human Th subset development were restricted to the analysis of naive T cells activated with anti-CD3 mAb in the absence of physiologic APC. In this study, we have analyzed the role of cytokines and physiologic APC on T cell maturation in an Ag-specific system, in which naive neonatal CD4 T cells were primed with allogeneic dendritic cells (DC). We found that the cytokine profile of primed cells was dependent upon 1) the ratio between T cells and allogeneic DC and 2) the endogenous production of IL-4 and IL-12. Neutralization of IL-4 during primary MLR increased IFN-gamma production at priming and shifted the phenotype of primed cells from Th0 to Th1. These effects were IL-12 dependent, in that they were suppressed by anti-IL-12 Abs. The production of IL-12 in primary MLR was further evidenced by the presence of IL-12 p40 in the culture supernatant fluids. IL-12 production was suppressed by exogenous IL-4 and increased by anti-IL-4 blocking mAbs, indicating that endogenous IL-4 down-regulated IL-12 production by DC. Finally, IL-12 was produced as a result of T cell/DC interaction involving the CD40/CD40 ligand and CD28/B7 costimulation pathways, as revealed by the inhibitory effect of anti-CD40 ligand mAb and CTLA-4Ig. These observations suggest that in neutral conditions, Ag presentation by DC results in the coordinate production of naive T cell-derived IL-4 and DC-derived IL-12 that in concert shape the cytokine profile of Th cells.     

Frequencies of T cells expressing interleukin-4 and interleukin-5 in atopic asthmatic children. Comparison with atopic asthmatic adults

T-cell-derived cytokines have been implicated in the pathogenesis of asthma and it has been suggested that Th2-type cytokines (interleukin-4 [IL-4], interleukin-5 [IL-5]) are pivotal in the allergic inflammation. However, there are little data on human cytokine production by individual T cells at the protein level, in particular in asthmatic children. In this study we analyzed the cytokine production at the single cell level in peripheral blood from mild atopic asthmatic (AA) children and adults and age-matched atopic nonasthmatic (AN) and nonatopic nonasthmatic (NN) control subjects (n = 9 in each group) using the technique of intracellular cytokine detection by flow cytometry. Comparing asthmatic children with atopic and nonatopic control subjects, an increased percentage of IL-5-producing T cells (AA: median 4.9% [range 1.1 to 8.9%]; AN: 0.3% [0.2 to 0.9%], p = 0.003; NN: 0.4% [0.1 to 3.8%], p = 0.001) was detectable, with a positive correlation to the number of peripheral eosinophils and to bronchial hyperresponsiveness. The frequency of IL-4-producing T cells was increased in both atopic groups compared with nonatopic controls (AA: 1.2% [0.2 to 2.6%], p = 0.011; AN: 0.8% [0.4 to 3.7%], p = 0.007; NN: 0.4% [0.2 to 0.9%]) with a positive correlation to total IgE concentration. In adults there were no differences in IL-5- or IL-4-producing T cells between all three groups. A substantial proportion of T cells coproducing IL-4 and IL-5 was not detectable in children and adults. These findings indicate that in asthmatic children the frequencies of Th2-type-producing T cells are increased and that expression of IL-4 and IL-5 is regulated independently.     

IL-4 induces functional cell-surface expression of CXCR4 on human T cells

Here we report that IL-4 specifically enhances cell surface expression of CXCR4 on resting peripheral and cord blood T cells. Whereas polarized Th2 clones express variable levels of CXCR4, expression of this receptor is undetectable on polarized Th1 clones but can be induced on the latter cells as well, following short-term culture in the presence of IL-4. The IL-4-induced CXCR4 is functional since interaction with its ligand, stromal-derived factor (SDF)-1, activates the p42 MAP-kinase ERK-2. In addition, although CXCR4 expression is down-regulated following stimulation of T cells and T cell clones via CD28 or CD3 and CD2 cell surface molecules, respectively, it is re-induced by IL-4. These data indicate an important role for IL-4 in rendering CD4+ T cells susceptible to infection with HIV via CXCR4, as well as in promoting SDF-1-induced migration of these cells.     

