A possible association of EMID2 polymorphisms with aspirin hypersensitivity in asthma

Aspirin-intolerant asthma (AIA) is an asthma phenotype characterized by the development of bronchoconstriction following ingestion of aspirin. Despite the well-defined pathological trigger, the underlying mechanisms of AIA are still unclear. With the biophysical characteristics of the human EMI domain-containing protein 2 (EMID2) gene in relation to the extracellular matrix deposition and epithelial-mesenchymal transition as pivotal characteristics of airway remodeling in asthma, we hypothesized that genetic polymorphisms of EMID2 might affect the development of AIA. In this study, the allelic associations of 49 single-nucleotide polymorphisms (SNPs) of the human EMID2 gene were evaluated from 163 AIA patients and 429 aspirin-tolerant asthma (ATA) subjects as controls in a Korean population. Logistic analysis showed that five SNPs (P?=?0.01-0.04, but P (corr)?>?0.05) and EMID2_BL2_ht2 haplotype (unique to the minor alleles of rs4727494 and rs13233066; P?=?0.02; P (corr)?=?0.02) were significantly associated with AIA. More interestingly, regression analysis of the decline of forced expiratory volume in one second (FEV(1)) by aspirin provocation revealed that 10 SNPs (P?=?0.003-0.04) and four relevant haplotypes (P?=?0.002-0.02) were significantly associated with the fall rate of FEV(1) by aspirin provocation, indicating that genetic polymorphisms of EMID2 could cause meaningful deficits in the upper and lower airways among AIA patients. These findings provide evidence that EMID2 may be a susceptible genetic factor for aspirin hypersensitivity among asthmatics in Korean population.     

Genome-wide and follow-up studies identify CEP68 gene variants associated with risk of aspirin-intolerant asthma

Aspirin-intolerant asthma (AIA) is a rare condition that is characterized by the development of bronchoconstriction in asthmatic patients after ingestion of non-steroidal anti-inflammatory drugs including aspirin. However, the underlying mechanisms of AIA occurrence are still not fully understood. To identify the genetic variations associated with aspirin intolerance in asthmatics, the first stage of genome-wide association study with 109,365 single nucleotide polymorphisms (SNPs) was undertaken in a Korean AIA (n = 80) cohort and aspirin-tolerant asthma (ATA, n = 100) subjects as controls. For the second stage of follow-up study, 150 common SNPs from 11 candidate genes were genotyped in 163 AIA patients including intermediate AIA (AIA-I) subjects and 429 ATA controls. Among 11 candidate genes, multivariate logistic analyses showed that SNPs of CEP68 gene showed the most significant association with aspirin intolerance (P values of co-dominant for CEP68, 6.0×10⁻⁵ to 4.0×10⁻⁵). All seven SNPs of the CEP68 gene showed linkage disequilibrium (LD), and the haplotype of CEP68_ht4 (T-G-A-A-A-C-G) showed a highly significant association with aspirin intolerance (OR  = 2.63; 95% CI  = 1.64–4.21; P = 6.0×10⁻⁵). Moreover, the nonsynonymous CEP68 rs7572857G>A variant that replaces glycine with serine showed a higher decline of forced expiratory volume in 1s (FEV₁) by aspirin provocation than other variants (P = 3.0×10⁻⁵). Our findings imply that CEP68 could be a susceptible gene for aspirin intolerance in asthmatics, suggesting that the nonsynonymous Gly74Ser could affect the polarity of the protein structure.     

Vasoactive intestinal peptide enhances wound healing and proliferation of human bronchial epithelial cells

In the present study, we investigated the effects of vasoactive intestinal peptide (VIP) on wound healing of bronchial epithelium. Wound healing of the mechanical damaged human bronchial epithelial cells (HBEC) was observed in the absence or presence of VIP. Effects of VIP on chemotactic migration, cell proliferation of HBEC were also tested. HBEC chemotaxis was assessed by the blind well chamber technique, the cell cycle was determined by flow cytometry, and cell proliferation was determined by measuring the expression of proliferating cell nuclear antigen Ki67. Effects of VIP on epithelial E-cadherins protein and mRNA were also measured by immunohistochemistry and RT-PCR. The results showed that VIP accelerated the recovery of wound area of HBEC. VIP increased the migration and proliferation of HBEC, and these effects were blocked by a VPAC1 receptor antagonist. VIP also increased the expression of E-cadherin mRNA and protein in HBEC, suggesting that protective effects of VIP on wound healing may be related to its ability to increase the expression of E-cadherin. In conclusion, VIP has protective effects against human bronchial epithelial cell damage, and the beneficial effects of VIP might be mediated, at least in part, by VPAC1, and associated with increased expression of E-cadherin.     

