Nitric oxide (.NO) plays a central role in the pathogenesis of diverse inflammatory and infectious disorders. The toxicity of .NO is thought to be engendered, in part, by its reaction with superoxide (O2.-), yielding the potent oxidant peroxynitrite (ONOO-). However, evidence for a role of ONOO- in vivo is based largely upon detection of 3-nitrotyrosine in injured tissues. We have recently demonstrated that nitrite (NO2-), a major end-product of .NO metabolism, readily promotes tyrosine nitration through formation of nitryl chloride (NO2Cl) and nitrogen dioxide (.NO2) by reaction with the inflammatory mediators hypochlorous acid (HOCl) or myeloperoxidase. We now show that activated human polymorphonuclear neutrophils convert NO2- into NO2Cl and .NO2 through myeloperoxidase-dependent pathways. Polymorphonuclear neutrophil-mediated nitration and chlorination of tyrosine residues or 4-hydroxyphenylacetic acid is enhanced by addition of NO2- or by fluxes of .NO. Addition of 15NO2- led to 15N enrichment of nitrated phenolic substrates, confirming its role in polymorphonuclear neutrophil-mediated nitration reactions. Polymorphonuclear neutrophil-mediated inactivation of endothelial cell angiotensin-converting enzyme was exacerbated by NO2-, illustrating the physiological significance of these reaction pathways to cellular dysfunction. Our data reveal that NO2- may regulate inflammatory processes through oxidative mechanisms, perhaps by contributing to the tyrosine nitration and chlorination observed in vivo.

The transcription factor NF-B, a central regulator of immunity, is subject to regulation by redox changes. We now report that cysteine-179 of the inhibitory B kinase (IKK) ␤-subunit of the IKK signalosome is a central target for oxidative inactivation by means of S-glutathionylation. S-glutathionylation of IKK-␤ Cys-179 is reversed by glutaredoxin (GRX), which restores kinase activity.

Background The interleukin (IL)-1 family member IL-33 plays a critical role in type-2 innate immune responses to allergens, and is an important mediator of allergic asthma. The mechanisms by which allergens provoke epithelial IL-33 secretion are still poorly understood. Objective Based on previous findings indicating involvement of the NADPH oxidase DUOX1 in epithelial wound responses, we explored the potential involvement of DUOX1 in allergen-induced IL-33 secretion and potential alterations in airways of subjects with asthma. Methods Cultured human or murine airway epithelial cells or mice were subjected to acute challenge with Alternaria alternata or house dust mite (HDM), and secretion of IL-33 and activation of subsequent type 2 responses were determined. The role of DUOX1 was explored using siRNA approaches and DUOX1-deficient mice. Cultured nasal epithelial cells from healthy or asthmatic subjects were evaluated for DUOX1 expression and allergen-induced responses. Results In vitro or in vivo allergen challenge resulted in rapid airway epithelial IL-33 secretion, which critically depended on DUOX1-mediated activation of epithelial epidermal growth factor receptor (EGFR) and the protease calpain-2, via a redox-dependent mechanism involving cysteine oxidation within EGFR and the tyrosine kinase Src. Primary nasal epithelial cells from subjects with allergic asthma were found to express elevated DUOX1 and IL-33, and demonstrated enhanced IL-33 secretion in response to allergen challenge compared to nasal epithelial cells from non-asthmatic subjects. Conclusion Our findings implicate epithelial DUOX1 as a pivotal mediator of IL-33-dependent activation of innate airway type 2 immune responses to common airborne allergens, and indicate that enhanced DUOX1 expression and IL-33 secretion may present important contributing features of allergic asthma.

