Intracellular proteins involved in Porphyromonas gingivalis-induced opsonophagocytic activities of a murine macrophage cell line (RAW264.7 cells).
The aim of this study was to determine the role of intracellular proteins in phagocytosis of opsonized Porphyromonas gingivalis by RAW264.7 cells, a murine macrophage-like cell line. This periodontopathogen was grown anaerobically and opsonized with an IgG2a murine monoclonal anti-P. gingivalis lipopolysaccharide antibody. RAW264.7 cells were preincubated with protein tyrosine kinase inhibitors (staurosporine and genistein), protein kinase C inhibitors (phorbol myristic acetate and bisindolylmaleimide), a serine/threonine phosphatase inhibitor (okadaic acid), a phosphatidylinositol 3-kinase inhibitor (worthmannin), phospholipase A2 inhibitors (bromophenacyl bromide and nordihydroguaiaretic acid), phospholipase C inhibitors (p-chloromercuriphenyl sulfonate and neomycin sulfate), an actin-filament depolymerizer (cytochalasin D), and a microtubule disrupting agent (colchicine). Inhibitor-treated macrophages were then incubated with the opsonized P. gingivalis and the phagocytosed cells determined microscopically. The results showed the percentage of the phagocytosed organisms decreased when the cells were preincubated with protein tyrosine kinase, protein kinase C, protein phosphatase and phosphatidylinositol 3-kinase inhibitors. Of interest, preincubation with phorbol myristic acetate for 30 min increased the ability of RAW264.7 cells to phagocytose the opsonized organisms. Phospholipase A2 and phospholipase C inhibitors only slightly reduced the number of phagocytosed organisms. The results indicated that opsonophagocytosis of P. gingivalis by RAW264.7 cells might be determined by the activation of protein tyrosine kinase, protein kinase C, protein phosphatases, and phosphatidylinositol 3-kinase inhibitor. Both phospholipase A2 and phospholipase C would appear to be involved to a lesser extent. The opsonophagocytosis of this periodontopathogen would also appear to be dependent upon actin and microtubule polymerization.
Sosroseno
Wihaskoro
W
Department of Oral Biology, School of Dental Sciences, Universiti Sains Malaysia, Kota Bharu, Malaysia. wihaskoro@kb.usm.my
Bird
Philip S
PS
Seymour
Gregory J
GJ
D000818:Animals; D002460:Cell Line; D015638:Cytochalasin D / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019833:Genistein / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000276:immunology* / Q000382:microbiology; D051379:Mice; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D010587:Phagocytosis / Q000187:drug effects; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D016966:Porphyromonas gingivalis / Q000276:immunology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D019311:Staurosporine / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D015638:Cytochalasin D; D004791:Enzyme Inhibitors; D019833:Genistein; D019384:Nucleic Acid Synthesis Inhibitors; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011493:Protein Kinase C; D011505:Protein-Tyrosine Kinases; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate