Differential activation of T cell cytokine production by the extracellular signal-regulated kinase (ERK) signaling pathway

Stimulation of T cells via the T cell receptor (TCR) activates a number of signaling pathways that are potentially involved in the elicitation of physiological responses, such as the production of cytokines. The extracellular signal-regulated kinases (ERK) are a group of molecules activated in response to TCR ligation, whose role in T cell cytokine production is controversial. In this study, we have asked whether ERK activation is coupled to the production of a number of T cell-derived cytokines, and whether particular cytokines are differentially affected by ERK activation. To address these questions, we have utilized a constitutively active version of the immediate upstream activator of both ERK1 and ERK2, mitogen-activated/extracellular signal-regulated kinase 1 (MEK1), to activate ERK signaling selectively in the absence of other TCR-activated signaling pathways. The effect of constitutive MEK/ERK activation on T cell cytokine production was measured by transiently co-transfecting newly activated mouse T cells with DNA encoding constitutively active MEK1 (CA-MEK1) and the human interleukin-2 (IL-2) receptor α chain (hCD25), purifying hCD25⁺ transfectants by flow-cytometric cell sorting, and measuring the production of IL-3, IL-4, interferon (IFN)-γ and granulocyte/macrophage-colony-stimulating factor (GM-CSF) either in the presence or absence of ionomycin stimulation. Newly activated T cells were used in these experiments as they more closely resemble T cells activated in vivo than do transformed T cells or long-term established T cell clones. CA-MEK1 expression led to constitutive ERK activation, which acted synergystically with ionomycin treatment to stimulate cytokine production. Furthermore, these experiments revealed a hierarchy of cytokine responsiveness to MEK/ERK activation, such that the production of IL-3 was most affected, followed by GM-CSF, IFN-γ, and IL-4.     

SAP regulates T(H)2 differentiation and PKC-theta-mediated activation of NF-kappaB1

XLP is caused by mutations affecting SAP, an adaptor that recruits Fyn to SLAM family receptors. SAP-deficient mice recapitulate features of XLP, including increased T cell activation and decreased humoral responses post-infection. SAP-deficient T cells also show increased TCR-induced IFN-gamma and decreased T(H)2 cytokine production. We demonstrate that the defect in IL-4 secretion in SAP-deficient T cells is independent of increased IFN-gamma production. SAP-deficient cells respond normally to polarizing cytokines, yet show impaired TCR-mediated induction of GATA-3 and IL-4. Examination of TCR signaling revealed normal Ca(2+) mobilization and ERK activation in SAP-deficient cells, but decreased PKC-theta recruitment, Bcl-10 phosphorylation, IkappaB-alpha degradation, and nuclear NF-kappaB1/p50 levels. Similar defects were observed in Fyn-deficient cells. SLAM engagement amplified PKC-theta recruitment in wt but not SAP- or Fyn-deficient cells, arguing that a SAP/Fyn-mediated pathway enhances PKC-theta/NF-kappaB1 activation and suggesting a role for this pathway in T(H)2 regulation.     

Induction of granulocyte-macrophage colony-stimulating factor by a new cytokine, ML-1 (IL-17F), via Raf I-MEK-ERK pathway

BACKGROUND: ML-1 (IL-17F) is a recently discovered cytokine, and its function remains elusive. GM-CSF is a crucial cytokine for the maturation of various cell types and regulates allergic airway inflammation. OBJECTIVE: The functional effect of ML-1 in the expression of GM-CSF was investigated. METHODS: The levels of gene and protein expression in normal human bronchial epithelial cells (NHBEs) in the presence or absence of various kinase inhibitors or, in some cases, of a Raf1 dominant-negative mutant were determined by RT-PCR and ELISA, respectively. Western blotting was performed to investigate kinase activation. RESULTS: The results showed first that ML-1 induces, in a time-dependent and dose-dependent manner, the gene and protein expression for GM-CSF NHBEs, which are associated with activation of Raf1 and MAP kinase kinase (MEK) kinases. Selective MEK inhibitors, PD98059 and U0126, and Raf1 kinase inhibitor I significantly inhibited ML-1-induced GM-CSF production. Furthermore, overexpression of Raf1 dominant-negative mutants inhibited IL-17F-induced GMCSF expression. The combination of PD98059 and Raf1 kinase inhibitor I completely blocked GM-CSF production, whereas 2 protein kinase C inhibitors, Ro-31-7549 and GF109203X, and a phosphatidylinositol 3-kinase inhibitor, LY294002, showed no inhibitory effect. CONCLUSION: These findings suggest that ML-1 induces GM-CSF expression through the activation of the Raf1-MEK-extracellular signal-regulated kinase 1/2 pathway.     

