MicroRNA-23a-3p Down-Regulation in Active Pulmonary Tuberculosis Patients with High Bacterial Burden Inhibits Mononuclear Cell Function and Phagocytosis through TLR4/TNF-α/TGF-ß1/IL-10 Signaling via Targeting IRF1/SP1.

The aim of this study is to explore the role of microRNAs (miR)-21/23a/146a/150/155 targeting the toll-like receptor pathway in active tuberculosis (TB) disease and latent TB infection (LTBI). Gene expression levels of the five miRs and predicted target genes were assessed in peripheral blood mononuclear cells from 46 patients with active pulmonary TB, 15 subjects with LTBI, and 17 non-infected healthy subjects (NIHS). THP-1 cell lines were transfected with miR-23a-3p mimics under stimuli with Mycobacterium TB-specific antigens. Both miR-155-5p and miR-150-5p gene expressions were decreased in the active TB group versus the NIHS group. Both miR-23a-3p and miR-146a-5p gene expressions were decreased in active TB patients with high bacterial burden versus those with low bacterial burden or control group (LTBI + NIHS). TLR2, TLR4, and interleukin (IL)10 gene expressions were all increased in active TB versus NIHS group. MiR-23a-3p mimic transfection reversed ESAT6-induced reduction of reactive oxygen species generation, and augmented ESAT6-induced late apoptosis and phagocytosis, in association with down-regulations of the predicted target genes, including tumor necrosis factor (TNF)-α, TLR4, TLR2, IL6, IL10, Notch1, IL6R, BCL2, TGF-ß1, SP1, and IRF1. In conclusion, the down-regulation of miR-23a-3p in active TB patients with high bacterial burden inhibited mononuclear cell function and phagocytosis through TLR4/TNF-α/TGF-ß1/IL-10 signaling via targeting IRF1/SP1.

Whole Genome DNA Methylation Analysis of Active Pulmonary Tuberculosis Disease Identifies Novel Epigenotypes: PARP9/miR-505/RASGRP4/GNG12 Gene Methylation and Clinical Phenotypes.

We hypothesized that DNA methylation patterns may contribute to the development of active pulmonary tuberculosis (TB). Illumina's DNA methylation 450 K assay was used to identify differentially methylated loci (DML) in a discovery cohort of 12 active pulmonary TB patients and 6 healthy subjects (HS). DNA methylation levels were validated in an independent cohort of 64 TB patients and 24 HS. Microarray analysis identified 1028 DMLs in TB patients versus HS, and 3747 DMLs in TB patients after versus before anti-TB treatment, while autophagy was the most enriched signaling pathway. In the validation cohort, PARP9 and miR505 genes were hypomethylated in the TB patients versus HS, while RASGRP4 and GNG12 genes were hypermethylated, with the former two further hypomethylated in those with delayed sputum conversion, systemic symptoms, or far advanced lesions. MRPS18B and RPTOR genes were hypomethylated in TB patients with pleural involvement. RASGRP4 gene hypermethylation and RPTOR gene down-regulation were associated with high mycobacterial burden. TB patients with WIPI2/GNG12 hypermethylation or MRPS18B/FOXO3 hypomethylation had lower one-year survival. In vitro ESAT6 and CFP10 stimuli of THP-1 cells resulted in DNA de-methylation changes of the PARP9, RASGRP4, WIPI2, and FOXO3 genes. In conclusions, aberrant DNA methylation over the PARP9/miR505/RASGRP4/GNG12 genes may contribute to the development of active pulmonary TB disease and its clinical phenotypes, while aberrant DNA methylation over the WIPI2/GNG12/MARPS18B/FOXO3 genes may constitute a determinant of long-term outcomes.

Altered pattern of monocyte differentiation and monocyte-derived TGF-ß1 in severe asthma.

