The alveolar compartment, the fundamental gas exchange unit in the lung, is critical for tissue oxygenation and viability. We explored hepatocyte growth factor (HGF), a pleiotrophic cytokine that promotes epithelial proliferation, morphogenesis, migration, and resistance to apoptosis, as a candidate mediator of alveolar formation and regeneration. Mice deficient in the expression of the HGF receptor Met in lung epithelial cells demonstrated impaired airspace formation marked by a reduction in alveolar epithelial cell abundance and survival, truncation of the pulmonary vascular bed, and enhanced oxidative stress. Administration of recombinant HGF to tight-skin mice, an established genetic emphysema model, attenuated airspace enlargement and reduced oxidative stress. Repair in the TSK/+ mouse was punctuated by enhanced akt and stat3 activation. HGF treatment of an alveolar epithelial cell line not only induced proliferation and scattering of the cells but also conferred protection against staurosporine-induced apoptosis, properties critical for alveolar septation. HGF promoted cell survival was attenuated by akt inhibition. Primary alveolar epithelial cells treated with HGF showed improved survival and enhanced antioxidant production. In conclusion, using both loss-of-function and gain-of-function maneuvers, we show that HGF signaling is necessary for alveolar homeostasis in the developing lung and that augmentation of HGF signaling can improve airspace morphology in murine emphysema. Our studies converge on prosurvival signaling and antioxidant protection as critical pathways in HGF–mediated airspace maintenance or repair. These findings support the exploration of HGF signaling enhancement for diseases of the airspace.
The airspace compartment of the mammalian lung, comprised of spherical sacs termed alveoli, harbors the architecture, cellular composition, and molecular armamentarium to perform the critical function of gas exchange or oxygen uptake. Despite the necessity of this alveolar compartment for organismal viability, the mechanism by which alveoli are formed and maintained is obscure. Furthermore, no treatments are currently available that can regenerate the airspace once damaged. In this manuscript, we sought to determine whether hepatocyte growth factor, a cytokine with a functional armamentarium that subserves the critical events of alveolar formation (epithelial proliferation, migration, resistance from apoptosis and angiogenesis), could be an important mediator of alveolar formation and airspace maintenance. Our simple paradigm was that critical homeostatic pathways for the lung should operate both in lung formation and in lung maintenance/regeneration. Using an informative battery of mouse models and cell lines, we show that hepatocyte growth factor is a determinant of alveolar formation and that the enhancement of hepatocyte growth factor signaling can both protect and repair the airspace from pathologic airspace enlargement or emphysema.
Theories of emphysema traditionally revolved around proteolytic destruction of extracellular matrix. Models have recently been developed that show airspace enlargement with the induction of pulmonary cell apoptosis. The purpose of this study was to determine the mechanism by which a model of epithelial cell apoptosis caused airspace enlargement. Mice were treated with either intratracheal microcystin (MC) to induce apoptosis, intratracheal porcine pancreatic elastase (PPE), or their respective vehicles. Mice from all groups were inflated and morphometry was measured at various time points. Physiology measurements were performed for airway resistance, tissue elastance, and lung volumes. The groups were further analyzed by air–saline quasistatic measurements, surfactant staining, and surfactant functional studies. Mice treated with MC showed evidence of reversible airspace enlargement. In contrast, PPE-treated mice showed irreversible airspace enlargement. The airspace enlargement in MC-treated mice was associated with an increase in elastic recoil due to an increase in alveolar surface tension. PPE-treated mice showed a loss of lung elastic recoil and normal alveolar surface tension, a pattern more consistent with human emphysema. Airspace enlargement that occurs with the MC model of pulmonary epithelial cell apoptosis displays physiology distinct from human emphysema. Reversibility, restrictive physiology due to changes in surface tension, and alveolar enlargement associated with heterogeneous alveolar collapse are most consistent with a mild acute lung injury. Inflation near total lung capacity gives the appearance of enlarged alveoli as neighboring collapsed alveoli exert tethering forces.
apoptosis; emphysema; physiology; murine model
Apoptosis of alveolar septal cells has been linked to emphysema formation. Nitrogen dioxide, a component of cigarette smoke, has been shown to induce alveolar epithelial cell apoptosis in vitro. It is hypothesised that exposure of rats to nitrogen dioxide may result in increased alveolar septal cell apoptosis in vivo with ensuing emphysema—that is, airspace enlargement and loss of alveolar walls.
Fischer 344 rats were exposed to 10 ppm nitrogen dioxide for 3, 7, 21 days or 21 days followed by 28 days at room air. Age‐matched control rats were exposed to room air for 3, 21 or 49 days. Lungs fixed at 20 cm fluid column, embedded in paraffin wax, glycol methacrylate and araldite, were analysed by design‐based stereology. Alveolar septal cell apoptosis (transferase dUTP nick end labelling assay, active caspase 3) and proliferation (Ki‐67), airspace enlargement, total alveolar surface area, and absolute alveolar septal volume as well as the ultrastructural composition of the alveolar wall were quantified.
Nitrogen dioxide resulted in an eightfold increase in alveolar septal cell apoptosis at day 3 and a 14‐fold increase in proliferation compared with age‐matched controls. Airspace enlargement, indicated by a 20% increase in mean airspace chord length, was evident by day 7 but was not associated with loss of alveolar walls. By contrast, nitrogen dioxide resulted in an increase in the total surface area and absolute volume of alveolar walls comprising all compartments. The ratio of collagen to elastin, however, was reduced at day 21. Lungs exposed to nitrogen dioxide for 21 days exhibited quantitative structural characteristics as seen in control lungs on day 49.
