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J D Crapo

Publications and source records attributed to J D Crapo.

At least 55 records · Page 3Linked to original sources
PubMed1996

Antioxidant enzyme expression in rat lungs during hyperoxia.

To understand the molecular mechanisms that upregulate the activities of pulmonary antioxidant enzymes in adult rats during exposure to 85% oxygen, the relative contents of corresponding mRNA in normal and hyperoxic lungs were determined. Hyperoxic exposure drastically induced the expression of lung manganese-containing superoxide dismutase (MnSOD) mRNA. Maximal induction of MnSOD mRNA occurred at days 3 and 5 of exposure to hyperoxia, reaching a 600 and a 340% increase over the levels of air-exposed rats, respectively. In addition, hyperoxia induced lung mRNA for glucose-6-phosphate dehydrogenase, glutathione peroxidase, glyceraldehyde-3-phosphate dehydrogenase, alpha-tubulin, and gamma-actin to different extends at various days of exposure. Hyperoxia had little or no effect on the levels of mRNA for copper/zinc-containing superoxide dismutase (CuZnSOD), catalase, heat shock protein (HSP70), and creatine kinase. Nuclear run-on experiments showed that the transcriptional rate of the MnSOD gene is enhanced in hyperoxic rat lungs by approximately 400% at day 3 of exposure compared with that of controls. The specific activities of CuZnSOD and MnSOD in these lung samples per unit of lung protein or DNA were also determined. The activity of CuZnSOD in hyperoxic lungs was found to be unchanged compared with controls, except a 20% decrease at day 7 of exposure when standardized against protein content of lung homogenate. Changes of CuZnSOD activity were more dramatic in hyperoxic lungs (a 40% increase at days 3, 5, 7, and 14 of exposure) when enzyme activity was normalized using lung DNA content. Surprisingly, no proportional increase of lung MnSOD enzyme activity was observed at days 3 and 5 of oxygen exposure. The increase of MnSOD activity per unit of lung protein also did not parallel the increase in MnSOD protein content at days 5, 7, and 14 of exposure. These data suggest that, in addition to transcriptional activation, translational and/or posttranslational regulation of the MnSOD gene expression may play a critical role in controlling lung MnSOD activity on hyperoxic exposure.

Animals↗
PubMed1996

Lung epithelial cell-released nitric oxide protects against PMN-mediated cell injury.

A calcium-independent type II nitric oxide (NO) synthase has been localized in lung epithelial cells; however, the function of NO. released by epithelial cells is unclear. We hypothesized that epithelial-derived NO may affect the interactions between polymorphonuclear neutrophils (PMN) and the alveolar epithelium and studied PMN adhesion and cytotoxicity to lung epithelial cells. A dose- and time-dependent production of NO. by L2 cells can be induced by a mixture of inflammatory mediators (cytomix) containing lipopolysaccharide, tumor necrosis factor-alpha, and interferon-gamma. Increased NO. production by L2 cells was associated with decreased 51Cr release by the epithelial cells after they were incubated with activated PMN. Addition of NG-monomethyl-L-arginine or oxyhemoglobin reversed the protective effects of cytomix, suggesting that increased NO. production by L2 cells was responsible for the decreased 51Cr release. However, PMN adhesion and intercellular adhesion molecule-1, a major adhesion molecule involved in PMN adhesion to epithelium, were increased by cytomix. We conclude that NO. released by lung epithelial cells was involved in protecting epithelial cells from PMN-mediated cytotoxicity. NO.-mediated protection of lung epithelial cells occurred in spite of PMN adhesion being increased, suggesting that reduced adhesion is not required for NO.-mediated inhibition of PMN cytotoxicity.

Animals↗
PubMed1996

Contrasting response of lung parenchymal cells to instilled TNF alpha and IFN gamma: the inducibility of specific cell ICAM-1 in vivo.

