Efficient depletion of alveolar macrophages using intratracheally inhaled aerosols of liposome-encapsulated clodronate.

Rat alveolar macrophages (AMs) were depleted via intratracheal inhalation (ITIH) of clodronate-containing liposomes. AM depletion following ITIH delivery of clodronate liposomes was 33.2 +/- 14.2 on day 1, 88.1 +/- 6.2 on day 3, and 91.4 +/- 1.8 on day 4 relative to control rats given saline-containing liposomes. Almost all (approximately 99%) of the AMs remaining at the 3-day time point were peroxidase negative, suggesting that immature macrophages were not recruited from the circulation to replace those undergoing cell death on that day. Only 0.5% +/- 0.5% of bronchoalveolar lavage (BAL) cells were neutrophils at this time (normalized to controls). Whole-body inhalation did not induce as much AM depletion at 3 days (37.6% +/- 10.1%) and required larger amounts of liposome-encapsulated clodronate compared to ITIH. Intratracheal instillation (as opposed to inhalation) of clodronate liposomes produced a significant inflammatory response characterized by the influx of both polymorphonuclear neutrophils (PMNs) and macrophages. In subsequent pilot studies, the response to intratracheally instilled crystalline silica (75 microg) was found to be markedly reduced in rats depleted of AMs by the ITIH method. We conclude that ITIH of clodronate liposomes in rats is both efficient and useful for examining the role of AMs in pulmonary toxicology.

Induction of adaptation to inhaled lipopolysaccharide in young and old rats and mice.

Lipopolysaccharide (LPS) is a component of the gram-negative bacterial cell wall that is known to activate inflammatory cells and enhance the production of inflammatory mediators in the lung. As it is a ubiquitous compound, inhalation exposure is highly likely in the human environment. Adaptation is a phenomenon by which a previous exposure results in improved survival or reduced injury as compared to a single exposure alone. We hypothesized that the basic proinflammatory effects of LPS in the lung could result in the development of adaptation in animals. Based on evidence of age- and species-related differences in lung injury, we used an acute lung injury model with inhaled LPS to compare the development of adaptation in young and old Fisher 344 rats and C57Bl/6J mice. Animals were exposed to low-dose (predicted lung deposition approximately 20 ng in rats and approximately 5 ng in mice) LPS aerosols for 10 min on 3 consecutive days; on day 4, a high dose (rats approximately 200 ng; mice approximately 25 ng) was delivered. Another group of animals received only the high LPS dose on day 4, whereas controls were unexposed. Twenty-four hours after the last exposure, cellular and inflammatory parameters in bronchoalveolar lavage (BAL) were determined. An adaptive response was found in both rats and mice. Adapted animals showed significantly fewer BAL neutrophils compared to nonadapted ones; there was also a significantly lower release of oxidants from phorbol methyl ester-stimulated BAL cells from adapted compared to nonadapted animals, which, in turn, showed a greater response than controls. Furthermore, studies in old animals (21 mo of age) showed that adaptation also occurs in this age group. The adaptive response is clear in old mice; in rats, there is greater variability in the response, but an adaptive trend is apparent. Therefore, we have demonstrated that inhaled low-dose LPS can induce adaptation to subsequent higher doses, much as has been shown for other toxicants that induce oxidative lung injury.

Acute pulmonary effects of ultrafine particles in rats and mice.

