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The effects of ozone (O3) on airway epithelia, inflammation, and expression of inflammatory stimuli were investigated to delineate the mechanisms of inflammatory reactions relevant to lung injury. Because the airway responses to O3 develop gradually, this investigation included a time-sequence analysis. Rats exposed for 3 h to 1 ppm O3 were studied at 4-h intervals up to 20 h postexposure. Bronchoalveolar lavage fluid (BAL) was analyzed for albumin as an indicator of increased permeability, polymorphonuclear leukocytes (PMNs) to assess the inflammatory status, macrophage inflammatory protein-2 (MIP-2, an inflammatory chemokine), and cell adhesion molecules for their role in inflammation and PMN functions. The time-related increase in albumin was matched by a similar significant increase for PMNs, MIP-2, and intercellular adhesion molecule-1 (ICAM-1). However, no marked change occurred for beta-2 integrin (CD-18) and leukotriene B4 (LTB4). The results establish a temporal correlation of epithelial permeability with changes in inflammatory activity and stimuli responsible for PMN recruitment in the lung. The observations of elevated MIP-2 and ICAM-1 levels are consistent with their role in injury and inflammation. An early expression of MIP-2 mRNA in BAL cells, that is, immediately post O3 exposure, and the peak increase in BAL MIP-2 levels 4 h later support the chemotactic role of MIP-2 in PMN recruitment at 4- and 12-h time points. The rapid drop in MIP-2 and ICAM-1 levels appears to signal the termination of inflammatory cell recruitment, which is accompanied by an onset of recovery.

Ozone (O3), an oxidant air pollutant, is capable of producing pulmonary inflammation and injury. Exposure to O3 results in the release of inflammatory cytokines including tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1) by alveolar macrophages. In addition, O3 exposure results in an increased expression of the inducible isoform of nitric oxide synthetase (iNOS). Interleukin-10 (IL-10) is an anti-inflammatory cytokine which inhibits the synthesis of TNF-alpha and IL-1 by macrophages and decreases the expression of iNOS. To test the protective properties of IL-10 in vivo, on the pulmonary injury induced by O3 exposure, we intratracheally instilled rat recombinant IL-10 1 h prior to O3 exposure (0.8 ppm x 3 h). Approximately 10-12 h following exposure, the animals were sacrificed and the bronchoalveolar lavage fluid (BALF) collected. The quantification of albumin, protein and fibronectin in the BALF provided a means of assessing pulmonary injury while the analysis of the BALF cells reflected the inflammatory response. Ozone exposure resulted in a significant (P<0.05) increase in BALF albumin, protein and fibronectin content as compared to air-exposed controls. In addition, significant increases in the percentage of BALF polymorphonuclear leukocytes (PMNs) and tissue expression of fibronectin mRNA were observed. The intratracheal instillation of IL-10 prior to O3 exposure resulted in a significant reduction in BALF albumin, protein and fibronectin content, and lung fibronectin mRNA as compared to O3 exposure alone. The data shows that IL-10, when given intratracheally, significantly reduces the pulmonary injury following O3 exposure in the rat. However, since the PMNs and the levels of albumin, protein and fibronectin in the IL-10 treated group did not reach baseline values, we conclude that other mediators of inflammation and injury not regulated by IL-10 also contribute to the pathophysiology of O3-induced lung injury.

The deleterious effects of ozone (O3), an oxidant air pollutant, in the lung are dependent on dose and exposure duration and generally evolve with time postexposure. This study characterized the time sequence of epithelial injury and fibronectin expression in the lungs of rats exposed to O3. Bronchoalveolar lavage (BAL) fluid was analyzed for alkaline phosphatase and total protein as markers of epithelial injury and increased permeability, and fibronectin for its role in inflammation and lung injury. The results revealed a time-related increase in total protein in the BAL fluid following a 3-h exposure of rats to 1 ppm O3. The increased protein concentrations peaked at 12 h and then declined, but remained significantly higher than control at 24 h postexposure. A similar time-related significant increase also occurred for BAL fibronectin and alkaline phosphatase activity. However, the return of alkaline phosphatase levels to baseline prior to a comparable reduction in protein levels suggests repair of injured cells, but a delay in the formation of epithelial junctions that limit the transfer of serum proteins to air spaces. By cytochemistry, alkaline phosphatase activity was detected in association with lung type II epithelial cells and in BAL polymorphonuclear leukocytes (PMNs), but not in macrophages. While a significant increase in cytochemically detectable alkaline phosphatase resulted from the increase in PMN number following O3 exposure, mononuclear cells constituted the primary cell type responsible for fibronectin mRNA upregulation. While the cytochemical observations support the role of inflammatory cells in the injury process, the comparability of temporal changes in BAL protein, fibronectin, and alkaline phosphatase suggests a mechanistic role for fibronectin in lung injury.