Opposing effects of sodium salicylate and haematopoietic cytokines IL-3, IL-5 and GM-CSF on mitogen-activated protein kinases and apoptosis of EoL-1 cells

Haematopoietic cytokines such as IL-3, IL-5 and GM-CSF not only activate eosinophils but also prolong their life span by inhibiting their apoptotic cell death. We have studied the effects of IL-3, IL-5 and GM-CSF on apoptosis and mitogen-activated protein kinases (MAPKs) in a human eosinophilic leukaemic cell line (EoL-1). Results demonstrated that all three cytokines could trigger the receptor-mediated activation of extracellular signal-regulated kinase (ERK) within one hour but not p38 MAPK activity in EoL-1 cells. In contrast, sodium salicylate (NaSal), a nonsteroidal anti-inflammatory drug (NSAID), could activate p38 MAPK but not ERK within one hour. Both cytokines and specific p38 MAPK inhibitor SB 203580 could partly block the NaSal-induced apoptosis in EoL-1 cells. A specific MAPK/ERK kinase (MEK) inhibitor, PD 098059, could induce apoptosis and eliminate the protective effect of IL-3, IL-5 and GM-CSF against NaSal-induced apoptosis in EoL-1 cells. Taken together, cytokines IL-3, IL-5 and GM-CSF could prolong EoL-1 cells survival through the transient activation of ERK. On the other hand, activation of p38 MAPK in EoL-1 cells by NaSal could lead to apoptosis. Activation of p38 MAPK and the resulting induction of apoptosis in EoL-1 cells may be important to explain the anti-inflammatory action of NSAID in allergic inflammation.     

Opposing Effects of Sodium Salicylate and Haematopoietic Cytokines IL-3, IL-5 and GM-CSF on Mitogen-Activated Protein Kinases and Apoptosis of EOL-1 Cells

Haematopoietic cytokines such as IL-3, IL-5 and GM-CSF not only activate eosinophils but also prolong their life span by inhibiting their apoptotic cell death. We have studied the effects of IL-3, IL-5 and GM-CSF on apoptosis and mitogen-activated protein kinases (MAPKs) in a human eosinophilic leukaemic cell line (EoL-1). Results demonstrated that all three cytokines could trigger the receptor-mediated activation of extracellular signal-regulated kinase (ERK) within one hour but not p38 MAPK activity in EoL-1 cells. In contrast, sodium salicylate (NaSal), a nonsteroidal anti-inflammatory drug (NSAID), could activate p38 MAPK but not ERK within one hour. Both cytokines and specific p38 MAPK inhibitor SB 203580 could partly block the NaSal-induced apoptosis in EoL-1 cells. A specific MAPK/ERK kinase (MEK) inhibitor, PD 098059, could induce apoptosis and eliminate the protective effect of IL-3, IL-5 and GM-CSF against NaSal-induced apoptosis in EoL-1 cells. Taken together, cytokines IL-3, IL-5 and GM-CSF could prolong EoL-1 cells survival through the transient activation of ERK. On the other hand, activation of p38 MAPK in EoL-1 cells by NaSal could lead to apoptosis. Activation of p38 MAPK and the resulting induction of apoptosis in EoL-1 cells may be important to explain the anti-inflammatory action of NSAID in allergic inflammation.     

Opposing Effects of Sodium Salicylate and Haematopoietic Cytokines IL-3, IL-5 and GM-CSF on Mitogen-Activated Protein Kinases and Apoptosis of EOL-1 Cells