The airway epithelium is continuously subjected to environmental pollutants, airborne pathogens, and allergens and relies on several intrinsic mechanisms to maintain barrier integrity and to promote epithelial repair processes following injury. Here, we report a critical role for dual oxidase 1 (Duox1), a newly identified NADPH oxidase homolog within the tracheobronchial epithelium, in airway epithelial cell migration and repair following injury. Activation of Duox1 during epithelial injury is mediated by cellular release of ATP, which signals through purinergic receptors expressed on the epithelial cell surface. Purinergic receptor stimulation by extracellular ATP is a critical determinant of epithelial cell migration and repair following injury and is associated with activation of extracellular signalregulated kinases (ERK1/2) and matrix metalloproteinase-9 (MMP-9). Stimulation of these integral features of epithelial cell migration and repair processes was found to require the activation of Duox1. Our findings demonstrate a novel role for Duox1 in the tracheobronchial epithelium, in addition to its proposed role in antimicrobial host defense, by participating in epithelial repair processes to maintain epithelial integrity and barrier function in the face of environmental stress.One of the primary functions of the airway epithelium is to provide a protective barrier against inhaled environmental toxins and airborne pathogens, and airway epithelial cells express a number of intrinsic factors that serve to minimize invasion by infectious agents and to promote repair processes following injury (1, 2). A newly discovered NADPH oxidase homolog, dual oxidase (Duox), 3 has been recently identified within the tracheobronchial epithelium (3-6) and is thought to contribute to innate epithelial host defense based on structural similarities with the phagocyte NADPH oxidase system (7,8). Of the two known Duox isoforms, Duox1 is primarily expressed within the ciliated epithelium and believed to be responsible for epithelial H 2 O 2 production after cell stimulation (5), whereas Duox2 has been localized to salivary or submucosal glands, thus constituting a functional host defense system with co-localized lactoperoxidase (4, 5). Other than recent observations in Drosophila (9), a direct role for Duox1/2 in host defense has not yet been directly demonstrated, and other functions of airway epithelial Duox1 have been postulated (6, 10).Both Duox isozymes contain two EF-hand Ca 2ϩ -binding domains and are activated by Ca 2ϩ -mobilizing stimuli (11, 12). A critical mechanism of Ca 2ϩ -mediated signaling within epithelial cells after bacterial infection or mechanical or oxidative injury involves the activation of purinergic receptors at the cell surface by release of ATP (13-15). ATP-mediated autocrine or paracrine signaling is known to regulate diverse processes involved in host defense, including anion transport, ciliary function, and mucin expression (16 -19), and some of these events have recently been associated with Duox1 act...

The respiratory epithelium is subject to continuous environmental stress and its responses to injury or infection are largely mediated by transactivation of the epidermal growth factor receptor (EGFR) and downstream signaling cascades. Based on previous studies indicating involvement of ATP-dependent activation of the NADPH oxidase homolog DUOX1 in epithelial wound responses, the present studies were performed to elucidate the mechanisms by which DUOX1-derived H2O2 participates in ATP-dependent redox signaling and EGFR transactivation. ATP-mediated EGFR transactivation in airway epithelial cells was found to involve purinergic P2Y2 receptor stimulation, and both ligand-dependent mechanisms as well as ligand-independent EGFR activation by the non-receptor tyrosine kinase Src. Activation of Src was also essential for ATP-dependent activation of the sheddase ADAM17, which is responsible for liberation and activation of EGFR ligands. Activation of P2Y2R results in recruitment of Src and DUOX1 into a signaling complex, and transient siRNA silencing or stable shRNA transfection established a critical role for DUOX1 in ATP-dependent activation of Src, ADAM17, EGFR, and downstream wound responses. Using thiol-specific biotin labeling strategies, we determined that ATP-dependent EGFR transactivation was associated with DUOX1-dependent oxidation of cysteine residues within Src as well as ADAM17. In aggregate, our findings demonstrate that DUOX1 plays a central role in overall epithelial defense responses to infection or injury, by mediating oxidative activation of Src and ADAM17 in response to ATP-dependent P2Y2R activation as a proximal step in EGFR transactivation and downstream signaling.