Augmentation of innate host defense by expression of a cathelicidin antimicrobial peptide - PubMed (original) (raw)

Augmentation of innate host defense by expression of a cathelicidin antimicrobial peptide

R Bals et al. Infect Immun. 1999 Nov.

Abstract

Antimicrobial peptides, such as defensins or cathelicidins, are effector substances of the innate immune system and are thought to have antimicrobial properties that contribute to host defense. The evidence that vertebrate antimicrobial peptides contribute to innate immunity in vivo is based on their expression pattern and in vitro activity against microorganisms. The goal of this study was to investigate whether the overexpression of an antimicrobial peptide results in augmented protection against bacterial infection. C57BL/6 mice were given an adenovirus vector containing the cDNA for LL-37/hCAP-18, a human cathelicidin antimicrobial peptide. Mice treated with intratracheal LL-37/hCAP-18 vector had a lower bacterial load and a smaller inflammatory response than did untreated mice following pulmonary challenge with Pseudomonas aeruginosa PAO1. Systemic expression of LL-37/hCAP-18 after intravenous injection of recombinant adenovirus resulted in improved survival rates following intravenous injection of lipopolysaccharide with galactosamine or Escherichia coli CP9. In conclusion, the data demonstrate that expression of an antimicrobial peptide by gene transfer results in augmentation of the innate immune response, providing support for the hypothesis that vertebrate antimicrobial peptides protect against microorganisms in vivo.

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Figures

FIG. 1

FIG. 1

LL-37 in BALF and serum after administration of the recombinant virus or synthetic peptide. (A) Levels of LL-37 in BALF as determined by quantitative dot blot analysis. Virus was injected on day 1 of the experiment, and concentrations in BALF were determined on the following days. (B) Levels of LL-37 in serum as determined by quantitative dot blot analysis. Virus or peptide was injected on day 1 of the experiment, and concentrations in serum were determined by bleeding the animals and using the serum for qualitative dot blot analysis. (C) Western blots following denaturing polyacrylamide gel electrophoresis under reducing conditions with Tricine gels of mouse serum and BALF, using a polyclonal antibody against LL-37/hCAP-18. Lanes: 1, 20 ng of synthetic LL-37 peptide; 2, serum from a mouse that received the control vector coding for β-galactosidase; 3, serum from a mouse that received the vector coding for LL-37/hCAP-18 (crude); 4, serum from a mouse that received the vector coding for LL-37/hCAP-18 (RP-HPLC purified); 5, 20 ng of synthetic LL-37 peptide; 6, BALF from a mouse that received the vector coding for β-galactosidase (crude); 7 and 8, BALF from a mouse that received the vector coding for LL-37/hCAP-18 (crude, lane 7; HPLC purified, lane 8).

FIG. 1

FIG. 1

LL-37 in BALF and serum after administration of the recombinant virus or synthetic peptide. (A) Levels of LL-37 in BALF as determined by quantitative dot blot analysis. Virus was injected on day 1 of the experiment, and concentrations in BALF were determined on the following days. (B) Levels of LL-37 in serum as determined by quantitative dot blot analysis. Virus or peptide was injected on day 1 of the experiment, and concentrations in serum were determined by bleeding the animals and using the serum for qualitative dot blot analysis. (C) Western blots following denaturing polyacrylamide gel electrophoresis under reducing conditions with Tricine gels of mouse serum and BALF, using a polyclonal antibody against LL-37/hCAP-18. Lanes: 1, 20 ng of synthetic LL-37 peptide; 2, serum from a mouse that received the control vector coding for β-galactosidase; 3, serum from a mouse that received the vector coding for LL-37/hCAP-18 (crude); 4, serum from a mouse that received the vector coding for LL-37/hCAP-18 (RP-HPLC purified); 5, 20 ng of synthetic LL-37 peptide; 6, BALF from a mouse that received the vector coding for β-galactosidase (crude); 7 and 8, BALF from a mouse that received the vector coding for LL-37/hCAP-18 (crude, lane 7; HPLC purified, lane 8).

