Glycosylphosphatidylinositol-specific phospholipase D is localized in keratinocytes.

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is abundant in mammalian plasma, but little is known of its cellular and tissue distribution. In this study frozen sections of perfused tissues from adult rats were stained with monoclonal antibodies against GPI-PLD. The most intense staining was observed in the stratified squamous epithelium of the forestomach. Staining was also observed in the esophagus, the tongue, the hard palate, and the skin but not in most other tissues including the columnar epithelium of the stomach or the lower gastrointestinal tract. GPI-PLD expression was also detected in several keratinocyte cell lines. Biochemical assays of glycosylphosphatidylinositol-degrading activity using [3H]myristate-labeled variant surface glycoprotein as substrate provided independent evidence for the presence of GPI-PLD. Expression of GPI-PLD by keratinocytes was not affected by culture in serum-free media, indicating that it does not originate by uptake of serum GPI-PLD in the media. These data suggest that keratinocytes are an important site of action of GPI-PLD and possibly a contributor to the plasma GPI-PLD pool.

Development and characterization of a rapid-onset rodent inhalation model of asbestosis for disease prevention.

A short-term inhalation model of asbestosis was developed in rodents to examine possible preventive approaches to lung disease. Fischer 344 (F344) rats were exposed for 10 and 20 days to National Institute of Environmental Health Sciences (NIEHS) crocidolite asbestos while sham controls were exposed to air only. To determine quantitative biochemical indicators of asbestos-induced lung disease, bronchoalveolar lavage (BAL) fluids were analyzed for lactic dehydrogenase (LDH), alkaline phosphatase, angiotensin-converting enzyme (ACE), and protein. Total and differential cell counts were performed on cell pellets from BAL. Lungs from additional rats were processed for histopathology, measurement of hydroxyproline, and autoradiography after injection of rats with 3H-thymidine. Exposure to asbestos for 10 and 20 days caused increases in LDH, alkaline phosphatase, and protein in BAL. In contrast, ACE was undetectable in BAL fluids from sham or asbestos-exposed rats. At both time periods, the percentages of polymorphonuclear leukocytes (PMNs) and lymphocytes in BAL were increased in asbestos-exposed rats. Total cell numbers in BAL were increased significantly at 20 days in animals inhaling asbestos. Exposure to asbestos for 10 and 20 days caused elevated amounts of hydroxyproline in lung and the development of fibrotic lesions. Asbestos-exposed rats exhibited increased numbers of interstitial cells and airspace epithelial cells incorporating 3H-thymidine, whereas labeled bronchiolar epithelial cells were not elevated significantly. The quantitative changes in asbestos-associated enzyme levels, cell types and protein in BAL, as well as increases in hydroxyproline and morphologic evidence of fibrosis, are useful indices of asbestos-related lung injury which enable preventive and therapeutic approaches to disease.

Hydrolysis of inositol phospholipids precedes cellular proliferation in asbestos-stimulated tracheobronchial epithelial cells.

Metabolism of inositol phospholipids and phosphatidylcholine was investigated in tracheobronchial epithelial cells exposed to mitogenic concentrations of crocidolite asbestos. Alterations in levels of diacylglycerol, the endogenous activator of protein kinase C, and inositol polyphosphates, presumed mobilizers of intracellular calcium, were examined. Cultures labeled with [3H]glycerol and exposed to proliferative concentrations of crocidolite asbestos demonstrated significant elevations in [3H]diacylglycerol. In contrast, crocidolite-exposed cells labeled with [3H]myristic acid or [3H]choline did not display elevated production of [3H]diacylglycerol or release of [3H]choline metabolites--i.e., evidence of phosphatidylcholine hydrolysis. The soluble tumor promoter phorbol 12-myristate 13-acetate catalyzed both of these changes. myo-[3H]Inositol-labeled cells exposed as briefly as 10 min to mitogenic concentrations of crocidolite demonstrated elevations in [3H]inositol mono-, tris-, and terakisphosphates, phenomena indicating turnover of inositol phospholipids. The detection of diacylglycerol and inositol phosphates in crocidolite asbestos-exposed cells suggests that this fibrous tumor promoter activates phospholipase C as it stimulates cellular proliferation.

Inhibition of lung injury, inflammation, and interstitial pulmonary fibrosis by polyethylene glycol-conjugated catalase in a rapid inhalation model of asbestosis.

Several in vitro studies suggest the involvement of active oxygen metabolites in cell damage caused by asbestos. To determine if lung injury, inflammation, and asbestosis could be inhibited in vivo in a rapid-onset, inhalation model of disease, a novel method of chronic administration of antioxidant enzymes was developed. In brief, Fischer 344 rats were treated with polyethylene glycol-conjugated (PEG-) superoxide dismutase or catalase in osmotic pumps over a 10-day (5 days/wk for 2 wk) or 20-day (5 days/wk for 2 wk) period of exposure to crocidolite asbestos. Control rats included sham-exposed animals and those exposed to asbestos but receiving chemically inactivated enzymes. After 10 days of exposure to asbestos, lactic dehydrogenase (LDH), alkaline phosphatase, and total protein in bronchoalveolar lavage (BAL) were measured in one group of rats. Total and differnetial cell counts in BAL also were assessed. After 20 days of exposure, lungs of an additional group of rats were evaluated by histopathology and by measurement of hydroxyproline. Asbestos-associated elevations in LDH, protein, and total cell numbers in BAL were reduced in rats receiving PEG-catalase. Decreases in numbers of alveolar macrophages, polymorphonuclear leukocytes, and lymphocytes occurred in these animals. Exposure to asbestos for 20 days caused significant increases in both the amount of hydroxyproline in lung and the severity and extent of fibrotic lesions as determined by histopathology. These indicators of asbestosis were inhibited in a dosage-dependent fashion in rats receiving PEG-catalase. Use of inactivated PEG-catalase failed to boost serum levels of catalase and did not inhibit asbestos-induced elevation of hydroxyproline in lung.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro assays to predict the pathogenicity of mineral fibers.

