eQTL of bronchial epithelial cells and bronchial alveolar lavage deciphers GWAS-identified asthma genes.

BACKGROUND: Genome-wide association studies (GWASs) have identified various genes associated with asthma, yet, causal genes or single nucleotide polymorphisms (SNPs) remain elusive. We sought to dissect functional genes/SNPs for asthma by combining expression quantitative trait loci (eQTLs) and GWASs. METHODS: Cis-eQTL analyses of 34 asthma genes were performed in cells from human bronchial epithelial biopsy (BEC, n = 107) and from bronchial alveolar lavage (BAL, n = 94). RESULTS: For TSLP-WDR36 region, rs3806932 (G allele protective against eosinophilic esophagitis) and rs2416257 (A allele associated with lower eosinophil counts and protective against asthma) were correlated with decreased expression of TSLP in BAL (P = 7.9 × 10(-11) and 5.4 × 10(-4) , respectively) and BEC, but not WDR36. Surprisingly, rs1837253 (consistently associated with asthma) showed no correlation with TSLP expression levels. For ORMDL3-GSDMB region, rs8067378 (G allele protective against asthma) was correlated with decreased expression of GSDMB in BEC and BAL (P = 1.3 × 10(-4) and 0.04) but not ORMDL3. rs992969 in the promoter region of IL33 (A allele associated with higher eosinophil counts and risk for asthma) was correlated with increased expression of IL33 in BEC (P = 1.3 × 10(-6) ) but not in BAL. CONCLUSIONS: Our study illustrates cell-type-specific regulation of the expression of asthma-related genes documenting SNPs in TSLP, GSDMB, IL33, HLA-DQB1, C11orf30, DEXI, CDHR3, and ZBTB10 affect asthma risk through cis-regulation of its gene expression. Whenever possible, disease-relevant tissues should be used for transcription analysis. SNPs in TSLP may affect asthma risk through up-regulating TSLP mRNA expression or protein secretion. Further functional studies are warranted.

An airway epithelial iNOS-DUOX2-thyroid peroxidase metabolome drives Th1/Th2 nitrative stress in human severe asthma.

Severe refractory asthma is associated with enhanced nitrative stress. To determine the mechanisms for high nitrative stress in human severe asthma (SA), 3-nitrotyrosine (3NT) was compared with Th1 and Th2 cytokine expression. In SA, high 3NT levels were associated with high interferon (IFN)-γ and low interleukin (IL)-13 expression, both of which have been reported to increase inducible nitric oxide synthase (iNOS) in human airway epithelial cells (HAECs). We found that IL-13 and IFN-γ synergistically enhanced iNOS, nitrite, and 3NT, corresponding with increased H(2)O(2). Catalase inhibited whereas superoxide dismutase enhanced 3NT formation, supporting a critical role for H(2)O(2), but not peroxynitrite, in 3NT generation. Dual oxidase-2 (DUOX2), central to H(2)O(2) formation, was also synergistically induced by IL-13 and IFN-γ. The catalysis of nitrite and H(2)O(2) to nitrogen dioxide radical (NO(2)(•)) requires an endogenous peroxidase in this epithelial cell system. Thyroid peroxidase (TPO) was identified by microarray analysis ex vivo as a gene distinguishing HAEC of SA from controls. IFN-γ induced TPO in HAEC and small interfering RNA knockdown decreased nitrated tyrosine residues. Ex vivo, DUOX2, TPO, and iNOS were higher in SA and correlated with 3NT. Thus, a novel iNOS-DUOX2-TPO-NO(2)(•) metabolome drives nitrative stress in HAEC and likely in SA.

Higher blood flow and circulating NO products offset high-altitude hypoxia among Tibetans.

