Pollen Proteases Play Multiple Roles in Allergic Disorders

Abstract

Allergic diseases are a major health concern worldwide. Pollens are important triggers for allergic rhinitis, conjunctivitis and asthma. Proteases released upon pollen grain hydration appear to play a major role in the typical immunological and inflammatory responses that occur in patients with allergic disorders. In this study, we aimed to identify specific proteolytic activity in a set of pollens with diverse allergenic potential. Diffusates from Chenopodium album, Plantago lanceolata and Eucalyptus globulus were added to a confluent monolayer of Calu-3 cells grown in an air-liquid interface system. We identified serine proteases and metalloproteinases in all pollen diffusates investigated. Proteases found in these pollen diffusates were shown to compromise the integrity of the lung epithelial barrier by disrupting transmembrane adhesion proteins E-cadherin, claudin-1 and Occludin, as well as, the cytosolic complex zonula occludens-1 (ZO-1) resulting in a time-dependent increase in transepithelial permeability. Tight junction disruption and increased transepithelial permeability facilitates allergen exposure to epithelial sub-layers contributing to the sensitization to a wide range of allergens. These pollen extracts also induced an increase in the release of interleukin 6 (IL-6) and interleukin 8 (IL-8) cytokines measured by flow cytometry possibly as a result of the activation of protease-activated receptors 2 (PAR-2).

Keywords: IL-6; IL-8 and PAR-2; allergy; pollen proteases; transepithelial permeability.

Figure 1

Specificity of pollen proteases from Chenopodium album, Plantago lanceolata and Eucalyptus globulus diffusates. (A) Substrate specificity of pollen proteolytic activity. The extracts were incubated for 20 min at 37 °C with 100 µM peptide-AMC. The black bars indicate the preferred substrate for each pollen diffusate, which was also the substrate used in the inhibition experiments. (B) Effect of class-specific inhibitors on the proteolytic activity of the pollen diffusates. The results represent the percentage of residual activity in comparison to the control condition (without inhibitor). The grey bars indicate statistically significant inhibition results. Dunnett’s post-test: *** p < 0.001; ** p < 0.01(n = 3). 1 unit of activity (U) = pmol AMC released/min/mg of protein.

Figure 2

Effect of pollen proteases from Chenopodium album (0.26 ± 0.06 mg/ml), Plantago lanceolata (0.18 ± 0.03 mg/ml) and Eucalyptus globulus (0.75 ± 0.18 mg/ml) on the paracellular permeability of epithelial cells. (A) Time-course effect of pollen diffusates on Calu-3 cells. (B) Effect of untreated pollen diffusates (black bars) and diffusates treated with 1 mM AEBSF (white bars) on the transepithelial permeability of Calu-3 cells. Cells incubated with culture medium were used as a control for this assay. Bonferroni post-test: *** p < 0.001; ** p < 0.01; * p < 0.05 (n = 3) in comparison to the control condition, and ^### p < 0.001; ^## p < 0.01 (n = 3) for comparisons between treated and untreated conditions.

Figure 3

Effect of pollen diffusates on intercellular junction integrity. Calu-3 cells were exposed to pollen diffusates from Chenopodium album (0.26 ± 0.06 mg/ml), Plantago lanceolata (0.18 ± 0.03 mg/ml) and Eucalyptus globulus (0.75 ± 0.18 mg/ml) (black bars) for 6 h. The cells were also incubated with pollen diffusates that were pre-treated with 1 mM AEBSF (white bars). The degradation of the transmembrane proteins E-cadherin (A) claudin-1 (B) occludin (C) and the cytosolic complex ZO-1 (D) was analyzed by immunoblotting. Cells incubated with culture medium were used as a control. The data were normalized and expressed as a percentage relative to each control. Representative blots are shown for each stimulus. Bonferroni post-test: *** p < 0.001 (n = 5) in comparison to the control condition, and ^### p < 0.001; ^## p < 0.01 (n = 5) for comparisons between treated and untreated conditions.

Figure 4

Functional characterization of the effect of pollen proteases from Chenopodium album (0.26 ± 0.06 mg/ml), Plantago lanceolata (0.18 ± 0.03 mg/ml) and Eucalyptus globulus (0.75 ± 0.18 mg/ml) on Calu-3 cells. (A) Calu-3 cultures were incubated for 240 s with Krebs solution and stimulated for 120 s with denatured pollen diffusates followed by 120 s incubation with pollen diffusates. (B) [Ca2+]_i variations in Calu-3 cells after exposure to C. album (left graph), P. lanceolata (middle graph) and E. globulus (right graph). Arrows in each panel indicate the time of addition of the pollen diffusate. The fluorescence profiles are representative of 10 cells. (C) The bar graph shows the maximal changes in [Ca²⁺]_i after stimulation with each pollen diffusate. Changes in [Ca²⁺]_i are depicted by the F340/F380 fluorescence ratios of Fura-2-loaded Calu-3 cells. The black bars correspond to each pollen diffusate, while the white bars correspond to denatured pollen diffusates (95 °C for 30 min). The positive control for this assay was a 0.25% trypsin solution. The data are expressed as the mean ±SEM values of at least 30 representative cell traces using Bonferroni’s post-test: *** p < 0.001; * p < 0.05 (n = 4) in comparison to the control condition and for comparisons between treated and untreated conditions ^### p < 0.001 (n = 4).

Figure 5

Effect of pollen proteases from Chenopodium album (0.26 ± 0.06 mg/ml), Plantago lanceolata (0.18 ± 0.03 mg/ml) and Eucalyptus globulus (0.75 ± 0.18 mg/ml) on IL-6 (A) and IL-8 (B) release. The black bars correspond to stimulation with each pollen diffusate, while the white bars correspond to the denatured pollen diffusates (95 °C for 30 min). Cells incubated with culture medium were used as the control condition for this assay. The data are expressed as the mean ± SEM values and were analyzed statistically using Bonferroni´s post-test: *** p < 0.001 (n = 3) in comparison to the control condition, and ^### p < 0.001 for comparisons between treated and untreated conditions (n = 3).