The airway epithelial lining serves as an efficient barrier against penetration of exogenous particles and macromolecules. Disruption of this barrier following O3 exposure represents a state of compromised epithelial defenses leading to increased transmucosal permeability. Although the barrier disruption following an acute exposure is transient in nature, the brief period of disruption caused by O3, an oxidant air pollutant, provides an opportunity for facilitated entry of a potentially toxic particulate copollutant(s) across the airway epithelia. The subsequent deposition and retention of the copollutant(s) in the subepithelial compartment for prolonged periods adds the risk of injury due to chronic exposure following an acute episode. Toxicological studies from several laboratories have demonstrated alterations in epithelial permeability, suggestive of barrier disruption, in animals and humans exposed to O3. Inflammatory cells represent another important component of pulmonary defenses, but upon activation these cells can both induce and sustain injury. The recruitment of these cells into the lung following O3 exposure presents a risk of tissue damage through the release of toxic mediators by activated inflammatory cells. Several studies have reported concomitant changes in permeability and recruitment of the inflammatory cells in the lung following O3 exposure. In these studies, an inflammatory response, as detected by an increase in the number of polymorphonuclear leukocytes in the bronchoalveolar lavage (BAL) or in lung parenchyma, was accompanied by either an increased tracer transport across the airway mucosa or an elevation in the levels of total protein and/or albumin in the BAL. The magnitude of response and the time at which the permeability changes and inflammatory response peaked varied with O3 concentration, exposure duration, and the mode of analysis. The responsiveness to O3 also appeared to vary with the animal species, and increased under certain conditions such as physical activity and pregnancy. Some of the effects seen after an acute exposure to O3 were modified upon repeated exposures. The responses following repeated exposures included attenuation, persistence, or elevation of permeability and inflammation. Mechanistic studies implicate chemotactic factors, cellular mediators, and cell-surface-associated molecules in the induction of inflammation and lung injury. In discussing these studies, this review serves to introduce the mucosal barrier functions in the lung, evaluates inflammatory and permeability consequences of O3, addresses mechanisms of inflammatory reactions, and offers alternate viewpoints.

This study investigated the relationship of fibronectin expression and induction of pulmonary inflammation by ozone (O3). Rats were exposed to 0.8 parts/million O3 to induce lung inflammation. A second inflammatory stimulus, rabbit serum, was applied intratracheally to augment O3-induced inflammation. Bronchoalveolar lavage fluid (BALF) and lung tissues were analyzed for fibronectin protein and mRNA expression. Blood plasma was analyzed to investigate the potential of a minimally invasive procedure in predicting lung inflammation and fibronectin levels. Significant increases in the levels of fibronectin protein in the BALF and lung tissue after O3 exposure were further enhanced by pretreatment with normal serum. An increase in fibronectin mRNA following O3 exposure was also enhanced by serum pretreatment, which by itself had no effect on lung fibronectin mRNA expression. Plasma fibronectin levels were comparable in air-PBS and O3-PBS groups but increased in the O3-serum group. The results suggest leakage of fibronectin from blood plasma into the lung following intratracheal application of rabbit serum and upregulation of local synthesis following O3 exposure.

