Ghrelin augments murine T-cell proliferation by activation of the phosphatidylinositol-3-kinase,extracellular signal-regulated kinase and protein kinase C signaling pathways

Thymic atrophy occurs during normal aging, and is accelerated by exposure to chronic stressors that elevate glucocorticoid levels and impair the naïve T cell output. The orexigenic hormone ghrelin was recently shown to attenuate age-associated thymic atrophy. Here, we report that ghrelin enhances the proliferation of murine CD4+ primary T cells and a CD4+ T-cell line. Ghrelin induced activation of the ERK1/2 and Akt signaling pathways, via upstream activation of phosphatidylinositol-3-kinase and protein kinase C, to enhance T-cell proliferation. Moreover, ghrelin induced expression of the cell cycle proteins cyclin D1, cyclin E, cyclin-dependent kinase 2 (CDK2) and retinoblastoma phosphorylation. Finally, ghrelin activated the above-mentioned signaling pathways and stimulated thymocyte proliferation in young and older mice in vivo.     

Role of interleukin (IL)-2 receptor beta-chain subdomains and Shc in p38 mitogen-activated protein (MAP) kinase and p54 MAP kinase (stress-activated protein Kinase/c-Jun N-terminal kinase) activation. IL-2-driven proliferation is independent of p38 and p54 MAP kinase activation

We have shown recently that interleukin (IL)-2 activates the mitogen-activated protein (MAP) kinase family members p38 (HOG1/stress-activated protein kinase II) and p54 (c-Jun N-terminal kinase/stress-activated protein kinase I). Furthermore, the p38 MAP kinase inhibitor SB203580 inhibited IL-2-driven T cell proliferation, suggesting that p38 MAP kinase might be involved in mediating proliferative signals. In this study, using transfected BA/F3 cell lines, it is shown that both the acidic domain and the membrane-proximal serine-rich region of the IL-2Rbeta chain are required for p38 and p54 MAP kinase activation and that, as for p42/44 MAP kinase, this activation requires the Tyr338 residue of the acidic domain, the binding site for Shc. It is well established that the acidic domain of the IL-2Rbeta chain is dispensable for IL-2-driven proliferation, and thus our observations suggest that neither p38 nor p54 MAP kinase activation is required for IL-2-driven proliferation of BA/F3 cells. In addition, the tetravalent guanylhydrazone inhibitor of proinflammatory cytokine production, CNI-1493, can block the activation of p54 and p38 MAP kinases by IL-2 but has no effect on IL-2-driven proliferation of BA/F3 cells, activated primary T cells, or a cytotoxic T cell line. Furthermore, our observations provide evidence for the existence of an additional, unknown target of the p38 MAP kinase inhibitor SB203580, the activation of which is essential for mitogenic signaling by IL-2.     

T cell effector function and anergy avoidance are quantitatively linked to cell division

We have shown previously that T cells activated by optimal TCR and CD28 ligation exhibit marked proliferative heterogeneity, and approximately 40% of these activated cells fail entirely to participate in clonal expansion. To address how prior cell division influences the subsequent function of primary T cells at the single cell level, primary CD4+ T cells were subjected to polyclonal stimulation, sorted based on the number of cell divisions they had undergone, and restimulated by ligation of TCR/CD28. We find that individual CD4+ T cells exhibit distinct secondary response patterns that depend upon their prior division history, such that cells that undergo more rounds of division show incrementally greater IL-2 production and proliferation in response to restimulation. CD4+ T cells that fail to divide after activation exist in a profoundly hyporesponsive state that is refractory to both TCR/CD28-mediated and IL-2R-mediated proliferative signals. We find that this anergic state is associated with defects in both TCR-coupled activation of the p42/44 mitogen-activated protein kinase (extracellular signal-related kinase 1/2) and IL-2-mediated down-regulation of the cell cycle inhibitor p27kip1. However, these defects are selective, as TCR-mediated intracellular calcium flux and IL-2R-coupled STAT5 activation remain intact in these cells. Therefore, the process of cell division or cell cycle progression plays an integral role in anergy avoidance in primary T cells, and may represent a driving force in the formation of the effector/memory T cell pool.     

