The costimulatory molecules CD80, CD86 and OX40L are up-regulated in Aspergillus fumigatus sensitized mice

Abstract

Aspergillus fumigatus (Af) is a fungus associated with allergic bronchopulmonary aspergillosis (ABPA) and other allergic diseases. Immune responses in these diseases are due to T and B cell responses. T cell activation requires both Af-specific engagement of the T-cell-receptor as well as interaction of antigen independent costimulatory molecules including CD28-CD80/CD86 and OX40-OX40L interactions. Since these molecules and their interactions have been suggested to have a potential involvement in the pathogenesis of ABPA, we have investigated their role in a model of experimental allergic aspergillosis. BALB/c mice were primed and sensitized with Af allergens, with or without exogenous IL-4. Results showed up-regulation of both CD86 and CD80 molecules on lung B cells from Af-sensitized mice (79% CD86+ and 24% CD80+) and Af/rIL-4-treated mice (90% CD86+ and 24% CD80+) compared to normal controls (36% and 17%, respectively). Lung macrophages in Af-sensitized mice treated or not with IL-4 showed enhanced expression of these molecules. OX40L expression was also up-regulated on lung B cells and macrophages from both Af-sensitized and Af/rIL-4 exposed mice as compared to normal controls. All Af-sensitized animals showed peripheral blood eosinophilia, enhanced total serum IgE and allergen-specific IgG1 antibodies and characteristic lung inflammation. The up-regulation of CD80, CD86 and OX40L molecules on lung B cells and macrophages from Af-allergen exposed mice suggests a major role for these molecules in the amplification and persistence of immunological and inflammatory responses in ABPA.

Fig. 1

Peripheral blood eosinophils and total serum IgE and Af-IgG antibodies levels detected in Af treated and control mice. (a) Eosinophils in the peripheral blood of mice treated with Af antigens, Af/rIL-4, rIL-4 alone or PBS (normal controls) before treatment (day 0), on day 30 after initial treatment and after the last Af antigen challenge (day 43). (b) Total serum IgE responses in the sera of mice before treatment (day 0), during treatment (day 35) and after last Af antigen challenge (day 43). (c) Aspergillus-specific IgG1 and IgG2a on day 43 post challenge. IgG values are presented as Log₁₀ titre. All pretreatment sera had < 1·6 Log₁₀ titre. The data is presented as mean + SEM, and P-values are designated as *P < 0·05, **P < 0·001 and ***P < 0·0001 versus the PBS controls. Five to 10 animals from each group were analysed. Data are representative of two identical experiments.

Fig. 2

Proliferative responses of spleen cells from Af sensitized and nonsensitized mice to A. fumigatus recombinant proteins, Asp f2, f3, f4 and f6. SI values are shown as means ± SEMs. The P-values less than <0·05 of sensitized versus PBS control mice were considered to be significant (*). Five animals from each group were studied. Data are representative of two identical experiments.

Fig. 3

Cytokine production by spleen cells from Af sensitized and control animals in response to Af allergens. (a) IFN-gamma, (b) IL-4, (c) IL-5 and (d) IL-10 were quantified by ELISA. Antigen induced cytokine expression was quantified after comparison with reference standards of mouse cytokines. Data represent the mean ± SEM of two separate experiments, and 5–10 mice were used in each group. *P-values < 0·05 were considered to be significant.

Fig. 4

Expression of CD86 and CD80 costimulatory molecules on cells from the lungs of sensitized and nonsensitized mice. Lung cells were pooled from mice in each group and analysed by flow cytometry. (a) Representative histograms comparing CD86 expression on gated B cells from the four groups tested are shown. (b) CD86 expression and (c) CD80 expression on lung B cells are depicted as percent positive cells. (d) Similarly, CD86 and CD80 expression on lung macrophages from experimental and control groups are shown. The results are from one of two independent experiments.

Fig. 5

Expression of OX40L molecules on pooled lung B cells and macrophages from four groups of mice. The percentages of (a) B cells and (b) macrophages expressing OX40L by flow cytometric analysis are shown. The data are representative from two independent experiments.

Fig. 6

Inflammatory changes in the lungs of Af sensitized mice. (a) Normal mouse lung architecture (40× HE stain). (b) Higher magnification of normal lung tissue shows an absence of inflammatory cell infiltration around vessels and bronchi, with a single layer of ciliated columnar epithelium (arrows), 400× HE stain. (c) Depicts the absence of goblet cells in the bronchial epithelium of lung tissue from PBS controls (400× PAS). (d) The lung tissue from an A. fumigatus antigen treated mouse showing the degree of perivascular cuffing (arrows), similar to that seen in Af/rIL-4 treated mice (6G). Few macrophages are present in alveoli or alveolar septae (40× HE stain). (e) A higher magnification of the lung tissue shows numerous eosinophils located in the perivascular interstitium (400× HE stain). Fewer PAS staining goblet cells are located in the mucosa lining the bronchial lumen in Af exposed mouse (f) as compared to Af/rIL-4 treated mouse (i) (400× PAS). Severe perivascular cuffing (arrows) with eosinophils, macrophages and lymphocytes are present in Af/rIL-4 treated mice (g,h). Lungs of rIL-4 treated mice did not show any abnormal histological changes (6 j–l). A, alveolar space; B, bronchial lumen; V, bronchial artery; M, macrophages; E, eosinophils.