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.
High-mobility group box 1 (HMGB1) is a nuclear protein that has been found to be a critical mediator of lethality in endotoxemia and sepsis. During the systemic inflammatory response, circulating levels of HMGB1 are increased, but in a delayed fashion compared with early inflammatory mediators. To counteract the inflammatory response of endotoxemia, a secondary anti-inflammatory response ensues in an attempt to prevent inflammation-induced tissue injury. One such cytoprotective gene that is induced during endotoxemia is heme oxygenase (HO)-1. HO-1, and its products of heme metabolism, possess anti-inflammatory and antioxidant properties to counter the damaging effects of endotoxemia. In the present study, we wanted to determine whether tissue and circulating levels of HMGB1 are increased further in the absence of HO-1 during endotoxemia, and whether this increase may contribute to the pathobiology of endotoxemia. Lung inflammation, HMGB1 protein levels, and expression of HMGB1 in inflammatory cells were increased in HO-1−/− mice compared with HO-1+/+ mice. After the administration of LPS, tissue levels of HMGB1 were not increased further in HO-1−/− mice; however, circulating levels of HMGB1 were higher when compared with HO-1+/+ mice. HO-1−/− mice treated with a carbon monoxide–releasing molecule or biliverdin showed a reduction in plasma HMGB1, which was associated with a marked improvement in survival. HO-1−/− mice given HMGB1-neutralizing antibody showed improvement in survival compared with control antibody. These data suggest that exaggerated circulating levels of HMGB1 contribute to endotoxin-induced mortality in the absence of HO-1.
endotoxemia; heme oxygenase-1; inflammation; high-mobility group box 1; oxidative stress
Mice infected for 60 days with Mycobacterium tuberculosis were treated with aerosolized XCL1-targeting small interfering RNA (siRNA) to induce local and transient suppression of XCL1/lymphotactin (an important chemokine in tuberculoid granuloma formation). The local pulmonary siRNA therapy resulted in a 50% decrease in the total amount of xcl1 gene transcripts at 3 days, and 40 to 50% protein suppression 3 and 5 days after treatment. Reduced XCL1 expression in the lungs was associated with decreased numbers of T lymphocytes, reduction in the IFN-γ response, disorganized granulomatous lesions, and higher fibrosis when compared with control mice treated with either PBS or nontargeting siRNA. This indicates that a transient but strong modulation of the production of XCL1 in the lungs has a significant effect on the influx of IFN-γ–secreting T cells, as well as local pathology, but without significantly altering containment of the infection.
tuberculosis; small interfering RNA; lymphotactin; XCL1; aerosol delivery
The induction of cyclooxygenase-2 (COX-2) in tissue macrophages (M∅) increases prostaglandin E2 (PGE2) release, potentially down-regulating granulomatous inflammation. In response to Mycobacteria, local M∅ express COX-2, which is either nuclear envelope (NE)-associated or NE-dissociated. Persistent mycobacterial pulmonary inflammation is characterized by alveolar M∅ expressing NE-dissociated (inactive) COX-2 without release of PGE2. In this study, we examined COX-2 in alveolar M∅ after intranasal exposure to heat-killed Mycobacterium bovis BCG (HK-BCG). After administration, whole lungs of C57Bl/6 mice were lavaged with saline; COX-2 expression and PGE2 release by alveolar M∅ and tumor necrosis factor (TNF)-α and nitric oxide levels in the lung lavage were monitored. Normal alveolar M∅ had undetectable levels of COX-2 on Western blots. However, 1 day after intranasal administration, almost all alveolar M∅ had phagocytosed HK-BCG and expressed NE-dissociated COX-2 without any increase in the release of PGE2. At 28 days after intranasal administration, 68% of alveolar M∅ still contained both BCG and the NE-dissociated form of COX-2. NE-associated (active) COX-2 was not observed in alveolar M∅. In contrast, 7 days after intraperitoneal injection of HK-BCG, peritoneal M∅ containing HK-BCG were no longer detected. At 28 days after intranasal administration, TNF-α and nitrite levels in the lung lavage fluid were significantly higher than those in controls. Our results indicate that mycobacterial pulmonary inflammation is associated with suppressed PGE2 production by alveolar M∅, with expression of COX-2 dissociated from the NE.
