Limits...
IL-4 and IL-13 exposure during mucociliary differentiation of bronchial epithelial cells increases antimicrobial activity and expression of antimicrobial peptides.

Zuyderduyn S, Ninaber DK, Schrumpf JA, van Sterkenburg MA, Verhoosel RM, Prins FA, van Wetering S, Rabe KF, Hiemstra PS - Respir. Res. (2011)

Bottom Line: Using sandwich ELISA we found that also hBD-2 in apical washes was increased by both IL-4 and IL-13.Apical wash obtained from IL-4- and IL-13-treated cultures displayed increased antimicrobial activity against Pseudomonas aeruginosa compared to medium-treated cultures.In addition, differentiation in the presence of Th2 cytokines resulted in increased MUC5AC production as has been shown previously.These data suggest that prolonged exposure to Th2 cytokines during mucociliary differentiation contributes to antimicrobial defence by increasing the expression and release of selected antimicrobial peptides and mucus.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pulmonology, Leiden University Medical Center, Leiden, The Netherlands. s.zuyderduyn@lumc.nl

ABSTRACT
The airway epithelium forms a barrier against infection but also produces antimicrobial peptides (AMPs) and other inflammatory mediators to activate the immune system. It has been shown that in allergic disorders, Th2 cytokines may hamper the antimicrobial activity of the epithelium. However, the presence of Th2 cytokines also affects the composition of the epithelial layer which may alter its function. Therefore, we investigated whether exposure of human primary bronchial epithelial cells (PBEC) to Th2 cytokines during mucociliary differentiation affects expression of the human cathelicidin antimicrobial protein (hCAP18)/LL-37 and human beta defensins (hBD), and antimicrobial activity.PBEC were cultured at an air-liquid interface (ALI) for two weeks in the presence of various concentrations of IL-4 or IL-13. Changes in differentiation and in expression of various AMPs and the antimicrobial proteinase inhibitors secretory leukocyte protease inhibitor (SLPI) and elafin were investigated as well as antimicrobial activity.IL-4 and IL-13 increased mRNA expression of hCAP18/LL-37 and hBD-2. Dot blot analysis also showed an increase in hCAP18/LL-37 protein in apical washes of IL-4-treated ALI cultures, whereas Western Blot analysis showed expression of a protein of approximately 4.5 kDa in basal medium of IL-4-treated cultures. Using sandwich ELISA we found that also hBD-2 in apical washes was increased by both IL-4 and IL-13. SLPI and elafin levels were not affected by IL-4 or IL-13 at the mRNA or protein level. Apical wash obtained from IL-4- and IL-13-treated cultures displayed increased antimicrobial activity against Pseudomonas aeruginosa compared to medium-treated cultures. In addition, differentiation in the presence of Th2 cytokines resulted in increased MUC5AC production as has been shown previously.These data suggest that prolonged exposure to Th2 cytokines during mucociliary differentiation contributes to antimicrobial defence by increasing the expression and release of selected antimicrobial peptides and mucus.

Show MeSH

Related in: MedlinePlus

Schematic representation of the air-liquid interface (ALI) culture model. Primary bronchial epithelial cells were seeded onto transwell filters and allowed to grow to confluence for 4-7 days. Cells were exposed to air at the apical side (air-liquid interface; ALI) and stimulated for 14 days at the basal side with medium (containing high concentrations of retinoic acid to induce mucociliary differentiation). To investigate effects of IL-4 or IL-13 on differentiating cells, IL-4 or IL-13 was added to the basal medium when cells were first exposed to air; medium with or without IL-4 or IL-13 was refreshed three times a week.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3113720&req=5

Figure 1: Schematic representation of the air-liquid interface (ALI) culture model. Primary bronchial epithelial cells were seeded onto transwell filters and allowed to grow to confluence for 4-7 days. Cells were exposed to air at the apical side (air-liquid interface; ALI) and stimulated for 14 days at the basal side with medium (containing high concentrations of retinoic acid to induce mucociliary differentiation). To investigate effects of IL-4 or IL-13 on differentiating cells, IL-4 or IL-13 was added to the basal medium when cells were first exposed to air; medium with or without IL-4 or IL-13 was refreshed three times a week.

Mentions: Primary bronchial epithelial cells (PBEC) were obtained from anonymized tumour-free lung tissue obtained at lung resection surgery for lung cancer by enzymatic digestion as described previously [19]. Cells from passage 2 were cultured at an air- liquid interface as described previously [9]. In short, cultures were grown submerged for 4-7 days until they reached confluence, after which they were cultured at an air-liquid interface (ALI) for another 2 weeks in medium containing a high concentration of retinoic acid (15 ng/ml; Lonza, Breda, The Netherlands) to induce mucociliary differentiation. IL-4 or IL-13 (Peprotech, Rocky Hill, NJ) was added during these two weeks of culture at the ALI (see Figure 1). Mucociliary differentiation was usually observed between day 7 and 10 after exposure to air-liquid interface. ALI cultures were maintained for 14 days at 37°C in a humidified atmosphere of 5% CO2. The apical side of the epithelial layers was washed with warm PBS three times a week; medium and stimuli were refreshed at the same time. At the end of the 14-day ALI culture period (48 hours after the last addition of stimulus), basal medium (BM) was harvested and frozen until further use. The apical side of the epithelial cultures was incubated with 100 μl of PBS containing 1% N-Acetyl-L-cysteine (Sigma-Aldrich) for 15 minutes at room temperature to dissolve mucus threads; this apical wash (AW) was frozen until further use.