Ozone (O3), a pulmonary irritant, and a major toxic component of photochemical smog, is capable of inducing pulmonary inflammation characterized by recruitment of polymorphonuclear leukocytes (PMNs) into the lung. The recruited PMNs, in turn, can release toxic mediators and produce lung injury. The mechanism of ozone-induced changes in lung permeability remains unknown. It is our hypothesis that PMNs migrating into the lung play a significant role in the pathophysiology following O3 exposure and that increasing the number of PMNs coming into the lung will exaggerate the changes in lung permeability. To test this hypothesis, we induced an influx of PMNs into the lungs of Sprague-Dawley rats by intratracheal instillation of 1% rabbit serum and then exposed the animals to either 0.8 ppm O3 or filtered air for 3 h. Control animals were intratracheally instilled with phosphate-buffered saline (PBS) and simultaneously exposed to O3 or filtered air in the same manner as the serum-treated animals. The animals were sacrificed and the lungs lavaged 10-12 h after exposure. The bronchoalveolar lavage fluid (BALF) was analyzed for albumin and protein, as indicators of permeability. In addition, BALF from the various groups was tested for its ability to alter epithelial resistance of pulmonary type II cells in culture. O3 exposure resulted in a significant increase in albumin and protein levels in the BALF as compared to air-exposed controls. The instillation of serum resulted in a significant increase in airway PMNs, but no significant elevations in albumin levels in both the O3 and air-exposed groups, as compared to PBS instillation. In vitro studies did not reveal a differential BALF effect on epithelial resistance. The data demonstrate that an excessive neutrophilia in the lung is not matched by a comparable amplification of epithelial injury. It is therefore suggested that a simple elevation in PMN number in the air spaces, as that induced by serum instillation, does not necessarily augment the lung pathophysiology, but that a more complex interaction with O3 may be required for cellular activation and release of toxic products.

Inhalation exposure to ozone (O3) is known to induce epithelial and inflammatory changes in the lungs, characterized by neutrophilia and changes in epithelial permeability. Several cell types and their soluble mediators, including interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha), are involved in the evolution of these responses. In this study, we have compared the effects of the combination of anti-IL-1 alpha and anti-TNF-alpha on in vitro and in vivo responses to inhaled O3. Male, Sprague-Dawley rats were exposed, nose-only, to 0.8 ppm O3 for 3 h and the in vitro and in vivo parameters were measured 8-12 h following exposure. The in vitro studies revealed that the adherence of inflammatory cells, primarily macrophages, harvested from the lungs of O3-exposed rats to cultured lung epithelial cells (ARL-14) was significantly greater than the adherence of macrophages from air-exposed controls. Furthermore, this adherence was significantly reduced in antibody-treated cells as compared to cells treated with preimmune rabbit serum. In vivo, elevations were found in the percentage of neutrophils in bronchoalveolar lavage fluid (BALF), transport of 99mTc-diethylenetriaminepentaacetate (DTPA) across the tracheal epithelium, and concentrations of total protein and albumin in BALF following O3 exposure. However, these effects were not significantly altered by treatment with the anti-IL-1 alpha/anti-TNF-alpha combination. Therefore, it was concluded that O3 affects the early stages of the inflammatory response, particularly with respect to macrophage activation and adherence to epithelial cells, and that this early response may be mediated by IL-1 alpha and/or TNF-alpha. The results also suggest that the in vivo effects of O3 are controlled by complex mechanisms involving factors other than IL-1 alpha and TNF-alpha, even though these cytokines are capable of modifying macrophage function as revealed by the in vitro adherence studies.

Ozone (O3) exposure of humans and animals induces an inflammatory response in the lung, which is associated with macrophage stimulation, release of chemotactic agents, and recruitment of polymorphonuclear leukocytes (PMNs). This study was designed to investigate the functional aspects of the macrophages that impact inflammatory processes in the lung. Macrophages recovered by bronchoalveolar lavage (BAL) from rats exposed to purified air or 0.8 ppm O3 were studied for their chemotactic activity, adhesive interactions with alveolar epithelial cells in culture, surface morphology, and surface expression of cell adhesion molecules. The macrophages isolated from O3-exposed rats exhibited a greater motility in response to a chemotactic stimulus than the macrophages isolated from rats exposed to purified air. The macrophages from O3-exposed animals also displayed greater adhesion when placed in culture with epithelial cells isolated from adult rat lung (ARL-14) than the macrophages from control rats. Both chemotactic motility and cell adhesion stimulated by O3 exposure were attenuated when the macrophages were incubated in the presence of monoclonal antibodies to leukocyte adhesion molecules, CD11b, or epithelial cell adhesion molecules, ICAM-1. Flow cytometry revealed a modest increase in the surface expression of CD11b but no change in ICAM-1 expression in macrophages from O3-exposed rats when compared to those from the air-exposed controls. The results demonstrate an alteration of macrophage functions following O3 exposure and suggest the dependence of these functions on the biologic characteristics, rather than the absolute expression, of the cell adhesion molecules.

