Luteolin, a flavonoid, inhibits AP-1 activation by basophils

Flavonoids including luteolin, apigenin, and fisetin are inhibitors of IL-4 synthesis and CD40 ligand expression by basophils. This study was done to search for compounds with greater inhibitory activity of IL-4 expression and to clarify the molecular mechanisms through which flavonoids inhibit their expression. Of the 37 flavonoids and related compounds examined, ayanin, luteolin, and apigenin were the strongest inhibitors of IL-4 production by purified basophils in response to anti-IgE antibody plus IL-3. Luteolin did not suppress Syk or Lyn phosphorylation in basophils, nor did suppress p54/46 SAPK/JNK, p38 MAPK, and p44/42 MAPK activation by a basophilic cell line, KU812 cells, stimulated with A23187 and PMA. However, luteolin did inhibit phosphorylation of c-Jun and DNA binding activity of AP-1 in nuclear lysates from stimulated KU812 cells. These results provide a fundamental structure of flavonoids for IL-4 inhibition and demonstrate a novel action of flavonoids that suppresses the activation of AP-1.     

KU812 cells provide a novel in vitro model of the human IL-33/ST2L axis: Functional responses and identification of signaling pathways

Activation of interleukin-1 family receptor ST2L by its ligand interleukin-33 (IL-33) is an important component in inflammatory responses. Peripheral blood basophils, recognized as major effector cells in allergic inflammation that play a role in both innate and adaptive immunity, are activated by IL-33 through ST2L. However, studies are challenging due to the paucity of this cell population, representing less than 1% of peripheral blood leukocytes. We identified a basophil-like chronic myelogenous leukemia cell line, KU812, that constitutively expresses ST2L and demonstrates functional responses to IL-33 stimulation. IL-33 induced production of multiple inflammatory mediators in KU812 cells that were blocked by anti-ST2L and anti-IL-33 antibodies. The interaction of IL-33 and ST2L activated NF-κB, JNK, and p38 MAPK, but not ERK1/2 signaling pathways. Studies using pharmacological inhibitors to IKK-2 and MAP kinases revealed that one of the functional responses, IL-33-induced IL-13 production, was regulated through NF-κB, but not JNK or p38 MAPK signaling. The requirement of NF-κB was confirmed by IKK-2 knockdown using shRNA. KU812 represents the first human cell line-based in vitro model of the IL-33/ST2L axis and provides a valuable tool to aid in understanding the mechanism and significance of IL-33 and ST2L interaction and function.     

IL-3 promotes basophilic differentiation of KU812 cells through high affinity binding sites

The myeloid precursor cell line KU812 exhibits a constitutive potential to differentiate into basophilic cells. In the present study, the influence of recombinant human (rh)IL-2, rhIL-3, and recombinant human granulocyte-macrophage-CSF on basophilic differentiation of KU812-F cells was studied. Of all cytokines tested, rhIL-3 induced a significant increase in formation of metachromatically granulated cells (from 10% in control cultures up to 30% in cultures supplemented with 100 U/ml of rhIL-3) as well as dose-dependent (1.5- to 3 fold) increase in cellular histamine in KU812-F cell cultures. In addition, KU812-F cells exposed to rhIL-3 bound more IgE antibody than cells cultured in control medium with up to 3.3-fold increases in the mean fluorescence intensity on days 2 and/or 5 compared with control (p less than 0.001). RhIL-3 failed to induce significant changes in expression of the Tac-reactive subunit of the IL-2R (CD25), surface aminopeptidase N (CD13), ICAM-1 Ag (CD54), or CD40 Ag on KU812-F cells. To investigate the mechanism of IL-3 action on KU812-F cells, receptor analyses were performed by using 125I-radiolabeled rhIL-3. Quantitative binding studies and Scatchard plot analyses revealed the presence of a single class of 1910 to 2460 high affinity IL-3-binding sites per KU812-F cell with an apparent dissociation constant of 1.22 to 2.35 x 10(-9) M. Together, these results show that rhIL-3 promotes basophilic differentiation of KU812-F cells through a specific receptor.     

Selective activation of p38 mitogen-activated protein kinase cascade in human neutrophils stimulated by IL-1beta.

