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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.
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______________________________________________________________________________
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
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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).
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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
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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
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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.
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