Anti-inflammatory effect of lysozyme from hen egg white on mouse peritoneal macrophages

Abstract

Lysozyme from hen egg has been reported to possess an anti-inflammatory effect. However, little is known about its detailed mechanism. The mechanism of anti-inflammatory effect of lysozyme was examined in this study. When mouse macrophage-like cell line RAW264.7 cells and mouse peritoneal macrophages were activated with lipopolysaccharide (LPS) and then treated with lysozyme, the production of tumor necrosis factor-α and interleukin-6 was significantly suppressed. The effect was induced by suppressing the gene expression levels of both cytokines. Phagocytosis activity of peritoneal macrophages was not altered by the treatment with lysozyme, suggesting that lysozyme shows the anti-inflammatory effect without inhibiting the phagocytotic response of macrophages. In addition, lysozyme inhibited phosphorylation of c-jun N-terminal kinase (JNK) and was taken up by macrophages within 1 h after treatment of the cells with lysozyme. Overall results suggest that lysozyme is taken up intracellularly and suppresses LPS-induced inflammatory responses by inhibiting JNK phosphorylation.

Keywords: Anti-inflammatory effect; Interleukin-6; Lysozyme; Peritoneal macrophages; Tumor necrosis factor-α.

Fig. 1

Effect of lysozyme on cytokine production and cell viability of peritoneal macrophages and RAW264.7 cells. For cytokine production assay, peritoneal macrophages and RAW264.7 cells were pretreated with 100 ng/mL of LPS. After washing, the cells were treated with various concentrations of lysozyme or 10 mM sodium phosphate buffer (control; open circle). After incubation for 11 h, the concentrations of IL-6 and TNF-α in the culture medium were measured. Data are represented as mean ± standard deviations (n = 6). *p < 0.05 or **p < 0.01 against control by Dunnett’s test. For cell viability assay, peritoneal macrophages and RAW264.7 cells were pretreated with 100 ng/mL of LPS. After washing, the cells were treated with various concentrations of lysozyme or 10 mM sodium phosphate buffer (control; open circle). After incubation for 11 h, cell viability was measured using a WST-8 assay kit. Data are represented as mean ± standard deviations (n = 9). n.s. indicates no statistical significance against control by Dunnett’s test

Fig. 2

Effect of lysozyme on transcription of cytokine genes in peritoneal macrophages. Peritoneal macrophages were pretreated with 100 ng/mL of LPS for 1 h. After washing, the cells were treated with 500 µg/mL of lysozyme or 10 mM sodium phosphate buffer (control) for 5 or 11 h. The mRNA levels of IL-6 and TNF-α genes were evaluated using real-time RT-PCR. Data are represented as mean ± standard deviations of three independent experiments. *p < 0.05 against control by Student’s t test

Fig. 3

Effect of lysozyme on phagocytotic activity of peritoneal macrophages. Peritoneal macrophages were pretreated with 100 ng/mL of LPS for 1 h. After washing, the cells were treated with 500 µg/mL of lysozyme or 10 mM sodium phosphate buffer (control) for 5 or 11 h. After washing, the cells were treated with or without Texas Red-labeled zymosan A for 1 h under dark condition, and the phagocytotic activity was measured on a flow cytometer. A representative histogram from three independent experiments is shown. Data are represented as mean ± standard deviations (n = 3). **p < 0.01 against control by Tukey–Kramer test. n.s. indicates no statistical significance against control

Fig. 4

Internalization of lysozyme. Peritoneal macrophages and RAW264.7 cells were pretreated with 100 ng/mL of LPS at 37 °C for 1 h. After washing, the cells were treated with FITC-labeled lysozyme for 1 h. After washing and fixing with 4% paraformaldehyde, images were taken using a confocal laser scanning microscope

Fig. 5

Effect of lysozyme on signaling pathways involved in macrophage activation. Peritoneal macrophages were pretreated with 100 ng/mL of LPS or with 10 mM sodium phosphate buffer (blank) for 15 min. After washing, the cells were treated with 500 µg/mL of lysozyme or 10 mM sodium phosphate buffer (blank, control) for 30 min. The protein levels of ERK, JNK, and p38 were then evaluated using immunoblot analysis. The p-ERK, p-JNK, and p-p38 represent phosphorylated ERK, phosphorylated JNK, and phosphorylated p38, respectively. A representative blot from three independent experiments is shown. The result of densitometric analysis is expressed as the ratio of (phosphorylated JNK protein amount)/(whole JNK protein amount). Data are represented as mean ± standard deviations of three independent experiments. *p < 0.01 against control by Tukey–Kramer test