Induction of cathelicidin in normal and CF bronchial epithelial cells by 1,25-dihydroxyvitamin D(3)

Abstract

Background: Antimicrobial peptides (AMPs) such as cathelicidins contribute to initial defense of the airway against inhaled pathogens. Recent studies have shown that the hormonally active form of vitamin D(3), 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) up-regulates AMP gene expression in several established cell lines. Furthermore, serum levels of vitamin D are often deficient in cystic fibrosis (CF) patients.

Methods: We investigated the effect of 1,25(OH)(2)D(3) on AMP mRNA levels in primary cultures of normal human bronchial epithelial (NHBE) cells by real-time PCR, and protein levels by Western blot. Antimicrobial activity of airway surface fluid from these cells was measured by in vitro assay against laboratory strains of bacteria.

Results: Treatment of NHBE cells with 1,25(OH)(2)D(3) (10(-8)M), resulted in a 10-fold up-regulation of cathelicidin mRNA levels after 12 h, which was augmented 2-fold with co-incubation of 1 mM Calcium. Moreover, 1,25(OH)(2)D(3) induced antimicrobial activity against the airway pathogens Bordetella bronchiseptica and Pseudomonas aeruginosa. 1,25(OH)(2)D(3) induced cathelicidin mRNA expression equally in both normal and CF bronchial epithelial cells.

Conclusions: Elucidation of the effect of 1,25(OH)(2)D(3) on cathelicidin expression in NHBE cells and CF bronchial epithelial cells will aid in the development of novel therapeutic agents for treatment of airway infections in CF.

Fig. 1

Induction of cathelicidin gene expression in NHBE cells. NHBE cells were treated with 10⁻⁸M 1,25(OH)₂D₃ for times indicated. (A) Total RNA was extracted, and RTQ-PCR was conducted to quantify hBD1, 2, 3, and cathelicidin mRNA levels. (B) Kinetics of cathelicidin mRNA induction was determined as described above. β-Actin was used as a house-keeping gene. Results are represented as the means±SEM (*p<0.05) from at least three independent experiments. (C) NHBE cells were treated with 10^-8M 1,25(OH)₂D₃ or with vehicle for 24 or 48 h. Total cell lysates were analyzed by Western blot to observe hCAP18 expression. The amount of protein from the lysates was determined by Bradford assay, and equal amount of protein was loaded in each well, and a parallel blot was performed with an antibody to β-actin. Lane 1, 24 h, vehicle; lane 2, 24 h, 1,25(OH)₂D₃; lane 3, 48 h, vehicle; lane 4, 48 h, 1,25(OH)₂D₃.

Fig. 2

Mechanism of induction of cathelicidin in NHBE cells. NHBE cells were pretreated in the absence (−CHX) or presence (+CHX) of cycloheximide (20 μg/ml) for 30 min followed by 10⁻⁸M 1,25(OH)2D3. Total RNA was extracted, and RTQ-PCR was conducted to determine cathelicidin mRNA levels, normalized to β-actin. Graphs show means±SEM (*p<0.05) from at least three experiments.

Fig. 3

Role of calcium in 1,25(OH)2D3-mediated induction of LL-37. NHBE cells were pretreated with 1.0 mM CaCl₂ (+calcium) or vehicle (−calcium) for 24 h followed by 10⁻⁸M 1,25(OH)₂D₃ exposure for increasing times. Total RNA was collected and RTQ-PCR was conducted. Cathelicidin mRNA levels were determined by RTQ-PCR and normalized to β-2-microglobulin. Results show means±SEM (*p<0.05) of three independent experiments.

Fig. 4

Induction of antibiotic activity of airway surface fluid by 1,25(OH)2D3. NHBE cells were grown on Transwell CM inserts and exposed to 10⁻⁸M 1,25(OH)₂D₃ or vehicle basolaterally for 24 h. Antimicrobial activity of ASL against B. bronchiseptica and P. aeruginosa was determined by counting the number of colonies on 5% BG plates supplemented with 5% sheep blood and LB plates, respectively, after incubation with the ASL collected from the NHBE cells cultured above. (B) Inhibition of antimicrobial activity with LL-37 antibody. ASF was pretreated with 1 µl anti LL-37 antibody (gift of R. Gallo, UCSD) or control serum for 1 h prior to addition of P. aeruginosa in antimicrobial assays as described above. Results are reported as means±SEM (*p<0.05) of at least three independent experiments.

Fig. 5

Induction of LL-37 mRNA in normal and CF cell lines. AA and KK cell lines were grown to confluence and exposed to 10⁻⁸M 1,25(OH)₂D₃ for 0, 3, 6, 12, 24, and 48 h. Total RNA was extracted, and RTQ-PCR was performed to determine cathelicidin mRNA levels. β-2-Microglobulin primer sets were used for normalization. Results are represented as the means±SEM (*p<0.05) from at least three independent experiments.