p38 MAPK is a critical regulator of the constitutive and the beta4 integrin-regulated expression of IL-6 in human normal thymic epithelial cells

Cytokines and adhesion receptors are key mediators in the dialog occurring between thymic epithelial cells (TEC) and thymocytes and regulating T cell maturation and epithelial embryonic differentiation. Among cytokines, IL-6 can be critical in the thymus, fostering proliferation, differentiation and/or survival of both TEC and thymocytes. We have previously reported in human normal TEC that clustering of the laminin receptor alpha6beta4 integrin induced by thymocyte contact or monoclonal antibody-mediated cross-linking regulates IL-6 gene expression via activation of NF-kappaB and NF-IL6 transactivators. Here we show that alpha6beta4 integrin activates p38 mitogen-activated protein kinase (MAPK) and that p38 is essential for IL-6 gene expression. In fact, beta4 cross-linking activated p38 and extracellular signal-regulated kinase (ERK) MAPK, Rac1, p21-activated protein kinase 1 (PAK1) and MAPK kinases (MKK) 3/MKK6. However, pharmacological blockade of p38 or ERK demonstrated that p38 inhibition abrogated both basal and beta4 integrin-induced production of IL-6 preventing NF-kappaB and NF-IL6 activation, whereas ERK inhibition reduced IL-6 production, hampering only NF-kappaB activation. Overall, our results indicate that p38 MAPK and alpha6beta4 integrin, expressed by TEC throughout their life, are critical regulators of the intrathymic availability of a cytokine controlling fate and functions of cells governing development and maintenance of thymic architecture and immune responses.     

Activation of p38 mitogen-activated protein kinase and nuclear factor-kappaB in tumour necrosis factor-induced eotaxin release of human eosinophils

The CC chemokine eotaxin is a potent eosinophil-specific chemoattractant that is crucial for allergic inflammation. Allergen-induced tumour necrosis factor (TNF) has been shown to induce eotaxin synthesis in eosinophils. Nuclear factor-kappaB (NF-kappaB) and mitogen-activated protein kinases (MAPK) have been found to play an essential role for the eotaxin-mediated eosinophilia. We investigated the modulation of NF-kappaB and MAPK activation in TNF-induced eotaxin release of human eosinophils. Human blood eosinophils were purified from fresh buffy coat using magnetic cell sorting. NF-kappaB pathway-related genes were evaluated by cDNA expression array system. Degradation of IkappaBalpha and phosphorylation of MAPK were detected by Western blot. Activation of NF-kappaB was determined by electrophoretic mobility shift assay. Eotaxin released into the eosinophil culture medium was measured by ELISA. TNF was found to up-regulate the gene expression of NF-kappaB and IkappaBalpha in eosinophils. TNF-induced IkappaBalpha degradation was inhibited by the proteasome inhibitor N-cbz-Leu-Leu-leucinal (MG-132) and a non-steroidal anti-inflammatory drug sodium salicylate (NaSal). Using EMSA, both MG-132 and NaSal were found to suppress the TNF-induced NF-kappaB activation in eosinophils. Furthermore, TNF was shown to induce phosphorylation of p38 MAPK time-dependently but not extracellular signal-regulated kinases (ERK). Inhibition of NF-kappaB activation and p38 MAPK activity decreased the TNF-induced release of eotaxin from eosinophils. These results indicate that NF-kappaB and p38 MAPK play an important role in TNF-activated signalling pathway regulating eotaxin release by eosinophils. They have also provided a biochemical basis for the potential of using specific inhibitors of NF-kappaB and p38 MAPK for treating allergic inflammation.     

