Mast Cell Proteases Tryptase and Chymase Induce Migratory and Morphological Alterations in Bronchial Epithelial Cells

Abstract

Chronic respiratory diseases are often characterized by impaired epithelial function and remodeling. Mast cells (MCs) are known to home into the epithelium in respiratory diseases, but the MC-epithelial interactions remain less understood. Therefore, this study aimed to investigate the effect of MC proteases on bronchial epithelial morphology and function. Bronchial epithelial cells were stimulated with MC tryptase and/or chymase. Morphology and epithelial function were performed using cell tracking analysis and holographic live-cell imaging. Samples were also analyzed for motility-associated gene expression. Immunocytochemistry was performed to compare cytoskeletal arrangement. Stimulated cells showed strong alterations on gene, protein and functional levels in several parameters important for maintaining epithelial function. The most significant increases were found in cell motility, cellular speed and cell elongation compared to non-stimulated cells. Also, cell morphology was significantly altered in chymase treated compared to non-stimulated cells. In the current study, we show that MC proteases can induce cell migration and morphological and proliferative alterations in epithelial cells. Thus, our data imply that MC release of proteases may play a critical role in airway epithelial remodeling and disruption of epithelial function.

Keywords: bronchial epithelial cells; chymase; holomonitor; mast cell; migration; morphology; proliferation; tryptase.

Figure 1

Tryptase induce mitogenic properties whereas chymase attenuate the proliferation rate in bronchial epithelial cells. (A) Percentage cell growth over 36 h for non-stimulated cells (NS) or cells treated with tryptase (T), chymase (C) or tryptase and chymase (TC) over time and the percentage of cell increase at 36 h relative to the starting point (B) was established using the holographic live cell imaging. LDH enzymatic activity was measured in a dose-response setup (C) to detect potential cytotoxicity. (D) Percentage of dividing cells at a 12 h interval. (E) Representation of MTT signal at 24 h, 48 h and 72 h after treatment. (F) Quantification of Ki67 expression (pixels⁺/cells^total) in BEAS-2B using fluorescence immunocytochemistry and ImageJ. (G) Representative micrographs of immunofluorescence stain for KI67 (FITC, green) and nuclei (DAPI, blue) in BEAS-2b stimulated with tryptase, chymase and in combination compared to non-stimulated cells. Scale bar in G: 50 µm. White arrowheads show Ki67 positive cells. Data represent mean ± SEM. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 using Mann–Whitney test. LDH, KI67, MTT: results are based on three independent experiments. Holomonitor: results are based on five focus points per experiment and stimulation, in three independent repeats.

Figure 2

Representative holographic images obtained with HolomonitorM4 at the starting point (A) and 36 h (B) of five focus points per experiment and stimulation, in three independent repeats. The top images in each panel represent high magnification images in 3D of the bottom 2D images. Scale bar (bottom panels): 100 µm. The colors representing the cell height, where yellow is the maximum cell height.

Figure 3

Representative images of epithelial cell morphology in scanning electron microscopy (SEM) (A) and confocal microscopy of immunofluorescence cytoskeletal staining using FITC-conjugated phalloidin (B). The upper panel in (A) and (B) represents high magnification images of BEAS-2B. White arrowheads indicate clusters of phalloidin-positive stainings located in the protrusions. Scale bar in A: upper panel 60 µm, lower panel 30 µm and B: upper panel 100 µm, lower panel 50 µm.

Figure 4

Holographical analysis of morphological alterations in airway epithelial cells stimulated with MC proteases. (A) Demonstration of single cell area (µm²) over time where each dot represents one cell. (B) Cell area at 36 h, relative to starting point. (C) Confluency at 36 h relative to the starting point. (D) The optical thicknesses and (E) optical volume. (F) Representation of the measurements of cell box length and box breadth. (G) The ratio of box breadth and box length plotted at different time points over time. (H) The percentage of cells in each group at different time point that were over the cut-off threshold. The cut-off threshold represents the 10% percentile of the ratio of all NS cells at all time points. Data represent mean ± SEM. Statistical analysis was tested using Mann–Whitney test (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001) and for (G) Chi-squared test * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. Data are based on the same number of cells at each time point and stimulation throughout the different analyzed parameters. Results are based on five focus points per experiment and stimulation in independent repeats.

Figure 5

Single-cell tracking showed enhanced cell migration, motility and speed in cells treated with mast cell proteases. (A) Representation of single-cell tracking XY-plots obtained from the HolomonitorM4 and compared migration at two different time intervals (0–12 h and 24–36 h) and between stimulation. Individual cells are seen in different colors. (B) Comparison of single-cell migration, (C) motility and (D) speed in tryptase (T), chymase (C) and in combination (TC) compared to controls (NS). Data represent mean ± SEM. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 using Mann-Whitney test. Results based on cell tracking of at least five cells per monitored position and from three randomly chosen focus positions in each well, giving a total of >15 cells per time point and stimulation.