Induction of intercellular adhesion molecule-1 (ICAM-1) by proinflammatory cytokines during inflammation plays an important role in regulating polymorphonuclear neutrophil (PMN) migration and localization. In this report, we examined the effects of tumor necrosis factor-alpha (TNF alpha) and interferon-gamma (IFN gamma) on specific lung cell expression of ICAM-1 in vivo and the accompanying morphological changes. Balb-c mice were treated with phosphate-buffered saline (PBS) alone or with PBS containing 5 micrograms TNF alpha or IFN gamma through intranasal instillation. Twenty-four hours after treatment, their lungs were processed for immunoblot analysis and electron microscope immunocytochemistry. In the normal lung, the ICAM-1 level is high on type I alveolar epithelial cells, medium on arterial and venous endothelial cells, low on type II epithelial cells and capillary endothelium, and not detectable on bronchial epithelium. Topical treatment of the lung with either TNF alpha or IFN gamma induced a 50-60% increase in total lung and alveolar ICAM-1. A dramatic increase of alveolar type II cell surface ICAM-1 was observed (> 20-fold). Both cytokines caused 2-3-fold higher ICAM-1 expression on capillary endothelial cells and a 40% increase of ICAM-1 on alveolar type I cells that was not uniform. However, due to the large total surface area of type I epithelium, type I cells contribute 70-86% of total alveolar septal ICAM-1 and > 90% of alveolar surface ICAM-1 in either treated or normal mouse lungs. Increased ICAM-1 expression was also observed on nonparenchymal endothelial and epithelial cells. Margination and sequestration of PMN in cytokine-treated lungs were observed by histologic examination, measurements of total lung myloperoxidase activity, and number of neutrophils recovered in bronchoalveolar lavage fluid. These results showed that TNF alpha and IFN gamma induce ICAM-1 expression and infiltration of neutrophils in the lung. The response of specific lung cells in terms of induction of ICAM-1 in response to cytokine stimulation varied significantly, particularly between type I and type II epithelial cells.

Administration, Intranasal↗
PubMed1995

Bronchiolarized metaplasia and interstitial fibrosis in rat lungs chronically exposed to high ambient levels of ozone.

The cellular and tissue changes in the lungs of rats were studied using electron microscopy following 20 months exposure to a range of ozone levels from 0.12 to 1.0 ppm. Male and female Fischer 344 rats were exposed and morphometric methods were used to determine the volume, surface area, and cellular changes observed in bronchiole-alveolar duct regions following chronic ozone exposure. No major gender-related effects were observed in response to chronic inhalation of ozone nor were significant effects of ozone exposure found in either terminal bronchioles or the proximal alveolar regions in animals chronically exposed to 0.12 ppm ozone. The proximal alveolar regions of animals exposed for 20 months to 0.5 and 1.0 ppm ozone were significantly altered with exposure. The high-dose, long-term exposure to ozone resulted in a pronounced increase in volume of both the interstitium and epithelium in the proximal alveolar regions. The thickening of the epithelium was due to a change in tissue type from the normal squamous epithelium to a cuboidal epithelium similar, but not identical, to that found in terminal bronchioles. This bronchiolar epithelial metaplasia of proximal alveolar ducts, which was dose related, was composed of differentiated ciliated and Clara cells similar to those found in terminal bronchioles. In addition, unique cells which contained morphologic features of many different cell types were observed. These cells, which may represent stem cells or differentiated but transformed cells, were found associated with the bronchiolar metaplasia of alveolar ducts. In conjunction with the epithelial changes, cellular and matrix components in the interstitium were increased with chronic exposure to 0.5 and 1.0 ppm ozone. All matrix components were increased including collagen, elastin, and basement membrane, as well as other acellular spaces which did not contain identifiable structures. The total volume of interstitial fibroblasts was also increased in the high-dose exposure group. Alveolar macrophages were increased only in the 1.0 ppm exposed animals. The cell and tissue changes in the terminal bronchioles were less pronounced indicating a relative resistance of this tissue to ozone and mainly consisted of a change in cell type from ciliated to Clara cells in the 1.0 ppm exposed animals. The relative resistance of bronchiolar tissue to high concentration ozone exposure and the extensive bronchiolar epithelial metaplasia may be an adaptive mechanism following chronic ozone exposure.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

An easy and economical method to prepare cells for cytologic analyses.