The low barometric pressure at high altitude causes lower arterial oxygen content among Tibetan highlanders, who maintain normal levels of oxygen use as indicated by basal and maximal oxygen consumption levels that are consistent with sea level predictions. This study tested the hypothesis that Tibetans resident at 4,200 m offset physiological hypoxia and achieve normal oxygen delivery by means of higher blood flow enabled by higher levels of bioactive forms of NO, the main endothelial factor regulating blood flow and vascular resistance. The natural experimental study design compared Tibetans at 4,200 m and U.S. residents at 206 m. Eighty-eight Tibetan and 50 U.S. resident volunteers (18-56 years of age, healthy, nonsmoking, nonhypertensive, not pregnant, with normal pulmonary function) participated. Forearm blood flow, an indicator of systemic blood flow, was measured noninvasively by using plethysmography at rest, after breathing supplemental oxygen, and after exercise. The Tibetans had more than double the forearm blood flow of low-altitude residents, resulting in greater than sea level oxygen delivery to tissues. In comparison to sea level controls, Tibetans had >10-fold-higher circulating concentrations of bioactive NO products, including plasma and red blood cell nitrate and nitroso proteins and plasma nitrite, but lower concentrations of iron nitrosyl complexes (HbFeIINO) in red blood cells. This suggests that NO production is increased and that metabolic pathways controlling formation of NO products are regulated differently among Tibetans. These findings shift attention from the traditional focus on pulmonary and hematological systems to vascular factors contributing to adaptation to high-altitude hypoxia.

Human airway epithelial cell determinants of survival and functional phenotype for primary human mast cells.

Mast cells (MCs) are found in increased numbers at airway mucosal surfaces in asthmatic patients. Because human airway epithelial cells (HAECs) actively participate in airway inflammatory responses and are in direct contact with MCs in the mucosa, we hypothesized that HAEC-MC interactions may contribute to the differentiation and survival of MCs in the airway mucosa. Here, we show that HAECs express mRNA and protein for soluble and membrane-bound stem cell factor, releasing soluble stem cell factor into the cell culture supernatant at a concentration of 5.9 +/- 0.1 ng per 10(6) HAEC. HAECs were able to support MC survival in coculture in the absence of any exogenous cytokines for at least 4 d. Before the initiation of coculture, MCs were uniformly tryptase and chymase (MC(TC)) double positive, but by 2 d of coculture the majority of MCs expressed tryptase (MC(T)) alone. MCs supported in coculture generated low amounts of cysteinyl-leukotrienes (cys-LT) after FcepsilonRI-dependent activation (0.2 +/- 0.1 ng of cys-LT per 10(6) cells) and required priming with IL-4 and IL-3 during coculture to achieve a quantity of cys-LT generation within the range expected for human lung mucosal MC (26.5 +/- 16 ng of cys-LT per 10(6) cells). In these culture conditions, HAECs were able to direct mucosal MC protease phenotype, but T cell-derived Th2 cytokines were required for the expression of a functional airway MC eicosanoid phenotype. Thus, distinct cell types may direct unique aspects of reactive mucosal MC phenotype in the airways.

Differential expression of manganese superoxide dismutase and catalase in lung cancer.

Reactive oxygen species (ROS) are important in the initiation and promotion of cells to neoplastic growth. In this context, cigarette smoke exposure, the primary risk factor in lung cancer development, leads to high levels of ROS within the human airway. Although well-equipped with an integrated antioxidant defense system consisting of low-molecular weight antioxidants such as glutathione and intracellular enzymes such as superoxide dismutase (SOD), catalase, and glutathione peroxidase, the lungs are vulnerable to increased endogenous and exogenous oxidative insults. Antioxidants increase in response to oxidative stress and minimize ROS-induced injury in experimental systems, indicating that antioxidant levels may determine whether ROS can initiate lung carcinogenesis. On this basis, we hypothesized that antioxidants would be decreased in lung carcinoma cells as compared with tumor-free adjacent lung tissues. Antioxidant expression was evaluated in 16 lung tumor and 21 tumor-free lung tissues collected between the years 1993 and 2001 from 24 individuals with surgically resectable non-small cell lung cancer, i.e., adenocarcinoma and squamous cell carcinoma. Total SOD activity was increased (P = 0.035), catalase activity decreased (P = 0.002), and glutathione and glutathione peroxidase were similar in tumors compared with tumor-free lung tissues. Alterations in antioxidant activities were attributable to increased manganese SOD and decreased catalase protein and mRNA expression in tumors. Immunohistochemical localization of catalase in the lung revealed decreased or no expression in the tumor cells, although healthy adjacent airway epithelial cells were strongly positive for catalase. Parallel changes in antioxidant activities, protein, and mRNA expression were noted in A549 lung carcinoma cell lines exposed to cytokines (tumor necrosis factor-alpha, interleukin 1beta, and IFN-gamma). Thus, inflammation in the lung may contribute to high levels of manganese SOD and decreased catalase, which together may lead to increased hydrogen peroxide intracellularly and create an intracellular environment favorable to DNA damage and the promotion of cancer.

