Fish-derived low molecular weight components modify bronchial epithelial barrier properties and release of pro-inflammatory cytokines

Abstract

The prevalence of fish allergy among fish-processing workers is higher than in the general population, possibly due to sensitization via inhalation and higher exposure. However, the response of the bronchial epithelium to fish allergens has never been explored. Parvalbumins (PVs) from bony fish are major sensitizers in fish allergy, while cartilaginous fish and their PVs are considered less allergenic. Increasing evidence demonstrates that components other than proteins from the allergen source, such as low molecular weight components smaller than 3 kDa (LMC) from pollen, may act as adjuvants during allergic sensitization. We investigated the response of bronchial epithelial cells to PVs and to LMC from Atlantic cod, a bony fish, and gummy shark, a cartilaginous fish. Polarized monolayers of the bronchial epithelial cell line 16HBE14o- were stimulated apically with fish PVs and/-or the corresponding fish LMC. Barrier integrity, transport of PVs across the monolayers and release of mediators were monitored. Intact PVs from both the bony and the cartilaginous fish were rapidly internalized by the cells and transported to the basolateral side of the monolayers. The PVs did not disrupt the epithelial barrier integrity nor did they modify the release of proinflammatory cytokines. In contrast, LMC from both fish species modified the physical and immunological properties of the epithelial barrier and the responses differed between bony and cartilaginous fish. While the barrier integrity was lowered by cod LMC 24 h after cell stimulation, it was increased by up to 2.3-fold by shark LMC. Furthermore, LMC from both fish species increased basolateral and apical release of IL-6 and IL-8, while CCL2 release was increased by cod but not by shark LMC. In summary, our study demonstrated the rapid transport of PVs across the epithelium which may result in their availability to antigen presenting cells required for allergic sensitization. Moreover, different cell responses to LMC derived from bony versus cartilaginous fish were observed, which may play a role in different allergenic potentials of these two fish classes.

Keywords: Allergic sensitization; Bronchial epithelial cells; Fish allergy; Food matrix; Low molecular weight components; Parvalbumin.

Fig. 1

PV transport across epithelial monolayers. A. Detection of 0.1 μg of PVs conjugated to Alexa Fluor 488 by CBB staining of an SDS-PAGE gel and fluorescence detection on a nitrocellulose membrane. B. CBB-stained gel and fluorescence detection of proteins in the basolateral medium from monolayers treated apically with 80 μg/mL or 160 μg/mL of labelled PVs for 24 h. C. Quantification of PVs in the basolateral medium after 1 h and 24 h of apical stimulation (n = 3, data shown as mean ± SD). D. Fish allergic patients’ IgE binding to cod PV in basolateral medium. Purified cod PV (0.2 μg) was used as a positive control.

Fig. 2

PVs do not significantly influence epithelial barrier integrity and the viability of bronchial epithelial cells. Polarized 16HBE14o- cell monolayers were exposed apically to (A) cod PV, (B) shark PV (80 or 160 μg/mL) or to (C) 5 mM EGTA and TEER was monitored for 24 h. Untreated monolayers were used as controls. Data are obtained from three independent experiments performed in duplicates and show means with ranges (min to max), normalized to control monolayers. D. Cell viability analyzed by an MTT assay 24 h after apical exposure to PVs. 1% SDS was used as a control for toxicity. Data show mean ± SD (n = 3).

Fig. 3

PVs are internalized by 16HBE14o- cells and do not co-localize with LAMP-2-positive late endosomes/lysosomes. The figure shows results of confocal microscopy of 16HBE14o- cell monolayers after apical exposure to cod or shark PV (100 μg/mL) for 1 h at 37 °C, or monolayers without treatment with PVs. Alexa Fluor 488-labelled PVs (green), late endosome/lysosome marker LAMP2 detected by a specific primary and Alexa Fluor 568-labelled secondary antibody (red), DRAQ5-labelled nuclei (blue). Bar = 10 μm (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Fig. 4

PVs do not modify the release of IL-6, IL-8 and CCL2 by 16HBE14o- cells. Epithelial cell monolayers were stimulated apically with cod or shark PV (80 or 160 μg/mL) and the concentrations of the cytokines released to the basolateral (A) or apical (B) media were measured after 24 h of stimulation. Cytokine concentrations were normalized to the concentrations measured for untreated monolayers. Data are representative of three independent experiments performed in duplicates and depict means with ranges (min to max).

Fig. 5

Fish LMC modify the epithelial barrier properties. A. 16HBE14o- cell monolayers were exposed apically to three doses of cod or shark LMC and TEER measured over time. TEER was normalized to untreated monolayers for each time point. Data show means with ranges (min to max) from three independent experiments performed in duplicates. B. Polarized monolayers were treated for 24 h with fish LMC (Dose III) and the permeability for FD10 was explored. Untreated cells were used as reference. n = 2, data show mean ± SD. C. Quantification of the PVs in basolateral cell culture medium after 24 h of apical stimulation in presence or absence of LMC. n = 3, data show mean ± SD. D. Cell viability analyzed by MTT assay 24 h after apical exposure to different doses of LMC. n = 3, data show mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 6

Fish LMC modify proinflammatory cytokine release. Protein levels of IL-6, IL-8 and CCL2 in basolateral (A) and apical (B) media were quantified 24 h after apical stimulation of the cells. Concentrations are normalized to concentrations of cytokines released from untreated cells. Data are representative of three independent experiments performed in duplicates and depict means with ranges (min to max). *p < 0.05.