Diesel exhaust particle-treated human bronchial epithelial cells upregulate Jagged-1 and OX40 ligand in myeloid dendritic cells via thymic stromal lymphopoietin

Ambient particulate matter, including diesel exhaust particles (DEP), promotes the development of allergic disorders. DEP increase oxidative stress and influence human bronchial epithelial cell (HBEC)-dendritic cell interactions via cytokines, including thymic stromal lymphopoietin (TSLP). Upregulation of TSLP results in Th2 responses. Using primary culture HBEC and human myeloid dendritic cell (mDC) cocultures, we show in this study that DEP upregulation of Th2 responses occurred via HBEC-dependent mechanisms that resulted from oxidative stress. Moreover, DEP-treated HBEC and ambient particulate matter-treated HBEC upregulated OX40 ligand (OX40L) and the Notch ligand Jagged-1 mRNA and expression on mDC. Upregulation of OX40L as well as Jagged-1 on mDC required HBEC and did not occur in the presence of N-acetylcysteine. Furthermore, OX40L and Jagged-1 upregulation was inhibited when HBEC expression of TSLP was silenced. Thus, DEP treatment of HBEC targeted two distinct pathways in mDC that were downstream of TSLP expression. Upregulation of OX40L and Jagged-1 by mDC resulted in mDC-driven Th2 responses. These studies expand our understanding of the mechanism by which ambient pollutants alter mucosal immunity and promote disorders such as asthma.     

TLR3, TLR4 and TLRs7–9 Induced Interferons Are Not Impaired in Airway and Blood Cells in Well Controlled Asthma

Defective Rhinovirus induced interferon-β and interferon-λ production has been reported in bronchial epithelial cells from asthmatics but the mechanisms of defective interferon induction in asthma are unknown. Virus infection can induce interferon through Toll like Receptors (TLR)3, TLR7 and TLR8. The role of these TLRs in interferon induction in asthma is unclear. This objective of this study was to measure the type I and III interferon response to TLR in bronchial epithelial cells and peripheral blood cells from atopic asthmatics and non-atopic non-asthmatics. Bronchial epithelial cells and peripheral blood mononuclear cells from atopic asthmatic and non-atopic non-asthmatic subjects were stimulated with agonists to TLR3, TLR4 & TLRs7–9 and type I and III interferon and pro-inflammatory cytokine, interleukin(IL)-6 and IL-8, responses assessed. mRNA expression was analysed by qPCR. Interferon proteins were analysed by ELISA. Pro-inflammatory cytokines were induced by each TLR ligand in both cell types. Ligands to TLR3 and TLR7/8, but not other TLRs, induced interferon-β and interferon-λ in bronchial epithelial cells. The ligand to TLR7/8, but not those to other TLRs, induced only type I interferons in peripheral blood mononuclear cells. No difference was observed in TLR induced interferon or pro-inflammatory cytokine production between asthmatic and non-asthmatic subjects from either cell type. TLR3 and TLR7/8,, stimulation induced interferon in bronchial epithelial cells and peripheral blood mononuclear cells. Interferon induction to TLR agonists was not observed to be different in asthmatics and non-asthmatics.     