alveolar macrophages; PGE2; COX-2; mycobacteria
Our previous studies revealed that the presence in lung fluids of anti–IL-8 autoantibody:IL-8 immune complexes is an important prognostic indicator for the development and outcome of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). Anti–IL-8:IL-8 complexes purified from lung edema fluids trigger chemotaxis of neutrophils, induce activation of these cells, and regulate their apoptosis, all via IgG receptor, FcγRIIa. Importantly, increased levels of FcγRIIa are present in lungs of patients with ARDS, where FcγRIIa is partially associated with anti–IL-8:IL-8 complexes. In the current study, we demonstrate the ability of anti–IL-8:IL-8 complexes to promote an inflammatory phenotype of human umbilical vein endothelial cells via interaction with FcγRIIa. Human umbilical vein endothelial cells cultured in the presence of the complexes become activated, as shown by increased phosphorylation of ERK, JNK, and Akt, and augmented nuclear translocation of NF-κB. Anti–IL-8:IL-8 complexes also up-regulate expression of intracellular adhesion molecule (ICAM)-1 on the cell surface. Furthermore, we detected increased levels of ICAM-1 on lung endothelial cells from mice in which lung injury was induced by generating immune complexes in alveolar spaces. On the other hand, ICAM-1 expression was unchanged in lungs of γ chain–deficient mice, lacking receptors that interact with immune complexes. Moreover, in lung tissues from patients with ARDS, anti–IL-8:IL-8 complexes were associated with endothelial cells that expressed higher levels of ICAM-1. Our current findings implicate that anti-chemokine autoantibody:chemokine immune complexes, such as IL-8:IL-8 complexes, may contribute to pathogenesis of lung inflammation by inducing activation of endothelial cells through engagement of IgG receptors.
chemokine; autoantibody; lung; IgG receptor
Mycobacterium abscessus is a rapidly growing environmental mycobacterium that can cause severe skin, soft tissue, and lung infections. M. abscessus grows inside macrophages, and these cells release a vast number of proinflammatory cytokines in response to infections. The metalloporphyrin, MnTE-2-PyP, is a broad antioxidant that reduces inflammatory cell signaling. Macrophage-like THP-1 cells were infected with M. abscessus in the presence or absence of MnTE-2-PyP. MnTE-2-PyP significantly decreased, in a dose-dependent manner, the number of M. abscessus organisms recovered from infected THP-1 cells 4 and 8 days after infection. Furthermore, when combined with clarithromycin, MnTE-2-PyP additively reduced the number of cells associated with M. abscessus. A mechanism of bacterial growth inhibition by MnTE-2-PyP was then elucidated. It was found that MnTE-2-PyP promoted the survival of infected THP-1 cells and increased fusion of M. abscessus–containing phagosomes with lysosomes.
antioxidant mimetic; MnTE-2-PyP; Mycobacterium abscessus; THP-1 cells
Chronic alcohol abuse impairs both alveolar epithelial and macrophage function, and renders individuals susceptible to acute lung injury, pneumonia, and other serious lung diseases. Zinc deficiency, which is known to impact both epithelial and immune cell functions, is also associated with alcohol abuse. In this study, chronic alcohol ingestion (6 wk) in rats altered expression of key zinc transporters and storage proteins in the small intestine and the lung, and decreased zinc levels in the alveolar compartment. Zinc supplementation of alveolar epithelial monolayers derived from alcohol-fed rats in vitro, or of the diets of alcohol-fed rats in vivo, restored alveolar epithelial barrier function, and these improvements were associated with salutary changes in tight junction protein expression and membrane localization. In parallel, dietary zinc supplementation increased intracellular zinc levels, GM-CSF receptor expression, and bacterial phagocytic capacity in the alveolar macrophages of alcohol-fed rats. Together, these studies implicate zinc deficiency as a novel mechanism mediating alcohol-induced alveolar epithelial and macrophage dysfunction. Importantly, these findings argue that dietary supplementation can overcome alcohol-induced zinc deficiency and restore alveolar epithelial and macrophage function, and therefore could be an effective treatment for the susceptible alcoholic lung phenotype.
GM-CSF; phagocytosis; tight junctions; zinc transporters; metallothionein
One host susceptibility factor for ozone identified in epidemiologic studies is NAD(P)H quinone oxidoreductase 1 (NQO1). We hypothesized that after ozone exposure, NQO1 is required to increase 8-isoprostane (also known as F2-isoprostane) production, a recognized marker of ozone-induced oxidative stress, and to enhance airway inflammation and hyperresponsiveness. In this report, we demonstrate that in contrast to wild-type mice, NQO1-null mice are resistant to ozone and have blunted responses, including decreased production of F2-isoprostane and keratinocyte chemokine, decreased airway inflammation, and diminished airway hyperreponsiveness. Importantly, these results in mice correlate with in vitro findings in humans. In primary human airway epithelial cells, inhibition of NQO1 by dicumarol blocks ozone-induced F2-isoprostane production and IL-8 gene expression. Together, these results demonstrate that NQO1 modulates cellular redox status and influences the biologic and physiologic effects of ozone.