Alterations in exhaled gas profile during allergen-induced asthmatic response.

The source of exhaled carbon monoxide (CO) and the relationship to airway inflammation are not clear. If CO is produced by the inflamed airway, we hypothesized that inflammation induced by allergen challenge would increase exhaled CO of atopic asthmatics. Eight atopic asthmatics underwent whole lung allergen challenge. CO, nitric oxide (NO), oxygen, and carbon dioxide (CO(2)) were measured simultaneously in exhaled breath which was collected into Mylar balloons before (baseline), immediately after, and at subsequent times after allergen. NO was higher in asthmatics than control subjects at baseline, increased further in seven of the eight asthmatics after allergen, and was inversely correlated to specific conductance. In contrast, exhaled CO of asthmatics was not higher than that of control individuals at baseline, decreased immediately after allergen, and returned to baseline levels during the late asthmatic response. Thus, allergen-induced airway inflammation did not lead to increased exhaled CO in asthma.

Flexible bronchoscopy in molecular biology.

Flexible fiberoptic bronchoscopy has allowed researchers to use the bench to bedside approach in the study and therapy of lung diseases. Through bronchoscopy, the lung is a relatively convenient source of samples for the direct evaluation of human gene expression and function. Sampling of respiratory epithelium is performed by brushing with a cytology brush, whereas the epithelial lining fluid and the inflammatory cells in the bronchoalveolar space are obtained by bronchoalveolar lavage. Furthermore, bronchoscopy has been a cornerstone essential to gene therapy trials for lung disease.

Double-stranded rna dependence of nitric oxide synthase 2 expression in human bronchial epithelial cell lines BET-1A and BEAS-2B.

The human airway epithelium expresses abundant nitric oxide synthase 2 (NOS2) in vivo. Although NOS2 is easily induced by cytokines in primary cultured human airway epithelial cells and lung adenocarcinoma cell line A549, the human bronchial epithelial cell lines BEAS-2B and BET-1A do not express NOS2 in response to cytokines. Mechanisms regulating NOS2 expression in human respiratory epithelial cells are complex, but we have recently shown that NOS2 expression in primary human airway epithelial cells occurs in response to double-stranded RNA (dsRNA) through activation of signaling proteins including nuclear factor (NF)-kappaB and interferon (IFN) regulatory factor (IRF)-1. In this context, we hypothesized that BEAS-2B and BET-1A cells may express NOS2 in response to dsRNA. Here, we show that although cytokines (IFN-gamma, tumor necrosis factor-alpha and interleukin-1beta) do not induce NOS2 expression in BEAS-2B or BET-1A cells, addition of dsRNA to this cytokine mix enables BEAS-2B cells to express NOS2. IFN-gamma and dsRNA induction of NOS2 in BET-1A cells occurs in a serum concentration-dependent manner, with a minimum of 3 d of serum treatment necessary for BET-1A cells to acquire the potential to induce NOS2. Importantly, dsRNA strongly activates NF-kappaB and IRF-1 in BEAS-2B cells, transcription factors essential for NOS2 gene expression in other cell lines. On the basis of these results, dsRNA-activated signaling pathways are clearly important for NOS2 expression in human respiratory epithelial cells. With conditions for NOS2 expression characterized, these cell lines are a convenient in vitro system to investigate the mechanisms regulating NOS2 expression in human respiratory epithelial cells.