Glucocorticoid-mediated inhibition of interleukin-2 receptor alpha and -beta subunit expression by human T cells

We have shown previously that vanadium ions (vanadate and vanadyl) inhibit autophosphorylation of histidine but not that of serine in ATP citrate lyase (ACL). Here we report the results concerning the effect of monovanadate (+ oligomers), decavanadate as well as vanadyl on the activity of ACL of the rat liver. Susceptibility of ACL to inhibition by vanadate was rather low. Vanadate at concentration 10(-4) mol/l inhibited ACL by only 10% and at 10(-3) mol/l concentration monovanadate inhibited ACL by 37%. Decavanadate had comparable potency to inhibit ACL. So was vanadyl which produced 20%, 32% and 66% inhibition at 10(-4) mol/l, 10(-3) mol/l and 10(-2) mol/l concentrations, respectively. From the kinetic data it appears that inhibition by mono- and deca-vanadate of ACL with respect to both ATP and citrate was of competitive nature. Vanadyl inhibited ACL noncompetitively with respect to these substrates. However, all three species of vanadium ions inhibited ACL noncompetitively with respect to CoA. Endogenous (auto)phosphorylation of the ACL histidine as well as its response to vanadate depended on the presence of he substrate (citrate + CoA). The kinetic characteristics of vanadium ions action of ACL was compared with that previously demonstrated for vanadium inhibition of succinyl-CoA synthetase. Plausibility of our hypothesis that inhibition of histidyl phosphorylation at the catalytic site may be a common mechanism by which vanadium ions suppress the activity of the histidyl containing enzymes catalyzing the phosphoryl transfer is discussed.     

Antigen-specific T lymphocytes regulate lipopolysaccharide-induced apoptosis of dendritic cells in vivo

The potent accessory properties of dendritic cells (DC) develop sequentially during a process termed "maturation." Splenic DC undergo functional maturation in vivo in response to the bacterial product LPS and migrate from the marginal zone to the T cell areas. The redistribution of fully mature DC, which present Ags encountered in the periphery, in the T cell area is likely to result in T cell priming. Unexpectedly, we found that DC rapidly die by apoptosis once they have entered the T cell zone. Injection of OVA peptide in OVA-specific, TCR-transgenic mice strongly delays the LPS-induced apoptosis of DC in situ. We conclude that mature DC are programmed to die unless they receive a survival signal from T cells and that the regulation of DC survival may be a mechanism aimed at controlling the initiation and the termination of the immune response.     

Cytokines regulate expression and function of the HIV coreceptor CXCR4 on human mature dendritic cells

HIV-infected dendritic cells (DC) efficiently transmit infection to CD4+ T cells during the process of T cell activation. To further understand interactions between DC and HIV, cytokine regulation of HIV coreceptors on cultured Langerhans cells (cLC, as prototypes of mature DC) was studied. Expression of cell surface CXCR4 on cLC was up-regulated by IL-4 and TGF-beta1 and inhibited by IFN-alpha, IFN-beta, and IFN-gamma, whereas cytokines did not appreciably regulate CCR5. Changes in cell surface CXCR4 expression on cLC correlated with T cell-tropic (X4)-HIV envelope-mediated syncytium formation and X4-HIV infection levels. A relative increase in the ratio of type 2/type 1 cytokine production, which can occur in HIV disease, may up-regulate CXCR4 expression on mature DC and promote infection by X4 viruses. Importantly, these findings suggest that cytokine dysregulation may be linked to the emergence of X4-HIV strains as HIV-infected individuals progress to AIDS.     

Microenvironmental factors regulate Ly-6 A/E expression on PyV-transformed BALB/c 3T3 cells

Ouabain-induced changes of the free cytoplasmic Na+ concentration ([Na+]i) were monitored in aggregates of cells prepared from beta-cell-rich pancreatic mouse islets and the results were compared with the total islet content of sodium. The steady-state [Na+]i was lower in 20 mM glucose (11 mM) than in 3 mM glucose (14 mM). In the presence of 3 mM glucose the addition of 1 mM ouabain resulted in a rise in [Na+]i with an initial rate of 1.5 mM/min. However, the increase of total sodium corresponded to 2.8 mM/min, suggesting that rapid binding and/or sequestration of Na+ are prominent features for pancreatic beta-cells. Elevation of the glucose concentration to 20 mM increased the rate of ouabain-dependent rise of [Na+]i. The effect of glucose was mimicked by 1 mM tolbutamide or 100 microM carbachol and was counteracted by 100 nM of the alpha 2-adrenergic agonist clonidine. Glucose also accelerated the lowering of [Na+]i after withdrawal of ouabain. In promoting not only the entry but also the extrusion of Na+, glucose actually enhances the turnover of the ion in pancreatic beta-cells.     

