Production of beta-defensins by human airway epithelia

Abstract

Human beta-defensins (HBDs) are antimicrobial peptides that may play a role in mucosal defense. Diminished activity of these peptides has been implicated in the pathogenesis of cystic fibrosis (CF) lung disease. We show that HBD-1 and HBD-2 mRNAs are expressed in excised surface and submucosal gland epithelia from non-CF and CF patients. The pro-inflammatory cytokine interleukin-1beta stimulated the expression of HBD-2 but not HBD-1 mRNA and peptide in primary cultures of airway epithelia. HBD-1 was found in bronchoalveolar lavage (BAL) fluid from normal volunteers, CF patients, and patients with inflammatory lung diseases, whereas HBD-2 was detected in BAL fluid from patients with CF or inflammatory lung diseases, but not in normal volunteers. Both HBD-1 and HBD-2 were found in BAL fluid in concentrations of several ng/ml, and both recombinant peptides showed salt-sensitive bactericidal activity. These data suggest that in the lung HBD-2 expression is induced by inflammation, whereas HBD-1 may serve as a defense in the absence of inflammation.

Figures

Figure 1

Expression of HBD-1 and HBD-2 mRNAs in cultured normal and CF airway epithelia measured by using the ribonuclease protection assay. Cells were studied under basal conditions (−) and after 24 h of IL-1β treatment (+; 100 ng/ml). For both non-CF and CF specimens, constitutive HBD-1 mRNA expression was detected and was unchanged by IL-1β treatment. In contrast, the mRNA for HBD-2 was markedly induced after IL-1β treatment. 18S rRNA was used as an internal control to standardize the mRNA signal. Shown is a representative assay from three separate experiments with similar results.

Figure 2

Localization of β-defensin mRNA expression in normal and CF airway by in situ hybridization. For each pair of images the left-hand panel is a bright-field image and the right-hand panel is dark-field. (A) HBD-1 is expressed in normal human bronchus in surface and submucosal gland epithelia (antisense probe, 8-week exposure). (B) HBD-1 is also expressed in CF bronchus in surface and submucosal gland epithelia (antisense probe, 11-week exposure). (C) HBD-2 is expressed in normal human bronchus in surface and submucosal gland epithelia (antisense probe, 8-week exposure). (D) HBD-2 is expressed in CF bronchus in surface and submucosal gland epithelia (antisense probe, 8-week exposure). (E) Representative control sense riboprobe for HBD-1 with nonspecific background. Similar results were found with the HBD-2 sense riboprobe. Arrows denote submucosal glands. (×10.)

Figure 3

Detection of HBD-1 and HBD-2 proteins in non-CF and CF ASL by acid/urea/PAGE and immunoblotting. Epithelia were cultured with (+) and without (−) stimulation. (Upper) Comparison of HBD-1 peptide abundance in non-CF and CF epithelia. No HBD-1 peptide was found in washings from non-CF or CF epithelia, but 5 ng of recombinant HBD-1 peptide (STD) was easily detected. No HBD-1 was detected in the cell lysates (not shown). (Lower) Comparison of HBD-2 abundance in non-CF and CF epithelia. The apical surface was washed with water to remove accumulated peptide prior to IL-1β treatment. HBD-2 peptide was easily detected in the washings from non-CF and CF epithelia. Little peptide was recovered with the subsequent NH4OAc wash, indicating good recovery of protein with aqueous washes (not shown). HBD-2 was also detected in the cell lysate (not shown). With IL-1β stimulation, protein recovery increased in all fractions. Results for CF cells are qualitatively similar to those from cultured non-CF airway epithelia. Control was 7.5 ng of HBD-2. Representative results are shown.

Figure 4

Concentration of β-defensin peptides in BAL fluid measured by Western immunoblotting. Human BAL samples were obtained and analyzed by acid/urea/PAGE and Western immunoblotting with specific antisera for HBD-1 and HBD-2. Samples from non-CF (NL), CF, and patients with inflammatory lung diseases (ILD) were studied. (A) Representative immunoblot results for lysozyme (LYSO), HBD-1, and HBD-2 (BAL, patient sample; Std, protein standard). (B) BAL β-defensin concentrations determined by comparison to peptide standards. Each open circle represents the value from a single patient sample. HBD-1 was detectable in some samples from all three groups but there were no statistically significant differences among the groups. HBD-2 was detected only in BAL from patients with CF or ILD (P < 0.05 for both groups compared with non-CF by ANOVA).

Figure 5

Antimicrobial activity and salt sensitivity of recombinant HBD-1 and HBD-2 peptides. (A) E. coli luminescence assay. HBD-1 and HBD-2 both kill E. coli in a dose-dependent fashion. The activity of HBD-2 was ≈10-fold greater than that of HBD-1. Each point represents mean ± SE for triplicate samples. (B) Salt sensitivity of HBD-1 and HBD-2. Increasing concentrations of NaCl inhibit the activity of both peptides against E. coli. The degree of inhibition by salt is also dependent on the concentration of peptide. For HBD-1, ○ = 12 μg/ml, □ = 6 μg/ml, and ⋄ = 1.2 μg/ml. For HBD-2, ○ = 0.5 μg/ml, ⋄ = 0.1 μg/ml, □ = 0.05 μg/ml, and ▵, 0.01 μg/ml. Results were replicated twice. (C) Colony-forming unit (CFU) assay for killing of P. aeruginosa by HBD-1 and HBD-2. Each point represents mean ± SE for triplicate samples.