Regulation of interleukin-2 production and phosphatidylserine synthesis in Jurkat T lymphocytes by K+ channel antagonists

Modification of phospholipid metabolism during T cell activation has been repeatedly reported. Recently, we have shown that phytohaemagglutinin, CD3 and CD2 mAbs, which are potent in vitro activators of helper T lymphocytes, markedly inhibit phosphatidylserine synthesis concomitantly as they induce the secretion of IL-2. In this paper, we show evidence that in T lymphocytes K+ channels, which have been shown to participate in the cell activation process, are also reciprocally related to phosphatidylserine synthesis. In fact, in resting T cells the drugs affecting the activity of K+ channels, such as quinine and 4-aminopyridine, induce a rise of phosphatidylserine synthesis. In activated cells, quinine and 4-amidopyridine also caused a rise in phosphatidylserine synthesis which paralleled a decreased production of IL-2, strongly suggesting that these two events are correlated in a reciprocal manner. More precisely, phosphatidylserine synthesis was stimulated by drugs which have been reported to inhibit potassium channels in lymphocytes, e.g. quinine, 4-aminopyridine, tetraethylammonium. These data suggest that the decreased PS synthesis observed during T cell activation intervenes in the cascade of events leading to IL-2 secretion. The decrease in the biosynthesis of this phospholipid seems to be dependent on the activity of K+ channels.     

Fungal malformins inhibit bleomycin-induced G2 checkpoint in Jurkat cells

A DNA-damaging agent, bleomycin, arrests the cell cycle at the G2 phase of Jurkat cells, which are defective in the G1 checkpoint, while microtubule-disrupting colchicine arrests it at M phase. Fungal cyclopeptides, malformin A1 and malformin C, were found to abrogate bleomycin-induced G2 arrest (IC(50); 0.48 microM and 0.9 nM, respectively), resulting in a drastic decrease in cells in G2 phase and increase in cells in subG1 phase. On the other hand, malformins showed little effect on the colchicine-induced M phase arrest in Jurkat cells (IC(50); 2.7 microM and 24 nM, respectively). Malformin C (0.026 microM) also abrogated bleomycin-induced G2 arrest in colon cancer-derived HCT-116 cells. These data strongly suggest that malformin C disrupted the cell cycle at the G2 checkpoint of cancer cells, leading to sensitization of the cancer cells to the anti-cancer reagent.     

Norcantharidin induces cell cycle arrest and inhibits progression of human leukemic Jurkat T cells through mitogen-activated protein kinase-mediated regulation of interleukin-2 production

Norcantharidin (NCTD) is a potential anti-cancer agent that inhibits proliferation and induces cell death through regulation of mitogen-activated protein kinases (MAPK). This study examined the effect of NCTD on tumor cells by using a model of phorbol 12-myristate 13-acetate plus ionomycin (PMAI)-activated leukemia Jurkat T cells. The results showed that NCTD significantly inhibited the viability of cells with and without PMAI treatment. NCTD induced cell cycle arrest at G2/M phase, down-regulated the expression of calcineurin and, by itself or in combination with Cyclosporine A, reduced calcineurin phosphatase activity. Furthermore, NCTD up-regulates the expression of phosphorylated (p)-P38 and p-ERK1/2, but not JNK in PMAI-activated Jurkat T cells, in accordance with the alteration in viability. Regarding major cytokine and chemokine secretion profile, NCTD attenuates PMAI-augmented production of IL-2, but slightly increases or has no effect on TNF-α and IL-8. By blockade of various MAPK, NCTD regulates PMAI-augmented IL-2 production through activation of P38 and ERK1/2, in accordance with the aforementioned MAPK expression. In conclusion, NCTD inhibited IL-2 production in PMAI-activated human leukemia Jurkat T cells through activation of P38 and ERK1/2, suggesting that NCTD might have the potential of being used as a chemopreventive agent to inhibit tumor progression in the future.     

Liver enzyme-mediated oxidation of Echinacea purpurea alkylamides: production of novel metabolites and changes in immunomodulatory activity

The medicinal plant Echinacea is widely used to treat upper respiratory infections and is reported to stimulate the human immune system. A major constituent class of Echinacea, the alkylamides, has immunomodulatory effects. Recent studies show that alkylamides are oxidized by cytochrome P450 enzymes, but the immunomodulatory activity of these products is unknown. The objectives of this study were to characterize the products formed by incubation of an Echinacea extract and an isolated alkylamide with human liver microsomes, and to evaluate the influence of Echinacea alkylamides and metabolites on cytokine production by Jurkat human T cells. A novel class of carboxylic acid alkylamide metabolites was identified and shown to be the major constituents present after 2-h incubation of alkylamides with human liver microsomes. Echinacea alkylamides suppressed IL-2 secretion by stimulated T cells, and this effect was significantly lessened upon oxidation of the alkylamides to carboxylic acids and hydroxylated metabolites. These findings highlight the importance of considering the influence of liver enzyme metabolism when evaluating the immunomodulatory effects of alkylamides.     

