Abstract
Periodontal disease, for which smoking is a known risk factor, is infectious, and is associated with oral biofilm. Cytokines mediate and regulate immune and inflammatory responses. Lipopolysaccharide produced by periodontopathic bacteria plays a role in the progression of periodontitis. The effect of nicotine on cytokine production in mice was evaluated in this study. Nicotine (10 or 200 µg mouse−1) was administered intraperitoneally to 4-week-old female BALB/c mice, once a day, for 49 days. Control mice received injections of phosphate-buffered saline. Blood was collected from all mice and serum IL-6, IL-10, tumor necrosis factor (TNF)-α and IFN-γ levels were measured by an enzyme-linked immunosorbent assay on the 42nd day. IL-6, IL-10 and IFN-γ levels in the nicotine-treated mice were higher than those in the control mice. However, no differences were found in TNF-α levels between nicotine-treated and control mice. Lipopolysaccharide (20 µg mouse−1) purified from Aggregatibacter actinomycetemcomitans (formerly Actinobacillus actinomycetemcomitans) Y4 was administered intraperitoneally on the 49th day. A rapid increase in TNF-α was observed in the control mice at 2 h after administration of lipopolysaccharide. In contrast, no increase was noted in the nicotine-treated groups. Significantly higher levels of IFN-γ were seen in the 200 µg nicotine-treated mice at 2 h after administration of lipopolysaccharide (P<0.05). The results showed that cytokine levels were influenced by nicotine in mice.
Introduction
The main cause of periodontal disease is bacterial infection, the immune response to which can result in the destruction of periodontal tissues if the ensuing inflammation persists. Periodontal disease is initiated by specific bacterial species. The local host response to these bacteria includes the recruitment of leukocytes and the subsequent release of inflammatory mediators and cytokines such as IL-1, IL-6, IL-8, IL-10, IL-12 and tumor necrosis factor-α (TNF-α), which are thought to play an important role in the pathogenesis of this disease. Increased levels of several of these cytokines are involved in periodontal tissue destruction (Genco, 1992). Over the past two decades, smoking has been identified as a major environmental risk factor in periodontal disease (Bergström & Eliasson, 1987; Genco & Löe, 1993; Bergström & Preber, 1994; Grossi et al, 1994; 1995). Nicotine is a major component of cigarette smoke. However, there is no consensus on the mechanisms of nicotine as a risk factor. Further research is needed to elucidate the role of nicotine in periodontal disease and its influence on host response. Nicotine has been shown to increase the release of IL-6 by cultured murine osteoblasts (Kamer et al, 2006).
Aggregatibacter actinomycetemcomitans (formerly Actinobacillus actinomycetemcomitans) is a Gram-negative, nonmotile capnophilic rod that has been implicated as a causative microorganism in human periodontal disease (Slots et al, 1980). It has been demonstrated that stimulation by A. actinomycetemcomitans serotype b lipopolysaccharide induces IL-4, IL-5 and IL-6 release from splenocytes in some strains of mice in vitro (Kato et al, 2000). Of particular significance is the ability of IL-6 to induce bone resorption, both by itself and in conjunction with other bone-resorbing agents (Ishimi et al, 1990).
Although a number of studies have investigated the effect of smoking on inflammatory components in the periodontium in subjects diagnosed with periodontal disease (Bostrom et al, 1998; Erdemir et al, 2004; Kamma et al, 2004), in general, no conclusive results have been reported. The objective of this study was to investigate the effect of nicotine on cytokine induction by A. actinomycetemcomitans lipopolysaccharide in mice.
Materials and methods
Lipopolysaccharide purification
Aggregatibacter actinomycetemcomitans Y4 bacterial cells were grown anaerobically at 37 ;°C for 3 days in trypticase soy broth (Becton Dickinson Microbiology System, Cockeysville, MD) with 0.4% yeast extract. Harvested cells were washed with phosphate-buffered saline (PBS; pH 7.2) and suspended in the same buffer. Lipopolysaccharide was prepared by the hot phenol-water method (Westphal & Jann, 1965) and purified as described previously (Kato et al, 2006). Lipopolysaccharide fractions were assessed for purity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using silver staining. The lipopolysaccharide fractions revealed slow migrating and repeating ladder bands, forming a typical lipopolysaccharide pattern. The lipopolysaccharide fraction was dialyzed against pyrogen-free water and lyophilized.
