IADR Abstract Archives
Complement receptor-1 detection in the dental pulp fibroblasts and its implication in phagocytosis
Objectives: Complement Receptor 1 (CR1) is the most versatile receptor due its dual nature to recognize both complement C3b and C4b opsonins. This receptor recognizes C3b attached on non-self surfaces (mainly pathogenic) and promotes their phagocytosis. In this work, we investigated CR1 expression in pulp tissues both in vivo and in vitro and its involvement in cariogenic bacteria phagocytosis.
Methods: Pulp cells were isolated from healthy human third molars extracted for orthodontic reasons. Fibroblasts were isolated and characterized by fibroblast surface antigen. Imunofluorescence (IF) was performed on histological sections of human pulps (in vivo) and cultured human pulp fibroblasts and inflammatory THP-1 cells. Briefly, after saturation (BSA1%) and immunostaining with anti-human CR1 (10μg/ml), followed by Alexa594 Fluor counterstaining (5μg/ml) and nuclei staining with DAPI (1μg/ml), the slides were mounted and observed under immunofluorescent microscope.
The phagocytic potential was investigated with a Fluorescent dye (pHrodo), fluorescent in acidic conditions.
Then, the labelled bacteria were incubated with pulp fibroblasts or THP-1 cells. After phagocytosis, intracellular bacteria were examined by fluorescence microscopy.
Results: CR1 was detected both in vivo and in cultured THP-1 cells and fibroblasts. While CR1 expression in THP-1 cells has been reported earlier. The expression of this receptor on pulp fibroblasts, both in vivo and in vitro is surprising. In order to investigate its possible role in bacteria phagocytosis pHrodo fluorescent dye was used. Bacteria were not fluorescent when incubated with pHrodo. However, after incubation with THP-1 cells or fibroblasts and phagocytosis by these cells, pHrodo-labeled bacteria appeared fluorescent within acidic intracellular compartments in pulp fibroblasts and THP-1 cells.
Conclusions: These results provide direct evidence that phagocytosed bacteria were directed to acidic intracellular compartments within both cell types and clearly demonstrate the pulp fibroblast phagocytosis potential.
Methods: Pulp cells were isolated from healthy human third molars extracted for orthodontic reasons. Fibroblasts were isolated and characterized by fibroblast surface antigen. Imunofluorescence (IF) was performed on histological sections of human pulps (in vivo) and cultured human pulp fibroblasts and inflammatory THP-1 cells. Briefly, after saturation (BSA1%) and immunostaining with anti-human CR1 (10μg/ml), followed by Alexa594 Fluor counterstaining (5μg/ml) and nuclei staining with DAPI (1μg/ml), the slides were mounted and observed under immunofluorescent microscope.
The phagocytic potential was investigated with a Fluorescent dye (pHrodo), fluorescent in acidic conditions.
Then, the labelled bacteria were incubated with pulp fibroblasts or THP-1 cells. After phagocytosis, intracellular bacteria were examined by fluorescence microscopy.
Results: CR1 was detected both in vivo and in cultured THP-1 cells and fibroblasts. While CR1 expression in THP-1 cells has been reported earlier. The expression of this receptor on pulp fibroblasts, both in vivo and in vitro is surprising. In order to investigate its possible role in bacteria phagocytosis pHrodo fluorescent dye was used. Bacteria were not fluorescent when incubated with pHrodo. However, after incubation with THP-1 cells or fibroblasts and phagocytosis by these cells, pHrodo-labeled bacteria appeared fluorescent within acidic intracellular compartments in pulp fibroblasts and THP-1 cells.
Conclusions: These results provide direct evidence that phagocytosed bacteria were directed to acidic intracellular compartments within both cell types and clearly demonstrate the pulp fibroblast phagocytosis potential.