Search

Genome-wide association studies (GWAS) have identified multiple associations with emphysema apicobasal distribution (EABD), but the biological functions of these variants are unknown. To characterize the functions of EABD-associated variants, we integrated GWAS results with 1) expression quantitative trait loci (eQTL) from the Genotype Tissue Expression (GTEx) project and subjects in the COPDGene (Genetic Epidemiology of COPD) study and 2) cell type epigenomic marks from the Roadmap Epigenomics project. On the basis of these analyses, we selected a variant near ACVR1B (activin A receptor type 1B) for functional validation. SNPs from 168 loci with P values less than 5 × 10(−5) in the largest GWAS meta-analysis of EABD were analyzed. Eighty-four loci overlapped eQTL, with 12 of these loci showing greater than 80% likelihood of harboring a single, shared GWAS and eQTL causal variant. Seventeen cell types were enriched for overlap between EABD loci and Roadmap Epigenomics marks (permutation P

Dl(CO) is a widely used pulmonary function test in clinical practice and a particularly useful measure for assessing patients with chronic obstructive pulmonary disease (COPD). We hypothesized that elucidating genetic determinants of Dl(CO) could lead to better understanding of the genetic architecture of COPD. We estimated the heritability of Dl(CO) using common genetic variants and performed genome-wide association analyses in four cohorts enriched for subjects with COPD (COPDGene [Genetic Epidemiology of COPD], NETT [National Emphysema Treatment Trial], GenKOLS [Genetics of Chronic Obstructive Lung Disease study], and TESRA [Treatment of Emphysema With a Gamma-Selective Retinoid Agonist study]) using a combined European ancestry white dataset and a COPDGene African American dataset. We assessed our genome-wide significant and suggestive associations for Dl(CO) in previously reported genome-wide association studies of COPD and related traits. We also characterized associations of known COPD-associated variants and Dl(CO). We estimated the SNP-based heritability of Dl(CO) in the European ancestry white population to be 22% (P = 0.0004). We identified three genome-wide significant associations with Dl(CO): variants near TGFB2, CHRNA3, and PDE11A loci (P

Genome-wide association studies have identified common variants associated with chronic obstructive pulmonary disease (COPD). Whole-genome sequencing (WGS) offers comprehensive coverage of the entire genome, as compared with genotyping arrays or exome sequencing. We hypothesized that WGS in subjects with severe COPD and smoking control subjects with normal pulmonary function would allow us to identify novel genetic determinants of COPD. We sequenced 821 patients with severe COPD and 973 control subjects from the COPDGene and Boston Early-Onset COPD studies, including both non-Hispanic white and African American individuals. We performed single-variant and grouped-variant analyses, and in addition, we assessed the overlap of variants between sequencing- and array-based imputation. Our most significantly associated variant was in a known region near HHIP (combined P = 1.6 × 10(−9)); additional variants approaching genome-wide significance included previously described regions in CHRNA5, TNS1, and SERPINA6/SERPINA1 (the latter in African American individuals). None of our associations were clearly driven by rare variants, and we found minimal evidence of replication of genes identified by previously reported smaller sequencing studies. With WGS, we identified more than 20 million new variants, not seen with imputation, including more than 10,000 of potential importance in previously identified COPD genome-wide association study regions. WGS in severe COPD identifies a large number of potentially important functional variants, with the strongest associations being in known COPD risk loci, including HHIP and SERPINA1. Larger sample sizes will be needed to identify associated variants in novel regions of the genome.

Chronic obstructive pulmonary disease (COPD) is caused by a complex interaction of environmental exposures, most commonly cigarette smoke, and genetic factors. Chronic cigarette smoke exposure in the mouse is a commonly used animal model of COPD. We aimed to expand our knowledge about the variable susceptibility of inbred strains to this model and test for genetic variants associated with this trait. To that end, we sought to measure differential susceptibility to cigarette smoke–induced emphysema in the mouse, identify genetic loci associated with this quantitative trait, and find homologous human genes associated with COPD. Alveolar chord length (CL) in 34 inbred strains of mice was measured after 6 months of exposure to cigarette smoke. After testing for association, we connected a murine candidate locus to a published meta-analysis of moderate-to-severe COPD. We identified deleterious mutations in a candidate gene in silico and measured gene expression in extreme strains. A/J was the most susceptible strain in our survey (Δ CL 7.0 ± 2.2 μm) and CBA/J was the least susceptible (Δ CL −0.3 ± 1.2 μm). By integrating mouse and human genome-wide scans, we identified the candidate gene Abi3bp. CBA/J mice harbor predicted deleterious variants in Abi3bp, and expression of the gene differs significantly between CBA/J and A/J mice. This is the first report of susceptibility to cigarette smoke–induced emphysema in 34 inbred strains of mice, and Abi3bp is identified as a potential contributor to this phenotype.

