A Study Of The Effects And Mechanisms Of LBP Inhibitory Peptide On The Activation Of Macrophage And Mice Induced By Lipopolysaccharide

Activation of macrophages can occur by recognition of the structural features of microbes,including the bacterial cell wall component lipopolysaccharide [LPS (endotoxin)]. After its release from bacteria, LPS can bind to a specific binding protein, known as LPS-binding protein (LBP). LBP can transfer monomeric LPS to CD14. The binding of the LPS/LBP complex to CD14 and toll like receptors (TLRs) may result in the transfer of a signal across the cell membrane and activation of nuclear factor kappa B (NF-kappa B), as well as extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase, leading to uncontrollable cytokine production and nitric oxide (NO) release. These mediators, which orchestrate inflammatory reactions in the lung, are also considered to be essential for the host defense system. LPS-mediated stimulation of CD14-positive cells can be enhanced 100-1000 folds when LBP is added to serum-free systems and therefore LBP has been thought to participate in the pathogenesis of septic shock. However, LBP can also detoxify LPS by transferring LPS into high-density lipoprotein (HDL) particles in vitro, and HDL-bound LPS lacks cell stimulatory activity. Therefore, LBP is a two-edge sword in inflammatory reaction. In our previous studies, we obtained phage clones with the sequence of WKXRKXFXKXXG (homologus with 91 -102 amino acids WKVRKSFFKLQG of LBP) and synthesized peptide with the sequence of WKVRKSFFKLQG (named as LBP inhibitory peptide, namely P12). In this study, we tested the hypothesis that blocking of LBP at the inflammation sites may protect against inflammatory responses associated with low-dose LPS, and thus reduce mortality. We examined the effects of LBP blockade with P12 on the human monocyte-like cell line U937 cells and a mouse model of LPS induced endotoxemia. Methods: 1. In vitro (1) The relative affinity of P12 to LPS was determined by enzyme-linked immunosorbent assay (ELISA). (2) Effect of P12 on the binding of FITC-conjugated LPS (FITC-LPS) to U937 cells was determined by flow cytometric analysis (FACS). (3) The mRNA and protein of CD14 and TLR4 in U937 cells were detected by semi-quantitative RT-PCR and Western blotting. (4) Nuclear factor kappa B (NF-kappa B) activation in U937 cells was evaluated with immuocytochemistry (ICC) and subunit P65 by Western blotting. (5) The productions of tumor necrosis factor-alpha (TNF-) and nitric oxide (NO) were measured by ELISA and enzymatic assay with nitrate reductase. 2. In vivo (1) Endotoxemic mice induced by LPS were made and alveolar macrophages (AMs) were isolated from bronchoalveolar lavage fluid (BALF). (2) Effect of P12 on the FITC-LPS to AMs was tested by FACS. (3) Effect of P12 on NF-kappa B activation in lung was determined with immunohistochemistry (IHC) and Western blotting. (4) The concentrations of TNF-αand NO in serum of mice were assayed with ELISA and nitrate reductase and enzymatic activity assay. (5) The mortality was also observed. Results: 1. In vitro (1) The relative binding activity to LPS of P12 was higher than that of LBP in the same mass concentration. (2) P12 markedly suppressed the binding of FITC-LPS to U937 cells. (3) We found elevated expressiong of CD14 and TLR4 in U937 cells challenged with LPS and P12 significantly reduced the expression of CD14 and TLR4. (4) In LPS group NF-kappa B activation was increased compared with that in control group, however NF-kappa B activation was inhibited in U937 cells challenged with low-dose LPS (100 ng/ml) after P12 treatment.(5) We found elevated concentrations of TNF-and NO in supernant of U937 cells induced by LPS. P12 treatment significantly reduced the production of TNF-and NO in U937 cells induced by low-dose LPS. 2. In vivo (1) Endotoxemic mice administered P12 revealed decreased inflammation in lung compare with the mice challenge with LPS. (2) P12 markedly suppressed the binding of FITC-LPS to AMs, inhibited NF-kappa B activation in lung of mice and reduced the concentration of TNF-and NO in serum of mice. (3) P12 reduced the mortality of endotoxemic mice induced by low-dose LPS. Conclusion: These studies suggest that LBP inhibitory peptide with the sequence of WKVRKSFFKLQG could inhibit the inflammatory function of LBP, attenuate LPS-induced circulatory shock and protect mice from death induced by LPS. The possible mechanism include some aspects as fellows, ①P12 significantly inhibiting the binding of FITC-LPS to U937 cells and AMs;②P12 significantly reducing the mRNA and protein expression of CD14 and TLR4, suppressing NF-κB activation and production of TNF-and NO, resulting in reducing the activation of U937 cells and AMs induced by low-dose LPS but not high-dose LPS. These results indicate that LBP inhibitory peptide play a protective role in the activation of U937 cells and mice induced by LPS through blocking the inflammatory site of LBP but not blocking the antiinflammatory site and provide new ideas for the treatment of endotoxemia.