We have investigated the possibility that tumor necrosis factor alpha (TNF) plays a role in the increased airway permeability and an inflammatory response following an acute ozone (O3) exposure. Male Sprague-Dawley rats were injected, intraperitoneally, with either rabbit anti-mouse antibody to TNF (anti-TNF) or preimmune rabbit serum, 2 h before a 3-h exposure to O3 or purified air. Permeability, as determined by [99mTc] diethylenetriamine pentaacetate (DTPA) transport, total protein and albumin concentrations in the bronchoalveolar lavage (BAL), and the inflammatory cell response in the BAL were assessed 10 h after the exposure was completed. The O3-exposed group that was injected with anti-TNF showed a significant decrease in permeability to DTPA in comparison to the O3- exposed group injected with preimmune rabbit serum. There was no difference between the anti-TNF group and the purified air group. In contrast, the total protein and albumin levels in the BAL were significantly greater in both of the O3-exposed groups than in the purified air group. The concentrations of protein and albumin in the anti-TNF group did, however, show an attenuating trend when compared to the preimmune O3-exposed group. The polymorphonuclear leukocytes (PMNs) in BAL of the anti-TNF group also showed an attenuating trend when compared to the preimmune O3-exposed group, but both of these O3-exposed groups were significantly greater than the purified air group. Lung sections stained with naphthol AS-D chloroacetate esterase showed an increase in the number of stained PMNs in the anti-TNF group in comparison to the preimmune O3- and air-exposed groups. These data suggest that TNF plays a role in the increase in tracheal permeability as determined by DTPA transport, while the contributing role that TNF plays in bronchoalveolar permeability and the inflammatory response seen following an acute exposure to 0.8 ppm O3 is less evident.

This study determined the interactive effects of O3 and enzymatically-generated oxidants and antioxidants in the lung. Rats treated with dimethylthiourea (DMTU) or H2O2, generated by glucose/glucose oxidase, were exposed for 2 h to 0.6 or 0.8 ppm O3. A significant increase in the flux of total albumin in the bronchoalveolar lavage (BAL) and a concomitant elevation in the transport of 99mTc-diethylenetriaminepentaacetate (99mTc-DTPA) from trachea to blood occurred after O3 exposure. Pretreatment of rats with DMTU prevented the albumin flux in the BAL. Intratracheal instillation of glucose/glucose oxidase produced a localized response in trachea, but it did not affect the broncho-alveolar permeability. The results demonstrate an additive effect of O3 and an enzymatically-generated oxidant, and an antagonistic effect of an antioxidant in rats exposed to O3. The observations support the suggestion that a balance of oxidant-antioxidant system may be critical in maintaining respiratory integrity following O3 exposure.

Ozone (O3) exposure of rats results in airway epithelial injury and an infiltration of polymorphonuclear leukocytes (PMNs) into the lungs, suggesting alteration of PMN functions. To identify the altered PMN functions and their possible effects on epithelia, rats were exposed to air or 0.8 ppm O3 for 2 hr. PMNs were isolated from the blood and incubated with an epithelial cell line derived from rat lung (ARL-14) or primary alveolar type II cell cultures. The PMNs from the O3-exposed rats exhibited stimulated motility and spontaneous redistribution of actin filaments and adhered in a greater number to the epithelial cells when compared with the PMNs from the air-exposed rats. Actin caps usually formed at the sites of contact between the PMNs and epithelial cells, suggesting a cytoskeletal role in the inflammatory-epithelial cell interaction. By scanning electron microscopy, PMNs from air-exposed rats had features of non-motile cells. In a striking contrast to this, PMNs from O3-exposed rats revealed surface modifications, which were quite prominent at the sites of PMN-epithelial cell contacts. Despite these morphological changes, the PMNs from O3-exposed rats did not alter the epithelial resistance, a measure of paracellular permeability. In contrast to this, PMNs stimulated by phorbol myristate acetate or N-formyl-methionyl-leucyl-phenylalanine not only exhibited greater adhesion to the epithelial cells, but also caused a reduction in epithelial resistance. The changes reflecting altered morphology, motility, and adhesion of PMNs from O3-exposed rats may represent important steps in the O3-induced inflammatory response that precedes barrier disruption in vivo, but they are not associated with increased epithelial permeability in an in vitro system. Besides their mechanistic relevance, the alterations of vascular PMNs may serve as important biomarkers for detecting O3 effects.