CD14+ monocytes contain precursors for macrophages and fibrocytes, known to be involved in regulating airway remodeling in human asthma and distinguishable by the PM-2K marker. We sought to identify circulating subsets of PM-2K+ macrophage-like cells and evaluate their relationships to lung function, severity and control status. Circulating PM-2K+ macrophage-like cells and fibrocytes could be identified and distinguished between normal individuals (N = 152) and asthmatic subjects (N = 133) using multi-parametric flow cytometry. PM-2K+ macrophage-like cells were found to be significantly lower in asthmatic subjects, particularly noted for the CD14-PM-2K+ subset and PM-2K+CCR7-CD86+ cells in subjects with poor lung function (FEV%/FVC% < 80%) as compared to those of normal subjects and asthmatics with normal lung function, whereas the frequency of fibrocytes was higher in asthmatics and the CCR7-CD86+ subset distribution was significantly different in subjects with varying severity. Moreover, exogenous transforming growth factor beta 1 (TGF-ß1) was found to inhibit the generation of PM-2K+ macrophage-like cells, but promote the growth of fibrocytes, from CD14+ monocytes, and monocyte-derived TGF-ß1 was found to correlate with the lung function, severity and control status in asthmatic patients. Collectively, aberrant differentiation of monocytes into PM-2K+ macrophage-like cell subsets and fibrocytes, together with increased monocyte-derived TGF-ß1, characterized patients with severe asthma.

Whole genome gene expression changes and hematological effects of rikkunshito in patients with advanced non-small cell lung cancer receiving first line chemotherapy.

It has been demonstrated that the traditional Chinese medicine rikkunshito, ameliorates anorexia in several types of human cancer and attenuates lung injury by inhibiting neutrophil infiltration. The current study investigated the clinical and hematological effects of rikkunshito and its underlying mechanisms of action in the treatment of advanced non-small cell lung cancer (NSCLC). The Illumina microarray BeadChip was used to analyze the whole-genome expression profiles of peripheral blood mononuclear cells in 17 patients with advanced NSCLC. These patients were randomized to receive combination chemotherapy (cisplatin and gemcitabine) with (n=9, CTH+R group) or without (n=8, CTH group) rikkunshito. The primary endpoint was the treatment response and the categories of the scales of anorexia, nausea, vomiting and fatigue; secondary endpoints included the hematological effect and whole genome gene expression changes. The results of the current study indicated that there were no significant differences in clinical outcomes, including treatment response and toxicity events, between the two groups. Median one-year overall survival (OS) was 12 months in the CTH group and 11 months in the CTH+R group (P=0.058 by log-rank test), while old age (>60 years old) was the only independent factor associated with one-year OS (hazard ratio 1.095, 95% confidence interval, 1.09-1.189, P=0.030). Patients in the CTH+R group experienced significantly greater maximum decreases in both white cell count (P=0.034) and absolute neutrophil count (P=0.030) from the baseline. A total of 111 genes associated with neutrophil apoptosis, the cell-killing ability of neutrophils, natural killer cell activation and B cell proliferation were up-regulated following rikkunshito treatment. A total of 48 genes associated with neutrophil migration, coagulation, thrombosis and type I interferon signaling were down-regulated following rikkunshito treatment. Rikkunshito may therefore affect the blood neutrophil count when used with combination chemotherapy in patients with NSCLC, potentially by down-regulating prostaglandin-endoperoxidase synthase 1, MPL, AMICA1 and junctional adhesion molecule 3, while up-regulating elastase, neutrophil expressed, proteinase 3, cathepsin G and cluster of differentiation 24.

Aberrant Toll-like receptor 2 promoter methylation in blood cells from patients with pulmonary tuberculosis.

OBJECTIVES: Toll-like receptor 2 (TLR2) is a major mediator of innate immunity against tuberculosis (TB). This study aimed to determine if TLR2 promoter DNA methylation is associated with pulmonary TB. METHODS: The DNA methylation levels of 20 CpG sites over the TLR2 promoter region and TLR2 gene/protein expressions of immune cells of the blood were examined in 99 sputum culture-positive pulmonary TB patients and 77 healthy subjects (HS). RESULTS: TB patients had higher methylation levels over five CpG sites (3, 7, 9, 13, and 18), lower TLR2 gene expression, lower TLR2 expression on monocyte, higher TLR2 expression on NK cell, and higher serum TNF-α/IFN-γ levels than HS after adjusting for confounding factors. Patients with a high bacillary load had lower methylation levels at CpG-15, -17, and -20. Patients with drug-resistant TB had higher CpG-18 methylation levels and lower TLR2 expression on NK cell. Patients with far advanced lesion on chest radiograph had higher serum TNF-α level and higher TLR2 expression on NK cell. Patients with a high TLR2 expression on NK cell had lower one-year survival. CpG-18 methylation level, TLR2 expressions on monocyte/NK cell, and TNF-α/IFN-γ levels were all reversed to normal after 6-month anti-TB treatment. CONCLUSIONS: Aberrant methylation of certain CpG sites over TLR2 promoter region is associated with active pulmonary TB or its phenotypes, probably through the down-regulation of TLR2 expression.