IL-4 and IL-13 exposure during mucociliary differentiation of bronchial epithelial cells increases antimicrobial activity and expression of antimicrobial peptides.

Zuyderduyn S, Ninaber DK, Schrumpf JA, van Sterkenburg MA, Verhoosel RM, Prins FA, van Wetering S, Rabe KF, Hiemstra PS - Respir. Res. (2011)

Schematic representation of the air-liquid interface (ALI) culture model. Primary bronchial epithelial cells were seeded onto transwell filters and allowed to grow to confluence for 4-7 days. Cells were exposed to air at the apical side (air-liquid interface; ALI) and stimulated for 14 days at the basal side with medium (containing high concentrations of retinoic acid to induce mucociliary differentiation). To investigate effects of IL-4 or IL-13 on differentiating cells, IL-4 or IL-13 was added to the basal medium when cells were first exposed to air; medium with or without IL-4 or IL-13 was refreshed three times a week.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3113720&req=5

Figure 1: Schematic representation of the air-liquid interface (ALI) culture model. Primary bronchial epithelial cells were seeded onto transwell filters and allowed to grow to confluence for 4-7 days. Cells were exposed to air at the apical side (air-liquid interface; ALI) and stimulated for 14 days at the basal side with medium (containing high concentrations of retinoic acid to induce mucociliary differentiation). To investigate effects of IL-4 or IL-13 on differentiating cells, IL-4 or IL-13 was added to the basal medium when cells were first exposed to air; medium with or without IL-4 or IL-13 was refreshed three times a week.
Mentions: Primary bronchial epithelial cells (PBEC) were obtained from anonymized tumour-free lung tissue obtained at lung resection surgery for lung cancer by enzymatic digestion as described previously [19]. Cells from passage 2 were cultured at an air- liquid interface as described previously [9]. In short, cultures were grown submerged for 4-7 days until they reached confluence, after which they were cultured at an air-liquid interface (ALI) for another 2 weeks in medium containing a high concentration of retinoic acid (15 ng/ml; Lonza, Breda, The Netherlands) to induce mucociliary differentiation. IL-4 or IL-13 (Peprotech, Rocky Hill, NJ) was added during these two weeks of culture at the ALI (see Figure 1). Mucociliary differentiation was usually observed between day 7 and 10 after exposure to air-liquid interface. ALI cultures were maintained for 14 days at 37°C in a humidified atmosphere of 5% CO2. The apical side of the epithelial layers was washed with warm PBS three times a week; medium and stimuli were refreshed at the same time. At the end of the 14-day ALI culture period (48 hours after the last addition of stimulus), basal medium (BM) was harvested and frozen until further use. The apical side of the epithelial cultures was incubated with 100 μl of PBS containing 1% N-Acetyl-L-cysteine (Sigma-Aldrich) for 15 minutes at room temperature to dissolve mucus threads; this apical wash (AW) was frozen until further use.

Bottom Line: Using sandwich ELISA we found that also hBD-2 in apical washes was increased by both IL-4 and IL-13.Apical wash obtained from IL-4- and IL-13-treated cultures displayed increased antimicrobial activity against Pseudomonas aeruginosa compared to medium-treated cultures.In addition, differentiation in the presence of Th2 cytokines resulted in increased MUC5AC production as has been shown previously.These data suggest that prolonged exposure to Th2 cytokines during mucociliary differentiation contributes to antimicrobial defence by increasing the expression and release of selected antimicrobial peptides and mucus.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pulmonology, Leiden University Medical Center, Leiden, The Netherlands. s.zuyderduyn@lumc.nl

ABSTRACT
The airway epithelium forms a barrier against infection but also produces antimicrobial peptides (AMPs) and other inflammatory mediators to activate the immune system. It has been shown that in allergic disorders, Th2 cytokines may hamper the antimicrobial activity of the epithelium. However, the presence of Th2 cytokines also affects the composition of the epithelial layer which may alter its function. Therefore, we investigated whether exposure of human primary bronchial epithelial cells (PBEC) to Th2 cytokines during mucociliary differentiation affects expression of the human cathelicidin antimicrobial protein (hCAP18)/LL-37 and human beta defensins (hBD), and antimicrobial activity.PBEC were cultured at an air-liquid interface (ALI) for two weeks in the presence of various concentrations of IL-4 or IL-13. Changes in differentiation and in expression of various AMPs and the antimicrobial proteinase inhibitors secretory leukocyte protease inhibitor (SLPI) and elafin were investigated as well as antimicrobial activity.IL-4 and IL-13 increased mRNA expression of hCAP18/LL-37 and hBD-2. Dot blot analysis also showed an increase in hCAP18/LL-37 protein in apical washes of IL-4-treated ALI cultures, whereas Western Blot analysis showed expression of a protein of approximately 4.5 kDa in basal medium of IL-4-treated cultures. Using sandwich ELISA we found that also hBD-2 in apical washes was increased by both IL-4 and IL-13. SLPI and elafin levels were not affected by IL-4 or IL-13 at the mRNA or protein level. Apical wash obtained from IL-4- and IL-13-treated cultures displayed increased antimicrobial activity against Pseudomonas aeruginosa compared to medium-treated cultures. In addition, differentiation in the presence of Th2 cytokines resulted in increased MUC5AC production as has been shown previously.These data suggest that prolonged exposure to Th2 cytokines during mucociliary differentiation contributes to antimicrobial defence by increasing the expression and release of selected antimicrobial peptides and mucus.

Show MeSH
Related in: MedlinePlus