Hyaluronan mediates ozone-induced airway hyperresponsiveness in mice

Abstract

Ozone is a common urban environmental air pollutant and significantly contributes to hospitalizations for respiratory illness. The mechanisms, which regulate ozone-induced bronchoconstriction, remain poorly understood. Hyaluronan was recently shown to play a central role in the response to noninfectious lung injury. Therefore, we hypothesized that hyaluronan contributes to airway hyperreactivity (AHR) after exposure to ambient ozone. Using an established model of ozone-induced airways disease, we characterized the role of hyaluronan in airway hyperresponsiveness. The role of hyaluronan in response to ozone was determined by using therapeutic blockade, genetically modified animals, and direct challenge to hyaluronan. Ozone-exposed mice demonstrate enhanced AHR associated with elevated hyaluronan levels in the lavage fluid. Mice deficient in either CD44 (the major receptor for hyaluronan) or inter-alpha-trypsin inhibitor (molecule that facilitates hyaluronan binding) show similar elevations in hyaluronan but are protected from ozone-induced AHR. Mice pretreated with hyaluronan-binding peptide are protected from the development of ozone-induced AHR. Overexpression of hyaluronan enhances the airway response to ozone. Intratracheal instillation of endotoxin-free low molecular weight hyaluronan induces AHR dependent on CD44, whereas instillation of high molecular weight hyaluronan protects against ozone-induced AHR. In conclusion, we demonstrate that hyaluronan mediates ozone-induced AHR, which is dependent on the fragment size and both CD44 and inter-alpha-trypsin inhibitor. These data support the conclusion that pulmonary matrix can contribute to the development of airway hyperresponsiveness.

FIGURE 1.

Ozone and hyaluronan in BALF. A, total lung lavage protein 24 h after either air- or ozone-exposed mice of all strains. B, lung lavage fluid hyaluronan level 24 h after either air or ozone exposure. C, agar gel, electrophoresis of concentrated lung lavage fluid hyaluronan, visualized with staining with Stains-All (Sigma). Lane 1, high molecular weight hyaluronan ladder. Lane 2, low molecular weight hyaluronan ladder. Lane 3, high molecular weight hyaluronan (Healon). Lane 4, sonicated Healon. Lane 5, C57BL/6, ozone-exposed. Lane 6, IaI wild-type ozone-exposed. Lane 7, IaI-deficient, ozone-exposed. Lane 8, CD44-deficient, ozone-exposed. Lane 9, HAS2 transgenic, ozone-exposed. Lane 10, representative free air exposed lavage for all strains (^*, p < 0.001, air versus ozone; #, p < 0.01 compared with C57BL/6/ozone).

FIGURE 2.

Hyaluronan and CD44 immunohistochemistry after air or ozone exposure. Expression and localization of hyaluronan (green) and CD44 (red) were identified immunohistochemically in naive (A and B) and ozone-exposed (C and D) mouse lungs. A, C57BL/6, air-exposed; B, CD44^-/-, air-exposed; C, C57BL/6, ozone-exposed; D, CD44^-/-, ozone-exposed. Hyaluronan is faintly visible in the subepithelial space in air-exposed mice (small arrows) but is more visible after ozone exposure (big arrows). CD44 (red) is localized in bronchial epithelial cells and macrophages (arrowheads). ×400 magnification.

FIGURE 3.

Airway staining of hyaluronan and CD44 after ozone exposure. In a higher magnification merged image, hyaluronan (green) is found adjacent to the basal membrane below bronchial epithelia (red) as well as surrounding subepithelial myocytes (small arrows). ×600 magnification.

FIGURE 4.

Macrophage staining of hyaluronan and CD44 after ozone exposure. C57BL/6J mice were exposed to either air or ozone and evaluated by immunohistochemistry for cellular distribution of CD44 and hyaluronan. A, after air exposure, alveolar macrophages (arrowheads) stain positive for CD44 (red) but not for hyaluronan (green). B, after ozone exposure, hyaluronan (green) and CD44 (red) colocalize on alveolar macrophages (arrows), yielding an orange appearance on merged image. ×600 magnification.