Mycophenolic acid differentially affects dendritic cell maturation induced by tumor necrosis factor-α and lipopolysaccharide through a different modulation of MAPK signaling

Mycophenolic acid (MPA) is an immunosuppressive drug which induces resistance to several maturation signals in human dendritic cells (DC) by unknown mechanisms. As mitogen-activated protein kinases (MAPK) are involved in the maturation process, we studied whether MPA affected p38MAPK and extracellular signal-regulated kinase (ERK1/2) in human DC. We first showed that MPA reduced TNFα-induced phenotype maturation, whereas it had no effect after LPS activation, suggesting that MPA preferentially affects the signaling pathway used by TNFα. We found that TNFα preferentially used p38MAPK to induce phenotype maturation in DC, whereas LPS preferentially activated NF-κB. Importantly, we showed that MPA more strongly inhibited p38MAPK phosphorylation induced by TNFα than by LPS. This difference in inhibition may therefore explain its different effect on DC phenotype. Interestingly, MPA inhibited the inflammatory cytokine synthesis and allostimulatory capacity induced by both stimuli. Exogenous guanosine antagonized the effect of MPA on the phenotype of TNFα-matured-DC as well as the IL-12p70 and IFNγ secretion induced by both stimuli, without affecting p38MAPK phosphorylation. The action of MPA on human DC phenotype maturation appears mainly to be due to its ability to inhibit p38MAPK. Furthermore, the difference between LPS and TNFα emphasizes that the DC microenvironment strongly influences DC sensitivity to MPA.     

RAC1/P38 MAPK signaling pathway controls beta1 integrin-induced interleukin-8 production in human natural killer cells

The MAP kinase (MAPK) p38 plays a key role in regulating inflammatory responses. Here, we demonstrate that beta1 integrin ligation on human NK cells results in the activation of the p38 MAPK signaling pathway, which is required for integrin-triggered IL-8 production. In addition, we identified some of the upstream events accompanying the beta1 integrin-mediated p38 MAPK activation, namely, the activation of the Rac guanine nucleotide exchange factor (GEF) p95 Vav, the small G protein Rac1, and the cytoplasmic kinases Pak1 and MKK3. Finally, we provide direct evidence that p95 Vav and Rac control the activation of p38 MAPK triggered by beta1 integrins.     

Intracellular JNK, p38 MAPK and NF-kappaB regulate IL-25 induced release of cytokines and chemokines from costimulated T helper lymphocytes

Novel Th2 cytokine IL-25 has been shown to be elevated in allergic inflammation. We investigated the intracellular mechanisms regulating IL-25-induced Th2 cytokines and chemokines from human Th lymphocytes upon costimulation by anti-CD3 and anti-CD28 antibodies. Cytokines, chemokines, and phosphorylated p38 mitogen activated protein kinases (MAPK), c-Jun amino-terminal kinase (JNK) and extracellular signal-regulated protein kinase were analyzed by bead-based array using flow cytometry. Nuclear factor (NF)-kappaB and total MAPK were assessed by electrophoretic mobility shift assay and Western blot, respectively. IL-25 could synergistically induce the release of Th2 cytokines IL-4, IL-5 and IL-10, inflammatory cytokine IL-6, Th1 related chemokines CXCL9 and CXCL10, and chemokine CCL5 from anti-CD3 and anti-CD28 antibodies costimulated Th cells, especially memory Th cells. Costimulation could also upregulate the cell surface expression of IL-25 receptor on Th cells. Costimulation with or without IL-25 treatment could activate JNK, p38 MAPK and NF-kappaB. The upregulation of costimulation-induced IL-25 receptors and release of cytokines and chemokines from IL-25 treated costimulated Th cells were differentially regulated by intracellular JNK, p38 MAPK and NF-kappaB activity. Therefore, the optimal activation of Th cells by IL-25 for the release of Th2 cytokines and chemokines requires the CD3 and CD28 mediated costimulation of Th cells via the upregulation of IL-25 receptors and the activation of intracellular signaling pathways. This mechanistic study shows that IL-25 and CD28 costimulation can play pathophysiological roles by inducing inflammation and hyperresponsiveness through the production of both Th2 cytokines and chemokines from memory Th cells.     

