Human gingival fibroblasts produce nitric oxide in response to proinflammatory cytokines.
BACKGROUND
Although nitric oxide (NO) synthesis is increased in periodontal disease (PD), little is known about the possible sources of production by gingival tissues. In fact, gingival tissues from patients with periodontitis demonstrate greater levels of inducible nitric oxide (iNOS) expression than healthy tissue. Macrophages are the source of the iNOS expression, with endothelial cells also contributing. In the present study, our hypothesis has been that human gingival fibroblasts (HGF) also have the ability to produce NO. We have established for the first time that HGF express increased levels of iNOS and modulate NO synthesis in response to proinflammatory cytokines that act synergistically.


METHODS
NO production under basal conditions or following incubation with tumor necrosis factor (TNF-alpha), interleukin (IL)-1beta, and interferon (IFN)-gamma was assessed by measurement of stable NO metabolites, nitrite, and nitrate, in a microplate adaptation of the Griess assay. Total RNA was isolated from HGF for determination of iNOS mRNA levels.


RESULTS
We have shown that NO production is elevated in HGF that are stimulated simultaneously by TNF-alpha, IL-1beta, and IFN-gamma. Northern blot analysis confirmed that the production of iNOS mRNA by HGF is upregulated in the presence of these cytokines. Addition of mercaptoethyl guanidine (MEG), a specific inhibitor of iNOS, profoundly reduced the production of NO in HGF. Non specific inhibitors of iNOS, L-NG-monomethyl arginine (L-NMMA), and L-arginine-methyl ester (L-NAME) had little or no effect on NO produced in HGF.


CONCLUSIONS
These results suggest that elevated NO production could be important in the pathogenesis of PD, and also suggest the ability of an iNOS inhibitor to modulate the disease. Treatments with drugs to block the production of nitric oxide or block its effects might be therapeutically valuable.
Daghigh
Farzaneh
F
Department of Biochemistry and Molecular Biology, Philadelphia College of Osteopathic Medicine, PA, USA.
Borghaei
Ruth C
RC
Thornton
Ruth D
RD
Bee
Joseph H
JH
D015152:Blotting, Northern; D002478:Cells, Cultured; D016207:Cytokines / Q000494:pharmacology*; D004357:Drug Synergism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D016166:Free Radical Scavengers / Q000378:metabolism*; D005881:Gingiva / Q000166:cytology / Q000378:metabolism*; D006146:Guanidines / Q000494:pharmacology; D006801:Humans; D018836:Inflammation Mediators / Q000494:pharmacology*; D007371:Interferon-gamma / Q000494:pharmacology; D007375:Interleukin-1 / Q000494:pharmacology; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009566:Nitrates / Q000378:metabolism; D009569:Nitric Oxide / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D009573:Nitrites / Q000378:metabolism; D012313:RNA / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D013223:Statistics as Topic; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D015854:Up-Regulation; D019323:omega-N-Methylarginine / Q000494:pharmacology
D016207:Cytokines; D004791:Enzyme Inhibitors; D016166:Free Radical Scavengers; D006146:Guanidines; D018836:Inflammation Mediators; D007375:Interleukin-1; D009566:Nitrates; D009573:Nitrites; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; D012313:RNA; D007371:Interferon-gamma; D019001:Nitric Oxide Synthase; C100078:2-mercaptoethylguanidine; D019331:NG-Nitroarginine Methyl Ester