Lactoferrin, a glycoprotein present in milk, mucosal secretions and neutrophils contributes to host defense. We have previously shown that orally given milk lactoferrin (LF) mediates anti-infectious and anti-inflammatory activities in vivo. Moreover, we have shown that LF could inhibit the LPS-induced IL-6 secretion in a human monocytic cell line, THP-1. This observation was expanded in the present study investigating the capacity of LF to inhibit cytokine mRNA expression and the involvement of nuclear transcription factor kappa B (NF-kappa B). Cells (THP-1 and Mono Mac 6 monocytic cell lines) were stimulated with Escherichia coli LPS (5-10 ng/10(6) cells) and LF was added (50-500 microg/10(6) cells) 30 min before, or after the LPS addition. By a semiquantitative RT-PCR lower levels of TNF-alpha, IL-1 beta, IL-6, and IL-8 mRNA expression were detected at the peak of the expression in THP-1 cells treated with LF. The reduction in the cytokine expression was followed by a similar reduction in the secreted cytokines as analyzed by ELISA. LF down-regulated also the IL-10 secretion (detected only in LPS-stimulated Mono Mac 6 cells). A similar level of inhibition of these cytokines was detected regardless of the time at which LF was added to the cells in relation to LPS. In addition, LF was internalized into cells and detected in the nucleoli as determined by immunostaining and immunofluorescence. Moreover, by electrophoretic mobility shift assay (EMSA) analysis LF decreased the LPS-induced binding of NF-kappa B to the TNF-alpha promoter. The results show that LF down-regulates the LPS-induced cytokine production in monocytic cells. The inhibitory mechanism is suggested to involve the interference of LF with NF-kappa B activation.