Corticosteroid insensitivity (CSI) represents a profound challenge in managing patients with asthma. We recently demonstrated that short exposure of airway smooth muscle cells (ASMCs) to proasthmatic cytokines drastically reduced their responsiveness to glucocorticoids (GCs), an effect that was partially mediated via interferon regulatory factor-1, suggesting the involvement of additional mechanisms (Am J Respir Cell Mol Biol 2008;38:463–472). Although GC receptor (GR) can be phosphorylated at multiple serines in the N-terminal region, the major phosphorylation sites critical for GR transcriptional activity are serines 211 (Ser211) and 226 (Ser226). We tested the novel hypothesis that cytokine-induced CSI in ASMCs is due to an impaired GR phosphorylation. Cells were treated with TNF-α (10 ng/ml) and IFN-γ (500 UI/ml) for 6 hours and/or fluticasone (100 nm) added 2 hours before. GR was constitutively phosphorylated at Ser226 but not at Ser211 residues. Cytokines dramatically suppressed fluticasone-induced phosphorylation of GR on Ser211 but not on Ser226 residues while increasing the expression of Ser/Thr protein phosphatase (PP)5 but not that of PP1 or PP2A. Transfection studies using a reporter construct containing GC responsive elements showed that the specific small interfering RNA–induced mRNA knockdown of PP5, but not that of PP1 or PP2A, partially prevented the cytokine suppressive effects on GR-meditated transactivation activity. Similarly, cytokines failed to inhibit GC-induced GR-Ser211 phosphorylation when expression of PP5 was suppressed. We propose that the novel mechanism that proasthmatic cytokine-induced CSI in ASMCs is due, in part, to PP5-mediated impairment of GR-Ser211 phosphorylation.
serine/threonine protein phosphatase; airway smooth muscle; asthma; corticosteroid insensitivity; airway remodeling
Glucocorticoid (GC) insensitivity represents a profound challenge in managing patients with asthma. The mutual inhibition of transcriptional activity between GC receptor (GR) and other regulators is one of the mechanisms contributing to GC resistance in asthma. We recently reported that interferon regulatory factor (IRF)-1 is a novel transcription factor that promotes GC insensitivity in human airway smooth muscle (ASM) cells by interfering with GR signaling (Tliba et al., Am J Respir Cell Mol Biol 2008;38:463–472). Here, we sought to determine whether the inhibition of GR function by IRF-1 involves its interaction with the transcriptional co-regulator GR-interacting protein 1 (GRIP-1), a known GR transcriptional co-activator. We here found that siRNA-mediated GRIP-1 depletion attenuated IRF-1–dependent transcription of the luciferase reporter construct and the mRNA expression of an IRF-1–dependent gene, CD38. In parallel experiments, GRIP-1 silencing significantly reduced GR-mediated transactivation activities. Co-immunoprecipitation and GST pull-down assays showed that GRIP-1, through its repression domain, physically interacts with IRF-1 identifying GRIP-1 as a bona fide transcriptional co-activator for IRF-1. Interestingly, the previously reported inhibition of GR-mediated transactivation activities by either TNF-α and IFN-γ treatment or IRF-1 overexpression was fully reversed by increasing cellular levels of GRIP-1. Together, these data suggest that the cellular accumulation of IRF-1 may represent a potential molecular mechanism mediating altered cellular response to GC through the depletion of GRIP-1 from the GR transcriptional regulatory complexes.
glucocorticoid; cytokine; airway smooth muscle; IRF-1; GRIP-1
The article by Yao and coworkers in this issue (Am. J. Respir. Cell Mol. Biol. 2008;39:7–18) reveals that the cyclin-dependent kinase inhibitor p21CIP1/WAF1/SDI1 (designated hereafter as p21), which has been linked to cell cycle growth arrest due to stress or danger cell responses, may modulate alveolar inflammation and alveolar destruction, and thus enlightens our present understanding of how the lung senses injury due to cigarette smoke and integrates these responses with those that activate inflammatory pathways potentially harmful to the lung (1). Furthermore, the interplay of p21 and cellular processes involving cell senescence and the imbalance of cell proliferation/apoptosis may provide us with a more logical explanation of how p21, acting as a sensor of cellular stress, might have such potent and wide roles in lung responses triggered by cigarette smoke. Molecular switches, ontologically designed for the protection of the host, are now hijacked by injurious stresses (such as cigarette smoke), leading to organ damage.
Fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) have been shown to differ from normal lung fibroblasts in functional behaviors that contribute to the pathogenesis of IPF, including the expression of contractile proteins and proliferation, but how such behaviors vary in matrices with stiffness matched to normal and fibrotic lung tissue remains unknown. Here, we tested whether pathologic changes in matrix stiffness control IPF and normal lung tissue–derived fibroblast functions, and compared the relative efficacy of mechanical cues to an antifibrotic lipid mediator, prostaglandin E2 (PGE2). Fibroblasts were grown on collagen I–coated glass or hydrogel substrates of discrete stiffnesses, spanning the range of normal and fibrotic lung tissue. Traction microscopy was used to quantify contractile function. The CyQuant Cell Proliferation Assay (Invitrogen, Carlsbad, CA) was used to assess changes in cell number, and PGE2 concentrations were measured by ELISA. We confirmed differences in proliferation and PGE2 synthesis between IPF and normal tissue–derived fibroblasts on rigid substrates. However, IPF fibroblasts remained highly responsive to changes in matrix stiffness, and both proliferative and contractile differences between IPF and normal fibroblasts were ablated on physiologically soft matrices. We also confirmed the relative resistance of IPF fibroblasts to PGE2, while demonstrating that decreases in matrix stiffness and the inhibition of Rho kinase both potently attenuate contractile function in IPF-derived fibroblasts. We conclude that pathologic changes in the mechanical environment control important IPF fibroblast functions. Understanding how mechanical cues control fibroblast function may offer new opportunities for targeting these cells, even when they are resistant to antifibrotic pharmacological agents or biological mediators.
pulmonary fibrosis; lung; extracellular matrix; fibroblast contractility
Periostin is an extracellular matrix protein that is up-regulated by T helper cell type 2 cytokines in the asthmatic airway and implicated in mouse studies as promoting eosinophil recruitment. We asked whether periostin modulates eosinophil adhesion and motility in vitro. Periostin adsorbed to polystyrene supported adhesion of purified human blood eosinophils stimulated by IL-5, IL-3, or granulocyte/macrophage colony–stimulating factor, but did not support adhesion of eosinophils treated with IL-4 or IL-13. The degree of adhesion depended on the concentrations of periostin during coating and activating cytokine during the adhesion assay. Both full-length periostin and alternatively spliced periostin, lacking C-terminal exons 17, 18, 19, and 21, supported adhesion. Adhesion was inhibited by monoclonal antibody to αM or β2 integrin subunits, but not by antibodies to other eosinophil integrin subunits. Adsorbed periostin also supported αMβ2-dependent random motility of IL-5–stimulated eosinophils with optimal movement at an intermediate coating concentration. In the presence of IL-5, eosinophils adherent on periostin formed punctate structures positive for filamentous actin, gelsolin, and phosphotyrosine. These structures fit the criteria for podosomes, highly dynamic adhesive contacts that are distinct from classical focal adhesions. The results establish αMβ2 (CD11b/CD18, Mac-1) as an adhesive and promigratory periostin receptor on cytokine-stimulated eosinophils, and suggest that periostin may function as a haptotactic stimulus able to guide eosinophils to areas of high periostin density in the asthmatic airway.
eosinophils; periostin; integrins; αMβ2 (CD11b/CD18; Mac-1); adhesion
Enhanced airway smooth muscle (ASM) contractility contributes to increased resistance to airflow in diseases such as bronchitis and asthma that occur in passive smokers exposed to secondhand smoke. Little information exists on the cellular mechanisms underlying such airway hyperreactivity. Sputum samples of patients with chronic sinusitis, bronchitis, and asthma show increased concentrations of growth factors called neurotrophins, including brain-derived growth factor (BDNF), but their physiological significance remains unknown. In human ASM, we tested the hypothesis that BDNF contributes to increased contractility with cigarette smoke exposure. The exposure of ASM to 1% or 2% cigarette smoke extract (CSE) for 24 hours increased intracellular calcium ([Ca2+]i) responses to histamine, and further potentiated the enhancing effects of a range of BDNF concentrations on such histamine responses. CSE exposure increased the expression of the both high-affinity and low-affinity neurotrophin receptors tropomyosin-related kinase (Trk)–B and p75 pan-neurotrophin receptor, respectively. Quantitative ELISA showed that CSE increased BDNF secretion by human ASM cells. BDNF small interfering (si)RNA and/or the chelation of extracellular BDNF, using TrkB-fragment crystallizable, blunted the effects of CSE on [Ca2+]i responses as well as the CSE enhancement of cell proliferation, whereas TrkB siRNA blunted the effects of CSE on ASM contractility. These data suggest that cigarette smoke is a potent inducer of BDNF and TrkB expression and signaling in ASM, which then contribute to cigarette smoke–induced airway hyperresponsiveness.