A number of mineral dusts are associated with the development of inflammation in lung and pulmonary interstitial fibrosis. In an effort to find alternative approaches to animal testing, cells (rat alveolar macrophages, hamster tracheal epithelial cells, rat lung fibroblasts) of the respiratory tract have been evaluated for cytotoxic and metabolic changes after exposure to fibers (defined as a greater than 3:1 length to diameter ratio) and particles. In all bioassays, fibrous materials provoked greater biological responses in cells in comparison to non-fibrous, chemically similar particulates at identical concentrations. For example, release of superoxide (O2-.) from alveolar macrophages (AMs) was increased (in comparison to that observed in untreated cells) after exposure to the fibers, crocidolite asbestos, erionite, Code 100 fiberglass and sepiolite. Riebeckite, mordenite and glass particles elicited minimal generation of O2-. at similar concentrations of dusts. In hamster tracheal epithelial (HTE) cells, fibers such as chrysotile asbestos, crocidolite asbestos, and Code 100 fiberglass caused increased release of 51Chromium in comparison to the particles antigorite, riebeckite and glass. Another area of exploration is the measurement of collagen and non-collagen protein in a cell line (RL-82) of rat lung fibroblasts as an indication of the fibrogenic potential of minerals. Crocidolite asbestos caused an increase in the ratio of cell-associated collagen to non-collagen protein in these cell types whereas glass beads did not affect biosynthesis of collagen. Results suggest that a battery of in vitro assays can be used to screen the capability of minerals to elicit cell damage and inflammatory changes in the respiratory tract.

Sensitivity of hamster tracheal epithelial cells to asbestiform minerals modulated by serum and by transforming growth factor beta 1.

A number of proliferative and cytotoxic responses similar to those induced by classical tumor promoters such as 12-O-tetradecanoylphorbol-13-acetate occur in tracheal epithelial cells and organ cultures exposed to asbestiform minerals. In these studies, cloned diploid hamster tracheal epithelial cells were examined in a number of short term (3-24 h) and long term (greater than 24 h) biological assays for their responsiveness to crocidolite asbestos in comparison to other fibers (chrysotile asbestos, Code 100 fiberglass) and particulates (riebeckite, antigorite, glass beads). In addition, the influence of serum on the biological effects of crocidolite were assessed. In 10% serum-containing medium, low concentrations of crocidolite (1.0 micrograms/cm2 dish) stimulated cellular proliferation (measured as increased [3H]thymidine incorporation), and concentrations greater than 5 micrograms/cm2 dish were cytotoxic (measured by decreased colony forming efficiency). In 2% serum-containing medium, lower concentrations of fibers (0.1-0.5 micrograms/cm2 dish) caused increases in [3H]thymidine incorporation and colony forming efficiency whereas higher concentrations of fibers were required for responses equitoxic to those observed in 10% serum. Crocidolite-induced [3H]thymidine incorporation was inhibited when 2% serum-containing medium was supplemented with transforming growth factor type beta at concentrations (3 ng/ml) which give rise to altered morphology of hamster tracheal epithelial cells. Furthermore, the profiles of colony forming efficiency and 51Cr release of cells grown in the presence of transforming growth factor beta resembled those of cells grown in 10% serum. Results suggest that the phenotypic changes induced by serum factors such as transforming growth factor beta influence the sensitivity of hamster tracheal epithelial cells to crocidolite asbestos.

Approaches to prevention of asbestos-induced lung disease using polyethylene glycol (PEG)-conjugated catalase.

Asbestos-associated damage to cells of the respiratory tract in vitro can be prevented by the simultaneous addition of scavengers of active oxygen species to cultures. To determine if administration of scavenger enzymes to animals and humans is a plausible approach to the prevention of asbestos-induced lung disease, osmotic pumps were filled with various concentrations of PEG-coupled catalase and implanted subcutaneously into Fischer 344 rats over a 28-day period. At 3, 14, and 28 days after implantation of the pumps, the animals were evaluated for levels of catalase in serum and lung. In addition, lung tissue and lavage fluids were examined at 28 days for biochemical and morphologic indications of cell injury, inflammation, and fibrotic lung disease. At all time points examined, the administration of PEG-catalase caused a dosage-dependent increase in serum levels of catalase. The levels of lung catalase were evaluated at 28 days but not at earlier time periods. In comparison to control rats, the amounts of enzymes (lactic dehydrogenase, alkaline phosphatase), protein, and cells in lavage fluids from treated animals were unaltered. Moreover, the lungs showed no evidence of inflammation or fibrotic disease as determined by differential cell counts in lavage and measurement of hydroxyproline. These studies suggest that administration of PEG-catalase does not cause injury or other alterations in lung tissue and can be pursued as a feasible approach to prevention of asbestosis.