Wound repair and proliferation of bronchial epithelial cells regulated by CTNNAL1

Adhesion molecules play vital roles in airway hyperresponsiveness (AHR) or airway inflammation. Our previous study indicated that adhesion molecule catenin alpha-like 1 (CTNNAL1) is relevant closely to asthma susceptibility, but its biological function or significance is still unclear. In the present study, we observed the temporal and spatial distribution of CTNNAL1 expression in mouse lung tissue with the OVA-sensitized asthma model and found that the level of CTNNAL1 mRNA showed a prominent negative correlation with pulmonary resistance (R(L)). To study the function of CTNNAL1 in airway, effects of CTNNAL1 on proliferation and wound repair activity of human bronchial epithelial cells (HBEC) was investigated with antisense oligonucleotide (ASO) technique. The results showed that: (1) CTNNAL1 ASO could decelerate the repairing velocity and proliferation of HBEC; (2) CTNNAL1 expression was increased on the edge cells of mechanic wounded area in culture; (3) extracellular matrix component fibronectin (Fn) obviously promoted wound repair activity and proliferation of HBEC, which could be blocked by CTNNAL1 ASO; (4) Western blot showed that Fn could promote FAK phosphorylation, which also be inhibited by CTNNAL1 ASO. In conclusion, the level of CTNNAL1 mRNA expression is highly correlated to airway resistance; CTNNAL1 may contribute to the wound repair and proliferation of HBEC. Furthermore, it may serve to Fn mediated cell-extracellular adhesion and its signal transduction.     

CXCR3 surface expression in human airway epithelial cells: cell cycle dependence and effect on cell proliferation

We recently demonstrated that human bronchial epithelial cells (HBEC) constitutively express the CXC chemokine receptor CXCR3, which when activated, induces directed cell migration. The present study in HBEC examined the relative expression of the CXCR3 splice variants CXCR3-A and -B, cell cycle dependence of CXCR3 expression, and the effects of the CXCR3 ligand, the interferon-gamma-inducible CXC chemokine I-TAC/CXCL11, on DNA synthesis and cell proliferation. Both CXCR3-A and -B mRNA, assessed by real-time RT-PCR, were expressed in normal HBEC (NHBEC) and the HBEC line 16-HBE. However, CXCR3-B mRNA was 39- and 6-fold greater than CXCR3-A mRNA in NHBEC and 16-HBE, respectively. Although most HBEC (>80%) assessed by flow cytometry and immunofluorescence microscopy contained intracellular CXCR3, only a minority (<40%) expressed it on the cell surface. In this latter subset of cells, most (>75%) were in the S + G(2)/M phases of the cell cycle. Stimulation of CXCR3 with I-TAC enhanced thymidine incorporation and cell proliferation and increased p38 and ERK1/2 phosphorylation. These data indicate that 1) human airway epithelial cells primarily express CXCR3-B mRNA, 2) surface expression of CXCR3 is largely confined to the S + G(2)/M phases of the cell cycle, and 3) activation of CXCR3 induces DNA synthesis, cell proliferation, and activation of MAPK pathways. We speculate that activation of CXCR3 exerts a mitogenic effect in HBEC, which may be important during airway mucosal injury in obstructive airway diseases such as asthma and chronic obstructive pulmonary disease.     

Contribution of the OBSCN nonsynonymous variants to aspirin exacerbated respiratory disease susceptibility in Korean population

Airway remodeling and exacerbated airway narrowing in asthma have been attributed to the regulation of intracellular Ca(2+) by sarcoplasmic reticulum (SR) of the airway smooth muscle cells. The protein encoded by obscurin, cytoskeletal calmodulin and titin-interacting RhoGEF (OBSCN) is a crucial factor in determining the SR architecture in Obscn(-/-) mice. This study genotyped a total of 55 common single-nucleotide polymorphisms (SNPs) in 592 Korean asthmatics including 163 aspirin exacerbated respiratory disease (AERD) cases and 429 aspirin-tolerant asthma (ATA) controls. Eight SNPs, including two nonsynonymous polymorphisms rs1188722C>T (Leu2116Phe) and rs1188729G>C (Cys4642Ser), and one haplotype BL2_ht1 showed statistically significant associations with AERD development (p=0.003-0.03). Two variants, rs1188722C>T (Leu2116Phe) and rs369252C>A, also revealed nominal association with FEV1 decline by aspirin provocation in asthmatics (p=0.03-0.04). Intriguingly, rs1188722C>T (Leu2116Phe) is a highly conserved amino acid residue among species, suggesting its functional relevance to AERD. In addition, the A allele of rs369252C>A, which was more prevalent in AERD than in ATA, was predicted as a potential branch point (BP) site for alternative splicing (BP score=4.29). Although further functional evaluation is required, our findings suggest that OBSCN polymorphisms, in particular, highly conserved nonsynonymous Leu2116Phe variant, might contribute to aspirin hypersensitivity in asthmatics.     

