Interleukin-1 and interleukin-8 in nicotine- and lipopolysaccharide-exposed gingival keratinocyte cultures.

BACKGROUND AND OBJECTIVE: Tobacco use is associated with increased periodontal destruction in both cigarette smokers and smokeless tobacco users. Gingival keratinocytes are the first cells in contact with microbial and tobacco components and play a key role in the innate immune response to these agents. The objective of this study was to evaluate the effect of nicotine and bacterial lipopolysaccharide (LPS) alone and in combination on gingival keratinocyte production of interleukin-1 alpha (IL-1 alpha) and interleukin-8 (IL-8). MATERIAL AND METHODS: Gingival keratinocyte cultures were established from 10 healthy, non-tobacco-using subjects. The cells were stimulated for 24 h with 1 mum or 1 mm nicotine and/or 10 microg/mL Escherichia coli or Porphyromonas gingivalis LPS. Interleukin-1 alpha and IL-8 proteins were quantified using ELISAs. RESULTS: Compared with untreated cultures, 1 mm nicotine stimulated production of IL-1 alpha (p < 0.001); E. coli and P. gingivalis LPS increased IL-8 production (p = 0.0014 and p = 0.0232, respectively). A combination of nicotine and LPS produced the highest cytokine quantities. Amounts of IL-1 alpha and IL-8 following 1 mm nicotine and LPS exposure were significantly greater than in untreated cultures (p < 0.001). Interleukin-8 was also responsive to 0.1 mum nicotine combined with E. coli or P. gingivalis LPS compared with control cultures (p < 0.0001 and p = 0.0029, respectively). Both cytokines tended to be elevated following the combined treatment relative to nicotine or LPS treatment alone. CONCLUSION: These results demonstrate that nicotine and LPS differentially regulate IL-1 and IL-8 production by gingival keratinocytes. Combined treatment tended to elevate cytokine production further, which may have implications for the progression of periodontitis in tobacco users.

Effect of smokeless tobacco extract on human gingival keratinocyte levels of prostaglandin E2 and interleukin-1.

Gingival recession and white mucosal lesions frequently occur at sites of smokeless tobacco (ST) placement. The etiology of these alterations is presumably related to the irritating effects of tobacco components. The purpose of this study was to examine the effect of an aqueous ST extract (STE) on gingival keratinocyte production of prostaglandin E2 (PGE2) and interleukin-1 (IL-1), mediators involved in periodontal destruction and keratinocyte proliferation. Keratinocyte cultures were established from healthy tissues discarded from 8 subjects undergoing crown lengthening procedures. Cells (passage 2-3) were seeded at 2.5 x 10(4) cells/well into 48 well tissue culture plates and maintained in serum-free media at 37 degrees C. On day 4 or 5, the wells were divided into 4 groups receiving either 10%, 5%, 2.5%, or 0% STE for time periods ranging from 30 to 240 minutes. PGE2 levels (pg/10(4) cells), as measured by enzyme immunoassay, were significantly (P < 0.05) increased in the 10% (215.66 +/- 34.58) and 5% STE (151.82 +/- 27.97) treated cultures compared to untreated cells (46.16 +/- 9.58). IL-1 alpha and IL-1 beta proteins were elevated (P < 0.05) in cell lysates (299.45 +/- 38.69 and 28.45 +/- 5.18, respectively) from 5% STE exposed cultures compared to control wells. At 10% STE, secreted IL-1 alpha was decreased (P < 0.05) relative to 2.5% STE. This may reflect a toxic effect, as 10% STE significantly (P < 0.05) depressed cell numbers and viability. Lower tobacco concentrations did not affect cell numbers or viability, but significantly (P < 0.05) increased PGE2 and IL-1 levels. Tobacco-induced synthesis of these mediators may play a role in the development of tobacco-related oral disease.

The effects of smokeless tobacco on clinical parameters of inflammation and gingival crevicular fluid prostaglandin E2, interleukin-1 alpha, and interleukin-1 beta.

