Acute pulmonary and hematological effects of two types of particle surrogates are influenced by their elemental composition.

Several epidemiological studies have consistently demonstrated significant associations between ambient levels of particulate matter and lung injury and cardiovascular events with increased morbidity and mortality. Particle surrogates (PS), such as residual oil fly ash (ROFA), have been widely used in experimental studies aimed at characterizing the mechanisms of particle toxicity. Since PS composition varies depending on its source, studies with different types of PS may provide clues about the relative toxicity of the components generated by high-temperature combustion process. In this work, we have studied the effects of nasal instillation of increasing doses of different PS in mice: saline, carbon, and two types of particle surrogates. PS type A (PSA) was the ROFA collected from the waste incinerator of our university hospital; PS type B (PSB) was collected from the electrostatic precipitator of a large steel company and thus had an elevated metal content. After 24h, we analyzed hematological parameters, fibrinogen, bronchoalveolar lavage, bone marrow, and pulmonary histology. Nasal instillation of the two types of PS-induced leucopenia. PSB elicited a greater elevation of plasma fibrinogen levels. Bone marrow and pulmonary inflammatory changes were more intense for PSA. We concluded that the PS composition modulates acute inflammatory changes more significantly than the mass for these two types of PS.

Differential ability of transition metals to induce pulmonary inflammation.

Transition metals are components of airborne particles and have been implicated in adverse health effects. The relative inflammatory potential of these metals is usually inferred from separate studies that focus on only one or a few individual metals. Comparisons of relative potency among several metals from these separate studies can be difficult. In one comprehensive study, we measured the pulmonary effects of equimolar doses of six metals in soluble form. Our purpose was to compare inflammatory potential and pulmonary toxicity among individual transition metals. Rats received saline, 0.1 or 1.0 micromol/kg of vanadium, nickel, iron(II), copper, manganese, or zinc as sulfates. Bronchoalveolar lavage (BAL) was performed at 0, 4, 16, or 48 h postinstillation. All treatments except V showed increased lactate dehydrogenase activity in BAL fluid; Cu- and Ni-exposed animals had the highest levels. Protein levels in BAL fluid were more than five times higher in Cu-exposed animals compared to other metal treatments at 16 and 48 h. At the 0.1 micromol/kg dose, only Cu induced significant neutrophilia at 16 and 48 h. For the 1.0 micromol/kg dose, all metals tested induced significant neutrophilia, with mean neutrophil numbers for Cu and Mn significantly higher compared to the other metals. At 48 h, neutrophil numbers were still elevated in all metal exposures. Only Mn caused substantial eosinophilia. At the 1.0 micromol/kg dose, only Cu induced macrophage inflammatory protein-2 (MIP-2) mRNA at 4 h. By 48 h, induction of MIP-2 mRNA was observed for all metal exposures except Cu, which subsequently returned to baseline levels. On an equimolar basis, Cu was the most proinflammatory metal, followed by Mn and Ni, while V, Fe(II), and Zn induced similar levels of inflammation. Overall, there were many similarities in the pulmonary responses of the metals we tested. However, we also observed divergent, metal-specific responses. These differential responses suggest that metals induce pulmonary inflammation by differing pathways or combinations of signals.

Transmission electron microscopy of intracellular particles of polyethylene from joint replacement prostheses: size distribution and cellular response.

The objectives of this transmission electron microscopy study of peri-implant tissues retrieved at revision arthroplasty were to (1) determine the size distribution of intracellular polyethylene particles, and (2) assess the cellular response to phagocytosed polyethylene particles as revealed by the condition of the cellular organelles. The frequency distributions of intracellular polyethylene particle sizes for 15 cases of total hip replacement showed that more than 75% of the particles had lengths of less than 0.5 microm. More than 90% of the particles were less than 1.0 microm in size. In comparison, the frequency distribution for the particles in cellscomprising tissue retrieved from three total knee replacement prostheses showed that only 43% of the particles were less than 0.5 microm in length and 72% were less than 1 microm in size. There was no statistically significant difference in the mean particle length between the specimens from the hip and knee patients. The majority of the cells containing polyethylene were without signs of degeneration. The cytoplasmic and nuclear membranes were intact. Several electron lucent voids which once contained polyethylene particles were seen surrounded by several healthy appearing mitochondria, which displayed sharp membranes and intact cristae. There were no signs of a cytotoxic response to polyethylene at the ultrastructural level.