Haematopoietic cytokines such as IL-3, IL-5 and GM-CSF not only activate eosinophils but also prolong their life span by inhibiting their apoptotic cell death. We have studied the effects of IL-3, IL-5 and GM-CSF on apoptosis and mitogen-activated protein kinases (MAPKs) in a human eosinophilic leukaemic cell line (EoL-1). Results demonstrated that all three cytokines could trigger the receptor-mediated activation of extracellular signal-regulated kinase (ERK) within one hour but not p38 MAPK activity in EoL-1 cells. In contrast, sodium salicylate (NaSal), a nonsteroidal anti-inflammatory drug (NSAID), could activate p38 MAPK but not ERK within one hour. Both cytokines and specific p38 MAPK inhibitor SB 203580 could partly block the NaSal-induced apoptosis in EoL-1 cells. A specific MAPK/ERK kinase (MEK) inhibitor, PD 098059, could induce apoptosis and eliminate the protective effect of IL-3, IL-5 and GM-CSF against NaSal-induced apoptosis in EoL-1 cells. Taken together, cytokines IL-3, IL-5 and GM-CSF could prolong EoL-1 cells survival through the transient activation of ERK. On the other hand, activation of p38 MAPK in EoL-1 cells by NaSal could lead to apoptosis. Activation of p38 MAPK and the resulting induction of apoptosis in EoL-1 cells may be important to explain the anti-inflammatory action of NSAID in allergic inflammation.     

Activation of T cells by superantigen: cytokine production but not apoptosis depends on MEK-1 activity

Engagement of the TCR may result in proliferation and cytokine release or programmed cell death. These two outcomes may be the consequence of distinct T cell receptor-coupled signal transduction pathways or may reflect quantitative differences in signaling strength via a single pathway. Here we show that genetic inhibition of MAP kinase kinase (MEK) by a dominant negative mutant or through chemical inhibition by PD98059 inhibits IL-2 secretion but not programmed cell death after TCR ligation by superantigen. This supports the hypothesis that T cell cytokine release and apoptosis result from signaling through distinct pathways and implies that the molecular signaling mechanisms regulating apoptosis of mature T cells and negative selection of thymocytes may be similar.     

Association of decreased phosphorylation of ERK-2 with costimulation of rat T cell activation by MEK-1 inhibitors and TGF-beta1

Transforming growth factor-beta (TGF-beta) is usually known as an immunosuppressive cytokine, but we and others have shown stimulatory effects of TGF-beta on activation of Th2 T-lymphocytes. In the present investigation we have studied the effect of TGF-beta1 on phosphorylation of ERK, a MAP-kinase downstream of the Ras pathway. ERK is phosphorylated by MEK-1 and PD098059 and U0126 are specific inhibitors for this kinase. We demonstrate in the present study that these inhibitors abrogate the inhibitory effect of adh-splc (adherent-spleen cells) on activation of primary rat T-cells and induce a strong costimulatory effect almost as strong as we have previously shown with TGF-beta1. When TGF-beta1 is acting stimulatory on T-cell activation, it decreases phosphorylation of ERK-2 and thereby its activation. To investigate whether TGF-beta1 and MEK-1 inhibitors influence the same pathways, we compared their effects on cytokine profiles associated with SEA-induced rat T cell activation. TGF-beta1 induced IL-10 production, slightly decreased TNF-alpha production and decreased IFN-gamma production. The PD098059 inhibitor decreased both IFN-gamma and TNF-alpha production and together with TGF-beta1, it totally blocked IFN-gamma, TNF-alpha and IL-10 production. Thus TGF-beta1 and PD098059 showed overlapping but not identical effects on the cytokine pattern.     

Role of defective ERK phosphorylation in the impaired GM-CSF-induced oxidative response of neutrophils in elderly humans

GM-CSF-induced oxidative responses are defective in neutrophils of elderly humans. In the present study we evaluated whether this phenomenon might be related to alterations in cytokine-dependent MAPK signalling. Neutrophils obtained from elderly humans and stimulated with GM-CSF showed a significant reduction in phosphorylated ERK1/2 levels and an even higher decrease in ERK1/2 activation with respect to baseline. No changes in GM-CSF-induced p38 MAPK phosphorylation were observed. Cell pretreatment with the MEK inhibitor PD98059 determined a marked suppression of GM-CSF-induced O2- release. Interestingly, under the above experimental condition, there was no longer any difference in O2- production observed between elderly and young subjects. Furthermore, despite the fact that the p38 MAPK pathway was activated less strongly by GM-CSF, the p38 MAPK inhibitor SB203580 reduced GM-CSF-induced O2- production in the neutrophils of the elderly to levels similar to those obtained with PD98059. TNF-alpha-triggered O2- production was not altered by ageing and in fact, a similar ERK1/2 or p38 MAPK activation was found in TNF-alpha-stimulated neutrophils from elderly and young individuals. In accordance with the different potency of TNF-alpha in activating ERK1/2 and p38 MAPK, the TNF-alpha-induced oxidative responses were more sensitive to the inhibitory effects of SB203580 than to those of PD98059 in young as well as elderly subjects. These results suggest that, along the GM-CSF-dependent ERK signalling pathway, a step proximal to MEK1/2 but distal to the connection with the p38 MAPK module likely becomes defective as a feature of age. The consequent decline in ERK1/2 activation could potentially account for the GM-CSF-dependent impairment of the neutrophil respiratory burst that occurs with ageing.     

