IADR Abstract Archives
Amelogenin Inhibits IFNγ-induced MHC Class II Expression in Macrophages
Objectives: It is suggested that enamel matrix derivatives suppress inflammatory response and promote wound healing after periodontal surgery, although precise mechanism is still uncertain. We previously demonstrated that amelogenin suppressed major histocompatibility complex class II (MHC II) gene expression in monocytes. In this study, therefore, we examined underlying mechanism as to how amelogenin suppresses IFNγ-induced MHC II expression in macrophages.
Methods: Human monocytic cell line, THP-1 cells, were incubated with PMA, and were differentiated into macrophages. These cells were pretreated with 10 μg/ml amelogenin for 24 hours and then stimulated with 2.5 ng/ml IFNγ for 24 hours. The cell surface expression of MHC II molecules was evaluated by flow cytometric analysis and signal transduction pathway of IFNγ-induced MHC II expression with or without amelogenin pre-treatment was analyzed by real-time PCR and western blotting. The uptake of amelogenin into macrophages was monitored by fluorescence confocal microscopy. Histone modification (acetylation and methylation) of class II transactivator (CIITA) promoter region was assessed by using chromatin immunoprecipitation. The capacity of macrophages to activate CD4-positive T cells was evaluated by mixed leukocyte reaction (MLR).
Results: Amelogenin accumulated in the nucleus of macrophages after 15 minutes of stimulation, and suppressed IFNγ-induced MHC II expression as well as CIITA expression at both gene and protein levels. In addition, H3K27ac, H3K4me3 in CIITA promoter IV region, both of which are known to promote CIITA transcription, were suppressed by amelogenin. The expression of T cell activation markers, CD25 and CD69, and IL-2 production from CD4 positive T cells were down-regulated in MLR assay when macrophages were pre-treated with amelogenin.
Conclusions: Amelogenin appeared to inhibit CIITA transcription by suppressing euchromatin in macrophages, and suppressed cell surface expression of MHC II. Thus, amelogenin downregulates the activation of immune responses by inhibiting antigen presentation and may promote wound healing after periodontal surgery.
Methods: Human monocytic cell line, THP-1 cells, were incubated with PMA, and were differentiated into macrophages. These cells were pretreated with 10 μg/ml amelogenin for 24 hours and then stimulated with 2.5 ng/ml IFNγ for 24 hours. The cell surface expression of MHC II molecules was evaluated by flow cytometric analysis and signal transduction pathway of IFNγ-induced MHC II expression with or without amelogenin pre-treatment was analyzed by real-time PCR and western blotting. The uptake of amelogenin into macrophages was monitored by fluorescence confocal microscopy. Histone modification (acetylation and methylation) of class II transactivator (CIITA) promoter region was assessed by using chromatin immunoprecipitation. The capacity of macrophages to activate CD4-positive T cells was evaluated by mixed leukocyte reaction (MLR).
Results: Amelogenin accumulated in the nucleus of macrophages after 15 minutes of stimulation, and suppressed IFNγ-induced MHC II expression as well as CIITA expression at both gene and protein levels. In addition, H3K27ac, H3K4me3 in CIITA promoter IV region, both of which are known to promote CIITA transcription, were suppressed by amelogenin. The expression of T cell activation markers, CD25 and CD69, and IL-2 production from CD4 positive T cells were down-regulated in MLR assay when macrophages were pre-treated with amelogenin.
Conclusions: Amelogenin appeared to inhibit CIITA transcription by suppressing euchromatin in macrophages, and suppressed cell surface expression of MHC II. Thus, amelogenin downregulates the activation of immune responses by inhibiting antigen presentation and may promote wound healing after periodontal surgery.