Previous studies have indicated that in adult smokers, a history of childhood pneumonia is associated with reduced lung function and chronic obstructive pulmonary disease. There have been few previous investigations using genome-wide association studies to investigate genetic predisposition to pneumonia. This study aims to identify the genetic variants associated with the development of pneumonia during childhood and over the course of the lifetime. Study subjects included current and former smokers with and without chronic obstructive pulmonary disease participating in the COPDGene Study. Pneumonia was defined by subject self-report, with childhood pneumonia categorized as having the first episode at

Cachexia, whether assessed by body mass index (BMI) or fat-free mass index (FFMI), affects a significant proportion of patients with chronic obstructive pulmonary disease (COPD), and is an independent risk factor for increased mortality, increased emphysema, and more severe airflow obstruction. The variable development of cachexia among patients with COPD suggests a role for genetic susceptibility. The objective of the present study was to determine genetic susceptibility loci involved in the development of low BMI and FFMI in subjects with COPD. A genome-wide association study (GWAS) of BMI was conducted in three independent cohorts of European descent with Global Initiative for Chronic Obstructive Lung Disease stage II or higher COPD: Evaluation of COPD Longitudinally to Identify Predictive Surrogate End-Points (ECLIPSE; n = 1,734); Norway-Bergen cohort (n = 851); and a subset of subjects from the National Emphysema Treatment Trial (NETT; n = 365). A genome-wide association of FFMI was conducted in two of the cohorts (ECLIPSE and Norway). In the combined analyses, a significant association was found between rs8050136, located in the first intron of the fat mass and obesity-associated (FTO) gene, and BMI (P = 4.97 × 10(-7)) and FFMI (P = 1.19 × 10(-7)). We replicated the association in a fourth, independent cohort consisting of 502 subjects with COPD from COPDGene (P = 6 × 10(-3)). Within the largest contributing cohort of our analysis, lung function, as assessed by forced expiratory volume at 1 second, varied significantly by FTO genotype. Our analysis suggests a potential role for the FTO locus in the determination of anthropomorphic measures associated with COPD.

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.

DNA methylation is a complex, tissue-specific phenomenon that can reflect both endogenous factors and exogenous exposures. Buccal brushings represent an easily accessible source of DNA, which may be an appropriate surrogate tissue in the study of environmental exposures and chronic respiratory diseases. Buccal brushings were obtained from a subset of current and former smokers from the COPDGene study. Genome-wide DNA methylation data were obtained in the discovery cohort (n = 82) using the Illumina HumanMethylation450K array. Empirical Bayes methods were used to test for differential methylation by current smoking status at 468,219 autosomal CpG sites using linear models adjusted for age, sex, and race. Pyrosequencing was performed in a nonoverlapping replication cohort (n = 130). Current smokers were significantly younger than former smokers in both the discovery and replication cohorts. Seven CpG sites were associated with current smoking at a false discovery rate less than 0.05 in the discovery cohort. Six of the seven significant sites were pyrosequenced in the replication cohort; five CpG sites, including sites annotated to CYP1B1 and PARVA, were replicated. Correlations between cumulative smoke exposure and time since smoking cessation were observed in a subset of the significantly associated CpG sites. A significant correlation between reduced lung function and increased radiographic emphysema with methylation at cg02162897 (CYP1B1) was observed among female subjects. Site-specific methylation of DNA isolated from buccal mucosa is associated with exposure to cigarette smoke, and may provide insights into the mechanisms underlying differential susceptibility toward the development of smoking-related chronic respiratory diseases.

