Characterization of acute ozone-induced leukopenia through flow cytometric quantification of immune cells
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Acute ozone (O3) inhalation concurrently increases circulating stress hormones, corticosterone and epinephrine, and decreases leukocytes (leukopenia) in rats. These two biological events correlate with the activation of the neuroendocrine stress response. We hypothesized that glucocorticoid-induced apoptosis in circulating lymphocytes was responsible for O3-induced leukopenia. To examine the dynamic changes in circulating leukocyte subpopulations we quantified live, apoptotic (early and late) and necrotic granulocytes, monocytes (classical and non-classical), and lymphocytes (B and T [T helper and T cytotoxic]) using flow cytometry (n=8) in 12-13 weeks old male WKY rats exposed to air or O3 (0.0, 0.4 and 0.8 ppm) for (30 min, 1h, 2h and 4h). To evaluate if circulating changes correlated with those taking place in immunological organs where maturation of leukocytes occurs, thymus and spleen cell populations were also analyzed. Generally, for those leukocyte subpopulations affected by O3 exposure, the responses were dose and time dependent. Circulating lymphocyte count was significantly decreased after O3 exposure (0.8 ppm, 2h and 4h). At 4h, this decrement was more extensive in B than in T cells (~50% and ~20% [for both T helper and T cytotoxic]). Circulating classical monocytes, also known as inflammatory monocytes, decreased after 4h of exposure (~60% and ~80% at 0.4 and 0.8 ppm respectively). Circulating granulocytes increased after 30 min of O3 exposure (at both 0.4 and 0.8 ppm) but tended to decrease after 2h of exposure in 0.8 ppm O3-exposed rats suggesting a rapid mobilization of these cells into peripheral organs. No significant ozone-induced changes were detected in cell populations for thymus and spleen at any time point. Also, the assessment of cells undergoing apoptosis or necrosis yielded no significant alterations in blood, thymus or spleen. Our results indicate that O3 -induced leukopenia is not mediated by cell death of lymphocytes or any other subpopulation, but rather by the mobilization of such cells. This study provides insights on how O3 exposure favored the decrease of specific leukocytes subpopulations such as classical monocytes and B lymphocytes that could explain the nature of O3 -induced immune effects in the respiratory system (This abstract does not reflect the US EPA policy).