Ultrafine particles in human lung macrophages.

As knowledge about size dependency of particle toxicity continues to grow, attention has been focused on ultrafine particles (i.e., < 0.1 microm in diameter). In recent studies with rats, investigators learned that ultrafine particles likely have greater pulmonary toxicity than larger particles, and it is possible that exposure to, and accumulation of, these particles in the human lung may be associated with adverse respiratory health effects. As part of an ongoing study, the authors performed bronchoalveolar lavage in 14 healthy current nonsmokers to investigate the extent to which ultrafine particles were present in lung macrophages. In addition, 10 of the 14 subjects performed pulmonary function tests. Eleven of the 14 subjects were utility workers, and 3 were nonmaintenance employees of a university. The authors used a Zeiss CEM902 electron microscope to study macrophages isolated from bronchoalveolar lavage fluid. Morphometric quantification revealed ultrafine particles in lung macrophages of all 14 volunteers; the average number of ultrafine particles/microm3 cytoplasm per cell (UFavg) ranged from 34 to 231 (mean = 95, standard deviation = 54). Regression analysis showed that the UFavg was associated inversely with percent predicted forced expiratory volume in 1 second (FEV1.0) (beta = -1.2 percent predicted FEV1.0/10 ultrafine particles x microm3 cytoplasm per cell [standard error = 0.45, p = .031). The demonstration of ultrafine particles in all 14 subjects, independent of occupational exposure, suggests that there is environmental exposure to ultrafine particles. The negative association between the number of ultrafine particles and ventilatory function demonstrates a need for further investigation into the pulmonary health effects of ultrafine particles.

Endocytosis of ultrafine particles by A549 cells.

Alveolar epithelium's capacity to ingest inhaled ultrafine particles is not well characterized. The objectives of this study were to use an in vitro model of type II lung epithelium and evaluate the cells' ability to take up ultrafine particles (titanium dioxide [TiO(2)], 50 nm diameter). The human epithelial cell line A549 was grown on aclar substrates and exposed to 40 microg/ml TiO(2) particles for 3, 6, and 24 h before imaging with energy-filtering transmission electron microscopy. Elemental mapping and electron energy loss spectroscopy were used to colocalize Ti/O with electron-dense particles. Particle endocytosis was compared in A549 cells with and without pretreatment with cytochalasin D (cyto D) (2 microg/ml). After 3 h of TiO(2) exposure, cells internalized aggregates of the ultrafine particles which were observed in cytosolic, membrane-bound vacuoles. After 24 h of exposure there were considerably more intracellular aggregates of membrane-bound particles, and aggregated particles were also enmeshed in loosely and tightly packed lamellar bodies. Throughout 24 h of exposure a preponderance of particles remained associated with the free surface of the cells and were not internalized. The majority of membrane-bound vacuoles contained aggregates of particles and only occasionally did they contain as few as two or three particles, despite the use of several different approaches to assure the possibility for individual particles to be ingested and detected. There was morphologic evidence of microfilament disturbance, but no evidence of a decrease in internalized particles in cells pretreated with cyto D. Thus, this model of type II epithelium is able to internalize aggregates of ultrafine particles.

Mechanisms of morbidity and mortality from exposure to ambient air particles.