The purpose of this study was to examine the effect of smokeless tobacco (ST) on gingival inflammation as assessed clinically and biochemically by gingival crevicular fluid (GCF) levels of PGE2, IL-1 alpha, and IL-1 beta. These parameters were compared in ST users (n = 12) and control, non-tobacco using subjects (n = 8) matched for plaque and probing depth levels. Both GI and GCF PGE2 concentration were significantly (P < 0.05) elevated at ST placement sites compared to sites in control subjects. A short-term longitudinal trial spanning 7 days also was undertaken within ST users (n = 18) to study the dynamics of gingival inflammation when ST is moved to a new site. At day 0 habitual ST placement sites exhibited a significantly (P < 0.05) higher GI compared to non-tobacco placement sites. Following transfer of ST to a new placement site, 48 hour GI scores were increased (P < 0.05) at this region. Although GCF parameters were not significantly altered, over 80% of the subjects developed dramatic inflammatory reactions in the alveolar mucosa, ranging from erythema to ulceration, at the new site of placement. This observation of a more severe response in alveolar mucosa compared to gingiva may be related to the physical location of ST or differences in tissue characteristics.

Prostaglandin E2 and interleukin-1 concentrations in nicotine-exposed oral keratinocyte cultures.

Oral keratinocytes are the first cells in contact with tobacco components and are capable of producing various inflammatory mediators, including PGE2 and IL-1. The purpose of this study was to examine PGE2 and IL-1 concentrations in nicotine-exposed oral keratinocyte cultures. Gingival keratinocyte cultures were established from healthy gingival tissues obtained from 7 subjects. Cultures were divided into 4 groups exposed to serum free medium (control), 0.1 microM, 10 microM or 1 mM nicotine for 4, 24 or 48 h. Using enzyme-linked immunosorbent assays, PGE2 and IL-1 alpha were quantified in culture supernatants; IL-1 alpha and beta were also measured in lysed cells. A repeated measures analysis of variance was used to identify significant differences over time and treatment. Nicotine exposure did not significantly alter PGE2 levels at any given time period; however, PGE2 quantities declined significantly (p = 0.0001) over time. At both 24 and 48 h, IL-1 alpha concentrations in lysates from 1 mM nicotine-exposed cells were significantly (p < 0.01) greater than those for all other treatments. Interleukin-1 alpha quantities also declined significantly (p = 0.037) over time in the cultures. Interleukin-1 beta concentrations were elevated, albeit not significantly, in the 1 mM treated cells at 24 and 48 h. Cell viability, mass and counts were not affected by nicotine treatment; these parameters increased significantly (p < 0.005) over time. In summary, nicotine treatment significantly increased IL-1 alpha concentrations in cultured keratinocytes; however, PGE2 synthesis was not altered. Elevated IL-1 production by keratinocytes may have implications in tobacco-induced lesions, given the central role IL-1 plays in tissue response to injury.

Effect of nicotine on arachidonic acid metabolites and epithelial parameters in rat oral mucosa.

This study examined the effects of nicotine on oral mucosal levels of eicosanoids and on histologic parameters, including keratinocyte proliferation. Surgically-created canals in the mandibular lips of 20 male Sprague Dawley rats received either nicotine or saline in a cotton pellet twice daily for six weeks. Thromboxane B2 (TxB2) levels were depressed (P < 0.05) in nicotine treated tissues compared to saline treatment (5.8 +/- 1.0 vs 13.4 +/- 2.1 pg/mg). Within the nicotine group, TxB2 concentrations were lower (P < 0.05) at the nicotine site compared to the posterior site (18.3 +/- 5.4 pg/mg). There was also a trend towards reduced 6-keto-PGF1 alpha in the nicotine-treated tissues compared to saline-exposed sites. These alterations in cyclooxygenase metabolites were not accompanied by changes in epithelial proliferation or histologic parameters. 12(S)-hydroxyeicosatetranoic acid (12-HETE) and leukotriene B4 (LTB4) were not affected by nicotine. Therefore, nicotine may not be directly responsible for the hyperplasia at habitual tobacco placement sites, but may contribute to alterations in cyclooxygenase products of arachidonic acid metabolism.