ozone; NAD(P)H quinone oxidoreductase 1; F2-isoprostane
Human mesothelial cells (LP9/TERT-1) were exposed to low and high (15 and 75 μm2/cm2 dish) equal surface area concentrations of crocidolite asbestos, nonfibrous talc, fine titanium dioxide (TiO2), or glass beads for 8 or 24 hours. RNA was then isolated for Affymetrix microarrays, GeneSifter analysis and QRT-PCR. Gene changes by asbestos were concentration- and time-dependent. At low nontoxic concentrations, asbestos caused significant changes in mRNA expression of 29 genes at 8 hours and of 205 genes at 24 hours, whereas changes in mRNA levels of 236 genes occurred in cells exposed to high concentrations of asbestos for 8 hours. Human primary pleural mesothelial cells also showed the same patterns of increased gene expression by asbestos. Nonfibrous talc at low concentrations in LP9/TERT-1 mesothelial cells caused increased expression of 1 gene Activating Transcription Factor 3 (ATF3) at 8 hours and no changes at 24 hours, whereas expression levels of 30 genes were elevated at 8 hours at high talc concentrations. Fine TiO2 or glass beads caused no changes in gene expression. In human ovarian epithelial (IOSE) cells, asbestos at high concentrations elevated expression of two genes (NR4A2, MIP2) at 8 hours and 16 genes at 24 hours that were distinct from those elevated in mesothelial cells. Since ATF3 was the most highly expressed gene by asbestos, its functional importance in cytokine production by LP9/TERT-1 cells was assessed using siRNA approaches. Results reveal that ATF3 modulates production of inflammatory cytokines (IL-1β, IL-13, G-CSF) and growth factors (VEGF and PDGF-BB) in human mesothelial cells.
mesothelioma; crocidolite asbestos; talc; titanium dioxide; gene profiling
Combinatorial therapies using the proteasome inhibitor, bortezomib, have been found to induce synergistic apoptosis in cancer cells grown as monolayers; however, three-dimensional spheroid culture may be a better model for the multicellular resistance found in solid tumors, such as lung cancer. We tested the combinatorial apoptotic strategy of using bortezomib together with TNF-related apoptosis–inducing ligand (TRAIL), both in monolayers and in spheroids of A549 lung cancer cells. Indeed, bortezomib plus TRAIL induced synergistic apoptosis in A549 cells grown as monolayers, but had little effect on A549 cells grown as three-dimensional multicellular spheroids. The acquired resistance of spheroids was not due to a limitation of diffusion, to survival pathways, such as NF-κB or PI3K/Akt/mTOR, or to the up-regulation of FLIPS (Fas-associated death domain–like IL-1β–converting enzyme inhibitory protein, short). We then investigated a role for the Bcl-2 family of anti- and proapoptotic proteins. When cells formed spheroids, antiapoptotic Bcl-2 increased, whereas antiapoptotic Mcl-1 decreased. ABT-737, a small molecule that inhibits Bcl-2, but not Mcl-1, abolished the multicellular resistance of A549 spheroids to bortezomib plus TRAIL. In another lung cancer cell line, H1299, acquisition of multicellular resistance in spheroids was also accompanied by an increase in Bcl-2 and decrease in Mcl-1. In H1299 spheroids compared with those of A549, however, Mcl-1 remained higher, and Mcl-1 knockdown was more effective than ABT-737 in removing multicellular resistance. Our study suggests that the balance of Bcl-2 family proteins contributes to the acquired multicellular resistance of spheroids, and suggests a possible target for improving the response of lung cancer to bortezomib therapies.