The studies reported here assessed pathophysiologic mechanisms that result from exposure to concentrated ambient particles (CAPs) in animals with and without cardiopulmonary compromise. These studies were carried out to determine the biologic plausibility of epidemiologic observations of increases in particulate air pollution associated with increases in human morbidity and mortality. Dogs were exposed two at a time to CAPs or filtered air via tracheostomy for six hours per day on three consecutive days. The electrocardiogram (ECG) and breathing pattern were recorded continuously, and indicators of inflammation were also assessed. In one experimental design, normal dogs were exposed in pairs to CAPs and subsequently to filtered air or to filtered air and subsequently CAPs (the double CAPs/double sham design). Comparisons were made between the CAPs measurements and each dog's own sham responses. In another design, one dog was exposed to CAPs while the chambermate received a sham exposure; these experiments were followed by crossover of the protocol the subsequent week (the crossover design). Comparisons were made between the CAPs exposure and both the chambermate's sham and each dog's own sham responses. The crossover experiments were conducted in normal animals and in animals who had undergone balloon occlusion of the left anterior descending (LAD) coronary artery to induce myocardial compromise. The effects of CAPs in animals with induced chronic bronchitis were part of the original specific aims; because these studies were not fully pursued, the results are presented only in Appendix A. In normal dogs, analyses of all double CAPs and crossover studies revealed low frequency (LF) and high frequency (HF) powers for heart rate variability (HRV) that were significantly higher for CAPs exposure compared to sham exposure. Variation in day-to-day exposure concentrations, aerosol composition, and pathophysiologic responses were also found. The crossover design, continuous measures of aerosol mass, and biologic responses were incorporated in the development of a statistical model that allowed isolation of changes associated with CAPs from changes due to animal variations. Comparison of individual exposures with this model revealed a range from no response in any measured parameter to statistically significant changes in cardiac autonomic balance, pulmonary air flow, and breathing pattern. On days in which dogs showed statistically significant changes in responses, the findings were consistent in both cardiac and respiratory parameters. Days associated with significant increases in LF and HF HRV, LF/ HF HRV ratio, and heart rate standard deviation (HR SD) were also associated with decreases in average heart rate. These same days had decreases in respiratory frequency, tidal volume, minute volume, and peak flows with corresponding increases in respiratory cycle times and enhanced pause (Pauenh), a measure of bronchoconstriction. These cardiac and respiratory changes suggest an effect mediated via both the sympathetic nervous system and the vagus nerve. Alternatively, days associated with increased heart rate had decreases in the HR SD; decreases or no change in HF and LF HRV; increases in respiratory flows and volumes; and decreases in breathing cycle times, all suggesting only sympathetic nervous system mediation. When all data from the crossover design experiments were assessed with this model, the heart rate and respiratory rate were significantly decreased in relation to both cumulative and actual exposure and the LF HRV, LF/HF HRV ratio, HR SD, and all other respiratory parameters were significantly increased (p < 0.0001 for all). When cardiac data were grouped by days in which the air mass trajectory came from the north or northwest (versus west, south, east, or northeast), significant increases in HR SD and HF HRV and significant decreases in average heart rate were associated with the northwest trajectory. (ABSTRACT TRUNCATED)

Inhaled concentrated ambient particles are associated with hematologic and bronchoalveolar lavage changes in canines.

Pulmonary inflammatory and hematologic responses of canines were studied after exposure to concentrated ambient particles (CAPs) using the Harvard ambient particle concentrator (HAPC). For pulmonary inflammatory studies, normal dogs were exposed in pairs to either CAPs or filtered air (paired studies) for 6 hr/day on 3 consecutive days. For hematologic studies, dogs were exposed for 6 hr/day for 3 consecutive days with one receiving CAPs while the other was simultaneously exposed to filtered air; crossover of exposure took place the following week (crossover studies). Physicochemical characterization of CAPs exposure samples included measurements of particle mass, size distribution, and composition. No statistical differences in biologic responses were found when all CAPs and all sham exposures were compared. However, the variability in biologic response was considerably higher with CAPs exposure. Subsequent exploratory graphical analyses and mixed linear regression analyses suggested associations between CAPs constituents and biologic responses. Factor analysis was applied to the compositional data from paired and crossover experiments to determine elements consistently associated with each other in CAPs samples. In paired experiments, four factors were identified; in crossover studies, a total of six factors were observed. Bronchoalveolar lavage (BAL) and hematologic data were regressed on the factor scores. Increased BAL neutrophil percentage, total peripheral white blood cell (WBC) counts, circulating neutrophils, and circulating lymphocytes were associated with increases in the aluminum/silicon factor. Increased circulating neutrophils and increased BAL macrophages were associated with the vanadium/nickel factor. Increased BAL neutrophils were associated with the bromine/lead factor when only the compositional data from the third day of CAPs exposure were used. Significant decreases in red blood cell counts and hemoglobin levels were correlated with the sulfur factor. BAL or hematologic parameters were not associated with increases in total CAPs mass concentration. These data suggest that CAPs inhalation is associated with subtle alterations in pulmonary and systemic cell profiles, and specific components of CAPs may be responsible for these biologic responses.