Cooperative regulation of the cell division cycle by the protein kinases RAF and AKT

The RAS-activated RAF-->MEK-->extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3'-kinase (PI3'-kinase)-->PDK1-->AKT signaling pathways are believed to cooperate to promote the proliferation of normal cells and the aberrant proliferation of cancer cells. To explore the mechanisms that underlie such cooperation, we have derived cells harboring conditionally active, steroid hormone-regulated forms of RAF and AKT. These cells permit the assessment of the biological and biochemical effects of activation of these protein kinases either alone or in combination with one another. Under conditions where activation of neither RAF nor AKT alone promoted S-phase progression, coactivation of both kinases elicited a robust proliferative response. Moreover, under conditions where high-level activation of RAF induced G(1) cell cycle arrest, activation of AKT bypassed the arrest and promoted S-phase progression. At the level of the cell cycle machinery, RAF and AKT cooperated to induce cyclin D1 and repress p27(Kip1) expression. Repression of p27(Kip1) was accompanied by a dramatic reduction in KIP1 mRNA and was observed in primary mouse embryo fibroblasts derived from mice either lacking SKP2 or expressing a T187A mutated form of p27(Kip1). Consistent with these observations, pharmacological inhibition of MEK or PI3'-kinase inhibited the effects of activated RAS on the expression of p27(Kip1) in NIH 3T3 fibroblasts and in a panel of bona fide human pancreatic cancer cell lines. Furthermore, we demonstrated that AKT activation led to sustained activation of cyclin/cdk2 complexes that occurred concomitantly with the removal of RAF-induced p21(Cip1) from cyclin E/cdk2 complexes. Cumulatively, these data strongly suggest that the RAF-->MEK-->ERK and PI3'K-->PDK-->AKT signaling pathways can cooperate to promote G(0)-->G(1)-->S-phase cell cycle progression in both normal and cancer cells.     

IL-2 activation of a PI3K-dependent STAT3 serine phosphorylation pathway in primary human T cells

Interleukin-2 (IL-2) is the major growth factor of activated T lymphocytes. By inducing cell cycle progression and protection from apoptosis in these cells, IL-2 is involved in the successful execution of an immune response. Upon binding its receptor, IL-2 activates a variety of signal transduction pathways, including the Ras/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) and Janus kinase (JAK)/STAT cascades. In addition, activation of phosphatidylinositol 3-kinase (PI3K) and several of its downstream targets has also been shown. However, the coupling of STAT3 serine phosphorylation to PI3K in response to IL-2 has yet to be shown in either T cell lines or primary human T cells. This report shows that the PI3K inhibitors LY294002 and wortmannin block activation of MEK and ERK by IL-2 in primary human T cells. Moreover, these inhibitors significantly reduce IL-2-triggered STAT3 serine phosphorylation without affecting STAT5 serine phosphorylation. Analysis of the effects of these inhibitors on cell cycle progression and apoptosis strongly suggests that PI3K-mediated events, which includes STAT3 activation, are involved in IL-2-mediated cell proliferation but not cell survival. Finally, results presented illustrate that in primary human T cells, activation of Akt is insufficient for IL-2-induced anti-apoptosis. Thus, these results demonstrate that IL-2 stimulates PI3K-dependent events that correlate with cell cycle progression, but not anti-apoptosis, in activated primary human T cells.     

