Montelukast inhibits tumour necrosis factor-α-mediated interleukin-8 expression through inhibition of nuclear factor-κB p65-associated histone acetyltransferase activity

Background Montelukast is a potent cysteinyl leukotriene‐1 receptor antagonist possessing some anti‐inflammatory effects although the molecular mechanism of these anti‐inflammatory effects is unknown. In this study, we aimed to investigate the effect of montelukast on nuclear factor (NF)‐κB‐associated histone acetylation activity in phorbol myristate acetate (PMA)‐differentiated U937 cells. Methods We examined the inhibitory effects of montelukast on TNF‐α‐induced IL‐8 production in PMA‐differentiated U‐937 cells. U‐937 cells were exposed to PMA (50 ng/mL) for 48 h to allow differentiation to macrophages. Macrophages were then exposed to TNF‐α (10 ng/mL) in the presence or absence of montelukast (0.01–10 μm ) for 24 h. After this time, the concentration of IL‐8 in the culture supernatant was measured by sandwich‐type ELISA kit. The effect of signalling pathways on TNF‐α‐induced IL‐8 release was examined pharmacologically using selective NF‐κB/IKK2 (AS602868, 3 μm ), (PD98059, 10 μm ) and p38 mitogen activated protein kinase (MAPK) (SB203580, 1 μm ) inhibitors. NF‐κB DNA binding activity was measured by a DNA‐binding ELISA‐based assay. NF‐κB‐p65‐associated histone acetyltransferase (HAT) activity was measured by immunoprecipitation linked to commercial flourescent HAT. Results TNF‐α‐induced IL‐8 release was suppressed by an NF‐κB inhibitor but not by MEK or p38 MAPK inhibitors. Montelukast induced a concentration‐dependent inhibition of TNF‐α‐induced IL‐8 release and mRNA expression that reached a plateau at 0.1 μm without affecting cell viability. Montelukast did not affect NF‐κB p65 activation as measured by DNA binding but suppressed NF‐κB p65‐associated HAT activity. Conclusion Montelukast inhibits TNF‐α‐stimulated IL‐8 expression through changes in NF‐κB p65‐associated HAT activity. Drugs targeting these enzymes may enhance the anti‐inflammatory actions of montelukast.     

Pranlukast, a cysteinyl leukotriene receptor 1 antagonist, attenuates allergen-specific tumour necrosis factor alpha production and nuclear factor kappa B nuclear translocation in peripheral blood monocytes from atopic asthmatics

BACKGROUND: The cysteinyl leukotriene receptor 1 (cysLTR1) antagonists are useful for oral treatment of bronchial asthma. The underlying mechanism of cysLTR1 antagonists on inhibition of inflammatory cytokine production is yet to be determined. OBJECTIVE: The present study was designed to determine the effect of pranlukast, a cysLTR1 antagonist, on production of inflammatory cytokines by allergen-stimulated peripheral blood monocytes (PBM) from atopic asthmatics. METHODS: PBM were obtained from normal control (n = 10) and Dermatophagoides farinae (Der f) allergen-sensitized atopic asthmatics (n = 12), and were cultured in the presence of Der f allergen. The production of TNF-alpha and nuclear-translocation of nuclear factor kappa B (NF-kappa B) was determined. In atopic asthmatics, pranlukast, tacrolimus or dexamethasone was added before stimulation by Der f. The additive effect of pranlukast and dexamethasone was also determined. RESULTS: PBM from atopic asthmatics cultured with Der f exhibited a significant increase in TNF-alpha production and nuclear translocation of NF-kappa B compared with normal control (P < 0.01). Pranlukast, tacrolimus and dexamethasone significantly inhibited production of TNF-alpha and nuclear-translocation of NF-kappa B in PBM of atopic asthmatics (P < 0.01). An additive effect of pranlukast on low-dose dexamethasone was also demonstrated. However, LTD4 did not induce TNF-alpha production or NF-kappa B nuclear translocation. CONCLUSION: Our results suggest that pranlukast may inhibit TNF-alpha production via suppression of NF-kappa B activation through pathways distinct from cysLTR1 antagonism.     