Effects of prototypical drug-metabolizing enzyme inducers on mRNA expression of housekeeping genes in primary cultures of human and rat hepatocytes

Quantitative real-time RT-PCR was used to investigate the effects of prototypical drug-metabolizing enzyme inducers rifampicin (Rif), dexamethasone (Dex), and omeprazole (Ome) on mRNA expression levels of the housekeeping genes beta-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-glucuronidase (GUSB), hypoxanthine phosphoribosyltransferase 1 (HPRT1), peptidylprolylisomerase A (PPIA), TATA box binding protein (TBP), and transferrin receptor (TFRC) in primary cultures of cryopreserved human and rat hepatocytes. The mRNA levels of ACTB, GAPDH, GUSB, PPIA, TBP, and TFRC relative to HPRT1 in human hepatocytes were constant at all concentrations of inducers. However, the mRNA level of GAPDH relative to HPRT1 in rat hepatocytes was markedly increased by Rif. The mRNA levels of GAPDH, GUSB, PPIA, TBP, and TFRC relative to HPRT1 in rat hepatocytes were significantly increased by Dex. ACTB and HPRT1 are suitable internal controls for evaluating mRNA expression levels in primary cultures of human and rat hepatocytes after Rif, Dex, or Ome exposure.     

Histological evidence: housekeeping genes beta-actin and GAPDH are of limited value for normalization of gene expression

Housekeeping genes are widely used as internal controls for gene expression normalization for western blotting, northern blotting, RT-PCR, etc. They are generally thought to be expressed in all cells of the organism at similar levels because it is assumed that these genes are required for the maintenance of basic cellular function as constitutive genes. However, real-time RT-PCR experiments revealed that their expression may vary depending on the developmental stage, type of tissue examined, experimental condition, and so on. To date, no histological data on their expression are available for embryonic development. In the present study, we compared the histological expression profile of two commonly used housekeeping genes, GAPDH and beta-actin, in the developing chicken embryo by using section and whole mount in situ hybridization supplemented by RT-PCR. Our results show that neither GAPDH mRNA nor beta-actin mRNA is expressed in all cell types or tissues at high levels. Strikingly, expression levels are very low in some organs. Moreover, the two genes show partially complementary expression patterns in the liver, the vascular system and the digestive tract. For example, GAPDH is more strongly expressed in the liver than beta-actin, but at lower levels in the arteries. Vice versa, beta-actin is more strongly expressed in the gizzard than GAPDH, but it is almost absent from cardiac muscle cells. Researchers should consider these histological results when using GAPGD and beta-actin for gene expression normalization in their experiments.     

Intracellular signaling molecules involved in vasoactive intestinal peptide-mediated wound healing in human bronchial epithelial cells

Vasoactive intestinal peptide (VIP), a non-adrenergic, non-cholinergic neuromediator, plays an important role in maintaining the bronchial tone of the airway and has anti-inflammatory properties. Recently, we reported that VIP enhances wound repair in human bronchial epithelial cells (HBEC). In the present study, we have identified the intracellular signaling molecules that are involved in VIP-mediated wound healing in HBEC. The effects of VIP on wound repair of HBEC were partially blocked by H-7 (a protein kinase C (PKC) inhibitor), W-7 (a calmodulin inhibitor), H-89 (a protein kinase A (PKA) inhibitor), and PD98059 (a specific extracellular signal-regulated kinase (ERK) inhibitor). VIP-induced chemotactic migration was inhibited in the presence of W-7, H-89, PD98059 or H-7. H-7, W-7, and H-89 were also found to decrease VIP-induced expression of Ki67 as well as the proliferation index in HBEC. Furthermore, H-7, W-7, H-89, and PD98059 inhibited the expression of E-cd protein and mRNA induced by VIP. These results suggest that intracellular signaling molecules such as PKA, PKC, ERK, and calmodulin play important role in VIP-mediated wound healing of HBEC.     

