Protective effects of a human 18-kilodalton cationic antimicrobial protein (CAP18)-derived peptide against murine endotoxemia

Abstract

CAP18 (an 18-kDa cationic antimicrobial protein) is a granulocyte-derived protein that can bind lipopolysaccharide (LPS) and inhibit various activities of LPS in vitro. The present study examined the protective effect of a synthetic 27-amino-acid peptide (CAP18(109-135)) from the LPS-binding domain of CAP18 against antibiotic-induced endotoxin shock, using highly LPS-sensitive D-(+)-galactosamine (D-GalN)-sensitized C3H/HeN mice. The antibiotic-induced endotoxin (CAZ-endotoxin) was prepared from the culture filtrate of Pseudomonas aeruginosa PAO1 exposed to ceftazidime (CAZ). Injection of CAP18(109-135) protected the mice injected with LPS or CAZ-endotoxin from death and lowered their tumor necrosis factor (TNF) levels in serum in a dose-dependent manner. Treatment with CAZ caused death of the D-GalN-sensitized P. aeruginosa PAO-infected mice within 48 h, while injection with CAP18(109-135) rescued the mice from death. In the mice rescued from death by injection with CAP18(109-135), endotoxin levels in plasma and TNF production by liver tissues were decreased but the numbers of viable infecting bacteria in their blood were not decreased significantly and remained at the levels in CAZ-treated mice. These results indicate that CAP18(109-135) is capable of preventing antibiotic-induced endotoxic shock in mice with septicemia and that the effect is due to its LPS-neutralizing activity rather than to its antibacterial activity.

FIG. 1

Protective effect of CAP18_109–135 against the lethal toxicity of LPS/endotoxin in d-GalN-sensitized mice. (A) C3H/HeN mice were injected i.p. with CAP18_109–135 at the indicated doses and immediately injected i.p. with LPS (100 ng/mouse) and d-GalN (18 mg/mouse). (B) C3H/HeN mice were injected i.p. with CAP18_109–135 (40 μg/mouse) or saline, CAZ-endotoxin (100 or 200 ng of LPS equivalent/mouse) or saline, and d-GalN (18 mg/mouse). Death was scored for 1 week after the injection.

FIG. 2

Effect of CAP18_109–135 on LPS/endotoxin-induced TNF levels in serum of mice. Mice were injected i.p. with CAP18_109–135 at the indicated doses or with saline and immediately injected i.p. with LPS (5 μg/mouse) or saline (A) or CAZ-endotoxin (2 μg of LPS equivalent/mouse) (B). Blood was collected 1 h after the injections. TNF levels in serum were determined by a functional cytotoxicity assay with L929 cells as the target cells.

FIG. 3

Protective effect of CAP18_109–135 against death in CAZ-treated, d-GalN-sensitized mice infected with P. aeruginosa. Mice were injected i.p. with CAZ (20 mg/kg in 0.2 ml per mouse), P. aeruginosa PAO organisms (3 × 10⁶ CFU/mouse), and d-galactosamine (18 mg/mouse). At 30, 90, and 150 min after the infection, the mice were injected with CAP18_109–135 (80 μg per mouse per injection) (○) or saline (•). Death was recorded every 24 h after the infection until day 7.

FIG. 4

Effects of CAZ and/or CAP18_109–135 on endotoxin levels in plasma and numbers of bacteria in the blood of P. aeruginosa-infected and d-GalN-sensitized mice. Mice were injected i.p. with CAZ (20 mg/kg in 0.2 ml per mouse) or saline, infected with P. aeruginosa PAO (3 × 10⁶ CFU/mouse in an experiment for endotoxin levels and 6 × 10⁶ CFU/mouse in an experiment for determination of the numbers of bacteria), and d-GalN (18 mg/mouse). At 30 and 90 min after the infection, the mice were injected with CAP18_109–135 peptide (80 μg per mouse per injection) or saline. The endotoxin levels (A) and number of bacteria (B) in the blood were determined 120 min after the infection.

FIG. 5

Effect of CAP18_109–135 peptide on the Limulus assay. In experiment 1, LPS (50 pg/ml) was mixed with CAP18_109–135 (400 μg/ml) and incubated at 37°C for 10 min. Then 50 μl of the Endospecy reagent was added to the mixture, which was incubated at 37°C for a further 30 min. At the end of the incubation, the absorbance of the reaction mixture was measured at 405 nm (A₄₀₅). In experiment 2, (1 → 3)-β-d-glucan (48.4 pg/ml in 25 μl) was incubated with CAP18_111–137 (200 μg/ml in 25 μl) at 37°C for 60 min. Then G-test reagent was added to the mixture, and the mixture was incubated at 37°C for a further 30 min. At the end of the incubation, the absorbance of the reaction mixture was measured at 405 nm. In experiment 3, LPS (10 μg/ml) was mixed with a solution of coagulation factors prepared from horseshoe crab (factor C, factor B, and proclotting enzyme) and the mixture was incubated at 37°C for 30 min. Then CAP18_111–137 (315 μg/ml) was added to the mixture, which was incubated at 37°C for a further 60 min. Finally, the substrate solution was added to the mixture, and the mixture was incubated at 37°C for a further 30 min. At the end of incubation, the absorbance of the reaction mixture was measured at 405 nm.

FIG. 6

Effects of CAP18_109–135 on TNF secretion in the liver tissues taken from P. aeruginosa-infected, CAZ-treated, d-GalN-sensitized mice. The mice were injected with CAZ (20 mg/kg in 0.2 ml per mouse) or saline, infected with P. aeruginosa (3 × 10⁶ CFU/mouse), and then injected with d-GalN (18 mg/mouse). At 30 and 90 min after the infection, the mice were treated with CAP18_109–135 peptide (80 μg per mouse per injection) or saline. The livers were removed from the mice 120 min after the infection, and liver slices were prepared as described in Materials and Methods. Each slice was incubated for 30 min, and the culture supernatants were collected. The TNF activity in the supernatants was determined by a functional cytotoxicity assay with L929 cells.