Question:

The main problem of inflammatory pulmonary diseases is the increased production of highly viscous mucus, which sticks to the respiratory epithelium and causes obstructive respiratory disorders. The bronchial mucus is a gel-like high molecular weight polymer consisting of different glycoproteins, the mucins. In the human respiratory tract four gel-forming mucins were identified: MUC2, MUC5AC, MUC5B and MUC19. MUC5AC is the dominant form during inflammatory processes. In addition to the high viscosity of the mucus, an interaction / adhesion between mucins and airway epithelium is supposed to occur, possibly resulting from the expression of a membrane-associated mucin receptor on airway epithelial cells in inflammatory processes. The aim of this work is to quantify the adhesion between the mucin MUC5AC and human airway epithelial cells at the single molecule level by force spectroscopy using atomic force microscopy (AFM). The results of this study should allow answering the question if a specific mucin-epithel interaction (receptor) exists during inflammatory processes. Identification of such a mucin receptor will open up new perspectives in the treatment of inflammatory pulmonary diseases.

Methods:

Lipopolysaccharide (LPS) treated human submucous gland cells (Calu-3 cells) were used to obtain mucin. These cells release MUC5AC into the supernatant after stimulation with Ca²⁺ ionophore. Since mucins polymerize spontaneously, acetylcysteine ??(ACC) was added in order to improve the yield of mucin monomers. An ethanolamine- or antibody- (anti-MUC5AC) modified cantilever (MLCT, k=0.01 N/m) was incubated in this supernatant in order to link mucin molecules to it. We used living human airway epithelial cells (16HBE14o^- cells) under normal and inflammatory conditions to quantify the adhesion of cantilever bound MUC5AC to the cell surface. Parameters of force spectroscopy (e.g. loading rate, tip velocity, contact time) had to be optimized thoroughly to achieve reliable data.

Results:

Measurements with unmodified cantilevers on 16HBE14o^- cells show detachment forces (unbinding forces) of approximately 140 pN with a detachment work of 0.14 fJ. The MUC5AC-modified tips reveal detachment forces with a peak at 270 pN and a detachment work of 0.45 fJ.

Conclusion:

Measured results link most likely to a specific binding between mucin MUC5AC and airway epithelium and indicate the presence of a MUC5AC receptor on airway epithelial cells. The molecular identification of a specific receptor for MUC5AC could be the first step towards the development of effective mucolytics and thus treatment of obstructive and inflammatory respiratory disorders.