Exposure of rats to ozone (O3) produces an increase in airway permeability and a concomitant influx of polymorphonuclear leukocytes in the lung. These observations raise the possibility that the inflammatory cells play a role in the cellular injury and increased airway permeability after O3 exposure. This study was therefore designed to determine if the inflammatory cells or their products are essential for the O3 effect. In a series of experiments, rats were rendered leukopenic with cyclophosphamide, treated with leukotriene B4 (LTB4), or with the inhibitors of lipoxygenase or cyclooxygenase products of arachidonic acid, followed by exposure to O3. A 2-h exposure to 0.8 ppm O3 caused a significant increase in the flux of proteins and albumin in bronchoalveolar lavage (BAL) and elevated the transport of 99mTc-diethylenetriaminepentaacetate (99mTc-DTPA) from trachea to blood. The treatment with cyclophosphamide caused a significant reduction in the circulating and pulmonary leukocytes and prevented an increase in tracheal mucosal permeability to 99mTc-DTPA and the protein and albumin flux in BAL. While the intratracheal instillation of LTB4 did not affect the permeability, tracheal permeability and albumin levels in BAL in rats treated with LTD4 antagonist FPL 55712 and exposed to O3 were lower than in the untreated O3-exposed rats. Pretreatment with indomethacin also prevented the O3 effects, as reflected by the decreased protein and albumin flux in BAL and 99mTc-DTPA transport from trachea to blood. These data show a reduction in the effect of O3 by agents that affect leukocytes or their products. The results support a mechanism of increased permeability that is dependent upon inflammatory cells and their products.

Changes in rat tracheal epithelial permeability and polymorphonuclear leukocyte (PMN) populations during a 24-hr time period following a 3-hr exposure to 0.8 ppm ozone (O3) were investigated. An increase in permeability to 99mTc-diethylene-triaminepentaacetate (DTPA) occurred immediately after the exposure, peaked at the 8-hr time point and decreased to control level by 24 hr. For correlation with tracheal permeability, tracheal cross sections were stained with naphthol AS-D chloroacetate and PMNs were scored by their location as well as staining characteristics (positive or negative). The total PMN population remained at the control value at the 0-, 4-, and 8-hr time points, and increased at 12 hr, followed by a rapid decline to below the control value for the remaining time points. There was a shift at the 8-hr time point in the population location, from the vasculature to the interstitium, which returned to control values at 12 hr. The percentage stained PMNs increased significantly at 16 hr while remaining at control values for all other time points. The data reveal that there is a significant increase in tracheal epithelial permeability immediately after the exposure, but the overall increase in the PMN population is preceded by a lag phase. A decrease in the vascular pool of PMNs concomitant with an increase in the interstitial pool of PMNs suggests their migration from blood to the interstitium after ozone exposure. These data indicate that while PMNs may play a role in permeability changes of tracheal epithelium, the initiation is most likely due to other factors.

Cytoskeletal perturbations and associated changes in transepithelial transport in rat airways were analyzed after in vivo treatment with cytochalasin D or vinblastine or exposure to ozone (O3). Exposure of O3 or cytochalasin D, but not vinblastine, increased permeability in the bronchoalveolar region. Combined treatment with cytochalasin D and O3 did not increase the effect seen with each agent alone. However, treatment with vinblastine plus 0.8 ppm O3 resulted in a slight enhancement of permeability over that seen with O3 alone. This enhancement was not seen with 2 ppm O3. When cytochalasin and vinblastine treatment were combined, a synergistic effect on bronchoalveolar permeability was seen, suggesting participation of both microfilamentous and microtubular cytoskeletal elements in maintaining the integrity of the bronchoalveolar epithelium. Potentially harmful effects of O3 on cytoskeletal elements were confirmed in rat lung epithelial cells in culture. O3 exposure produced reversible changes in microfilamentous structures comparable to those produced by cytochalasin D. The results of these studies support the hypotheses that the cytoskeleton has a central role in maintenance of respiratory epithelial integrity and that a target for O3 toxicity may be the components of cytoskeleton. These results, however, do not rule out the possibility that treatment with cytoskeleton destabilizing drugs leads to the release of mediators, which in turn contribute to the airway epithelial dysfunction and increased permeability.

