Carbon monoxide inhibits IL-17-induced IL-6 production through the MAPK pathway in human pulmonary epithelial cells

Interleukin (IL)-17 is a proinflammatory cytokine that is produced by activated memory CD4 T cells, which regulates pulmonary neutrophil emigration by the induction of CXC chemokines and cytokines. IL-17 constitutes a potential target for pharmacotherapy against exaggerated neutrophil recruitment in airway diseases. As a cytoprotective and anti-inflammatory gaseous molecule, carbon monoxide (CO) may also regulate IL-17-induced inflammatory responses in pulmonary cells. Herein, we examine the production of cytokine IL-6 induced by IL-17 and the effect of CO on IL-17-induced IL-6 production in human pulmonary epithelial cell A549. We first show that IL-17 can induce A549 cells to release IL-6 and that CO can markedly inhibit IL-17-induced IL-6 production. IL-17 activated the ERK1/2 MAPK pathway but did not affect p38 and JNK MAPK pathways. CO exposure selectively attenuated IL-17-induced ERK1/ERK2 MAPK activation without significantly affecting either JNK or p38 MAPK activation. Furthermore, in the presence of U0126 and PD-98059, selective inhibitors of MEK1/2, IL-17-induced IL-6 production was significantly attenuated. We conclude that CO inhibits IL-17-stimulated inflammatory response via the ERK1/2-dependent pathway.     

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.     

Regulation of IL-17A responses in human airway smooth muscle cells by Oncostatin M

Background

Regulation of human airway smooth muscle cells (HASMC) by cytokines contributes to chemotactic factor levels and thus to inflammatory cell accumulation in lung diseases. Cytokines such as the gp130 family member Oncostatin M (OSM) can act synergistically with Th2 cytokines (IL-4 and IL-13) to modulate lung cells, however whether IL-17A responses by HASMC can be altered is not known.

Objective

To determine the effects of recombinant OSM, or other gp130 cytokines (LIF, IL-31, and IL-6) in regulating HASMC responses to IL-17A, assessing MCP-1/CCL2 and IL-6 expression and cell signaling pathways.

Methods

Cell responses of primary HASMC cultures were measured by the assessment of protein levels in supernatants (ELISA) and mRNA levels (qRT-PCR) in cell extracts. Activation of STAT, MAPK (p38) and Akt pathways were measured by immunoblot. Pharmacological agents were used to assess the effects of inhibition of these pathways.

Results

OSM but not LIF, IL-31 or IL-6 could induce detectable responses in HASMC, elevating MCP-1/CCL2, IL-6 levels and activation of STAT-1, 3, 5, p38 and Akt cell signaling pathways. OSM induced synergistic action with IL-17A enhancing MCP-1/CCL-2 and IL-6 mRNA and protein expression, but not eotaxin-1 expression, while OSM in combination with IL-4 or IL-13 synergistically induced eotaxin-1 and MCP-1/CCL2. OSM elevated steady state mRNA levels of IL-4Rα, OSMRβ and gp130, but not IL-17RA or IL-17RC. Pharmacologic inhibition of STAT3 activation using Stattic down-regulated OSM, OSM/IL-4 or OSM/IL-13, and OSM/IL-17A synergistic responses of MCP-1/CCL-2 induction, whereas, inhibitors of Akt and p38 MAPK resulted in less reduction in MCP-1/CCL2 levels. IL-6 expression was more sensitive to inhibition of p38 (using SB203580) and was affected by Stattic in response to IL-17A/OSM stimulation.

Conclusions

Oncostatin M can regulate HASMC responses alone or in synergy with IL-17A. OSM/IL-17A combinations enhance MCP-1/CCL2 and IL-6 but not eotaxin-1. Thus, OSM through STAT3 activation of HASMC may participate in inflammatory cell recruitment in inflammatory airway disease.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0164-4) contains supplementary material, which is available to authorized users.     