Nitrogen dioxide exposure of rats results in increased alveolar septal cell turnover leading to accelerated lung growth, which is associated with an imbalance in the relative composition of the extracellular matrix, but fails to induce emphysema.
Pulmonary emphysema is a connective tissue disease characterized by the progressive destruction of alveolar walls leading to airspace enlargement and decreased elastic recoil of the lung. However, the relationship between microscopic tissue structure and decline in stiffness of the lung is not well understood. In this study, we developed a 3D computational model of lung tissue in which a pre-strained cuboidal block of tissue was represented by a tessellation of space filling polyhedra, with each polyhedral unit-cell representing an alveolus. Destruction of alveolar walls was mimicked by eliminating faces that separate two polyhedral either randomly or in a spatially correlated manner, in which the highest force bearing walls were removed at each step. Simulations were carried out to establish a link between the geometries that emerged and the rate of decline in bulk modulus of the tissue block. The spatially correlated process set up by the force-based destruction lead to a significantly faster rate of decline in bulk modulus accompanied by highly heterogeneous structures than the random destruction pattern. Using the Karhunen-Loève transformation, an estimator of the change in bulk modulus from the first four moments of airspace cell volumes was setup. Simulations were then obtained for tissue destruction with different idealized alveolar geometry, levels of pre-strain, linear and nonlinear elasticity assumptions for alveolar walls and also mixed destruction patterns where both random and force-based destruction occurs simultaneously. In all these cases, the change in bulk modulus from cell volumes was accurately estimated. We conclude that microscopic structural changes in emphysema and the associated decline in tissue stiffness are linked by the spatial pattern of the destruction process.
Current standards for characterizing microscopic structural changes in emphysema are based on estimating the amount of tissue loss using stereological techniques. However, several previous studies reported that, in emphysema, there is a lack of correlation between stereological indices of tissue structure and increases in lung compliance, which is the inverse of tissue stiffness. In this study, we developed a novel three-dimensional computational model to show that the amount of tissue loss is not the sole determinant of increased lung compliance in emphysema. A key component that needs to be considered is the pattern of tissue destruction, which we demonstrate has a significant effect on the rate of decline in stiffness. Our findings also indicate that the heterogeneity observed at the microscopic scale in emphysema is a signature of the spatial history of the destruction process. These results highlight the importance of characterizing the heterogeneity of lung tissue structure in order to be able to relate microscopic structural changes to macroscopic functional measures such as lung compliance.
Rationale: Cigarette smoke (CS) exposure is an important risk factor for chronic obstructive pulmonary disease; however, not all smokers develop disease, suggesting that other factors influence disease development.
Objectives: We sought to determine whether neuropilin-1 (Nrp1), an integral component of receptor complexes mediating alveolar septation and vascular development, was involved in maintenance of normal alveolar structure, and/or altered susceptibility to the effects of CS.
Methods: Transgenic mice were generated to achieve inducible lung-specific deletion of epithelial Nrp1. We determined whether conditional Nrp1 deletion altered airspace size, then compared the effects of chronic CS or filtered air exposure on airspace size, inflammation, and the balance between cell death and proliferation in conditionally Nrp1–deficient adult mice and littermate controls. Finally, we evaluated the effects of Nrp1 silencing on cell death after acute exposure of A549 cells to cigarette smoke extract or short chain ceramides.
Measurements and Main Results: Genetic deletion of epithelial Nrp1 in either postnatal or adult lungs resulted in a small increase in airspace size. More notably, both airspace enlargement and apoptosis of type I and type II alveolar epithelial cells were significantly enhanced following chronic CS exposure in conditionally Nrp1-deficient adult mice. Silencing of Nrp1 in A549 cells did not alter cell survival after vehicle treatment but significantly augmented apoptosis after exposure to cigarette smoke extract or ceramide.
Conclusions: These data support a role for epithelial Nrp1 in the maintenance of normal alveolar structure and suggest that dysregulation of Nrp1 expression may promote epithelial cell death in response to CS exposure, thereby enhancing emphysema development.
chronic obstructive pulmonary disease; genetically modified mice; apoptosis
Rationale: Matrix metalloprotease (MMP)-9 is an elastolytic endopeptidase produced by activated macrophages that may be involved in the development of human pulmonary emphysema and could be inhibited with existing compounds. Mouse models have demonstrated that excess MMP-9 production can result in permanent alveolar destruction.
Objectives: To determine if MMP-9 causes cigarette smoke–induced emphysema using MMP-9 knockout mice and human samples.
Methods: Mouse lungs were analyzed for inflammation and airspace enlargement using a mainstream smoke-exposure model. Human macrophage mRNA was isolated from subjects with emphysema by laser capture microdissection. Human blood monocyte mRNA was isolated from subjects with greater than 30 pack-year smoking history. Human gene expression was determined by quantitative polymerase chain reaction and compared with emphysema severity determined by automated computed tomography analysis. Plasma Clara cell secretory protein and surfactant protein-D were quantified to measure ongoing lung injury.
Measurements and Main Results: Mice deficient in MMP-9 develop the same degree of cigarette smoke–induced inflammation and airspace enlargement as strain-matched controls. Macrophages are the predominant source of MMP-9 production in human emphysema specimens and similar quantities of macrophage MMP-9 mRNA is present in areas of lung with and without emphysema. Circulating monocytes produce more MMP-9 in individuals with advanced emphysema severity despite no correlation of MMP-9 with markers of ongoing lung damage.
