Mucus secretion and cytoskeletal modifications in cultured nasal epithelial cells exposed to wall shear stresses

Abstract

The nasal epithelium is continuously subjected to wall shear stresses (WSS) induced by respiratory airflows. An in vitro experimental model was developed to expose nasal epithelial cells cultured under air-liquid interface conditions to steady airflow-induced WSS. Mucus secretion from epithelial goblet cells was quantified using an enzyme-linked lectinosorbent assay, and modifications of the cytoskeletal structure were qualitatively evaluated from fluorescent stains of actin and beta-tubulin fibers. The results show increased mucus secretion from cells subjected to WSS of 0.1 and 1.0 dyne/cm(2) for more than 15 min in comparison with unstressed cells. The integrity levels of beta-tubulin fibers were significantly lower in cells subjected to WSS than in unstressed cells. The increased mucus secretion in response to WSS was approximately the same in Taxol-free and Taxol-treated cultures, which indicates that there is no direct connection between beta-tubulin fragmentation and mucus secretion. The stressed cells regained their normal cytoskeletal appearance 24 h after the exposure to WSS. The results of this study suggest that WSS have an important role in the mechanical regulation of the nasal surface epithelium function.

FIGURE 1

(a) Custom-designed well for ALI cultures. (b) The disassembled well. The well-bottom allows application of a planar field of WSS in the flow chamber on the membrane with the cells. (c) Scheme of the flow chamber for exposing ALI cultures to WSS. The flow chamber can hold three well-bottoms. The perspective cross-sectional view shows the membrane on which the cells are seeded in the well-bottom and the space for culture medium.

FIGURE 2

Examples of the levels of β-tubulin fiber integrity used in this work. (a) Good integrity: <20% of the cells appeared with fragmented fibers. (b) Moderate integrity: 20–60% of the cells showed fragmented fibers. (c) Poor integrity: >60% of the cells in the field were observed with fragmented fibers. Bar = 10 μm.

FIGURE 3

Mucus secretions from culture lengths that were kept under static conditions for different time lengths. All of the cultures were measured 0.25, 24, and 48 h after the beginning of the experiment. Cultures 2, 3, and 4 were also measured after 2, 6, and 12 h, respectively. Culture 5 was measured together with all the other cultures, i.e., after 15 min and 2, 6, 12, 24, and 48 h. The results are expressed as a percentage of the baseline measurement of each culture, as described in the Materials and Methods section. These results are from a representative experiment and show the same trends as the two other experiments.

FIGURE 4

Accumulative data for mucus secretion measurements presented in Fig. 3, for cultures kept under static conditions (*p < 0.05).

FIGURE 5

Mucus secretions measured immediately after exposure of cultured nasal epithelial cells to WSS of (a) 0.1 dyne/cm² and (b) 1.0 dyne/cm², and after 24 h recovery from WSS of (c) 0.1 dyne/cm² and (d) 1.0 dyne/cm². The results are expressed as a percentage of the baseline measurement of each culture, as described in the Materials and Methods section (*p < 0.05, with respect to the unstressed control culture).

FIGURE 6

Representative images of β-tubulin (red) and actin (green) fibers stained immediately (a–d, i–l) and 24 h (e–h, m–p) after exposure of the cultures to WSS of 0.1 and 1.0 dyne/cm² for 5, 15, and 30 min. Bar = 10 μm.

FIGURE 7

Level of integrity of β-tubulin fibers in cultures exposed to (a) 0.1 dyne/cm² and (b) 1.0 dyne/cm². The results are from a representative experiment and show the same trends as the two other experiments. Dotted bars represent good integrity, horizontal lines bars represent moderate integrity, and gray bars represent poor integrity.

FIGURE 8

Images of β-tubulin in (a) unstressed Taxol-free culture, (b) unstressed Taxol-treated culture, (c) stressed Taxol-free culture, and (d) stressed Taxol-treated culture. The stressed cultures were exposed to WSS of 1.0 dyne/cm² for 15 min. Bar = 10 μm.

FIGURE 9

Mucus secretions measured in unstressed Taxol-free and Taxol-treated cultures in comparison with Taxol-free and Taxol-treated cultures that were exposed to WSS of 1.0 dyne/cm² for 15 min. The results are expressed as a percentage of the baseline measurement of each culture, as described in the Materials and Methods section.

FIGURE 10

Resistance coefficient (λ) as a function of Re for a smooth rectangular pipe. Reproduced from Schlichting (36).