The mycotoxins citrinin and gliotoxin differentially affect production of the pro-inflammatory cytokines tumour necrosis factor-α and interleukin-6, and the anti-inflammatory cytokine interleukin-10

BACKGROUND: Microbial growth is considered one of the major causes of indoor air problems. Moulds have been associated with asthma, allergy and a wide range of diffuse indoor air-related symptoms. However, mechanisms of the adverse health effects are not well understood. OBJECTIVE: We hypothesized that the mycotoxins citrinin and gliotoxin could cause an imbalance between the secretion of the pro-inflammatory cytokines TNF-alpha and IL-6 and the anti-inflammatory cytokine IL-10. METHODS: We investigated the influence of citrinin and gliotoxin on the human monocytic cell line Mono-Mac-6 (MM6) with and without lipopolysaccharide -stimulation. The levels of IL-10, IL-6 and TNF-alpha were analysed in cell culture supernatants by ELISA. Cell viability and cell apoptosis were measured by flow cytometry. RESULTS: The strongest inhibition of cytokine secretion was found for IL-10. IL-6 levels were found to decrease in a dose-dependent manner along with reduced cell viability. TNF-alpha levels increased with low gliotoxin exposure (less than 100 ng/mL), but decreased significantly at 375 ng/mL and higher along with increased cell apoptosis and reduced cell viability. TNF-alpha levels were not reduced by citrinin exposure. CONCLUSION: We observed a cytokine imbalance with a more pronounced reduction of IL-10 concentrations compared with those of TNF-alpha and IL-6. We suggest that low exposure doses of citrinin and gliotoxin (corresponding to less than 100 ng/mL gliotoxin and less than 10 mug/mL citrinin) may inhibit IL-10 and lead to increased risk of an inflammatory response with relative overproduction of TNF-alpha and IL-6. The findings and their clinical implications must be verified by human studies. However, we speculate that the observed biological effects may be of importance as they may partly explain the occurrence of diffuse general indoor air-related symptoms as well as the worsening of asthmatic inflammatory reactions experienced in mouldy environments.     

Combined effect of tumour necrosis factor-α and interleukin-13 polymorphisms on bronchial hyperresponsiveness in Korean children with asthma

Background TNF‐α and IL‐13, two pivotal pro‐inflammatory cytokines, are increased in asthmatic airways and may be linked to asthma susceptibility and/or bronchial hyperresponsiveness (BHR). Objective We investigated the association between the TNF‐α?308G/A polymorphism and asthma susceptibility or asthma‐related phenotypes in Korean children with asthma, and tested for a combined effect with IL‐13 polymorphisms. Methods Asthmatic children (n=719) and non‐atopic healthy control children (n=243) were evaluated for asthma phenotypes including total serum IgE and BHR to methacholine. Genotypes were determined by PCR‐restriction fragment length polymorphism analysis. Results The allele frequency of TNF‐α?308A in asthmatics (14.1%) was higher than that in control children [8.7%, odds ratio (OR) 1.72, 95% confidence interval (CI) 1.05–2.82]. Significantly lower PC₂₀ values were found in asthmatic children carrying one or two copies of the TNF‐α risk allele (?308A) vs. those homozygous for the common allele (P=0.026). Combined analysis revealed that atopic asthmatic children co‐inherited the risk alleles of TNF‐α?308G/A and IL‐13 +2044G/A more frequently than control children (aOR 1.91, 95% CI 1.00–3.65), and asthmatic children co‐inheriting both risk alleles had significantly lower PC₂₀ values vs. asthmatic children homozygous for the common alleles (P=0.024). Conclusion The TNF‐α promoter polymorphism (?308G/A) may be associated with asthma susceptibility and BHR in Korean children with asthma. In addition, there appears to be a synergistic effect between the TNF‐α promoter polymorphism and an IL‐13 coding region polymorphism in terms of asthma susceptibility and BHR in this population.     