Exposure of rats to ozone (O3), 0.8 ppm increases the tracheal permeability to 99mTc-diethylenetriaminepentaacetate (99mTc-DTPA) about twofold but to 125I-bovine serum albumin (125I-BSA) to a lesser extent. It is generally believed that exposure to air pollutants causes perturbation of tight junctions and formation of intercellular channels for the passage of molecules from airway lumen to blood. We now report that a second mechanism, vesicular transport, is operative in the transepithelial movement of molecules, that this mechanism is speeded in tracheas of O3-exposed rats, and that there is a concurrent delay in movement of BSA from connective tissue to capillaries after O3 exposure. Horseradish peroxidase (HRP) instilled in trachea was taken up by endocytic vesicles, which could be localized in apical as well as basal regions of ciliated and nonciliated cells. A count of HRP-positive vesicles and measurement of their surface area revealed an approximate twofold increase in O3-exposed rats over that in control animals breathing clean air; this paralleled a twofold increase in transport of 99mTc-DTPA from tracheal lumen to blood. An autophagocytic process induced in tracheal epithelial cells by O3 is proposed. Despite the difference in the size of HRP and BSA, the 2 molecules migrated through common pathways and were colocalized in the luminal membranes as well as in endocytic vesicles and intercellular spaces in double labeling experiments involving simultaneous detection of HRP by cytochemistry and 125I-BSA by autoradiography. This procedure proved particularly useful in detecting a dramatic accumulation of 125I-BSA autoradiographic grains in subepithelial connective tissue and HRP accumulation in intercellular spaces and at the basal membrane-connective tissue junction in O3-exposed rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Permeability of tracheal and bronchoalveolar airways of rats was measured and used to examine the effects of inhaled oxidant-containing atmospheres. The atmospheres studied were (a) ozone (O3) at 0.6 ppm (1.2 mg/m3) or 0.8 ppm (1.6 mg/m3); (b) nitrogen dioxide (NO2) at 6 ppm (11.3 mg/m3) or 12 ppm (22.6 mg/m3); (c) O3 + NO2 at 0.6 ppm (1.2 mg/m3) and 2.5 ppm (4.7 mg/m3), respectively; and (d) a 7-component particle and gas mixture (complex atmosphere) representing urban air pollution in a photochemical environment. The rats were exposed for 2 h. The effects of exercise during exposure were evaluated by exposing additional groups in an enclosed treadmill. Exposure of resting rats to 0.8 ppm O3 increased tracheal permeability to DTPA and bronchoalveolar permeability to diethylenetriamine pentaacetate (DTPA) and bovine serum albumin (BSA) at 1 h after the exposure. Bronchoalveolar, but not tracheal, permeability remained elevated at 24 h after the exposure. Exercise during exposure to O3 increased permeability to both tracers in the tracheal and the bronchoalveolar zones, and prolonged the duration of increased permeability in the tracheal zone from 1 h to 24 h, and in the bronchoalveolar zone from 24 h to 48 h. Permeability in the tracheal and bronchoalveolar zones of rats exposed at rest to 6 or 12 ppm NO2 did not differ from controls. However, rats exposed during exercise to 12 ppm NO2 for 2 h developed a significant increase in tracheal and bronchoalveolar permeability to DTPA and BSA at 1 h, but not at 24 or 48 h, after exposure. Exposure at rest to 0.6 ppm O3 plus 2.5 ppm NO2 significantly increased bronchoalveolar permeability at 1 and 24 h after exposure, although exposure at rest to 0.6 ppm O3 alone increased bronchoalveolar permeability only at 1 h after exposure. Exposure to O3 + NO2 during exercise led to significantly greater permeability to DTPA than did exercising exposure to O3 alone. Resting rats exposed to a complex gas/aerosol atmosphere composed of the above O3 and NO2 concentrations, plus 5 ppm (13.1 mg/m3) sulfur dioxide (SO2) and an aerosol of insoluble colloidal Fe2O3 with an aerosol of manganese, ferric, and ammonium salts, demonstrated increased permeability at 1 and 24 h after exposure. Nitric acid vapor was formed in both the O3 + NO2 atmosphere and the complex gas/aerosol atmosphere.(ABSTRACT TRUNCATED AT 400 WORDS)