Weak TCR stimulation induces a calcium signal that triggers IL-4 synthesis, stronger TCR stimulation induces MAP kinases that control IFN-gamma production

Th1 and Th2 cells produce different cytokines and have distinct functions. Th1/Th2 cell differentiation is influenced, among other factors, by the nature of TCR-MHC interactions. However, how the TCR transduces a signal resulting in IFN-gamma or IL-4 production is a matter of debate. For example, some authors reported a loss of calcium signaling pathway in Th2 cells. We used a T cell hybridoma producing IL-4 upon weak TCR stimulation and both IL-4 and IFN-gamma for strong TCR engagement as a model to study how TCR signaling pathways are differentially activated in both conditions of stimulation and how this influences the production of cytokines. We show that: (1) the calcium response is identical following weak and strong TCR stimulation; (2) mitogen-activated protein kinase(MAPK) activation is a gradual phenomenon depending upon the strength of TCR activation; (3) a calcium response, even weak, triggers IL-4 expression; (4) IFN-gamma synthesis requires not only a calcium response but also MAPK activation. The MAPK pathway is dispensable for IL-4 production, although it amplifies IL-4 synthesis upon strong TCR stimulation; (5) TCR-induced IL-4 production also depends on calcium signaling in Th2 cells, while IFN-gamma synthesis is dependent, in addition, on MAPK activation in Th1 cells.     

Conversion of p56(lck) to p60(lck) in human peripheral blood T lymphocytes is dependent on co- stimulation through accessory receptors: involvement of phospholipase C, protein kinase C and MAP-kinases in vivo

Activation of T cells requires co-stimulation of the TCR and accessory receptors like CD2, CD4, CD8, CD11a or CD28. Engagement of the TCR without co-stimulation results in anergy / apoptosis. Here we show that induction of the shift of the tyrosine kinase p56lck from 56 kDa to apparent 60 kDa in resting human peripheral blood T cells (PBT) is strictly dependent on co-stimulation through both TCR and accessory receptors. In contrast, triggering of the TCR alone is only sufficient to induce the lck shift in preactivated cells like T cell clones or the T lymphoma line Jurkat. Our studies predict an involvement of a phospholipase C isoform which surprisingly acts downstream of a phorbolester-sensitive, H7-insensitive protein kinase C. Inhibition of the lck shift in vivo by U73122, a specific inhibitor of phospholipase C, correlates with reduced activation of the MAP-kinases ERK1 / 2. Moreover, the MEK1-specific inhibitor PD98059 blocks the lck shift in vivo. These findings demonstrate that activation of the MEK1-ERK1 / 2 pathway is required for lck conversion in vivo. The lck shift is not inducible by co-stimulation through acidic sphingomyelinase or ceramides which even prevent ERK2 activation in PBT. Moreover, it is resistant to treatment with W7, KN62 and cyclosporin A.     

Control of Borrelia burgdorferi-specific CD4+-T-cell effector function by interleukin-12- and T-cell receptor-induced p38 mitogen-activated protein kinase activity

