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The correlation between smoking and the concentration of IL-1β in gingival crevice fluid of patients suffering from chronic periodontitis 1 Tabibzadeh Z. DDS. MS., 2Roayaee Ardekani M. DDS., 3Ghaforian Brojerdnia M. MS., 4Akbarzadeh Bagheban AR. MS 1 Assistant Professor, Dept. of Periodontics, Dental School, Shahid Beheshti University of Medical Sciences, TehranIran 2 Dentist 3 Associate Professor, Dept. of Immunology, Medical School, Jondi Shapoor University of Medical Sciences, AhvazIran 4 Assistant Professor, Dept. of Biostatistics, Dental School & Iranian Center for Endodontic Research, , Shahid Beheshti University of Medical Sciences, Tehran- Iran Abstract Background and Aim: Cigarette smoking is a significant risk factor in pathogenesis and progression of periodontal diseases. Smoking could interfere with pre-inflammatory cytokines in inflammatory process. IL-1β is a main inflammatory cytokine in gingival crevicular fluid (GCF) which contributes in periodontitis. In fact it is a key molecule in pathogenesis of periodontitis. This study aimed to investigate the correlation between cigarette smoking and concentration of cytokine interleukin (IL)-1β in GCF of patients with moderate-to-severe chronic periodontitis. Materials & Methods: Sixty subjects were entered into this analytical case-control study, divided equally into smokers and non smokers.The two groups were matched in clinical parameters, age and gender. GCF samples were collected in one diseased and one healthy site from each subject. The IL-1β concentration in all 120 samples was determined by ELISA kits, specific for IL-1β. The observed data were analyzed with SPSS13 software using t, Paired t, Chi-square and MannWhitney tests. Results: Mean concentration of GCF IL-1β in healthy sites of the smokers was significantly more than non smokers (P<0.01). But in diseased sites no significant differences were shown between the two groups. The differences between concentration of IL-1β in smokers and nonsmokers were not significant. Conclusion: Although no significant differences were found in concentration of IL-1β between all smokers and all non smokers, there were significant differences between two groups in healthy sites, which require more investigations. 1 ______________________________________________________________________________ Key words: Interleukine-1β, Smoking, Periodontal disease Received on:October 2007 Final revision :August 2008 Accepted on: December 2008 Introduction Periodontitis is inflammation of periodontium which not only afflicts gingivae but also destroys the tissues around a tooth (1-3). Although the key factor for periodontitis to occur is microorganism, the emergence of the symptom of periodontitis depends upon the response of the host to the bacterial attack . In response to periodontal pathogens and their endotoxin, immune cells produce pre–inflammatory mediators. Among many known immune and inflammatory mediators present in GCF , Cytokine has drawn special attention. Cytokines such as IL-1, IL-4, 6, IL-8 and TNF α have been observed in the GCF of the patients suffering from periodontal disease (4-9). Among these Cytokines IL-1 has the key role . IL –1 is a very powerful pre-inflammatory cytokine that is produced by most of the body cells , especially macrophages (10). Almost all body cells are receptive to IL-1 and can respond to it. This molecule stimulates T and B cells which in turn stimulate immunized responses and it also destroys body tissues by stimulationg the production of tissue decomposer proteinases. IL-1 is produced in two forms: α and ß IL-1ß is stronger and its catabolic effect on bones is 10 times more than that of IL-1α . IL-1ß is the main inflammatory cytokine which is in relation with periodontitis i.e. It's the key molecule in pathogenic periodontitis. This cytokine is released when immune system respond to bacteria and other tissue decomposing products. Bacterial attack is influenced by several environmental risk factors. Among these factors which are in relation with periodontitis, smoking is regarded as the most important risk factor. It has been discovered that smoking can intervene the inflammatory processes by affecting the production of preinflammatory cytokines. Rawlinson (2003)(1) and Zhong et al(13) (2007) proposed that there is a meaningful relationship between smoking and the concentration of IL-1ß; although