proteasome; TNF-related apoptosis–inducing ligand; mitochondria; bortezomib; ABT-737; Mcl-1
Glucocorticoids (GCs) and protein kinase A (PKA)–activating agents (β-adrenergic receptor agonists) are mainstream asthma therapies based on their ability to prevent or reverse excessive airway smooth muscle (ASM) constriction. Their abilities to regulate another important feature of asthma—excessive ASM growth—are poorly understood. Recent studies have suggested that GCs render agents of inflammation such as IL-1β and TNF-α mitogenic to ASM, via suppression of (antimitogenic) induced cyclooxygenase-2–dependent PKA activity. To further explore the mechanistic basis of these observations, we assessed the effects of epidermal growth factor and IL-1β stimulation, and the modulatory effects of GC treatment and PKA inhibition, on the ASM transcriptome by microarray analysis. Results demonstrate that ASM stimulated with IL-1β, in a manner that is often cooperative with stimulation with epidermal growth factor, exhibit a profound capacity to function as immunomodulatory cells. Moreover, results implicate an important role for induced autocrine/paracrine factors (many whose regulation was minimally affected by GCs or PKA inhibition) as regulators of both airway inflammation and ASM growth. Induction of numerous chemokines, in conjunction with regulation of proteases and agents of extracellular matrix remodeling, is suggested as an important mechanism promoting upregulated G protein–coupled receptor signaling capable of stimulating ASM growth. Additional functional assays suggest that intracellular PKA plays a critical role in suppressing the promitogenic effects of induced autocrine factors in ASM. Finally, identification and comparison of GC- and PKA-sensitive genes in ASM provide insight into the complementary effects of β-agonist/GC combination therapies, and suggest specific genes as important targets for guiding the development of new generations of GCs and adjunct asthma therapies.
airway smooth muscle; protein kinase A; glucocorticoid; gene expression; G protein–coupled receptors
We recently reported that induction of metallothionein (MT) was critical in limiting nickel (Ni)-induced lung injury in intact mice. Nonetheless, the mechanism by which Ni induces MT expression is unclear. We hypothesized that the ability of Ni to mobilize zinc (Zn) may contribute to such regulation and therefore, we examined the mechanism for Ni-induced MT2A expression in human airway epithelial (BEAS-2B) cells. Ni induced MT2A transcript levels and protein expression by 4 hours. Ni also increased the activity of a metal response element (MRE) promoter luciferase reporter construct, suggesting that Ni induces MRE binding of the metal transcription factor (MTF-1). Exposure to Ni resulted in the nuclear translocation of MTF-1, and Ni failed to induce MT in mouse embryonic fibroblasts lacking MTF-1. As Zn is the only metal known to directly bind MTF-1, we then showed that Ni increased a labile pool of intracellular Zn in cells as revealed by fluorescence-activated cell sorter using the Zn-sensitive fluorophore, FluoZin-3. Ni-induced increases in MT2A mRNA and MRE-luciferase activity were sensitive to the Zn chelator, TPEN, supporting an important role for Zn in mediating the effect of Ni. Although neither the source of labile Zn nor the mechanism by which Ni liberates labile Zn was apparent, it was noteworthy that Ni increased intracellular reactive oxygen species (ROS). Although both N-acetyl cysteine (NAC) and ascorbic acid (AA) decreased Ni-induced increases in ROS, only NAC prevented Ni-induced increases in MT2A mRNA, suggesting a special role for interactions of Ni, thiols, and Zn release.
nickel; metallothionein; zinc; epithelium
IL-10 is a potent, endogenous anti-inflammatory cytokine known to decrease cytokine and keratinocyte-derived chemokine (KC) expression. Traditionally, in vivo effects of IL-10 were extrapolated from studies employing systemic antibody neutralization. As a result, divergent data regarding the protective and/or harmful roles of IL-10 have been reported. In this study, we used a lung-specific, tetracycline-inducible IL-10 overexpression–transgenic (IL-10 OE) mouse to study the effects of IL-10 overexpression on Pseudomonas aeruginosa–induced lung inflammation and corresponding survival in mice. Overexpression of IL-10 in the lung significantly increased mortality. During the early phase after infection (6-hours after infection), neutrophil recruitment as well as cytokine (TNF-α) and chemokine (KC) expression were significantly decreased in the IL-10 OE mice, which resulted in attenuated bacterial clearance. In contrast, overzealous production of KC and TNF-α intensified neutrophil infiltration and increased vascular leakage in IL-10 OE mice at the later stage of infection (24 hours after infection). Neutrophil depletion showed impaired bacterial clearance in both control and IL-10 OE mice, and further enhanced mouse mortality, whereas exogenous administration of KC reversed this finding. Our data indicate that early neutrophil recruitment is important for combating bacterial infection, and that the inhibition of neutrophil recruitment by IL-10 results in insufficient bacteria clearance in the lung, leading to excessive development of inflammation and increased mortality.
