It has been postulated that reactive oxygen species (ROS) may act as second messengers leading to nuclear factor (NF)-kB activation. This hypothesis is mainly based on the ®ndings that N-acetyl-L-cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC), compounds recognized as potential antioxidants, can inhibit NF-kB activation in a wide variety of cell types. Here we reveal that both NAC and PDTC inhibit NF-kB activation independently of antioxidative function. NAC selectively blocks tumor necrosis factor (TNF)-induced signaling by lowering the af®nity of receptor to TNF. PDTC inhibits the IkB± ubiquitin ligase activity in the cell-free system where extracellular stimuli-regulated ROS production does not occur. Furthermore, we present evidence that endogenous ROS produced through Rac/NADPH oxidase do not mediate NF-kB signaling, but instead lower the magnitude of its activation.

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When supercoiled plasmid DNA was incubated with 2,2'-azobis (2-amidinopropane)hydrochloride (AAPH) at pH 7.4 in the presence and absence of oxygen, the DNA single strands were effectively cleaved. The breaking in the presence of oxygen was not inhibited by superoxide dismutase and catalase, but inhibited by mannitol, ethanol, butyl hydroxyanisole, thiol compounds, tertiary amines and spin trapping agents N-tert-butyl-alpha-phenylnitrone (PBN) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The breaking in the absence of oxygen was inhibited by ethanol, a tertiary amine and PBN. By electron spin resonance spin-trapping with PBN, the carbon-centered radical was detected both in the presence and the absence of oxygen. Hydroxyl radical was detected by use of DMPO only in the presence of oxygen. The DNA breaking activity of AAPH was found to be due primarily to the aliphatic carbon-centered radical. While the reactivity of carbon-centered radicals have received little attention, the aliphatic carbon-centered radical generated from AAPH was found to be highly reactive to break the DNA strands.

Lactoferrin (LF) has been implicated in innate immunity. Here we reveal the signal transduction pathway responsible for human LF (hLF)‐triggered nuclear factor‐κB (NF‐κB) activation. Endotoxin‐depleted hLF induces NF‐κB 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‐κB activation at a level comparable to that seen in wild‐type MEFs, whereas TRAF6‐deficient MEFs show significantly impaired NF‐κB activation in response to hLF. TRAF6 is known to be indispensable in leading to NF‐κB 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‐κB activation in MyD88‐deficient MEFs, delayed, but remarkable, NF‐κB 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‐κB 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.

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