Mechanisms of mucin stimulation in response to inflammatory mediators in human airway epithelial cells in vitro

Stimulation of mucin hypersecretion by airway epithelial cells is associated with asthma, chronic bronchitis, and cystic fibrosis. The precise mechanisms of mucin hypersecretion are not fully known. Mucin secretion has been stimulated via a nitric oxide (NO)-dependent mechanism in differentiated guinea pig tracheal epithelial cells maintained in air-liquid interface (ALI) cultures. The hypothesis of the current studies was that cytokine-induced mucin secretion and/or expression of MUC5AC and MUC5B genes in normal human bronchial epithelial (NHBE) cells in ALI culture occurs via a NO-dependent signaling mechanism. NHBE cells were found to express mRNA of the three isoforms of nitric oxide synthase (NOS), detected by reverse transcriptase-polymerase chain reaction (RT-PCR). The isoforms were expressed based on the retinoic acid (RA) status of the cultures: Cultures grown in RA+ media undergo mucous differentiation, while RA-- cultures undergo squamous differentiation. Mucous differentiated NHBE cultures strongly expressed the inducible form of NOS (iNOS), while squamous cultures weakly expressed the gene. Endothelial NOS (eNOS) was only expressed by squamous cultures. Brain NOS (bNOS) was expressed in both types of cultures. Only the iNOS protein was detected in the cultures, but only after stimulation with cytomix, a mixture of 10 ng/ml each of tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma) and interleukin-1beta (IL-1beta). Culture conditions of NHBE cells were modified to offset high basal levels of mucin secretion plus MUC5AC and MUC5B gene expression. NHBE cells cultured in media composed of a 1:1 mixture of Dulbecco's modified Eagle's medium and bronchial epithelial cell basal medium (DMEM/BEBM) supplemented with ten hormones and/or growth factors were defined as complete (C) cultures. The first modification of the culture system was to nurture NHBE cells in 4-factor quiescence (4F-Q) media, composed of 1:1 DMEM/BEBM with insulin, bovine serum albumin, and RA, from day (D) 10--12 of culture; defined as early 4F-Q cultures. Levels of basal mucin secretion and gene expression were suppressed below those of C cultures. Mucin levels were considered to be too low (<10mug mucin equivalents/well) to classify early 4F-Q cultures as mucous cultures. Therefore, late stage 4F-Q cultures were established from D14--16. Basal levels of mucin secretion and gene expression were determined to be similar to those of C cultures. Therefore, studies of NHBE cells cultured in media composed of 1:1 DMEM/BEBM, without hormones/growth factors or with RA, insulin or bovine serum albumin for 48 h were done. These cultures were defined as quiescent (Q) cultures. Q cultures were found to secrete significantly lower basal levels of mucin and express MUC5AC and MUC5B at lower levels than C cultures. (Abstract shortened by UMI.).