Ambient fine particles consist of ultrafine particles (< 100 nm) and accumulation-mode particles (approximately 100 to 1,000 nm). Our hypothesis that ultrafine particles can have adverse effects in humans is based on results of our earlier studies with particles of both sizes and on the finding that urban ultrafine particles can reach mass concentrations of 40 to 50 micrograms/m3, equivalent to number concentrations of 3 to 4 x 10(5) particles/cm3. The objectives of the exploratory studies reported here were to (1) evaluate pulmonary effects induced in rats and mice by ultrafine particles of known high toxicity (although not occurring in the ambient atmosphere) in order to obtain information on principles of ultrafine particle toxicology; (2) characterize the generation and coagulation behavior of ultrafine particles that are relevant for urban air; (3) study the influence of animals' age and disease status; and (4) evaluate copollutants as modifying factors. We used ultrafine Teflon (polytetrafluoroethylene [PTFE]*) fumes (count median diameter [CMD] approximately 18 nm) generated by heating Teflon in a tube furnace to 486 degrees C to evaluate principles of ultrafine particle toxicity that might be helpful in understanding potential effects of ambient ultrafine particles. Teflon fumes at ultrafine particle concentrations of approximately 50 micrograms/m3 are extremely toxic to rats when inhaled for only 15 minutes. We found that neither the ultrafine Teflon particles alone when generated in argon nor the Teflon fume gas-phase constituents when generated in air were toxic after 25 minutes of exposure. Only the combination of both phases when generated in air caused high toxicity, suggesting the existence of either radicals on the particle surface or a carrier mechanism of the ultrafine particles for adsorbed gas-phase compounds. We also found rapid translocation of the ultrafine Teflon particles across the epithelium after their deposition, which appears to be an important difference from the behavior of larger particles. Furthermore, the pulmonary toxicity of the ultrafine Teflon fumes could be prevented by adapting the animals with short 5-minute exposures on 3 days prior to a 15-minute exposure. This shows the importance of preexposure history in susceptibility to acute effects of ultrafine particles. Aging of the fresh Teflon fumes for 3.5 minutes led to a predicted coagulation resulting in particles greater than 100 nm that no longer caused toxicity in exposed animals. This result is consistent with greater toxicity of ultrafine particles compared with accumulation-mode particles. When establishing dose-response relationships for intratracheally instilled titanium dioxide (TiO2) particles of the size of the urban ultrafine particles (20 nm) and of the urban accumulation-mode particles (250 nm), we observed significantly greater pulmonary inflammatory response to ultrafine TiO2 in rats and mice. The greater toxicity of the ultrafine TiO2 particles correlated well with their greater surface area per mass. Ultrafine particles of carbon, platinum, iron, iron oxide, vanadium, and vanadium oxide were generated by electric spark discharge and characterized to obtain particles of environmental relevance for study. The CMD of the ultrafine carbon particles was approximately 26 nm, and that of the metal particles was 15 to 20 nm, with geometric standard deviations (GSDs) of 1.4 to 1.7. For ultrafine carbon particles, approximately 100 micrograms/m3 is equivalent to 12 x 10(6) particles/cm3. Homogeneous coagulation of these ultrafine particles in an animal exposure chamber occurred rapidly at 1 x 10(7) particles/cm3, so that particles quickly grew to sizes greater than 100 nm. Thus, controlled aging of ultrafine carbon particles allowed the generation of accumulation-mode carbon particles (due to coagulation growth) for use in comparative toxicity studies. We also developed a technique to generate ultrafine particles consisting of the stable isotope 13C by using 13C-graphite electrodes made in our laboratory from amorphous 13C powder. These particles are particularly useful tools for determining deposition efficiencies of ultrafine carbon particles in the respiratory tracts of laboratory animals and the translocation of particles to extrapulmonary sites. For compromised animals, we used acute and chronic pulmonary emphysema; a low-dose endotoxin inhalation aimed at priming target cells in the lung was also developed. Other modifying factors were age and copollutant (ozone) exposure. Exposure concentrations of the generated ultrafine particles for acute rodent inhalation studies were selected on the basis of lung doses predicted to occur in people inhaling approximately 50 micrograms/m3 urban ultrafine particles. Concentrations that achieved the same predicted lung burden per unit alveolar surface were used in rodents. (ABSTRACT TRUNCATED)

Pulmonary inflammatory response to inhaled ultrafine particles is modified by age, ozone exposure, and bacterial toxin.

Epidemiological studies demonstrate associations between increasing levels of ambient particles and morbidity in the elderly with cardiopulmonary disease. Such findings have been challenged partly because particles may not act alone to cause these effects. We hypothesized that carbonaceous ambient ultrafine particles and ozone can act together to induce greater oxidative stress and inflammation in the lung than when administered alone and that these effects would be amplified in the compromised, aging lung. Two models of a compromised lung were used: endotoxin priming and old-age emphysema (TSK mice). Young (10 wk) and old (22 mo) male F344 rats and male TSK mice (14-17 mo) were exposed to ultrafine carbon particles (count median diameter 25 nm, 110 micrograms/m3) and to ozone (1 ppm) alone and in combination for 6 h. Inhalation of low-dose endotoxin (70 and 7.5 units estimated alveolar deposited dose in rats and mice, respectively) was used to model respiratory-tract infection. Cellular and biochemical lavage parameters and oxidant release from lung lavage cells were assessed 24 h after exposure. Inflammatory cell influx into the alveolar space was observed for both species and age groups: The combination of inhaled ultrafine carbon and ozone after endotoxin priming resulted in the greatest increase in lavage-fluid neutrophils. In general, the unstimulated and stimulated release of reactive oxygen species (ROS) from lavage inflammatory cells correlated well with the neutrophil response. There were significant effects of carbon particles as well as a consistent interaction between carbon and ozone as determined by analysis of variance (ANOVA). However, this interaction was in the opposite direction in young rats versus old rats and old TSK mice: Carbon and ozone interacted such that ROS activity was depressed in young rats, whereas it was enhanced in old rats and old TSK mice, indicating age-dependent functional differences in elicited pulmonary inflammatory cells. These results demonstrate that ultrafine carbonaceous particles inhaled for short periods of time can induce significant pulmonary inflammation and oxidative stress that are modified by age, copollutants, and a compromised respiratory tract.