Distinct roles of PI3Kδ and PI3Kγ in a toluene diisocyanate-induced murine asthma model.

TDI-induced asthma is characterized by neutrophil-dominated airway inflammation and often associated with poor responsiveness to steroid treatment. Both PI3Kδ and PI3Kγ have been demonstrated to play important proinflammatory roles in ovalbumin-induced asthma. We've already reported that blocking pan PI3K effectively attenuated TDI-induced allergic airway inflammation. Yet the specific functions of PI3Kδ and PI3Kγ in TDI-induced asthma are still unclear. Male BALB/c mice were first dermally sensitized and then challenged with TDI to generate an asthma model. Sellective inhibitors of PI3Kδ (IC-87114, AMG319) and PI3Kγ (AS252424, AS605240) were respectively given to the mice after each airway challenge. Treatment with IC-87114 or AMG319 after TDI exposure led to significantly decreased airway hyperresponsiveness (AHR), less neutrophil and eosinophil accumulation, attenuated airway smooth muscle (ASM) thickening, less M1 and M2 macrophages in lung, as well as lower levels of IL-4, IL-5, IL-6 and IL-18 in bronchoalveolar lavage fluid (BALF) and recovered IL-10 production. While mice treated with AS252424 or AS605240 had increased AHR, more severe ASM thickening, larger numbers of neutrophils and eosinophils, more M1 but less M2 macrophages, and higher BALF levels of IL-4, IL-5, IL-6, IL-10, IL-12, IL-18 when compared with those treated with vehicle. These data revealed that pharmacological inhibition of PI3Kδ attenuates TDI-induced airway inflammation while PI3Kγ inhibition exacerbates TDI-induced asthma, indicating distinct biological functions of PI3Kδ and PI3Kγ in TDI-induced asthma.

Protective effects of N-acetylcysteine on a chemical-induced murine model of asthma.

INTRODUCTION: Oxidative stress is involved in the pathophysiology of inflammatory airway diseases, including asthma. In this study, we elucidated the possible protective effects of the antioxidant N-acetylcysteine (NAC) on a toluene diisocyanate (TDI)-induced murine asthma model. METHODS: Male BALB/c mice were sensitized and challenged with TDI to generate a chemical-induced asthma model. NAC was given intraperitoneally to mice immediately after each TDI challenge. Airway reactivity to methacholine and bronchoalveolar lavage fluid was analyzed. Lungs were examined by histology. RESULTS: NAC treatment dramatically reduced the increased airway hyperresponsiveness, inflammatory infiltration, and goblet cell metaplasia in TDI-exposed mice. Numbers of total cells, neutrophils, and eosinophils in the bronchoalveolar lavage fluid of TDI-challenged mice were significantly higher than vehicle control, but the administration of NAC decreased these inflammatory cell counts. TDI exposure led to significantly increased levels of interleukin 4 (IL-4) and IL-5, which were also suppressed by NAC. In addition, diminished lung reduced oxidized glutathione ratio and superoxide dismutase activity were observed after TDI challenge, and these changes were attenuated by NAC. CONCLUSION: NAC treatment has beneficial effects in TDI-induced asthma.

Phosphatidylinositol 3-Kinase Mediates ß-Catenin Dysfunction of Airway Epithelium in a Toluene Diisocyanate-Induced Murine Asthma Model.

Cell-cell junctions are critical for the maintenance of cellular as well as tissue polarity and integrity. Yet the role of phosphatidylinositol 3-kinase (PI3K) in dysregulation of airway epithelial adherens junctions in toluene diisocyanate (TDI)-induced asthma has not been addressed. Male BALB/c mice were first dermally sensitized and then challenged with TDI by means of compressed air nebulization. The mice were treated intratracheally with PI3K inhibitor LY294002. Levels of phospho-Akt in airway epithelium and whole lung tissues were markedly increased in TDI group compared with control mice, which decreased after administration of LY294002. The dilated intercellular spaces of airway epithelium induced by TDI were partially recovered by LY294002. Both the protein expression and distribution of adherens junction proteins E-cadherin and ß-catenin were altered by TDI. Treatment with LY294002 rescued the distribution of E-cadherin and ß-catenin at cell-cell membranes, restored total ß-catenin pool, but had no effect on protein level of E-cadherin. At the same time, LY294002 also inhibited phosphorylation of ERK, glycogen synthase kinase3ß and tyrosine 654 of ß-catenin induced by TDI. In summary, our results showed that the PI3K pathway mediates ß-catenin dysregulation in a TDI-induced murine asthma model, which may be associated with increased tyrosine phosphorylation of ß-catenin.

Ethyl pyruvate decreases airway neutrophil infiltration partly through a high mobility group box 1-dependent mechanism in a chemical-induced murine asthma model.

