Tracheobronchial air-liquid interface cell culture: a model for innate mucosal defense of the upper airways?

Abstract

Human tracheobronchial epithelial cells grown in air-liquid interface culture have emerged as a powerful tool for the study of airway biology. In this study, we have investigated whether this culture system produces "mucus" with a protein composition similar to that of in vivo, induced airway secretions. Previous compositional studies of mucous secretions have greatly underrepresented the contribution of mucins, which are major structural components of normal mucus. To overcome this limitation, we have used a mass spectrometry-based approach centered on prior separation of the mucins from the majority of the other proteins. Using this approach, we have compared the protein composition of apical secretions (AS) from well-differentiated primary human tracheobronchial cells grown at air-liquid interface and human tracheobronchial normal induced sputum (IS). A total of 186 proteins were identified, 134 from AS and 136 from IS; 84 proteins were common to both secretions, with host defense proteins being predominant. The epithelial mucins MUC1, MUC4, and MUC16 and the gel-forming mucins MUC5B and MUC5AC were identified in both secretions. Refractometry showed that the gel-forming mucins were the major contributors by mass to both secretions. When the composition of the IS was corrected for proteins that were most likely derived from saliva, serum, and migratory cells, there was considerable similarity between the two secretions, in particular, in the category of host defense proteins, which includes the mucins. This shows that the primary cell culture system is an important model for study of aspects of innate defense of the upper airways related specifically to mucus consisting solely of airway cell products.

Fig. 1.

Schematic summary of the approach used in this study. AS, apical secretions; IS, induced sputum; GuHCl, guanidine HCl; MS, mass spectrometry; LC, liquid chromatography.

Fig. 2.

Group separation of mucins and proteins by density-gradient centrifugation. After CsCl/4 M GuHCl density-gradient centrifugation (60 h, 40,000 rpm), fractions (2 ml) were emptied from the top and assayed for protein by amido black (○) and carbohydrate by periodic acid-Schiff (PAS, ▴) stain (A) and for the mucins MUC5B (□) and MUC5AC (•) by immunodetection with mucin-specific antisera (B). Dashed lines, density gradient. Fractions were pooled to yield a protein-rich (fractions 1–6) and a mucin-rich (fractions 7–20) pool. Typical relative mass recoveries in 2 pools of the gradient are shown (measured by refractometry).

Fig. 3.

Identification of proteins in protein-rich pool by SDS-PAGE in combination with tandem MS (MS/MS). A: aliquots from AS and IS protein-rich pools after density-gradient centrifugation were dialyzed against water, freeze-dried, and solubilized in 8 M urea. Proteins were separated by SDS-PAGE on a 4–20% gradient gel under reducing conditions and visualized by Coomassie blue staining. Positions of molecular mass markers (kDa) are indicated at left. Major bands (1–7 for AS and 1–6 for IS) were digested in-gel with trypsin, and proteins were identified by MS/MS (B). First number in parentheses is number of peptides identified by gel digestion, and second number represents number of peptides identified for the same protein by shotgun analysis. Albumin detected in AS is from a bovine source and is derived from culture medium.

Fig. 4.

Mucins (A) and protein groups (B) in AS and IS. Values in A are averages over 4 independent experiments; error bars represent SD. All peptides from proteins from extraneous sources, e.g., saliva, serum, and lung, were excluded from B (see supplemental Table 2 for excluded proteins).

Fig. 5.

Analysis and comparison of cellular origin of proteins in AS and IS. Presence of secretory signals was checked by SignalP 3.0 and SecretomeP 2.0 server. Nonsecreted proteins were classified according to UniProtKB/Swiss-Prot database annotations. Potential salivary- and blood-sourced proteins were excluded.