Susceptibility to ozone-induced airway inflammation is associated with decreased levels of surfactant protein D

Abstract

Background: Ozone (O3), a common air pollutant, induces exacerbation of asthma and chronic obstructive pulmonary disease. Pulmonary surfactant protein (SP)-D modulates immune and inflammatory responses in the lung. We have shown previously that SP-D plays a protective role in a mouse model of allergic airway inflammation. Here we studied the role and regulation of SP-D in O3-induced inflammatory changes in the lung.

Methods: To evaluate the effects of O3 exposure in mouse strains with genetically different expression levels of SP-D we exposed Balb/c, C57BL/6 and SP-D knockout mice to O3 or air. BAL cellular and cytokine content and SP-D levels were evaluated and compared between the different strains. The kinetics of SP-D production and inflammatory parameters were studied at 0, 2, 6, 12, 24, 48, and 72 hrs after O3 exposure. The effect of IL-6, an O3-inducible cytokine, on the expression of SP-D was investigated in vitro using a primary alveolar type II cell culture.

Results: Ozone-exposed Balb/c mice demonstrated significantly enhanced acute inflammatory changes including recruitment of inflammatory cells and release of KC and IL-12p70 when compared with age- and sex-matched C57BL/6 mice. On the other hand, C57BL/6 mice had significantly higher levels of SP-D and released more IL-10 and IL-6. Increase in SP-D production coincided with the resolution of inflammatory changes. Mice deficient in SP-D had significantly higher numbers of inflammatory cells when compared to controls supporting the notion that SP-D has an anti-inflammatory function in our model of O3 exposure. IL-6, which was highly up-regulated in O3 exposed mice, was capable of inducing the expression of SP-D in vitro in a dose dependent manner.

Conclusion: Our data suggest that IL-6 contributes to the up-regulation of SP-D after acute O3 exposure and elevation of SP-D in the lung is associated with the resolution of inflammation. Absence or low levels of SP-D predispose to enhanced inflammatory changes following acute oxidative stress.

Figure 1

A decrease in BAL SP-D levels was associated with significantly increased inflammation in Balb/c mice 6 hrs after O3 exposure. Groups of Balb/c (grey bars) and C57BL/6 (black bars) mice were exposed to O₃ or room air for 2 hrs and BAL was performed 6 hrs later. (A) Ozone exposed Balb/c mice had significantly reduced SP-D levels as compared to air exposed Balb/c or O₃ exposed C57BL/6 mice. SP-D was detected by Western blot analysis of the cell-free supernatant of the BAL (top panel) and was performed using our in-house rabbit polyclonal anti-SP-D antibody. Two representative samples out of a total of six are shown in each group. SP-D expression was quantified by densitometric analysis. Results are expressed as % of naïve control levels. * p = 0.0249 vs. room air; p = 0.0027 vs. C57BL/6. (B) Balb/c mice had significantly higher numbers of inflammatory cells in their BAL as compared to C57BL/6 mice. Cells were counted using a Coulter counter and results are expressed as cell number/ml (*p = 0.0316). (C): The BAL total protein content was significantly higher in Balb/c mice compared to C57BL/6 mice. Total protein was measured by Bradford assay in the cell-free supernatant *p = 0.028. Absolute protein contents were 919,6 (± 51,4) and 3262,3 (± 281.0) in air and O₃ exposed Balb/c mice, respectively, and 772 (± 26.4) and 2110 (± 36.4) in air and O₃ exposed C57BL/6 mice, respectively.(D-G): Cytokine expression was studied by Endogen^®SearchLight™ and Luminex^®100™ technologies. O₃ induced the release of IL-12p70 (34 pg/ml ± 4 in Balb/c, 18.7 pg/ml ± 1 in C57BL/6), IL-6 (2,393 pg/ml ± 119 in Balb/c, 412 pg/ml ± 68.7 in C57BL/6), IL-10 (110 pg/ml ± 16 in Balb/c, 14.2 pg/ml ± 1.6 in C57BL/6) and KC (1,896 pg/ml ± 224 in Balb/c, 136.8 pg/ml ± 27.7 in C57BL/6). Cytokine and chemokine levels are expressed as % increase from control levels. The O₃- induced pro-inflammatory cytokine IL-12p70 and KC levels were significantly higher (*p = 0.0134 and *p = 0.0001, respectively) whereas the immunosuppressive IL-10 and the immunoregulatory IL-6 levels were significantly lower (*p < 0.0001 and *p < 0.0001, respectively) in Balb/c mice than in C57BL/6 mice. (A-G): Mean ± SEM of n = 6 in each groups.

Figure 2

Kinetics of O₃-induced SP-D and inflammatory changes in C57BL/6 mice. Groups of C57BL/6 mice were exposed to 3 ppm O₃ or room air and studied 0, 2, 6, 12, 24, 48, and 72 hrs later. (A): SP-D and SP-A recovered from the BAL were quantified by an in-house ELISA. Results are expressed as % change from naïve controls. SP-D levels gradually increased until the 72 hrs time point (p = 0.0022, ANOVA and post test for linear trend) whereas SP-A levels did not change significantly. (B): The number of neutrophilic granulocytes was assessed by counting total number of BAL cells in the BAL fluid and performing differential cell counting on Kwick™Diff cytospin preparations. Results are expressed as absolute cell number/ml of BAL fluid. Neutrophilic inflammation peaked around 12 hrs post exposure (p < 0.0001) and largely resolved by 72 hrs after O₃ exposure. (C-D) IL-6 and IL-10 levels were assessed as part of a Luminex^®100™ assay and showed significant increases 2–6 hrs after O₃ exposure (p < 0.0001) and 6–24 hrs after O₃ exposure (p = 0.0007), respectively. (A-D): Mean ± SEM of n = 6 in each groups.

Figure 3

SP-D deficient mice have increased airway inflammation following O₃ exposure. (A) SP-D -/- mice and age-matched C57BL/6 controls were exposed to 3 ppm O₃ for 2 hrs or (B) to 0.5 ppm for 24 hrs. Influx of neutrophilic granulocytes was assessed on cytospin preparations stained with Kwick™Diff. In both models cellular inflammation in SP-D -/- mice was significantly higher compared to wt mice (A) Student t-test *p = 0.0106 (B) ANOVA and Barlett's test *p = 0.0082.

Figure 4

In vitro production of SP-D by alveolar type II cells after IL-6 stimulation. (A) Primary rat alveolar type II cells were cultured in the presence or absence of DCI and IL-6 for up to 4 days and intra-cellular SP-D and SP-A levels were assayed by Western blot analysis. Day 0 SP-D and SP-A signals were obtained from freshly isolated alveolar type II cells. To maintain the SP-D producing phenotype, cells have to be cultured in the presence of DCI (10 nM dexamethason, 100 nM IBMX, 10 nM cAMP) that in turn stimulates SP-D production. IL-6 selectively stimulated the production of SP-D but did not induce up-regulation of SP-A. (B) Increase of SP-D after IL-6 stimulation at day 4 was slightly dose dependent. All levels were compared to non-stimulated alveolar type II cells cultured in DCI containing medium (DCI control).