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Comparative Study
. 2003 Dec 8;4(1):15.
doi: 10.1186/1465-9921-4-15. Print 2003.

Attenuated allergic airway hyperresponsiveness in C57BL/6 mice is associated with enhanced surfactant protein (SP)-D production following allergic sensitization

Elena N Atochina  1 , Michael F Beers, Yaniv Tomer, Seth T Scanlon, Scott J Russo, Reynold A Panettieri Jr, Angela Haczku
Affiliations
Comparative Study

Attenuated allergic airway hyperresponsiveness in C57BL/6 mice is associated with enhanced surfactant protein (SP)-D production following allergic sensitization

Elena N Atochina et al. Respir Res. .

Abstract

Background: C57BL/6 mice have attenuated allergic airway hyperresponsiveness (AHR) when compared with Balb/c mice but the underlying mechanisms remain unclear. SP-D, an innate immune molecule with potent immunosuppressive activities may have an important modulatory role in the allergic airway response and the consequent physiological changes. We hypothesized that an elevated SP-D production is associated with the impaired ability of C57BL/6 mice to develop allergic AHR.

Methods: SP-D mRNA and protein expression was investigated during development of allergic airway changes in a model of Aspergillus fumigatus (Af)-induced allergic inflammation. To study whether strain dependency of allergic AHR is associated with different levels of SP-D in the lung, Balb/c and C57BL/6 mice were compared.

Results: Sensitization and exposure to Af induced significant airway inflammation in both mouse strains in comparison with naïve controls. AHR to acetylcholine however was significantly attenuated in C57BL/6 mice in spite of increased eosinophilia and serum IgE when compared with Balb/c mice (p < 0.05). Af challenge of sensitized C57BL/6 mice induced a markedly increased SP-D protein expression in the SA surfactant fraction (1,894 +/- 170% of naïve controls) that was 1.5 fold greater than the increase in Balb/c mice (1,234 +/- 121% p < 0.01). These changes were selective since levels of the hydrophobic SP-B and SP-C and the hydrophilic SP-A were significantly decreased following sensitization and challenge with Af in both strains. Further, sensitized and exposed C57BL/6 mice had significantly lower IL-4 and IL-5 in the BAL fluid than that of Balb/c mice (p < 0.05).

Conclusions: These results suggest that enhanced SP-D production in the lung of C57BL/6 mice may contribute to an attenuated AHR in response to allergic airway sensitization. SP-D may act by inhibiting synthesis of Th2 cytokines.