Regulation and phenotype of an innate Th1 cell: role of cytokines and the p38 kinase pathway

We have explored the phenotype and regulation of Th1 cell activation by the cytokines IL-12 and IL-18. We demonstrate that these two cytokines selectively induce IFN-gamma in a differentiated Th1 cell population through the previously described p38 mitogen-activated protein (MAP) kinase pathway. Using a highly selective p38 MAP kinase inhibitor, we demonstrate that it is possible to block IFN-gamma induction from activated, differentiated Th1 cells via p38 MAP kinase without disrupting the activation and differentiation of naive T cells or the proliferation of naive or differentiated T cells. In addition, IL-12 and IL-18 provide an Ag and IL-2-independent survival signal to this uniquely differentiated Th1 cell population. We hypothesize that this Ag-independent survival of Th1 cells may participate in an innate inflammatory loop with monocytes at the sites of chronic inflammation. In addition, p38 MAP kinase inhibition of this cytokine-regulated pathway may be a unique mechanism to inhibit chronic inflammation without disruption of Ag-driven activation and function of naive T cells.     

Antiapoptotic microenvironment of acute myeloid leukemia

We showed previously that tumor-derived supernatant (TSN) from acute myeloid leukemia (AML) myeloblasts inhibits peripheral blood T cell activation and proliferation, rendering the T cells functionally incompetent. We show here that the AML TSN also significantly delays apoptosis of both resting and stimulated T cells, as judged by reduction in annexin V/propidium iodide staining. In addition, we show that this is not unique to T cells and that AML TSN inhibits apoptosis of peripheral B cells, neutrophils, and monocytes. Furthermore, it also enhances the survival of other AML myeloblasts with lower viability. Investigations into the mechanism demonstrate a reduction in the cleavage of procaspase-3, -8, and -9 and the caspase substrate, poly(ADP-ribose)polymerase (PARP). This may be due to Bcl-2, which is normally down-regulated in CD3/CD28-stimulated T cells, but is maintained in the presence of AML TSN. We conclude that AML cells generate an antiapoptotic microenvironment that favors the survival of malignant cells, but also inhibits apoptosis of other normal hemopoietic cells. Reversal of these immunosuppressive effects and restoration of normal immune responses in patients with AML would improve the success of immunotherapy protocols.     

MiR-522-3p inhibits proliferation and activation by regulating the expression of SLC31A1 in T cells

We investigated the role of miR-522-3p in thymoma-associated myasthenia gravis (TAMG), and the mechanism of action in T cells. The miR-522-3p expression in normal serum, non-thymoma MG patient serum and TAMG patient serum and tissues was detected by quantitative real-time PCR (qRT-PCR), respectively. We assessed miR-522-3p expression in Jurkat cells and human CD4⁺ T cells after activation by anti-CD3 and anti-CD28 using qRT-PCR. The viability, proliferation, cycle distribution and the levels of CD25, CD69, interleukin-2 (IL-2) and IL-10 in transfected Jurkat cells were detected by Cell counting kit-8, 5-ethynyl-2′-deoxyuridine (EdU), flow cytometry, qRT-PCR, respectively. Targeting relationships of miR-522-3p and SLC31A1 were predicted and validated by bioinformatics analysis and dual-luciferase reporter. The viability, proliferation, cycle distribution and the levels of SLC31A1, CD25, CD69, IL-2 and IL-10 in transfected Jurkat cells were detected by above methods and western blot. The miR-522-3p expression was declined in TAMG and activated T cells. MiR-522-3p inhibitor promoted cell viability, EdU positive cells, cycle progression, and the level of CD25, CD69, IL-2 and IL-10 in Jurkat cells, while the effect of miR-522-3p mimic was the opposite. SLC31A1 was targeted by miR-522-3p, and miR-522-3p inhibited SLC31A1 expression. Overexpressed SLC31A1 reversed the inhibitory effects of miR-522-3p mimic on cell viability, EdU positive cell, cycle progression, and the levels of IL-2 and IL-10 in transfected Jurkat cells. MiR-522-3p expression was down-regulated in TAMG, and miR-522-3p inhibited proliferation and activation by regulating SLC31A1 expression in T cells.