many of the biological mechanisms which are the subsequent results of smoking are unknown yet. Although the relationship between the amount of IL-1ß and the presence of periodontal disease has been generally proven, some contradictory results show that this factor may be influeced by some probably ignored factors . To be assured of diagnostic value of cytokine, we should evaluate it by studying wide spectrum of people who are under the same environmental situation . Figuered et al (1999)(8), Bostrom et al (2000)(14) and Giannopoulou et al (2003) (5,6) have observed significant difference between the concentration of IL-1 ß in smokers and that of non- smokers but Rawlinson et al (2003) observed that the density of IL-1ß in non-smokers is greater than that 2 of smokers while Zhong et al (2007) found more density in IL-1ß of smokers (1,13). The purpose of this research was to specify the correlation between smoking and the amount of IL-1ß in GCF of the patients suffering from mild to severe chronic periodontitis under clinically identical situation . Method and Materials: The sample under this analytical study consisted of 60 people divided into two groups. The smokers were those who, according to Center of Disease Control has consumed 100 cigarettes in their life time and now they are in the habit of smoking. This group consisted of 9 women and 21 men whose ages ranged from 22 to 66 with the average of age 44.8 Non-smokers were those who have never been in the habit of smoking so far and consisted of 15 women and 15 men whose ages ranged from 22 to 63 years. The average age in this group was 44.2. These people were selected among the patients referring to dental school of Shahid Beheshti Medical University .They lacked any systematic disease. They did not take any special drugs, were not pregnant or menopaused and they didn’t use contraceptive drugs. Also none of them had been taking anti -biotic in the past three months before sampling. They were not under periodontal treatment in the past one year. In terms of every smoker in the group, the number of packets of cigarettes and the period during which they smoked were recorded. Those who had been accustomed to smoking but quitted their habit were not studied. All the people in the group had at least one intact and one mild-to-severe chronic periodontal infected area in their mouth. By means of Willam's probe and radiography a chronic periodontitis infected areas were and a intact area were selected in every individuals mouth. The selected areas were isolated by means of cotton roll and suction. By means of a curette the supra-gingival plaque was removed carefully without touching the gingiva. The surface of the tooth was cleaned and dried with cotton roll. Then a perio-paper strip was placed in cervical area and sampling process was completed in about 2 minutes. After sampling, periodontal evaluations such as CAL (clinical attachment level) PPD (probing pocket depth) and PL(plaque index) were carried out on the basis of Loe & Silness and tooth mobility index on the selected teeth. The perio-paper strips were collected and placed into micro-tubes and promptly transferred into a-70ºC fridge and kept there until the time of experiment. Strips with observable sign of blood or saliva on were not studied. All laboratorial processes were done in oral biology laboratory of Shahid Beheshti Medical University. In the day of the experiment the samples were removed from the fridge and defreezed and 800 ul of PBS was added to each of the samples and all of them were centrifuged for 5 3 minutes in 4ºC at 3000g. Then the obtained solutions were evaluated by means of ELISA kits (Bender Med System Com,Vienna, Astria) which are exclusive to IL-1ß to specify the density the density of IL-1ß. The intensity of the color change of the hales in each plate of ELISA was read at a wave length of 450 nm and reference of 620 nm. To analyze the data SPSS 13 software and independent statistical t tests were used. Furthermore; to compare the concentration of IL-1ß intact area of the mouth with that of the infected area paired t test and to compare a few variables between the two groups Chi-square and Mann-Whitney tests were used. Results: One hundred and twenty samples were collected in this study 60 of which were from smokers and the other 60 from non-smokers .Each of these 60 samples consisted of 30 healthy samples and 30 unhealthy ones. In this study two