Involvement of p42/p44 MAPK, JNK, and NF-kappaB in IL-1beta-induced ICAM-1 expression in human pulmonary epithelial cells

Interleukin-1beta (IL-1beta) has been shown to induce the expression of intercellular adhesion molecule-1 (ICAM-1) on airway epithelial cells and contributes to inflammatory responses. However, the mechanisms regulating ICAM-1 expression by IL-1beta in human A549 cells was not completely understood. Here, the roles of mitogen-activated protein kinases (MAPKs) and NF-kappaB pathways for IL-1beta-induced ICAM-1 expression were investigated in A549 cells. IL-1beta induced expression of ICAM-1 protein and mRNA in a time- and concentration-dependent manner. The IL-1beta induction of ICAM-1 mRNA and protein were partially inhibited by U0126 and PD98059 (specific inhibitors of MEK1/2) and SP600125 [a specific inhibitor of c-Jun-N-terminal kinase (JNK)]. U0126 was more potent than other inhibitors to attenuate IL-1beta-induced ICAM-1 expression. Consistently, IL-1beta stimulated phosphorylation of p42/p44 MAPK and JNK which was attenuated by pretreatment with U0126 or SP600125, respectively. Moreover, transfection with dominant negative mutants of MEK1/2 (MEK K97R) or ERK2 (ERK2 K52R) also attenuated IL-1beta-induced ICAM-1 expression. The combination of PD98059 and SP600125 displayed an additive effect on IL-1beta-induced ICAM-1 gene expression. IL-1beta-induced ICAM-1 expression was almost completely blocked by a specific NF-kappaB inhibitor helenalin. Consistently, IL-1beta stimulated translocation of NF-kappaB into the nucleus and degradation of IkappaB-alpha which was blocked by helenalin, U0126, or SP600125. Taken together, these results suggest that activation of p42/p44 MAPK and JNK cascades, at least in part, mediated through NF-kappaB pathway is essential for IL-1beta-induced ICAM-1 gene expression in A549 cells. These results provide new insight into the mechanisms of IL-1beta action that cytokines may promote inflammatory responses in the airway disease.     

Concerted expression of eotaxin-1, eotaxin-2, and eotaxin-3 in human bronchial epithelial cells

Eotaxin-1/CCL11, eotaxin-2/CCL24, and eotaxin-3/CCL26 bind specifically and exclusively to CC chemokine receptor (CCR) 3, which is a potential therapeutic target in treating the peribronchial eosinophilia associated with allergic airway diseases. Bronchial epithelial cells represent an important source of chemokines, and thus we investigated in vitro and in vivo expression of eotaxin-2 and eotaxin-3 in bronchial epithelial cells in comparison with that of eotaxin-1. Immunohistochemistry showed increased expression of both eotaxin-2 and eotaxin-3 in addition to eotaxin-1 in asthmatics. Considerable amounts of eotaxins were secreted by bronchial epithelial lineage. As with eotaxin-1 production, generation of eotaxin-2 and eotaxin-3 by bronchial epithelial cells was up-regulated by IL-4 and IL-13, and attenuated by IFN-gamma and glucocorticoids. In addition to eotaxin-1 expression, but also eotaxin-2 and eotaxin-3 expression in the bronchial epithelium should be taken into consideration when developing the therapeutic strategies to treat eosinophilic airway diseases.     

Involvement of p42/p44 MAPK, p38 MAPK, JNK, and NF-kappaB in IL-1beta-induced VCAM-1 expression in human tracheal smooth muscle cells