Chromatin complexes as aperiodic microcrystalline arrays that regulate genome organisation and expression

Chemoresistance is of outstanding importance for the limited results of chemotherapy in solid tumors. Chemoresistance of multicellular tumor tissues is more pronounced than that of single cells in vivo and in vitro. The enzyme hyaluronidase is able to loosen the cell-cell contact and the interstitial connective tissue and as such, in a number of preclinical and clinical trials, was shown to enhance the efficacy of cytostatic agents. Although proven to be very effective as additive to local chemotherapy, the systemic efficacy is not documented as well. We present a randomized trial done in high-grade astrocytomas with combined chemotherapy and radiation therapy with and without hyaluronidase. After very promising pilot results with systemic hyaluronidase in various tumor entities and also astrocytomas, this randomized study failed to show synergy to chemotherapy and radiation therapy in high-grade astrocytomas concerning survival. The promising preclinical data and the rather well documented activity in therapeutic use as additive to local chemotherapy seem to be an adequate motive to further elucidate the complex manner in which hyaluronidase is active in the interstitial tumor matrix and to obtain more information concerning the optimal route of application, the optimal dosage and the spectrum of tumor entities where it is synergistic with cytostatic chemotherapy and perhaps even radiation therapy.     

Linkage of protein kinase C-beta activation and intracellular interleukin-2 accumulation in human naive CD4 T cells

A critical role for protein kinase C (PKC) in signal transduction events has been well established. Moreover, studies of regulation in PKC levels suggest participation in mediating long-term cellular functions. Protein kinase C-beta (PKC-beta) has been reported to be involved in interleukin-2 (IL-2) synthesis in T lymphocytes. In this study, the role of PKC-beta in intracellular accumulation of IL-2 was investigated using specific inhibitors. Preincubation with two different PKC inhibitors, one specific for classical isotypes (alpha and beta I) Go6976, and one which inhibits both classical and non-classical isotypes, GF109203X, caused a complete block in cytoplasmic IL-2 accumulation when naive CD4 T cells were stimulated in the presence of CD2+CD28+phorbol myristate acetate (PMA). In contrast, preincubation with up to 1000 ng/ml of cyclosporin A (CsA) resulted in a reduction in the intracellular IL-2 detected, as observed by a decrease in the proportion of positive cells as well as a fall in the mean fluorescence intensity (MFI). CsA did not influence PKC-beta translocation. Flow cytometric assessments of PKC-beta and its isoforms beta I and beta II correlated with Western blotting analysis and these results were further supported by the use of PKC-beta-positive (HUT 78) and -negative (BW5147) T-cell lines. Using the specific inhibitors, Go6976 and GF109203X, the findings in this study suggest that activation and translocation of PKC-beta is critical for accumulation of intracellular IL-2. The influence of CsA in reducing but not blocking IL-2 synthesis is discussed. PMA-induced down-regulation of the CD4 antigen was observed in the presence of Go6976 and but not GF109203X, suggesting regulation by non-classical PKC isoforms.     

The effect of different corticosteroids and cyclosporin A on interleukin-4 and interleukin-5 release from murine TH2-type T cells

By secretion of interleukin-4 and interleukin-5, TH2-type T cells are thought to play an important role in the pathogenesis of asthma. Corticosteroids are currently the most effective therapy available for asthma, but recently it has been demonstrated that cyclosporin A improves lung function in patients with severe corticosteroid-dependent asthma. In order to examine the effects of corticosteroids and cyclosporin A on anti-CD3-induced production of interleukin-4 and interleukin-5 we used the murine TH2-type cell clone D10.G4.1. Interleukin-4/interleukin-5 release was inhibited by all drugs tested with the following IC50 values (nmol/l) for interleukin-4 and interleukin-5, respectively: budesonide (0.32/0.22), beclomethasone (0.65/0.33), dexamethasone (4.70/3.52), 6 alpha-methyl-prednisolone (24.04/17.02), hydrocortisone (34.27/22.55), and cyclosporin A (72.59/242.21). In conclusion, corticosteroids exert strong inhibitory effects on cytokine production by TH2-cells, which may explain, at least partly, its clinical efficacy in asthma. Cyclosporin A also showed a concentration-dependent inhibition; however, in relation to corticosteroids the inhibitory activity of cyclosporin A was found to be weaker.