     

Role of TNF receptor-associated factor 2 and p38 mitogen-activated protein kinase activation during 4-1BB-dependent immune response

4-1BB is a costimulatory member of the TNFR family, expressed on activated CD4(+) and CD8(+) T cells. Previous results showed that 4-1BB-mediated T cell costimulation is CD28-independent and involves recruitment of TNFR-associated factor 2 (TRAF2) and activation of the stress-activated protein kinase cascade. Here we describe a role for the p38 mitogen-activated protein kinase (MAPK) pathway in 4-1BB signaling. Aggregation of 4-1BB alone induces p38 activation in a T cell hybridoma, whereas, in normal T cells, p38 MAPK is activated synergistically by immobilized anti-CD3 plus immobilized 4-1BB ligand. 4-1BB-induced p38 MAPK activation is inhibited by the p38-specific inhibitor SB203580 in both a T cell hybridoma and in murine T cells. T cells from TRAF2 dominant-negative mice are impaired in 4-1BB-mediated p38 MAPK activation. A link between TRAF2 and the p38 cascade is provided by the MAPK kinase kinase, apoptosis-signal-regulating kinase 1. A T cell hybrid transfected with a kinase-dead apoptosis-signal-regulating kinase 1 fails to activate p38 MAPK in response to 4-1BB signaling. To assess the role of p38 activation in an immune response, T cells were stimulated in an MLR in the presence of SB203580. In a primary MLR, SB203580 blocked IL-2, IFN-gamma, and IL-4 secretion whether the costimulatory signal was delivered via 4-1BB or CD28. In contrast, following differentiation into Th1 or Th2 cells, p38 inhibition blocked IL-2 and IFN-gamma without affecting IL-4 secretion. Nevertheless, IL-4 secretion by Th2 cells remained costimulation-dependent. Thus, critical T cell signaling events diverge following Th1 vs Th2 differentiation.     

Mycophenolic acid inhibits IL-2-dependent T cell proliferation,but not IL-2-dependent survival and sensitization to apoptosis

Mycophenolic acid (MPA), the active metabolite of the immunosuppressive drug mycophenolate mofetil, is a selective inhibitor of inosine 5'-monophosphate dehydrogenase type II, a de novo purine nucleotide synthesis enzyme expressed in T and B lymphocytes and up-regulated upon cell activation. In this study, we report that the blockade of guanosine nucleotide synthesis by MPA inhibits mitogen-induced proliferation of PBL, an effect fully reversed by addition of guanosine and shared with mizoribine, another inhibitor of inosine 5'-monophosphate dehydrogenase. Because MPA does not inhibit early TCR-mediated activation events, such as CD25 expression and IL-2 synthesis, we investigated how it interferes with cytokine-dependent proliferation and survival. In activated lymphoblasts that are dependent on IL-2 or IL-15 for their proliferation, MPA does not impair signaling events such as of the extracellular signal-regulated kinase 2 and Stat5 phosphorylation, but inhibits down-regulation of the cyclin-dependent kinase inhibitor p27(Kip1). Therefore, in activated lymphoblasts, MPA specifically interferes with cytokine-dependent signals that control cell cycle and blocks activated T cells in the mid-G(1) phase of the cell cycle. Although it blocks IL-2-mediated proliferation, MPA does not inhibit cell survival and Bcl-x(L) up-regulation by IL-2 or other cytokines whose receptors share the common gamma-chain (CD132). Finally, MPA does not interfere with IL-2-dependent acquisition of susceptibility to CD95-mediated apoptosis and degradation of cellular FLIP. Therefore, MPA has unique functional properties not shared by other immunosuppressive drugs interfering with IL-2R signaling events such as rapamycin and CD25 mAbs.     

Expression of the murine homologue of the cell cycle control protein p34cdc2 in T lymphocytes.