Infection with Borrelia burgdorferi, the causative agent of Lyme disease, results in a Th1 response and proinflammatory cytokine production. Mice deficient for MKK3, an upstream activator of p38 mitogen-activated protein (MAP) kinase, develop a lower Th1 response and exhibit an impaired ability to produce proinflammatory cytokines upon infection with the spirochete. We investigated the contribution of p38 MAP kinase activity in gamma interferon (IFN-gamma) production in CD4+ T cells in response to specific antigen through T-cell receptor (TCR)- and interleukin-12 (IL-12)-mediated signals. The specific inhibition of p38 MAP kinase in T cells and the administration of a pharmacological inhibitor of the kinase during the course of infection with the spirochete resulted in reduced levels of IFN-gamma in the sera of infected mice. Our results also demonstrate that although p38 MAP kinase activity is not required for the differentiation of B. burgdorferi-specific CD4+ T cells, the production of IFN-gamma by Th1 effector cells is regulated by the kinase. Both TCR engagement and IL-12 induced the production of the Th1 cytokine through the activation of the p38 MAP kinase pathway. Thus, the inhibition of this pathway in vitro resulted in decreased levels of IFN-gamma during restimulation of B. burgdorferi-specific T cells in response to anti-CD3 and IL-12 stimulation. These results clarify the specific contribution of the p38 MAP kinase in the overall immune response to the spirochete and its role in the effector function of B. burgdorferi-specific T cells.     

Degree of ERK activation influences both positive and negative thymocyte selection

Considerable evidence suggests that the ERK pathway is required for positive but not negative thymocyte selection. Here, we report that ERK is highly activated in double-positive (DP) thymocytes expressing an MHC class I-restricted TCR (P14) in response to negatively selecting conditions, whereas ligands that trigger positive selection induced weaker ERK activation. Biochemical evidence also shows that death by neglect is associated with a further reduction in ERK activation. These findings are consistent with the affinity / avidity model of thymocyte selection. To further examine the role of ERK in negative selection we used the MEK-1 inhitibor, PD98059, a specific pharmacological inhibitor of the ERK pathway. Biochemical data demonstrated a reduction of ERK activity by PD98059 in the presence of the negatively selecting ligand. Analysis of P14 TCR-transgenic fetal thymic lobes cultured with PD98059 under negatively selecting conditions showed impaired clonal deletion of DP thymocytes and a concomitant increase in positive selection of functional mature, TCR(hi) transgenic T cells. This demonstrates that altering ERK activity switched negative to positive selection. Contrary to previous reports that show an exclusive role for ERK signaling in positive selection, our data demonstrate that negative selection is also sensitive to the degree of ERK activation.     

IL-2 triggers specific signaling pathways in human NKT cells leading to the production of pro- and anti-inflammatory cytokines

NKT cells belong to a conserved T lymphocyte subgroup that has been implicated in the regulation of various immune responses, including responses to viruses, bacteria, and parasites. They express a semi-invariant TCR that recognizes glycolipids presented by the nonpolymorphic MHC class I-like molecule CD1d, and upon activation, they produce various pro- and anti-inflammatory cytokines. Recent studies have shed light on the nature of glycolipids and the environmental signals that may influence the production of cytokines by NKT cells and thus, modulate the immune response. To better understand the regulation mechanisms of NKT cells, we explored their behavior following activation by IL-2 and investigated the signaling pathways and biological responses triggered. We demonstrated that IL-2 activates not only STAT3 and -5 and the PI-3K and ERK-2 pathways as in all IL-2 responder cells but also STAT4 as in NK cells and the p38 MAPK pathway as in alphabeta T cells. We also showed that STAT6 is activated by IL-2 in NKT cells. Moreover, IL-2 induces the production of IFN-gamma and IL-4. The ability of IL-2 to induce pro- and anti-inflammatory cytokine production, in addition to proliferation, could open new therapeutic approaches for use in combination with molecules that activate NKT cells through TCR activation.     

c-Rel-dependent priming of naive T cells by inflammatory cytokines

The intrinsic refractoriness of naive T cells for cytokine production is counteracted by cells of the innate immune system. Upon sensing danger via Toll-like receptors, these cells upregulate T cell costimulatory molecules and secrete cytokines that enhance T cell activation. We show that cytokine-mediated priming of naive T cells requires the NF-kappaB family member c-Rel. In resting naive cells c-Rel is associated primarily with IkappaBbeta, an inhibitory molecule that is not effectively degraded by TCR signals. Exposure of T cells to proinflammatory cytokines, TNF-alpha and IL-1beta, shifts c-Rel to IkappaBalpha-associated complexes that are readily targeted by the TCR. As a consequence, IL-2 and IFN-gamma mRNA are produced more quickly, and at higher levels, in cytokine-primed T cells. This mechanism does not operate in effector T cells where cytokine gene expression is c-Rel-independent. We propose that c-Rel plays a crucial role as a target of innate signals in T cells.