BACKGROUND: Diisocyanates are one of the leading causes of occupational asthma, which is dominated by granulocytic inflammation in the airway. In this study, we intended to explore the role of ethyl pyruvate (EP) on neutrophil infiltration in a toluene-2,4-diisocyanate (TDI)-induced murine asthma model. METHODS: The experimental mice were first dermally sensitized and then challenged with TDI via oropharyngeal aspiration. The mice were treated intraperitoneally with 100, 50 or 10mg/kg EP 1h before each challenge. One day after the last challenge, airway reactivity to methacholine was measured by a barometric plethysmographic chamber. Total and differential cell counts, along with levels of macrophage inflammatory protein-2 (MIP-2), TNF-α in bronchoalveolar lavage (BAL) fluid and mRNA expression of CXCR2 in the lung were assessed. To depict neutrophils, a naphthol AS-D chloroacetate esterase kit was used. High mobility group box 1 (HMGB1) was determined by western blot and immunohistochemistry. RESULTS: Treatment with EP dramatically decreased airway hyperresponsiveness in TDI-challenged mice, as well as numbers of neutrophils in BAL fluid and peribronchovascular regions. Both the TDI-induced raised protein level and abnormal distribution of HMGB1 were significantly recovered by EP in a dose-dependent manner. The concentration of MIP-2 in TDI-induced asthma mice was significantly higher than that of the control ones, while EP had few effects on MIP-2. The mRNA expression of CXCR2 didn't change significantly, and TNF-α was not detected in BAL fluids. CONCLUSION: EP reduces airway neutrophil infiltration partly through downregulating HMGB1 in a chemical-induced murine asthma model.

Mechanism of E-cadherin redistribution in bronchial airway epithelial cells in a TDI-induced asthma model.

E-cadherin (epithelial cadherin), a transmembrane protein, provides essential architecture and immunological function to the airway epithelium, a barrier structure that plays an essential role in asthma pathogenesis. Toluene diisocyanate (TDI) is currently one of the leading causes of occupational asthma. However, relatively few studies have been undertaken to determine the biological effects of TDI on the barrier properties of airway epithelium, but it is known that TDI can damage airway epithelial tight junctions in vitro. Here, we hypothesize that TDI can injure E-cadherin both in normal and allergic-induced airway epithelium. To test this, we developed a murine model of TDI-induced asthma characterized by neutrophil-dominated airway inflammation, epithelial shedding, and obvious aberrant distribution of E-cadherin. Pretreatment with dexamethasone (DEX) significantly rescued the immunoreactivity of E-cadherin, accompanied by increased neutrophils in bronchoalveolar lavage fluid (BALF). In vitro, TDI-human serum albumin (HSA)-induced redistribution of E-cadherin was associated with extracellular signal-regulated kinase (ERK)1/2 activation. The inhibition of phospho-ERK (p-ERK)1/2 by DEX can partly reverse this reaction. These results indicate that E-cadherin redistribution may be an important contributor in the generation of TDI-induced asthma.

Induction of thymic stromal lymphopoietin expression in 16-HBE human bronchial epithelial cells by 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3.

Vitamin D exerts profound effects on airway epithelial cells. Thymic stromal lymphopoietin (TSLP) derived from airway epithelial cells plays a role in the innate and antigen­specific adaptive immune responses. However, the effect of vitamin D on TSLP expression in airway epithelial cells is unclear. In this study, 16-HBE human bronchial epithelial (HBE) cells were cultured with various concentrations of 25-hydroxyvitamin D(3) (25 D(3)) and 1,25-dihydroxyvitamin D(3) (1,25 D(3)). The expression of TSLP in the 16-HBE human bronchial epithelial cell line was analyzed by PCR and enzyme-linked immunosorbent assay (ELISA). We found that the 16-HBE cells converted inactive 25 D(3) to active 1,25 D(3) and that TSLP mRNA and protein expression levels were significantly increased, peaking at 2 or 12 h in the cells exposed to 500 nM 25 D(3) and 50 nM 1,25 D(3) respectively. Since vitamin D(3) upregulated protein 1 (VDUP1) plays a multifunctional role in a variety of cellular responses, we hypothesized that VDUP1 is involved in the induction of TSLP production by 25 D(3). The results showed that the mRNA and protein levels of VDUP1 were significantly upregulated by vitamin D. Furthermore, the silencing of VDUP1 by small interfering RNA (siRNA) significantly inhibited the 25 D(3)- and 1,25 D(3)-mediated induction of TSLP expression. To characterize the metabolic properties of vitamin D in airway epithelial biology, we used the chemical inhibitor of 1α-hydroxylase, itraconazole. The results revealed that itraconazole (10-6 M) reduced the 25 D(3)- but not the 1,25 D(3)-induced TSLP expression in 16-HBE cells. Based on these data, it can be concluded that vitamin D increases TSLP expression in 16-HBE cells through the VDUP1 pathway, which suggests a novel mechanism by which vitamin D alters immune function in the lungs.

[High-mobility group box protein 1 in synergy with interleukin-1ß promotes interleukin-8 expression in human airway epithelial cells in vitro].

OBJECTIVE: To test the effect of high-mobility group box protein 1 (HMGB1) alone or in synergy with interleukin-1ß (IL-1ß) on the expression of IL-8 in human airway epithelial cells in vitro. METHODS: Human airway epithelial 16HBE and A549 cell lines were incubated with HMGB1 (100 ng/ml) in the absence or presence of IL-1ß (10 ng/ml) for 24 h, and the changes of IL-8 mRNA and protein expressions were assessed using quantitative PCR and enzyme-linked immunosorbent assay (ELISA). RESULTS: In the two human airway epithelial cell lines, HMGB1 alone did not produce obvious effect on the expression of IL-8, but in the presence of IL-1ß, HMGB1 caused a significant increase of IL-8 expressions at both the mRNA and protein levels. CONCLUSION: HMGB1 in synergy with IL-1ß increases the expression of IL-8 in human airway epithelial cells, which provides new evidence that HMGB1 contributes to neutrophilic airway inflammation by regulating IL-8 expression.