Urban air particulate inhalation alters pulmonary function and induces pulmonary inflammation in a rodent model of chronic bronchitis.

Epidemiological studies have reported increased morbidity in human populations following inhalation of elevated levels of urban particulate matter. These responses are especially prevalent in populations with chronic obstructive pulmonary diseases, including chronic bronchitis. Toxicological studies have reported altered pulmonary function and increased pulmonary inflammation following particulate inhalation in the laboratory setting. However, most of these studies have utilized artificial particles that may not accurately mimic outdoor air pollutant conditions. Few studies have utilized actual urban air particle samples in inhalation studies. In the present study, the effects of inhaled concentrated urban air particulates on pulmonary function and pulmonary inflammation are addressed. Normal rats and rats with chronic bronchitis induced by approximately 200 ppm SO(2) for 6 wk were subsequently subjected to filtered air or concentrated air particles (CAPs). Twelve rats per group in 4 groups (48 rats total) were exposed for 5 h/day for 3 consecutive days. The CAPs aerosol levels were 206, 733, and 607 microg/m(3) (MMAD = 0.18 microm, sigma(g) = 2.9) on days 1, 2, and 3, respectively. Following the final day of exposure, pulmonary function parameters, including peak expiratory flow (PEF), tidal volume (TV), respiratory frequency (RF), and minute volume (MV), were measured and compared to preexposure baseline levels. Twenty-four hours following the final day of exposure, bronchoalveolar lavage was performed for total cell counts, differential cell counts, and total lavage protein levels. Pulmonary responses to CAPs in chronic bronchitic animals indicated a significant increase in tidal volume as well as peak expiratory flow. In CAPs-exposed animals without underlying bronchitis, significantly increased tidal volume was observed. Significant pulmonary inflammation was observed in the CAPs-exposed animals, particularly those with chronic bronchitis. Significant increases in neutrophils, lymphocytes, and total lavage protein were observed. These results suggest two distinct mechanistic responses to inhaled particles: a stress-type pulmonary function response marked by increases in flow and volume, that is, deeper breathing; and acute pulmonary inflammation marked by cellular influx, particularly neutrophils. From these data it is concluded that inhaled urban air particles alter pulmonary breathing parameters and increase pulmonary inflammation.

Regulation of macrophage inflammatory protein-2 gene expression by oxidative stress in rat alveolar macrophages.

Chemokines are important mediators in the development of inflammation. Our previous work demonstrated that an oxidative stress can up-regulate mRNA expression of a CC chemokine macrophage inflammatory protein (MIP)-1alpha in rat alveolar macrophages. In the present study, we further investigate whether an oxidative stress can regulate the gene expression of a related CXC chemokine MIP-2, involved in both neutrophil chemotaxis and activation. A rat alveolar macrophage cell line (NR8383) was exposed to 10 microg/ml bacterial lipopolysaccharide (LPS) and MIP-2 mRNA levels dramatically increased after 4 hr of stimulation. This increase by LPS was attenuated by co-treatment with the antioxidants N-acetylcysteine and dimethylsulphoxide, suggesting that the induction of MIP-2 mRNA is mediated via the generation of reactive oxygen species. To assess directly the role of oxidative stress on regulation of MIP-2 mRNA expression, macrophages were exposed to H2O2. MIP-2 mRNA levels had significantly increased after 1 hr exposure to 0.5 mm H2O2, were maximally increased after 4 hr and decreased after 6 hr. Co-treatment of macrophages with the transcriptional inhibitor actinomycin D eliminated the H2O2-induction of MIP-2 mRNA, implicating a role for transcriptional activation in increased expression of MIP-2. Genomic cloning of the rat MIP-2 gene 5'-flanking region has identified a consensus nuclear factor-kappaB (NF-kappaB) binding site. Gel-mobility shift assays revealed NF-kappaB binding to the MIP-2 promoter/enhancer sequence was induced by H2O2. LPS treatment for 4 hr also significantly activated NF-kappaB binding, which could also be attenuated by pretreatment with N-acetylcysteine at the doses that reduced MIP-2 mRNA expression. The half-life of MIP-2 mRNA transcripts was also increased by H2O2 treatment. These observations indicate that MIP-2 gene expression is subject to both transcriptional and post-transcriptional control in response to an H2O2 oxidative stress.