IL-10; Pseudomonas; neutrophils; chemokines
Carbon monoxide (CO) is a biologically active molecule produced in the body by the stress-inducible enzyme, heme oxygenase. We have previously shown that CO suppresses fibrosis in a murine bleomycin model. To investigate the mechanisms by which CO opposes fibrogenesis, we performed gene expression profiling of fibroblasts treated with transforming growth factor-β1 and CO. The most highly differentially expressed categories of genes included those related to muscular system development and the small proline-rich family of proteins. We confirmed in vitro, and in an in vivo bleomycin model of lung fibrosis, that CO suppresses α–smooth muscle actin expression and enhances small proline-rich protein-1a expression. We further show that these effects of CO depend upon signaling via the extracellular signal–regulated kinase pathway. Our results demonstrate novel transcriptional targets for CO and further elucidate the mechanism by which CO suppresses fibrosis.
carbon monoxide; heme oxygenase-1; lung fibrosis; small proline-rich protein; α-smooth muscle actin
Prenatal nicotine exposure impairs normal lung development and leads to diminished pulmonary function after birth. Previous work from our laboratory has demonstrated that nicotine alters lung development by affecting a nonneuronal cholinergic autocrine loop that is expressed in lung. Bronchial epithelial cells (BECs) express choline acetyltransferase, the choline high-affinity transporter and nicotinic acetylcholine (ACh) receptor (nAChR) subunits. We now demonstrate through a combination of morphological and electrophysiological techniques that nicotine affects this autocrine loop by up-regulating and activating cholinergic signaling. RT-PCR showed the expression of α3, α4, α7, α9, α10, β2, and β4 nAChR mRNAs in rhesus monkey lung and cultured BECs. The expression of α7, α4, and β2 nAChR was confirmed by immunofluorescence in the cultured BECs and lung. The electrophysiological characteristics of nAChR in BECs were determined using whole-cell patch–clamp on cultured BECs. Both ACh and nicotine evoked an inward current, with a rapid desensitizing current. Nicotine induced inward currents in a concentration-dependent manner, with an EC50 of 26.7 μM. Nicotine-induced currents were reversibly blocked by the nicotinic antagonists, mecamylamine, dihydro-β-erythroidine, and methyllcaconitine. Incubation of BECs with 1 μM nicotine for 48 hours enhanced nicotine-induced currents by roughly 26%. The protein tyrosine phosphorylation inhibitor, genistein, increased nicotine-induced currents by 58% and enhanced methyllcaconitine-sensitive currents (α7 nAChR activities) 2.3-fold, whereas the protein tyrosine phosphatase inhibitor, pervanadate, decreased the effects of nicotine. These results demonstrate that chronic nicotine exposure up-regulates nAChR activity in developing lung, and that nAChR activity can be further modified by tyrosine phosphorylation.
nicotinic acetylcholine receptors; electrophysiology; bronchial epithelial cells; nicotine; lung
Defective epithelial repair in the setting of chronic lung disease has been suggested to contribute to uncontrolled extracellular matrix (ECM) deposition and development of fibrosis. We sought to directly test this hypothesis through gene expression profiling of total lung RNA isolated from mouse models of selective epithelial cell injury that are associated with either productive or abortive repair. Analysis of gene expression in repairing lungs of naphthalene-exposed mice revealed prominent clusters of up-regulated genes with putative roles in regulation of the extracellular matrix and cellular proliferation. Further analysis of tenascin C (Tnc), a representative matrix protein, in total lung RNA revealed a transient 4.5-fold increase in mRNA abundance 1 day after injury and a return to steady-state levels by Recovery Day 3. Tnc was deposited by the peribronchiolar mesenchyme immediately after injury and was remodeled to basement membrane subtending the bronchiolar epithelium during epithelial repair. Epithelial restitution was accompanied by a decrease in Tnc mRNA and protein expression to steady-state levels. In contrast, abortive repair using a transgenic model allowing ablation of all reparative cells led to a progressive increase in Tnc mRNA within lung tissue and accumulation of its gene product within the subepithelial mesenchyme of both conducting airways and alveoli. These data demonstrate that the ECM is dynamically remodeled in response to selective epithelial cell injury and that this process is activated without resolution in the setting of defective airway epithelial repair.