neurotrophin; asthma; TrkB; environmental tobacco exposure; secondhand smoke
Sulfur mustard (SM) inhalation causes the rare but life-threatening disorder of plastic bronchitis, characterized by bronchial cast formation, resulting in severe airway obstruction that can lead to respiratory failure and death. Mortality in those requiring intubation is greater than 80%. To date, no antidote exists for SM toxicity. In addition, therapies for plastic bronchitis are solely anecdotal, due to lack of systematic research available to assess drug efficacy in improving mortality and/or morbidity. Adult rats exposed to SM analog were treated with intratracheal tissue plasminogen activator (tPA) (0.15–0.7 mg/kg, 5.5 and 6.5 h), compared with controls (no treatment, isoflurane, and placebo). Respiratory distress and pulse oximetry were assessed (for 12 or 48 h), and arterial blood gases were obtained at study termination (12 h). Microdissection of fixed lungs was done to assess airway obstruction by casts. Optimal intratracheal tPA treatment (0.7 mg/kg) completely eliminated mortality (0% at 48 h), and greatly improved morbidity in this nearly uniformly fatal disease model (90–100% mortality at 48 h). tPA normalized plastic bronchitis–associated hypoxemia, hypercarbia, and lactic acidosis, and improved respiratory distress (i.e., clinical scores) while decreasing airway fibrin casts. Intratracheal tPA diminished airway-obstructive fibrin–containing casts while improving clinical respiratory distress, pulmonary gas exchange, tissue oxygenation, and oxygen utilization in our model of severe chemically induced plastic bronchitis. Most importantly, mortality, which was associated with hypoxemia and clinical respiratory distress, was eliminated.
plastic bronchitis; tissue plasminogen activator; airway obstruction; sulfur mustard; fibrin
Earlier studies indicated a role for reactive oxygen species (ROS) in host defense against Pseudomonas aeruginosa infection. However, the role of nicotinamide adenine dinucleotide phosphate–reduced (NADPH) oxidase (NOX) proteins and the mechanism of activation for NADPH oxidase in P. aeruginosa infection are not well-defined. Here, we investigated the role of NOX2 and NOX4 proteins in P. aeruginosa infection, ROS generation, and endothelial barrier function in murine lungs and in human lung microvascular endothelial cells (HLMVECs). Airway instillation of P. aeruginosa strain 103 (PA103) significantly increased ROS concentrations in bronchial alveolar lavage (BAL) fluid, along with the expression of NOX2 and NOX4, but not NOX1 and NOX3, in lung tissue. In addition, PA103-infected HLMVECs revealed elevated concentrations of ROS, NOX2, and NOX4. In murine lungs and HLMVECs, PA103 induced the NF-κB pathway, and its inhibition blocked PA103-dependent NOX2 and NOX4 expression. Barrier function analysis showed that heat-killed PA103 induced endothelial permeability in a dose-dependent manner, which was attenuated by treatment with small interfering (si)RNA specific for NOX4, but not NOX2. Furthermore, the knockdown of NOX4, but not NOX2, with siRNA reduced PA103-mediated apoptosis in HLMVECs. In vivo, the down-regulation of NOX4 with NOX4 siRNA attenuated PA103-induced lung vascular permeability. The deletion of NOX2 in mice exerted no effect on permeability, but offered significant resistance to P. aeruginosa–induced lung inflammation. These data show that P. aeruginosa lung infection up-regulates NOX2 and NOX4 expression and ROS generation, which play distinct roles in regulating lung inflammation, apoptosis, and permeability.
Pseudomonas aeruginosa; NADPH oxidase; reactive oxygen species; apoptosis; vascular permeability
Rheumatoid arthritis–related interstitial lung disease (RA-ILD) is associated with significant morbidity and mortality. Studies in humans have found that the incidence of bronchus-associated lymphoid tissue (BALT) correlates with the severity of lung injury. However, the mechanisms underlying the development of BALT during systemic autoimmunity remain unknown. We have determined whether systemic autoimmunity in a murine model of autoimmune arthritis can promote the development of BALT by generating a novel murine model derived from K/BxN mice. Transgenic mice with the KRN T-cell receptor specific for the autoantigen, glucose-6–phosphate isomerase (GPI), were crossed with GPI-specific immunoglobulin heavy and light chain knock-in mice, producing mice with a majority of T and B cells specific for the same autoantigen. We found that 67% of these mice demonstrated lymphocytic infiltration in the lungs, localized to either the perivascular or peribronchial regions. Fifty percent of the mice with lymphocytic infiltration manifested lymphoid-like lesions resembling BALT, with distinct T and B cell follicles. The lungs from mice with lymphoid infiltrates had increased numbers of cytokine-producing T cells, including IL-17A+ T cells and increased major histocompatibility complex Class II expression on B cells. Interestingly, challenge with bleomycin failed to elicit a significant fibrotic response, compared with wild-type control mice. Our data suggest that systemic autoreactivity promotes ectopic lymphoid tissue development in the lung through the cooperation of autoreactive T and B cells. However, these BALT-like lesions may not be sufficient to promote fibrotic lung disease at steady state or after inflammatory challenge.