We investigated activation of mitogen-activated protein kinase (MAPK) subtype cascades in human neutrophils stimulated by IL-1beta. IL-1beta induced phosphorylation and activation of p38 MAPK and phosphorylation of MAPK kinase-3/6 (MKK3/6). Maximal activation of p38 MAPK was obtained by stimulation of cells with 300 U/ml IL-1beta for 10 min. Extracellular signal-regulated kinase (ERK) was faintly phosphorylated and c-Jun N-terminal kinase (JNK) was not phosphorylated by IL-1beta. IL-1beta primed neutrophils for enhanced release of superoxide (O(2)(-)) stimulated by FMLP in parallel with increased phosphorylation of p38 MAPK. IL-1beta also induced O(2)(-) release and up-regulation of CD11b and CD15, and both responses were inhibited by SB203580 (p38 MAPK inhibitor), suggesting that p38 MAPK activation mediates IL-1beta-induced O(2)(-) release and up-regulation of CD11b and CD15. Combined stimulation of neutrophils with IL-1beta and G-CSF, a selective activator of the ERK cascade, resulted in the additive effects when the priming effect and phosphorylation of p38 MAPK and ERK were assessed. IL-1beta induced phosphorylation of ERK and JNK as well as p38 MAPK in human endothelial cells. These findings suggest that 1) in human neutrophils the MKK3/6-p38 MAPK cascade is selectively activated by IL-1beta and activation of this cascade mediates IL-1beta-induced O(2)(-) release and up-regulation of CD11b and CD15, and 2) the IL-1R-p38 MAPK pathway and the G-CSF receptor-ERK pathway work independently for activation of neutrophils.     

Visfatin induces MUC8 and MUC5B expression via p38 MAPK/ROS/NF-κB in human airway epithelial cells

Background

Among a variety of inflammatory mediators, visfatin is a proinflammatory adipocytokine associated with inflammatory reactions in obesity, metabolic syndrome, chronic inflammatory disease, and autoimmune disease. However, the biological role of visfatin in secretion of major mucins in human airway epithelial cells has not been reported. Therefore, this study was conducted in order to investigate the effect and the brief signaling pathway of visfatin on MUC8 and MUC5B expression in human airway epithelial cells.

Results

Visfatin significantly induced MUC8 and MUC5B expression. Visfatin significantly activated phosphorylation of p38 MAPK. Treatment with SB203580 (p38 MAPK inhibitor) and knockdown of p38 MAPK by siRNA significantly blocked visfatin-induced MUC8 and MUC5B expression.Visfatin significantly increased ROS formation. Treatment with SB203580 significantly attenuated visfatin-induced ROS formation. Treatment with NAC (ROS scavenger) and DPI (NADPH oxidase inhibitor) significantly attenuated visfatin-induced MUC8 and MUC5B expression. However, treatment with NAC and DPI did not attenuate visfatin-activated phosphorylation of p38 MAPK. Visfatin significantly activated the phosphorylation of NF-κB. Treatment with PDTC (NF-κB inhibitor) significantly attenuated visfatin-induced MUC8 and MUC5B expression.

Conclusions

These results suggest that visfatin induces MUC8 and MUC5B expression through p38 MAPK/ROS/NF-κB signaling pathway in human airway epithelial cells.     

Paracoccidioides brasiliensis induces secretion of IL-6 and IL-8 by lung epithelial cells. Modulation of host cytokine levels by fungal proteases

Paracoccidioides brasiliensis is a pathogenic, dimorphic fungus that causes paracoccidioidomycosis, a systemic human mycosis that is highly prevalent in Latin America. In this study, we demonstrated that P. brasiliensis yeasts induced interleukin (IL)-8 and IL-6 secretion by human lung epithelial A549 cells. However, tumor necrosis factor-α and interferon-γ were undetectable in these cultures. Moreover, P. brasiliensis yeasts induced activation of p38 mitogen-activated protein kinase (MAPK), c-Jun NH(2)-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) 1/2 in A549 cells, and IL-8 and IL-6 secretion promoted by this fungus was dependent on activation of p38 MAPK and ERK 1/2. In addition, IL-8 and IL-6 levels were significantly higher in culture supernatants of A549 cells that were incubated with formaldehyde-fixed P. brasiliensis compared to cultures of cells that were infected with live yeasts. Our results indicate that the observed cytokine level differences were due to protease expression, in live yeasts, that degraded these cytokines. Degradation of human recombinant IL-8 and IL-6 by live P. brasiliensis was inhibited by AEBSF and aprotinin, suggesting that these proteases belong to a family of serine proteases. This is the first report showing that P. brasiliensis may modulate host inflammation by expressing proteases that degrade proinflammatory cytokines.     

