cytochalasin-d and Periodontal-Diseases

Desulfovibrio are sulfate-reducing anaerobic gram-negative rods that have been proposed as potential periodontopathogens. We investigated the capacity of Desulfovibrio to invade epithelial cells and induce cytokine secretion from these cells. Desulfovibrio strains were co-cultured with KB cells and counts of intracellular bacteria evaluated up to 3 days after infection. Desulfovibrio desulfuricans and Desulfovibrio fairfieldensis were able to survive within epithelial cells. Intracytoplasmic location of both bacterial species was confirmed by confocal laser scanning microscopy and transmission electron microscopy. Invasion was sensitive to nocodazole, an inhibitor of microtubule polymerization, but not to cytochalasin D, a microfilament inhibitor, suggesting that microtubule rearrangements were involved in the internalization of Desulfovibrio strains by KB cells. Infection by Desulfovibrio resulted in increased production of IL-6 and IL-8 by KB cells. The ability of D. desulfuricans and D. fairfieldensis to survive within oral epithelial cells and to modulate the epithelial immune response may contribute to the initiation and progression of periodontal diseases.

Topics: Antibodies, Bacterial; Coculture Techniques; Cytochalasin D; Cytoplasm; Desulfovibrio; Endocytosis; Epithelial Cells; Host-Pathogen Interactions; Humans; Inflammation Mediators; Interleukin-1beta; Interleukin-6; Interleukin-8; KB Cells; Microscopy, Confocal; Microscopy, Electron; Microtubules; Nocodazole; Nucleic Acid Synthesis Inhibitors; Periodontal Diseases; Tubulin Modulators

The gingival sulcus is the shallow crevice around the tooth, and its epithelium is a gateway for initial bacterial infection in periodontal disease. Recent studies have shown that Actinobacillus actinomycetemcomitans invades an epithelial cell line, KB cells, in vitro. The aim of the present study was to clarify the changes in KB cells after A. actinomycetemcomitans infection. The cytotoxic effects of A. actinomycetemcomitans on KB cells were determined at 72, 96 and 120 h after infection by an MTT assay. Nuclear morphological changes were observed by staining with Hoechst 33258. Cytoplasmic histone-associated DNA fragmentation in the infected KB cells was determined by ELISA. A. actinomycetemcomitans was cytotoxic on KB cells, and condensation and degradation of the nuclei were observed. DNA fragmentation was increased after the infection. In addition, A. actinomycetemcomitans showed similar cytotoxic effects on human gingival epithelial cells. The present study demonstrated that A. actinomycetemcomitans induces apoptotic cell death of oral epithelial cells in an in-vitro culture system. This induced apoptosis might be involved in the initiation and progression of periodontitis.

Topics: Aggregatibacter actinomycetemcomitans; Apoptosis; Cytochalasin D; DNA Fragmentation; Gingiva; Humans; KB Cells; Periodontal Diseases

Human gingival fibroblasts (HGFs) degrade collagen fibrils in physiological processes by phagocytosis. Since Treponema denticola outer membrane (OM) extract perturbs actin filaments, important structures in phagocytosis, we determined whether the OM affects collagen phagocytosis in vitro by HGFs. Phagocytosis was measured by flow cytometric assessment of internalized collagen-coated fluorescent latex beads. Confluent HGFs pretreated with T. denticola ATCC 35405 OM exhibited an increase in the percentage of collagen phagocytic cells (phagocytosis index [PI]) and in the number of beads per phagocytosing cell (phagocytic capacity [PC]) compared with untreated controls. The enhancement was swift (within 15 min) and was still evident after 1 day. PI and PC of HGFs for bovine serum albumin (BSA)-coated beads were also increased, indicating a global increase in phagocytic processes. These results contrasted those for control OM from Veillonella atypica ATCC 17744, which decreased phagocytosis. The T. denticola OM-induced increase in bead uptake was eliminated by heating the OM and by depolymerization of actin filaments by cytochalasin D treatment of HGFs. Fluid-phase accumulation of lucifer yellow was enhanced in a saturable, concentration-dependent, transient manner by the T. denticola OM. Our findings were not due to HGF detachment or cytotoxicity in response to the T. denticola OM treatment since the HGFs exhibited minimal detachment from the substratum; they did not take up propidium iodide; and there was no change in their size, granularity, or content of sub-G1 DNA. We conclude that a heat-sensitive component(s) in T. denticola OM extract stimulates collagen phagocytosis and other endocytic processes such as nonspecific phagocytosis and pinocytosis by HGFs.

Topics: Cell Membrane; Cell Survival; Collagen; Cytochalasin D; Fibroblasts; Gingiva; Hot Temperature; Humans; Periodontal Diseases; Phagocytosis; Treponema