Leukotriene B4 enhances tumour necrosis factor-α-induced CCL27 production in human keratinocytes

BACKGROUND: A chemokine CCL27 recruits skin-homing T cells. CCL27 production by epidermal keratinocytes is dependent on nuclear factor-kappaB (NF-kappaB) activity and is enhanced in lesions with atopic dermatitis or allergic contact dermatitis. A lipid mediator leukotriene B(4) (LTB(4)) may be involved in the development of these allergic dermatoses. LTB(4) acts on cell surface G-protein-coupled receptors, BLT1 and BLT2. OBJECTIVE: The aim of this study was to investigate the in vitro effects of LTB(4) on CCL27 production in human keratinocytes. METHODS: Keratinocytes were incubated with TNF-alpha and LTB(4). CCL27 secretion and mRNA levels were analysed by ELISA and RT-PCR, respectively. NF-kappaB activities were analysed by luciferase assays. Protein levels or phosphorylation status were analysed by cell-based ELISA. RESULTS: LTB(4) alone did not enhance CCL27 production and modestly enhanced NF-kappaB activity in human keratinocytes. However, LTB(4) potently enhanced TNF-alpha-induced CCL27 secretion and mRNA expression and NF-kappaB activity. LTB(4) alone or together with TNF-alpha, induced phosphorylation and degradation of inhibitory NF-kappaB alpha (IkappaBalpha) and phosphorylation of NF-kappaB p65. These effects of LTB(4) were suppressed by BLT1 antagonist U75302, pertussis toxin, phosphoinositide-3 kinase (PI3K) inhibitor LY294002 and extracellular signal-regulated kinase (ERK) kinase inhibitor U0126, but not by BLT2 antagonist LY255283. LTB(4) induced phosphorylation of ERK and Akt, downstream kinase of PI3K; LY294002 suppressed phosphorylation of both kinases while U0126 suppressed only the former. CONCLUSION: These results suggest that LTB(4) may enhance TNF-alpha-induced CCL27 production by activating NF-kappaB via the BLT1/G(i/o)/PI3K/ERK pathway in human keratinocytes. LTB(4) may contribute to the enhanced CCL27 production of keratinocytes in lesions with atopic dermatitis or allergic contact dermatitis.     

Tumour necrosis factor-α-induced expression of intercellular adhesion molecule-1 on human eosinophilic leukaemia EoL-1 cells is mediated by the activation of nuclear factor-κB pathway

BACKGROUND: Intercellular adhesion molecule-1 (ICAM-1) has been shown to mediate the adhesion and migration of eosinophils to the site of allergic inflammation. However, molecular mechanisms regulating the expression of ICAM-1 in eosinophils are still being elucidated. We investigated the effect of tumour necrosis factor-alpha (TNF-alpha) on ICAM-1 expression of eosinophils. METHODS: The surface expression of ICAM-1 on a human eosinophilic leukaemic cell line, EoL-1, was assessed by immunocytochemical staining. The phosphorylation of inhibitor kappa B-alpha (IkappaB-alpha) and p38 mitogen-activated protein kinase (MAPK) was detected by Western blot. Nuclear factor kappa-B (NF-kappaB) pathway-related genes were evaluated by the cDNA expression array system, whereas the activity of NF-kappaB was measured by electrophoretic mobility shift assay (EMSA). RESULTS: TNF-alpha was found to induce the cell surface expression of ICAM-1. A specific proteasome inhibitor N-cbz-Leu-Leu-leucinal (MG-132), but not a p38 MAPK inhibitor (SB 203580), was found to suppress the TNF-alpha-induced expression of ICAM-1 on EoL-1 cells. The gene expressions of ICAM-1, NF-kappaB and IkappaBalpha were up-regulated after the stimulation with TNF-alpha. Further, TNF-alpha was shown to induce IkappaB-alpha phosphorylation and degradation, thereby indicating the activation of NF-kappaB. In EMSA, there was a shifted NF-kappaB band on TNF-alpha-treated cells with or without SB 203580, but no shifted band was observed on MG-132-treated cells. CONCLUSION: In vitro studies of EoL-1 cells, an eosinophilic leukaemic cell line, confirmed that NF-kappaB plays an important role in the expression of ICAM-1 and recruitment of eosinophils in allergic inflammation.     