Astilbin selectively facilitates the apoptosis of interleukin-2-dependent phytohemagglutinin-activated Jurkat cells.

The present study examined the relationship between the activation of T cells and the apoptosis-facilitating effect of astilbin on them. By the stimulation of PHA, a remarkable IL-2 production was detected in the supernatant of Jurkat cells after 120 h among 72--144 h incubation. This kinetics was quite in accordance with that of astilbin-induced apoptosis of Jurkat cells, where 1 h-exposure of the PHA-activated cells to astilbin caused a significantly increased apoptosis in a dose-dependent manner. To the Jurkat cells that had been cultivated for 72--144 h without PHA, however, astilbin did not show any facilitation of the cell apoptosis. Pre-treatment by cyclosporine A simultaneously with PHA dose-dependently lowered the IL-2 production and susceptibility of the cells to astilbin, while the treatment after 120 h of PHA-activation did not. The exogenous IL-2 treatment after 72 h of PHA-activation significantly and dose-dependently raised the susceptibility of the Jurkat cells to astilbin. These results indicated the dependency of the apoptosis-facilitating effect of astilbin on appropriate status of activated T lymphocytes with a relation to IL-2 production. This characteristic of astilbin may be of great significance for the treatment of a variety of immunologically related diseases.     

Manganese promotes phorbol ester-induced interleukin-2 production via AP-1 activation in Jurkat T-cells

Mn²⁺ is a minor nutrient, but is essential for numerous enzymatic activities and thus, for many cellular functions. However, its physiological roles and toxicity remain to be elucidated. In this study, we assessed the pharmacological potential and toxicity of Mn²⁺ in the immune system by examining the effects of Mn²⁺ on interleukin-2 (IL-2) production by Jurkat T-cells. Mn²⁺ at 0.1–1 mM did not significantly induce IL-2 production, whereas phorbol 12-myristate 13-acetate (PMA) at 1 μM slightly induced IL-2 production. Interestingly, Mn²⁺ at 0.3–0.7 mM promoted PMA-induced IL-2 production in a dose-dependent manner. A reporter gene assay revealed that Mn²⁺ promoted the activity of AP-1 (activator protein-1, a complex of c-Fos and c-Jun) in the presence of PMA. Western blot analysis showed that Mn²⁺ promoted the activation of JNK2 (c-Jun N-terminal kinase 2) and p38 MAPK (mitogen-activated protein kinase), which are both activators of AP-1, and upregulated the production of c-Fos and c-Jun within 4 h in the presence of PMA. These results suggest that Mn²⁺ promotes PMA-induced IL-2 production by inducing the production and activation of AP-1, at least in part.     

Inhibitory effect of berberine on interleukin-2 secretion from PHA-treated lymphocytic Jurkat cells

Berberine is an isoquinoline alkaloid isolated from herb plants, such as Cortex phellodendri (Huangbai) and Rhizoma coptidis (Huanglian). Huanglian and Huangbai have been used as “heat-removing” agents. In addition, berberine has been reported to exert anti-inflammatory effect both in vivo and in vitro, where mitogen-activated protein kinase (MAPK) and cyclooxygenase-2 (COX-2) expressions are critically implicated. We herein tested the hypothesis that berberine exerts an anti-inflammatory effect through MAPK and COX-2 signaling pathway in T-cell acute lymphoblastic leukemia (T-ALL). In Jurkat cells, we found that PHA exposure caused elevation on interleukin-2 (IL-2) production in a time-dependent manner. PHA-stimulated reactions were steeply suppressed by berberine, such as IL-2 mRNA expression and protein secretion. However, berberine did not exert any cytotoxic effect at doses of 40 μg/ml. In addition, the possible molecular mechanism of anti-inflammation effect of berberine could be the inhibition of PHA-evoked phosphorylation of p38, since c-Jun N-terminal kinases (JNK) and extracellular signal-regulated kinase (ERK) expressions did not alter. Consistent with above results, berberine inhibition on PHA-induced IL-2 secretion could be reversed by treatment of SB203580, a specific inhibitor of p38-MAPK. Interestingly, upregulation of PHA-induced COX-2 expression was also observed following berberine treatment of Jurkat cells. Furthermore, flow cytometry analysis showed berberine-induced cell cycle arrest at G1 phase after PHA stimulation and decreased percentage of G2/M phase. In conclusion, our study demonstrated that the anti-inflammatory effect of berberine largely potentially results from its ability to attenuate p38 MAPK expression, and does not exclude a positive action of berberine on cell cycle arrest. These results provide an innovative medicine strategy to against or treat T-ALL patients.     

