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Inspired oxygen, an essential therapy for cardiorespiratory disorders, has the potential to generate reactive oxygen species that damage cellular DNA. Although DNA damage is implicated in diverse pulmonary disorders, including neoplasia and acute lung injury, the type and magnitude of DNA lesion caused by oxygen in vivo is unclear. We used single-cell gel electrophoresis (SCGE) to quantitate two distinct forms of DNA damage, base adduction and disruption of the phosphodiester backbone, in the lungs of mice. Both lesions were induced by oxygen, but a marked difference between the two was found. With 40 h of oxygen exposure, oxidized base adducts increased 3- to 4-fold in the entire population of lung cells. This lesion displayed temporal characteristics (a progressive increase over the first 24 h) consistent with a direct effect of reactive oxygen species attack upon DNA. DNA strand breaks, on the other hand, occurred in < 10% of pulmonary cells, which acquired severe levels of the lesion; dividing cells were preferentially affected. Characteristics of these cells suggested that DNA strand breakage was secondary to cell death, rather than a primary effect of reactive oxygen species attack on DNA. By analysis of IL-6- and IL-11-overexpressing transgenic animals, which are resistant to hyperoxia, we found that DNA strand breaks, but not base damage, correlated with acute lung injury. Analysis of purified alveolar type 2 preparations from hyperoxic mice indicated that strand breaks preferentially affected this cell type.

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Proliferation and differentiation of the pulmonary epithelium after injury is a critical process in the defense against the external environment. Defects in this response can result in airway remodeling, such as mucus cell metaplasia (MCM), commonly seen in patients with chronic lung disease. We have previously shown that amphiregulin (AREG), a ligand to the epidermal growth factor receptor (EGFR), is induced during the repair/differentiation process elicited by naphthaleneinduced lung injury. Thus, we hypothesized that AREG signaling plays an important role in epithelial proliferation and differentiation of the repairing airway. Mice deficient in AREG and lung epithelial EGFR were used to define roles for AREG-dependent EGFR signaling in airway repair and remodeling. We show that AREG and epithelial EGFR expression is dispensable to pulmonary epithelial repair after naphthalene-induced lung injury, but regulates secretory cell differentiation to a mucusproducing phenotype. We show that the pulmonary epithelium is the source of AREG, suggesting that naphthalene-induced MCM is mediated through an autocrine signaling mechanism. However, induction of MCM resulting from allergen exposure was independent of AREG. Our data demonstrate that AREG-dependent EGFR signaling in airway epithelial cells contributes to MCM in naphthaleneinduced lung injury. We conclude that AREG may represent a determinant of nonallergic chronic lung diseases complicated by MCM.Keywords: Clara cells; epidermal growth factor receptor; amphiregulin; mucus cell metaplasiaThe epithelium lining the lungs plays a critical role in defense against inhaled particles, viruses, and xenobiotics. In health, the pulmonary epithelium can adapt to these insults by the induction of defensive mechanisms, which include increased secretory cells and the secretions they produce, such as mucins (1). Even though these changes typically resolve after acute airway injury, in humans with chronic lung diseases, such as asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis, these mechanisms may be compromised, resulting in pathologic remodeling of airways, including mucus cell metaplasia (MCM) and mucus hypersecretion (2-4). Much attention has been placed on understanding roles for T helper cell type 2 cytokines and inflammatory mediators in regulating epithelial remodeling and mucus production. Growth factors, such as epidermal growth factor (EGF) and related ligands for the EGF receptor (EGFR), have been shown to contribute to cellular proliferation and MCM that accompanies exposure to allergens and pneumotropic viruses (5). However, it is not clear what mechanisms drive epithelial remodeling and MCM in nonallergic lung diseases.We previously demonstrated that amphiregulin (AREG), a ligand that binds to EGFR, was significantly up-regulated during repair from naphthalene-induced lung injury (6). Parenterally delivered naphthalene is bioactivated to a toxic naphthalene dihydrodiol by cytochrome P450 mono-oxygenases, which results in depletion of airwa...

