Regulation effects of LBP on activating macrophages to secrete cytokines

Objective To study the regulatory effects of lipopolysaccharide (LPS) binding protein (LBP) on the process of LPS transference and activation effects,and observe the secretion balance of macrophage cells in secreting pro-inflammatory cytokines and anti-inflammatory cytokines as a result of stimulation by LPS. Methods After proliferation and passage,the human THP-1 cells were induced into macrophages by PMA. Experiment was divided into four groups:LPS group,LBP group,LBP/LPS pre-incubation group,and LBP/LPS without pre-incubation group. Cells were cultured for 4,12 and 24 hours,respectively,and the supernatants were then removed. TNF-α,IL-10 and IL-6 contents were determined by luminescence assay,the results were then statistically analyzed. Results a) THP-1 cells grew as suspended cells,and transferred as adherent macrophages after PMA inducement. b) LPS in respective concentration of 1ng/ml,10ng/ml,100ng/ml and 1000ng/ml was used alone in stimulating macrophages,the secretion levels of TNF-α differed significantly between 1ng/ml group and the other 3 groups (P0.05),while no significant difference was found among the other 3 groups (P0.05). c) TNF-α and IL-6 secretion reached a maximum value when macrophages were stimulated by LBP in concentration of 1000ng/ml. d) In LBP/LPS without pre-incubation group,the peak value of TNF-α and IL-6 secretion was fourd when the LBP/LPS was equal to 10; the TNF-α and IL-6 secretion curve was more or less flat without a peak in LBP/LPS pre-incubation group; IL-10 was less than 5pg/ml. e) Factorial analysis revealed that no interaction existed between LPS and LBP (F=3.425,P0.01),incubation and stimulation time (F=4.240,P=0.026),as well as between LBP and incubation time (F=4.896,P0.01). Conclusions LPS plays an important role in activating macrophages. LBP,which may stimulate macrophages in a high concentration,shows positive regulatory effect in low dose,while high dose of LPS shows a negative regulatory effert. After PMA differentiation in vitro from mononuclear cells,macrophages do not show significant IL-10 secretion by LPS stimulation,thus leading to an imbalance of pro-inflammatory cytokines and anti-inflammatory cytokines.