Interleukin-1beta (IL-1beta) has been shown to induce the expression of adhesion molecules on airway epithelial and smooth cells and contributes to inflammatory responses. Here, the roles of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappaB (NF-kappaB) pathways for IL-1beta-induced vascular cell adhesion molecule (VCAM)-1 expression were investigated in human tracheal smooth muscle cells (HTSMC). IL-1beta induced expression of VCAM-1 protein and mRNA in a time-dependent manner, which was significantly inhibited by inhibitors of MEK1/2 (U0126 and PD-98059), p38 (SB-202190), and c-Jun NH(2)-terminal kinase (JNK; SP-600125). Consistently, IL-1beta-stimulated phosphorylation of p42/p44 MAPK, p38, and JNK was attenuated by pretreatment with U0126, SB-202190, or SP-600125, respectively. IL-1beta-induced VCAM-1 expression was significantly blocked by the specific NF-kappaB inhibitors helenalin and pyrrolidine dithiocarbamate. As expected, IL-1beta-stimulated translocation of NF-kappaB into the nucleus and degradation of IkappaB-alpha were blocked by helenalin but not by U0126, SB-202190, or SP-600125. Moreover, the resultant enhancement of VCAM-1 expression increased the adhesion of polymorphonuclear cells to a monolayer of HTSMC, which was blocked by pretreatment with helenalin, U0126, SB-202190, or SP-600125 before IL-1beta exposure or by anti-VCAM-1 antibody. Together, these results suggest that in HTSMC, activation of p42/p44 MAPK, p38, JNK, and NF-kappaB pathways is essential for IL-1beta-induced VCAM-1 gene expression. These results provide new insight into the mechanisms of IL-1beta action that cytokines may promote inflammatory responses in airway disease.     

Constitutive and inducible thymic stromal lymphopoietin expression in human airway smooth muscle cells: role in chronic obstructive pulmonary disease

Thymic stromal lymphopoietin (TSLP) is a novel cytokine that triggers dendritic cell-mediated T helper (Th)-2 inflammatory responses. Previous studies have demonstrated that human airway smooth muscle cells (HASMC) play a critical role in initiating or perpetuating airway inflammation by producing chemokines and cytokines. In this study, we first evaluated the expression of TSLP in primary HASMC and investigated how proinflammatory cytokines (TNF-alpha and IL-1beta) and Th-2 cytokines (IL-4, IL-9) regulate TSLP production from HASMC. TSLP mRNA and protein were assessed by real-time RT-PCR, ELISA, and immunofluorescence from primary HASMC cultures. Primary HASMC express constitutive level of TSLP. Incubation of HASMC with IL-1 or TNF-alpha resulted in a significant increase of TSLP mRNA and protein release from HASMC. Furthermore, combination of IL-1beta and TNF-alpha has an additive effect on TSLP release by HASMC. Primary HASMC pretreated with inhibitors of p38 or p42/p44 ERK MAPK, but not phosphatidylinositol 3-kinase, showed a significant decrease in TSLP release on IL-1beta and TNF-alpha treatment. Furthermore, TSLP immunoreactivity was present in ASM bundle from chronic obstructive pulmonary disease (COPD) and to lesser degree in normal subjects. Taken together, our data provide the first evidence of IL-1beta- and TNF-alpha-induced TSLP expression in HASMC via (p38, p42/p44) MAPK signaling pathways. Our results raise the possibility that HASMC may play a role in COPD airway inflammation via TSLP-dependent pathway.     

Differential requirement of members of the MAPK family for CCL2 expression by hepatic stellate cells

Hepatic stellate cells (HSC) coordinate the liver wound-healing response through secretion of several cytokines and chemokines, including CCL2 (formerly known as monocyte chemoattractant protein-1). In this study, we evaluated the role of different proteins of the MAPK family (ERK, p38(MAPK), and JNK) in the regulation of CCL2 expression by HSC, as an index of their proinflammatory activity. Several mediators activated all three MAPK, including TNF, IL-1, and PDGF. To assess the relative role of the different MAPKs, specific pharmacological inhibitors were used; namely, SB203580 (p38(MAPK)), SP600125 (JNK), and PD98059 (MEK/ERK). The efficacy and specificity of the different inhibitors in our cellular system were verified analyzing the enzymatic activity of the different MAPKs using in vitro kinase assays and/or testing the inhibition of phosphorylation of downstream substrates. SB203580 and SP600125 dose-dependently inhibited CCL2 secretion and gene expression induced by IL-1 or TNF. In contrast, inhibition of ERK did not affect the upregulation of CCL2 induced by the two cytokines. Finally, activin A was also found to stimulate CCL2 expression and to activate ERK, JNK, p38, and their downstream targets. Unlike in cells exposed to proinflammatory cytokines, all three MAPKs were required to induce CCL2 secretion in response to activin. We conclude that members of the MAPK family differentially regulate cytokine-induced chemokine expression in human HSC.     