Elevation of Eosinophil-Derived Neurotoxin in Plasma of the Subjects with Aspirin-Exacerbated Respiratory Disease: A Possible Peripheral Blood Protein Biomarker

Aspirin-exacerbated respiratory disease (AERD) remains widely underdiagnosed in asthmatics, primarily due to insufficient awareness of the relationship between aspirin ingestion and asthma exacerbation. The identification of aspirin hypersensitivity is therefore essential to avoid serious aspirin complications. The goal of the study was to develop plasma biomarkers to predict AERD. We identified differentially expressed genes in peripheral blood mononuclear cells (PBMC) between subjects with AERD and those with aspirin-tolerant asthma (ATA). The genes were matched with the secreted protein database (http://spd.cbi.pku.edu.cn/) to select candidate proteins in the plasma. Plasma levels of the candidate proteins were then measured in AERD (n = 40) and ATA (n = 40) subjects using an enzyme-linked immunosorbent assay (ELISA). Target genes were validated as AERD biomarkers using an ROC curve analysis. From 175 differentially expressed genes (p-value <0.0001) that were queried to the secreted protein database, 11 secreted proteins were retrieved. The gene expression patterns were predicted as elevated for 7 genes and decreased for 4 genes in AERD as compared with ATA subjects. Among these genes, significantly higher levels of plasma eosinophil-derived neurotoxin (RNASE2) were observed in AERD as compared with ATA subjects (70(14.62∼311.92) µg/ml vs. 12(2.55∼272.84) µg/ml, p-value <0.0003). Based on the ROC curve analysis, the AUC was 0.74 (p-value = 0.0001, asymptotic 95% confidence interval [lower bound: 0.62, upper bound: 0.83]) with 95% sensitivity, 60% specificity, and a cut-off value of 27.15 µg/ml. Eosinophil-derived neurotoxin represents a novel biomarker to distinguish AERD from ATA.     

Validation of endogenous controls for gene expression studies in human adipocytes and preadipocytes.

Quantitative gene expression protocols require adequate controls to monitor intersample variation. Quantitative approaches to describe relative changes in gene expression use endogenous controls--"housekeeping" genes. Given the low amounts of mRNA in fat cells, RT-PCR is the method of choice, and housekeeping genes are widely used as endogenous controls. However, literature reports suggest changes in gene expression of typical housekeeping genes (e. g. GAPDH, beta-actin, 18S rRNA) upon hormonal stimulation or during adipogenic differentiation. Thus, we tested the influence of 6 hormones and adipogenic differentiation on gene expression levels of 11 commonly used housekeeping genes in primary cultured mature human adipocytes and preadipocytes. Using the TaqMan RT-PCR technique and "Human Endogenous Control Assays" (PE Biosystems), we found several housekeeping genes with at least twice the difference in expression levels between stimulated and unstimulated cells (such as acidic ribosomal protein, beta-actin, beta(2)-microglobulin and beta-glucuronidase). Only GAPDH and transferrin receptor gene expression levels did not change under any of the stimuli tested, thus appeared best suited for gene expression studies in human adipose cells across a wide range of experimental settings.     

Fatty acid binding protein 1 is related with development of aspirin-exacerbated respiratory disease

Background

Aspirin-exacerbated respiratory disease (AERD) refers to the development of bronchoconstriction in asthmatics following the ingestion of aspirin. Although alterations in eicosanoid metabolites play a role in AERD, other immune or inflammatory mechanisms may be involved. We aimed to identify proteins that were differentially expressed in nasal polyps between patients with AERD and aspirin-tolerant asthma (ATA).

Methodology/Principal Findings

Two-dimensional electrophoresis was adopted for differential display proteomics. Proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS). Western blotting and immunohistochemical staining were performed to compare the amount of fatty acid-binding protein 1 (FABP1) in the nasal polyps of patients with AERD and ATA. Fifteen proteins were significantly up- (seven spots) or down-regulated in the nasal polyps of patients with AERD (n = 5) compared to those with ATA (n = 8). LC-MS revealed an increase in seven proteins expression and a decrease in eight proteins expression in patients with AERD compared to those with ATA (P = 0.003–0.045). FABP1-expression based on immunoblotting and immunohistochemical analysis was significantly higher in the nasal polyps of patients with AERD compared to that in patients with ATA. FABP1 was observed in epithelial, eosinophils, macrophages, and the smooth-muscle cells of blood vessels in the polyps.