Reactive oxygen species‐induced activation of ERK and p38 MAPK mediates PMA‐induced NETs release from human neutrophils

Neutrophils/polymorphonuclear leukocytes (PMNs), an important component of innate immune system, release extracellular traps (NETs) to eliminate invaded pathogens; however understanding of the role of signaling molecules/proteins need to be elucidated. In the present study role of p38 MAPK and extracellular signal regulated kinase (ERK) against phorbol 12‐myristate 13‐acetate (PMA) induced reactive oxygen species (ROS) generation and NETs formation has been investigated. Human neutrophils were treated with PMA to induce free radical generation and NETs release, which were monitored by NBT reduction and elastase/DNA release, respectively. PMA treatment led to the time dependent phosphorylation of p38 MAPK and ERK in PMNs. Pretreatment of PMNs with SB202190 or U0126 did not significantly reduce PMA induce free radical generation, but prevented NETs release. Pretreatment of PMNs with NADPH oxidase inhibitor (diphenyleneiodonium chloride) significantly reduced free radical generation, p38 MAPK and ERK phosphorylation as well as NETs release, suggesting that p38 MAPK and ERK activation was downstream to free radical generation. The present study thus demonstrates ROS dependent activation of ERK and p38 MAPK, which mediated PMA induced NETs release from human neutrophils. J. Cell. Biochem. 114: 532–540, 2013. © 2012 Wiley Periodicals, Inc.     

JNK-dependent NFATc1 pathway positively regulates IL-13 gene expression induced by (–)-epigallocatechin-3-gallate in human basophilic KU812 cells

(–)-Epigallocatechin-3-gallate (EGCG) has been reported to possess a wide range of biological and pharmacological properties. In this study, we investigated the effects of EGCG on IL-13 gene expression in human basophilic KU812 cells. The IL-13 mRNA expression level was dose-dependently increased by treatment with EGCG (5–20 μM) for 1 h and additional incubation in a medium for 23 h. EGCG significantly increased the intracellular peroxide level as detected by the peroxide-sensitive probe 2′,7′-dichlorodihydrofluorescein diacetate. A pharmacological experiment using catalase and a structure–activity relationship study revealed that the exogenously produced H₂O₂ significantly, but partially, contributed to the IL-13 expression as well as the intracellular oxidative status. Furthermore, EGCG at the concentration required for IL-13 up-regulation activated c-Jun NH₂-terminal kinase (JNK), but not extracellular signal-regulated protein kinase or p38 mitogen-activated protein kinase in KU812 cells. Transfection of a JNK-specific siRNA as well as treatment with a JNK-specific inhibitor, SP600125, significantly reduced the EGCG-induced IL-13 mRNA expression, by 47.1 and 44.6%, respectively. In addition, we observed the nuclear translocation, mRNA up-regulation, and activation of DNA binding with the IL-13 promoter of nuclear factor of activated T cells (NFATc1) in the EGCG-treated cells. These data provide biological evidence that EGCG induces IL-13 mRNA expression via the JNK-dependent NFATc1 pathway in KU812 cells.     

The presence of serotonin in cigarette smoke – a possible mechanistic link to 5-HT-induced airway inflammation

We previously reported the involvement of serotonin (5-HT) metabolism in cigarette smoke-induced oxidative stress in rat lung in vivo. Here, we report cigarette smoke as a source of serotonin (5-HT) to the airways and aim at investigating the effects of 5-HT on oxidative stress and inflammation in human bronchial epithelial cells (BEAS-2B). A 5-HT analog was identified to be present in aqueous phase cigarette smoke using the LC-MS/MS approach, which was later confirmed by a 5-HT enzyme-linked immune assay (EIA). Furthermore, exposure to 5-HT caused a time-dependent elevation of intracellular ROS level, which was blocked in the presence of apocynin (a NOX inhibitor). In support, the immunoblot analysis indicated that there was an increase in the expression of NOX2 time-dependently. 5-HT-induced elevation of IL-8 at both mRNA and protein levels was observed, which was inhibited by TEMPOL (a free radical scavenger), and inhibitors for p38 MAPK (SB203580) and ERK (U0126), in line with the time-dependent phosphorylation of p38 MAPK and ERK. In conclusion, our findings suggest that 5-HT presented in bronchial epithelium of smokers may be involved in cigarette smoke-induced oxidative stress and inflammation via activation of p38 MAPK and ERK pathway after the formation of free radicals.     