Peripheral immune cell gene expression changes in advanced non-small cell lung cancer patients treated with first line combination chemotherapy.

INTRODUCTION: Increasing evidence has shown that immune surveillance is compromised in a tumor-promoting microenvironment for patients with non-small cell lung cancer (NSCLC), and can be restored by appropriate chemotherapy. METHODS: To test this hypothesis, we analyzed microarray gene expression profiles of peripheral blood mononuclear cells from 30 patients with newly-diagnosed advanced stage NSCLC, and 20 age-, sex-, and co-morbidity-matched healthy controls. All the patients received a median of four courses of chemotherapy with cisplatin and gemcitabine for a 28-day cycle as first line treatment. RESULTS: Sixty-nine differentially expressed genes between the patients and controls, and 59 differentially expressed genes before and after chemotherapy were identified. The IL4 pathway was significantly enriched in both tumor progression and chemotherapy signatures. CXCR4 and IL2RG were down-regulated, while DOK2 and S100A15 were up-regulated in the patients, and expressions of all four genes were partially or totally reversed after chemotherapy. Real-time quantitative RT-PCR for the four up-regulated (S100A15, DOK2) and down-regulated (TLR7, TOP1MT) genes in the patients, and the six up-regulated (TLR7, CRISP3, TOP1MT) and down-regulated (S100A15, DOK2, IL2RG) genes after chemotherapy confirmed the validity of the microarray results. Further immunohistochemical analysis of the paraffin-embedded lung cancer tissues identified strong S100A15 nuclear staining not only in stage IV NSCLC as compared to stage IIIB NSCLC (p = 0.005), but also in patients with stable or progressive disease as compared to those with a partial response (p = 0.032). A high percentage of S100A15 nuclear stained cells (HR 1.028, p = 0.01) was the only independent factor associated with three-year overall mortality. CONCLUSIONS: Our results suggest a potential role of the IL4 pathway in immune surveillance of advanced stage NSCLC, and immune potentiation of combination chemotherapy. S100A15 may serve as a potential biomarker for tumor staging, and a predictor of poor prognosis in NSCLC.

High tidal volume mechanical ventilation elicits increased activity in protein kinase B and c-Jun NH2-terminal kinase pathways in mouse diaphragm.

PURPOSE: Unloading of the diaphragm via mechanical ventilation for more than 5 days leads to weaning difficulties. Mechanical ventilation can induce production of inflammatory cytokines and extracellular matrix proteins. The mechanisms regulating interactions between mechanical ventilation and diaphragmatic injury are unclear. We hypothesized that high tidal volume mechanical stretch augmented diaphragmatic injury via serine/threonine kinase/protein kinase B (Akt) and c-Jun NH(2)-terminal kinase (JNK) pathways. METHODS: Male C57BL/6, either wild type or Akt deficient, weighing between 20 and 25 g, were exposed to high tidal volume (30 ml/kg) or low tidal volume (6 ml/kg) mechanical ventilation with room air for 2-8 h. RESULTS: High tidal volume mechanical ventilation induced Akt, JNK, and class O of forkhead box transcription factor 4 (Foxo4) activation in a time-dependent manner. Disruption and atrophy of muscle fibers in the diaphragm, positive staining of phospho-Akt in the myofiber membrane, and increased production of free radicals were also found. Mechanical ventilation of Akt-deficient mice resulted in attenuated diaphragmatic injury, Akt, JNK, and Foxo4 activation, and free radical production. CONCLUSIONS: Our data suggest that high tidal volume mechanical ventilation produces diaphragmatic muscle damage and free radical production through activation of the Akt and JNK pathways.

Down-regulation of insulin-like growth factor I (IGF-I) in the mouse diaphragm during sepsis.

BACKGROUND: Diaphragmatic muscle impairment is an important cause of respiratory failure during sepsis. Insulin-like growth factor I (IGF-I) is an anabolic growth factor which prevents muscle degradation and wasting during sepsis, but its role in the diaphragmatic muscle during sepsis is unknown. The aim of this study was to investigate the expression of IGF-I in the diaphragmatic muscle in a murine model of sepsis induced by lipopolysaccharide (LPS). METHODS: Male B57 mice were peritoneally injected with LPS, and were killed and studied at different time-points, 24 h, 48 h, 72 h, and 96 h after injection. Diaphragm sarcolemmal damage was visualized by orange tracer dye infusion, and the expression of IGF-I, interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) in diaphragm tissue extracts were measured using ELISA. RESULTS: LPS induced sarcolemmal damage in diaphragm myofibers from 24 h to 96 h, which was accompanied by a significant increase in IL-1ß expression in the tissues while IGF-I levels were down-regulated. No change in TNF-α was observed. Body weights of animals were also reduced, especially at 96 h. CONCLUSIONS: The expression of IGF-I in diaphragm tissues was down-regulated during sepsis- induced diaphragm myofiber damage, suggesting that IGF-I may be an important factor in the regulation of diaphragm myofiber repair. Further studies are needed to examine the mechanisms involved.

