Immunomodulatory activities of small host defense peptides

Abstract

Recent studies have demonstrated that in addition to their antimicrobial activity, cationic host defense peptides, like the human cathelicidin LL-37, perform many activities relating to innate immunity, including the induction or modulation of chemokine and cytokine production, alteration of gene expression in host cells, and inhibition of proinflammatory responses of host cells to bacterial components such as lipopolysaccharide (LPS) in vitro and in vivo. To investigate if these properties are shared by smaller peptides, two cathelicidin peptides derived from bovine neutrophils, the 13-mer indolicidin and Bac2A, a linear 12-amino-acid derivative of bactenecin, were compared to the 37-amino-acid peptide LL-37. Indolicidin, like LL-37, inhibited LPS-induced tumor necrosis factor alpha (TNF-alpha) secretion, even when added up to an hour after the addition of Escherichia coli O111:B4 LPS to the human macrophage/monocyte-like THP-1 cell line. In contrast, Bac2A demonstrated no significant antiendotoxin activity. At low concentrations, indolicidin and LL-37 acted synergistically to suppress LPS-induced production of TNF-alpha. Indolicidin was analogous to LL-37 in its ability to induce the production of the chemokine interleukin-8 (IL-8) in a human bronchial cell line, 16HBE14o(-), but it was unable to induce production of IL-8 in THP-1 cells. In contrast, Bac2A was unable to induce IL-8 in either cell type. Conversely, Bac2A was chemotactic for THP-1 cells at concentrations between 10 and 100 mug/ml, while indolicidin and LL-37 were not chemotactic at these concentrations for THP-1 cells. This indicates that in addition to the potential for direct microbicidal activity, cationic host defense peptides may have diverse and complementary abilities to modulate the innate immune response.

FIG. 1.

LL-37, Bac2A, and indolicidin are not cytotoxic to THP-1 cells (A) or HBE cells (B) at concentrations ≤100 μg/ml. At 300 μg/ml, significant cytotoxicity was observed in indolicidin-treated HBE cells and in indolicidin- and LL-37-treated THP-1 cells. Mean values ± standard errors of the mean of three independent experiments are represented. A Student's t test was performed, and asterisks denote significance at a P value of <0.005 compared to cells treated with a vehicle control only.

FIG. 2.

Dose-dependent inhibition of LPS-induced TNF-α by addition of peptides. Indolicidin and LL-37 significantly reduced the amount of TNF-α produced by differentiated THP-1 cells upon stimulation with 100 ng/ml of E. coli O111:B4 LPS. Bac2A did not reduce the amount of TNF-α at the concentrations tested (N/D, no detectable decrease in TNF-α). Mean values ± standard error of the mean of one representative experiment of at least three are represented. A Student's t test was performed, and asterisks denote significance at a P value of <0.001 compared to controls treated with LPS only.

FIG. 3.

The addition of indolicidin and LL-37 in combination produces a greater-than-additive inhibition of LPS-induced TNF-α. Indolicidin and LL-37 significantly reduced the amount of TNF-α produced by differentiated THP-1 cells upon stimulation with 100 ng/ml of E. coli O111:B4 LPS. The combination of very small amounts of indolicidin (1 μg/ml) or LL-37 (1 μg/ml or 100 ng/ml) resulted in a greater reduction of LPS-induced TNF-α than would be predicted to occur if this effect was strictly additive. Mean values of the averages of three independent experiments ± the standard error of the mean are shown. A one-tailed Student's t test was performed, and the results significant at a P value of <0.05 are marked with an asterisk.

FIG. 4.

Bac2A is a more potent chemoattractant for undifferentiated THP-1 cells than indolicidin. Indolicidin, Bac2A, LL-37, or water (vehicle control) was added to the bottom well of a chemotaxis chamber at the concentrations shown. Undifferentiated THP-1 cells were added to the top well, and after 3 hours, chemotaxis was assessed. Chemotaxis was measured by calculating the total number of cells that migrated as a percentage of the total number of cells added. Mean values of the averages of three independent experiments ± the standard error of the mean are shown. A two-tailed t test was performed, and the results significant at a P value of <0.05 are marked with an asterisk, and those significant at a P value of <0.001 are marked by a double asterisk.

FIG. 5.

Indolicidin induces IL-8 production in a dose-dependent manner in the human bronchial epithelial cell line 16HBE4o⁻. Cells were grown to confluency and subsequently incubated with indolicidin for 8 hours. The presence of IL-8 in the supernatant was detected by ELISA. Mean values ± standard errors of the means of one representative experiment of at least three are represented (n = 4 replicates for each condition). A two-tailed t test was performed, and results significant at a P value of <0.05 are marked with an asterisk.