Nitric oxide synthase 2 through an autocrine loop via respiratory epithelial cell-derived mediator.

Respiratory epithelium expresses nitric oxide synthase 2 (NOS2) continuously in vivo; however, mechanisms responsible for its expression are only partially understood. We definitively identify an autocrine mechanism of induction and maintenance of NOS2 in human airway epithelial cells through the synthesis and secretion of a soluble mediator. Short exposure of human airway cells to interferon (IFN)-gamma leads to prolonged NOS2 expression. Transfer of the overlying culture medium (conditioned medium) induces NOS2 expression in other airway epithelial cells, suggesting the presence of an intermediary substance regulating NOS2 expression in an autocrine loop. Characterization of the soluble mediator reveals that it is stable and transferable in conditioned medium for up to 7 days. However, soluble mediator does not induce NOS2 mRNA in human alveolar macrophages, indicating that the response to soluble mediator is unique to human respiratory epithelium. Soluble mediator is heat labile but is not inactivated by acid treatment, unlike IFN-gamma itself. Importantly, IFN regulatory factor-1, which is critical for murine NOS2 expression, is expressed and activated by soluble mediator through the signal transducer and activator of transcription-1-dependent pathway. Based on these findings, we propose novel regulatory mechanisms for NOS2 expression in human airway epithelium.

Eosinophils are a major source of nitric oxide-derived oxidants in severe asthma: characterization of pathways available to eosinophils for generating reactive nitrogen species.

Eosinophil recruitment and enhanced production of NO are characteristic features of asthma. However, neither the ability of eosinophils to generate NO-derived oxidants nor their role in nitration of targets during asthma is established. Using gas chromatography-mass spectrometry we demonstrate a 10-fold increase in 3-nitrotyrosine (NO(2)Y) content, a global marker of protein modification by reactive nitrogen species, in proteins recovered from bronchoalveolar lavage of severe asthmatic patients (480 +/- 198 micromol/mol tyrosine; n = 11) compared with nonasthmatic subjects (52.5 +/- 40.7 micromol/mol tyrosine; n = 12). Parallel gas chromatography-mass spectrometry analyses of bronchoalveolar lavage proteins for 3-bromotyrosine (BrY) and 3-chlorotyrosine (ClY), selective markers of eosinophil peroxidase (EPO)- and myeloperoxidase-catalyzed oxidation, respectively, demonstrated a dramatic preferential formation of BrY in asthmatic (1093 +/- 457 micromol BrY/mol tyrosine; 161 +/- 88 micromol ClY/mol tyrosine; n = 11 each) compared with nonasthmatic subjects (13 +/- 14.5 micromol BrY/mol tyrosine; 65 +/- 69 micromol ClY/mol tyrosine; n = 12 each). Bronchial tissue from individuals who died of asthma demonstrated the most intense anti-NO(2)Y immunostaining in epitopes that colocalized with eosinophils. Although eosinophils from normal subjects failed to generate detectable levels of NO, NO(2-), NO(3-), or NO(2)Y, tyrosine nitration was promoted by eosinophils activated either in the presence of physiological levels of NO(2-) or an exogenous NO source. At low, but not high (e.g., >2 microM/min), rates of NO flux, EPO inhibitors and catalase markedly attenuated aromatic nitration. These results identify eosinophils as a major source of oxidants during asthma. They also demonstrate that eosinophils use distinct mechanisms for generating NO-derived oxidants and identify EPO as an enzymatic source of nitrating intermediates in eosinophils.

High levels of exhaled nitric oxide (NO) and NO synthase III expression in lesional smooth muscle in lymphangioleiomyomatosis.