ASK1-p38 MAPK/JNK signaling cascade mediates anandamide-induced PC12 cell death

Anandamide is a neuroimmunoregulatory molecule that triggers apoptosis in a number of cell types including PC12 cells. Here, we investigated the molecular mechanisms underlying anandamide-induced cell death in PC12 cells. Anandamide treatment resulted in the activation of p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), and p44/42 MAPK in apoptosing cells. A selective p38 MAPK inhibitor, SB203580, or dn-JNK, JNK1(A-F) or SAPKbeta(K-R), blocked anandamide-induced cell death, whereas a specific inhibitor of MEK-1/2, U0126, had no effect, indicating that activation of p38 MAPK and JNK is critical in anandamide-induced cell death. An important role for apoptosis signal-regulating kinase 1 (ASK1) in this event was also demonstrated by the inhibition of p38 MAPK/JNK activation and death in cells overexpressing dn-ASK1, ASK1 (K709M). Conversely, the constitutively active ASK1, ASK1DeltaN, caused prolonged p38 MAPK/JNK activation and increased cell death. These indicate that ASK1 mediates anandamide-induced cell death via p38 MAPK and JNK activation. Here, we also found that activation of p38 MAPK/JNK is accompanied by cytochrome c release from the mitochondria and caspase activation (which can be inhibited by SB203580), suggesting that anandamide triggers a mitochondrial dependent apoptotic pathway. The caspase inhibitor, zVAD, and the mitochondrial pore opening inhibitor, cyclosporine A, blocked anandamide-induced cell death but not p38 MAPK/JNK activation, suggesting that activation of these kinases may occur upstream of mitochondrial associated events.     

Role of caveolin-1 in p42/p44 MAP kinase activation and proliferation of human airway smooth muscle

Chronic airways diseases, including asthma, are associated with an increased airway smooth muscle (ASM) mass, which may contribute to chronic airway hyperresponsiveness. Increased muscle mass is due, in part, to increased ASM proliferation, although the precise molecular mechanisms for this response are not completely clear. Caveolae, which are abundant in smooth muscle cells, are membrane microdomains where receptors and signaling effectors can be sequestered. We hypothesized that caveolae and caveolin-1 play an important regulatory role in ASM proliferation. Therefore, we investigated their role in p42/p44 MAPK signaling and proliferation using human ASM cell lines. Disruption of caveolae using methyl-beta-cyclodextrin and small interfering (si)RNA-knockdown of caveolin-1 caused spontaneous p42/p44 MAPK activation; additionally, caveolin-1 siRNA induced ASM proliferation in mitogen deficient conditions, suggesting a key role for caveolae and caveolin-1 in maintaining quiescence. Moreover, caveolin-1 accumulates twofold in myocytes induced to a contractile phenotype compared with proliferating ASM cells. Caveolin-1 siRNA failed to increase PDGF-induced p42/p44 MAPK activation and cell proliferation, however, indicating that PDGF stimulation actively reversed the antimitogenic control by caveolin-1. Notably, the PDGF induced loss of antimitogenic control by caveolin-1 coincided with a marked increase in caveolin-1 phosphorylation. Furthermore, the strong association of PDGF receptor-beta with caveolin-1 that exists in quiescent cells was rapidly and markedly reduced with agonist addition. This suggests a dynamic relationship in which mitogen stimulation actively reverses caveolin-1 suppression of p42/p44 MAPK signal transduction. As such, caveolae and caveolin-1 coordinate PDGF receptor signaling, leading to myocyte proliferation, and inhibit constitutive activity of p42/p44 MAPK to sustain cell quiescence.     