Conclusions: These results suggest that MMP-9 in humans who smoke is similar to smoke-exposed mice, where MMP-9 is present in emphysematous lung but not correlated with the emphysema. To the degree that the mechanisms of emphysema in humans who smoke resemble the mouse model, these data suggest specific inhibition of MMP-9 is unlikely to be an effective therapy for cigarette smoke–induced emphysema.
Clinical trial registered with www.clinicaltrials.gov (NCT 00757120).
pulmonary disease, chronic obstructive; laser capture microdissection; mice, knockout
Peroxisome proliferator-activated receptor (PPAR)-γ is a nuclear hormone receptor that regulates gene expression, cell proliferation and differentiation. We previously described airway epithelial cell PPARγ deficient mice that develop airspace enlargement with decreased tissue resistance and increased lung volumes. We sought to understand the impact of airspace enlargement in conditionally targeted mice upon the physio-mechanical properties of the lung.
We measured elastic recoil and its determinants, including tissue structure and surface forces. We measured alveolar number using radial alveolar counts, and airspace sizes and their distribution using computer-assisted morphometry.
Air vs. saline-filled pressure volume profiles demonstrated loss of lung elastic recoil in targeted mice that was contributed by both tissue components and surface tension, but was proportional to lung volume. There were no significant differences in surfactant quantity/function nor in elastin and collagen content between targeted animals and littermate controls. Importantly, radial alveolar counts were significantly reduced in the targeted animals and at 8 weeks of age there were 18% fewer alveoli with 32% more alveolar ducts. Additionally, the alveolar ducts were 19% larger in the targeted animals.
Our data suggest that the functional abnormalities, including loss of recoil are secondary to altered force transmission due to differences in the structure of alveolar ducts, rather than changes in surfactant function or elastin or collagen content. These data further define the nature of abnormal lung maturation in the absence of airway epithelial cell PPARγ and identify a putative genetic determinant of dysanapsis, which may serve as a precursor to chronic lung disease.
Reactive oxygen species (ROS) participate in the pathogenesis of emphysema. Among ROS-producing enzymes, NOX NADPH oxidases are thought to be responsible for tissue injury associated with several lung pathologies. To determine whether NOX2 and/or NOX1 participate in the development of emphysema, their expression patterns were first studied by immunohistochemistry in the lungs of emphysematous patients. Subsequently, we investigated their contribution to elastase-induced emphysema using NOX2- and NOX1-deficient mice. In human lung, NOX2 was mainly detected in macrophages of control and emphysematous lungs, while NOX1 was expressed in alveolar epithelium and bronchial cells. We observed an elevated number of NOX2-positive cells in human emphysematous lungs, as well as increased NOX2 and NOX1 mRNA expression in mouse lungs following elastase exposure. Elastase-induced alveolar airspace enlargement and elastin degradation were prevented in NOX2-deficient mice, but not in NOX1-deficient mice. This protection was independent of inflammation and correlated with reduced ROS production. Concomitantly, an elevation of sirtuin 1 (SIRT1) level and a decrease of matrix metalloproteinase-9 (MMP-9) expression and activity were observed in alveolar macrophages and neutrophils. We addressed the specific role of macrophage-restricted functional NOX2 in elastase-induced lung emphysema using Ncf1 mutant mice and Ncf1 macrophage rescue mice (Ncf1 mutant mice with transgenic expression of Ncf1 only in CD68-positive mononuclear phagocytes; the MN mouse). Compared to WT mice, the lack of functional NOX2 led to decreased elastase-induced ROS production and protected against emphysema. In contrast, ROS production was restored specifically in macrophages from Ncf1 rescue mice and contributes to emphysema. Taken together, our results demonstrate that NOX2 is involved in the pathogenesis of human emphysema and macrophage-specific NOX2 participates in elastase-induced emphysema through the involvement of SIRT1/MMP-9 pathways in mice.
ROS; NOX2; MMP-9; SIRT1; emphysema; macrophages
BACKGROUND--Emphysema is currently defined as "a condition of the lung characterised by abnormal, permanent enlargement of the airspaces distal to the terminal bronchiole, accompanied by destruction of their walls, and without obvious fibrosis." The functional and morphological changes that occur in emphysema have largely been attributed to changes in alveolar elastin rather than in collagen. A study was performed to determine whether the amount of collagen in the alveolar wall changes with age in the lungs of non-smokers and of smokers with different types of macroscopically defined emphysema in relation to a microscopic measurement of lung structure. METHODS--Total alveolar wall collagen was measured (as hydroxyproline) in known volumes of distended lung tissue (by reverse phase high pressure liquid chromatography) in the lungs of non-smokers (n = 23) and in regions sampled away from emphysematous lesions in the lungs of 36 smokers (four with no emphysema, 13 with centriacinar emphysema (CAE), nine with panacinar emphysema (PAE), and 10 with a mixture (MIX) of both PAE and CAE). Mean lung airspace wall surface area per unit volume (AWUV) was calculated from at least six random blocks per lung and on histological sections immediately adjacent to those prepared for collagen measurement with a rapid scanning device (fast interval processor). RESULTS--In non-smokers there was no significant correlation between the amount of collagen in the alveolar wall tissue and either mean lung AWUV or increasing patient age when amounts of collagen were expressed either per unit volume of distended lung (40 mm3 sample) or per unit surface area of airspace wall tissue. Smokers without emphysema had similar amounts of collagen to non-smokers. Lungs with PAE and MIX, but not CAE alone, contained significantly more collagen than normal when expressed per unit volume of airspace wall tissue whereas all groups, including CAE, contained significantly raised amounts of collagen when expressed per unit surface area. CONCLUSIONS--There is no significant age related change in the collagen content of the lungs of non-smokers which suggests that, as AWUV is lost with age, the main collagenous framework is maintained. However, in smokers with emphysema there is a loss of airspace wall tissue in regions remote from the macroscopic lesions that is accompanied by a net increase in collagen mass. The greater accumulation of collagen in MIX lungs than in CAE lungs suggests a greater degree of structural damage, indicative of an alternative pathogenetic mechanism operating between the different types of emphysema. Our results suggest an active alveolar wall fibrosis in emphysema as a consequence of cigarette smoking. It is suggested that the definition of emphysema may require further revision to include such change.