Pulmonary hypertension is associated with advanced chronic obstructive pulmonary disease (COPD), although pulmonary vascular changes occur early in the course of the disease. Pulmonary artery (PA) enlargement (PAE) measured by computed tomography correlates with pulmonary hypertension and COPD exacerbation frequency. Genome-wide association studies of PAE in subjects with COPD have not been reported. To investigate whether genetic variants are associated with PAE within subjects with COPD, we investigated data from current and former smokers from the COPDGene Study and the Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints study. The ratio of the diameter of the PA to the diameter of the aorta (A) was measured using computed tomography. PAE was defined as PA/A greater than 1. A genome-wide association study for COPD with PAE was performed using subjects with COPD without PAE (PA/A ≤ 1) as a control group. A secondary analysis used smokers with normal spirometry as a control group. Genotyping was performed on Illumina platforms. The results were summarized using fixed-effect meta-analysis. Both meta-analyses revealed a genome-wide significant locus on chromosome 15q25.1 in IREB2 (COPD with versus without PAE, rs7181486; odds ratio [OR] = 1.32; P = 2.10 × 10(-8); versus smoking control subjects, rs2009746; OR = 1.42; P = 1.32 × 10(-9)). PAE was also associated with a region on 14q31.3 near the GALC gene (rs7140285; OR = 1.55; P = 3.75 × 10(-8)). Genetic variants near IREB2 and GALC likely contribute to genetic susceptibility to PAE associated with COPD. This study provides evidence for genetic heterogeneity associated with a clinically important COPD vascular subtype.

Oxidative stress plays an important role in the development of fibrotic responses in the lung. However, it is not clear whether inhibiting oxidative stress with antioxidants can attenuate fibrotic processes in the lung. The objective of these studies was to test whether the catalytic antioxidant porphyrin manganese (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP) could protect mice against bleomycin-induced lung fibrosis. A 10 mg/kg intraperitoneal dose of MnTBAP was established as safe and had a serum and lung half-life of 9.5 h in mice. Based on this data, four groups of mice were given one dose of bleomycin (3.2 U/kg, intratracheal) or saline and MnTBAP (5 mg/kg, intraperitoneal) or saline twice daily for 14 d. Lung fibrosis was assessed by measuring (1) lung hydroxyproline content as an index of collagen accumulation, (2) airway dysfunction by whole body plethysmography, and (3) histopathology. Bleomycin produced a 20% loss in body weight that was only 10% in the bleomycin/MnTBAP group. Bleomycin produced a twofold increase in hydroxyproline content that was decreased 23% by MnTBAP. Bleomycin produced a twofold increase in airway dysfunction that was also attenuated 30% by MnTBAP. Histopathologic analysis of the lungs of mice treated with bleomycin demonstrated a severe fibrotic response that was attenuated 28% by MnTBAP. Future studies on the oxidant mechanisms that MnTBAP is affecting in this bleomycin model of lung fibrosis may shed light on potential new therapeutic approaches for treating interstitial lung diseases.

The antioxidant enzymes copper/zinc (Cu-Zn) and manganese (Mn) superoxide dismutase (SOD) have been implicated in protection of the lungs from oxidant damage. Mn SOD in particular may be related to acquired tolerance in cells following chronic ozone exposure. In order to study these protective and adaptive phenomena in oxidant injury, the cellular location and relative abundance of Mn SOD and Cu-Zn SOD were examined in the lungs of Fischer 344 rats following exposure to 0.0 and 1.0 ppm ozone for up to 3 mo using immunolabeling and morphometric techniques. Cu-Zn SOD labeling was found to be markedly reduced following ozone exposure in epithelial cells within airways and parenchyma. In contrast, a significant increase was noted in Mn SOD labeling in the centriacinar regions of exposed lungs for both alveolar macrophages and epithelial type II cells. Mn SOD labeling per epithelial type II cell was significantly increased in alveoli 0-400 microm beyond the bronchiole-alveolar duct junction (BADJ), while type II cell Mn SOD labeling was similar to control values with greater distance down the alveolar duct. No induction of Mn SOD was noted in type I epithelial cells, fibroblasts, or Clara cells. Thus, alterations in Cu-Zn and Mn SOD are both site and cell specific in the lungs. The differential increase in Mn SOD in type II cells of the proximal alveolar duct may reflect the ability of these cells to acquire tolerance and to resist further injury to repeated ozone exposure.

