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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.

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.

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.

Pulmonary macrophages (PM) play a key role in the immune defenses of the lung. When stimulated, PM express Fc receptors (FcR) that regulate the immune response. PM were assayed for FcR expression following subchronic inhalation exposure of adult Fischer 344 rats to either 90 micrograms/m3 nitrate (NH4NO3), 300 micrograms/m3 road dust, or clean air, for 4 h/day, 4 days/week, for 8 weeks. PM were lavaged from the lungs and attached to glass coverslips for 18 h. PM FcR were labelled with rat IgG conjugated with cyanine-3. For each exposure, FcR were determined with a Meridian ACAS 570 confocal cytometer by imaging the fluorescence of 50 cells. We found that the IgG binding to FcR (in arbitrary fluorescence units, FU, per cell) for PM from road dust exposed rats was less (835 +/- 39.3 FU/cell) than that for PM from both ammonium nitrate or clean air-exposed rats (1115 +/- 58.0 FU/cell and 1123 +/- 46.6 FU/cell, respectively). While acid incubation conditions in vitro (pH 5.5 for 30 min to simulate the acid environment of ammonium nitrate inhalation) resulted in a 16% decrease in IgG binding (P < 0.05), IgG binding to PM from acid aerosol exposed rats was no different than the IgG bound to PM from clean air-exposed rats. PM exposed to road dust in vivo expressed 25% fewer FcR (P < 0.05). Three-dimensional images of PM failed to show any major alterations in FcR distribution. These preliminary results indicate cellular recognition of antibody-immune complexes may be impaired by subchronic exposure to road dust, which could decrease the immune response of road dust exposed animals.

Intimate interaction occurs between M cells which take up antigens in Peyer's patch epithelium and underlying lymphocytes but the migratory pathway and subtypes of these lymphocytes have remained uncertain. Lymphocyte homing to Peyer's patches was investigated by separating mouse splenic B and T lymphocytes, labeling with [3H]adenosine, reinjecting into syngeneic mice, and localizing them by autoradiography after 17, 40, and 68 hr. Both B and T lymphocytes migrated across postcapillary venules through intercellular spaces. Most labeled cells of both types localized around postcapillary venules in the initial time period, then migrated through the lymphoid mass to follicle epithelium. T-cell number in follicle epithelium peaked at 17 hr but B cells peaked at 40 hr. At each time point more labeled B than T lymphocytes were found below M cells and in follicle epithelium but the differences were only significant (P less than 0.01) at 40 hr. Endogenous B lymphocytes, identified by antibody staining, were also associated with M cells, possibly reflecting a role for secretory antibody in modulating antigen uptake.