Key host responses to the stress induced by environmental exposure to cigarette smoke (CS) are responsible for initiating pathogenic effects that may culminate in emphysema development. CS increases lung ceramides, sphingolipids involved in oxidative stress, structural alveolar cell apoptosis, and inhibition of apoptotic cell clearance by alveolar macrophages, leading to the development of emphysema-like pathology. RTP801, a hypoxia and oxidative stress sensor, is also increased by CS, and has been recently implicated in both apoptosis and inflammation. We investigated whether inductions of ceramide and RTP801 are mechanistically linked, and evaluated their relative importance in lung cell apoptosis and airspace enlargement in vivo. As reported, direct lung instillation of either RTP801 expression plasmid or ceramides in mice triggered alveolar cell apoptosis and oxidative stress. RTP801 overexpression up-regulated lung ceramide levels 2.6-fold. In turn, instillation of lung ceramides doubled the lung content of RTP801. Cell sorting after lung tissue dissociation into single-cell suspension showed that ceramide triggers both endothelial and epithelial cell apoptosis in vivo. Interestingly, mice lacking rtp801 were protected against ceramide-induced apoptosis of epithelial type II cells, but not type I or endothelial cells. Furthermore, rtp801-null mice were protected from ceramide-induced alveolar enlargement, and exhibited improved static lung compliance compared with wild-type mice. In conclusion, ceramide and RTP801 participate in alveolar cell apoptosis through a process of mutual up-regulation, which may result in self-amplification loops, leading to alveolar damage.
emphysema; sphingolipids; apoptosis; cigarette smoke; stress response
Despite the importance of airspace integrity in vertebrate gas exchange,
the molecular pathways that instruct distal lung formation are poorly
understood. Recently, we found that fibrillin-1 deficiency in mice impairs
alveolar formation and recapitulates the pulmonary features of human Marfan
syndrome. To further elucidate effectors involved in distal lung formation, we
performed expression profiling analysis comparing the fibrillin-1-deficient
and wild-type developing lung. NeuroD, a basic helix-loop-helix transcription
factor, fulfilled the expression criteria for a candidate mediator of distal
lung development. We investigated its role in murine lung development using
genetically targeted NeuroD-deficient mice. We found that NeuroD deficiency
results in both impaired alveolar septation and altered morphology of the
pulmonary neuroendocrine cells. NeuroD-deficient mice had enlarged alveoli
associated with reduced epithelial proliferation in the airway and airspace
compartments during development. Additionally, the neuroendocrine compartment
in these mice manifested an increased number of neuroepithelial bodies but a
reduced number of solitary pulmonary neuroendocrine cells in the neonatal
lung. Overexpression of NeuroD in a murine lung epithelial cell line conferred
a neuroendocrine phenotype characterized by the induction of neuroendocrine
markers as well as increased proliferation. These results support an
unanticipated role for NeuroD in the regulation of pulmonary neuroendocrine
and alveolar morphogenesis and suggest an intimate connection between the
neuroendocrine compartment and distal lung development.
Alveolar growth abnormalities and severe respiratory dysfunction are often fatal. Identifying mechanisms that control epithelial proliferation and enlarged, poorly septated airspaces is essential in developing new therapies for lung disease. The membrane-bound ligand ephrin-B2 is strongly expressed in lung epithelium, and yet in contrast to its known requirement for arteriogenesis, considerably less is known regarding the function of this protein in the epithelium. We hypothesize that the vascular mediator ephrin-B2 governs alveolar growth and mechanics beyond the confines of the endothelium. We used the in vivo manipulation of ephrin-B2 reverse signaling to determine the role of this vascular mediator in the pulmonary epithelium and distal lung mechanics. We determined that the ephrin-B2 gene (EfnB2) is strongly expressed in alveolar Type 2 cells throughout development and into adulthood. The role of ephrin-B2 reverse signaling in the lung was assessed in Efnb2LacZ/6YFΔV mutants that coexpress the intracellular truncated ephrin-B2–β-galactosidase fusion and an intracellular point mutant ephrin-B2 protein that is unable to become tyrosine-phosphorylated or to interact with either the SH2 or PDZ domain–containing downstream signaling proteins. In these viable mice, we observed pulmonary hypoplasia and altered pulmonary mechanics, as evidenced by a marked reduction in lung compliance. Associated with the reduction in lung compliance was a significant increase in insoluble fibronectin (FN) basement membrane matrix assembly with FN deposition, and a corresponding increase in the α5 integrin receptor required for FN fibrillogenesis. These experiments indicate that ephrin-B2 reverse signaling mediates distal alveolar formation, fibrillogenesis, and pulmonary compliance.
arterial; fibronectin; α5β1 integrin; alveoli; pulmonary mechanics
We previously reported that joint swelling, synovial thickening, and cartilage matrix depletion induced by the injection of anti-collagen monoclonal antibodies and lipopolysaccharide (LPS) in BALB/c mice are increased in the absence of inhibitory leukocyte immunoglobulin (Ig)-like receptor B4 (LILRB4; formerly gp49B1) in a neutrophil-dependent manner. Because both mast cells and neutrophils express LILRB4, we sought a mast cell requirement with mast cell–deficient mouse strains, but unexpectedly obtained full arthritis in KitW-sh mice and full resistance in KitW/KitW-v mice. KitW-sh mice were indeed mast cell deficient as assessed by histology and the absence of IgE/mast cell–dependent passive cutaneous anaphylaxis in the ear and joint as well as passive systemic anaphylaxis. Deletion of LILRB4 in KitW-sh mice exacerbated anti-collagen/LPS-induced joint swelling that was abolished by neutrophil depletion, establishing a counterregulatory role for LILRB4 in the absence of mast cells. Whereas blood neutrophil levels and LPS-elicited tissue neutrophilia were equal in KitW-sh and Kit+ mice, both were impaired in KitW/KitW-v mice. Although both strains are mast cell deficient and protected from IgE-mediated anaphylactic reactions, their dramatically different responses to autoantibody-mediated, neutrophil-dependent immune complex arthritis suggest that other host differences determine the extent of mast cell involvement. Thus, a conclusion for an absolute mast cell role in a pathobiologic process requires evidence from both strains.
Rationale: Pathogenic T cells drive, or sustain, a number of inflammatory diseases. Chronic obstructive pulmonary disease (COPD) is an inflammatory lung disease associated with the accumulation of activated T cells. We previously demonstrated that chronic cigarette smoke (CS) exposure causes oligoclonal expansion of lung CD4+ T cells and CD8+ T cells in a mouse model of COPD, thus implicating these cells in disease pathogenesis.
Objectives: To determine whether T cells are pathogenic in a CS-induced mouse model of COPD.
Methods: We transferred lung CD3+ T cells from filtered air (FA)- and CS-exposed mice into Rag2−/− recipients. Endpoints associated with the COPD phenotype were then measured.
Measurements and Main Results: Here, we demonstrate that chronic CS exposure generates pathogenic T cells. Transfer of CD3+ T cells from the lungs of CS-exposed mice into Rag2−/− recipients led to substantial pulmonary changes pathognomonic of COPD. These changes included monocyte/macrophage and neutrophil accumulation, increased expression of cytokines and chemokines, activation of proteases, apoptosis of alveolar epithelial cells, matrix degradation, and airspace enlargement reminiscent of emphysema.
Conclusions: These data formally demonstrate, for the first time, that chronic CS exposure leads to the generation of pathogenic T cells capable of inducing COPD-like disease in Rag2−/− mice. This report provides novel insights into COPD pathogenesis.
mouse model; inflammation; lung; emphysema
Purpose: Reduction in the level of vascular endothelial growth factor (VEGF) has been implicated in the pathogenesis of pulmonary emphysema. To this end, pharmacological VEGF receptor blockade, and the Cre-lox system models have been utilized to study the effects of VEGF depletion in the lung. These models generally reproduce air space enlargement resembling clinical emphysema. Here we report a potentially more readily available model of lung targeted VEGF depletion by airway administration of VEGF small inhibitory RNA oligonucleotides (siRNAs) in mice.
Methods: Airway administration of VEGF siRNAs were done in C57BL/6 mice. The lungs were removed for histology and protein analysis 2, and 4 days later. Airspace enlargement was evaluated by lung volume measurement, and histological analyses. VEGF levels were analyzed by western blot and immunohistochemistry.
Results: Airway administration of VEGF siRNAs induced transient air space enlargement in the mouse lung morphologically resembling the previously reported models of pulmonary emphysema. VEGF expression was significantly reduced in the lung, particularly in the alveolar septal cells. We also found that in this particular model, sequential airway administration of recombinant VEGF protein attenuated this air space enlargement. Additionally, we found that airway administration of DCI, a combination of dexamethasone, 3'-5'-cyclic adenosine monophosphate, and isobutylmethylxanthine attenuated the air space enlargement in this particular model, at least in part through the recovery of lung VEGF expression.
Conclusions: The pathogenesis of pulmonary emphysema is likely to be multifaceted, but the present mouse model may be useful in dissecting the involvement of VEGF in pulmonary emphysema.
vascular endothelial growth factor; emphysema; mouse model.
Rationale: As the smallest free-living bacteria and a frequent cause of respiratory infections, mycoplasmas are unique pathogens. Mice infected with Mycoplasma pulmonis can develop localized, life-long airway infection accompanied by persistent inflammation and remodeling.
Objective: Because mast cells protect mice from acute septic peritonitis and gram-negative pneumonia, we hypothesized that they defend against mycoplasma infection. This study tests this hypothesis using mast cell–deficient mice.
Methods: Responses to airway infection with M. pulmonis were compared in wild-type and mast cell–deficient KitW-sh/KitW-sh mice and sham-infected control mice.