Risks of exposure to occupational asthmogens in atopic and nonatopic asthma: a case-control study in Taiwan.

RATIONALE: Asthma is often work-related and can be classified as atopic or nonatopic on the basis of its pathogenesis. Few studies have reported an association between exposure to occupational asthmogens and asthma with and without atopy. OBJECTIVES: We investigated, in adults with asthma, whether occupational exposure to asthmogens influenced the risk of having atopic or nonatopic asthma, and their level of lung function. METHODS: We recruited 504 hospital-based adults with current asthma, 504 community-based control subjects, and 504 hospital-based control subjects in southern Taiwan. Asthma with atopy was defined as having asthma in combination with an increase in total IgE (≥100 U/ml) or a positive Phadiatop test (≥0.35 Pharmacia arbitrary unit/L) (Pharmacia ImmunoCAP; Pharmacia, Uppsala, Sweden). Occupational exposure to asthmogens was assessed with an asthma-specific job exposure matrix. MEASUREMENTS AND MAIN RESULTS: We found a significant association between atopic asthma and exposure to high molecular weight asthmogens (adjusted odds ratio [AOR], 4.0; 95% confidence interval [CI], 1.8-8.9). Nonatopic asthma was significantly associated with exposure to low molecular weight asthmogens (AOR, 2.6; 95% CI, 1.6-4.3), including industrial cleaning agents and metal sensitizers. Agriculture was associated with both atopic and nonatopic asthma (AOR, 7.8; 95% CI, 2.8-21.8; and AOR, 4.1; 95% CI, 1.3-13.0, respectively). The ratio of FEV1 to FVC in the high-risk group was significantly lower than in the no-risk group (P = 0.026) in currently employed patients with asthma. CONCLUSIONS: In adults with asthma, occupational exposure to high and low molecular weight asthmogens appears to produce differential risks for atopic and nonatopic asthma.

Role of cathepsin S in ozone-induced airway hyperresponsiveness and inflammation.

Ambient ozone has been linked to the worsening of symptoms of patients with obstructive diseases such as chronic obstructive pulmonary disease (COPD) and asthma. We investigated the role of cathepsin S on ozone-induced airway hyperresponsiveness (AHR) and inflammation, using the selective cathepsin S inhibitor, Compound A. Balb/c mice were exposed to ozone at a concentration of 3 ppm or air for 3 h, following administration by gavage of Compound A or vehicle. Bronchoalveolar lavage (BAL) was performed 3 h and 20-24 h following exposure, AHR was measured at 20-24 h only. Ozone exposure, compared to air exposure increased BAL cathepsin S levels, AHR and BAL inflammatory cells. Compound A (30 mg kg(-1) p.o.) dosing compared to vehicle dosing inhibited ozone-induced AHR (-logPC100 vehicle: -0.70+/-0.12, n=8 vs. cathepsin S inhibitor: -1.30+/-0.06, P<0.001, n=8) at 20-24 h and BAL neutrophilia at 3 h and 20-24 h (P<0.05, n=6). Ozone exposure increased levels of BAL cytokines IL-6, TNF-alpha and IFN-gamma. Compound A reduced IL-6 at 3 h and 20-24 h (P<0.05, n=5) and TNF-alpha, at 20-24 h (P<0.05, n=6). These data indicate an important role for cathepsin S in the regulation of ozone-induced AHR and neutrophil cell recruitment and suggest that cathepsin S may be a target in the treatment of oxidative stress-induced AHR and inflammation.

Role of TLR2, TLR4, and MyD88 in murine ozone-induced airway hyperresponsiveness and neutrophilia.