groups of smokers and non-smokers were assessed with regard to age, gender and periodontal clinical parameters (CAL, PPD, PL, and mobility) and no significant difference was seen in any of these factors. The results of the evaluation of clinical indices, demographic and average pack-year have been tabulated in table 1. Table 1. The descriptive data and the mean concentration of IL –1 ß in the smoker and nonsmoker groups Smoker Non-smoker 30 30 9.21 15.15 0.08 44.83 (8) 44.28 (9.1) 0.8 Number Female/ Male Age *Pack-year P-value 17.32 (13.1) **CAL 7.10 (2.6) 6.62 0.42 ***PPD 6.16 (1.9) 5.79 (1.3) 0.39 *Number of cigarette packets used in a day multiply by years of usage. **Clinical Attachment Level ***Probing Pocket Depth The average concentration of IL-1ß in healthy and unhealthy areas of non – smokers and smokers was 11.075 and 26.05 pg/ml respectively and no significant difference was observed to give smokers an advantage over non-smokers (P< 0.01). Also the average concentration of this Cytoakine in unhealthy sites of non–smokers and smokers was respectively 52.21 and 62.364 pg/ml but the difference between the two groups was not significant statistically (P<0.05). The average Concentration of IL-1ß in the two groups was 44.50 and 31.64 respectively (Table 2). 4 Table 2- Comparison of mean concentration of IL-1ß pg /ml in the intact and the diseased sites of smokers and non – smokers Smoker Non-smoker P-value Intact site 26.05(23.7) 11.57(9) <0.01 Diseased site 62.03(56.2) 52.21(56.05) <0.02 Total area 44.50(36.07) 31.64(24.49) ** <0.136 *The mean concentration of two intact and diseased areas in one individual. **P-value in comparing IL-1β concentration between the two sites in each group is <0.001 Although the average Concentration of IL-1ß in smokers was greater than that of non–smokers, but the difference was not significant .In comparing intact and diseased sites of each groups separately, significant difference was observed which was to the benefit of unhealthy sites in each group (P<0.001). Discussion: Since clinical parameters (such as PPD, mobility, PL, CAL) and the extremity of ailment in general can influence the amount of IL-1ß (13,15,16), this study aimed to equalize these factors in the two groups as far as possible. Also, all the unhealthy sites from which samples were taken, suffered from BOP and bone loss. Since age and gender can be effective in this regard, the two groups were matched for age and gender. In this study, IL-1ß was found in all samples of GCF which is in consistent with most of the studies (1,6,13,15) . Though in studies carried out by lshibara (1997) (9) and Wilson et al (1992) (17) IL-1ß was not found in healthy sites and in another reseach by Bostrom et al (2000)(16) IL-1ß factor was found in GCF of 95% of people under study. It is not peculiar to say that IL-1ß exists in healthy sites, too, because some investigations revealed that a few macrophages and mononuclear cells exist in gingival tissue and some neutrophils in GCF in tissues that are clinically normal. These cells can produce IL-1ß .In the present research like most of the recent researches, IL-1ß was seen in all samples. This can be due to taking samples with a completely modern method and using accurate laboratorial method which is exclusive to IL-1ß. Probably, laboratorial methods in previous researches was not so accurate so they had not been able to recognize the presence of little amount of IL-1ß in the GCF of healthy sites. It is also probable that the old methods were not exclusive to IL-1ß. Due to variation among the population under study, methodology of research and evaluation procedures, it is not safe to compare the results of this study with those of the old 5 ones. For example the amount of IL-1ß has been reported in different forms through different researches. Also effective factors such as age, gender and the extremity of disease have not been matched between the two groups and the number of samples has also varied through different researches. In this study it was observed that the density of IL-1ß in unhealthy sites of the two groups was significantly greater than that of the intact sites (P<0.001).Also Chi-cheng (1995), Figueredo et al (8) (1999) and Bostrom et al (14) by by studying the people suffering from periodontitis and healthy ones; Ishibara et al (2003) (9) by studying the people with varied extremity of periodontal disease; Giannopoulou et al (2003) by