Surfactant proteins A and D protect mice against pulmonary hypersensitivity induced by Aspergillus fumigatus antigens and allergens

Abstract

Allergic bronchopulmonary aspergillosis (ABPA) is an allergic disorder caused by an opportunistic fungal pathogen, Aspergillus fumigatus (AFU:). Lung surfactant proteins SP-A and SP-D can interact with the glycosylated antigens and allergens of AFU:, inhibit specific IgE binding to these allergens, and block histamine release from sensitized basophils. We have now examined the therapeutic effect of exogenous administration of human SP-A, SP-D, and a recombinant fragment of SP-D (rSP-D), in a murine model of pulmonary hypersensitivity induced by AFU: antigens and allergens, which resembles human ABPA immunologically. The ABPA mice exhibited high levels of AFU:-specific IgG and IgE, blood eosinophilia, extensive infiltration of lymphocytes and eosinophils in the lung sections, and a Th2 cytokine response. Treatment with SP-A, SP-D, and rSP-D lowered blood eosinophilia, pulmonary infiltration, and specific Ab levels considerably, which persisted up to 4 days in the SP-A-treated ABPA mice, and up to 16 days in the SP-D- or rSP-D-treated ABPA mice. The levels of IL-2, IL-4, and IL-5 were decreased, while the level of IFN-gamma was raised in the splenic supernatants of the treated mice, indicating a marked shift from Th2 to Th1 response. These results clearly implicate pulmonary SP-A and SP-D in the modulation of allergic reactions.

Figure 1

(a) SDS-PAGE (15% wt/vol) analysis of purified preparations of rSP-D under reducing as well as nonreducing conditions (Coomassie staining). A recombinant, homotrimeric fragment composed of the eight Gly-Xaa-Yaa repeats, α-helical coiled-coil neck region, and CRD of human SP-D was expressed in E. coli as the inclusion bodies and purified. The recombinant protein behaved as a homotrimer of about 60 kDa when examined by gel filtration chromatography and chemical cross-linking (data not shown). Under reducing conditions (lane 2), it ran as a monomer of about 18 kDa. No higher oligomers were seen when rSP-D was run under nonreducing conditions (lane 3), confirming that the trimerization was not a result of aberrant disulfide bridges between CRD regions. The rSP-D was also assessed for correct folding using disulfide mapping, and its crystallographic structure complexed with maltose in the carbohydrate-binding pockets (A.K. Shrive et al., unpublished data). (b) SDS-PAGE (10% wt/vol) analysis of purified preparations of SP-D and SP-A under reducing conditions (Coomassie staining). The majority of SP-D is composed of a 43-kDa polypeptide chain (lane 1) with faint bands corresponding to dimers and trimers of the 43-kDa chain (confirmed by immunoblotting). Two bands are seen, a major band corresponding to the 32-kDa polypeptide chain of SP-A (lane 2), together with a proportion of nonreducible dimers (64 kDa). Traces of higher oligomers and some aggregates (confirmed by immunoblotting) can also been seen. The nonreduced preparations of SP-D and SP-A behaved on SDS-PAGE as described previously (13).

Figure 2

Ratio of Afu-IgG Ab’s (estimated by indirect ELISA as described in Methods) of the untreated ABPA mice (black bars), SP-A–treated ABPA mice (white bars), SP-D–treated ABPA mice (dark gray bars), rSP-D–treated ABPA mice (light gray bars), and BSA-treated ABPA mice (medium gray bars) to their respective controls observed on day 0, 4, 10, and 16 of the treatment study. Each value represents a mean of nine readings (triplicate values from three animals of each group). Absorbance values ± SD for each control group observed on day 16 are as follows: untreated control, 0.158 ± 0.020; SP-A–treated control, 0.13 ± 0.013; SP-D–treated control, 0.118 ± 0.011; rSP-D–treated control, 0.120 ± 0.009; and BSA-treated control, 0.158 ± 0.015. ^AP < 0.05 compared with the values of the untreated ABPA mice on the same day.

Figure 3

Ratio of Afu-IgE Ab’s (estimated by indirect ELISA as described in Methods) of the untreated ABPA mice (black bars), SP-A–treated ABPA mice (white bars), SP-D–treated ABPA mice (dark gray bars), rSP-D–treated ABPA mice (light gray bars), and BSA-treated ABPA mice (medium gray bars) to their respective controls observed on day 0, 4, 10, and 16 of the treatment study. Each value represents a mean of nine readings (triplicate values from three animals of each group). Absorbance values ± SD for the control groups observed on day 16 are as follows: untreated control, 0.095 ± 0.011; SP-A–treated control, 0.072 ± 0.014; SP-D–treated control, 0.068 ± 0.012; rSP-D–treated control, 0.071 ± 0.010; and BSA-treated control (0.105 ± 0.018). AP < 0.05 compared with the values of the untreated ABPA mice on the same day.