airway epithelium; repair; Clara cell; extracellular matrix; fibrosis
Idiopathic pulmonary fibrosis is a lethal parenchymal lung disease characterized by denudation of the lung epithelium, fibroblast proliferation, and collagen deposition. Cellular changes underlying disease progression involve injury to alveolar epithelial cells, epithelial to mesenchymal transition, proliferation of α-smooth muscle actin (α-SMA)–expressing myofibroblasts and of fibroblasts resulting in enhanced deposition of extracellular matrix proteins. Hepatocyte growth factor (HGF) inhibits progression of bleomycin-induced pulmonary fibrosis in mice. The mechanism underlying the inhibitory effect of HGF was investigated in an in vitro model. We show that HGF markedly antagonizes basal and transforming growth factor (TGF)-β–induced expression of myofibroblast markers such as α-SMA, collagen type 1, and fibronectin in rat alveolar epithelial cells. HGF also inhibited TGF-β–induced α-SMA expression in primary murine alveolar epithelial cells. Since TGF-β is known to regulate α-SMA expression, the effect of HGF on components of TGF-β signaling was investigated. HGF induced expression of Smad7, an inhibitor of TGF-β signaling, in a mitogen-activated protein kinase–dependent manner. HGF also induced the nuclear export of Smad7 and Smad ubiquitin regulatory factor 1 (Smurf1) to the cytoplasm. HGF-dependent decrease in α-SMA was abolished with specific siRNAs targeted to Smad7. Thus, induction of Smad7 by HGF serves to limit acquisition of the myofibroblast phenotype in alveolar epithelial cells.
EMT; HGF; Smad7
Protein kinase C (PKC) is known to regulate ryanodine receptor (RyR)–mediated local Ca2+ signaling (Ca2+ spark) in airway and vascular smooth muscle cells (SMCs), but its specific molecular mechanisms and functions still remain elusive. In this study, we reveal that, in airway SMCs, specific PKCɛ peptide inhibitor and gene deletion significantly increased the frequency of Ca2+ sparks, and decreased the amplitude of Ca2+ sparks in the presence of xestospogin-C to eliminate functional inositol 1,4,5-triphosphate receptors. PKCɛ activation with phorbol-12-myristate-13-acetate significantly decreased Ca2+ spark frequency and increased Ca2+ spark amplitude. The effect of PKCɛ inhibition or activation on Ca2+ sparks was completely lost in PKCɛ−/− cells. PKCɛ inhibition or PKCɛ activation was unable to affect Ca2+ sparks in RyR1−/− and RyR1+/− cells. Modification of RyR2 activity by FK506-binding protein 12.6 homozygous or RyR2 heterozygous gene deletion did not prevent the effect of PKCɛ inhibition or activation. RyR3 homogenous gene deletion did not block the effect of PKCɛ inhibition and activation, either. PKCɛ inhibition promotes agonist-induced airway muscle contraction, whereas PKCɛ activation produces an opposite effect. Taken together, these results indicate that PKCɛ regulates Ca2+ sparks by specifically interacting with RyR1, which plays an important role in the control of contractile responses in airway SMCs.
protein kinase C; local calcium signaling; ryanodine receptor; contraction; airway myocytes
Previous studies have shown that leukotriene B4 (LTB4), a proinflammatory lipid mediator, is linked to the development of airway hyperresponsiveness through the accumulation of IL-13–producing CD8+ T cells, which express a high affinity receptor for LTB4, BLT1 (Miyahara et al., Am J Respir Crit Care Med 2005;172:161–167; J Immunol 2005;174:4979–4984). By using leukotriene A4 hydrolase–deficient (LTA4H−/−) mice, which fail to synthesize LTB4, we determined the role of this lipid mediator in allergen-induced airway responses. Two approaches were used. In the first, LTA4H−/− mice and wild-type (LTA4H+/+) mice were systemically sensitized and challenged via the airways to ovalbumin. In the second, mice were passively sensitized with anti-ovalbumin IgE and exposed to ovalbumin via the airways. Mast cells were generated from bone marrow of LTA4H+/+ mice or LTA4H−/− mice. After active sensitization and challenge, LTA4H−/− mice showed significantly lower airway hyperresponsiveness compared with LTA4H+/+ mice, and eosinophil numbers and IL-13 levels in the bronchoalveoloar lavage of LTA4H−/− mice were also significantly lower. LTA4H−/− mice also showed decreased airway reactivity after passive sensitization and challenge. After LTA4H+/+ mast cell transfer, LTA4H−/− mice showed increased airway reactivity after passive sensitization and challenge, but not after systemic sensitization and challenge. These data confirm the important role for LTB4 in the development of altered airway responses and suggest that LTB4 secretion from mast cells is critical to eliciting increased airway reactivity after passive sensitization with allergen-specific IgE.