Extracellular superoxide dismutase (EC-SOD) is the major extracellular antioxidant enzyme. We have determined the primary structure of mouse EC-SOD by characterization of complementary DNA (cDNA) clones and by amino-acid sequence analysis of purified protein. cDNA sequence analysis indicates that mouse EC-SOD is synthesized as a 251-amino-acid precursor protein with a predicted molecular weight of 27,400 D. Amino-terminal micro sequence analysis of purified mature mouse lung EC-SOD demonstrated the sequence to begin with SSFDLADRLDPV-. These results indicate that EC-SOD as initially synthesized contains a 24-amino-acid precursor peptide, and that the mature protein is 227 amino acids in length. Computer algorithms that predict the most likely site of cotranslational signal peptidase cleavage suggest that processing will occur between amino acids 18 and 19 or 20 and 21, which implies that EC-SOD may be initially synthesized as a pre-pro-protein. Like human EC-SOD, mature mouse EC-SOD is glycosylated. The full-length mouse EC-SOD cDNA is 1,834 base pairs long and is 82% (79% for protein) identical to rat EC-SOD, but only 60% (60% for protein) identical to human EC-SOD. The mouse EC-SOD gene locus (Sod3) was mapped by interspecific backcross haplotype analysis as being 0.9 +/- 0.9 centimorgans distal to the Qdpr locus on mouse Chromosome 5, a position suggesting that the human homologue of EC-SOD will map close to the human QDPR locus (4p15.3). Of nine tissues examined by Northern blot analysis, those of the kidney and lung are by far the major tissues that express EC-SOD messenger RNA. Using in situ hybridization in the mouse lung, we demonstrate EC-SOD gene expression to be highly localized to alveolar Type II epithelial cells. These data suggest that alveolar Type II cells play a central role in mediating EC-SOD antioxidant function in the lung.

The extracellular form of superoxide dismutase (EC-SOD), SOD3, is contained in the human lung in relatively high amounts when compared to other organs. It has not been previously shown whether or not EC-SOD is synthesized and secreted by specific lung cells. We examined the expression of EC-SOD mRNA in human lung cells by in situ hybridization using a digoxigenin-labeled EC-SOD cRNA probe. Strong signals of EC-SOD synthesis were found in the epithelium of all airways. Secretory and basal cells, but not ciliated cells, were labeled for EC-SOD mRNA. Expression of EC-SOD mRNA was found in endothelial cells lining both arteries and veins. Many cells in the alveolar septum exhibited strong expression of EC-SOD mRNA. In addition, epithelial cells lining the outer wall of intrapulmonary airways and vessels were heavily labeled for EC-SOD mRNA. The lung parenchymal epithelial cells containing EC-SOD mRNA were identified as alveolar type II cells by colocalization with surfactant protein-A. Human alveolar macrophages were found to contain a substantial amount of EC-SOD mRNA expression. Alveolar type I epithelial cells and capillary endothelial cells did not display detectable signals of EC-SOD mRNA. Smooth muscle cells in muscular arteries were not labeled by the EC-SOD mRNA probe. These results show that airway epithelial cells and alveolar type II cells are the major cell types that synthesize fibroblasts EC-SOD in the human lung. EC-SOD has been shown by immunocytochemistry to be associated with the extracellular matrix around airway epithelium and in the walls of intrapulmonary arterioles. The site of EC-SOD localization, therefore, is closely related to the site of its synthesis.

Because alveolar macrophages generate and release reactive oxygen metabolites but also contain antioxidative enzymes, they have the potential of either damaging or protecting tissues. We investigated the relative role of the hydrogen peroxide (H2O2)-scavenging antioxidative enzymes in H2O2 disposal and cell protection using freshly isolated (5 h ex vivo) and overnight (24 h ex vivo) cultured human alveolar macrophages. Cell protection was assessed on the basis of maintenance of cellular high-energy phosphates, leakage of intact nucleotides into the extracellular medium, and appearance of the nucleotide catabolic products xanthine, hypoxanthine, and uric acid. To investigate the relative importance of catalase and the glutathione redox cycle, the experiments were conducted in cells pretreated with amino-triazole (ATZ) to inactivate catalase or with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) to inactivate glutathione reductase. Catalase, glutathione peroxidase, and glutathione reductase activities did not change significantly during overnight culture of the cells. Both freshly isolated and cultured cells consumed exogenous H2O2 mainly by the catalase-dependent pathway. When the cells were exposed to H2O2 (100 microM), catalase and the glutathione redox cycle equally participated in maintaining cellular high-energy nucleotides. However, when cultured cells were exposed to formylated peptide (FMLP) (10(-7) M), the glutathione redox cycle was responsible for the maintenance of high-energy nucleotides. Furthermore, in both exposures, the glutathione redox cycle was more important in maintaining cell membrane integrity and preventing nucleotide leakage from the cells. Immunocytochemical labeling showed that catalase was primarily localized in the peroxisomal compartment of these cells.(ABSTRACT TRUNCATED AT 250 WORDS)