Abundant populations of epithelial progenitor cells maintain the epithelium along the proximal-to-distal axis of the airway. Exposure of lung tissue to ionizing radiation leads to tissue remodeling and potential cancer initiation or progression. However, little is known about the effects of ionizing radiation on airway epithelial progenitor cells. We hypothesized that ionizing radiation exposure will alter the behavior of airway epithelial progenitor cells in a radiation dose- and quality-dependent manner. To address this hypothesis, we cultured primary airway epithelial cells isolated from mice exposed to various doses of 320 kVp X-ray or 600 MeV/nucleon 56Fe ions in a 3D epithelial-fibroblast co-culture system. Colony-forming efficiency of the airway epithelial progenitor cells was assessed at culture day 14. In vivo clonogenic and proliferative potentials of airway epithelial progenitor cells were measured after exposure to ionizing radiation by lineage tracing and IdU incorporation. Exposure to both X-rays and 56Fe resulted in a dose dependent decrease in the ability of epithelial progenitors to form colonies in vitro. In vivo evidence for increased clonogenic expansion of epithelial progenitors was observed after exposure to both X-rays and 56Fe. Interestingly, we found no significant increase in the epithelial proliferative index, indicating that ionizing radiation does not promote increased turnover of the airway epithelium. Therefore, we propose a model in which radiation induces a dose-dependent decrease in the pool of available progenitor cells, leaving fewer progenitors able to maintain the airway long-term. This work provides novel insights into the effects of ionizing radiation exposure on airway epithelial progenitor cell behavior.

BackgroundEpidemiologic studies associate childhood exposure to traffic-related air pollution with increased respiratory infections and asthmatic and allergic symptoms. The strongest associations between traffic exposure and negative health impacts are observed in individuals with respiratory inflammation. We hypothesized that interactions between nitric oxide (NO), increased during lung inflammatory responses, and reactive oxygen species (ROS), increased as a consequence of traffic exposure ─ played a key role in the increased susceptibility of these at-risk populations to traffic emissions.MethodsDiesel exhaust particles (DEP) were used as surrogates for traffic particles. Murine lung epithelial (LA-4) cells and BALB/c mice were treated with a cytokine mixture (cytomix: TNFα, IL-1β, and IFNγ) to induce a generic inflammatory state. Cells were exposed to saline or DEP (25 μg/cm2) and examined for differential effects on redox balance and cytotoxicity. Likewise, mice undergoing nose-only inhalation exposure to air or DEP (2 mg/m3 × 4 h/d × 2 d) were assessed for differential effects on lung inflammation, injury, antioxidant levels, and phagocyte ROS production.ResultsCytomix treatment significantly increased LA-4 cell NO production though iNOS activation. Cytomix +  DEP-exposed cells incurred the greatest intracellular ROS production, with commensurate cytotoxicity, as these cells were unable to maintain redox balance. By contrast, saline + DEP-exposed cells were able to mount effective antioxidant responses. DEP effects were mediated by: (1) increased ROS including superoxide anion (O2˙-), related to increased xanthine dehydrogenase expression and reduced cytosolic superoxide dismutase activity; and (2) increased peroxynitrite generation related to interaction of O2˙- with cytokine-induced NO. Effects were partially reduced by superoxide dismutase (SOD) supplementation or by blocking iNOS induction. In mice, cytomix +  DEP-exposure resulted in greater ROS production in lung phagocytes. Phagocyte and epithelial effects were, by and large, prevented by treatment with FeTMPyP, which accelerates peroxynitrite catalysis.ConclusionsDuring inflammation, due to interactions of NO and O2˙-, DEP-exposure was associated with nitrosative stress in surface epithelial cells and resident lung phagocytes. As these cell types work in concert to provide protection against inhaled pathogens and allergens, dysfunction would predispose to development of respiratory infection and allergy. Results provide a mechanism by which individuals with pre-existing respiratory inflammation are at increased risk for exposure to traffic-dominated urban air pollution.