rodent; T cells; cytokines; lipid mediators; lung
Bacterial pneumonia remains a serious disease. Pattern recognition receptors play an integral role in neutrophil accumulation during pneumonia. Although myeloid differentiation protein (MD)-2 has been recognized as a key molecule for LPS signaling, the role of MD-2 in neutrophil accumulation in the lung during bacterial infection has not been explored. Here, we investigate the role of MD-2 in Escherichia coli LPS–induced lung inflammation and E. coli–induced pneumonia. LPS-induced CD14-independent neutrophil accumulation was abolished in CD14/MD-2−/− mice. MD-2−/− mice challenged with LPS displayed attenuated neutrophil influx, NF-κB activation, cytokine/chemokine expression, and lung histopathology. MD-2−/− mice transplanted with MD-2+/+ bone marrow demonstrated decreased neutrophil influx and cytokine/chemokine expression in the lungs when challenged by LPS. MD-2−/− mice infected with E. coli demonstrated reduced neutrophil influx and cytokine/chemokine expression in the lungs, whereas heat-killed E. coli did not induce either neutrophil accumulation or cytokine/chemokine expression in MD-2−/− mice infected with E. coli. Furthermore, MD-2−/− mice displayed increased bacterial burden in the lungs and enhanced bacterial dissemination. Toll-like receptor (TLR)-5−/− mice infected with E. coli exhibited attenuated neutrophil accumulation, whereas MD-2/TLR5−/− mice inoculated with E. coli showed further attenuated neutrophil influx and impaired bacterial clearance. Taken together, these new findings demonstrate: (1) the important role of MD-2 in the CD14-independent LPS-mediated cascade of neutrophil influx; (2) the relative importance of bone marrow– and non–bone marrow cell–derived MD-2 in LPS-induced inflammation; and (3) the essential role of MD-2–dependent and MD-2–independent (TLR5) signaling in E. coli–induced neutrophil accumulation and pulmonary host defense.
neutrophil; host defense; mouse model
The destruction of elastic fibers has been implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). Emphysema has been described in autosomal dominant cutis laxa, which can be caused by mutations in the elastin gene. Previously, a rare functional mutation in the terminal exon of elastin was found in a case of severe, early-onset COPD. To test the hypothesis that other similar elastin mutations may predispose to COPD, we screened 90 probands from the Boston Early-Onset COPD Study and 90 smoking control subjects from the Normative Aging Study for mutations in elastin exons using high-resolution DNA melt analysis followed by resequencing. Rare nonsynonymous single-nucleotide polymorphisms (SNPs) seen only in cases were examined for segregation with airflow obstruction within pedigrees. Common nonsynonymous SNPs were tested for association with COPD in a family-based analysis of 949 subjects from the Boston Early-Onset COPD Study, and in a case–control analysis in 389 COPD cases from the National Emphysema Treatment Trial and 472 control subjects from the Normative Aging Study. Of 28 elastin variants found, 3 were nonsynonymous SNPs found only in cases. The previously described Gly773Asp mutation was found in another proband. The other two SNPs did not clearly segregate with COPD within families. Two common nonsynonymous SNPs did not demonstrate significant associations in either a family-based or case–control analysis. Exonic SNPs in the elastin gene do not appear to be common risk factors for severe COPD.
elastin; chronic obstructive pulmonary disease; emphysema; genetic polymorphism
Toluene diisocyanate (TDI), a reactive, hazardous irritant, causes respiratory symptoms such as cough, rhinitis, dyspnea, and chest tightness in exposed workers. Although previous animal studies have shown that TDI causes respiratory reflexes that are abolished by desensitization of capsaicin-sensitive sensory nerves, the specific molecular identity of the transducer(s) responsible for sensing this noxious stimulus has, to date, remained elusive. Recent studies have demonstrated that transient receptor potential ankyrin 1 (TRPA1), an ion channel largely restricted to a subset of capsaicin-sensitive sensory nerves, functions as a transducer capable of initiating reflex responses to many reactive chemical stimuli. We therefore hypothesized that TRPA1 is the primary molecular transducer through which TDI causes sensory nerve activation and respiratory reflexes. Consistent with this hypothesis, TDI activated TRPA1, but not the capsaicin-sensitive transient receptor potential vanilloid 1 channel, in heterologous expression systems. TDI also activated a subset of dissociated trigeminal sensory neurons from wild-type but not TRPA1-deficient mice. In vivo, TDI mimicked known TRPA1 agonists by causing a pronounced decrease in breathing rate, indicative of respiratory sensory irritation, and this reflex was abolished in TRPA1-deficient mice. Together, our data suggest that TDI causes sensory nerve activation and airway sensory irritation via the activation of the ion channel, TRPA1.