autoimmunity; bronchus-associated lymphoid tissue; T cells; B cells
Lung epithelial cell death is a prominent feature of hyperoxic lung injury, and has been considered a very important underlying mechanism of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Here we report on a novel mechanism involved in epithelial cytoprotection and homeostasis after oxidative stress. p62 (sequestosome 1; SQSTM1) is a ubiquitously expressed cellular protein. It interacts with ubiquitinated proteins and autophagic marker light chain 3b (LC3b), thus mediating the degradation of selective targets. In this study, we explored the role of p62 in mitochondria-mediated cell death after hyperoxia. Lung alveolar epithelial cells demonstrate abundant p62 expression, and p62 concentrations are up-regulated by oxidative stress at both the protein and mRNA levels. The p62/LC3b complex interacts with Fas and truncated BID (tBID) physically. These interactions abruptly diminish after hyperoxia. The deletion of p62 robustly increases tBID and cleaved caspase-3, implying an antiapoptotic effect. This antiapoptotic effect of p62 is further confirmed by measuring caspase activities, cleaved poly ADP ribose polymerase, and cell viability. The deletion of the p62 PBI domain or the ubiquitin-associated domain both lead to elevated tBID, cleaved caspase-3, and significantly more cell death after hyperoxia. Moreover, p62 traffics in an opposite direction with LC3b after hyperoxia, leading to the dissociation of the p62/Cav-1/LC3b/BID complex. Subsequently, the LC3b-mediated lysosomal degradation of tBID is eliminated. Taken together, our data suggest that the p62/LC3b complex regulates lung alveolar epithelial cell homeostasis and cytoprotection after hyperoxia.
p62/SQSTM1; hyperoxia; tBid; LC3b; apoptosis
In vitro assays of biological activity induced by particulate matter (PM) are a tool for investigating mechanisms of PM health effects. They have potential application to exposure assessment in chronic disease epidemiology. However, there has been little reporting of the impact of real-world PM collection techniques on assay results. Therefore, we examined the effect of sampling duration and postsampling delays in freezing on PM-induced biological activity. Duplicate samples of respirable ambient Los Angeles PM were collected on polyurethane foam filters during 17 days and during three contemporaneous consecutive shorter periods. After collection, one duplicate was stored at ambient temperature for 24 hours before freezing; the other was frozen immediately. Cytokine response (IL-1β, IL-6, IL-8, and TNF-α) to PM aqueous extract was assessed in THP-1 cells, a model for evaluating monocyte/macrophage lineage cell responses. There was consistent 3- to 4-fold variation in PM-induced cytokine levels across the three collection intervals. Compared with levels induced by PM pooled across the three periods, continuously collected PM-induced levels were reduced by 25% (IL-6) to 39% (IL-8). The pattern of cytokine gene expression response was similar. Cytokine level variation by time to freezing was not statistically significant. PM-induced inflammatory response varied substantially over a weekly time scale. We conclude that long PM sampling interval induced less activity than the average of equivalent shorter consecutive sampling intervals. Time to freezing was less important. Implications for development of metrics of long-term spatial variation in biological exposure metrics for study of chronic disease merit further investigation.
air pollution; toxicology; exposure assessment; epidemiology
Excessive alcohol use increases the risk of acute lung injury and pneumonia. Chronic alcohol ingestion causes oxidative stress within the alveolar space, including near depletion of glutathione (GSH), which impairs alveolar epithelial and macrophage function, in experimental animals and human subjects. However, the fundamental mechanism(s) by which alcohol induces such profound lung oxidative stress is unknown. Nuclear factor (erythroid-derived 2)–like 2 (Nrf2) is a redox-sensitive master transcription factor that regulates activation of the antioxidant response element (ARE). As the alveolar epithelium controls GSH levels within the alveolar space, we hypothesized that alcohol also decreases Nrf2 expression and/or activation within the alveolar epithelium. In this study, we determined that alcohol ingestion in vivo or direct alcohol exposure in vitro down-regulated the Nrf2–ARE pathway in lung epithelial cells, decreased the expression of antioxidant genes, and lowered intracellular GSH levels. RNA silencing of Nrf2 gene expression in alveolar epithelial cells in vitro decreased expression of these same antioxidant genes, and likewise lowered intracellular GSH levels, findings that mirrored the effects of alcohol. In contrast, treating alcohol-exposed alveolar epithelial cells in vitro with the Nrf2 activator, sulforaphane, preserved Nrf2 expression, ARE activation, intracellular GSH levels, and epithelial barrier function. These new experimental findings implicate down-regulation of the Nrf2–ARE signaling pathway as a fundamental mechanism by which alcohol causes profound oxidative stress and alveolar epithelial dysfunction, and suggest that treatments, such as sulforaphane, that activate this pathway could mitigate the pathophysiological consequences of alcohol on the lung and other organs.