The mammalian homologue of the cdc2 gene of the fission yeast Schizosaccharomyces pombe encodes a p34cdc2 cyclin-dependent kinase that regulates the cell cycle of a wide variety of cell types. Resting murine T lymphocytes contained no detectable p34cdc2 protein, histone kinase activity, or specific mRNA for the cdc2 gene. Activation of the T cells by immobilized anti-CD3 resulted in the expression of specific mRNA late in the G1 phase of the cell cycle, and p34cdc2 protein was detectable at or near G1/S. At this point in the cell cycle, the protein was phosphorylated at tyrosine and displayed no H1 histone kinase activity. As the cells progressed through the cycle, the amount of specific mRNA and p34cdc2 increased, and H1 histone kinase activity was detectable when the cells were blocked at G2/M by nocodazole. The activation of T cells by phorbol dibutyrate induced the expression of IL-2R but failed to induce the synthesis of IL-2 or the expression of cdc2-specific mRNA. Under these conditions, the activated cells failed to enter the S phase of the cell cycle. Because the presence of IL-2 added exogenously during activation by phorbol dibutyrate resulted in the expression of cdc2-specific mRNA and progression through the cell cycle, either IL-2 or the interaction with IL-2R may be involved in the expression of cdc2 and regulation of the G1/S transition.     

Porcine testicular extract inhibits T cell proliferation by blocking cell cycle transition from G1 phase to S phase

Since T cells express diverse sex steroid hormone receptors, they might be a good model to evaluate the effects of sex steroid hormones on immune modulation. Porcine testicular extract contains several sex steroid hormones and may be useful to study the effects of sex steroid hormones during T cell activation. We have examined the effects of the porcine testicular extract on T cell activation: proliferation and secretion of cytokines (IL-2 and IFN-γ) by activated T cells were severely decreased after treatment with porcine testicular extract. The extract produced an immunosuppressive effect and inhibited the proliferation of activated T cells by blocking the cell cycle transition from the G(1) phase to S phase. These effects were mediated by a decrease in the expression of cyclin D1 and cyclin E and constitutive expression of p27(KIP1) after T cell activation.     

Generation of cytotoxic T lymphocytes from peripheral blood of leukaemic patients

Peripheral blood mononuclear cells from 13 patients with acute leukaemia were used to establish long-term interleukin-2-dependent cytotoxic T lymphocytes. Cells were grown in RPMI medium containing interleukin-2 (IL-2, 100 U/ml) and 2.5% conditioned medium prepared by activating normal lymphocytes with phytohaemagglutinin. Proliferation of IL-2-dependent CD3-positive lymphocytes was seen in 1 of 2 acute lymphocytic leukaemia cases (ALL), 1 of 4 acute myelogeneous leukaemia cases (AML) (M1) and 8 of 8 more differentiated AML. In 2 cases with detectable leukaemic cell markers (1 ALL and 1 AML) passageable cells were developed, that expressed normal T cell phenotypes (namely CD3, CD4, and CD8) at the expense of leukaemic cells. In 1 of 2 cases, long-term IL-2-cultured cells showed specific cytotoxic activity against autologous leukemic cells. The percentage killing against autologous and two allogeneic target cell lines at a 50/1 effector/target (E/T) ratio was 42%, 9% and 19% respectively. Similarly the cytotoxic activity of IL-2 activated from 4 different individuals against conventional tumour targets K562 and Daudi at a ratio of 50/1 was 29%–68% (median=55%) and 34%–78% (median=61%) respectively. It was also found that this killing potential of the activated cells was maintained for as long as culture was continued (median 23 days, range 17–75 days). The mechanism(s) of T cell proliferation at the expense of leukaemic blast cells in the case of a minority of leukaemic patients and the possible clinical therapeutic potential of these cells following in vitro IL-2 activation deserve further investigation.     