Prostaglandins E1 and E2 inhibit lipopolysaccharide-induced interleukin-18 production in monocytes

The purpose of this present study was to explore the therapeutic potential of prostaglandins E1 and E2 on the systemic inflammatory response evoked by endotoxin. Since interleukin-18, a monocyte-derived cytokine, is increased during sepsis, decreasing the production of interleukin-18 is important in treating this condition. Prostaglandin E1 and E2 inhibited interleukin-18 production in human monocytes treated with lipopolysaccharide and prostanoid IP-, EP2- and EP4-receptor agonists mimicked the effects of prostaglandins E1 and E2. Therefore, prostanoid IP, EP2- and EP4-receptors might be involved in the decrease in interleukin-18 production during sepsis.     

A selective type 4 phosphodiesterase inhibitor,T-440, modulates intracellular cyclic AMP level and interleukin-2 production of Jurkat cells

Effect of a selective type 4 phosphodiesterase (PDE4) inhibitor, T-440, on intracellular cyclic AMP (cAMP) level and interleukin (IL)-2 production of Jurkat cells was investigated. T-440 suppressed both cAMP-PDE activities in cytosolic and membrane fractions of Jurkat cells. Intracellular cAMP level in Jurkat cells was elevated by PGE₂ and forskolin but not by T-440. T-440, however, significantly enhanced the increase of cAMP by PGE₂. PGE₂ and forskolin inhibited IL-2 production of Jurkat cells stimulated with concanavalin A. T-440 by itself did not affect IL-2 production, but significantly enhanced the effect of PGE₂ on IL-2 production. The increase of intracellular cAMP by T-440, PGE₂, forskolin and T-440 plus PGE₂ was well correlated with the inhibition of IL-2 production. These results indicate that IL-2 production of T cells is regulated by cAMP-PDE activity. Immunomodulatory effects of PDE4 inhibitors like T-440 should further be explored in vivo.     

Multiple actions of lysophosphatidylcholine in human Jurkat T cells

AIM: To obtain pathophysiological meanings of lysophosphatidylcholine (LPC)through the investigation of the effects of LPC in Jurkat T cells . METHODS: We measured ROS generation, [Ca(2+)](i), and mitochondrial membrane potential (MMP)by fluorescent spectrometry in Jurkat T cells. RESULTS: We observed that LPC significantly increased the reactive oxygen species (ROS) level in human Jurkat T cells. Among structurally-related lysolipids and eleven synthetic LPCs with different acyl chain lengths, palmitoyl LPC increased ROS to the highest level. alpha-Tocopherol, an antioxidant, and rottlerin PKCdelta inhibitor were inhibitory effects on LPC-induced ROS generation. LPC rapidly depolarized MMP and markedly elevated [Ca(2+)](i) by Ca(2+) influx across the plasma membrane. However, LPC-induced ROS increase seemed to not be related with LPC-induced depolarization of MMP or [Ca(2+)](i) increase. G2A family G protein-coupled receptors (GPCR) for lysolipids were expressed in Jurkat T cells, however, evidence indicated that GPCR was not involved in LPC actions. CONCLUSION: LPC induced several cellular changes in Jurkat T cells, including an increase of ROS generation in a PKCdelta-dependent and GPCR-independent manner, increase of [Ca(2+)](i) through Ca(2+) influx, and decrease of MMP. LPC-induced actions in Jurkat T cells represent novel action modes of LPC that do not involve GPCR and multiple independent changes of intracellular signaling molecules.     

Chalcones from Chinese liquorice inhibit proliferation of T cells and production of cytokines

Licochalcone A (LicA), an oxygenated chalcone, has been shown to inhibit the growth of both parasites and bacteria. In this study, we investigated the effect of LicA and four synthetic analogues on the activity of human peripheral blood mononuclear cell proliferation and cytokine production. Four out of five chalcones tested inhibited the proliferation of lymphocytes measured by thymidine incorporation and by flow cytometry. The production of pro- and anti-inflammatory cytokines from monocytes and T cells was also inhibited by four of five chalcones. Furthermore, intracellular detection of cytokines revealed that the chalcones inhibited the production rather than the release of the cytokines. Taken together, these results indicate that LicA and some analogues may have immunomodulatory effects, and may thus be candidates not only as anti-microbial agents, but also for the treatment of other types of diseases.     