acute respiratory distress syndrome; glutathione; lung; redox signaling; oxidative stress
The respiratory epithelium plays a critical role in innate defenses against airborne pathogens and pollutants, and alterations in epithelial homeostasis and repair mechanisms are thought to contribute to chronic lung diseases associated with airway remodeling. Previous studies implicated the nicotinamide adenine dinucleotide phosphate–reduced oxidase dual oxidase–1 (DUOX1) in redox signaling pathways involved in in vitro epithelial wound responses to infection and injury. However, the importance of epithelial DUOX1 in in vivo epithelial repair pathways has not been established. Using small interfering (si)RNA silencing of DUOX1 expression, we show the critical importance of DUOX1 in wound responses in murine tracheal epithelial (MTE) cells in vitro, as well as its contribution to epithelial regeneration in vivo in a murine model of epithelial injury induced by naphthalene, a selective toxicant of nonciliated respiratory epithelial cells (club cells [Clara]). Whereas naphthalene-induced club-cell injury is normally followed by epithelial regeneration after 7 and 14 days, such airway reepithelialization was significantly delayed after the silencing of airway DUOX1 by oropharyngeal administration of DUOX1-targeted siRNA. Wound closure in MTE cells was related to DUOX1-dependent activation of the epidermal growth factor receptor (EGFR) and the transcription factor signal transducer and activator of transcription–3 (STAT3), known mediators of epithelial cell migration and wound responses. Moreover, in vivo DUOX1 silencing significantly suppressed naphthalene-induced activation of STAT3 and EGFR during early stages of epithelial repair. In conclusion, these experiments demonstrate for the first time an important function for epithelial DUOX1 in lung epithelial regeneration in vivo, by promoting EGFR–STAT3 signaling and cell migration as critical events in initial repair.
airway injury; redox signaling; EGFR; STAT3; naphthalene
Epithelial injury and airway hyperresponsiveness are prominent features of asthma. We have previously demonstrated that laser ablation of single epithelial cells immediately induces global airway constriction through Ca2+-dependent smooth muscle shortening. The response is mediated by soluble mediators released from wounded single epithelial cells; however, the soluble mediators and signaling mechanisms have not been identified. In this study, we investigated the nature of the epithelial-derived soluble mediators and the associated signaling pathways that lead to the L-type voltage-dependent Ca2+ channel (VGCC)–mediated Ca2+ influx. We found that inhibition of adenosine A1 receptors (or removal of adenosine with adenosine deaminase), cyclooxygenase (COX)-2 or prostaglandin E receptor 3 (EP3) receptors, epidermal growth factor receptor (EGFR), or platelet-derived growth factor receptor (PDGFR) all significantly blocked Ca2+ oscillations in smooth muscle cells and airway contraction induced by local epithelial injury. Using selective agonists to activate the receptors in the presence and absence of selective receptor antagonists, we found that adenosine activated the signaling pathway A1R→EGFR/PDGFR→COX-2→EP3→VGCCs→calcium-induced calcium release, leading to intracellular Ca2+ oscillations in airway smooth muscle cells and airway constriction.
ATP; epidermal growth factor receptor; platelet-derived growth factor receptor; cyclooxygenase-2; L-type voltage-dependent Ca2+ channels
The regeneration of alveolar epithelial cells is a critical aspect of alveolar reorganization after lung injury. Although alveolar Type II (AT2) cells have been described as progenitor cells for alveolar epithelia, more remains to be understood about how their progenitor cell properties are regulated. A nuclear, chromatin-bound green fluorescence protein reporter (H2B-GFP) was driven from the murine surfactant protein–C (SPC) promoter to generate SPC H2B-GFP transgenic mice. The SPC H2B-GFP allele allowed the FACS-based enrichment and gene expression profiling of AT2 cells. Approximately 97% of AT2 cells were GFP-labeled on Postnatal Day 1, and the percentage of GFP-labeled AT2 cells decreased to approximately 63% at Postnatal Week 8. Isolated young adult SPC H2B-GFP+ cells displayed proliferation, differentiation, and self-renewal capacity in the presence of lung fibroblasts in a Matrigel-based three-dimensional culture system. Heterogeneity within the GFP+ population was revealed, because cells with distinct alveolar and bronchiolar gene expression arose in three-dimensional cultures. CD74, a surface marker highly enriched on GFP+ cells, was identified as a positive selection marker, providing 3-fold enrichment for AT2 cells. In vivo, GFP expression was induced within other epithelial cell types during maturation of the distal lung. The utility of the SPC H2B-GFP murine model for the identification of AT2 cells was greatest in early postnatal lungs and more limited with age, when some discordance between SPC and GFP expression was observed. In adult mice, this allele may allow for the enrichment and future characterization of other SPC-expressing alveolar and bronchiolar cells, including putative stem/progenitor cell populations.
