Figure 1.

SPLUNC1 reduces surface tension in airway epithelial secretions. A: Linear relationship between SPLUNC1 abundance and surface activity in airway epithelial fluids. Airway secretions were collected by rinsing the apical surfaces of primary cultured human airway epithelia with PBS. Secretions were then immunoblotted to assess SPLUNC1 expression levels and relative differences in SPLUNC1 abundance were quantitated using densitometry as described in Materials and Methods. In parallel, surface tension was measured for each sample using a pulsating bubble surfactometer. Surface tension values represent the minimum surface tension achieved after 2 minutes of pulsation. Each data point represents a secretion sample from a different human donor specimen. We observed a significant linear relationship between relative SPLUNC1 levels and surface tension (*P < 0.001). For each one-unit increase in SPLUNC1 abundance (expressed as normalized pixel intensity), surface tension was decreased by an estimated 15.1 ± 7.3 mNm. B: SPLUNC1 expression confers surface tension-reducing activity to airway epithelial secretions. Calu-3 cultures stably secreting SPLUNC1 protein, as well as negative control cells (empty vector), were grown at an air-liquid interface and rinsed apically with PBS to collect secretions. The presence of SPLUNC1 in the secretions was confirmed by immunoblot (top panel). In this blot, 250 ng recombinant human SPLUNC1 protein (rhSPLUNC1) serves as a positive control for the SPLUNC1 antibody. Overall surface tension in the wash samples was measured using the pulsating bubble surfactometer (bottom panel). Minimum surface tension after 10 minutes pulsation was significantly reduced in the SPLUNC1-containing washes, supporting the idea that surfactant activity by SPLUNC1 contributes significantly to the surface activity in airway epithelial washes (n = 3). *P = 0.01.