A crucial step in any cytology application begins with the placement of cells on slides in a manner that provides good preservation of cell morphology. Commercial cytocentrifuges are the most commonly used devices for this purpose. However, there are instances when these devices are either not available or not applicable. We devised an easy and economical alternative method to prepare cells for cytologic analysis. The device (cytograv) was easily assembled from common laboratory and office supplies, and produced cell preparations of similar quality as those produced with a cytocentrifuge (cytospin-3). Cellular analyses of biological fluids is one of the most common applications for these kinds of devices. The cytograv device was successfully employed in the identification and verification of a PMN isolation procedure from whole blood. The cytograv device was also successfully used to quantitative increases in the number of PMNs in bronchoalveolar lavage fluid recovered from mice treated with increasing doses of paraquat. These two examples illustrate some of the many possible uses of the cytograv device to provide high-quality preparations for cytologic analyses.

Animals↗
PubMed1995

Artificial surfactant attenuates hyperoxic lung injury in primates. II. Morphometric analysis.

Diffuse lung injury from hyperoxia is accompanied by low compliance and hypoxemia with disruption of endothelial and alveolar epithelial cell layers. Because both function and content of surfactant in diffuse lung injury decrease in animals and in humans, changes in the extent of injury during continuous hyperoxia were evaluated after treatments with a protein-free surfactant in primates. Ten baboons were ventilated with 100% O2 for 96 h and five were intermittently given an aerosol of an artificial surfactant (Exosurf). Physiological and biochemical measurements of the effects of the surfactant treatment are presented in a companion paper (Y.-C. T. Huang, A. C. Sane, S. G. Simonson, T. A. Fawcett, R. E. Moon, P. J. Fracica, M. G. Menache, C. A. Piantadosi, and S. L. Young. J. Appl. Physiol. 78: 1823-1829, 1995.) After O2 exposures, lungs were fixed and processed for electron microscopy. The cellular responses to O2 included epithelial and endothelial cell injuries, interstitial edema, and inflammation. Morphometry was used to quantitate changes in lungs of animals treated with the artificial surfactant during O2 exposure and to compare them with the untreated animals. The surfactant decreased neutrophil accumulation, increased fibroblast proliferation, and decreased changes in the volume of type I epithelial cells. Surfactant-treated animals also demonstrated better preservation of endothelial cell integrity. These responses indicate ameliorating effects of the surfactant on the pulmonary response to hyperoxia, including protection against epithelial and endothelial cell destruction. Significant interstitial inflammation and fibroblast proliferation remained, however, in surfactant-treated lungs exposed to continuous hyperoxia.

Aerosols↗
PubMed1995

Catalase and glutathione reductase protection of human alveolar macrophages during oxidant exposure in vitro.

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)

Adenine Nucleotides↗
PubMed1995

Effects of prolonged exposure to low doses of nitric oxide or nitrogen dioxide on the alveolar septa of the adult rat lung.

BACKGROUND: Nitric oxide (NO) and nitrogen dioxide (NO2) are common copollutants resulting from combustion processes such as the burning of fossil fuels and tobacco smoke. The relative toxicity of these two pollutants has not been adequately addressed. Separate low level exposures to each of these two pollutants were carried out to allow comparisons of relative health risks. EXPERIMENTAL DESIGN: Male rats were exposed to either NO or NO2 for 9 weeks at 0.5 ppm with twice daily, 1-hour spikes to 1.5 ppm. Lungs from five rats in each exposed group and from rats from a clean air control group were preserved by vascular perfusion of fixative and were embedded for sectioning. The number of fenestrations in alveolar septa of the lung was determined by using serial sections to directly count the number of fenestrae in a known volume of lung. RESULTS: The average number of fenestrae was 328 +/- 156 x 10(3) (mean +/- SE, n = 5) per lung in the NO group. In the NO2 exposure group, there were 99 +/- 42 x 10(3) fenestrae per lung. The number of fenestrae per lung in the controls (9 +/- 9 x 10(3)) was not statistically different from zero. The number of fenestrae in the NO group was significantly greater than that in the control or NO2 groups. Analyses of total parenchymal cells per lung demonstrated a statistically significant 29% reduction in the number of interstitial cells in the NO group. There were no significant differences in the numbers of other types of cells between the control and exposed groups. The thickness of the interstitial space was reduced in the NO group (0.24 +/- 0.02 microns versus 0.32 +/- 0.02 microns in controls) but not in the NO2 group (0.29 +/- 0.02 microns). Epithelial cell thickness did not differ between groups. CONCLUSIONS: Focal degeneration of interstitial cells, interstitial matrix, and connective tissue fibers is the principal injury resulting from low level NO exposure. NO is significantly more potent than NO2 in the production of these defects in the interstitial spaces of alveolar septa. Although limited in number and size, the formation of fenestrae by atrophy of the interstitial spaces is similar to the initial steps in an emphysema-like destruction of alveolar septa.