FIG. 1

FIG. 1

LL-37 in BALF and serum after administration of the recombinant virus or synthetic peptide. (A) Levels of LL-37 in BALF as determined by quantitative dot blot analysis. Virus was injected on day 1 of the experiment, and concentrations in BALF were determined on the following days. (B) Levels of LL-37 in serum as determined by quantitative dot blot analysis. Virus or peptide was injected on day 1 of the experiment, and concentrations in serum were determined by bleeding the animals and using the serum for qualitative dot blot analysis. (C) Western blots following denaturing polyacrylamide gel electrophoresis under reducing conditions with Tricine gels of mouse serum and BALF, using a polyclonal antibody against LL-37/hCAP-18. Lanes: 1, 20 ng of synthetic LL-37 peptide; 2, serum from a mouse that received the control vector coding for β-galactosidase; 3, serum from a mouse that received the vector coding for LL-37/hCAP-18 (crude); 4, serum from a mouse that received the vector coding for LL-37/hCAP-18 (RP-HPLC purified); 5, 20 ng of synthetic LL-37 peptide; 6, BALF from a mouse that received the vector coding for β-galactosidase (crude); 7 and 8, BALF from a mouse that received the vector coding for LL-37/hCAP-18 (crude, lane 7; HPLC purified, lane 8).

FIG. 2

FIG. 2

Expression of LL-37/hCAP-18 in mouse liver and lungs after gene transfer. Organs were harvested 5 days after gene transfer and analyzed for transgene expression by RT-PCR and immunohistochemistry. Immunohistochemistry with polyclonal antibodies to LL-37/hCAP-18 revealed signals in hepatocytes (B) or epithelial cells of airways (D) of mice that received LL-37 vector but not in those of mice treated with Ad.AlkPhos vector (A and C). Bar, 100 μm. (E) RT-PCR was performed with LL-37/hCAP-18-specific primers and revealed the presence of transcripts only in mice after application of LL-37 vector. Amplification of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was used as a positive control. The PCR products were blotted and hybridized to an LL-37/hCAP-18-specific probe. Lane LL-37, positive control with plasmid DNA; lanes 1 and 2, PCR on RNA extracted from lungs (lane 1) or liver (lane 2) obtained from a mouse treated with lacZ vector; lanes 3 and 4, PCR on RNA extracted from lungs (lane 3) or liver (lane 4) obtained from a mouse treated with LL-37 vector.

FIG. 3

FIG. 3

Effect of gene transfer of LL-37/hCAP-18 to the respiratory tracts of mice on the bacterial load and inflammatory response. Mice were injected intratracheally on day 1, challenged with bacteria on day 5, and euthanized on day 6. Individual data points are presented. The bar represents the mean. (A) The bacterial load was significantly decreased in mice that received the LL-37/hCAP-18-encoding vector (Ad.LL-37) (P < 0.005). (B) Levels of TNF-α were significantly lower in mice that received the LL-37/hCAP-18-encoding vector (P < 0.05) (40 mice per group).

FIG. 4

FIG. 4

Effect of the systemic overexpression of LL-37/hCAP-18 in mice on survival after intraperitoneal injection of LPS (in galactosamine-sensitized mice) or gram-negative bacteria. (A) Mice received either lacZ or LL-37 vector on day 1 and were injected with LPS plus galactosamine or E. coli CP9 on day 5. Survival of the animals that were treated with LL-37 vector (Ad.LL-37) (5 × 1010 particles) was significantly (∗∗, P < 0.05) increased compared to survival of the mice that received the same dose of lacZ control vector (Ad.lacZ) (20 mice in each group). (B) Dose-dependent survival of animals treated with different amounts of LL-37/hCAP-18-encoding virus. Whereas the control group treated with β-galactosidase-encoding vector showed high mortality after injection, the animals that received LL-37 vector showed increased survival rates that correlated with the amount of virus applied and therefore with the level of the LL-37 peptide in serum (10 mice in each group).

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