Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2012 Mar 1.
Published in final edited form as: J Clin Periodontol. 2010 Dec 29;38(3):219–228. doi: 10.1111/j.1600-051X.2010.01684.x

Influence of smoking on gingival crevicular fluid cytokines in severe chronic periodontitis

Keelen D Tymkiw 1, Daniel H Thunell 1, Georgia K Johnson 1,2, Sophie Joly 2, Kindra K Burnell 2, Joseph E Cavanaugh 3, Kim A Brogden 1,2, Janet M Guthmiller 4
PMCID: PMC3038432  NIHMSID: NIHMS261018  PMID: 21198766

Abstract

Aim

The aim of this study was to compare the expression of 22 chemokines and cytokines in gingival crevicular fluid (GCF) from smokers and non-smokers with periodontitis and periodontally healthy control subjects.

Materials and Methods

Forty subjects with generalized severe chronic periodontitis (20 smokers and 20 non-smokers) and 12 periodontally healthy control subjects participated in this study. Four diseased and 2 healthy sites were selected from each of the periodontitis subjects. GCF samples were collected and cytokines analyzed utilizing a multiplexed immunoassay (Luminex®). Statistical analyses employed non-parametric tests including the Mann-Whitney and Wilcoxon matched-pairs signed-rank tests.

Results

Compared to healthy control subjects, GCF in subjects with chronic periodontitis contained significantly higher amounts of IL-1α, IL-1β, IL-6, IL-12 (p40) (pro-inflammatory cytokines); IL-8, MCP-1, MIP-1α, RANTES (chemokines); IL-2, IFN-γ, IL-3, IL-4 (Th1/Th2 cytokines); IL-15 (regulator of T-cells and NK cells). Smokers displayed decreased amounts of pro-inflammatory cytokines (IL-1α, IL-6, IL-12 (p40)), chemokines (IL-8, MCP-1, MIP-1, RANTES) and regulators of T-cells and NK cells (IL-7, IL-15).

Conclusions

Periodontitis subjects had significantly elevated cytokine and chemokine profiles. Smokers exhibited a decrease in several pro-inflammatory cytokines and chemokines and certain regulators of T-cells and NK-cells. This reflects the immunosuppressant effects of smoking which may contribute to an enhanced susceptibility to periodontitis.

Keywords: Smoking, periodontal disease, immune response, periodontitis/etiology, chronic periodontitis, gingival crevicular fluid

Introduction

Gingival crevicular fluid (GCF) in periodontitis subjects contains inflammatory cells, serum proteins, bacteria, tissue breakdown products, enzymes, antibodies, complement, and numerous inflammatory mediators (Cimasoni, 1983, Armitage, 1996). GCF volume increases with periodontal inflammation (Loe and Holm-Pedersen, 1965, Oliver et al., 1969), its flow rate increasing up to 30-fold in periodontitis sites compared to healthy sites (Goodson, 2003). Collection of GCF is relatively noninvasive, making it a convenient tool to evaluate markers of periodontal inflammation. The magnitude of inflammation can be determined by measuring pro-inflammatory and immuno-regulatory cytokines and chemokines in the GCF. Sites with clinical inflammation have elevated levels of many different cytokines and chemokines including interleukin (IL)-1, IL-6, IL-8, and tumor necrosis factor (TNF)-α that play an important role in the pathogenesis of the periodontitis (Genco, 1992).

Cigarette smoking is a significant risk factor for periodontitis (Bergstrom and Preber, 1994). Tobacco use induces alterations in microbial populations (Zambon et al., 1996, Umeda et al., 1998, Haffajee and Socransky, 2001, van Winkelhoff et al., 2001, Eggert et al., 2001). It is well accepted that smoking alters the host response, including vascular function, neutrophil/monocyte activities, adhesion molecule expression, antibody production, as well as cytokine and inflammatory mediator release (Barbour et al., 1997, Kinane, 2000, Palmer et al., 2005, Ryder, 2007). These changes likely contribute to the negative impact of smoking on the reparative and regenerative potential of the periodontium.