Smooth-muscle proliferation is the hallmark of lymphangioleiomyomatosis (LAM). Although little is known about the pathogenesis of LAM, nitric oxide (NO) is a key regulator of smooth-muscle proliferation. NO is linked to the pathogenesis of other lung diseases such as asthma, in part by the finding of higher-than-normal levels of exhaled NO. If NO were involved in the abnormal smooth-muscle proliferation in LAM, we reasoned that exhaled NO from individuals with LAM would also differ from that of healthy control subjects. To evaluate this hypothesis, we studied exhaled NO in individuals with LAM in comparison with healthy and asthmatic women using a chemiluminescent NO analyzer. Women with LAM had higher exhaled NO than did healthy women but lower than asthmatic women (NO [parts per billion] median (25 to 75%): LAM 8 [7 to 15] [n = 28], control 6 [5 to 8] [n = 21], asthma 14 [8 to 25] [n = 22]; Kruskal-Wallis P < 0.001). Immunohistochemical studies on formalin-fixed, paraffin-embedded sections of surgical and autopsy material from lungs of individuals with LAM showed diffuse NO synthase III (NOSIII) expression in the lesional smooth muscle of LAM similar to that in the vascular endothelium. NOSIII expression was limited to the vascular endothelium and bronchial smooth muscle in healthy control lungs. The increased NO and the presence of NOSIII expression in lesional smooth muscle warrants further study into the potential role for NO in the pathogenesis of LAM.

NO chemical events in the human airway during the immediate and late antigen-induced asthmatic response.

A wealth of evidence supports increased NO (NO.) in asthma, but its roles are unknown. To investigate how NO participates in inflammatory airway events in asthma, we measured NO. and NO. chemical reaction products [nitrite, nitrate, S-nitrosothiols (SNO), and nitrotyrosine] before, immediately and 48 h after bronchoscopic antigen (Ag) challenge of the peripheral airways in atopic asthmatic individuals and nonatopic healthy controls. Strikingly, NO(3)(-) was the only NO. derivative to increase during the immediate Ag-induced asthmatic response and continued to increase over 2-fold at 48 h after Ag challenge in contrast to controls [P < 0.05]. NO(2)(-) was not affected by Ag challenge at 10 min or 48 h after Ag challenge. Although SNO was not detectable in asthmatic airways at baseline or immediately after Ag, SNO increased during the late response to levels found in healthy controls. A model of NO. dynamics derived from the current findings predicts that NO. may have harmful effects through formation of peroxynitrite, but also subserves an antioxidant role by consuming reactive oxygen species during the immediate asthmatic response, whereas nitrosylation during the late asthmatic response generates SNO, safe reservoirs for removal of toxic NO. derivatives.

Extracellular glutathione peroxidase induction in asthmatic lungs: evidence for redox regulation of expression in human airway epithelial cells.

A critical first-line antioxidant defense on the airway epithelial surface against reactive oxygen and nitrogen species (ROS and RNS) is extracellular glutathione peroxidase (eGPx). Little is known about the regulation of eGPx or its role in ROS-mediated lung diseases such as asthma. Here we show that eGPx is increased in the asthmatic airway in comparison to healthy controls. Higher levels of eGPx mRNA in asthmatic airway epithelium verified bronchial epithelial cells as the source for the increased eGPx. The eGPx mRNA in bronchial epithelial cells in vitro increased eightfold after exposure to ROS and glutathione, an essential cofactor for eGPx activity. Alterations in intracellular and extracellular oxidized and reduced glutathione were temporally associated with eGPx induction, further supporting redox mechanisms in gene expression. Overexpression of superoxide dismutase, but not catalase, inhibited induction and identified superoxide as a key intermediary. The eGPx mRNA half-life was not affected by ROS, suggesting a transcriptional mechanism for eGPx regulation. Fusion genes of deletion fragments of the eGPx gene 5' flanking region driving a reporter gene conclusively identified the ROS-responsive region, which contained the consensus DNA binding site for the redox-regulated transcription factor, activator protein 1.

High levels of nitric oxide in individuals with pulmonary hypertension receiving epoprostenol therapy.