Selective regulation of tumor necrosis factor-induced Erk signaling by Src family kinases and the T cell protein tyrosine phosphatase

The proinflammatory cytokine tumor necrosis factor (TNF) modulates cellular responses through the mitogen-activated protein kinase (MAPK) and nuclear factor-kappaB (NF-kappaB) signaling pathways, but the molecular mechanisms underlying MAPK activation are unknown. T cell protein tyrosine phosphatase (TCPTP) is essential for hematopoietic development and negatively regulates inflammatory responses. Using TCPTP-deficient fibroblasts, we show here that TCPTP regulates TNF-induced MAPK but not NF-kappaB signaling. TCPTP interacted with the adaptor protein TRAF2, and dephosphorylated and inactivated Src tyrosine kinases to suppress downstream signaling through extracellular signal-regulated kinases and production of interleukin 6. These results link TCPTP and Src tyrosine kinases to the selective regulation of TNF-induced MAPK signaling and identify a previously unknown mechanism for modulating inflammatory responses mediated by TNF.     

Role of the Phosphatidylinositol 3-Kinase and Mitogen-Activated Protein Kinase Pathways in the Secretion of Tumor Necrosis Factor-α and Interleukin-10 by the PPD Antigen of Mycobacterium tuberculosis

Here we investigated the role of the phosphatidylinositol 3-kinase (PI 3-K) and mitogen-activated protein kinase (MAPK) pathways in the secretion of tumor necrosis factor (TNF)-α and interleukin (IL)-10 in human primary monocytes after stimulation with the PPD antigen of Mycobacterium tuberculosis. MAPK [extracellular signal-regulated kinase (ERK) 1/2 and p38] and Akt are rapidly phosphorylated in human monocytes stimulated with PPD. We found that the PI 3-K-Akt pathway stimulated by PPD is essential for both IL-10 and TNF-α production, although the inhibition of IL-10 production was more pronounced. The analysis of cytokine production using specific inhibitors of the MAPK pathway revealed that both p38 and ERK activation are essential for PPD-induced TNF-α production, whereas p38, but not ERK, activation is essential for IL-10 secretion. The inhibition of PI 3-K did not significantly activate p38 MAPK or ERK 1/2 in PPD-stimulated human monocytes. Further, the Src inhibitor PP2 inhibited the release of TNF-α but enhanced IL-10 release, suggesting the differential regulation of Src kinase in upstream signaling. Collectively, these data suggest that the PI 3-K and MAPK pathways play a central role in the regulation of both pro- and anti-inflammatory cytokines by the PPD antigen of M. tuberculosis.     

Distinct regulation of CD40-mediated interleukin-6 and interleukin-12 productions via mitogen-activated protein kinase and nuclear factor kappaB-inducing kinase in mature dendritic cells

The role of mitogen-activated protein kinase (MAPK) and nuclear factor kappaB (NF-kappaB) pathways, especially NF-kappaB-inducing kinase (NIK)-mediated alternative pathway, in CD40-mediated interleukin (IL)-6 and IL-12 productions by immature or mature dendritic cells (DCs) was investigated. Murine myeloid DCs were matured by treatment with lipopolysaccharide. CD40 ligation induced modest or vigorous cytokine productions in immature or mature DCs, respectively. After CD40 ligation, p38 MAPK was significantly activated in either immature or mature DCs. SB203580, a p38 MAPK inhibitor, markedly decreased CD40-mediated IL-6 and IL-12 productions in immature DCs. In mature DCs, SB203580 significantly decreased CD40-mediated IL-6 but not IL-12 production. On the other hand, CD40 ligation induced vigorous activation of the NF-kappaB alternative pathway including p100 phosphorylation and subsequent nuclear translocations of p52, a processed form of p100, and RelB in mature but not immature DCs. The CD40-mediated phosphorylation of p100 was completely abolished in NIK-mutated mature DCs. The NIK mutation markedly reduced CD40-mediated IL-12 but not IL-6 production by mature DCs. Taken together, we concluded that IL-6 and IL-12 productions in response to CD40 ligation were controlled by p38 MAPK and NIK mediated alternative pathway, respectively, in mature DCs.     