Anatomic localization of 24- and 96-h particle retention in canine airways.

Long-term retention of particles in airways is controversial. However, precise anatomic localization of the particles is not possible in people. In this study the anatomic location of retained particles after shallow bolus inhalation was determined in anesthetized, ventilated beagle dogs. Fifty 30-cm(3) boluses containing monodisperse 2.5-micron polystyrene particles (PSL) were delivered to a shallow lung depth of 81-129 cm(3). At 96 h before euthanasia, red fluorescent PSL were used; at 24 h, green fluorescent PSL and (99m)Tc-labeled PSL were used. Clearance of (99m)Tc-PSL was measured during the next 24 h. Sites of particle retention were determined in systematic, volume-weighted random samples of microwave-fixed lung tissue. Precise particle localization and distribution was analyzed by using gamma counting, conventional fluorescence microscopy, and confocal microscopy. Within 24 h after shallow bolus inhalation, 50-95% of the deposited (99m)Tc-PSL were cleared, but the remaining fraction was cleared slowly in all dogs, similar to previous human results. The three-dimensional deposition patterns showed particles across the entire cross-sectional plane of the lungs at the level of the carina. In these locations, 33 +/- 9.9% of the retained particles were found in small, nonrespiratory airways (0.3- to 1-mm diameter) and 49 +/- 10% of the particles in alveoli; the remaining fraction was found in larger airways. After 96 h, a similar pattern was found. These findings suggest that long-term retention in airways is at the bronchiolar level.

Health effects of sulfur-related environmental air pollution. V. Lung structure.

The lungs of 8 male beagle dogs were examined morphologically and morphometrically after exposure for 13 mo to a respirable sulfur(IV) aerosol at a mass concentration of 1.53 mg m(-3) (16.5 h/day), and to an acidic sulfate aerosol carrying 15.2 micromol m(-3) hydrogen ions into the lungs (6 h/day). An additional eight dogs served as unexposed controls. Standard morphometric analyses of both the surface epithelia of the conducting airways and the alveolar region were performed. These analyses showed no difference between the exposure group and control group. However, there was a tendency to an increase in the volume density of bronchial glands in the exposure group. Five of eight exposed animals showed thickened ridges (knob-like structures) at the entrance to alveoli in the alveolar duct and alveolar sac. Transmission electron microscopy revealed that the thickening was mainly due to type II cell proliferation. As the previous experiment using sulfite aerosol only showed no alterations in the proximal alveolar regions, the changes observed may be considered as effects of acidic sulfate aerosol alone or in combination with sulfite. These findings suggest that sulfur aerosols have the potential to induce epithelial alterations in the proximal alveolar region, which is a primary target for air pollutants.

Mediating phosphorylation events in the vanadium-induced respiratory burst of alveolar macrophages.

Occupational exposure by inhalation to vanadium-containing particles such as residual oil fly ash results in respiratory tract inflammation. This inflammation, characterized by abundant neutrophilia, appears to be initiated by alveolar macrophages (AMs) encountering particles and the subsequent release of proinflammatory cytokines. Intracellular signaling events in these cells in response to particles or their components are largely unknown. We investigated two immediate responses of AMs to vanadium exposure in vitro, the production of reactive oxygen intermediates (ROI) or respiratory burst (RB), and the tyrosine phosphorylation of cellular proteins. Macrophages exposed in vitro to 100 microM vanadyl chloride/1 microCi 48V incorporated 8.3% of the metal after 30 min. Exposure of AMs to increasing concentrations of sodium metavanadate resulted in a dose-dependent increase in production of ROI as measured by dichlorofluorescin oxidation. The lowest dose yielding a significant response was 50 microM, whereas 1000 microM increased RB activity by 173%. NADPH oxidase inhibitors deoxy-D-glucose (100 mM) and diphenylene iodonium (25 microM) reduced the metavanadate-induced RB by 62 and 71%, respectively, implicating NADPH oxidase as the primary cellular source of ROI. Enhanced cerium chloride oxidation in response to metavanadate localized to the plasma membrane consistent with increased NADPH oxidase activity. Pretreatment of AMs with the epidermal growth factor receptor inhibitor, tryphostin B50 (10 microM), reduced the metavanadate-induced RB, but did not influence overall tyrosine phosphorylation. Metavanadate and H2O2 exposure greatly increased overall tyrosine phosphorylation, yielding a similar but distinguishable pattern of phosphorylation in these cells. These observations demonstrate that in vitro metavanadate exposure regulates two distinct, yet related intracellular signaling pathways important in initiating inflammatory responses in these cells: (1) activation of the NADPH oxidase complex with subsequent increased ROI synthesis, and (2) enhanced tyrosine phosphorylation of cellular proteins.

