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

Kesimer M, Kirkham S, Pickles RJ, Henderson AG, Alexis NE, Demaria G, Knight D, Thornton DJ, Sheehan JK - Am. J. Physiol. Lung Cell Mol. Physiol. (2008)

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Biochemistry and Biophysics, Univ. of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. kesimer@med.unc.edu

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.

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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).
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f2: 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).

Mentions: Other studies on the composition of mucous secretions (induced sputum, saliva, and cervical mucus) have highlighted their complexity. However, the mucins, which are major components of these secretions and key determinants of mucus properties, are not well represented in these analyses (8, 25, 33, 36, 37, 43, 44). Therefore, we sought to gain a more comprehensive analysis of mucin-containing secretions from the apical surface of human airway cells in culture (AS) and sputum induced from the airways of normal individuals (IS). To this end, CsCl/4 M GuHCl density-gradient centrifugation was performed to separate mucins from the other proteins in the AS and IS. A typical density-gradient profile (Fig. 2) shows that the majority of the proteins were present in the low-density fractions (Fig. 2A; assessed by amido black staining) while the gel-forming mucins were present in the higher-density fractions (Fig. 2; assessed by periodic acid-Schiff staining and immunoreactivity with mucin-specific antisera). Fractions were pooled to yield a protein- and a mucin-rich fraction, and the weight of material in each fraction was determined by refractometry after gel filtration chromatography. This analysis showed that, for AS, ∼62% of the total biomolecules are found in the protein-rich fraction and 38% are found in the mucin-rich fraction. Similar values were observed for IS: 65% in the protein-rich fraction and 35% in the mucin-rich fraction. The proteins in the two pools from each sample were digested with trypsin and identified by MS/MS (see materials and methods).


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

Kesimer M, Kirkham S, Pickles RJ, Henderson AG, Alexis NE, Demaria G, Knight D, Thornton DJ, Sheehan JK - Am. J. Physiol. Lung Cell Mol. Physiol. (2008)

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).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2636953&req=5

f2: 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).
Mentions: Other studies on the composition of mucous secretions (induced sputum, saliva, and cervical mucus) have highlighted their complexity. However, the mucins, which are major components of these secretions and key determinants of mucus properties, are not well represented in these analyses (8, 25, 33, 36, 37, 43, 44). Therefore, we sought to gain a more comprehensive analysis of mucin-containing secretions from the apical surface of human airway cells in culture (AS) and sputum induced from the airways of normal individuals (IS). To this end, CsCl/4 M GuHCl density-gradient centrifugation was performed to separate mucins from the other proteins in the AS and IS. A typical density-gradient profile (Fig. 2) shows that the majority of the proteins were present in the low-density fractions (Fig. 2A; assessed by amido black staining) while the gel-forming mucins were present in the higher-density fractions (Fig. 2; assessed by periodic acid-Schiff staining and immunoreactivity with mucin-specific antisera). Fractions were pooled to yield a protein- and a mucin-rich fraction, and the weight of material in each fraction was determined by refractometry after gel filtration chromatography. This analysis showed that, for AS, ∼62% of the total biomolecules are found in the protein-rich fraction and 38% are found in the mucin-rich fraction. Similar values were observed for IS: 65% in the protein-rich fraction and 35% in the mucin-rich fraction. The proteins in the two pools from each sample were digested with trypsin and identified by MS/MS (see materials and methods).

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Biochemistry and Biophysics, Univ. of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. kesimer@med.unc.edu

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.

Show MeSH
Related in: MedlinePlus