Inflammatorv Cytokines Induced By Leptospiral Lipopolvsaccharide And Mononuclear-macrophage Infiltration In Tissues Of Infected Mice

Objective To understand the role of Leptospira interrogans lipopolysaccharide(L-LPS)on inducing human monocytes or mouse mononuclear-macrophages to produce inflammatory cytokines and the infiltration of peripheral blood-derived mononuclear-macrophages in tissues of L.interrogans-infected mice.Methods L-LPS of L.interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai was extracted using phenol-water method.LPS concentration and endotoxic activity of the L-LPS extract were determined using LPS quantitative detection kit and limulus test kit,respectively.Cytokine antibody array was used to detect the cytokines produced by human THP-1 monocytes or mouse J774A.1 mononuclear-macrophages after treatment with L-LPS or infection with L.nterrogans strain Lai.Cytokines in sera of L.interrogans strain Lai-infected C3H/HeJ mice were detected by cytokine antibody array.Using peripheral blood mononuclear-macrophage-specific CD68-IgG as the primary antibody and HRP-labelling donkey antirabit-IgG as the secondary antibody,infiltration of peripheral blood-derived CD68+ mononuclear-macrophages in lung,liver and kidney tissues of L.interrogans L.strain Lai-infected C3H/HeJ mice were detected by immunohistochamical examination.Results Approximate 1μg of L-LPS could be obtained from 109 leptospires.The minimal concentraon of L-LPS to solidify limulus amebocyte lysate was 0.5 ng/mL,which was 1/5 compared to that of Escherichia coli LPS(E-LPS).The levels of 29 cytokines in the supernatants of the THP-1 cells treated with L-LPS for 24 or 48 h were significantly increased,in which eotaxin,GM-CSF,ICAM-1,1-309,IL-lα,IL-6,IL-12p40,MCP-2,TNF-a or MIG level was increased with over 5-folds(p<0.01),while eotaxin-2,IFN-γ,IL-3,IL-10,IL-11,IL-12p70,MCP-1,MIP-1β,G-CSF,IL-1β,IL-7,IL-13,IL-15,IP-10,M-CSF,MIP-1δ,TGF-β1,sTNF RI or PDGF-BB level was increased with 1.5～<5-folds(p<0.05).The levels of 9 cytokines in the supernatants of the J774A.1 cells treated with L-LPS for 24 or 48 h were significantly increased,in which G-CSF,IL-6,IL-10,TNF-a or RANTES increased with over 5-folds(p<0.01)while MCP-1,IL-12p40,IL-1β or sTNF RII was increased with 1.5～<5-folds(p<0.05).The results of cytokine detection in the supernatants of L.interrogans strain Lai-infected THP-1 or J774A.1 cells and sera of L.interrogans strain Lai-infected mice were similar to that of L-LPS-treated THP-1 or J774A.1 cells as described above.A great number of the CD68+ mononuclear-macrophages were presented in the lung,liver and kidney tissues of the mice infected with L.interrogans strain Lai for 5 d.However,the CD68+ mononuclear-macrophages in the animal tissues were continuously increased when the mice were infected with the spirochete for 7 or 10 d as well as the structural ambiguity and cell disruption in the lung and kidney tissues could be observed.Conclusion L-LPS has a powerful role in induction of mononuclear-macrophages to up-regulate the expression of many inflammatory cytokines and mononuclear-macrophage chemotatic factors.IL-6 and TNF-a are the major induced inflammatory cytokines.There is an infiltration of numerous mononuclear-macrophages in the tissues of internal organs from L.interrogans-infected mice.