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Isolation, Processing and Analysis of Murine Gingival Cells
Published on: July 2, 2013
Soluble CD14-dependent intercellular adhesion molecular-1 induction by Porphyromonas gingivalis lipopolysaccharide in
T Masaka1, J Hayashi, I Ishikawa
1Department of Periodontology, Faculty of Dentistry, Tokyo Medical and Dental University, Japan.
Insights
Porphyromonas gingivalis lipopolysaccharide (LPS) induces intercellular adhesion molecule-1 (ICAM-1) expression on human gingival fibroblasts, but requires soluble CD14 (sCD14) for this effect. This suggests a role for sCD14 in periodontitis pathogenesis.
Area of Science:
- Oral microbiology
- Immunology
- Cell biology
Background:
- Intercellular adhesion molecule-1 (ICAM-1) mediates leukocyte adhesion and activation at inflammatory sites.
- Leukocyte recruitment involves ICAM-1 binding to beta2 integrins.
- Periodontopathic bacterium Porphyromonas gingivalis produces lipopolysaccharide (LPS).
Purpose of the Study:
- To investigate the effect of P. gingivalis LPS on ICAM-1 expression in human gingival fibroblasts (HGF).
- To examine the role of serum and soluble CD14 (sCD14) in modulating this response.
Main Methods:
- HGF were stimulated with P. gingivalis LPS and E. coli LPS.
- Soluble CD14 (sCD14) was purified from normal human serum (NHS).
- ICAM-1 expression was quantified using cell enzyme-linked immunosorbent assay.
Main Results:
- P. gingivalis LPS induced ICAM-1 expression on HGF in a dose-dependent manner, requiring serum.
- The induction was dependent on soluble CD14 (sCD14) and inhibited by anti-CD14 antibody.
- sCD14 alone restored the responsiveness of HGF to P. gingivalis LPS in serum-free conditions.
Conclusions:
- P. gingivalis LPS induces ICAM-1 expression in HGF via an sCD14-dependent mechanism.
- Overexpressed ICAM-1 on gingival fibroblasts may contribute to inflammatory cell retention in periodontitis lesions.
Background:
Intercellular adhesion molecule-1 (ICAM-1) is involved in the accumulation and activation of leukocytes in inflammatory sites through binding to beta2 integrins expressed on leukocytes. We investigated whether or not lipopolysaccharide (LPS) derived from the periodontopathic bacterium Porphyromonas gingiualis affects ICAM-1 expression on human gingival fibroblasts (HGF). CD14 is a receptor for LPS on monocytes and macrophages and is also present in serum as a soluble protein. We further examined the effects of serum and soluble CD14 (sCD14) on ICAM-1 expression in HGF stimulated with P. gingivalis LPS.
Methods:
HGF were prepared from explants of human gingival tissues and incubated in 96-well culture plates before LPS stimulation. LPS derived from Escherichia coli O55:B5 and P. gingivalis ATCC 33277 LPS were employed. sCD14 was purified from normal human serum (NHS) by affinity chromatography using an anti-CD14 monoclonal antibody. ICAM-1 expression on HGF was measured by a cell enzyme-linked immunosorbent assay.
Results:
P. gingivalis LPS induced ICAM-1 on HGF in a dose-dependent manner in the presence of either 10% fetal calf serum or 2% NHS. The ability of P. gingivalis LPS to induce ICAM-1 was comparable to that of LPS from E. coli at high LPS concentrations. In the absence of NHS, ICAM-1 induction was negligible in HGF stimulated with P. gingivalis LPS, reaching a maximum at 2% NHS. The ICAM-1 expression induced by P. gingivalis LPS was inhibited by a monoclonal antibody to CD14. Supplementation of serum-free medium with sCD14 alone restored the capacity of HGF to respond to P. gingivalis LPS.
Conclusions:
These results indicate that P. gingivalis LPS induces ICAM-1 expression in HGF in an sCD14-dependent manner. The overexpression of ICAM-1 on fibroblasts in gingiva induced by P. gingivalis LPS seems to be involved in the retention of inflammatory cells in periodontitis lesions.