Measurements of pulmonary epithelial permeability were made in rats exposed for 2 h to either purified air or ozone (O3) at concentrations of 0.8 or 2 ppm. [99mTc]Diethylenetriaminepentaacetate ([99mTc]DTPA) (492 daltons) and 125I-labeled bovine serum albumin ([125I]BSA) (69,000 daltons) were injected intravenously, and the lungs were lavaged 6 min later. In rats exposed to air, transfer of the larger tracer molecule (BSA) from blood to the lavage fluid was less than that of the smaller molecule (DTPA) when the amount of tracer in the lavage fluid was calculated as percent of the counts in femoral artery blood 5 min after injection, i.e., 1 min prior to lavage. In rats exposed to O3, alveolar permeability increased in a dose-related fashion, the increase under all exposure conditions was greater for the smaller molecule than for the larger one, and the permeability was reversible with time. The increase in permeability from blood to air was comparable to the increase from air to blood reported earlier (Bhalla et al., 1986). The increased permeability provided an early and reliable indicator of short-term O3 exposure effect in rats. Autoradiography by electron microscopy identified multiple pathways for BSA transfer from blood to the alveolar space. Grains produced by [125I]BSA were localized over endothelial and epithelial cell surfaces, were associated with cytoplasmic vesicles, were over cell surface invaginations, and were found in the cytoplasm of apparently degenerating cells. Although defects in tight junctions of alveolar type I cells were observed in lungs of rats exposed to O3, autoradiographic grains also appeared in intercellular spaces, with the intercellular junctions remaining intact.

Ozone (O3) exposure of rats increases airway epithelial permeability. We hypothesized that this increased permeability may be mediated by the epithelial cell cytoskeleton. To test this hypothesis, we studied the effect of cytoskeletal disruption on the transmucosal transport of tracers from airway lumen to blood and compared the results with the effects of O3 exposure. No increase in transport occurred following disruption of microtubules by vinblastine, but disruption of microfilaments with cytochalasin D resulted in increased transport of radiolabeled tracers [99mTc- and 111In-labeled diethylenetriamine-pentacetate (DTPA) and 125I-labeled bovine serum albumin (BSA)]. In control rats, both horseradish peroxidase (HRP) and BSA, localized by cytochemistry and autoradiography, respectively, were detected on the epithelial cell surfaces and in endocytic vesicles. In rats treated with cytochalasin D or exposed to O3, the tracer molecules also penetrated the intercellular spaces, though the apical tight junctions remained devoid of the tracers. Increased numbers of endocytic vesicles containing HRP and aggregation of 125I-labeled BSA autoradiographic grains in the subepithelial region were also seen after either treatment. We conclude that destabilization of cytoskeletal elements following O3 exposure is a possible mechanism of increased transmucosal transport, which may be a combined effect of accelerated transport through both endocytic and paracellular pathways.

Full-length tracheas from Sprague-Dawley rats were exposed to cytoskeleton-active drugs in short-term organ culture, and the permeability of the tracheal epithelium was measured by instilling radiotracers into the lumen and assay of the radioactivity appearing in the external bathing medium. In vitro treatment with cytochalasin D (cyto D, 2-10 x 10(-6) M) increased the rate of movement of [14C]mannitol across the epithelium. Exposure to vinblastine (VB, 10(-4) M) alone had no significant effect. However, VB in combination with cyto D increased the permeability in a dose-dependent manner. In vivo exposure to ozone (O3, 0.8 or 2.0 ppm, 2 h) had only a slight effect on the rate of movement of the tracer as measured in vitro immediately after exposure. At 24 h postexposure there was no significant difference in permeability between ozone- and air-exposed tracheas. Prior in vivo O3 exposure sensitized the tracheas to the in vitro effects of cyto D; treatment of O3-exposed tracheas with cyto D immediately after O3 exposure produced a greater than additive effect on permeability measured in vitro. VB at concentrations up to 10(-4) M had no enhancing effect on permeability in O3-exposed tracheas. Sham exposure to clean air did not affect permeability compared to untreated (shelf) controls. Electron microscopic studies demonstrated penetration of horseradish peroxidase into intercellular spaces in the tracheas treated in vitro with cyto D or cyto D plus VB. Cyto D is known to affect intracellular microfilaments that have attachments at or near the cell surface, while VB affects microtubules associated with internal cellular structures. Therefore, the synergistic effect on tracheal permeability observed with O3 and cyto D, but not with O3 and VB, suggests that O3 may change cell surface structures associated with the microfilamentous cytoskeleton.