Inhibition of interleukin-2 production in the human T cell line JURKAT by nonpolar maleimides

The immunosuppressive properties of polar and nonpolar maleimides were studied by measuring their ability to inhibit mitogen-induced interleukin-2 (IL-2) production by JURKAT T cells. The nonpolar maleimides N-ethylmaleimide (NEM) and N-phenylmaleimide (NPM) inhibited IL-2 production by 85-99%, but only when added to JURKAT cells prior to the mitogen. The polar maleimides N-hydroxymaleimide (NHM) and 4-maleimidosalicylic acid (M84) did not suppress IL-2 production significantly, even though NHM reacted with more cellular thiols (12%) than did NPM (8%). Both NEM and NPM suppressed IL-2 production at doses that did not affect proliferation. NEM inhibited IL-2 production induced by PHA, anti-CD3 (alpha CD3) monoclonal antibodies or PMA, and A23187, but did not interfere with the binding of alpha CD3 to the cells. NEM inhibited IL-2 production at concentrations that did not interfere with the PHA-induced increase in intracellular free calcium [( Ca]i). Neither NPM nor NHM inhibited the rise in [Ca]i, even at the highest concentrations tested. Although JURKAT T cells require both PMA and A23187 to induce IL-2 production, we found that cells pretreated with PMA could respond to A23187 added 18 hr later. PMA-treated cells were not resistant to the immunosuppressive effects of NEM or NPM. However, PMA-pretreated cells became resistant to the inhibitory effects of NEM upon the addition of A23187, suggesting that nonpolar maleimides inhibit activation events induced by the rise in [Ca]i.     

Histamine augments interleukin-2 production and the activation of cytotoxic T lymphocytes

Histamine (0.5 g/kg) was found to augment the activation of cytotoxic T lymphocytes in vivo, if injected in the late phase (day 4) of the response. The production of interleukin-2 in concanavalin A-activated spleen cell cultures was also strongly augmented by 1 X 10(-2) M histamine or by a combination of 2 X 10(-3) M histamine and histaminase (diamine oxidase). This suggests the possibility that the augmentation in vivo is mediated by the oxidized histamine derivative imidazolylacetaldehyde, since diamine oxidase occurs in many tissues. The interleukin-2-dependent proliferation of a T cell clone, on the other hand, was not affected by histamine with or without diamine oxidase. The experiments suggest that histamine supports the late phase of the cytotoxic T lymphocyte response by augmenting the interleukin-2 production.     

Relationship between thallium(I)-mediated plasma membrane fluidification and cell oxidants production in Jurkat T cells

The effects of thallous cation (Tl(+)) on: (a) the production of oxidant species and (b) membrane fluidity were evaluated in human leukemia T cells (Jurkat). After 72 h of incubation in the presence of Tl(+) (5-100 microM), no significant changes in cell viability were observed, although the average cell size was decreased as evaluated by steady-state light scattering. Tl(+) (5-100 microM) caused a significant increase in the concentration of cellular oxidants as measured with the probe 5(6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate (DCDCDHF). Similarly, a higher lipid oxidation products release was observed as measured by TBARS production. Both Tl(+)-mediated DCDCDHF oxidation and TBARS production were prevented when cells were supplemented with 2mM Trolox. Tl(+) (5-100 microM) also induced a concentration-dependent increase in plasma membrane fluidity, evaluated with the probe 6-(9-anthroyloxy)stearic acid (6-AS). This effect of Tl(+) was neither associated to the externalization of phosphatidylserine, nor observed in Trolox-supplemented cells. Significant correlations were found between the increase in plasma membrane fluidity and TBARS production and DCDCDHF oxidation. Together, the present results suggest that the increase in cellular oxidants caused by Tl(+) could oxidize membrane fatty acids, resulting in an increase in membrane fluidity. These effects could underlie the pathology associated with Tl(+) toxicity.     

The flavones luteolin and apigenin inhibit in vitro antigen-specific proliferation and interferon-gamma production by murine and human autoimmune T cells

Plant-derived flavonoids are inhibitors of various intracellular processes, notably phosphorylation pathways, and potential inhibitors of cellular autoimmunity. In this study, the inhibiting effects of various flavonoids on antigen-specific proliferation and interferon-gamma (IFN-gamma) production by human and murine autoreactive T cells were evaluated in vitro. T-cell responses were evaluated for the human autoantigen alpha B-crystallin, a candidate autoantigen in multiple sclerosis, and for the murine encephalitogen proteolipid protein peptide PLP (139-151). The flavones apigenin and luteolin were found to be strong inhibitors of both murine and human T-cell responses while fisitin, quercitin, morin and hesperitin, members of the subclasses of flavonoles and flavanones, were ineffective. Antigen-specific IFN-gamma production was reduced more effectively by flavones than T-cell proliferation, suggesting that the intracellular pathway for IFN-gamma production in T cells is particularly sensitive to flavone inhibition. These results indicate that flavones but not flavanoles or flavanones are effective inhibitors of the potentially pathogenic function of autoreactive T cells. The effects of flavones were the same for human and murine autoreactive T cells, stressing the usefulness of animal models of autoimmunity for further studies on the effects of flavonones on autoimmune diseases.