Exposure to air pollutants such as ozone (O(3)) induces airway hyperresponsiveness (AHR) and airway inflammation. Toll-like receptors (TLR) are first-line effector molecules in innate immunity to infections and signal via adapter proteins, including myeloid differentiation factor-88 (MyD88). We investigated the sensing of ozone by TLR2, TLR4, and MyD88. Ozone induced AHR in wild-type (WT) C57BL/6 mice, but AHR was absent in TLR2(-/-), TLR4(-/-), and MyD88(-/-) mice. Bronchoalveolar lavage neutrophilia induced by ozone was inhibited at 3 h but not at 24 h in TLR2(-/-) and TLR4(-/-) mice, while in MyD88(-/-) mice, this was inhibited at 24 h. We investigated the expression of inflammatory cytokines and TLR2, TLR4, and MyD88 in these mice. Ozone induced time-dependent increases in inflammatory gene expression of keratinocyte chemoattractant (KC) and IL-6 and of TLR2, TLR4, and MyD88 in WT mice. IL-6 and KC expression induced by ozone was inhibited in TLR2(-/-), TLR4(-/-), and MyD88(-/-) mice. Expression of MyD88 was increased in TLR2(-/-) and TLR4(-/-) mice, while induction of TLR2 or TLR4 was reduced in TLR2(-/-) and TLR4(-/-) mice, respectively. TLR2 and TLR4 mediate AHR induced by oxidative stress such as ozone, while the adapter protein MyD88, but not TLR2 or TLR4, is important in mediating ozone-induced neutrophilia. TLR2 and TLR4 may also be important in regulating the speed of neutrophilic response. Therefore, ozone may induce murine AHR and neutrophilic inflammation through the activation of the Toll-like receptor pathway that may sense noninfectious stimuli such as oxidative stress.

Attenuation of ozone-induced airway inflammation and hyper-responsiveness by c-Jun NH2 terminal kinase inhibitor SP600125.

Ozone has potent oxidizing properties, and exposure to ozone causes airway hyper-responsiveness (AHR) and lung inflammation. We determined the importance of c-Jun NH(2) terminal kinase (JNK), a member of the mitogen-activated protein kinase pathway, in ozone-induced AHR and inflammation. SP600125 [anthra[1,9-cd] pyrazol-6 (2H)-one], a specific JNK inhibitor (30 mg/kg) or vehicle, was administered by intraperitoneal injection before and after ozone exposure (3 ppm for 3 h). SP600125 significantly reduced total cells, and neutrophils in bronchoalveolar fluid recovered at 20 to 24 h after exposure and inhibited ozone-induced AHR. Ozone exposure induced activation of JNK in the lung as measured by the expression of phosphorylated-c-Jun, an effect abolished by SP600125. Gene-microarray analysis revealed that ozone increased the expression of over 400 genes by more than 2-fold, including interleukin-6 (IL-6), CXCL1 (keratinocyte cytokine), and CCL2 (monocyte chemoattractant protein-1). SP600125 modulated the expression of a subset of 29 ozone-induced genes; IL-6 and CCL2 expression were further increased, whereas the expression of metallothionein 1, hemopexin, and mitogen-activated 3 kinase 6 was decreased in SP600125-treated ozone-exposed mice. Changes in mRNA for IL-6, CXCL1, and CCL2 were confirmed by real-time polymerase chain reaction. Ozone also decreased the expression of over 500 genes, with the most potent effect on angiopoietin-1. SP600125 modulated the expression of 15 of these genes, and in particular, SP600125 reversed ozone-induced decrease in expression of the redox-sensitive transcription factor, hypoxia-induced factor-1alpha. This study highlights an important role for JNK in response to oxidative stress through modulation of specific inflammatory and redox mediators. Inhibition of JNK with small molecule kinase inhibitors may be a means of reducing ozone-induced inflammation and AHR.

Repeated allergen inhalation induces cytoskeletal remodeling in smooth muscle from rat bronchioles.

