| Objective: This study investigated the expression of membrane receptor CD14, TLR4, SR-A and MHC-Ⅱin healthy and hyperlipidemic rabbit tissue-specific monocytes/ macrophages stimulated by Porphyromonas gingivalis lipopolysaccharide, with or without the additional of exogenous IL-10. The aim of the study is to provide a theoretic foundation for understanding the relationship of periodontal diseases and systemic disorders.Methods Peripheral mononuclear cells (Mo) were separated by Ficoll-Hypaque density gradient centrifugation; alveolar macrophages (AM) were isolated by alveolar lavage; peritoneal macrophages were isolated by peritoneal irrigation; and liver kupffer cells (KC) were isolated by enzyme digestion plus Percoll density gradient centrifugation. Cellular morphology and microstructure were evaluated under microscope and transmission electron microscope. The phagocytosis ability was examined by ink phagocytosis test and monocyte/macrophages surface marker MAC387 expression was evaluated by immunocytochemistry. Real-time fluorescence quantitative polymerase chain reaction and double immune-fluorescence staining were respectively performed to detect four kinds of monocytes / macrophages membrane receptors including CD14, TLR4, SR-A and MHC-Ⅱgene and protein expression in 6 healthy and 6 hyperlipidemic rabbit samples. The phagocyte from four tissues was investigated in all 6 healthy and 6 hyperlipidemic rabbits. Four combinations were carried out including: RPMI1640 + macrophages, 1μg/ml E.coli-LPS + macrophages, 1μg / ml P.gingivalis-LPS + macrophages, 100ng/ml IL-10 +1μg / ml P.gingivalis-LPS + macrophages. After the treatment with IL-10 for 2 hours, P. gingivalis-LPS or E. coli-LPS was applied for 24hours to all the samples, then the mRNA expression levels of CD14, TLR4, SR-A and MHC-Ⅱ.were detected using real-time fluorescence quantitative polymerase chain reaction in each treatment cells Results The adherent cells obtained from different tissues showed typical characteristics in morphology, immunophenotype and strong phagocytic capacity. of monocyte / macrophage. In regarding to phagocytosis ability, AM showed highest, whereas, PM and KC showed lower and Mo was lowest (P<0.05). It was noted that MAC387 expression was highest in KC, followed by AM, PM, and Mo (P<0.05). Double immune-fluorescence staining showed all kinds of monocytes / macrophages from both healthy and hyperlipidemic groups expressed different degrees of fluorescence. Realtime PCR results showed that the levels of SR-A and MHC -Ⅱin Mo, AM, KC , and the TLR4 in AM, PM, KC were significantly increased (P<0.05) in hyperlipidemic groups compared with healthy groups; however, all CD14, the TLR4 in Mo and the MHC-Ⅱin PM were significantly decreased(P<0.05), Comparing with negative control groups, the treatment with Pg-LPS resulted in that the SR-A in AM, PM, KC and the CD14 in AM were significantly higher (P <0.05); the CD14 in Mo, KC and the TLR4 in AM , KC were little difference (P> 0.05); the other expressions were decreased (P <0.05). The E.coli-LPS treatment had the same direction as Pg-LPS groups, but the intensity was higher (P <0.05). Comparing with negative control groups, in hyperlipidemic Pg-LPS groups: the CD14 in AM and KC, the SR-A in AM and PM, and the TLR4 in Mo and AM were significantly increased (P <0.05); whereas the TLR4 in PM and KC showed little difference (P> 0.05); all others decreased (P <0.05). In E.coli-LPS groups, gene expressions basically had the same direction as Pg-LPS groups. In both healthy and hyperlipidemic groups, exogenous IL-10 caused the increased SR-A mRNA expression and decreased CD14, MHC-II, TLR4 mRNA expression.Conclusion 1. Monocytes/macrophages originated from different rabbit tissues had functional and phonotypical heterogeneity in ex vivo culture. 2. In both normal and hyperlipidemic groups, monocytes/macrophages from different locations showed different gene and protein expression profile for CD14, MHC-II, SR-A and TLR4. Furthermore,, the proteins expression was correlated with the same direction as mRNA expression. Without LPS participation, monocytes / macrophages response to hyperlipidemia was mainly by the increase of SR-A, TLR4, and MHC-Ⅱ.. 3. AM, PM, KC were mainly in defensive immunity response to the stimulation by 1μg/ml Pg-LPS for 24 hrs in healthy rabbit as specific immune response and inflammation had not be detected. At the same time, in hyperlipidemic rabbits, AM was in a state of defensive immune response and inflammatory effects, however PM was in the stage of immune defense. In the mean time, Mo and KC had entered inflammatory stage, showing decreased protective receptors. In addition, the specific immunity of these cells from hyperlipidemic rabbits was suppressed. 4. Exogenous IL-10 showed anti-inflammatory effect by increasing SR-A levels and decreasing CD14, MHC-Ⅱ, TLR4 levels. |
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