Functional characterization of recombinant rat macrophage inflammatory protein-1 alpha and mRNA expression in pulmonary inflammation.

Chemokines are important inflammatory mediators that function by activating and recruiting leukocytes to an inflamed tissue. We have recently cDNA cloned the rat chemokine macrophage inflammatory protein-1 alpha (MIP-1 alpha) (1). In the present study, we characterize the biological function of recombinant MIP-1 alpha protein and describe expression of its mRNA both in vitro and in a rat model of lung inflammation. In vitro rat rMIP-1 alpha protein was chemotactic for both polymorphonuclear leukocytes (PMNs) and macrophages with maximal activity at 50 nM for both cell types. In in vivo studies, we found that intratracheal instillation of 1 and 5 micrograms of rMIP-1 alpha resulted in a significant (P < 0.05) influx of cells, primarily monocytes/macrophages, into the airspace of the lungs after 6 h. Mean numbers of lavagable PMNs were not elevated significantly (P < 0.05) for either dose of MIP-1 alpha. As a model of inflammation, rats were intratracheally instilled with 0.1 mg/kg bacterial lipopolysaccharide (LPS). Bronchoalveolar lavage (BAL) was performed 3 h later. Instillation of LPS resulted in an acute neutrophilia, but no significant change in lavagable macrophages. BAL cells from control animals (saline instilled) displayed no basal mRNA expression of either MIP-1 alpha or MIP-2 (positive control). In contrast, both MIP-1 alpha and MIP-2 mRNA levels increased markedly in BAL cells from rats instilled with LPS. The rat alveolar macrophage cell line (NR8383) also showed increased MIP-1 alpha mRNA levels in response to LPS (10 micrograms/ml) with a maximal increase after 6-8 h. The induction of MIP-1 alpha mRNA expression by LPS in NR8383 cells was attenuated by cotreatment with the antioxidants N-acetylcysteine and dimethylsulfoxide, suggesting that the induction of MIP-1 alpha mRNA by LPS is mediated via the generation of reactive oxygen species. We conclude that MIP-1 alpha is a potent chemoattractant for macrophages in vivo, and its mRNA expression in macrophages and BAL cells in response to inflammatory stimuli suggests a fundamental role in acute pulmonary inflammation.

The elephant's respiratory system: adaptations to gravitational stress.

Elephants have had to adapt to gravitational stresses imposed on their very large respiratory structures. We describe some unusual features of the elephant's respiratory system and speculate on their functional significance. A distensible network of collagen fibers fills the pleural space, loosely connects lung to chest wall but appears not to constrain lung-chest wall movements. Myriad spaces within the network and its rich supply of capillaries suggest effective local sources and sinks for pleural fluid that may replace the gravity-dependent flows of smaller mammals. The lung is partitioned into approximately equal to 1 cm3 parenchymal units by a system of thick, elastic septa that ramify throughout the lung from origins on the lung's elastic external capsule. Parenchymal units suspended upon the elastic septal system protect dependent alveoli from compression, thereby reducing the usual gravitational gradient of lung expansion. Intra-pulmonary airways are devoid of cartilage, instead they appear to derive resistance to collapse from tethering forces of the attached septa.

Vanadium-induced chemokine mRNA expression and pulmonary inflammation.