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Figures

Figure 1
Figure 1
Allergen challenge of sensitized C57BL/6 mice induced a significantly attenuated airway hyperresponsiveness to ACh in comparison with Balb/c mice. Mice were sensitized and exposed to Af and their lung function was assessed by measuring Lung Resistance (RL, panel A) and Dynamic Compliance (Cdyn, panel B) by a BUXCO online system as described. RL and Cdyn values were obtained in response to increasing concentrations of intravenous (i.v.) ACh in both strains of mice. Data are expressed as % changes from baseline. The Naïve group (open square, C57BL/6 or open circle, Balb/c) received intranasal glycerol treatment alone. The Sensitized group (closed squares, C57BL/6 or closed circle, Balb/c) received intraperitoneal (i.p.) sensitization and intranasal (i.n.) challenge with Af extract as described. Baseline RL values in the Naïve and in the Sensitized groups were 0.84 ± 0.04 and 1.25 ± 0.11 Hgmm/cmH2O/min respectively, in the C57BL/6 mice and 1.26 ± 0.03 and 1.48 ± 0.06 Hgmm/cmH2O/min, respectively in the Balb/c mice. Results are expressed as Mean ± SEM of n = 6–7. ANOVA was used to compare the dose response curves followed by Student's t test for comparisons between individual data points. *p < 0.05 Sensitized vs Naive; #p < 0.05 C57BL/6 vs Balb/c.
Figure 2
Figure 2
C57BL/6 mice developed significant systemic IgE and IgG1 responses following allergenic sensitization and challenge with Af comparable to the responses of Balb/c mice. Total serum IgE and Af-specific IgE and IgG1 profiles were analyzed by ELISA as described. Total serum IgE (panel A): expressed as ng/ml. Af-specific IgG1 and IgE (panel B and C) expressed as optical density (O.D.). Naïve mice (open bar: Balb/c (n = 15); light gray bar: C57BL/6 (n = 17)) received i.n. glycerol treatment alone. Sensitized mice (black bars: Balb/c (n = 22); dark gray bar: C57BL/6 (n = 22)) received i.p. sensitization and i.n. treatment with Af extract as described. Data are expressed as Mean ± SEM, *p < 0.05 Sensitized vs Naive; #p < 0.05 C57BL/6 vs Balb/c.
Figure 3
Figure 3
Upon allergen challenge, sensitized C57BL/6 mice developed significantly augmented airway eosinophilia when compared with Balb/c mice. The absolute number of BAL cells (panel B) was derived from the differential counts in Giemsa-stained cytospin preparations and the total cell counts (panel A) in each BAL sample as described. Naïve mice (open bar: Balb/c (n = 15); light gray bar: C57BL/6 (n = 17)) received i.n. glycerol treatment alone. Sensitized mice (black bars: Balb/c (n = 22); dark gray bar: C57BL/6 (n = 22)) received i.p. sensitization and i.n. treatment with Af extract as described. Data are expressed as Mean ± SEM, *p < 0.05 Sensitized vs Naive; #p < 0.05 C57BL/6 vs Balb/c.
Figure 4
Figure 4
Following allergic challenge of sensitized C57BL/6 mice there was a significantly increased production of SP-D in the lung in comparison with similarly treated Balb/c mice. Western blot of three representative SP-D samples from the SA surfactant fraction of the BAL fluid of Balb/c and C57BL/6 mice (A). Nitrocellulose blots with samples of SA surfactant from BAL naïve and sensitized mice were probed with polyclonal antisera against SP-D antibody as described. Each lane contains 10 μg total protein. The relative content of SP-D bands in each sample was determined by densitometric scanning of the 43-kD bands from multiple blots and quantified as described (B). Open bars: Naive mice; Closed bars: Sensitized mice. Data are expressed as % of naïve controls levels. N = 3–5 samples were used in each group. Mean ± SEM was calculated after deriving the average of the results from two independent experiments. *p < 0.05 Sensitized vs Naive; #p < 0.05 C57BL/6 vs Balb/c. Autoradiographs of three representative SP-D mRNA samples of Balb/c and C57BL/6 mice (C). Total RNA for northern blot analysis was prepared from the lungs of naïve and sensitized mice as described. Intensity was quantified by densitometric scanning and values were normalized to 28S mRNA (D). Intensity of 28S band was similar in each sample (data not shown). SP-D mRNA content is expressed as % of naïve Balb/c level. Open bars: Naive mice; Closed bars: Sensitized mice. N = 7–8 samples were used. Mean ± SEM was calculated after deriving the average of the results from two independent experiments.
Figure 5
Figure 5
SP-A protein levels in the lung of C57BL/6 mice were not increased following allergic challenge of sensitized mice. Western blot of three representative SP-A samples from the LA surfactant fraction of the BAL fluid of Balb/c and C57BL/6 mice (A). Nitrocellulose blots with samples of LA surfactant from naïve and sensitized mice were probed with polyclonal anti-SP-A antibody as described. Each lane contains 5 μg total protein. The relative content of SP-A doublet bands in each sample was determined by densitometric scanning of the 29–35 kD bands from multiple blots and quantified as described (B). Open bars: Naive mice; Closed bars: Sensitized mice. Data are expressed as % of naïve Balb/c levels. Mean ± SEM of n = 3–5 was calculated after deriving the average of the results from two independent experiments. # p < 0.05 C57BL/6 vs naïve Balb/c mice. The SP-A content in the LA and SA fractions of naïve mice was also determined by using an ELISA protocol as described. Total BAL SP-A content (C) was expressed as total ng. Gray bars: LA fraction of BAL; Black bars: SA fractions of BAL. Data are expressed as Mean ± SEM of n = 5 samples in each group. #p < 0.05 C57BL/6 vs naïve Balb/c mice. Total RNA for northern blot analysis was prepared from the lungs of naïve and sensitized mice as described. Intensity was quantified by densitometric scanning and values were normalized to 28S mRNA (D). SP-A mRNA contents are expressed as % of naïve Balb/c level. Open bars: Naive mice; Closed bars: Sensitized mice. Mean ± SEM of n = 3–5 samples was calculated after deriving the average of the results from two independent experiments.
Figure 6
Figure 6
Allergen challenge of sensitized C57BL/6 mice induced impaired IL-4 and IL-5 release in comparison with Balb/c mice. Cytokine levels in BAL were determined by ELISA. Data are expressed as pg per μg of protein level. The Naïve group (open bar, Balb/c (n = 9) or light gray bar, C57BL/6 (n = 9)) received intranasal glycerol treatment alone. The Sensitized group (black bar, Balb/c (n = 12) or dark gray bar, C57BL/6 (n = 12)) received i.p. sensitization and i.n. treatment with Af extract. Data are expressed as Mean ± SEM, *p < 0.05 Sensitized vs Naive; #p < 0.05 C57BL/6 vs Balb/c.
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