We have investigated factors that regulate hydrogen peroxide (H2O2) release from vascular endothelial cells. Endothelial cells produce H2O2 at an intracellular site in the vicinity of peroxisomes and at a second site near the cell surface that is inaccessible to intracellular catalase or glutathione peroxidase. Regulation of H2O2 generation at the intracellular site was studied using aminotriazole, which inactivates catalase in the presence of H2O2. Regulation of H2O2 generation at the second site was studied by measuring H2O2 release into the medium. The rate of H2O2 release was constant over 2 h when cells were incubated in room air. Changing O2 levels in the atmosphere from 0% to 10% O2 resulted in a threefold increase in the rate of H2O2 release. Elevation of O2 levels from 10% to 95% O2 produced no further enhancement in the rate of release. Preincubation of cells under hypoxic conditions did not lead to an exaggerated rate of H2O2 release when cells were returned to room air. Pretreatment of cells with exogenous H2O2 inhibited subsequent H2O2 release while pretreatment with catalase enhanced H2O2 release. Although arachidonic acid transiently enhanced the rate of H2O2 release through a mechanism dependent on PGH synthase, basal H2O2 release was independent of this enzyme. Neither hypoxia, hyperoxia, or hypoxia followed by reoxygenation altered H2O2 generation at the intracellular site accessible to peroxisomal catalase. These data demonstrate that H2O2 release from endothelial cells is responsive to changes in O2 concentrations over a narrow range. The mechanisms involved are subject to product inhibition and appear to be saturated at 10% O2 in the atmosphere.

The role of different antioxidant pathways in cultured rat pleural mesothelial cells was studied by exposing the cells to various hydrogen peroxide (H2O2) concentrations and by measuring H2O2 cell cytotoxicity and the capacity of the cells to scavenge H2O2. The antioxidant enzymes, glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase, and catalase were analyzed biochemically. Catalase and CuZn superoxide dismutase were localized by immunocytochemistry. To enable investigation of the glutathione redox cycle and catalase pathways, glutathione reductase was inactivated with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and catalase was inactivated with aminotriazole. When the cells were exposed to a low, sublethal (0.030 mM) H2O2 concentration, glutathione reductase but not catalase inactivation resulted in a decreased capacity to remove H2O2 from the extracellular medium. When the cells were exposed to a high (0.25 mM) H2O2 concentration, H2O2-scavenging capacity decreased remarkably when catalase was inactivated. When the cells were exposed to 0.1 to 0.5 mM H2O2, cell cytotoxicity (lactate dehydrogenase release) increased significantly if glutathione reductase was inactivated; catalase inactivation resulted in a significant cytotoxicity only at high (greater than or equal to 0.25 mM) H2O2 concentrations. Immunocytochemical studies showed that the cells, both in situ and in vitro, contained low amounts of catalase. This suggests that the results of the catalase-inhibition studies are probably not due to a change in the characteristics of the cells in culture. 3-Aminobenzamide is a compound that is known to prevent NAD depletion through inhibition of poly(ADP-ribose) polymerase during oxidant stress. When intact cells were treated with different antioxidants and exposed to 0.5 mM H2O2, both catalase and 3-aminobenzamide protected the cells completely.(ABSTRACT TRUNCATED AT 250 WORDS)

