Ozone-induced lung injury and sterile inflammation. Role of toll-like receptor 4

Abstract

Inhalation of toxic doses of ozone is associated with a sterile inflammatory response characterized by an accumulation of macrophages in the lower lung which are activated to release cytotoxic/proinflammatory mediators that contribute to tissue injury. Toll-like receptor 4 (TLR4) is a pattern recognition receptor present on macrophages that has been implicated in sterile inflammatory responses. In the present studies we used TLR4 mutant C3H/HeJ mice to analyze the role of TLR4 in ozone-induced lung injury, oxidative stress and inflammation. Acute exposure of control C3H/HeOuJ mice to ozone (0.8ppm for 3h) resulted in increases in bronchoalveolar lavage (BAL) lipocalin 24p3 and 4-hydroxynonenal modified protein, markers of oxidative stress and lipid peroxidation. This was correlated with increases in BAL protein, as well as numbers of alveolar macrophages. Levels of surfactant protein-D, a pulmonary collectin known to regulate macrophage inflammatory responses, also increased in BAL following ozone inhalation. Ozone inhalation was associated with classical macrophage activation, as measured by increased NF-κB binding activity and expression of TNFα mRNA. The observation that these responses to ozone were not evident in TLR4 mutant C3H/HeJ mice demonstrates that functional TLR4 contributes to ozone-induced sterile inflammation and macrophage activation.

Fig. 1

Effects of loss of functional TLR4 on ozone-induced markers of lung injury and inflammation. BAL was collected 0.5–48 hr following exposure of C3H/HeOuJ or C3H/HeJ mice to air or ozone. Upper panel: Protein content was analyzed by the BCA assay with bovine serum albumin as the standard. Each bar represents the mean ± SE (n=14–18). Lower panel: Viable cells were enumerated using trypan blue exclusion. Each bar represents the mean + SE (n=7–11). *Significantly different (p≤0.05) from air control.

Fig. 2

Effects of ozone on markers of oxidative stress. BAL was collected 12–48 hr after exposure of C3H/HeOuJ or C3H/HeJ mice to air or ozone. Upper panel: BAL was analyzed for the presence of lipocalin 24p3 and 4-HNE-modified proteins by western blotting. Note that only a single 50 kDa molecular weight protein was detected in BAL following ozone inhalation. Lower panel: Gels were scanned and analyzed by densitometry. Each bar represents the mean ± SE in (n=3–5). *Significantly different (p≤0.05) from air control.

Fig. 3

Effects of ozone on SP-D levels. BAL was collected 12–48 hr after exposure of C3H/HeOuJ or C3H/HeJ mice to air or ozone. Upper panel: BAL was analyzed for the presence of SP-D by western blotting. Lower panel: Gels were scanned and analyzed by densitometry using ImageJ. Each bar represents the mean ± SE (n=3–5). *Significantly different (p≤0.05) from air control.

Fig. 4

Effects of ozone on NF-κB nuclear binding activity. Alveolar macrophages were isolated 0.5–24 hr following exposure of C3H/HeOuJ or C3H/HeJ mice to air or ozone. NF-κB nuclear binding activity was quantified by EMSA. One representative of three independent experiments is shown. Cold competitor (CC) oligo was incubated with nucleic extracts 2 hr before addition of ³²P-labeled NF-κB consensus oligonucleotide.

Fig. 5

Effects of ozone on TNFα mRNA expression. Alveolar macrophages were isolated 0.5–48 hr following exposure of C3H/HeOuJ or C3H/HeJ mice to air or ozone. TNFα mRNA was quantified by relative quantitative RT-PCR. Upper panel: Representative PCR gel. Lower panel: TNFα was quantified by scintillation counting of excised bands. Each bar represents the mean ± SE (n=3). *Significantly different (p≤0.05) from air control.

Fig. 6

Effects of ozone on proSP-C expression. Lung sections, collected 12–48 hr following exposure of C3H/HeOuJ or C3H/HeJ mice to air or ozone, were stained with anti-proSP-C antibody. Binding was visualized using a Vector DAB peroxidase substrate kit. Slides were counterstained with hematoxylin. Original magnification, 1000x.