FIGURE 5.

Ozone-induced airway hyperresponsiveness. A, CD44 and IaI are essential for the development of ozone-induced AHR (^*, p < 0.01 compared with CD44- and IaI-deficient). B, IaI but not UTI/bikunin injection reconstitutes AHR in IaI-deficient mice (^*, p < 0.05 compared with other groups).

FIGURE 6.

HABP effect on CD44-hyaluronan binding after ozone exposure. Confocal immunohistochemistry shows staining for hyaluronan (green, far left panels), CD44 (red, left middle panels), or merged images (right middle panels), and differential interference contrast images (right panels). A, saline-pretreated mouse lungs demonstrate significant hyaluronan and CD44 colocalization (yellow in merged image). B, scrambled peptide pretreated mouse lungs also show significant hyaluronan staining and colocalization with CD44. C, HABP-pretreated mouse lungs demonstrate significantly decreased hyaluronan staining on macrophages. ×630 magnification. There is no difference in hyaluronan or CD44 staining in respective air controls (far right panels).

FIGURE 7.

Blockade of hyaluronan attenuates ozone-induced AHR. A, treatment with vehicle, SBP, or hyaluronan-binding peptide does not alter ozone-induced increases in total protein in the lung lavage. B, treatment with vehicle, SBP, or HABP does not affect AHR in air-exposed animals. C, hyaluronan-binding protein, but not scrambled protein, significantly decreased AHR after ozone exposure (HABP versus other groups ^*, p < 0.05).

FIGURE 8.

Overexpression of hyaluronan enhances ozone-induced AHR. A, animals, which overexpress HAS2 in airway epithelia, have similar levels of total protein in the lung lavage after exposure to ozone. B, both strains of mice have increased levels of soluble hyaluronan after exposure to ozone when compared with air exposure. HAS2 transgenic mice have significantly more soluble hyaluronan when compared with littermate controls (^*, p < 0.01). C, HAS2 overexpressing animals are no different from littermate controls after exposure to filtered air but have enhanced AHR response after exposure to ozone (^*, p < 0.01 compared with all other groups).

FIGURE 9.

Airway hyperresponsiveness in mice after intratracheal instillation of hyaluronan. A, sHA but not HMW-HA or vehicle induces AHR in naive C57BL/6 mice (^*, p < 0.05 compared with other groups; #, p < 0.05 compared with HMW-HA). B, response to sHA is dose-dependent. Both low dose (25 μg, 0.5 mg/ml) and high dose (87.5 μg, 3.5 mg/ml) sHA induce AHR. C, CD44-deficient mice are resistant to sHA-induced AHR compared with C57BL/5 mice (^*, p < 0.05, sHA treated C57 versus sHA treated CD44^-/-). D, instillation of HMW-HA but not vehicle before and after ozone exposure to ozone significantly ameliorates AHR (^*, p < 0.01 vehicle versus HMW-HA; #, p < 0.05, vehicle versus HMW-HA).

FIGURE 10.

Lung lavage cells in ozone-treated mice. A, 24 h after ozone exposure IaI (vertical stripes) and CD44 (horizontal stripes) deficiency leads to significantly decreased numbers of inflammatory cells in the lung lavage fluid, which are mostly macrophages (top panel), with a few neutrophils (bottom panel). Injection of IaI-deficient mice with IaI (bold cross-stripes) but not equimolar bikunin (fine cross-stripes) reconstitutes the C57BL/6 phenotype. (^*, p < 0.001 compared with C57BL/6 and IaI-deficient + IaI, Bonferroni multiple comparisons testing.) B, instillation of HABP (right hatched) but not scrambled SBP (left hatched) reduces lavage cells, which are mostly macrophages (top panel) with a few neutrophils (bottom panel) (^*, p < 0.01 compared with saline and SBP-treated). C, HAS2 transgene-positive animals (hatched) have decreased lung lavage cells after ozone exposure compared with control littermates. The difference is because of macrophages (top panel) (^*, p < 0.05 compared with HAS2 transgenic ozone-exposed).