Retinoids play an important role in lung development and repair. We showed that retinoic acid (RA) inhibits O 2 -induced fibroblast proliferation in rat lung explants. IGF-1, which enhances the proliferation of human fetal lung fibroblasts and stimulates collagen production during lung injury, has an important role in the lung injury/repair process. Interactions of IGF-1 with its receptor are modulated by IGF-binding proteins IGFBPs. We hypothesized that RA alters IGFBP-2 and -3 in hyperoxiaexposed neonatal lung and alters collagen production. Neonatal rat lungs were cultured in room air or 95% O 2 and 5% CO 2 for 3 d with or without RA. IGFBP-2 and -3 were measured both in culture medium and in lung tissue. Type I collagen and procollagen propeptide were analyzed in the lung tissue. Hyperoxia induced an increase in type I collagen that was significantly inhibited in the presence of RA. IGFBP-2 and IGFBP-3 in the lungs were decreased in hyperoxia but significantly increased in hyperoxia plus RA. In the culture medium, IGFBP-2 and -3 were not increased with hyperoxia but significantly increased in the presence of RA plus hyperoxia. There was no increase in IG-FBP-3 RNA transcript after RA treatment in either room air or O 2 exposure. In conclusion, RA modulates the secreted IGFBP-2 and -3 during O 2 exposure and inhibits the increase in collagen that occurs during lung injury. We speculate that RA protects against O 2 -induced neonatal lung injury through modulation of the IGFBPs. Bronchopulmonary dysplasia (BPD) is the most common chronic pulmonary disease of premature infants who require prolonged ventilation and/or O 2 therapy, usually after the presence of respiratory distress syndrome (1). Airway hyperreactivity, which can persist for several years, is one of the long-term sequelae of BPD. Improved perinatal and neonatal care, surfactant therapy, and new ventilation modalities have had a modest impact on the incidence of BPD in very small neonates, indicating a need for greater understanding of the mechanisms that lead to BPD.Histologic features of infants who die of BPD include chronic airway inflammation and squamous metaplasia of epithelial cells in airways (2). These changes are observed in animal models of hyperoxic damage to developing lung. For example, neonatal rats that are exposed to high O 2 concentrations develop alveolar epithelial thickening as well as an increased thickness of smooth muscle (3) and interstitial matrix, with abundant myofibroblasts (4). The increase in myofibroblasts in lung tissue correlates with increased type III collagen synthesis and fibrosis (5).A recent study of biopsy and autopsy material from lungs of infants who had BPD and had received both antenatal steroids and surfactant treatment showed developmental arrest, enlarged airspaces with minimal alveolarization and variable alveolar wall cellularity, and fibrosis (6). BPD in very low birth weight infants may result from inflammatory mediators' interfering with the signaling molecules in late lung development (7). Overe...

Rationale Bone marrow transplant (BMT) recipients experience frequent and severe respiratory viral infections (RVI). However, the immunological mechanisms predisposing to RVIs are uncertain. Therefore, we hypothesized that antiviral T cell immunity is impaired as a consequence of allogeneic BMT, independent of pharmacologic immunosuppression, and is responsible for increased susceptibility to RVI. Methods Bone marrow and splenocytes from C57BL/6(H2b) mice were transplanted into B10.BR(H2k) (Allo) or C57BL/6(H2b) (Syn) recipients. Five weeks after transplantation, these mice were inoculated intranasally with mouse parainfluenza virus type 1 (mPIV-1), commonly known as Sendai virus (SeV), and monitored for relevant immunological and disease endpoints. Main Results Severe and persistent airway inflammation, epithelial injury, and enhanced mortality are found after viral infection in Allo mice but not in control Syn and non-transplanted mice. In addition, viral clearance is delayed in Allo mice as evidenced by prolonged detection of viral transcripts at Day 15 post-inoculation (p.i.) but not in control mice. In concert with these events, we also detected decreased levels of total and virus-specific CD8+ T cells, as well as increased T cell-expression of inhibitory receptor programmed death-1 (PD-1), in the lungs of Allo mice at Day 8 p.i. Adoptive transfer of CD8+ T cells from non-transplanted mice recovered from SeV infection into Allo mice at Day 8 p.i. restored normal levels of viral clearance, epithelial repair and lung inflammation. Conclusions Taken together these results indicate that allogeneic BMT results in more severe RVI based on the failure to develop an appropriate pulmonary CD8+ T cell response, providing an important potential mechanism to target in improving outcomes of RVI after BMT.