Effects of nicotine on serum cytokine levels in mice
Nicotine (Product Number N5511) was obtained from Sigma Chemical Co. (St Louis, MO). Four-week-old female BALB/c mice were divided into two nicotine-treated groups (10 ;µg ;mouse−1 group, five mice; 200 ;µg ;mouse−1 group, six mice) and a non-nicotine-treated control group (five mice). Nicotine (10 or 200 ;µg ;mouse−1) was administered intraperitoneally once a day for 49 days. Control mice received injections of PBS. Blood was collected from all mice, serum was separated and serum IL-6, IL-10, TNF-α and IFN-γ levels were measured using the Endogen ELISA kit on the 42nd day (Pierce Biotechnology, Inc., Rockford, IL). Next, A. actinomycetemcomitans Y4 lipopolysaccharide (20 ;µg mouse−1) was administered on the 49th day in all groups, including the controls, and the effect of nicotine on lipopolysaccharide cytokine induction was determined. Blood was collected at 2 and 24 ;h after lipopolysaccharide administration and serum was separated. All animals were treated in accordance with ‘The Guidelines for the Treatment of Experimental Animals at Tokyo Dental College’.
Statistical analyses
Data are expressed as means±SDs. Statistical analysis was performed with the Mann–Whitney U-test, in which a probability of <0.05 was considered to be statistically significant.
Results
No mice died after nicotine injection during the course of the study. The body weight of each mouse was measured at 6 weeks of age. No significant difference was found between the body weights of the mice treated with nicotine and those of the control mice.
IL-6, IL-10 and IFN-γ levels in the 200 ;µg nicotine-treated mice were higher than those in the control mice, at P<0.01, P<0.05 and P<0.02, respectively (Fig. 1). However, no differences were found between the nicotine-treated and control mice in terms of the TNF-α level.
Effects of nicotine on serum IL-6, IL-10, TNF-α and IFN-γ levels. *, P<0.01 compared with control (Mann–Whitney U-test), **, P<0.05 compared with control (Mann–Whitney U-test), ***, P<0.02 compared with control (Mann–Whitney U-test).
Figure 2 shows the effects of nicotine on A. actinomycetemcomitans lipopolysaccharide-induced cytokine levels. IL-6 levels showed a remarkable increase after stimulation with lipopolysaccharide. Although IL-6 levels were higher at 2 and 24 ;h after lipopolysaccharide administration in 10 and 200 ;µg nicotine-treated mice, the differences were not significant. A rapid increase in TNF-α was observed in the control mice at 2 ;h after administration of lipopolysaccharide. In contrast, no increase was noted in the nicotine-treated groups. In addition, TNF-α levels in the nicotine-treated groups were significantly lower than those in the control group (nicotine-treated group, P<0.0001 in comparison with the controls). At 24 ;h after administration of lipopolysaccharide, TNF-α levels showed a decrease compared with at baseline in all mice tested. No marked change was seen in IFN-γ levels in the controls or 10 ;µg nicotine-treated mice at 2 ;h after administration of lipopolysaccharide. However, significantly higher levels of IFN-γ were seen in the 200 ;µg nicotine-treated mice (P<0.05). IL-10 levels showed an increase after stimulation with lipopolysaccharide, although no significant difference was seen between the nicotine-treated and control mice.
Effects of nicotine on lipopolysaccharide-induced serum IL-6, IL-10, TNF-α and IFN-γ levels. *, P<0.0001 compared with control (Mann–Whitney U-test), **, P<0.05 compared with control (Mann–Whitney U-test).