Animals↗
PubMed1995

Immunocytochemical localization of the sites of superoxide dismutase induction by hyperoxia in rat lungs.

BACKGROUND: Rats exposed to 85% O2 can survive subsequent exposures to 100% O2. The development of this tolerance to oxygen toxicity is associated with an increase in total pulmonary content of both CuZn and Mn superoxide dismutases (SOD). It is not known, however, in which cells increases in these antioxidant enzymes occur. We carried out an electron microscopic immunocytochemical study to define the distribution of CuZnSOD and MnSOD and their sites of induction in rat lungs. EXPERIMENTAL DESIGN: The lungs of rats exposed to either air or 85% O2 were fixed by instillation of fixative 7 and 14 days after the exposures were started. Cryoultrathin sections were prepared from randomly selected, frozen tissue blocks and were immunolabeled for either CuZnSOD or MnSOD. The labeling densities of the two enzymes in the major alveolar parenchymal cells (type I and type II epithelial cells, interstitial fibroblasts, and endothelial cells), were evaluated. RESULTS: All of the alveolar septal cells studied displayed similar concentrations of CuZnSOD. After 7 or 14 days of exposure to 85% O2, the total lung content of CuZnSOD was increased because of cell hyperplasia and hypertrophy, but the intracellular concentrations of CuZnSOD did not increase. MnSOD was present in the mitochondria of all lung cells in control rat lungs but was highest in alveolar type II epithelial cells. Exposure to 85% O2 increased the concentration of MnSOD in the mitochondria of interstitial fibroblasts by 197 and 139% after O2 exposure of 7 days and 14 days, respectively. A significant induction of MnSOD also occurred in the mitochondria of alveolar type II cells after 7 days of hyperoxia exposure. Biochemical analyses showed that a significant fraction of the hyperoxia-induced MnSOD was in the form of an enzymatically inactive protein. CONCLUSIONS: Hyperoxia induced total lung CuZnSOD in proportion to lung cell hypertrophy and hyperplasia. Total lung MnSOD was increased in association with specific inductions of MnSOD protein expression in the mitochondria of type II alveolar epithelial cells and interstitial fibroblasts. Hyperoxia appeared to result in inactivation and accumulation of the inactive MnSOD as well as induction of enzymatically active MnSOD. The ability of fibroblasts and alveolar type II cells to replace inactive MnSOD or to augment active MnSOD in the mitochondria appears to play important roles in the development of tolerance to hyperoxia by rats.

Animals↗
PubMed1995

Consequences of prolonged inhalation of ozone on F344/N rats: collaborative studies. Part VIII: Morphometric analysis of structural alterations in alveolar regions.

Morphometric techniques were used to examine cellular and tissue changes occurring in male and female rat lungs exposed to ozone for a prolonged time. F344/N rats were exposed to 0.0, 0.12, 0.5, or 1.0 parts per million (ppm)* ozone for six hours per day, five days per week, for 20 months. Changes in cell volume, cell surface ratios, and cellular characteristics were studied in the terminal bronchioles and in the proximal alveolar regions of the lungs. Animals exposed for 20 months to 0.5 or 1.0 ppm ozone demonstrated dramatic increases in the volume of interstitium and epithelium along the alveolar ducts. The thickening of the epithelium was caused by an epithelial metaplasia in which the normal squamous epithelium was modified to a cuboidal epithelium similar, but not identical, to the type found in terminal bronchioles. This bronchiolar epithelial metaplasia was directly related to dose of ozone, and was characterized by differentiated ciliated cells and Clara cells similar to those found in terminal bronchioles; undifferentiated cuboidal cells also were found in the animals exposed to 0.5 and 1.0 ppm ozone. A mild fibrotic response was seen in the animals exposed to 1.0 ppm ozone, with increases in both the interstitial matrix and cellular interstitium. The individual components of the interstitial matrix, including collagen, elastin, basement membrane, and acellular spaces, all were increased. The increase in cellular interstitium was due to an increase in the volume of interstitial fibroblasts. A slight inflammatory response, identified by an increase in alveolar macrophages, was observed in the animals exposed to 1.0 ppm. The terminal bronchioles were less affected than the proximal alveolar region by the ozone exposures, which may indicate a resistance of this tissue to ozone damage. The changes in the terminal bronchioles mainly consisted of a shift in cell type from ciliated to Clara cells in the animals exposed to 1.0 ppm ozone. The bronchiolar epithelial metaplasia observed in the proximal alveolar ducts may indicate that a protective mechanism develops in response to prolonged exposure to high concentrations of ozone.