Measurements and Main Results: Endpoints include mortality, body and lymph node weight, mycoplasma antibody titer, and lung mycoplasma burden and histopathology at intervals after infection. The results reveal that infected KitW-sh/KitW-sh mice, compared with other groups, lose more weight and are more likely to die. Live mycoplasma burden is greater in KitW-sh/KitW-sh than in wild-type mice at early time points. Four days after infection, the difference is 162-fold. Titers of mycoplasma-specific IgM and IgA appear earlier and rise higher in KitW-sh/KitW-sh mice, but antibody responses to heat-killed mycoplasma are not different compared with wild-type mice. Infected KitW-sh/KitW-sh mice develop larger bronchial lymph nodes and progressive pneumonia and airway occlusion with neutrophil-rich exudates, accompanied by angiogenesis and lymphangiogenesis. In wild-type mice, pneumonia and exudates are less severe, quicker to resolve, and are not associated with increased angiogenesis.
Conclusions: These findings suggest that mast cells are important for innate immune containment of and recovery from respiratory mycoplasma infection.
angiogenesis; bronchitis; innate immunity; lymphangiogenesis; pneumonia
The pathogenesis of chronic obstructive pulmonary disease (COPD) remains unclear, but involves loss of alveolar surface area (emphysema) and airway inflammation (bronchitis) as the consequence of cigarette smoke (CS) exposure. Previously, we demonstrated that autophagy proteins promote lung epithelial cell death, airway dysfunction, and emphysema in response to CS; however, the underlying mechanisms have yet to be elucidated. Here, using cultured pulmonary epithelial cells and murine models, we demonstrated that CS causes mitochondrial dysfunction that is associated with a reduction of mitochondrial membrane potential. CS induced mitophagy, the autophagy-dependent elimination of mitochondria, through stabilization of the mitophagy regulator PINK1. CS caused cell death, which was reduced by administration of necrosis or necroptosis inhibitors. Genetic deficiency of PINK1 and the mitochondrial division/mitophagy inhibitor Mdivi-1 protected against CS-induced cell death and mitochondrial dysfunction in vitro and reduced the phosphorylation of MLKL, a substrate for RIP3 in the necroptosis pathway. Moreover, Pink1–/– mice were protected against mitochondrial dysfunction, airspace enlargement, and mucociliary clearance (MCC) disruption during CS exposure. Mdivi-1 treatment also ameliorated CS-induced MCC disruption in CS-exposed mice. In human COPD, lung epithelial cells displayed increased expression of PINK1 and RIP3. These findings implicate mitophagy-dependent necroptosis in lung emphysematous changes in response to CS exposure, suggesting that this pathway is a therapeutic target for COPD.
Pulmonary inflammation, abnormalities in type II cell and macrophage morphology, and pulmonary fibrosis are features of Hermansky-Pudlak Syndrome (HPS), a recessive disorder associated with intracellular trafficking defects. We have previously reported that “Pearl” (HPS2) and “Pale Ear” (HPS1) mouse models have pulmonary inflammatory dysregulation and constitutive alveolar macrophage (AM) activation (Young LR et al., J Immunol 2006;176:4361–4368). In the current study, we used these HPS models to investigate mechanisms of lung fibrosis. Unchallenged HPS1 and HPS2 mice have subtle airspace enlargement and foamy AMs, but little or no histologic evidence of lung fibrosis. Seven days after intratracheal bleomycin (0.025 units), HPS1 and HPS2 mice exhibited increased mortality and diffuse pulmonary fibrosis compared to strain-matched C57BL/6J wild-type (WT) mice. HPS mice had significantly increased collagen deposition, and reduced quasi-static and static compliance consistent with a restrictive defect. The early airway and parenchymal cellular inflammatory responses to bleomycin were similar in HPS2 and WT mice. Greater elevations in levels of TGF-β and IL-12p40 were produced in the lungs and AMs from bleomycin-challenged HPS mice than in WT mice. TUNEL staining revealed apoptosis of type II cells as early as 5 h after low-dose bleomycin challenge in HPS mice, suggesting that type II cell susceptibility to apoptosis may play a role in the fibrotic response. We conclude that the trafficking abnormalities in HPS promote alveolar apoptosis and pulmonary fibrosis in response to bleomycin challenge.
alveolar type II cells; alveolar macrophage; lung fibrosis; Hermansky-Pudlak; adaptor protein 3
Chronic obstructive pulmonary disease (COPD) is known to greatly affect ventilation (V) and perfusion (Q) of the lung through pathologies such as inflammation and emphysema. However, there is little direct evidence regarding how these pathologies contribute to the V/Q mismatch observed in COPD and models thereof. Also, little is known regarding how smoking cessation affects V/Q relationships after inflammation and airspace enlargement have become established. To this end, we have quantified V/Q on a per-voxel basis using single photon emission computed tomography (SPECT) in mouse models of COPD and lung obstruction.
Three distinct murine models were used to investigate the impact of different pathologies on V/Q, as measured by SPECT. Lipopolysaccharide (LPS) was used to produce neutrophilic inflammation, porcine pancreatic elastase (PPE) was used to produce emphysema, and long-term cigarette smoke (CS) exposure and cessation were used to investigate the combination of these pathologies.