High serum levels of tumour necrosis factor‐α and interleukin‐8 in severe asthma: markers of systemic inflammation?

BACKGROUND: Severe asthma is characterized by elevated levels of pro-inflammatory cytokines and neutrophilic inflammation in the airways. Blood cytokines, markers of 'systemic' inflammation, may be a feature of amplified inflammation in severe asthma. OBJECTIVE: To detect differences in IL-8, TNF-alpha, IL-16 and IL-13 levels in the serum(s) of stable severe and mild-moderate asthmatics related to blood leucocytes proportion, airway calibre and exhaled nitric oxide (NO) levels. METHODS: We assessed cytokine serum levels by ELISA and blood leucocyte counts by an alkaline peroxidase method in 20 healthy controls, 22 mild-moderate [forced expiratory volume in 1 s (FEV1)(%pred): 89+/-3] and 14 severe asthmatics [FEV1(%pred): 49+/-2]. RESULTS: IL-8 and TNF-alpha levels were higher in severe asthmatics than in mild-moderate asthmatics or in controls (P<0.05). No differences in IL-16 and IL-13 levels were detected. Severe asthmatics showed higher circulating neutrophil and eosinophil number than controls (P<0.05). In severe asthmatics, exhaled NO levels were superior than in controls (P<0.05), but inferior than in mild-moderate asthmatics (P<0.05). We found positive correlation between TNF-alpha levels and exhaled NO (r=0.67; P=0.01) or circulating neutrophil counts (r=0.57; P=0.03) in severe asthmatics. CONCLUSION: sTNF-alpha and sIL-8 are markers of 'systemic' inflammation in severe asthmatics, in conjunction with augmented circulating neutrophils, suggesting the involvement of neutrophil-derived cytokine pattern in severe asthma.     

Intestinal inflammation in children with atopic eczema: faecal eosinophil cationic protein and tumour necrosis factor-α as non-invasive indicators of food allergy

Background Food allergy is contemplated in atopic eczema. Early recognition of food allergies is difficult and the diagnosis is often missed because of the non-specificity of symptoms. New non-invasive tests are clearly needed. Objeetive and methods We measured the concentrations of tumour necrosis factor-α, eosinophil cationic protein and α-1 antitrypsin in faeces as indicators of intestinal inflammation induced by double-blind placebo-controlled oral cow's milk challenge in infants and young children with atopic eczema. Results An increased α-l antitrypsin concentration (>2mg/g) after cow's milk challenge was detected in 43% of the infants positive as compared with 11% of the infants negative to challenge P= 0.02. The concentration of eosinophil cationic protein in faeces increased after cow's milk challenge in patients positive to challenge (P=0.02) but not in those negative to challenge (P=0.79). The concentration of eosinophil cationic protein was enhanced particularly in patients manifesting immediate-type reactions to the cow's milk challenge. The concentration of tumour necrosis factor-α increased after cow's milk challenge in patients positive to challenge (P=0.005) but not in those negative to challenge (P =0.25). The concentration of tumour necrosis factor-α in faeces was enhanced particularly in patients manifesting delayed-type reactions to the cow's milk challenge. Conclusion We conclude that in children with atopic eczema food allergy is associated with intestinal inflammation indicating that more general immunologic disturbances than previously thought take place in these patients. We further suggest that faecal eosinophil cationic protein, tumour necrosis factor-α and α-1 antitrypsin distinctly indicate various reaction types of food allergy. Parallel testing with eosinophil cationic protein and tumour necrosis factor-α may signiticantly enhance the accuracy in diagnosis of food allergy in patients with atopic eczema.     