Discussion
The results of this study demonstrated that cytokine levels were influenced by nicotine. Aggregatibacter actinomycetemcomitans is a widely studied human periodontopathogen, and is believed to play a role in periodontal disease. Aggregatibacter actinomycetemcomitans-induced periodontal disease is an interesting model for the investigation of the mechanisms of both tissue destruction and control of infection in the peridontium. Therefore, this microorganism was selected for use in this study. IL-6 levels in all groups showed a remarkable increase with A. actinomycetemcomitans lipopolysaccharide stimulation. At the physiological level, IL-6 plays an important role in the cytokine network. However, excessive production of IL-6 in response to exposure to lipopolysaccharide has an inflammatory effect, resulting in injury. Many cell types produce IL-6, but it is not clear whether the IL-6 produced by different cell types acts in the same way. Lipopolysaccharide-induced IL-6 may be mainly derived from macrophages. Nicotine-induced IL-6 may be derived from different cells. The possibility of inhibition of TNF-α production by IL-6 has been reported in several studies (Fiers, 1991; Mizuhara et al, 1994; Matthys et al, 1995). In this study, the significantly lower level of TNF-α in the nicotine-treated group with A. actinomycetemcomitans lipopolysaccharide stimulation may have been due to inhibition of TNF-α production by nicotine-induced IL-6.
Although IFN-γ plays a pivotal role in host defense mechanisms, its excessive release has been associated with the pathogenesis of chronic inflammatory and autoimmune diseases (Farrar & Schreiber, 1993; Feldmann et al, 1998; Tilg & Kaser, 1999). Previous reports (Baker et al, 1999; Kawai et al, 2000; Gorska et al, 2003) have suggested that IFN-γ+ Th1 cells are strongly associated with enhanced alveolar bone loss during periodontal infections, and that high absolute levels of proinflammatory cytokines, including IFN-γ, are closely associated with the degree of severity in periodontal disease. In addition, it has been reported that IFN-γ plays an important role in modulating alveolar bone loss under inflammatory conditions induced by microbial challenge in humans and mice, in vivo (Teng et al, 2005). In contrast, it has also been shown that IFN-γ may directly inhibit receptor activator of nuclear factor (NF) κ B ligand (RANKL)-mediated osteoclastogenesis in in vitro and animal studies (Takayanagi et al, 2000; De Klerck et al, 2004). Teng (2005) clearly demonstrated a positive coexpression relationship between IFN-γ and RANKL-mediated osteoclastogenesis in a mouse periodontitis model. Yonezawa (2005) suggested that the restraint of IFN-γ production elicited by gingipain DNA vaccine played a significant role in protection against periodontopathic Porphyromonas gingivalis infection in mice. In the present study, significantly higher levels of IFN-γ were seen in the 200 ;µg nicotine-treated mice (P<0.02), suggesting that this factor is involved in periodontal tissue destruction.
We observed an increase in IL-10 levels in the nicotine-treated mice. The ability of IL-10 to inhibit cytokine synthesis by helper T cells was found to be due to its inhibitory effect on macrophage-monocytes (Fiorentino et al, 1991). IL-10 has been shown to reduce neutrophil-dependent bacterial killing (Laichalk et al, 1996). High levels of IL-10 may benefit periodontopathic bacteria survival.
This paper is the first to demonstrate changes in cytokine levels in mice continuously treated with nicotine for around 1 month. Nicotine may play an important role in the mechanism by which smoking induces periodontal disease. Further study is required to evaluate the impact of these findings on the understanding of the mechanisms by which smoking affects periodontal disease.
In conclusion, the results of this study suggest that nicotine affects the immune response through disturbance of the cytokine network that plays a crucial role in the regulation of periodontal tissue inflammation.
Acknowledgements
This research was supported by Oral Health Science Center Grant HRC7 from Tokyo Dental College, a ‘High-Tech Research Center’ Project for Private Universities: matching fund subsidy from MEXT (Ministry of Education, Culture, Sports, Science and Technology) of Japan, 2006–2010. The authors would like to thank Associate Professor Jeremy Williams, Tokyo Dental College, for his assistance with the English of this manuscript.
References
Author notes
Editor: Johannes G. Kusters