Occupational exposure to vanadium is common in petrochemical, mining, steel, and utilities industries and results in toxic effects largely confined to the respiratory system. Vanadium exposure has been associated with inflammatory changes in the upper and lower respiratory tracts in addition to changes in pulmonary function. We investigated the abilities of several vanadium compounds to increase mRNA levels for selected cytokines in bronchoalveolar lavage (BAL) cells and also to induce pulmonary inflammation. Rats (200-250 g) were intratracheally instilled with either sodium metavanadate (NaVO3), vanadyl sulfate (VOSO4), vanadium pentoxide (V2O5) at several concentrations, or vehicle alone. Pulmonary inflammation was assessed by cytologic analysis of cells recovered from the respiratory tract (1 hr to 10 days postexposure). All three vanadium compounds were capable of inducing pulmonary inflammation in a dose-dependent manner. Neutrophil influx was greatest following exposure to VOSO4 (peaked at approximately 40% of cell population) and lowest following exposure to V2O5 (peaked at approximately 20 %). Significant neutrophil influx was detected as early as 4 hr following the instillation of NaVO3 and VOSO4 but not until 24 hr upon exposure to V2O5. The VOSO4-induced inflammatory response persisted longer (5 days) than that induced by NaVO3 and V2O5. Analysis of inflammatory cytokine mRNA expression closely followed these cytologic observations. Levels of mRNA for macrophage inflammatory protein-2 (MIP-2) and KC, considered the principal neutrophil chemotactic factors expressed in the rat, were rapidly induced as early as 1 hr following exposure, continued to be expressed throughout 48 hr, and were low but detectable at 5 and 10 days. NaVO3 and VOSO4, both very soluble forms of vanadium, tended to induce pulmonary inflammation and inflammatory cytokine mRNA expression more rapidly and more intensely than the less soluble form, V2O5. Analysis of KC mRNA expression in BAL cells 24 hr after instillation of NaVO3 by PCR in situ hybridization confirmed the increase in KC mRNA levels and indicated that alveolar macrophages have the highest expression level observed. Vanadium content of lavage fluid, BAL cells, and lung indicated rapid clearance of the metal from the lung surface and substantial accumulation by BAL cells and lung tissue. The rapid expression of MIP-2 and KC mRNA in BAL cells prior to the observed neutrophilia implicate them as important in the initiation of inflammation.

Regulation of macrophage inflammatory protein-1alpha mRNA by oxidative stress.

Accumulation of inflammatory cells within the lung has been implicated in oxidative injury. Recruitment of these cells to a tissue site is a complex process that depends in part upon the local expression of appropriate proinflammatory chemokines. Macrophage inflammatory protein-1alpha (MIP-1alpha), a member of the CC subfamily of chemokines, has been shown to contribute to monocyte/macrophage and neutrophil chemotaxis and activation. Our previous work demonstrated that MIP-1alpha mRNA expression in macrophages is induced by bacterial endotoxin. The objective of this study was to test the hypothesis that an oxidative stress alone may trigger expression of MIP-1alpha mRNA in macrophages and to determine the mechanism leading to increased expression. A rat alveolar macrophage cell line (NR8383) was exposed to H2O2 or menadione (2-methyl-1,4-naphthoquinone (MQ)), a quinone compound that undergoes redox cycling and generates reactive oxygen species continuously. Steady-state mRNA levels encoding MIP-1alpha were markedly increased (3-fold) in these cells after 1 h of exposure to 0.5 mM H2O2, remained higher than control levels after 4 h, and decreased after 6 h. Similarly, MQ (25 or 50 microM) caused a significant increase of MIP-1 alpha mRNA with a maximal induction after 4 h of exposure (5-fold). Both H2O2 and MQ-induced up-regulation of MIP-1 alpha mRNA was suppressed by co-treatment with N-acetylcysteine, a synthetic antioxidant. Co-treatment with actinomycin D reduced the MQ induction of MIP-1alpha mRNA to a greater extent than the H2O2-induced increase. Transcription of the MIP-1alpha gene was increased by exposure to both H2O2 and MQ. H2O2 treatment also induced a marked increase of the MIP-1alpha mRNA half-life, indicating post-transcriptional stabilization. These observations indicate that an oxidative stress can regulate MIP-1alpha mRNA expression by two distinct mechanisms: transcriptional activation of the MIP-1alpha gene and post-transcriptional stabilization of MIP-1alpha mRNA.

Imaging biliary lipid secretion in the rat: ultrastructural evidence for vesiculation of the hepatocyte canalicular membrane.