Lack of vasodilator substances, such as nitric oxide (NO), has been implicated in the development of pulmonary hypertension, but the pathogenesis of the disease remains speculative. We hypothesized that NO plays a role in the pathogenesis of primary pulmonary hypertension (PPH), and may serve as a sensitive and specific marker of disease progression and/or severity. To test this, exhaled NO and pulmonary artery pressure were measured in individuals with PPH and secondary pulmonary hypertension (SPH) on various therapies, including the potent vasodilator epoprostenol (prostacyclin), compared with healthy controls. NO in exhaled breath of individuals with PPH was lower than SPH or control (p<0.05). In contrast, exhaled NO of individuals with PPH or SPH receiving epoprostenol was strikingly higher than PPH or SPH individuals not receiving epoprostenol, or controls. Concomitant with higher NO levels, right ventricular systolic pressure of individuals significantly decreased with epoprostenol. Importantly, in paired measures of exhaled NO before and after epoprostenol, NO increased in all pulmonary hypertensive individuals 24 h after initiation of epoprostenol therapy (p<0.05). NO may be a useful noninvasive marker of pulmonary hypertension severity and response to prostacyclin therapy.

Differential induction of extracellular glutathione peroxidase and nitric oxide synthase 2 in airways of healthy individuals exposed to 100% O(2) or cigarette smoke.

Reactive oxygen species (ROS) is increased in the airway during the inhalation of 100% O(2) or cigarette smoke and participates in the development of tracheobronchitis. We hypothesized that inhaled ROS upregulates local extracellular ROS scavenging systems or reactive molecules, e.g., nitric oxide (NO). Extracellular glutathione peroxidase (eGPx) is synthesized by airway epithelium and alveolar macrophages, secreted into the surface epithelial lining fluid, and functions as a first-line defense against inhaled ROS. NO, produced by NO synthase 2 (NOS2), combines rapidly with ROS to form reactive nitrogen species (RNS). In this study, human airway epithelial cells and alveolar macrophages from healthy individuals before and after exposure to 100% O(2) for 12 h, or from cigarette-smoking individuals, were evaluated for eGPx and NOS2 messenger RNA (mRNA) expression. Hyperoxia increased NOS2 mRNA in airway epithelial cells by 2.5-fold but did not increase eGPx mRNA. In contrast, cigarette smoke upregulated eGPx mRNA over 2-fold in airway epithelial cells and alveolar macrophages but did not affect NOS2 expression. In vitro exposure of respiratory epithelial cells to ROS or RNS also increased eGPx expression. These findings define distinct molecular responses in the airway to different inhaled ROS, which likely influences the susceptibility of the airway to oxidative injury.

Central role of double-stranded RNA-activated protein kinase in microbial induction of nitric oxide synthase.

NO synthase 2 (NOS2) is induced in airway epithelium by influenza virus infection. NOS2 induction late in the course of viral infection may occur in response to IFN-gamma, but early in infection gene expression may be induced by the viral replicative intermediate dsRNA through the dsRNA-activated protein kinase (PKR). Since PKR activates signaling pathways important in NOS2 gene induction, we determined whether PKR is a component in the signal transduction pathway leading to NOS2 gene expression after viral infection of airway epithelium. We show that NOS2 gene expression in human airway epithelial cells occurs in response to influenza A virus or synthetic dsRNA. Furthermore, dsRNA leads to rapid activation of PKR, followed by activation of signaling components including NF-kappaB and IFN regulatory factor 1. NOS2 expression is markedly diminished and IFN regulatory factor 1 and NF-kappaB activation are substantially impaired in PKR null cells. Strikingly, NOS2 induction in response to LPS is abolished in PKR null cells, confirming a central role for PKR in the general signaling pathway to NOS2.

Interleukin-9 receptor expression in asthmatic airways In vivo.