Figure 4

Ratio of eosinophil counts in the peripheral blood of the untreated ABPA mice (black bars), SP-A–treated ABPA mice (white bars), SP-D–treated ABPA mice (dark gray bars), rSP-D–treated ABPA mice (light gray bars), and BSA-treated ABPA mice (medium gray bars) to their respective controls observed on day 0, 4, 10, and 16 of treatment study. Each value represents a mean of nine readings (triplicate values from three animals of each group). Numbers of eosinophils × 10⁶/ml ± SD for the control groups observed on day 16 are as follows: untreated control, 58 ± 7 × 10⁶/ml; SP-A–treated control, 45 ± 6 × 10⁶/ml; SP-D–treated control, 42 ± 8 × 10⁶/ml; rSP-D–treated control, 44 ± 4 × 10⁶/ml; and BSA-treated control, 61 ± 5 × 10⁶/ml. ^AP < 0.05 compared with the values of the untreated ABPA mice on the same day.

Figure 5

Histopathological examination of the lung sections, stained with hematoxylin and eosin (H&E) and observed at ×200, from the groups of untreated and treated ABPA mice and their respective control groups on day 16 of the treatment study. (a) Untreated ABPA mice; (b) untreated control; (c) BSA-treated ABPA mice; (d) BSA-treated control; (e) SP-A–treated ABPA mice; (f) SP-A–treated control; (g) SP-D–treated ABPA mice; (h) SP-D–treated control; (i) rSP-D–treated ABPA mice; (j) rSP-D–treated control. The nature of cellular infiltration, which includes lymphocytes and eosinophils (with characteristic bilobed nuclei), is clearly seen in the inset (×500) of each panel.

Figure 6

Ratio of lung EPO activity (estimated in the lung cell suspensions by a colorimetric substrate assay as described in Methods) of the untreated ABPA mice (black bars), SP-A–treated ABPA mice (white bars), SP-D–treated ABPA mice (dark gray bars), rSP-D–treated ABPA mice (light gray bars), and BSA-treated ABPA mice (medium gray bars) to their respective controls as observed on day 0, 4, 10, and 16 of the treatment study. Each value represents a mean of nine readings (triplicate values from three animals of each group). Absorbance values ± SD for the control groups observed on day 16 are as follows: untreated control, 0.252 ± 0.012; SP-A–treated control, 0.236 ± 0.015; SP-D–treated control, 0.242 ± 0.012; rSP-D–treated control, 0.225 ± 0.007; and BSA-treated control, 0.226 ± 0.001. ^AP < 0.05 compared with the values of the untreated ABPA mice on the same day.

Figure 7

Ratios of IL-2, IL-4, IL-5, and IFN-γ in splenic supernatants from untreated ABPA mice (black bars), SP-A–treated ABPA mice (white bars), SP-D–treated ABPA mice (dark gray bars), rSP-D–treated ABPA mice (light gray bars), and BSA-treated ABPA mice (medium gray bars) groups to their respective control groups as observed on day 10 of the treatment study. Each value represents a mean of nine readings (triplicate values from three animals of each group). Cytokine levels ± SD for the control groups observed on day 10 are as follows: untreated control, IL-2: 19.286 ± 0.827 pg/ml; IL-4: 42.036 ± 2.101 pg/ml; IL-5: 22.436 ± 1.121 pg/ml; IFN-γ: 33.281 ± 1.691 ng/ml; SP-A–treated control, IL-2: 14.529 ± 0.726 pg/ml; IL-4: 39.048 ± 1.952 pg/ml; IL-5: 18.229 ± 0.911 pg/ml; IFN-γ: 29.476 ± 1.473 ng/ml; SP-D–treated control, IL-2: 15.016 ± 0.750 pg/ml; IL-4: 36.725 ± 1.837 pg/ml; IL-5: 17.548 ± 0.877 pg/ml; IFN-γ: 28.359 ± 1.417 ng/ml; rSP-D–treated control, IL-2: 14.856 ± 0.742 pg/ml; IL-4: 37.543 ± 1.877 pg/ml; IL-5: 19.212 ± 0.960 pg/ml; IFN-γ: 29.021 ± 1.451 ng/ml; and BSA-treated control, IL-2: 19.893 ± 0.815 pg/ml; IL-4: 49.135 ± 1.962 pg/ml; IL-5: 28.431 ± 0.989 pg/ml; IFN-γ: 28.941 ± 1.216 ng/ml. ^AP < 0.05 compared with the values of the untreated ABPA mice on the same day.