Endogenous hydrogen peroxide (H2O2) release from aortic endothelial cells was studied in the presence of antioxidant enzyme inhibitors, mitochondrial inhibitors, a microsomal cytochrome P-450 inhibitor, and after oxidative stress induced with H2O2 or menadione. Extracellular H2O2 generation was determined spectrofluorometrically using 3-methoxy-4-hydroxy phenylacetic acid, and intracellular H2O2 production (in or near peroxisomes) was measured indirectly using aminotriazole, which inactivates catalase in the presence of H2O2. Extracellular H2O2 release was 0.079 +/- 0.005 nmol/min/mg protein in Hanks' balanced salt solution, was constant during a 120-min incubation period, and was not affected by the cell passage number. The half-life for catalase inactivation with aminotriazole was 23 min. Inhibition of catalase, glutathione reductase, or gamma-glutamylcysteine synthetase did not change the rate of extracellular release of H2O2. Furthermore, inhibition of the mitochondrial respiratory chain (rotenone, antimycin A) or microsomal cytochrome P-450 (8-methoxypsoralen) did not change extracellular H2O2 release or intracellular H2O2 production (at peroxisomes) by endothelial cells or cells in which glutathione reductase was inactivated. When the cells were exposed to exogenous H2O2 (30 microM), extracellular H2O2 was scavenged primarily by the glutathione redox pathway. Exogenously added H2O2 (100 microM) changed intracellular H2O2 production (in or near peroxisomes) only when the glutathione redox cycle was inactivated. Menadione (20 microM), which undergoes intracellular redox cycling, increased extracellular H2O2 release almost 4-fold to 0.3 nmol/min/mg protein. Furthermore, menadione increased peroxisomal H2O2 levels and decreased the half-life for catalase inactivation in the presence of aminotriazole to 13 min. Catalase inhibition increased extracellular H2O2 release during menadione treatment, indicating that H2O2 can diffuse across the plasma membrane during oxidant stress.(ABSTRACT TRUNCATED AT 250 WORDS)

The time course and nature of the cellular response to left pneumonectomy, with or without prior adrenalectomy, were evaluated in the right lungs of male Sprague-Dawley rats using morphometric techniques. Animals were studied at days 2, 5, and 14 following pneumonectomy, intervals prior to, during the course of, and following significant compensatory changes in right lung mass. The postoperative increase in right lung mass and volume in pneumonectomized animals involved minimal changes in the ratios of most tissue components, when compared to the lungs of sham-operated controls. A transient disproportionate increase in type II cell volume and epithelial thickness was evident on day 14. Postpneumonectomy changes in the type II epithelium were accentuated in the lungs of adrenalectomized-pneumonectomized animals. Adrenalectomy 5 days prior to pneumonectomy resulted in a substantial increase in the volume of all right lung tissue components, associated with thickening of the alveolar wall and with increases in the volume of both cellular and noncellular interstitium. Effects of adrenalectomy on the endothelium also were evident. In both adrenal-intact and adrenalectomized animals, pneumonectomy increased alveolar number by day 14 but had no effect on the volume of individual alveoli. These results confirm a coordinated pattern of compensatory growth following pneumonectomy in the adrenal-intact rat. The data further suggest that in adrenalectomized animals compensatory lung growth is more poorly synchronized, with pronounced postoperative elevations in volume of the interstitial and type II epithelial compartments leading to increased thickness of the alveolar wall. Adrenal hormones thus appear to be required for coordination and control of compensatory lung growth and for rapid restoration of normal tissue structure.

The manganese-containing superoxide dismutase (MnSOD) constitutes one of the major cellular defense mechanisms against the toxic effects of superoxide radical. The development of tolerance in adult rats to lethal exposure of O2 (100%) after pre-exposing them to a sublethal concentration of O2 (85%) was found to be closely associated with the increased activity of this enzyme in the lungs. Further experiments have shown that the transcriptional rate of the gene coding for MnSOD in rat lungs is increased at day 3 of 85% O2 exposure. To elucidate the nature of this transcriptional activation during hyperoxic insults, we chose to first understand the structure of the rat MnSOD gene. Three overlapping rat genomic fragments were isolated, and the DNA sequence containing the whole MnSOD gene was completely determined. The rat MnSOD gene contains at least five exons and is located in one piece of 16.4-kb EcoRI genomic DNA fragment. However, Southern blot analysis of total rat genomic DNA probed with MnSOD cDNA revealed an additional hybridizing 8.6-kb EcoRI genomic fragment besides the 16.4-kb one. To clarify the origin of this unexpected hybridizing genomic fragment, three unique genomic sequences derived from the promoter, intron 2, and the 3' untranslated region of the genomic clones were used to rehybridize the same Southern blot filter and were found to only hybridize to the 16.4-kb but not 8.5-kb EcoRI genomic fragment. These data suggest: (1) two MnSOD genes are present per haploid rat genome, and (2) all three cloned genomic fragments are derived from the MnSOD gene, which is located in the 16.4-kb EcoRI genomic fragment.(ABSTRACT TRUNCATED AT 250 WORDS)