Inflammation of the airway wall is a defining feature in asthma and is likely the cause of the hyperreactivity and variable airflow limitation found in asthma. Immune response biased toward production of Th2 cytokines has been proposed as a mechanism in the pathogenesis of airway inflammation in asthma. The Th2 cytokine interleukin-9 (IL-9) is one candidate gene for asthma on the basis of position cloning and animal models of airway inflammation. To determine whether IL-9 is involved in the chronic inflammation of the asthmatic airway, we investigated the expression of IL-9 and the IL-9 specific receptor chain in asthmatic airways compared with healthy airways. IL-9 and IL-9 receptor expression in airway epithelial cells and bronchoalveolar lavage cells obtained at bronchoscopy of healthy (n = 9) and mild intermittent asthmatic individuals (n = 7) were studied by Northern analyses and reverse-transcription polymerase chain reaction technique. Primary and transformed human airway epithelial cells were also evaluated for IL-9 specific receptor chain expression in vitro. IL-9 was not detected in airways of healthy or mild asthmatic individuals. In contrast, IL-9 specific receptor chain expression was found in asthmatic airway samples but not in healthy controls. In vitro, airway epithelial cells did not express IL-9 specific receptor chain until stimulation with interferon gamma. Our results support that IL-9 may play a role in the mechanism leading to chronic airway inflammation and asthma.

Molecular mechanisms of increased nitric oxide (NO) in asthma: evidence for transcriptional and post-translational regulation of NO synthesis.

Evidence supporting increased nitric oxide (NO) in asthma is substantial, although the cellular and molecular mechanisms leading to increased NO are not known. Here, we provide a clear picture of the events regulating NO synthesis in the human asthmatic airway in vivo. We show that human airway epithelium has abundant expression of NO synthase II (NOSII) due to continuous transcriptional activation of the gene in vivo. Individuals with asthma have higher than normal NO concentrations and increased NOSII mRNA and protein due to transcriptional regulation through activation of Stat1. NOSII mRNA expression decreases in asthmatics receiving inhaled corticosteroid, treatment effective in reducing inflammation in asthmatic airways. In addition to transcriptional mechanisms, post-translational events contribute to increased NO synthesis. Specifically, high output production of NO is fueled by a previously unsuspected increase in the NOS substrate, l -arginine, in airway epithelial cells of asthmatic individuals. Finally, nitration of proteins in airway epithelium provide evidence of functional consequences of increased NO. In conclusion, these studies define multiple mechanisms that function coordinately to support high level NO synthesis in the asthmatic airway. These findings represent a crucial cornerstone for future therapeutic strategies aimed at regulating NO synthesis in asthma.

Eosinophils generate brominating oxidants in allergen-induced asthma.

Eosinophils promote tissue injury and contribute to the pathogenesis of allergen-triggered diseases like asthma, but the chemical basis of damage to eosinophil targets is unknown. We now demonstrate that eosinophil activation in vivo results in oxidative damage of proteins through bromination of tyrosine residues, a heretofore unrecognized pathway for covalent modification of biologic targets in human tissues. Mass spectrometric studies demonstrated that 3-bromotyrosine serves as a specific "molecular fingerprint" for proteins modified through the eosinophil peroxidase-H(2)O(2) system in the presence of plasma levels of halides. We applied a localized allergen challenge to model the effects of eosinophils and brominating oxidants in human lung injury. Endobronchial biopsy specimens from allergen-challenged lung segments of asthmatic, but not healthy control, subjects demonstrated significant enrichments in eosinophils and eosinophil peroxidase. Baseline levels of 3-bromotyrosine in bronchoalveolar lavage (BAL) proteins from mildly allergic asthmatic individuals were modestly but not statistically significantly elevated over those in control subjects. After exposure to segmental allergen challenge, lung segments of asthmatics, but not healthy control subjects, exhibited a >10-fold increase in BAL 3-bromotyrosine content, but only two- to threefold increases in 3-chlorotyrosine, a specific oxidation product formed by neutrophil- and monocyte-derived myeloperoxidase. These results identify reactive brominating species produced by eosinophils as a distinct class of oxidants formed in vivo. They also reveal eosinophil peroxidase as a potential therapeutic target for allergen-triggered inflammatory tissue injury in humans.