Studies on lactoferrin-induced activation of macrophage activation and endotoxin tolerance

Abstract

Lactoferrin (LF) is an iron-binding glycoprotein that is highly found in milk, and also present in exocrine secretions. LF is well known as a multifunctional protein including antibacterial, bactericidal, and antiviral activities. Although LF also functions in the regulation of inflammation and immunomodulation, its mode of action has not been fully elucidated. Previous studies suggested activity of LPS is inhibited by direct binding to LF. However, here we show that when the LPS and purified LF was mixed and formed complex (termed as LF-LPS), it was found to induce production of inflammatory mediators in macrophages to some extent rather than inhibit LPS activity. In this study, we examined the effect of LF-LPS on macrophages and investigated its mechanisms. Nitric oxide and TNF-α were induced by LF-LPS treatment in a dose dependent manner. Also, mRNA level of inflammatory cytokines and chemokines were increased with LFLPS dose dependently. Phosphorylation of MAP kinases was induced, and NF-κB DNA binding activity was increased by LF-LPS treatment. These serial results of activation were mediated by a cellular transmembrane receptor termed Toll-like receptor (TLR) 4: Comparative studies with C3H/HeN and C3H/HeJ mice demonstrated the strong dependency of the LF-LPS signal on TLR4. Production of nitric oxide or TNF-α was not affected by LF in the peritoneal macrophages from C3H/HeJ, a strain of tlr4 -/-. TLR4-dependent IFN-regulated gene 1 (IRG1) gene expression was shown only in RAW 264.7 cells or macrophages those are obtained from C3H/HeN mice. In addition, when macrophages were pretreated with LF-LPS, cells were rendered a tolerant state to lipopolysaccharide (LPS) challenge. The induction of tolerance by LF-LPS was accompanied by an inhibition of the production of inflammatory mediators, such as TNF-α and nitric oxide. LPSinduced gene expression of inflammatory cytokines IL-1β and IL-6 were also inhibited in LF-LPS-pretreated macrophages. When LF-LPS-pretre...