This study analyzed rats' nasal, tracheal and bronchoalveolar epithelial permeability to macromolecules after they were exposed, in 2- or 4-hour periods of rest or exercise, to ozone (O3) (0.6, 0.8 or 2 ppm), nitrogen dioxide (NO2) (2.5, 6 or 12 ppm) or formaldehyde (10 ppm). Exercise was performed on a treadmill operated at a speed that led to a 2-fold increase in oxygen consumption. Histopathologic and electron microscopic cytochemical and autoradiographic studies were performed to identify the structural aspects of mucosal response. In rats not exposed to pollutants, the quantity of macromolecular tracers (99mTc-DTPA, 125I-BSA) in blood sampled 6, 7, 8, 9 and 10 minutes after a slow 5-minute instillation of comparable quantities of tracer molecules in the lumen of each zone, was lowest in nasal, highest in tracheal, and intermediate in the bronchoalveolar region. Exposure of resting rats to O3 did not affect nasal permeability, but tracheal and bronchoalveolar permeabilities increased by 2-fold 1 hour after the exposure. In rats exposed at rest to O3, tracheal permeability was no longer elevated 24 hours after exposure, but bronchoalveolar permeability remained elevated at 24 hours after exposure and was normal at 48 hours. Exposure during exercise increased the effect of O3 in the trachea and in the bronchoalveolar zone. However, exercise also prolonged the duration of the O3 effect on the tracheal zone from 1 hour to 24 hours and, in the bronchoalveolar zone, from 24 hours to 48 hours. Histologically, focal inflammatory lesions in the alveolar zone were maximal at 48 hours after a 4-hour resting exposure to O3. After exposure during exercise, the area of lung involved by lesions increased 4- to 7-fold above the lesion-bearing area in rats exposed while resting. By electron microscopy, horseradish peroxidase (HRP) was localized in epithelial intercellular spaces, but not in the apical tight junctions, of tracheal epithelial cells from O3-exposed rats; no HRP was found in intercellular spaces in controls. The number of HRP-containing endocytic vesicles in tracheal epithelial cells was 2-fold greater in rats exposed to O3 than in control rats. This 2-fold increase in vesicles presumed to be transporting HRP matches the 2-fold increase in transfer of DTPA from the tracheal lumen to the blood. Electron microscopic autoradiography revealed 125I-BSA accumulation in subepithelial connective tissue, and electron microscopic cytochemistry identified accumulation of HRP not only between cells but also at the basal lamina.(ABSTRACT TRUNCATED AT 400 WORDS)

Nasal, tracheal and bronchoalveolar injuries resulting from acute ozone exposure of rats were investigated by permeability changes. 99mTc-labeled diethylenetriaminepentaacetate (DPTA) and 125I-labeled bovine serum albumin (BSA) were selectively instilled into localized airway regions of anesthetized rats exposed to 0.8 ppm 03 or clean air for 2 h. Transmucosal transfer of the radiolabeled tracers was detected by counting the radioactivity in blood samples collected at short postinstillation time intervals. Permeability measurements were made on d 0, 1, and 2 after O3 exposure to analyze the extent and persistence of tissue injury in the nasal, tracheal, and bronchoalveolar regions. Normal mucosal permeability was low in nose, intermediate in bronchoalveolar zone, and high in trachea. The O3-related injury, reflected by elevated permeability, was substantial in the trachea and bronchoalveolar zone but was minimal in the nose immediately after the exposure. Abnormal permeability persisted for less than 24 h in the trachea but for more than 24 h in the bronchoalveolar zone. The results are consistent with the properties of O3 of causing greater injury in the smaller airways and the alveolar zone than in the trachea.