The Effect of (-)-Epigallocatechin-3-Gallate on IL-1β Induced IL-8 Expression in Orbital Fibroblast from Patients with Thyroid-Associated Ophthalmopathy

Orbital fibroblasts have been reported to be an important effector cells for the development of thyroid-associated ophthalmopathy (TAO). Orbital fibroblasts secrete various inflammatory cytokines in response to an inflammatory stimulation, leading to TAO-related tissue swelling. It has also been reported that (-)-epigallocatechin-3-gallate (EGCG), a major polyphenolic constituent of green tea, has antioxidant and anti-inflammatory properties. In the current study, we investigated the issue of whether or how EGCG affects the interleukin (IL)-1β-induced secretion of IL-8 in human orbital fibroblasts from TAO patients. Treatment with EGCG significantly reduced the level of IL-1β-induced secretion of IL-8 and the expression of IL-8 mRNA. IL-1β-induced the degradation of IκBα, and the phosphorylation of p38 and ERK, and the IL-1β-induced expression of IL-8 mRNA was inhibited by specific inhibitors, such as BAY-117085 for NF-kB, SB203580 for p38, and PD98059 for ERK. In addition, treatment with EGCG inhibited the IL-1β-induced degradation of IκBα, and the phosphorylation of p38 and ERK. However, pre-treatment with antioxidants, NVN and NAC, which suppressed ROS generation, did not reduce IL-8 expression in IL-1β-treated orbital fibroblasts, suggesting that the IL-1β-induced IL-8 expression is not mediated by the generation of ROS. These results show that EGCG suppresses the IL-1β-induced expression of IL-8 through inhibition of the NF-κB, p38, and ERK pathways. These findings could contribute to the development of new types of EGCG-containing pharmacological agents for use in the treatment of TAO.     

Role of SIRT1 in Streptococcus pneumoniae-induced human β-defensin-2 and interleukin-8 expression in A549 cell

Streptococcus pneumoniae is an important pathogen of pneumonia in human. Human alveolar epithelium acts as an effective barrier and is an active participant in host defense against invasion of bacterial by production of various mediators. Sirtuin 1 (SIRT1), the prototypic class III histone deacetylase, is involved in the molecular control of lifespans and immune responses. This study aimed at examining the role of SIRT1 in mediating S. pneumoniae-induced human β-defensin-2 (hBD2) and interleukin-8(IL-8) expression in the alveolar epithelial cell line A549 and the underlying mechanisms involved. A549 cells were infected with S. pneumoniae for indicated times. Exposure of A549 cells to S. pneumoniae increased the expressions of SIRT1 protein, hBD2 and IL-8 mRNA, and protein. The SIRT1 activator resveratrol enhanced S. pneumoniae-induced gene expression of hBD2 but decreased IL-8 mRNA levels. Blockade of SIRT1 activity by the SIRT1 inhibitors nicotinamide reduced S. pneumoniae-induced hBD2 mRNA expression but increased its stimulatory effects on IL-8 mRNA. S. pneumoniae-induced activation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). SIRT1 expression was attenuated by selective inhibitors of ERK and p38 MAPK. The hBD2 mRNA production was decreased by pretreatment with p38 MAPK inhibitor but not with ERK inhibitor, whereas the IL-8 mRNA expression was controlled by phosphorylation of ERK. These results suggest that SIRT1 mediates the induction of hBD2 and IL-8 gene expression levels in A549 cell by S. pneumoniae. SIRT1 may play a key role in host immune and defense response in A549.     

IGF-I stimulates IL-8 production in the promyelocytic cell line HL-60 through activation of extracellular signal-regulated protein kinase

Interleukin (IL)-8 serves as a major chemoattractant for neutrophils and has also been proposed to affect cancer progression. In the present study, we show that IGF-I stimulates IL-8 mRNA expression and IL-8 secretion in the leukemic cell line HL-60. Stimulation of IL-8 expression was completely attenuated by two inhibitors of mitogen-activated protein kinase (MAPK) kinase (MEK), which phosphorylates the MAPKs extracellular-regulated kinase (ERK)1 and ERK2, and by the c-Jun NH2-terminal kinase (JNK) inhibitor SP600125. In contrast, inhibitors of p38 MAPK and phosphatidylinositol-3 kinase (PI3K) did not abrogate the effect of IGF-I. We also show that IGF-I stimulates the activation of ERK1 and ERK2, but we could not detect any effect of IGF-I on the phosphorylation of p38, JNK^p46 or JNK^p54. Collectively, our results suggest that basal JNK activity and activation of the MEK–ERK pathway are required for upregulation of IL-8 by IGF-I in HL-60 cells.