Physical-chemical and biological studies of hepatic bile suggest that biliary phospholipid molecules are secreted as unilamellar vesicles. Systematic ultrastructural studies of bile canaliculi were undertaken to visualize this event. Liver tissue was obtained from normal adult male rats (control), from bile salt-depleted rats (by overnight biliary diversion), and from depleted rats infused intravenously with a hydrophilic-hydrophobic congener series of common taurine-conjugated bile salts. Livers were fixed in situ either by modified chemical methods or by ultrarapid cryofixation. In control rats, chemical fixation revealed unilamellar vesicles 63 +/- 17 (+/- SD) nm in diameter, mostly free within canalicular lumena. Vesicles were infrequent in canaliculi of bile salt-depleted rats, but were present in canaliculi of rats infused with taurocholate. In cryofixed liver tissue, vesicles 67 +/- 13 nm in diameter were observed in canaliculi of control rats and bile-salt depleted rats infused with common bile salts. The majority of these vesicles were affixed to the luminal side of the canalicular membrane. The average number of vesicles per bile canaliculus was in agreement with that estimated on the basis of biliary phospholipid secretion rates, mean vesicle size, and area of close-packed phosphatidylcholine molecules. By immunoelectron microscopy, canalicular vesicles were free of actin and of a 100 kDa canalicular membrane protein. We conclude that biliary phospholipid molecules are secreted from hepatocytes into bile canalicular lumena as unilamellar vesicles approximately 63-67 nm in average diameter. We postulate that this secretion mechanism involves lumenal bile salt-induced vesiculation of lipid microdomains in the exoplasmic hemileaflet of the canalicular membrane.

Molecular cloning and posttranscriptional regulation of macrophage inflammatory protein-1 alpha in alveolar macrophages.

Macrophage inflammatory protein-1 alpha (MIP-1 alpha) belongs to the "chemokine" superfamily of chemoattractant pro-inflammatory cytokines. MIP-1 alpha is chemotactic for monocytes and neutrophils and thus, plays an important role in initiation and control of inflammation. We have isolated and sequenced a cDNA clone encoding rat MIP-1 alpha. This 0.75 kb cDNA includes a single open reading frame of 92 amino acids. Expression of MIP-1 alpha mRNA was characterized in NR8383, a rat alveolar macrophage cell line (RAM). In resting RAM cells, MIP-1 alpha mRNA decayed rapidly, with a half life of less than 2 hours. Lipopolysaccharide (LPS) treatment of RAM cells resulted in a dose-dependent increase in MIP-1 alpha steady state mRNA expression. The induction of MIP-1 alpha mRNA by LPS was partially the result of mRNA stabilization, as half life increased to over 6 hours.

Chronic bronchitis alters the pattern of aerosol deposition in the lung.

Knowledge of the local and regional doses of inhaled particulates is crucial for inhalation therapy and for understanding the progression of pulmonary disease. We studied the deposition pattern of radioactively tagged particles in rats with chronic bronchitis. Rats were exposed to sulfur dioxide (SO2; 236 +/- 14 ppm) for 5 h/d, 5 d/wk for 7 wk to produce chronic bronchitis (CB). Control rats were exposed to room air. The control animals gained 85% more weight over the 7-wk period than did the CB rats. Five control and five CB rats were then exposed for 30 min to an insoluble 99mTc-labeled aerosol. The animals were killed within 5 min after the exposure period. The lungs were excised, dried at total lung capacity (TLC), and sliced into 1 mm sections. The distribution of the radiolabeled particles retained in the lungs was determined in two ways. First, autoradiographs were made of the distribution of the radioactivity throughout a lung slice. Autoradiographs were quantified by image analysis to determine the amount of radioactivity (relative density of the film) associated with airway versus parenchyma (ratio of airway to parenchyma density). The lung slices were then dissected into pieces, the weight and radioactivity content of each piece was measured, and its evenness index (EI) was calculated. This type of analysis enables the homogeneity of particle deposition throughout the lungs to be assessed. If deposition were totally uniform, the average EI would be 1.0 with an SD = 0. The total amount of radioactivity retained in the lungs was similar in control and CB rats.(ABSTRACT TRUNCATED AT 250 WORDS)