alveolar Type II cells; SPC; epithelial; GFP; differentiation
Mucous cell metaplasia is a hallmark of airway diseases, such as asthma and chronic obstructive pulmonary disease. The majority of human airway epithelium is pseudostratified, but the cell of origin of mucous cells has not been definitively established in this type of airway epithelium. There is evidence that ciliated, club cell (Clara), and basal cells can all give rise to mucus-producing cells in different contexts. Because pseudostratified airway epithelium contains distinct progenitor cells from simple columnar airway epithelium, the lineage relationships of progenitor cells to mucous cells may be different in these two epithelial types. We therefore performed lineage tracing of the ciliated cells of the murine basal cell–containing airway epithelium in conjunction with the ovalbumin (OVA)-induced murine model of allergic lung disease. We genetically labeled ciliated cells with enhanced Yellow Fluorescent Protein (eYFP) before the allergen challenge, and followed the fate of these cells to determine whether they gave rise to newly formed mucous cells. Although ciliated cells increased in number after the OVA challenge, the newly formed mucous cells were not labeled with the eYFP lineage tag. Even small numbers of labeled mucous cells could not be detected, implying that ciliated cells make virtually no contribution to the new goblet cell pool. This demonstrates that, after OVA challenge, new mucous cells do not originate from ciliated cells in a pseudostratified basal cell–containing airway epithelium.
mucous cell metaplasia; pseudostratified airway epithelium; ovalbumin; ciliated cell; goblet or mucous cell
In asthma, basic fibroblast growth factor (FGF-2) plays an important (patho)physiological role. This study examines the effects of FGF-2 on the transforming growth factor–β (TGF-β)–stimulated differentiation of airway smooth muscle (ASM) cells in vitro. The differentiation of human ASM cells after incubation with TGF-β (100 pM) and/or FGF-2 (300 pM) for 48 hours was assessed by increases in contractile protein expression, actin-cytoskeleton reorganization, enhancements in cell stiffness, and collagen remodeling. FGF-2 inhibited TGF-β–stimulated increases in transgelin (SM22) and calponin gene expression (n = 15, P < 0.01) in an extracellular signal-regulated kinase 1/2 (ERK1/2) signal transduction–dependent manner. The abundance of ordered α–smooth muscle actin (α-SMA) filaments formed in the presence of TGF-β were also reduced by FGF-2, as was the ratio of F-actin to G-actin (n = 8, P < 0.01). Furthermore, FGF-2 attenuated TGF-β–stimulated increases in ASM cell stiffness and the ASM-mediated contraction of lattices, composed of collagen fibrils (n = 5, P < 0.01). However, the TGF-β–stimulated production of IL-6 was not influenced by FGF-2 (n = 4, P > 0.05), suggesting that FGF-2 antagonism is selective for the regulation of ASM cell contractile protein expression, organization, and function. Another mitogen, thrombin (0.3 U ml−1), exerted no effect on TGF-β–regulated contractile protein expression (n = 8, P > 0.05), α-SMA organization, or the ratio of F-actin to G-actin (n = 4, P > 0.05), suggesting that the inhibitory effect of FGF-2 is dissociated from its mitogenic actions. The addition of FGF-2, 24 hours after TGF-β treatment, still reduced contractile protein expression, even when the TGF-β–receptor kinase inhibitor, SB431542 (10 μM), was added 1 hour before FGF-2. We conclude that the ASM cell differentiation promoted by TGF-β is antagonized by FGF-2. A better understanding of the mechanism of action for FGF-2 is necessary to develop a strategy for therapeutic exploitation in the treatment of asthma.
airway wall remodeling; α–smooth muscle actin; asthma; cytoskeleton; transgelin
Pulmonary arterial hypertension (PAH) is increased in HIV, but its pathogenesis is not fully understood. Nonhuman primates infected with simian immunodeficiency virus (SIV) or SIV-HIV chimeric virus (SHIV) exhibit histologic changes characteristic of human PAH, but whether hemodynamic changes accompany this pathology is unknown. Repeated measurements of pulmonary artery pressures would permit longitudinal assessments of disease development and provide insights into pathogenesis. We tested the hypothesis that SIV-infected and SHIV-infected macaques develop physiologic manifestations of PAH. We performed right heart catheterizations, echocardiography, and computed tomography (CT) scans in macaques infected with either SIV (ΔB670) or SHIV (89.6P), and compared right heart and pulmonary artery pressures, as well as pulmonary vascular changes on CT scans, with those in uninfected control animals. Right atrial, right ventricular systolic, and pulmonary artery pressures (PAPs) were significantly elevated in 100% of macaques infected with either SIV or SHIV compared with control animals, with no difference in pulmonary capillary wedge pressure. PAPs increased as early as 3 months after SIV infection. Radiographic evidence of pulmonary vascular pruning was also found. Both SIV-infected and SHIV-infected macaques exhibited histologic changes in pulmonary arteries, predominantly consisting of intimal and medial hyperplasia. This report is the first to demonstrate SHIV-infected and SIV-infected macaques develop pulmonary hypertension at a high frequency, with physiologic changes occurring as early as 3 months after infection. These studies establish an important nonhuman primate model of HIV-associated PAH that will be useful in studies of disease pathogenesis and the efficacy of interventions.
