Induction of Porphyromonas gingivalis GroEL signaling via binding to Toll-like receptors 2 and 4.
OBJECTIVE
Heat shock protein 60 (HSP60) has been recognized as an important molecule in infectious and autoimmune diseases. Although Porphyromonas gingivalis GroEL, a homologue of HSP60, is a potent stimulator of inflammatory cytokines, its receptor and signaling mechanisms are not yet understood in detail. In this study, we investigated whether the Toll-like receptor (TLR) family plays a functional role as a P. gingivalis GroEL receptor.


METHODS
Human macrophage-like THP-1 cells were used and the nuclear factor-kappaB (NF-kappaB) activity of cells stimulated with a recombinant P. gingivalis GroEL was measured with a luciferase assay. Flow cytometry analysis was used to determine the binding to THP-1 cells of fluorescein isothiocyanate (FITC)-labeled GroEL. In addition, anti-human TLR (anti-hTLR)2 and anti-hTLR4 monoclonal antibodies were used to assess the functional role of TLR2 and TLR4 as the receptors for GroEL.


RESULTS
We observed by luciferase assay that the purified recombinant GroEL was able to stimulate NF-kappaB transcriptional activity in THP-1 cells. Flow cytometry analysis showed that the FITC-labeled GroEL bound to THP-1 cells in a dose-dependent fashion. Our binding competition analysis with FITC-labeled and unlabeled GroEL showed that it bound to the cells as a specific mode of action. On the other hand, GroEL-stimulated NF-kappaB transcriptional activity was significantly inhibited by anti-hTLR2 and anti-hTLR4 antibodies and was inhibited more strongly by a combination of both antibodies.


CONCLUSIONS
Our present study demonstrates that P. gingivalis GroEL induces its intracellular signaling cascade in THP-1 cells via TLR2 or TLR4 and via a combination of both receptors.
Argueta
J G M
JG
Division of Oral Infectious Diseases and Immunology, Faculty of Dental Science, Kyushu University, Fukuoka, Japan.
Shiota
S
S
Yamaguchi
N
N
Masuhiro
Y
Y
Hanazawa
S
S
D000911:Antibodies, Monoclonal; D018834:Chaperonin 60 / Q000494:pharmacology / Q000502:physiology*; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D008156:Luciferases; D049408:Luminescent Agents; D008264:Macrophages / Q000382:microbiology; D009000:Monocytes / Q000382:microbiology; D016328:NF-kappa B / Q000502:physiology; D016966:Porphyromonas gingivalis / Q000502:physiology*; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D051195:Toll-Like Receptor 2 / Q000187:drug effects / Q000502:physiology*; D051197:Toll-Like Receptor 4 / Q000187:drug effects / Q000502:physiology*; D014158:Transcription, Genetic / Q000235:genetics
D000911:Antibodies, Monoclonal; D018834:Chaperonin 60; D005456:Fluorescent Dyes; D008070:Lipopolysaccharides; D049408:Luminescent Agents; D016328:NF-kappa B; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D008156:Luciferases; D016650:Fluorescein-5-isothiocyanate