CS exposure resulted in an increase in mononuclear cells and neutrophils, an increase in airspace enlargement, and an increase in V/Q mismatching. The inflammation produced by LPS was more robust and predominantly neutrophilic, compared to that of cigarette smoke; nevertheless, inflammation alone caused V/Q mismatching similar to that seen with long-term CS exposure. The emphysematous lesions caused by PPE administration were also capable of causing V/Q mismatch in the absence of inflammation. Following CS cessation, inflammatory cell levels returned to those of controls and, similarly, V/Q measures returned to normal despite evidence of persistent mild airspace enlargement.
Both robust inflammation and extensive airspace enlargement, on their own, were capable of producing V/Q mismatch. As CS cessation resulted in a return of V/Q mismatching and inflammatory cell counts to control levels, lung inflammation is likely a major contributor to V/Q mismatch observed in the cigarette smoke exposure model as well as in COPD patients. This return of V/Q mismatching to control values also took place in the presence of mild airspace enlargement, indicating that emphysematous lesions must be of a larger volume before affecting the lung significantly. Early smoking cessation is therefore critical before emphysema has an irreversible impact on gas exchange.
Chronic obstructive pulmonary disease; Gas exchange; Lung function; Perfusion; Single photon emission computed tomography; Ventilation
Persistent macrophage accumulation and alveolar enlargement are hallmark features of chronic obstructive pulmonary disease (COPD). A role for CD8+ lymphocytes in the development of COPD is suggested based on observations that this T cell subset is increased in the airways and parenchyma of smokers that develop COPD with airflow limitation. In this study, we utilize a mouse model of COPD to examine the contributions of CD8+ T cells in the persistent macrophage accumulation and airspace enlargement resulting from chronic irritant exposure.
We analyzed pulmonary inflammation and alveolar destruction in wild-type and Cd8-deficient mice chronically exposed to acrolein, a potent respiratory tract irritant. We further examined cytokine mRNA expression levels by RNase protection assay, matrix metalloproteinase (MMP) activity by gelatin zymography, and epithelial cell apoptosis by active caspase3 immunohistochemistry in wild-type and Cd8-deficient mice exposed chronically to acrolein.
These studies demonstrate that CD8+ T cells, are important mediators of macrophage accumulation in the lung and the progressive airspace enlargement in response to chronic acrolein exposures. The expression of several inflammatory cytokines (IP-10, IFN-γ, IL-12, RANTES, and MCP-1), MMP2 and MMP9 gelatinase activity, and caspase3 immunoreactivity in pulmonary epithelial cells were attenuated in the Cd8-deficient mice compared to wild-type.
These results indicate that CD8+ T cells actively contribute to macrophage accumulation and the development of irritant-induced airspace enlargement.
Rationale: Chronic obstructive pulmonary disease is a leading cause of death worldwide, but its pathogenesis is not well understood. Previous studies have shown that airway surface dehydration in β-epithelial Na+ channel (βENaC)–overexpressing mice caused a chronic lung disease with high neonatal pulmonary mortality and chronic bronchitis in adult survivors.
Objectives: The aim of this study was to identify the initiating lesions and investigate the natural progression of lung disease caused by airway surface dehydration.
Methods: Lung morphology, gene expression, bronchoalveolar lavage, and lung mechanics were studied at different ages in βENaC-overexpressing mice.
Measurements and Main Results: Mucus obstruction in βENaC-overexpressing mice originated in the trachea in the first days of life and was associated with hypoxia, airway epithelial necrosis, and death. In surviving βENaC-overexpressing mice, mucus obstruction extended into the lungs and was accompanied by goblet cell metaplasia, increased mucin expression, and airway inflammation with transient perinatal increases in tumor necrosis factor-α and macrophages, IL-13 and eosinophils, and persistent increases in keratinocyte-derived cytokine (KC), neutrophils, and chitinases in the lung. βENaC-overexpressing mice also developed emphysema with increased lung volumes, distal airspace enlargement, and increased lung compliance.
Conclusions: Our studies demonstrate that airway surface dehydration is sufficient to initiate persistent neutrophilic airway inflammation with chronic airways mucus obstruction and to cause transient eosinophilic airway inflammation and emphysema. These results suggest that deficient airway surface hydration may play a critical role in the pathogenesis of chronic obstructive pulmonary diseases of different etiologies and serve as a target for novel therapies.