Hyperoxia has been shown to cause extensive lung injury, which involves all components of the alveolar septum, although the type I epithelium has generally been reported to be resistant to significant injury. Electron microscopic morphometry was performed to define changes in volumes of subcellular components of alveolar epithelial cells in rats exposed to 85% O2 for 0, 7, and 14 d. Because of their large size, type I cells in control animals actually contain a greater volume of most of the organelles involved in cell metabolism than do type II cells. Hyperoxic exposure causes a dramatic change in the subcellular composition of the average type I cell, suggesting significant injury and/or response. Injury was suggested by the finding that lysosomes plus peroxisomes increased 1,250% after 7 d in hyperoxia and remained elevated by 200% after 14 d of exposure. Volumes of mitochondria, rough endoplasmic reticulum, smooth endoplasmic reticulum, and Golgi apparatus increased by 100%, 51%, 91%, and 500%, respectively, after hyperoxia. Qualitative analysis showed an altered, ruffled air border with focal areas of cytoplasmic translucency (suggesting injury) and focal areas of subcellular hypertrophy. Exposure to hyperoxia was associated with more organelles being found in peripheral or attenuated portions of type I alveolar cells. Since the increase in type I organelles exceeds the volume of these organelles in its progenitor, the type II cell, it is likely that hyperoxia causes hypertrophy of the type I alveolar epithelium itself, independent of simple type II cell differentiation. Because of the large size and wide distribution of the type I cell, dramatic shifts in cell substructure caused by hyperoxia are more difficult to detect and require quantitative analysis to fully ascertain the extent of cell alterations.

To investigate the role of manganese-containing superoxide dismutase (MnSOD) in lung antioxidant defense, lines of transgenic B6C3 hybrid mice carrying human MnSOD transgenes under the transcriptional control of a human beta-actin promoter were established. Expression studies demonstrated that the human MnSOD transgene in line TgHMS66 is expressed and functional. The cellular distribution of the transgene product in the lungs was further examined by immunocytochemical analysis. Increased immunoreactive MnSOD was found in mitochondria of lung type I epithelial cells, type II epithelial cells, capillary endothelial cells, and fibroblasts. Furthermore, the magnitude of increase in mitochondrial labeling density of type II cells of nontransgenic, hemizygous, and homozygous transgenic littermates was proportional to the increased lung activity of MnSOD found in these mice. Transgenic mice over-expressing MnSOD did not have enhanced survival relative to controls when exposed to99% oxygen. However, when exposed to 90% oxygen, the transgenic mice had a small but statistically significant increase in survival time. Our results indicate that when the beta-actin promoter is used to increase activity of MnSOD it provides modest protection to B6C3 mice against hyperoxic lung injury.

