Baseline Goblet Cell Mucin Secretion in the Airways Exceeds Stimulated Secretion over Extended Time Periods, and Is Sensitive to Shear Stress and Intracellular Mucin Stores.

Carla M P Ribeiro,Michael I Lethem,Sean P Doyle,Kristine Nguyen,M Gregory Forest,Paula A Vasquez,Burton F Dickey,C William Davis,Lubna H Abdullah,Yunxiang Zhu

doi:10.1371/journal.pone.0127267

Abstract

Airway mucin secretion studies have focused on goblet cell responses to exogenous agonists almost to the exclusion of baseline mucin secretion (BLMS). In human bronchial epithelial cell cultures (HBECCs), maximal agonist-stimulated secretion exceeds baseline by ~3-fold as measured over hour-long periods, but mucin stores are discharged completely and require 24 h for full restoration. Hence, over 24 h, total baseline exceeds agonist-induced secretion by several-fold. Studies with HBECCs and mouse tracheas showed that BLMS is highly sensitive to mechanical stresses. Harvesting three consecutive 1 h baseline luminal incubations with HBECCs yielded equal rates of BLMS; however, lengthening the middle period to 72 h decreased the respective rate significantly, suggesting a stimulation of BLMS by the gentle washes of HBECC luminal surfaces. BLMS declined exponentially after washing HBECCs (t1/2 = 2.75 h), to rates approaching zero. HBECCs exposed to low perfusion rates exhibited spike-like increases in BLMS when flow was jumped 5-fold: BLMS increased >4 fold, then decreased within 5 min to a stable plateau at 1.5–2-fold over control. Higher flow jumps induced proportionally higher BLMS increases. Inducing mucous hyperplasia in HBECCs increased mucin production, BLMS and agonist-induced secretion. Mouse tracheal BLMS was ~6-fold higher during perfusion, than when flow was stopped. Munc13-2 null mouse tracheas, with their defect of accumulated cellular mucins, exhibited similar BLMS as WT, contrary to predictions of lower values. Graded mucous metaplasia induced in WT and Munc13-2 null tracheas with IL-13, caused proportional increases in BLMS, suggesting that naïve Munc13-2 mouse BLMS is elevated by increased mucin stores. We conclude that BLMS is, [i] a major component of mucin secretion in the lung, [ii] sustained by the mechanical activity of a dynamic lung, [iii] proportional to levels of mucin stores, and [iv] regulated differentially from agonist-induced mucin secretion.

Highlights

Mucus in the airways represents the first line of innate defense in the airways against inhaled aerosols and pathogens [1]
Baseline versus agonist-stimulated mucin secretion in human bronchial epithelial cell cultures (HBECCs). These first studies began with a methodological effort to define a rigorous, but practical, wash procedure for HBECCs that would minimize mechanical stress during removal of accumulated mucus, while preserving goblet cell mucin stores [15,23]. This procedure allows a comparison of the relative amounts of mucin secreted at baseline and during agonist stimulation of HBECCs (ATPγS, 100 μM) [5], as well as the mucins removed from the luminal surfaces during preparatory wash periods (i.e., a ‘Careful Wash’)
Was the ratio of baseline/stimulated mucin release modest, but the quantity of mucins removed from the cultures prior to the experiment, 5.98 μg, exceeded those released in response to agonist by 5.7-fold. These data are consistent with a relatively robust release of mucins at baseline; any conclusion regarding the relative rates of baseline and agonist-induced mucin secretion over macroscopic periods of time requires knowing the time required for intracellular mucin store recharge following stimulation by agonist

Summary

Introduction

Mucus in the airways represents the first line of innate defense in the airways against inhaled aerosols and pathogens [1]. The focus of research on goblet cell mucin secretion has been on agonist-induced mucin secretion, to the virtual exclusion of consideration of mucin secretion at baseline This focus may have been short-sighted: in 11 studies from 6 different laboratories working with goblet cells in native airways or primary airway epithelial cell cultures from human and other mammalian sources [7,8,9,10,11,12,13,14,15,16], the average increase of ATP-induced mucin release was just 3.2 ± 0.5 fold higher than baseline when determined over equal periods of time (mean ± SE). This modest stimulation suggests a hypothesis that the mucins secreted at baseline may be significant, a prospect investigated in this paper

Methods

Results

Conclusion