Allergen particle binding by human primary bronchial epithelial cells is modulated by surfactant protein D

Abstract

Background: Allergen-containing subpollen particles (SPP) are released from whole plant pollen upon contact with water or even high humidity. Because of their size SPP can preferentially reach the lower airways where they come into contact with surfactant protein (SP)-D. Our previous work demonstrated that SP-D increases the uptake of SPP by alveolar macrophages. In the present study, we investigated the uptake of SPP in human primary epithelial cells and the potential modulation by SP-D. The patho-physiological consequence was evaluated by measurement of pro-inflammatory mediators.

Methods: SPP were isolated from timothy grass and subsequently fluorescently labelled. Human primary bronchial epithelial cells were incubated with SPP or polystyrene particles (PP) in the presence and absence of surfactant protein D. In addition, different sizes and surface charges of the PP were studied. Particle uptake was evaluated by flow cytometry and confocal microscopy. Soluble mediators were measured by enzyme linked immunosorbent assay or bead array.

Results: SPP were taken up by primary epithelial cells in a dose dependent manner. This uptake was coincided with secretion of Interleukin (IL)-8. SP-D increased the fraction of bronchial epithelial cells that bound SPP but not the fraction of cells that internalized SPP. SPP-induced secretion of IL-8 was further increased by SP-D. PP were bound and internalized by epithelial cells but this was not modulated by SP-D.

Conclusions: Epithelial cells bind and internalize SPP and PP which leads to increased IL-8 secretion. SP-D promotes attachment of SPP to epithelial cells and may thus be involved in the inflammatory response to inhaled allergen.

Figure 1

Diameter distribution of SPP. Approximately 300 SPP were analysed by scanning electron microscopy. The frequency distribution of diameters is shown.

Figure 2

Confocal microscopy of SPP uptake into normal human bronchial epithelial cells. Cells were incubated for 8 hours with 5 × 10⁶/cm² Alexa Fluor 488 labelled subpollen particles (green) in chamber slides. Cell cytoskeletons were stained with an antibody against F-Actin (Rhodamine phalloidin, red) and nuclei staining was performed by TO-PRO-3 Iodide (blue). Three dimensions along the white lines are shown. The intersection displays an internalized SPP.

Figure 3

Association of particles with primary epithelial cells. A) Percentage of normal human bronchial epithelial cells (NHBE) which participated in uptake and attachment of polydisperse subpollen particles (SPP) and monodisperse 1 μm polystyrene particles (PP) incubated for 8 hours as measured by flow cytometry. B) Percentage of human primary bronchial epithelial cells (HPBEC) which participated in uptake and attachment of PP in different sizes (0.5 μm - 1 μm - 3 μm). C) Percentage of HPBEC which participated in uptake and attachment of PP with different surface charge. Each value represents the mean ± SEM of four to five experiments. * p < 0.05; ** p < 0.01; *** p < 0.001 for within group comparison against 5 × 10⁶ particles/cm² ; # p < 0.05; ## p < 0.01 for comparison between two groups.

Figure 4

Association of particles with A549 cells. A) Percentage of A549 cells which participated in uptake and attachment of polydisperse subpollen particles (SPP) and monodisperse 1 μm polystyrene particles (PP) after 8 hours incubation as measured by flow cytometry. B) Percentage of A549 cells which participated in uptake and attachment of differently sized PP (0.5 μm - 1 μm - 3 μm). C) Percentage of A549 cells which participated in uptake and attachment of PP with different surface charge. Each value represents the mean and SEM from five to eight experiments. * p < 0.05; ** p < 0.01; *** p < 0.001 versus respective 5 × 10⁶ particles/cm² value; # p < 0.05 for inter-group comparison.

Figure 5

Representative confocal images visualizing the influence of surfactant protein D (SP-D) on subpollen particle (SPP) uptake and attachment. Normal human bronchial epithelial cells (NHBE) after incubation with 5 × 10⁶ SPP/cm² for 8 hours in A) the absence or B) presence of SP-D (10 μg/ml). White arrows show SPP (Alexa Fluor 488; green); cell cytoskeleton (F-Actin; red); nuclei (TO-PRO-3 iodide; blue).

Figure 6

Effect of surfactant protein D (SP-D) on the percentage of particle-positive human primary bronchial epithelial cells. Influence of SP-D on the uptake and binding of A+B) subpollen particles (SPP) and C) polystyrene particles (PP) in normal human bronchial epithelial cells (NHBE, A+C) and human primary bronchial epithelial cells (HPBEC, B) that were found particle-positive as measured by flow cytometry. Each value represents the mean ± SEM of five experiments. * p < 0.05 versus control.

Figure 7

Differentiation of uptake and attachment of subpollen particles (SPP) in normal human bronchial epithelial cells (NHBE). Cells which at least had internalized one SPP were defined as "internalized". These cells could also have attached SPP. Cells which only had attached SPP but no internalized SPP were defined as "attached". At least 10 pictures each with approximately 30 cells were analyzed by confocal microscopy. Each cell was individually assessed for uptake and attachment of SPP. Each value represents the mean ± SEM. ** p < 0.01 versus respective control.

Figure 8

Interleukin 8 (IL-8) in supernatants of normal human bronchial epithelial cells. Cells were incubated with particles in the presence of absence of SP-D for 8 hours (A, B) and subsequently cultured in medium for further 72 hours (C, D) using either subpollen particles (SPP, A and C) or polystyrene particles (PP, B and D). Each value represents the mean ± SEM from three experiments. * p < 0.05; ** p < 0.01; *** p < 0.001 versus control (CO). # p < 0.05 versus 5 × 10⁶ particles/cm². Control represents untreated cells.