17Beta-estradiol downregulates Kupffer cell TLR4-dependent p38 MAPK pathway and normalizes inflammatory cytokine production following trauma-hemorrhage

Although studies have shown that 17beta-estradiol (estradiol) normalized Kupffer cell function following trauma-hemorrhage, the mechanism by which E2 maintains immune function remains unclear. Activation of Toll-like receptor 4 (TLR4) initiates an inflammatory cascade, involving activation of p38 mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K), and nuclear factor-kappaB (NF-kappaB). This leads to the release of proinflammatory cytokines. Thus, we hypothesized that the salutary effects of estradiol on Kupffer cell function following trauma-hemorrhage are mediated via negative regulation of TLR4-dependent p38 MAPK and NF-kappaB. TLR4 mutant (C3H/HeJ) and wild type (C3H/HeOuJ) mice were subjected to trauma-hemorrhage (mean BP 35+/-5 mmHg approximately 90 min, then resuscitation) or sham operation. Administration of estradiol following trauma-hemorrhage in wild type mice decreased Kupffer cell TLR4 expression as well as prevented the phosphorylation of p38 MAPK and NF-kappaB. This was accompanied by normalization of Kupffer cell production capacities of IL-6, TNF-alpha, macrophage inflammatory protein (MIP)-1alpha, and MIP-2 and the decrease in plasma cytokine levels. In contrast, TLR4 mutant mice did not exhibit the increase in Kupffer cell p38 MAPK and NF-kappaB activation, cytokine production, or the increase in circulating cytokine levels following trauma-hemorrhage. No difference was observed in activation of PI3K among groups. These results suggest that the protective effect of estradiol on Kupffer cell function is mediated via downregulation of TLR4-dependent p38 MAPK and NF-kappaB signaling following trauma-hemorrhage, which prevents the systemic release of cytokines.     

Sesamin inhibits lipopolysaccharide-induced cytokine production by suppression of p38 mitogen-activated protein kinase and nuclear factor-kappaB

Sesame seed oil increases the survival after cecal ligation and puncture in mice and the increased IL-10 levels with non-lethal lipopolysaccharides (LPS) challenge. We showed that sesamin and sesamolin, major lignans of sesame oil, regulated LPS-induced nitric oxide production in the murine microglia and BV-2 cell line. In this study, we studied the effect of sesamin on cytokine production by LPS stimulation. The result showed that sesamin significantly inhibited LPS-stimulated IL-6 mRNA and protein, and to a lesser degree TNF-alpha, in BV-2 microglia. Sesamin and sesamolin also reduced LPS-activated p38 mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-kappaB activations. Furthermore, SB203580, a specific inhibitor of p38 MAP kinase, specifically inhibited LPS-induced IL-6 production. These results suggest that sesamin inhibited LPS-induced IL-6 production by suppression of p38 MAPK signal pathway and NF-kappaB activation.     

RAGE is the major receptor for the proinflammatory activity of HMGB1 in rodent macrophages

Abstract High-mobility group box chromosomal protein 1 (HMGB1) is a protein with both intranuclear functions and extracellular cytokine-like effects. In this report, we study possible candidate receptors for HMGB1 on macrophages (Mphi) and define pathways activated by HMGB1 binding. Bone marrow Mphi were prepared from Dark Agouti (DA) rats and stimulated in vitro with HMGB1. The kinetics of tumour necrosis factor (TNF) production, NO production, activation of p38 mitogen-activated protein kinase (MAPK), p44/42 MAPK- and SAPK/JNK-signalling pathways, nuclear translocation of nuclear factor kappa B (NF-kappaB) and HMGB1-induced upregulation of major histocompatibility complex (MHC) class II and CD86 were analysed. Mphi from interleukin (IL)-1 receptor type I-/-, Toll-like receptor 2 (TLR2-/-) and RAGE-/- mice were used to investigate the role of these receptors in HMGB1 signalling. HMGB1 induced TNF and NO production by Mphi, phosphorylation of all investigated MAP kinase pathways and NF-kappaB translocation, and expression of MHC class II was increased. Mphi from RAGE-/- mice produced significantly lower amounts of TNF, IL-1beta and IL-6, while IL-1RI-/- and TLR2-/- Mphi produced cytokine levels comparable with wildtype controls in response to HMGB1 stimulation. We conclude that HMGB1 has the potential to induce a proinflammatory phenotype in Mphi, with RAGE as the major activation-inducing receptor.