Prevotella intermedia lipopolysaccharide stimulates release of nitric oxide by inducing expression of inducible nitric oxide synthase.
OBJECTIVE
The purpose of this study was to examine the effects of lipopolysaccharide from Prevotella intermedia, a major cause of inflammatory periodontal disease, on the production of nitric oxide (NO) and expression of inducible nitric oxide synthase (iNOS) in the murine macrophage cell line RAW264.7. We also attempted to throw light on the signaling mechanisms involved in P. intermedia lipopolysaccharide-induced NO production.


METHODS
Lipopolysaccharide from P. intermedia ATCC 25611 was prepared by the standard hot phenol-water method. NO production was assayed by measuring the accumulation of nitrite in culture supernatants. Western blot analysis of iNOS and analysis of reverse transcription-polymerase chain reaction (RT-PCR) products were carried out.


RESULTS
We found that P. intermedia lipopolysaccharide can induce iNOS expression and stimulate the release of NO without additional stimuli and demonstrated an important role of the transcription factor nuclear factor-kappaB (NF-kappaB) and microtubule polymerization in NO production. The production of NO required l-arginine but not activation of protein kinase C or protein tyrosine kinase.


CONCLUSIONS
The present study clearly shows that P. intermedia lipopolysaccharide fully induced iNOS expression and NO production in RAW264.7 cells in the absence of other stimuli. The ability of P. intermedia lipopolysaccharide to promote the production of NO may be important in the pathogenesis of inflammatory periodontal disease.
Kim
Sung-Jo
SJ
Department of Periodontology, College of Dentistry, Pusan National University, Busan, South Korea. sungjokim@pusan.ac.kr
Ha
Mi-Suk
MS
Choi
Eun-Young
EY
Choi
Jeom-Il
JI
Choi
In-Soon
IS
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D004790:Enzyme Induction / Q000187:drug effects; D015870:Gene Expression; D019833:Genistein / Q000633:toxicity; D007211:Indoles / Q000633:toxicity; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000187:drug effects / Q000201:enzymology* / Q000382:microbiology; D008301:Maleimides / Q000633:toxicity; D051379:Mice; D008870:Microtubules / Q000502:physiology; D009569:Nitric Oxide / Q000096:biosynthesis* / Q000633:toxicity; D019001:Nitric Oxide Synthase / Q000096:biosynthesis*; D052247:Nitric Oxide Synthase Type II; D018713:Prevotella intermedia / Q000472:pathogenicity*; D047428:Protein Kinase Inhibitors / Q000633:toxicity; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction
D007211:Indoles; D008070:Lipopolysaccharides; D008301:Maleimides; D047428:Protein Kinase Inhibitors; D012333:RNA, Messenger; D009569:Nitric Oxide; D019833:Genistein; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; C088060:bisindolylmaleimide