Antagonistic Effects Of Limulus Anti-lipopolysaccharide Factor On The Expression And Functions Of HMGB1

Objective This study aims to investigate the effect of Limulus Anti-lipopolysaccharide Factor ( LALF ) on the expression and immune functions of a newly found late proinflammatory cytokine named high mobility group box 1 (HMGB1) so as to find the new target of LALF and the mechanisms of animal death and survival in sepsis.Methods Murine macrophage-like RAW 264.7 cells were stimulated by LPS and TNF-αrespectively, with or without the presence of LALF. The expression of HMGB1 mRNA was analyzed by RT-PCR. The primary mouse macrophages were isolated and stimulated by HMGB1, with or without the presence of LALF. The culture supernatants were quantitated by ELISA for TNF-αproduction and macrophage migration was determined evaluated by the use of chemotaxis chamber. The effect of LALF on the expression of TLR2 on murine macrophage surface was measured by Flow cytometry (FCM).Results1. The expression of HMGB1 mRNA in RAW264.7 cells was increased by LPS and TNF-αstimulation respectively.2. The chemotactic index of macrophages and the concentration of TNF-αin the supernatant evidently increased in dose-dependent manner in HMGB1 treated groups.3. LALF markedly reduced the up-regulation of HMGB1 expression induced by LPS and TNF-α.4. The chemotactic index of macrophages and the concentration of TNF-αin the supernatant prominently decreased in the LALF co-incubated group.5. LALF greatly raised the percentage of TLR2-positive macrophages in a dose-dependent manner surveyed by FCM. Moreover, LALF synergistically enhanced LPS-induced TLR2 expression.Conclusion These results suggest that: 1. LPS or TNF-αalone can increase the expression of HMGB1 mRNA in RAW264.7 cells, while LALF plays an inhibitory role in LPS or TNF-αinduced HMGB1 mRNA expression. 2. High mobility group box-1 (HMGB1) protein acts as a chemoattractant and activator of murine macrophages. LALF can markedly reduce the migration and the TNF-αsecretion of macrophages. This study reveals a reciprocal functional relationship between the activities of the early (TNF-α) and late (HMG-1) cytokines and demonstrates that LALF confers protection against lethal experimental sepsis partly by inhibiting the expression and the pro-inflammatory activities of HMGB1. 3. LALF not only suppresses immune and inflammatory response, but also enhances the expression of the host defense receptor TLR2 on murine macrophage surface. Thus, our studies may bring new insights into the novel roles of LALF in orchestrating and optimizing host immune and defense responses during bacterial infections.