Chlamydia pneumoniae infection enhances cellular proliferation and reduces steroid responsiveness of human peripheral blood mononuclear cells via a tumor necrosis factor-α-dependent pathway

BACKGROUND: Although epidemiological studies have found an association between Chlamydia pneumoniae infection and severe asthma, the causality and underlying mechanism are largely unknown. We hypothesized that C. pneumoniae infection increases the proliferation and enhances the survival of immune and inflammatory cells, resulting in reduced responsiveness to corticosteroids and suggesting that the underlying mechanism is related to a TNF-alpha-dependent pathway. METHODS: Human peripheral blood mononuclear cells (PBMCs) were cultured in vitro in the presence or absence of C. pneumoniae infection. Responsiveness to corticosteroids was assayed by adding dexamethasone, and the underlying mechanism was investigated by treating cells with infliximab that is a chimeric anti-TNF-alpha monoclonal antibody. Cellular proliferation and apoptosis was assessed by thymidine uptake and counting apoptotic cells using flow cytometry. RESULTS: Cellular proliferation was significantly higher in C. pneumoniae-infected PBMCs than in uninfected PBMCs, which is more prominent in Th2-dominant microenvironment. The anti-proliferative and pro-apoptotic effect of corticosteroid were significantly reduced in C. pneumoniae-infected PBMCs compared with uninfected PBMCs. The proliferative effect of C. pneumoniae infection and the reduced response to corticosteroid were effectively reversed by blocking the TNF-alpha pathway at least partially. CONCLUSION: C. pneumoniae infection enhanced the proliferation and survival of immune and inflammatory cells, resulting in steroid resistance. The reversal of these phenomena by the TNF-alpha inhibitor suggests that TNF-alpha may play an important role in the induction of steroid dependence or resistance. A TNF-alpha inhibitor may therefore be a candidate agent for managing steroid-dependent or -resistant severe asthma.     

Involvement of tumor necrosis factor-α, interleukin-I β, interleukin-8, and interleukin-I receptor antagonist in acute lung injury caused by local Shwartzman reaction

A local Shwartzman reactlon (LSR) was prepared in rabbit lung as a model of acute lung injury. To induce LSR, intratracheal injection of lipopolysaccharide (LPS) 10 μg into the lower lobe of the right lung, followed 24 h later by I.v. injectlon of LPS (10 μg/kg). In the lung with the LSR, myeloperoxidase activity, representing neutrophil accumulation, peaked at 1–2 h and was sustained for 48 h after challenge with i.v. LPS. The lung water content peaked at 12 h, and decreased gradually. Histological findings showed diffuse interstitial widenlng, lntra-alveolar leukocyte infiltration with hemorrhage, and alveolar exudate formation. The production of tumor necrosis factor-α (TNF-α), interleukin-1 β (IL-1β), interleukin 8 (IL-8), and IL-1 receptor antagonist (IL-1Ra) in the lung was analyzed. TNF-α first elevated and peaked at 0.5h (66.±16.7ng/g.lung), subsequently, 11–1β and 11–8 increased and peaked at 2h (17.8 ± 3.4 ng/g. lung and 336.9±49.6ng/g.lung, respectively). IL-1Ra was present even before the challenge, and the production increased to show a dual peak (0.5 h, 1.5±0.2 μg/g.lung; and 2h, 1.6±0.1 μg/g.lung), and a large concentration of IL-1 Ra was sustained for 48 h. lmmunohistochemistry showed that the cellular source of these cytokines was alveolar macrophages and infiltrating neutrophils. Thus, disclosing the kinetics of the generation of cytokines led to a better understanding of thelr roles, namely TNF-α as an initiator, IL-1 and IL-8 as amplitier and effector, and IL-1 Ra as regulator of the intensity of acute inflammation.