Analysis of Variance↗
PubMed1995

A metalloporphyrin superoxide dismutase mimetic protects against paraquat-induced endothelial cell injury, in vitro.

There is an increased interest in development of therapeutic agents to treat disease states that involve reactive oxygen species in their pathophysiology. The metalloporphyrins, MnTBAP and ZnTBAP, were found to be active in a superoxide dismutase (SOD) assay. The efficacy of these compounds were tested against a well established model of intracellular superoxide-mediated cell injury. Paraquat generates superoxide by redox cycling with intracellular diaphorases and molecular oxygen. SOD mimetics were employed in a cell culture model with calf pulmonary artery endothelial cells (CPA-47) grown to near confluence in 24-well plates. Cell injury was assessed by measuring the release of cytosolic lactate dehydrogenase into the cell medium and by the number of adherent cells remaining after treatment. Exposure to various concentrations of paraquat for 4 h produced a dose-dependent injury response that was attenuated by 50 microM SOD mimetic, MnTBAP. MnTBAP also protected endothelial cells in a dose-dependent manner (EC50 approximately 40 microM) against paraquat (2 mM), whereas its less active analog (ZnTBAP) did not protect them. The protective effect of MnTBAP appears to be due to its intracellular superoxide-scavenging activity because neither the zinc form or exogenous CuZnSOD protected the cells against paraquat-induced injury. These studies suggest that metalloporphyrin-based SOD mimetics may be useful agents in preventing injury and disease states associated with the generation of intracellular reactive oxygen species.

Animals↗
PubMed1994

Extracellular superoxide dismutase (SOD3): tissue-specific expression, genomic characterization, and computer-assisted sequence analysis of the human EC SOD gene.

We have isolated and characterized over 10,000 bp of the human EC SOD gene (SOD3 or EC 1.15.1.1) and its 5'- and 3'-flanking regions. Human genomic Southern blot analysis supports the existence of a single gene, without evidence for pseudogenes. The human EC SOD gene spans approximately 5900 bp. The gene can be divided into 3 exons and 2 introns. The 720-bp coding region is uninterrupted and located within exon 3. The 560 bp 5' to the transcription start site were sequenced. No obvious TATA box was identified. A variety of conserved cis elements were identified by database searching. Exon 3 is surrounded by an Alu-J repetitive element in reverse orientation at the 5' and by an Alu-Sx repetitive element in the 3'-flanking DNA. The relative levels of EC SOD tissue-specific expression were determined by RNA gel blot analysis. Adult heart, placenta, pancreas, and lung had the most expression, followed by kidney, skeletal muscle, and liver. Little EC SOD message was found in the brain. A second unique mRNA, approximately 4.2 kb in length, was highly expressed in skeletal muscle. When tissue enzyme activity is compared to relative mRNA levels, there is a marked disparity in the brain, pancreas, and lung, suggesting that these tissues have enhanced affinity for circulating EC SOD or translate the EC SOD message more efficiently than other tissues. These results indicate that the EC SOD gene contains unique transcriptional regulatory elements and that its expression may be regulated at the post-transcriptional or post-translational level. The characterization of the human EC SOD gene should now allow the development of further insights into its biology and provide the basis for studies of its role in human heritable disorders.

Amino Acid Sequence↗