pulmonary hypertension; HIV; SIV; SHIV; macaque model
Mechanical ventilation with supraphysiological concentrations of oxygen (hyperoxia) is routinely used to treat patients with respiratory distress. However, a significant number of patients on ventilators exhibit enhanced susceptibility to infections and develop ventilator-associated pneumonia (VAP). Pseudomonas aeruginosa (PA) is one of the most common species of bacteria found in these patients. Previously, we demonstrated that prolonged exposure to hyperoxia can compromise the ability of alveolar macrophages (AMs), an essential part of the innate immunity, to phagocytose PA. This study sought to investigate the potential molecular mechanisms underlying hyperoxia-compromised innate immunity against bacterial infection in a murine model of PA pneumonia. Here, we show that exposure to hyperoxia (≥ 99% O2) led to a significant elevation in concentrations of airway high mobility group box–1 (HMGB1) and increased mortality in C57BL/6 mice infected with PA. Treatment of these mice with a neutralizing anti-HMGB1 monoclonal antibody (mAb) resulted in a reduction in bacterial counts, injury, and numbers of neutrophils in the lungs, and an increase in leukocyte phagocytic activity compared with mice receiving control mAb. This improved phagocytic function was associated with reduced concentrations of airway HMGB1. The correlation between phagocytic activity and concentrations of extracellular HMGB1 was also observed in cultured macrophages. These results indicate a pathogenic role for HMGB1 in hyperoxia-induced impairment with regard to a host’s ability to clear bacteria and inflammatory lung injury. Thus, HMGB1 may provide a novel molecular target for improving hyperoxia-compromised innate immunity in patients with VAP.
hyperoxia; bacterial infection; macrophage function; HMGB1
Recently, we reported that diesel exhaust particles (DEPs) disrupt tight junctions (TJs) in alveolar epithelial cells (AECs) via an increase in reactive oxygen species (ROS). In this study, we investigated the role of protein kinase C (PKC)–ζ activation in DEP-induced lung injury. C57/bl6 mice were instilled intratracheally with 50 μl of saline containing 100 μg of DEPs or titanium dioxide (TiO2). Twenty-four hours later, bronchoalveolar lavage was performed to assess neutrophil counts and protein concentrations. In addition, in vitro experiments were performed in primary rat and human AECs exposed to DEPs (50 μg/cm2) for 3 hours. Transepithelial electrical conductance was measured, and TJ protein association was analyzed by immunoprecipitation. To determine whether the overexpression of antioxidants prevented DEP-induced lung injury, AECs and mice were infected with adenoviruses containing catalase and manganese superoxide dismutase (MnSOD) plasmids. In vivo, the overexpression of catalase and MnSOD prevented DEP-induced neutrophil recruitment. The inhibition of PKC-ζ activation also prevented DEP-induced neutrophil recruitment in vivo. In vitro, DEPs activated PKC-ζ in AECs, but not in alveolar macrophages. Using a specific myristolated PKC-ζ pseudosubstrate pepetide (PKC-ζ ps), we showed that PKC-ζ mediated the DEP-induced dissociation of occludin and zonula occludin–1 (ZO1) in rat and human AECs. In addition, the overexpression of constitutively active PKC-ζ induced the dissociation of occludin and ZO1 in AECs. DEP-induced TJ disruption occurs via PKC-ζ. TJ disruption seems to be in part responsible for DEP-induced lung injury.
diesel exhaust particles; PKC-ζ; occludin; ZO1; ROS
Matrix metalloproteinase–7 (MMP7) expression is quickly up-regulated after injury, and functions to regulate wound repair and various mucosal immune processes. We evaluated the global transcriptional response of airway epithelial cells from wild-type and Mmp7-null mice cultured at an air–liquid interface. The analysis of differentially expressed genes between genotypes after injury revealed an enrichment of functional categories associated with inflammation, cilia, and differentiation. Because these analyses suggested that MMP7 regulated ciliated cell formation, we evaluated the recovery of the airway epithelium in wild-type and Mmp7-null mice in vivo after naphthalene injury, which revealed augmented ciliated cell formation in the absence of MMP7. Moreover, in vitro studies evaluating cell differentiation in air–liquid interface cultures also showed faster ciliated cell production under Mmp7-null conditions compared with wild-type conditions. These studies identified a new role for MMP7 in attenuating ciliated cell differentiation during wound repair.
