Human lactoferrin activates NF-kappaB through the Toll-like receptor 4 pathway while it interferes with the lipopolysaccharide-stimulated TLR4 signaling.

Lactoferrin (LF) has been implicated in innate immunity. Here we reveal the signal transduction pathway responsible for human LF (hLF)-triggered nuclear factor-kappaB (NF-kappaB) activation. Endotoxin-depleted hLF induces NF-kappaB activation at physiologically relevant concentrations in the human monocytic leukemia cell line, THP-1, and in mouse embryonic fibroblasts (MEFs). In MEFs, in which both tumor necrosis factor receptor-associated factor 2 (TRAF2) and TRAF5 are deficient, hLF causes NF-kappaB activation at a level comparable to that seen in wild-type MEFs, whereas TRAF6-deficient MEFs show significantly impaired NF-kappaB activation in response to hLF. TRAF6 is known to be indispensable in leading to NF-kappaB activation in myeloid differentiating factor 88 (MyD88)-dependent signaling pathways, while the role of TRAF6 in the MyD88-independent signaling pathway has not been clarified extensively. When we examined the hLF-dependent NF-kappaB activation in MyD88-deficient MEFs, delayed, but remarkable, NF-kappaB activation occurred as a result of the treatment of cells with hLF, indicating that both MyD88-dependent and MyD88-independent pathways are involved. Indeed, hLF fails to activate NF-kappaB in MEFs lacking Toll-like receptor 4 (TLR4), a unique TLR group member that triggers both MyD88-depependent and MyD88-independent signalings. Importantly, the carbohydrate chains from hLF are shown to be responsible for TLR4 activation. Furthermore, we show that lipopolysaccharide-induced cytokine and chemokine production is attenuated by intact hLF but not by the carbohydrate chains from hLF. Thus, we present a novel model concerning the biological function of hLF: hLF induces moderate activation of TLR4-mediated innate immunity through its carbohydrate chains; however, hLF suppresses endotoxemia by interfering with lipopolysaccharide-dependent TLR4 activation, probably through its polypeptide moiety.

Interaction of nitric oxide with glutathione or cysteine generates reactive oxygen species causing DNA single strand breaks.

It was found that reactive oxygen species (ROS) were generated in the interactions of nitric oxide (NO) with glutathione (GSH) or cysteine (CySH) under aerobic conditions. When supercoiled DNA was incubated with a mixture of NO/GSH, NO/CySH, NOC-7 (a NO donor)/GSH or NOC-7/CySH under aerobic conditions, DNA single-strand breaks were observed on agarose gel electrophoresis. The strand breaks were inhibited by common ROS scavengers: superoxide dismutase+catalase, the spin trapping agent 5,5-dimethyl-1-pyrroline-N oxide (DMPO), ethanol, and EDTA. The strand breaks were also caused by incubation with a mixture of S-nitrosoglutathione (GSNO) with GSH or CySH, which was inhibited by ROS scavengers. In the reaction of NO/GSH, GSNO rapidly formed and then gradually decreased, and in the reaction of GSNO/GSH, GSNO was gradually decreased. The decrease inf GSNO was accelerated in the presence of superoxide+catalase. Hydroxyl radical was detected in the mixtures of NO with GSH or CySH under aerobic conditions, and thiyl radicals were detected in the mixtures of GSNO with GSH or CySH under anaerobic conditions as examined in electron spin resonance studies using DMPO as a spin trap. The results indicate that the interaction of NO with thiols in the presence of O2 generates ROS that caused DNA single-strand breaks.