Deepak K. Bhalla

Alteration of epithelial integrity, alkaline phosphatase activity, and fibronectin expression in lungs of rats exposed to ozone.

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.

Attenuation of ozone-induced lung injury by interleukin-10

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.

Alteration of alveolar macrophage chemotaxis, cell adhesion, and cell adhesion molecules following ozone exposure of rats

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.

The influence of polymorphonuclear leukocytes on altered pulmonary epithelial permeability during ozone 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.

In Utero Tobacco Smoke Exposure Alters Pulmonary Responses of Newborn Rats to Ozone

Prenatal tobacco smoke (TS) exposure has been implicated in various adverse health outcomes in the offspring, including poor development of lung and immune system, which in turn can alter the response of neonates to environmental challenges. This study was performed to determine whether in utero exposure to TS influences the pulmonary response of newborn rat pups to ozone (O3). Timed pregnant Sprague-Dawley (SD) rats were exposed to TS or air for 3 h/d from gestation d 7 through 21. The pulmonary response of pups was assessed following a single 3-h exposure to air or 0.6 ppm O3 on d 13 after birth. In all, 4 exposure groups were evaluated: (1) Air/Air (in utero air and postnatal air), (2) Air/O3 (in utero air and postnatal O3), (3) TS/Air (in utero TS and postnatal air), and (4) TS/O3 (in utero TS and postnatal O3). Bronchoalveolar lavage (BAL) was performed, and BAL cells and fluid were analyzed. Data revealed a significant increase in polymorphonuclear leukocytes (PMN) and total BALF protein in the Air/O3 group compared to the Air/Air control, reflecting the inflammatory and cytotoxic effects of O3. However, in utero exposure to TS attenuated PMN infiltration into the air spaces for recovery in the BAL of TS/O3 pups. Lung tissue myeloperoxidase activity significantly increased only in the TS/O3 group but not in Air/O3 pups, thus suggesting that PMN are sequestered in the lung tissue and that the in utero TS likely inhibits O3-mediated influx of PMN into the air spaces. Lung tissue analyses further showed a significant rise in manganese superoxide dismutase (SOD) protein and a decrease in extracellular SOD protein in the Air/O3 group, suggesting oxidative effects of O3. Interestingly, in utero TS exposure again suppressed these effects in the TS/O3 group. Overall, results suggest that in utero exposure to TS alone produced minimal acute pulmonary effects in newborn rats, but modulated adverse effects of postnatal O3 exposure. The results are contrary to the interactive toxic responses predicted for sequential exposures to TS and O3, and may represent the development of “cross-tolerance.”

Toxicity of chemical components of fine particles inhaled by aged rats: effects of concentration

Abstract This study tested the hypothesis that exposure to mixtures containing fine particles and ozone (O3) would cause pulmonary injury and decrements in functions of immunological cells in exposed rats (22–24 months old) in a dose-dependent manner. Rats were exposed to high and low concentrations of ammonium bisulfate and elemental carbon and to 0.2 ppm O3. Control groups were exposed to purified air or O3 alone. The biological end points measured included histopathological markers of lung injury, bronchoalveolar lung fluid proteins, and measures of the function of the lung’s innate immuno-logical defenses (macrophage antigen-directed phagocytosis and respiratory burst activity). Exposure to O3 alone at 0.2 ppm did not result in significant changes in any of the measured end points. Exposures to the particle mixtures plus O3 produced statistically significant changes consistent with adverse effects. The low-concentration mixture produced effects that were statistically significant compared to purified air but, with the exception of macrophage Fc receptor binding, exposure to the high-concentration mixture did not. The effects of the low- and high-concentration mixtures were not significantly different. The study supports previous work that indicated that particle + O3 mixtures were more toxic than O3 alone.

Inflammation and Fibrosis

Inflammatory cells represent an important component of the pulmonary defenses, but they also play a critical role in the pathogenesis of lung disorders such as adult respiratory distress syndrome (Patterson et al., 1989), asthma (Bigby and Nadel, 1988), silicosis (Lugano et al., 1984; Sjostrand et al., 1991; Li et al., 1992), interstitial pulmonary fibrosis, and asbestosis (Sibille and Reynolds, 1990; Rochester and Elias, 1993; Gee and Mossman, 1995). Because of the generally recognized adverse effects of airway inflammation and contribution of inflammatory cells to lung fibrosis, the factors involved in inflammatory reactions, mechanisms of toxicity and development of chronic lung disease have received considerable attention. The initial steps in the cascade of events, that ultimately result in the development of chronic lung disease, include stimulation of resident cells, release of chemotactic agents and recruitment of inflammatory cells. These events can be set in motion by an intrapulmonary insult in the form of an acute or chronic inhalation exposure to either environmental or occupational pollutants.

Tobacco smoke modulates ozone-induced toxicity in rat lungs and central nervous system

Adult Sprague-Dawley (SD) male rats were exposed for a single 3 h period to air, ozone (O3) or O3 followed by tobacco smoke (O3/TS). For pulmonary effects, bronchoalveolar lavage (BAL) cells and fluid were analyzed. Data revealed a significant increase in polymorphonuclear leukocytes (PMN), total protein and albumin concentrations in the O3 group, reflecting inflammatory and toxic responses. A subsequent exposure to TS attenuated PMN infiltration into the airspaces and their recovery in the BAL. A similar reduction was observed for BAL protein and albumin in the O3/TS group, but it was not statistically significant. We also observed a significant increase in BAL total antioxidant capacity following O3 exposure, suggesting development of protective mechanisms for oxidative stress damage from O3. Exposure to TS attenuated the levels of total antioxidant capacity. Lung tissue protein analysis showed a significant reduction of extracellular superoxide dismutase (EC-SOD) in the O3 or O3/TS group and catalase in the O3/TS group. TS further altered O3-induced EC-SOD and catalase protein expression, but the reductions were not significant. For effects in the central nervous system (CNS), we measured striatal dopamine levels by HPLC with electrochemical detection. O3 exposure produced a nonsignificant decrease in the striatal dopamine content. The effect was partially reversed in the O3/TS group. Overall, the results show that the toxicity of O3 in the lung is modulated by TS exposure, and the attenuating trend, though nonsignificant in many cases, is contrary to the synergistic toxicity predicted for TS and O3, suggesting limited cross-tolerance following such exposures.