IADR Abstract Archives
Human pulp fibroblast involvement in bacteria opsonization via complement C3b secretion
Objectives: The complement mediated phagocytosis includes the opsonization of microorganisms with complement protein derived peptides. These are recognized by specific receptors on phagocytic cell membranes for subsequent intracellular destruction. Previous works have shown that the pulp fibroblasts produce complement components required for complement activation. Here, we investigate the human pulp fibroblast ability to produce the C3b opsonin, its attachment Complement Receptor 1 (CR1) and implication 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. To simulate carious injury, fibroblasts were stimulated with lipoteichoic acid (LTA). C3b secretion in MEM fibroblast culture media was investigated by ELISA with C3b specific antibodies (10µg/mL). Expression of its receptor (CR1) was investigated in human tissue sections in inflammatory THP-1 cells as well as in fibroblasts. C3b fixation on CR1 on both cultured fibroblasts and inflammatory THP-1 was investigated by immunofluorescence. C3b role in cariogenic bacteria opsonization was studied with LTA-Stimulated fibroblast supernatants and the detection of C3b fixation on bacteria with specific anti-C3b antibodies. Controls were incubated with the respective primary antibody isotypes.
Results: Fibroblasts secrete an increasing concentration of C3b in the culture medium in function of time and peaked at 24 hours of LTA stimulation. CR1 receptor is expressed in human pulp fibroblasts in vivo. It is also expressed in both THP-1 and fibroblasts. The C3b produced after LTA-stimulation opsonizes cariogenic bacteria and can be fixed on the CR1 receptor of both THP1 and fibroblasts.
Conclusions: Pulp fibroblasts produce complement C3b component significantly after LTA-stimulation. These cells express the receptor of this component which can recognize and fixe the C3b. These results show that the pulp fibroblast may mediate the process of phagocytosis by opsonizing cariogenic bacteria within the pulp.
Methods: Pulp cells were isolated from healthy human third molars extracted for orthodontic reasons. Fibroblasts were isolated and characterized by fibroblast surface antigen. To simulate carious injury, fibroblasts were stimulated with lipoteichoic acid (LTA). C3b secretion in MEM fibroblast culture media was investigated by ELISA with C3b specific antibodies (10µg/mL). Expression of its receptor (CR1) was investigated in human tissue sections in inflammatory THP-1 cells as well as in fibroblasts. C3b fixation on CR1 on both cultured fibroblasts and inflammatory THP-1 was investigated by immunofluorescence. C3b role in cariogenic bacteria opsonization was studied with LTA-Stimulated fibroblast supernatants and the detection of C3b fixation on bacteria with specific anti-C3b antibodies. Controls were incubated with the respective primary antibody isotypes.
Results: Fibroblasts secrete an increasing concentration of C3b in the culture medium in function of time and peaked at 24 hours of LTA stimulation. CR1 receptor is expressed in human pulp fibroblasts in vivo. It is also expressed in both THP-1 and fibroblasts. The C3b produced after LTA-stimulation opsonizes cariogenic bacteria and can be fixed on the CR1 receptor of both THP1 and fibroblasts.
Conclusions: Pulp fibroblasts produce complement C3b component significantly after LTA-stimulation. These cells express the receptor of this component which can recognize and fixe the C3b. These results show that the pulp fibroblast may mediate the process of phagocytosis by opsonizing cariogenic bacteria within the pulp.