Airway hyperresponsiveness (AHR) is associated with airway wall structural remodeling and alterations in airway smooth muscle (ASM) function. Previously, in bronchioles from Brown Norway rats challenged by repeated ovalbumin (OVA) inhalation, we have reported increased force generation and depletion of smooth muscle contractile proteins. Here, we investigated if cytoskeletal changes in smooth muscle could account for this paradox. Sensitized rats (n = 5/group) were repeatedly challenged with OVA or saline, and the lungs were removed 24 h after the last challenge. Levels of globular (G) and filamentous (F) actin in bronchioles were determined by DNase I inhibition and contraction assessed in intact small bronchioles using a myograph. DNase I inhibition assays showed that G-actin monomers were more abundant ( approximately 1F:2G) in extracts from resting small bronchioles from OVA- or saline-challenged animals. However, while contractile protein levels in bronchioles were reduced by OVA (P < 0.05), the proportion of F:G actin was 1.8-fold greater compared with saline challenge (P < 0.05). Consistent with induction of F-actin after OVA challenge, increases in maximum tension development to carbachol or KCl in small bronchioles from OVA-challenged animals were abrogated (P < 0.01) by actin cytoskeleton disruption with 0.5 microM latrunculin A. Cytoskeletal stabilization of F-actin with 0.1 microM jasplakinolide potentiated maximum contractions to carbachol or KCl (P < 0.05) in bronchioles from OVA- but not saline-treated rats. We conclude that alterations in the composition and/or arrangement of the contractile apparatus after OVA exposure confer enhanced contractile responses, possibly as a result of increased F-actin content. Such a mechanism may have relevance for AHR found in allergic asthma.

Effects of ciclesonide and fluticasone propionate on allergen-induced airway inflammation and remodeling features.

BACKGROUND: Several topical corticosteroids are available as anti-inflammatory treatment for asthma. Their comparative effects on allergic inflammation and airway remodeling are unclear. OBJECTIVE: We compared the effects of ciclesonide with those of fluticasone propionate in a Brown Norway rat model of chronic allergic asthma. METHODS: Rats sensitized and exposed to ovalbumin (OVA) were treated with dry powder vehicle, ciclesonide, or fluticasone (0.01, 0.03, and 0.1 mg/kg administered intratracheally) 24 hours and 1 hour before each of 6 OVA exposures. In a second protocol we administered 0.1 mg/kg ciclesonide or fluticasone only after the third OVA exposure. RESULTS: Ciclesonide at all doses inhibited the allergen-induced increase in airway eosinophils and T cells, reduced goblet cell hyperplasia, and decreased 5-bromo-2'-deoxyuridine-immunoreactive airway smooth muscle (ASM) and epithelial cells. At 0.03 and 0.1 mg/kg ciclesonide, bronchial hyperresponsiveness (BHR) was also inhibited. Fluticasone did not attenuate allergen-induced BHR, despite inhibiting airway eosinophils and T cells, goblet cell hyperplasia, and 5-bromo-2'-deoxyuridine-immunoreactive ASM and epithelial cells. Fluticasone (0.1 mg/kg) caused a significant reduction in body weight (9%) compared with ciclesonide (0.1 mg/kg). Ciclesonide did not change plasma corticosterone levels, whereas fluticasone (0.1 mg/kg) reduced them. In the second protocol both fluticasone and ciclesonide inhibited BHR, bronchial inflammation, goblet cell hyperplasia, and ASM proliferation. CONCLUSION: Ciclesonide potently inhibited chronic allergic inflammation, remodeling, and BHR without having an effect on body weight and the hypothalamic-pituitary-adrenal axis. Fluticasone prevented airway inflammation but not BHR, but both fluticasone and ciclesonide are effective at reversal of BHR, inflammation, and remodeling features.

Effect of an inhibitor of Jun N-terminal protein kinase, SP600125, in single allergen challenge in sensitized rats.

Jun N-terminal kinase (JNK) has been implicated in the pathogenesis of inflammatory diseases including asthma. We examined the effect of SP600125 (anthra [1,9-cd] pyrazol-6 (2H)-one), a novel inhibitor of JNK in a model of asthma. Brown-Norway rats were sensitized to ovalbumin and treated with SP600125 intraperitoneally (90 mg/kg in total). SP600125 inhibited allergen-induced, increased activity of phosphorylated c-jun but not of phosphorylated-MAPKAPK2, indicative of activation of p38 MAPK, in the lung. SP600125 inhibited macrophage (P < 0.04), lymphocyte (P < 0.05), eosinophil (P < 0.04) and neutrophil (P < 0.005) numbers in bronchoalveolar lavage. Eosinophil and T-cell accumulation in the airways, mRNA expression for interleukin-1beta, tumour necrosis factor-beta, interleukin-3, interleukin-4 and interleukin-5, serum levels of allergen-specific immunoglobulin E and bronchial hyperresponsiveness were not affected by SP600125. Selective inhibition of JNK reduced inflammatory cell egress into the airway lumen after single allergen exposure. The role of JNK mitogen-activated protein kinase activation may be limited in the pathogenesis of bronchial hyperresponsiveness after single allergen exposure.