Increased Nitric Oxide Production Prevents Airway Hyperresponsiveness in Caveolin-1 Deficient Mice Following Endotoxin Exposure

Abstract

Background: Caveolin-1, the hallmark protein of caveolae, is highly expressed within the lung in the epithelium, endothelium, and in immune cells. In addition to its classical roles in cholesterol metabolism and endocytosis, caveolin-1 has also been shown to be important in inflammatory signaling pathways. In particular, caveolin-1 is known to associate with the nitric oxide synthase enzymes, downregulating their activity. Endotoxins, which are are composed mainly of lipopolysaccharide (LPS), are found ubiquitously in the environment and can lead to the development of airway inflammation and increased airway hyperresponsiveness (AHR).

Methods: We compared the acute responses of wild-type and caveolin-1 deficient mice after LPS aerosol, a well-accepted mode of endotoxin exposure, to investigate the role of caveolin-1 in the development of environmental lung injury.

Results: Although the caveolin-1 deficient mice had greater lung inflammatory indices compared to wild-type mice, they exhibited reduced AHR following LPS exposure. The uncoupling of inflammation and AHR led us to investigate the role of caveolin-1 in the production of nitric oxide, which is known to act as a bronchodilator. The absence of caveolin-1 resulted in increased nitrite levels in the lavage fluid in both sham and LPS treated mice. Additionally, inducible nitric oxide synthase expression was increased in the lung tissue of caveolin-1 deficient mice following LPS exposure and administration of the potent and specific inhibitor 1400W increased AHR to levels comparable to wild-type mice.

Conclusions: We attribute the relative airway hyporesponsiveness in the caveolin-1 deficient mice after LPS exposure to the specific role of caveolin-1 in mediating nitric oxide production.

Keywords: Airway hyperresponsiveness; Caveolin-1; Cytokines; Lipopolysaccharide; Lung injury; Nitric oxide.

Figure 1

Lung inflammation in WT and cav-1^−/− mice following sham or LPS exposure BAL was collected 4 hours after LPS aerosol exposure (cav-1^−/− LPS and WT LPS). Cav-1^−/− (cav-1^−/− sham) and WT (WT sham) mice were exposed to PBS as a sham treatment. A) Total cell counts were determined from BAL fluid of cav-1^−/− and WT mice. B) Cell differentials were determined from H&E stained cytospins of cells recovered from BAL fluid. C) Total protein in the BAL was determined by BCA (***, p < 0.001, *, p < 0.05, n = 8 - 12).

Figure 2. Inflammatory infiltration into lung tissue following LPS treatment

Following BAL, lungs were inflated with 4% formaldehyde by gravity. Paraffin embedded sections were cut and stained with H&E. Sham treated cav-1^−/− mice (cav-1^−/− sham) have a baseline pathology of increased cellularity (non-inflammatory) compared to WT (WT sham). Following LPS treatment, perivascular and peribronchiolar infiltrates are found in WT mice (WT LPS) and this inflammation is increased in cav-1^−/− mice (cav-1^−/− LPS). Representative images taken at 10X by light microscopy and are representative of sections from three mice per group. The scale bar is 100 μm in all photomicrographs.

Figure 3. Levels of proinflammatory cytokines and chemokines in the BAL of WT and cav-1^−/− mice

Cytokine and chemokine levels in the BAL were measured by Multiplex. Cav-1^−/− mice (cav-1^−/− LPS) had increased cytokine and chemokine levels following LPS exposure including A) MIP-1α, B) IL-6, C) IL-1β, and D) TNF-α in their BAL compared to WT mice (WT LPS). There were no differences in cytokine and chemokine levels in the BAL of PBS treated WT and cav-1^−/− mice (WT sham and cav-1^−/− sham), which were used as controls (***, p < 0.001, **, p < 0.01, n = 8 – 11).

Figure 4. AHR following LPS aerosol exposure

Total airway resistance (R_T) of wild type (WT LPS) and cav-1^−/− (cav-1^−/− LPS) mice to increasing doses of aerosolized Mch was measured using the flexi-Vent 4 hours after the initiation of LPS aerosol exposure. As a control, WT (WT sham) and cav-1^−/− mice (cav-1^−/− sham) were exposed to aerosolized PBS (***, p < 0.001 WT LPS vs. cav-1^−/− LPS and cav-1^−/− LPS vs. cav-1^−/− sham, *, p < 0.05 cav-1^−/− LPS vs. cav-1^−/− sham, n = 12 - 15).

Figure 5. Nitrite levels in the BAL and iNOS expression in lung tissue of WT and cav-1^−/− mice

BAL and lung tissue were collected 4 hours after LPS aerosol exposure (cav-1^−/− LPS and WT LPS). Cav-1^−/− (cav-1^−/− sham) and WT (WT sham) mice were exposed to PBS as a sham treatment. A) Nitrite levels in the BAL were assessed by the Greiss assay. Data is reported as fold change over WT sham. (*, p < 0.05, n = 11 - 14) Total RNA was extracted from 1 lung lobe and B) iNOS expression was assessed using quantitative real-time PCR analysis. The data were normalized to the WT sham group within each individual experiment before being combined (**, p < 0.01, n = 7 - 14). C) Lung tissue homogenate from WT sham(*,p<0.05, n = 11-14) treated mice was subjected to density separation on a sucrose gradient and the resulting fractions were immunoblotted for iNOS and cav-1. A representative blot is shown.

Figure 6. AHR in WT and cav-1^−/− mice following treatment with 1400W and LPS exposure

Total airway resistance (R_T) was measured as before in WT and cav-1^−/− mice treated with saline or LPS (WT sham, cav-1^−/− sham, WT LPS, cav-1^−/− LPS) and the same trends were observed as were reported in Figure 3. In addition, R_T of wild type (WT LPS 1400W) and cav-1^−/− (cav-1^−/− LPS 1400W) mice to increasing doses of aerosolized Mch was measured using the flexiVent 4 hours after the initiation of LPS aerosol exposure and IP administration of the iNOS specific inhibitor, 1400W. As a control, WT (WT sham 1400W) and cav-1^−/− mice (cav-1^−/− sham 1400W) were exposed to aerosolized PBS and were given IP injections of 1400W (***, p < 0.001 cav-1^−/− LPS + 1400W vs. cav-1^−/− LPS, cav-1^−/− LPS + 1400W and WT LPS + 1400W, and WT LPS vs. WT LPS + 1400W, **, p < 0.01 cav-1^−/− sal + 1400W vs. cav-1^−/− saline, *, p < 0.05 cav-1^−/− LPS + 1400W vs. WT LPS + 1400W, n = 6 - 11).