Conclusions/Significance

Our results indicate that alterations in 15 proteins, including FABP1, may be related to the development of AERD.     

Alveolar macrophage-epithelial cell interaction following exposure to atmospheric particles induces the release of mediators involved in monocyte mobilization and recruitment

Background

Studies from our laboratory have shown that human alveolar macrophages (AM) and bronchial epithelial cells (HBEC) exposed to ambient particles (PM₁₀) in vitro increase their production of inflammatory mediators and that supernatants from PM₁₀-exposed cells shorten the transit time of monocytes through the bone marrow and promote their release into the circulation.

Methods

The present study concerns co-culture of AM and HBEC exposed to PM₁₀ (EHC-93) and the production of mediators involved in monocyte kinetics measured at both the mRNA and protein levels. The experiments were also designed to determine the role of the adhesive interaction between these cells via the intercellular adhesion molecule (ICAM)-1 in the production of these mediators.

Results

AM/HBEC co-cultures exposed to 100 μg/ml of PM₁₀ for 2 or 24 h increased their levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), M-CSF, macrophage inflammatory protein (MIP)-1β, monocyte chemotactic protein (MCP)-1, interleukin (IL)-6 and ICAM-1 mRNA, compared to exposed AM or HBEC mono-cultures, or control non-exposed co-cultures. The levels of GM-CSF, M-CSF, MIP-1β and IL-6 increased in co-cultured supernatants collected after 24 h exposure compared to control cells (p < 0.05). There was synergy between AM and HBEC in the production of GM-CSF, MIP-1β and IL-6. But neither pretreatment of HBEC with blocking antibodies against ICAM-1 nor cross-linking of ICAM-1 on HBEC blocked the PM₁₀-induced increase in co-culture mRNA expression.

Conclusion

We conclude that an ICAM-1 independent interaction between AM and HBEC, lung cells that process inhaled particles, increases the production and release of mediators that enhance bone marrow turnover of monocytes and their recruitment into tissues. We speculate that this interaction amplifies PM₁₀-induced lung inflammation and contributes to both the pulmonary and systemic morbidity associated with exposure to air pollution.     

A novel anti‐apoptotic role for apolipoprotein L2 in IFN‐γ‐induced cytotoxicity in human bronchial epithelial cells

Airway epithelium functions not only as a physical barrier, but also a regulator of lung inflammation. IFN‐γ plays a critical role in airway inflammation associated with respiratory viral infection. We investigated differential protein profiling in IFN‐γ‐stimulated normal human bronchial epithelial cells (HBEC) using a 2‐dimensional gel electrophoresis followed by MALDI‐TOF‐MS/MS. IFN‐γ markedly stimulated apolipoprotein L2 (ApoL2) protein expression in normal HBEC. ApoL2 mRNA expression was also elevated in normal human lung fibroblasts and smooth muscle cells stimulated with IFN‐γ, in lung tissues from an IFN‐γ‐predominant influenza A virus‐infected mouse lung injury model, and in cancer lung tissues from human patients. Normal HBEC showed strong resistance to IFN‐γ‐induced cytotoxicity. ApoL2 knockdown by siRNA promoted IFN‐γ‐induced cytotoxicity as revealed by a significant drop in cell viability using MTT and CyQUANT NF cell proliferation assays, and a marked increase in hypodiploid sub‐G1 cell population in cell cycle analysis. Furthermore, depletion of ApoL2 facilitated IFN‐γ‐induced membrane damage and chromatin condensation as observed in Hoechst and propidium iodide‐double staining and in transmission electron microscopy, and DNA fragmentation using a DNA laddering assay, in a caspase‐dependent manner. Our results reveal a novel function for ApoL2 in conferring anti‐apoptotic ability of human bronchial epithelium to the cytotoxic effects of IFN‐γ, in maintaining airway epithelial layer integrity. J. Cell. Physiol. 226: 397–406, 2011. © 2010 Wiley‐Liss, Inc.