An in vitro model system for the study of modulatory effects of ozone on the alveolar macrophage in response to SP-A stimulation

Ozone (O3), a major component of air pollution and a very strong oxidizing agent, is known to affect various aspects of the lung defense mechanism. The resulting ozone-induced changes in lung physiology are manifested as an overall increase in inflammatory response that can lead to lung injury. Ozone-induced lung injury is associated with edema, inflammation, and epithelial cell damage as well as surfactant derangement. The observed inflammatory process is a result of a complex interaction of various components of the innate immunological system. The effects of ozone on pulmonary immune cells have been studied by various in vivo and in vitro systems. Specific aim 1 of this thesis defines a model system of cells of monocyte/macrophage lineage (THP-1 cells) exposed to ozone in vitro, by studying cell viability, cell proliferation and cell surface marker expression. THP-1 cells exposed to ozone in concentrations ranging from 0.1 ppm to 0.5 ppm for 1h, were analyzed for cell viability and apoptosis (Annexin V/7-Amino-actinomycin D flow cytometric assay) either immediately after ozone exposure or at later time points. This analysis showed absence of apoptosis and a small decrease in cell viability (5--17%) in ozone exposed THP-1 cells. In addition, ozone did not induce a significant proliferation in THP-1 cells. Cell surface protein expression (CD14 and CD11b), did not change following ozone exposure, but the effect of LPS on TNF-alpha production following ozone exposure changed compared to filtered air-exposed cells. These findings indicate that this in vitro ozone cell-exposure system may be used in studies where the effects of various agents (physiological and non-physiological) on phagocytic cells can be analyzed. This model system offers conditions where the experimental results are not due to cell death, but rather due to the effects of ozone and/or agents under investigation.; Specific aim 2 of this thesis describes effects of ozone on alveolar macrophages and surfactant protein A (SP-A) as well as on their interaction. We exposed to ozone cells of monocyte/macrophage lineage (THP-1 cells) and/or native SP-A obtained from bronchoalveolar lavage of patients with alveolar proteinosis, and studied cytokine production and NF-kappaB signaling. The results showed: (1) Exposure of THP-1 cells to ozone significantly decreased their ability to express TNF-alpha in response to SP-A; TNF-alpha production, under these conditions, was still significantly higher than basal (unstimulated) levels in filtered air (FA)-exposed THP-1 cells; (2) Exposure of both, THP-1 cells and SP-A, to ozone did not result in any significant differences in TNF-alpha expression compared to basal levels (i.e. FA exposed and untreated cells), although each, ozone exposure or SP-A stimulation, altered cytokine levels, compared to basal levels; (3) ozone exposure of SP-A resulted in a decreased ability of SP-A to activate the NF-kappaB signaling pathway, as assessed by the lack of significant increase and decrease of nuclear p65 subunit of NF-kappaB and cytoplasmic IkappaB, respectively; (4) ozone exposure of THP-1 cells, on the other hand, resulted in a decrease in S-P-A mediated responsiveness and this decrease did not seem to be mediated via the NF-kappaB pathway. These findings indicate that ozone exposure may mediate its effect on macrophage function both directly and indirectly (via SP-A oxidation) and by involving different mechanisms.