Lipopolysaccharide (LPS) stimulates the production of tumor necrosis factor (TNF)-alpha and expression of inducible nitric oxide synthase (iNOS) by osteoclasts (OCL) in murine bone marrow cell culture.
Osteoclasts (OCL) resorb bone. They are essential for the development of normal bones and the repair of impaired bones. The function of OCL is presumed to be supported by cytokines and other biological mediators, including tumor necrosis factor (TNF)-alpha and nitric oxide (NO). Bacterial lipopolysaccharide (LPS) is a potent inducer of TNF-alpha and inducible nitric oxide synthase (iNOS), which is the specific enzyme for synthesizing NO from L-arginine. To obtain direct evidence on LPS-induced TNF-alpha production and iNOS expression by OCL, OCL-enriched cultures were prepared by 7-day cocultures of bone marrow cells of adult BALB/c mice and osteoblastic cells (OBs) derived from calvaria of newborn BALB/c mice, and the generation of TNF-alpha and iNOS in OCL stimulated with LPS was examined immunocytochemically. When the cultured cells were stimulated with 100 ng/ml of LPS, OCL clearly showed TNF-alpha and iNOS expression. Without LPS-stimulation, no expression was observed. TNF activity in the culture supernatants of the OCL-enriched cultures in the presence of LPS was also detected by cytotoxic assay that used TNF-sensitive L929 cells. The dentin resorption activity of OCL was estimated by area and number of pits formed on dentin slices, which were covered by the OCL fraction and cultured in the presence or absence of LPS, sodium nitroprusside (SNP; a NO generating compound), N(G)-monomethyl L-arginine acetate (L-NMMA; a competitive inhibitor of NO synthase (NOS)), or LPS plus L-NMMA. Pit formation was obviously inhibited in the presence of SNP and slightly inhibited in the presence of L-NMMA, but it was not affected in the presence of LPS or LPS plus L-NMMA. These findings indicate that OCL produces TNF and expresses iNOS in response to LPS, but the LPS-activation of OCL scarcely affects pit formation by them.
Kikkawa
I
I
Department of Orthopedic Surgery, Jichi Medical School, Tochigi, Japan.
Saito
S
S
Tominaga
K
K
Hoshino
Y
Y
Ooi
Y
Y
Nakano
M
M
D000818:Animals; D001853:Bone Marrow; D001862:Bone Resorption; D002478:Cells, Cultured; D007150:Immunohistochemistry; D008070:Lipopolysaccharides / Q000494:pharmacology*; D051379:Mice; D008807:Mice, Inbred BALB C; D019001:Nitric Oxide Synthase / Q000096:biosynthesis*; D052247:Nitric Oxide Synthase Type II; D009599:Nitroprusside / Q000494:pharmacology; D010010:Osteoclasts / Q000187:drug effects* / Q000201:enzymology; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis*; D019323:omega-N-Methylarginine / Q000494:pharmacology
D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha; D009599:Nitroprusside; D019323:omega-N-Methylarginine; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse