Structure-dependent functional properties of human defensin 5

Abstract

The mucosal epithelium secretes a variety of antimicrobial peptides that act as part of the innate immune system to protect against invading microbes. Here, we describe the functional properties of human defensin (HD) 5, the major antimicrobial peptide produced by Paneth cells in the ileum, in relation to its structure. The antimicrobial activity of HD-5 against Escherichia coli proved to be independent of its structure, whereas the unstructured peptide showed greatly reduced antimicrobial activity against Staphylococcus aureus. We find that HD-5 binds to the cell membrane of intestinal epithelial cells and induced secretion of the chemokine interleukin (IL)-8 in a concentration- and structure-dependent fashion. Incubation of HD-5 in the presence of tumor necrosis factor alpha further increased IL-8 secretion synergistically, suggesting that HD-5 may act as a regulator of the intestinal inflammatory response.

Figure 1

Folded and purified HD-5 and HD-5Abu analyzed by reversed phase high-performance liquid chromatography (RP-HPLC) and electrospray ionization mass spectrometry (ESI-MS). The HPLC analysis was carried out at 40 °C using a linear gradient of 15–60% (solvent A: water + 0.1% TFA; solvent B: acetonitrile + 0.1% TFA) at a flow rate of 1 ml/min over 30 min. The determined molecular masses were within experimental error of the expected values based on calculations of the average isotopic compositions.

Figure 2

(A) Survival curves of E. coli ATCC 25922 (left) and S. aureus ATCC 29213 (right) exposed to HD-5 (filled symbols) or HD-5Abu (open symbols). Strains were exposed to the peptides at concentrations varying twofold from 0.12 to 125 μg/ml. (B) Strains were exposed to fixed peptide concentrations (100 μg/ml for E. coli; 50 μg/ml for S. aureus) in the absence or presence of the indicated concentrations of sodium chloride. Each curve is the mean of three separate experiments. Points scored as zero survival could not be plotted.

Figure 3

Confocal laser scanning microscopy images of Caco-2 cells incubated with rhodamine-HD-5. Cells were incubated in serum-free RPMI medium for 3 hours with 10 μg/ml of the peptide and were gently washed twice with HBSS prior to imaging. Left panels show the bright field image, middle panels show the fluorescence image of the rhodamine-labeled peptide, and right panels are a superposition of the two images. The fluorescence image is a 1-μm optical section acquired approximately at the equator of the largest cells, which are typically 14 – 15 μm in thickness. Filled arrowheads indicate examples of surface labeling; open arrowheads indicate examples of internalization.

Figure 4

IL-8 secretion by Caco-2 cells in the absence (light grey bar) or presence of HD-5 (white bars) or HD-5Abu (black bars) at final concentrations of 50 or 100 μg/ml. TNFα (100 ng/ml; dark grey bar) served as a positive control. Following incubation for 18 hours, culture supernatants were analyzed for IL-8 using the Luminex-100 system in duplicate. Data represent mean and standard deviation of three individual experiments.

Figure 5

IL-8 secretion by Caco-2 cells in the presence of HD-5 (100 μg/ml; white bars) or HD-5Abu (100 μg/ml; black bars), with and without TNFα (100 ng/ml) as indicated. No peptides (c) and TNFα alone (dark grey bar) served as controls. Following incubation for 18 hours, culture supernatants were analyzed for IL-8 using the Luminex-100 system in duplicate. Data represent mean and standard deviation of three individual experiments.