Surfactant protein-A inhibits Aspergillus fumigatus-induced allergic T-cell responses

Abstract

Background: The pulmonary surfactant protein (SP)-A has potent immunomodulatory activities but its role and regulation during allergic airway inflammation is unknown.

Methods: We studied changes in SP-A expression in the bronchoalveolar lavage (BAL) using a murine model of single Aspergillus fumigatus (Af) challenge of sensitized animals.

Results: SP-A protein levels in the BAL fluid showed a rapid, transient decline that reached the lowest values (25% of controls) 12 h after intranasal Af provocation of sensitized mice. Decrease of SP-A was associated with influx of inflammatory cells and increase of IL-4 and IL-5 mRNA and protein levels. Since levels of SP-A showed a significant negative correlation with these BAL cytokines (but not with IFN-gamma), we hypothesized that SP-A exerts an inhibitory effect on Th2-type immune responses. To study this hypothesis, we used an in vitro Af-rechallenge model. Af-induced lymphocyte proliferation of cells isolated from sensitized mice was inhibited in a dose-dependent manner by addition of purified human SP-A (0.1-10 microg/ml). Flow cytometric studies on Af-stimulated lymphocytes indicated that the numbers of CD4+ (but not CD8+) T cells were significantly increased in the parental population and decreased in the third and fourth generation in the presence of SP-A. Further, addition of SP-A to the tissue culture inhibited Af-induced IL-4 and IL-5 production suggesting that SP-A directly suppressed allergen-stimulated CD4+ T cell function.

Conclusion: We speculate that a transient lack of this lung collectin following allergen exposure of the airways may significantly contribute to the development of a T-cell dependent allergic immune response.

Figure 4

SP-A has a significant inhibitory effect on antigen specific lymphocyte proliferation from mice sensitized and challenged with Af. (A): Baseline (unstimulated) proliferation of splenic lymphocytes. Endotoxin in concentration equivalent to the endotoxin content of the 10 μg/ml SP-A samples did not affect cell proliferation. (B): In vitro Af stimulated proliferation of cells from mice sensitized and challenged with Af was inhibited by presence of SP-A, in a dose-dependent manner. (C): The Af dose-response curve of sensitized lymphocytes was abrogated in the presence of SP-A (10 μg/ml). (D) Presence of SP-A inhibited murine lymphocytes stimulated with PMA (2 ng/ml) and ionomycin (100 ng/ml). Background cpm (from wells that contain no cells): 0–16. Mean ± SEM of n = 4 independent experiments presented, each performed in triplicates. * p < 0.05: 0 vs. 10 μg/ml

Figure 1

Allergen challenge of sensitized mice with Af induced a transient decrease in the BAL SP-A protein levels. (A) SP-A Western blots from two representative samples of BAL obtained 1, 6, 12, 24, 48 and 72 h after Af challenge. (B): SP-A protein from BAL (open circles) and lung tissue mRNA (closed circles); % of the naïve control values; n = 4–6. (C): SP-A protein levels measured 12 h after Af challenge Mean ± SEM of n = 8 each; ** p < 0.01 (Af/Af vs. Af/gly).

Figure 2

Single intranasal provocation of sensitized mice with Af induced increase in Penh, inflammatory cell influx and Th2 but not Th1 cytokine release, 12 h After intranasal challenge. (A): Lung function measured by Penh. (B): BAL total and differential cell counts were used to calculate the absolute cell number. MP: macrophages EP: eosinophils, NP: neutrophils, LC: lymphocytes. (C): BAL Cytokine levels measured by ELISA (pg/ml). Mean ± SEM of n = 8 each; ** p < 0.01 (Af/Af vs. Af/gly) (D): Lung tissue cytokine mRNA levels measured by real-time PCR (fold increase in comparison to naïve control levels). ** p < 0.01 (vs. 0 h)

Figure 3

Purification and biological activity of SP-A from human PAP material. (A): Coomassie Blue staining. (B): Western blot comparison of endotoxin-depleted (ED) SP-A preparations (1 μg) with human PAP material (1 μg) used as a positive control. (C): Effect of purified human SP-A on normal human PBMC proliferation. Cells were stimulated with PMA (2 ng/ml) and ionomycin (100 ng/ml). Stimulation Index (cpm of stimulated cells divided by cpm of corresponding unstimulated cells). Mean ± SEM of n = 12

Figure 5

SP-A inhibited Af-induced CD4+ T cell proliferation and release of IL-4 and IL-5. (A): SP-A (10 μg/ml) increased the number of cells in the G1 (black bars) and decreased them in the G3-G4 populations of CD3/CD4+ lymphocytes (% change from the medium-treated samples). Average of 3 independent experiments. (B): Af-induced production of IL-4 and IL-5 by sensitized lymphocytes (S/Medium) was reduced in the presence of 10μg/ml of SP-A (S/SP-A) to levels similar to naïve lymphocytes (N/Medium). Lymphocytes were stimulated by Af (1 μg/ml). IL-4 (grey bars), IL-5 (white bars) and IFN-γ (black bars). * p < 0.05 S/SP-A vs. S/Medium. Mean ± SEM of n = 4 experiments performed in triplicates..