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Abstract
Background
Particulate matters (PM) when inhaled is known to induce pulmonary diseases including asthma and chronic bronchitis when inhaled. Despite the epidemiological proofevidence, the pathogenesis of PM-related pulmonary diseases is unclearremain poorly understood.
Methods
Primary alveolar macrophages were harvested from the SPF and inflammatory rats by bronchioalveolar lavage (BAL). The cultured primary alveolar macrophages were treated with the medium only, PM only (5~40µg/cm2), LPS (5ng/ml) only, and PM with LPS for 24 and 48 hours. The level of secreted nitric oxide (NO) was assayed from the cultured medium by using the Griess reaction. The cultured cells were utilized for the western blotting against the inducible nitric oxide synthase (iNOS) proteins. Immunocyto- chemical staining against the iNOS and NT-proteins were performed in cells that cultured in the Lab-Tek® chamber slide after treatments.
Results
The PM that utilizein this experiments induced NO formation with iNOS expression in the cultured SPF and inflammatory rats alveolar macrophages, by itself. When the cells were co-treated with PM and LPS, there was a statistically significant synergistic effect on NO formation and iNOS expression over the LPS effect. The cells from the sham control showed minimal immunoreactivity for the NT-proteins. Significantly higher quantities of NT-proteins were detected in the PM and PM with LPS co-treated cells than from the sham control.
Figures and Tables
Figure 1
Composition and morphology of BAL cells (×400). Diff-Quick stained alveolar macrophages from the SPF rat (a) and from the inflammatory rat (b).
Figure 2
Figure 2A. Treatment-response relation for NO2- formation in cultured SPF rat alveolar macrophages at 24 hours. Cells were cultured with medium only (sham control), LPS (5ng/ml), various concentrations of PM (5, 10, 20µg/cm2) and various concentrations of PM with LPS.
*P<0.05 versus LPS; †P<0.01 versus control. Mean ± SEM of four independent experiments per category (each experiment's n=3).
Figure 2B. Treatment-response relation for NO2- formation in cultured SPF rat alveolar macrophages at 48 hours. Cells were cultured with medium only (sham control), LPS (5ng/ml), various concentrations of PM (5, 10, 20µg/cm2) and various concentrations of PM with LPS.
**P<0.01 versus LPS; †P<0.01 versus control. Mean ± SEM of four independent experiments per category (each experiment's n=3).
Figure 3
Time-response relation for NO2- formation in cultured SPF rat alveolar macrophages. Cells were cultured with medium only (control), LPS only, PM only and PM with LPS for 12-48 hours.
*P<0.05, **P<0.01 versus LPS; †P<0.01 versus control. Mean ± SEM of four independent experiments per category.
Figure 4
Treatment-response relation for NO2- formation in cultured inflammatory BAL cells. Cells were cultured with medium only, LPS (10ng/ml) only and various concentrations of PM (5~20µg/cm2) for 24 hours and 48 hours.
*P<0.05, **P<0.01 versus control. Mean ± SEM of four independent experiments per category.
Figure 5
Immunocytochemical stain for iNOS in the cultured rat alveolar macrophages (×400). Cells were cultured with (a) medium only, (b) LPS (5ng/ml) only, (c) PM(20µg/cm2) only and (d) LPS with PM for 48 hours.
Figure 6
Immunocytochemical stain for nitrotyrosiliated-protein (NP) in cultured rat alveolar macrophages (×400). Cells were cultured with (a) medium, (b) LPS (5ng/ml), (c) PM (20µg/cm2) and (d) LPS with PM (20µg/cm2) for 48 hours.
Figure 7
Western blot for iNOS in cultured rat alveolar macrophages. Cells were cultured with medium only, LPS only, PM only and PM with LPS for 24 hours. The level of β-actin was similar in all of the tested samples.
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