toluene diisocyanate; formaldehyde; sensory irritation; transient receptor potential ankyrin-1
The primary function of neutrophils in host defense is to contain and eradicate invading microbial pathogens. This is achieved through a series of swift and highly coordinated responses culminating in ingestion (phagocytosis) and killing of invading microbes. While these tasks are usually performed without injury to host tissues, in pathologic circumstances such as sepsis, potent antimicrobial compounds can be released extracellularly, inducing a spectrum of responses in host cells ranging from activation to injury and death. In the lung, such inflammatory damage is believed to contribute to the pathogenesis of diverse lung diseases, including acute lung injury and the acute respiratory distress syndrome, chronic obstructive lung disease, and cystic fibrosis. In these disorders, epithelial cells are targets of leukocyte-derived antimicrobial products, including proteinases and oxidants. Herein, we review the mechanisms involved in the physiologic process of neutrophil transepithelial migration, including the role of specific adhesion molecules on the leukocyte and epithelial cells. We examine the responses of the epithelial cells to the itinerant leukocytes and their cytotoxic products and the consequences of this for lung injury and repair. This paradigm has important clinical implications because of the potential for selective blockade of these pathways to prevent or attenuate lung injury.
inflammation; acute lung injury; tight junctions; adherens junctions; proteolytic enzymes
Pleural effusions are commonly clinical disorders, resulting from the imbalance between pleural fluid turnover and reabsorption. The mechanisms underlying pleural fluid clearance across the mesothelium remain to be elucidated. We hypothesized that epithelial Na+ channel (ENaC) is expressed and forms the molecular basis of the amiloride-sensitive resistance in human mesothelial cells. Our RT-PCR results showed that three ENaC subunits, namely, α, β, γ, and two δ ENaC subunits, are expressed in human primary pleural mesothelial cells, a human mesothelioma cell line (M9K), and mouse pleural tissue. In addition, Western blotting and immunofluorescence microscopy studies revealed that α, β, γ, and δ ENaC subunits are expressed in primary human mesothelial cells and M9K cells at the protein level. An amiloride-inhibitable short-circuit current was detected in M9K monolayers and mouse pleural tissues when mounted in Ussing chambers. Whole-cell patch clamp recordings showed an ENaC-like channel with an amiloride concentration producing 50% inhibition of 12 μM in M9K cells. This cation channel has a high affinity for extracellular Na+ ions (Km: 53 mM). The ion selectivity of this channel to cations follows the same order as ENaC: Li+ > Na+ > K+. The unitary Li+ conductance was 15 pS in on-cell patches. Four ENaC subunits form a functional Na+ channel when coinjected into Xenopus oocytes. Furthermore, we found that both forskolin and cGMP increased the short-circuit currents in mouse pleural tissues. Taken together, our data demonstrate that the ENaC channels are biochemically and functionally expressed in human pleural mesothelial cells, and can be up-regulated by cyclic AMP and cyclic GMP.
M9K mesothelioma cells; Ussing chamber; protein kinase A; protein kinase G; human primary mesothelial cells
Asthma is a chronic inflammatory disorder of the airways. Type 2 T helper (Th) cell–dominated inflammation in the lung is a hallmark of asthma. Src homology 2 domain–containing protein tyrosine phosphatase (SHP)-1 is a negative regulator in the signaling pathways of many growth factor and cytokine receptors. However, a direct role of SHP-1 in the IL-4/IL-13 signaling pathway has not been established. In this study, we sought to define the function of SHP-1 in the lung by characterizing the pulmonary inflammation of viable motheaten (mev) mice, and to investigate the molecular mechanisms involved. Pulmonary histology, physiology, and cytokine expression of mev mice were analyzed to define the nature of the inflammation, and the gene-deletion approach was used to identify critical molecules involved. In mev mice, we observed spontaneous Th2-like inflammatory responses in the lung, including eosinophilia, mucus metaplasia, airway epithelial hypertrophy, pulmonary fibrosis, and increased airway resistance and airway hyperresponsiveness. The pulmonary phenotype was accompanied by up-regulation of Th2 cytokines and chemokines. Selective deletion of IL-13 or signal transducer and activator of transcription 6, key genes in the Th2 signaling pathway, significantly reduced, but did not completely eliminate, the inflammation in the lung. These findings suggest that SHP-1 plays a critical role in regulating the IL-4/IL-13 signaling pathway and in maintaining lung homeostasis.
Src homology 2 domain–containing protein tyrosine phosphatase-1; protein tyrosine phosphatase; motheaten mouse; type 2 T helper cell inflammation; lung