Role of mitogen-activated protein kinases in Thy-1-induced T-lymphocyte activation

Thy-1 (CD90) crosslinking by monoclonal antibodies (mAb) in the context of costimulation causes the activation of mouse T-lymphocytes; however, the associated signal transduction processes have not been studied in detail. In this study we investigated the role of mitogen-activated protein kinases (MAPKs) in Thy-1-mediated T-lymphocyte activation using mAb-coated polystyrene microspheres to crosslink Thy-1 and costimulatory CD28 on murine T-lymphocytes. Concurrent Thy-1 and CD28 crosslinking induced DNA synthesis by T-lymphocytes, as well as interleukin (IL)-2 and IL-2 receptor (IL-2R) α chain (CD25) expression. Increased phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, p38 MAPK, and c-Jun N-terminal protein kinase (JNK) was also observed. Pharmacologic inhibition of ERK1/2 or JNK activation inhibited Thy-1-induced DNA synthesis and IL-2 production by T-lymphocytes. p38 MAPK inhibition also decreased DNA synthesis in Thy-1-stimulated T-lymphocytes; however, IL-2 production was increased in these cells. Inhibition of JNK, but not ERK1/2 or p38 MAPK, caused a marked reduction in Thy-1-induced CD25 expression. In addition, inhibition of p38 MAPK or JNK, but not ERK1/2, impaired the growth of IL-2-dependent CTLL-2 T-lymphocytes but did not substantially affect CD25 expression. Finally, exogenous IL-2 reversed the inhibitory effect of ERK1/2 or JNK inhibition on Thy-1-stimulated DNA synthesis by T-lymphocytes but did not substantially reverse JNK inhibition of CD25 expression. Collectively, these results suggest that during Thy-1-induced T-lymphocyte activation, ERK1/2 and JNK promoted IL-2 production whereas p38 MAPK negatively regulated IL-2 expression. JNK signalling was also required for CD25 expression. IL-2R signalling involved both p38 MAPK and JNK in CTLL-2 cells, whereas p38 MAPK was most important for IL-2R signalling in primary T-lymphocytes. MAPKs are therefore essential signalling intermediates for the Thy-1-driven proliferation of mouse T-lymphocytes.     

Moxifloxacin but not ciprofloxacin or azithromycin selectively inhibits IL-8, IL-6, ERK1/2, JNK, and NF-kappaB activation in a cystic fibrosis epithelial cell line

Cystic fibrosis (CF) is associated with severe neutrophilic airway inflammation. We showed that moxifloxacin (MXF) inhibits IL-8 and MAPK activation in monocytic and respiratory epithelial cells. Azithromycin (AZM) and ciprofloxacin (CIP) are used clinically in CF. Thus we now examined effects of MXF, CIP, and AZM directly on CF cells. IB3, a CF bronchial cell line, and corrected C38 cells were treated with TNF-alpha, IL-1beta, or LPS with or without 5-50 microg/ml MXF, CIP, or AZM. IL-6 and IL-8 secretion (ELISA), MAPKs ERK1/2, JNK, p38, and p65 NF-kappaB (Western blot) activation were measured. Baseline IL-6 was sixfold higher in IB3 than C38 cells but IL-8 was similar. TNF-alpha and IL-1beta increased IL-6 and IL-8 12- to 67-fold with higher levels in IB3 than C38 cells post-TNF-alpha (P < 0.05). Levels were unchanged following LPS. Baseline phosphorylated form of ERK1/2 (p-ERK1/2), JNK, and NF-kappaB p65 were higher in IB3 than C38 cells (5-, 1.4-, and 1.4-fold), and following TNF-alpha increased, as did the p-p38, by 1.6- to 2-fold. MXF (5-50 microg/ml) and CIP (50 microg/ml), but not AZM, suppressed IL-6 and IL-8 secretion by up to 69%. MXF inhibited TNF-alpha-stimulated MAPKs ERK1/2, 46-kDa JNK, and NF-kappaB up to 60%, 40%, and 40%, respectively. In contrast, MXF did not inhibit p38 activation, implying a highly selective pretranslational effect. In conclusion, TNF-alpha and IL-1beta induce an exaggerated inflammatory response in CF airway cells, inhibited by MXF more than by CIP or AZM. Clinical trials are recommended to assess efficacy in CF and other chronic lung diseases.