chronic obstructive lung disease; epithelial Na+ channels; airway surface liquid; inflammation; mucus
BACKGROUND: Increased epithelial permeability of the airspaces occurs commonly in the lungs of cigarette smokers. It is likely to be important in augmenting the inflammatory response in the airspaces and hence may have a role in the pathogenesis of emphysema. It has previously been shown that intratracheal instillation of cigarette smoke condensate induces increased epithelial permeability in vivo in rats and in vitro in epithelial cell monolayers, associated with a disturbance in the lung antioxidant, glutathione (GSH). The aim of this study was to assess the role of neutrophils, GSH, and tumour necrosis factor (TNF) in the increased epithelial permeability following intratracheal instillation of cigarette smoke condensate. METHODS: Epithelial permeability of the airspaces was measured in rat lungs as the passage of intratracheally instilled 125-iodine labelled bovine serum albumin (BSA) into the blood. The permeability of a monolayer of human type II alveolar epithelial cells to 125I-BSA was also measured. RESULTS: Cigarette smoke condensate produced a 59.7% increase in epithelial permeability over control values peaking six hours after instillation and returning to control values by 24 hours. Depletion of neutrophils and, to a lesser extent, macrophages by an intraperitoneal injection of antineutrophil antibody did not influence the increased epithelial permeability induced by cigarette smoke condensate. Although instillation of human recombinant TNF alpha produced an increase in epithelial permeability in the rat lung from 0.62 (0.61)% to 1.27 (0.08)%, only a trivial amount of TNF alpha was detected in bronchoalveolar lavage (BAL) fluid in vivo or in culture medium from BAL leucocytes obtained from animals treated with cigarette smoke condensate (94.9 (28.8) units/ml). Furthermore, antiTNF antibody did not abolish the increased epithelial permeability produced by cigarette smoke condensate. The role of GSH was assessed by measuring the changes in both the reduced (GSH) and oxidised form (GSSG) in lung tissue and in BAL fluid. One hour after instillation of cigarette smoke condensate there was a marked fall in the GSH content in the lung (from 809.8 (31.8) to 501.7 (40.5) nmol/g) in association with increased GSSG levels (from 89.8 (2.7) to 148.7 (48.8) nmol/g). This was followed by a return of GSH levels to control values, with a concomitant decrease in GSSG levels six hours after instillation. GSH levels in BAL fluid fell dramatically following cigarette smoke condensate (from 2.56 (0.30) to 0.31 (0.21) nmol/ml) and this fall was sustained up to six hours after instillation of cigarette smoke condensate. CONCLUSIONS: These studies suggest that neutrophils and TNF do not have a major role in the increased epithelial permeability induced by cigarette smoke condensate. However, the data support a role for the depletion of the antioxidant glutathione in the increased epithelial permeability caused by cigarette smoke condensate.
Emphysema is a progressive disease characterized by the destruction of peripheral airspaces and subsequent decline in lung function. However, the relation between structure and function during disease progression is not well understood. The objective of this study was to assess the time course of the structural, mechanical, and remodeling properties of the lung in mice after elastolytic injury. At 2, 7, and 21 days after treatment with porcine pancreatic elastase, respiratory impedance, the constituents of lung extracellular matrix, and histological sections of the lung were evaluated. In the control group, no changes were observed in the structural or functional properties, whereas, in the treatment group, the respiratory compliance and its variability significantly increased by Day 21 (P < 0.001), and the difference in parameters decreased with increasing positive end-expiratory pressure. The heterogeneity of airspace structure gradually increased over time. Conversely, the relative amounts of elastin and type I collagen exhibited a peak (P < 0.01) at Day 2, but returned to baseline levels by Day 21. Structure–function relations manifested themselves in strong correlations between compliance parameters and both mean size and heterogeneity of airspace structure (r2 > 0.9). Similar relations were also obtained in a network model of the parenchyma in which destruction was based on the notion that mechanical forces contribute to alveolar wall rupture. We conclude that, in a mouse model of emphysema, progressive decline in lung function is sensitive to the development of airspace heterogeneity governed by local, mechanical, force-induced failure of remodeled collagen.
lung compliance; morphology; elastin; collagen; network model
Chronic obstructive pulmonary disease (COPD) is characterized by the presence of airflow obstruction and lung destruction with airspace enlargement. In addition to cigarette smoking, respiratory pathogens play a role in pathogenesis, but specific organisms are not always identified. Recent reports demonstrate associations between the detection of Pneumocystis jirovecii DNA in lung specimens or respiratory secretions and the presence of emphysema in COPD patients. Additionally, human immunodeficiency virus-infected individuals who smoke cigarettes develop early emphysema, but a role for P. jirovecii in pathogenesis remains speculative. We developed a new experimental model using immunocompetent mice to test the interaction of cigarette smoke exposure and environmentally acquired Pneumocystis murina infection in vivo. We hypothesized that cigarette smoke and P. murina would interact to cause increases in total lung capacity, airspace enlargement, and pulmonary inflammation. We found that exposure to cigarette smoke significantly increases the lung organism burden of P. murina. Pulmonary infection with P. murina, combined with cigarette smoke exposure, results in changes in pulmonary function and airspace enlargement characteristic of pulmonary emphysema. P. murina and cigarette smoke exposure interact to cause increased lung inflammatory cell accumulation. These findings establish a novel animal model system to explore the role of Pneumocystis species in the pathogenesis of COPD.
Although both animal and human studies suggested the association between placenta growth factor (PlGF) and chronic obstructive pulmonary disease (COPD), especially lung emphysema, the role of PlGF in the pathogenesis of emphysema remains to be clarified. This study hypothesizes that blocking PlGF prevents the development of emphysema.
Pulmonary emphysema was induced in PlGF knock-out (KO) and wild type (WT) mice by intra-tracheal instillation of porcine pancreatic elastase (PPE). A group of KO mice was then treated with exogenous PlGF and WT mice with neutralizing anti-VEGFR1 antibody. Tumor necrosis factor alpha (TNF-α), matrix metalloproteinase-9 (MMP-9), and VEGF were quantified. Apoptosis measurement and immuno-histochemical staining for VEGF R1 and R2 were performed in emphysematous lung tissues.
After 4 weeks of PPE instillation, lung airspaces enlarged more significantly in WT than in KO mice. The levels of TNF-α and MMP-9, but not VEGF, increased in the lungs of WT compared with those of KO mice. There was also increased in apoptosis of alveolar septal cells in WT mice. Instillation of exogenous PlGF in KO mice restored the emphysematous changes. The expression of both VEGF R1 and R2 decreased in the emphysematous lungs.
In this animal model, pulmonary emphysema is prevented by depleting PlGF. When exogenous PlGF is administered to PlGF KO mice, emphysema re-develops, implying that PlGF contributes to the pathogenesis of emphysema.