Antioxidant enzymes located in the bronchial epithelium can be expected to be important in protecting these cells against both endogenous and exogenous oxidants. In this study, human bronchial epithelial cells were isolated and cultured from specimens obtained from donors for lung transplantation. The levels and relative importance of different antioxidant enzymes were also assessed using an immortalized human bronchial epithelial cell line (BEAS 2B cells). Immunocytochemical studies showed a similar pattern of intracellular localization with the moderate degrees of labeling for Mn superoxide dismutase (SOD), CuZn SOD, and catalase in freshly isolated bronchial epithelial cells, bronchial epithelial cells in primary culture, and BEAS 2B cells. CuZn SOD and catalase decreased in labeling density whereas Mn SOD was unchanged when bronchial epithelial cells were placed in primary cultures. In contrast, Mn SOD and catalase were decreased in BEAS 2B cells compared with primary cultures. Although Mn SOD was low in BEAS 2B cells, it could be significantly induced by tumor necrosis factor treatment. Biochemical analysis showed remarkably similar catalase and glutathione reductase activities in primary cultured epithelial cells and BEAS 2B cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphometric procedures were used to determine the number of cells, cell volume, cell diameter, and surface areas of the airways in human and rat lungs. Nuclear sizes of epithelial cells from human bronchi were significantly larger than other lung cell nuclei. The average volume of human ciliated cell nuclei was 310 +/- 30 microns 3 and 167 +/- 12 microns 3 in bronchi and bronchioles, respectively. The smaller nuclei of human bronchioles were comparable to those of alveolar cells. In the pseudostratified epithelium of human bronchi, basal cells had a large surface area in contact with the basement membrane (51.3 +/- 4.6 microns 2 per cell) when compared with ciliated (1.1 +/- 0.1 microns 2), goblet (7.6 +/- 1.2 microns 2), or other secretory cells (12.0 +/- 2.1 microns 2). In the first four airway generations distal to the trachea, basal cells account for 30% of the cells in human airway epithelium and 2% of the cells in rat airway epithelium. Total airway surface area from trachea to bronchioles was 2,471 +/- 320 and 27.2 +/- 1.7 cm2 in human and rat lungs, respectively. These direct measurements of airway surface area are less than half of the estimates based on current lung models. The total number of airway epithelial cells were 10.5 x 10(9) for human and 0.05 x 10(9) for rat lungs. For both species, there were 18 times more alveolar cells than bronchial epithelial cells.

The distribution of intercellular adhesion molecule-1 (ICAM-1) on alveolar epithelial cells and the effects of exposure to 100% O2 on ICAM-1 expression in mouse lungs were studied by EM immunocytochemistry and immunoblot analysis. Cryoultrathin sections from mouse lungs exposed to air or 100% O2 for 84 h were labeled with a monoclonal rat anti-mouse ICAM-1 antibody. In the normal lung, abundant ICAM-1 expression was found on the alveolar surface of type I epithelial cells. Furthermore, ICAM-1 is highly concentrated on the surfaces near cell junctions. ICAM-1 was also found on the capillary surface of endothelial cells and alveolar surface of type II cells at densities considerably lower than that found on type I epithelial cells. After exposure to O2, the labeling density of ICAM-1 on the central surface of type I epithelial cells was not changed significantly. However, the gradient of ICAM-1 on the surfaces near cell junctions was nearly abolished. ICAM-1 labeling on the capillary surface of endothelial cells remained low. ICAM-1 was also markedly induced on the alveolar surface of type II epithelial cells after hyperoxic exposure. These results show that ICAM-1 is expressed primarily on type I epithelial cell surfaces near cell junctions. Exposure to hyperoxia causes a dramatic change in the distribution pattern of ICAM-1 on alveolar type I epithelial cells and induces expression of ICAM-1 on alveolar type II epithelial cells. These hyperoxia-induced changes may influence the associated neutrophil invasion/retention in the alveolar air spaces or alveolar walls.

Allometric studies have shown that lung volume, alveolar surface area, and diffusing capacity increase proportionally with body weight across a broad range of mammalian species. Changes in the number of cells and in average cell size and surface areas with increasing body weight have not been defined. We speculated that cell size is determined more by cell function than by species and body weight. To test this hypothesis, nine species ranging in size from shrew (2 to 3 g) to horse (510 kg) were studied. Random sites from the distal alveolar region of each species were analyzed using morphometric techniques. Six to 10 nuclei from each of the major classes of parenchymal lung cells were three-dimensionally reconstructed to determine their average diameter, volume, and surface area. To calculate the cell density, nuclear profiles were counted using electron microscopy. The number of cells per lung increased with body mass and lung volume with a slope of 1.01 (r2 = 0.99). The lung is unique among organs in the diversity and function of individual cell types, such as mechanical, sensory, secretory, transporting, and circulating cells. Excluding the circulatory cells, the lung has greater than 60 different cell types, making it an ideal organ for examining the varieties in cell characteristics across different species. Up to 6-fold differences in size were found between different lung cell types within a single species; however, for cells having secretory functions, such as type II cells, there was no detectable change in cell size with increasing lung surface area or body mass.(ABSTRACT TRUNCATED AT 250 WORDS)