Roles And Mechanisms Of Endotoxin Tolerance On Neutrophils:a Preliminary Study

Periodontitis is a chronic inflammatory disease induced by bacteria,and is characterized by the loss of supporting tissues.Lipopolysaccharide(LPS)is the outer membrane of G’ bacteria.It can activate host immune responses,including the production of pro-inflammatory cytokines,anti-inflammatory cytokines and chemokines,which might be important in the development of periodontitis.Exposure of the host to periodontal pathogens and their virulence factors induces a state of hyporesponsiveness to subsequent stimulations,which is termed endotoxin tolerance.Neutrophils are the first line of defense against pathogenic bacteria in periodontal tissues,and can kill bacteria through phagocytosis,respiratory burst and cytokine production.In the process of endotoxin tolerance,changes of neutrophil responses are currently unknown.Monocytes/macrophages are another important effector cells against intruding bacteria in innate immune system.Potential influences and mechanisms of tolerized-macrophages/monocytes on neutrophils still unclear.Objective:The aim of this study is to observe the effects of endotoxin tolerance on the production of reactive oxygen species(ROS),levels of apoptosis and production of inflammatory cytokines in neutrophils and to explore the roles and mechanisms of Porphyromonas gingivalis(P.gingivalis)lipopolysaccharide(LPS)-tolerized monocytes on chemotaxis in neutrophils.Methods:1.Neutrophils were obtained from venous blood from healthy donors by density gradient centrifugation.THP-1 cells were purchased from Shanghai Institute of Cell of Chinese Academy of Science.2.Freshly isolated neutrophils were pretreated with 1μg/ml P.gingivalis LPS(12h),washed and stimulated with the same LPS again.Escherichia coli(E.coli)LPS was served as positive control.Production of ROS and Caspase 3 expression levels were measured by flow cytometer,and levels of TNF-α in supernatants were detected by ELISA.3.THP-1 cells were pretreated with 1μg/ml P.gingivalisLPS or 1μg/ml E.coli LPS,washed and stimulated with the same LPS again.Neutrophils recruited by the supernatants from THP-1 cells and then migrated through Transwell membrane were counted.Levels of epithelial neutrophil-activating peptide 78(ENA-78)in supernatants from THP-1 cells were detected by ELISA.Results:1.No difference was found in ROS generation in neutrophils stimulated with 1μg/ml P.gingivalis LPS compared with those without any stimulations(p>0.05),while ROS levels in the cells challenged by E.coli LPS were increased significantly(p<0.05).There was a marked decrease in ROS production in neutrophils with repeated P.gingivalis LPS or E.coli LPS stimulations compared with those following single challenge(p<0.05).2.No difference was found in Caspase 3 expressions in neutrophils stimulated with 1μg/ml P.gingivalis LPS or 1μg/ml E.coli LPS compared with those stimulated without any stimulations(p>0.05).In addition,after repeated P.gingivalis LPS or E.coli LPS stimulations,expression levels of Caspase 3 were decreased significantly compared with those following single challenge(p<0.05).3.Compared with those without any stimulations,the amounts of TNF-αsecreted by the cells treated with 1μg/ml P.gingivalis LPS or 1μg/ml E.coli LPS were increased significantly(p<0.05).In addition,secretions of TNF-α were also decreased significantly after repeated P.gingivalis LPS or E.coli LPS stimulations(p<0.05).4.There was an increase in the numbers of neutrophils migrated to the supernatants from THP-1 cells stimulated with P.gingivalis LPS or E.coli LPS only once(p<0.05).Moreover,a decrease in the numbers of neutrophils migrated to the supernatants from two kinds of LPS-tolerized THP-1 cells could be observed compared with those stimulated with supernatants from LPS-stimulated THP-1 cells(p<0.05).There were no differences in ENA-78 production in THP-1 cells stimulated with P.gingivalis LPS and culture medium(p>0.05),while the amounts of ENA-78 produced by THP-1 cells challenged by E.coli LPS were increased significantly(p<0.05).In addition,secretions of ENA-78 were decreased significantly after repeated P.gingivalis LPS or E.coli LPS stimulations compared with those following single challenge(p<0.05).Conclusion:Endotoxin tolerance might coctribute to depressing respratory burst,apoptosis and secretions of TNF-α in neutrophils,and the suppressed production of chemokkine ENA-78 from tolerized THP-1 cells might be responsible for the impaired migration of neutrophils.