NF-kappa B-inducing kinase is a common mediator of IL-17-, TNF-alpha-, and IL-1 beta-induced chemokine promoter activation in intestinal epithelial cells

IL-17 expression is restricted to activated T cells, whereas the IL-17R is expressed in a variety of cell types including intestinal epithelial cells. However, the functional responses of intestinal epithelial cells to stimulation with IL-17 are unknown. Moreover, the signal transduction pathways activated by the IL-17R have not been characterized. IL-17 induced NF-kappa B protein-DNA complexes consisting of p65/p50 heterodimers in the rat intestinal epithelial cell line IEC-6. The induction of NF-kappa B correlated with the induction of CXC and CC chemokine mRNA expression in IEC-6 cells. IL-17 acted in a synergistic fashion with IL-1 beta to induce the NF-kappa B site-dependent CINC promoter. Induction of the CINC promoter by IL-17 in IEC-6 cells was TNF receptor-associated factor-6 (TRAF6), but not TRAF2, dependent. Furthermore, IL-17 induction of the CINC promoter could be inhibited by kinase-negative mutants of NF-kappa B-inducing kinase and I kappa B kinase-alpha. In addition to activation of the NF-kappa B, IL-17 regulated the activities of extracellular regulated kinase, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinases in IEC-6 cells. Whereas the IL-17-mediated activation of extracellular regulated kinase mitogen-activated protein kinases was mediated through ras, c-Jun N-terminal kinase activation was dependent on functional TRAF6. These data suggest that NF-kappa B-inducing kinase serves as the common mediator in the NF-kappa B signaling cascades triggered by IL-17, TNF-alpha, and IL-1 beta in intestinal epithelial cells.     

Lck is involved in interleukin-2 induced proliferation but not cell survival in human T cells through a MAP kinase-independent pathway

The role of Lck in IL-2-induced proliferation and cell survival is still controversial. Here, we show that the Src family kinase inhibitor, PP1, reduced the IL-2-induced proliferation of human T cells significantly without inhibiting the anti-apoptotic effect of IL-2. As Lck is the only Src family kinase activated upon IL-2 stimulation in T cells, this indicates that Lck is involved in IL-2-induced proliferation but not survival. IL-2-induced MAP kinase activation was only slightly inhibited by PP1, suggesting that Lck is not essential for IL-2-induced MAP kinase activation in human T cells. We found that an IL-2-sensitive, human mycosis fungoides-derived tumor T cell line is Lck negative, and that the IL-2-induced MAP kinase activation is comparable to non-cancerous T cells, although a little delayed in kinetics. An Lck expressing clone was established by transfecting Lck into mycosis fungoides tumor T cells, but Lck had no influence on the delayed kinetics of MAP kinase activation, indicating that Lck is not essential for MAP kinase activation in mycosis fungoides tumor T cells or in non-cancerous T cells. Taken together, this indicates that Lck is involved in IL-2-induced proliferation, but not cell survival, through a pathway not involving MAP kinase.     

Diacylglycerol kinase inhibition prevents IL-2-induced G1 to S transition through a phosphatidylinositol-3 kinase-independent mechanism.

Stimulation via IL-2R ligation causes T lymphocytes to transit through the cell cycle. Previous experiments by our group have demonstrated that, in human T cells, IL-2 binding induces phosphatidic acid production through activation of the alpha isoform of diacylglycerol kinase. In this study, using the IL-2-dependent mouse T cell line CTLL-2, we demonstrate that pharmacological inhibition of IL-2-induced diacylglycerol kinase activation is found to block IL-2-induced late G1 to S transition without affecting cell viability. Herein, we demonstrate that diacylglycerol kinase inhibition has a profound effect on the induction of the protooncogenes c-myc, c-fos, and c-raf by IL-2, whereas expression of bcl-2 and bcl-xL are not affected. When the IL-2-regulated cell cycle control checkpoints are examined in detail, we demonstrate that inhibition of diacylglycerol kinase activation prevents IL-2 induction of cyclin D3 without affecting p27 down-regulation. The strict control of cell proliferation exerted by phosphatidic acid through activation of diacylglycerol kinase is independent of other well-characterized IL-2R-derived signals, such as the phosphatidylinositol-3 kinase/Akt pathway, indicating the existence of a different and important mechanism to control cell division.