comparing patients suffering from gingivitis with healthy people and by comparing the patients suffering from adult gingivitis periodontitis and early progressive periodontitis with healthy people through another research in the same year: and Kamma (2004)(4) by comparing the patients suffering from periodontitis with healthy people, all reported that the density of IL-1ß in unhealthy sites is greater than that of the healthy site. All of the studies support the outcome of the present study. In comparing the two groups of smokers and non-smokers in terms of the concentration of IL-1ß in healthy sites, significant difference was seen which was to the advantage of smokers (P<0.01) while, this difference was not significant in terms of unhealthy sites (P<0.501). Also in comparing the concentration of IL-1ß in the two groups, although the average amount of IL-1ß in smokers was a bit more, the difference was not significant. Figueredo et al (1999)(8) and Bostrom et al (2000)(14) suggested that there is no significant relationship between the density of IL-1ß in the two groups of smokers and non–smokers. Also, Giannopoulou et al (8,6) , in both of their researches in 2003, didn’t find any significant relationship between the concentration of IL-1ß in smokers and that of non–smokers, however they reported that IL-1ß secretion in smokers is more than that of non–smokers although this difference is not significant. Kamma et al (2004)(4), by comparing smokers and non-smokers suffering from EOP with healthy people found that, there is no significant difference between smoker and non – smoker groups but in terms of healthy people , the difference is to the benefit of the smokers (P<0.001). Since this study obtained a significant relationship between smokers and non-smokers only in terms of healthy sites, it's outcome is in consistent with the present study. Since on the basis of several investigations, using nicotin didn’t prove to have any effect on lymphocyte, monocyte, mononuclear cells of gingiva of patients suffering from periodontal disease, and on the secretion of IL-1ß, in laboratorial situation, we can conclude that nicotin can not activate more cell in periodontal disease and this is probably due to prior maximum stimulation of the cells caused by periodontal disease itself. However, these investigations have been carried out in laboratorial 6 situation and their outcome can not be generalized to human situation. Among various investigations, there are studies that reported a close relationship between smoking and IL-1ß in heavy smokers with that of non- smokers, reported significant difference to the benefit of smokers; however in this study several variables affecting the amount of IL-1ß such as smoking and diabetes were studied (13). Also Rawlinson et al (2003) (1) stated that the density of IL-1ß in bleeding sites of non-smokers was more than that of smokers. It is worth noting that the extremity of the disease had not been balanced out between the two groups and this is probably due to the presence of little amount of IL-1ß in smokers. Conclusion: More amount of IL-1ß in unhealthy site than to healthy one supports the idea that the concentration of IL-1ß has relationships with sites and periodontal disease. In studying various sites, significant differences were seen between the amount of IL-1ß in healthy intact sites of smokers and that of non-smokers. On the basis of some contradictory results obtained from various studies in this regard, we can infer that smoking has a complicated effect on GCF. Since there are other unknown effective factors in this respect more investigations are needed to discover these factors. References 1. Rawlinson A, Grammitt JM, Walsh TF, Ian Douglas CW: Interleukin 1 and reseptor antagonist level in gingival crevicular fluid in heavy smokers versus non-smokers. J Clin Periodontal 2003;30:42-48. 2. Chi-Cheng Tsai, Yea-Pyng Ho, Ching-Charng Chen: Levels of Interleukin-1β and Interleukin8 in gingival crevicular fluids in adult periodontitis. J Periodontol 1995;66:852-859. 3. Newman MG, Takei HH, Carranza AF: Clinical periodontology, 9th Ed. WB Saunders Co. Philadelphia, USA. 2002;Chaps4,8,14:70-71,126-136,208-210. 4. 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Payne JB, Johnson GK, Reinhardt RA, Dyer JK, Maze CA, Dunning DG: Nicotine effects on PGE2 and IL-1 beta release by LPS-treated human monocyte. J Periodontal Res 1996;31:99104. 9