Effect of lactoferrin hydrolysates on cytokine expression in Raw264.7 cells

Abstract text: Lactoferrin (LF) is an iron-binding glycoprotein which is present in colostrum, milk, and other body secretions. LF is associated with human infants’ activities, inflammatory and immune responses. The objective of this study was to determine the effects of alkaline protease generated lactoferrin hydrolysates (LH) on immunomodulatory activities of nitric oxide (NO) production and cytokines productions, including anti-inflammatory cytokines [interleukin(IL)-4], pro-inflammatory cytokines (interleukin-6, tumor necrosis factor-α, interferon-γ), Th2 cytokines (interleukin-4 or interleukin-6), and Th1 cytokines [tumor necrosis factor (TNF)-α, interferon (IFN)-γ] in immune cells. The presence of LH was confirmed by SDS-PAGE and HPLC analyses. The LH above 10 kDa and below 10 kDa were isolated from the extracted LH using 10 kDa cut-off centricon. Raw264.7 cells were treated with 3 different LH concentrations (1, 50, 100 μg/ml) for three types of LH (whole, above and below 10 kDa) treatments at 37^oC for 3hr, and then the culture supernatants were quantified by TNF-α and IL-1β ELISA kit. Cytokine expression levels in Raw264.7 cells were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). Results showed that one µg/ml of three types of LH treatments produced 1,500-2,000 ng/ml TNF-α, whereas the positive LPS (lipopolysaccharide) and negative controls produced 2,450 and 1,000 ng/ml TNF-α, respectively. The 50 µg/ml treatments of the three types of LH produced about 20-28 ng/ml IL-1β during at 3, 6, 9 hr, while negative control had 7 ng/ml. TNF-α expression was decreased dose-dependently by the 3 LH groups, while none of the LH treated groups  affected IL-6. The mRNA expression of IL-13 appeared in all LH concentration. In RAW264.7 cells treated with the 1, 50, 100 μg/ml for 3 hr, the mRNA expression induced a remarkable increase in nitric oxides synthesis (INOS) with dose dependent manner. NO was secreted dose-dependently from macrophages which were activated by all concentrations of the 3 LH treated groups. The results of RT-PCR revealed that LH caused INOS and inhibited the production of TNF-α in Raw264.7 cells. It was concluded that lactoferrin hydrolysates had immunomodulating effects on anti-, pro-inflammatory and anti-allergic reactions.

Keywords: Lactoferrin hydrolysates, cytokines, Raw264.7 cells