MMP7; matrilysin; microarray; wound repair
Myristoylated alanine-rich C kinase substrate (MARCKS) is a ubiquitously expressed protein kinase C substrate that has emerged as a potential therapeutic target for the amelioration of mucin secretion and inflammation in patients with chronic obstructive pulmonary disease. MARCKS also plays a key role in regulating the adhesion, migration, and degranulation of neutrophils. Moreover, given its biological role in epithelial and immune cells, we hypothesized that MARCKS may play an integral role in cytokine secretion by neutrophils. Because the amino terminus of MARCKS is highly conserved across vertebrate species, we successfully applied the well-characterized human MARCKS inhibitory peptide, myristoylated N-terminal sequence (MANS), to attenuate the function of MARCKS in isolated canine neutrophils. Pretreatment of canine neutrophils with MANS peptide significantly reduced both mRNA and protein expression in a broad range of LPS-induced cytokines, including IL-8, a chemokine (C-X-C motif) ligand–1 orthologue, and TNF-α, in comparison with untreated cells or those treated with a control peptide. This reduction in cytokine expression was observed even when neutrophils were treated with MANS 2 hours after LPS exposure. The observed reduction in cytokine secretion was not attributable to protein retention or cell death, but was associated with reduced cytokine transcript synthesis. These observations identify MARCKS protein as a promising therapeutic target in the treatment of inflammatory diseases or syndromes attributed to neutrophil influx and inflammatory cytokine production, such as sepsis, acute lung injury, and acute respiratory distress syndrome.
MARCKS; cytokine; inflammation; neutrophil
Previous observations by our laboratory indicate that the presence of anti–IL-8 autoantibody:IL-8 immune complexes in lung fluids from patients with acute lung injury/acute respiratory distress syndrome (ALI/ARDS) comprises an important prognostic indicator in the development and ultimate outcome of ALI/ARDS. We also showed that these complexes display proinflammatory activity toward neutrophils through the engagement of FcγRIIa receptors. Because sepsis is one of the most common risk factors for ALI/ARDS, the initial goal of our present study involved investigating the effects of LPS on the expression of FcγRIIa receptors in neutrophils. Our results indicate that LPS triggers an increase in the expression of FcγRIIa on the neutrophil surface, which leads to shortening of the molecular distance between FcγRIIa and Toll-like receptor–4 (TLR4). When such neutrophils are stimulated with anti–IL-8:IL-8 complexes, the TLR4 cascade becomes activated via the engagement of FcγRIIa. The underlying molecular mechanism has been subsequently examined and involves Bruton’s tyrosine kinase (Btk). In conclusion, our study reveals the existence of Btk-dependent molecular cooperation between FcγRIIa and TLR4 signaling cascades in LPS-“primed” human neutrophils. Furthermore, we used fluorescence lifetime imaging to study the interactions between TLR4 and FcγRIIa in human alveolar neutrophils from patients with ALI/ARDS. The results from these experiments confirm the existence of the molecular cooperation between TLR4 and FcγRIIa.
neutrophil; FcуRIIa; TLR4; Btk
β2-Adrenoceptor (β2AR) agonists are the most effective class of bronchodilators and a mainstay of asthma management. The first potent β2AR agonist discovered and widely used in reversing the airway constriction associated with asthma exacerbation was the endogenous activator of the β2AR, epinephrine. In this study, we demonstrate that activation of the β2AR by epinephrine is paradoxically required for development of the asthma phenotype. In an antigen-driven model, mice sensitized and challenged with ovalbumin showed marked elevations in three cardinal features of the asthma phenotype: inflammatory cells in their bronchoalveolar lavage fluid, mucin over production, and airway hyperresponsiveness. However, genetic depletion of epinephrine using mice lacking the enzyme to synthesize epinephrine, phenylethanolamine N-methyltransferase, or mice that had undergone pharmacological sympathectomy with reserpine to deplete epinephrine, had complete attenuation of these three cardinal features of the asthma phenotype. Furthermore, administration of the long-acting β2AR agonist, formoterol, a drug currently used in asthma treatment, to phenylethanolamine N-methyltransferase–null mice restored the asthma phenotype. We conclude that β2AR agonist–induced activation is needed for pathogenesis of the asthma phenotype. These findings also rule out constitutive signaling by the β2AR as sufficient to drive the asthma phenotype, and may help explain why chronic administration of β2AR agonists, such as formoterol, have been associated with adverse outcomes in asthma. These data further support the hypothesis that chronic asthma management may be better served by treatment with certain “β-blockers.”
β2-adrenoceptor agonists; formoterol; epinephrine; murine model; asthma