In cell culture systems, nicotine treatment increases production of IL-6 by murine osteoblasts (Kamer et al., 2006) and fibroblasts (Wendell and Stein, 2001) and IL-1 by keratinocytes (Johnson and Organ, 1997). Although these studies are useful in understanding mechanisms of tobacco related periodontal destruction, nicotine is only one component of tobacco. In addition, there are a variety of cell types involved in the in vivo response to smoking. GCF provides an avenue to evaluate the multiple effects of smoking on the host response in the periodontium. The existing studies have focused on smoking’s impact on a limited number of pro-inflammatory cytokines and chemokines. Smokers have elevated GCF levels of TNF-α (Bostrom et al., 1998) and IL-8 (Giannopoulou et al., 2003a, Giannopoulou et al., 2003b). In other studies, smoking decreases the levels of certain cytokines in GCF, such as IL-1β, IL-1 receptor antagonist (ra) (Rawlinson, Grummitt et al. 2003), IL-1α (Petropoulos, McKay et al. 2004), and IL-8 (Kamma et al., 2004), although others have shown no effect on IL-1β amounts (Giannopoulou et al., 2003b, Bostrom et al., 2000). Smoking’s effect on a wider spectrum of biomarkers in GCF has not been well characterized. Therefore, this study evaluated the impact of smoking on a panel of biomarkers.

GCF was obtained from smokers and nonsmokers with generalized severe chronic periodontitis and periodontally healthy controls. Twenty two different biomarkers were evaluated: Th1 cytokines (IL-2, IL-12(p70), and interferon (IFN)-γ); Th2 cytokines (IL-3, IL-4, IL-5, IL-10, and IL-13); pro-inflammatory cytokines (IL-1α, IL-1β, IL-6, granulocyte macrophage colony stimulating factor (GM-CSF), TNF-α, and IL-12(p40)); chemokines (IL-8, interferon inducible protein-10 (IP-10), macrophage chemotactic protein-1 (MCP-1), macrophage inflammatory protein (MIP)-1α, regulated on activation normal T cell expressed and secreted (RANTES) and Eotaxin); and regulators of T and natural killer cell activation and proliferation (IL-7 and IL-15). Through the evaluation of this extensive panel of GCF biomarkers, this study aimed to characterize the impact of smoking and periodontitis on the host response.

Materials and Methods

Study population

Fifty-two subjects, including 40 periodontally diseased subjects (20 smokers and 20 non-smokers) and 12 periodontally healthy non-smokers, participated in this study (Table 1). The mean age of all 52 subjects was 55 ± 9.6 years. All subjects were Caucasian with the exception of one Asian and one Hispanic subject. Periodontally diseased subjects had a diagnosis of generalized severe chronic periodontitis (> 30% of sites with a clinical attachment level (CAL) and probing depth (PD) ≥ 5 mm) (Table 2).

Table 1.

Demographic characteristics of the fifty-two subjects

Healthy Controls
(N = 12)		 Smokers
(N = 20)		 Non-smokers
(N = 20)
Age (years, mean ± SEM) 51.2 ± 5.2 51.2 ± 7.4 61.2 ± 9.7
Female 10 8 12
Male 2 12 8
Open in a new tab

Table 2.

Site characteristics (mean ± SEM) of the subjects.

NH SH ND SD Con H (pooled) D (pooled)
Probing depth (mm) 2.6 ± 0.6 2.8 ± 0.4 5.8 ± 0.9* 5.6 ± 0.5* 2.3 ± 0.6 2.7 ± 0.5 5.7 ± 0.8*
Recession (mm) 0.3 ± 0.7 0.3 ± 0.4 0.7 ± 0.9* 1.0 ± 1.1* 0.1 ± 0.4 0.3 ± 0.6 0.8 ± 1.0*
Attachment level (mm) 2.9 ± 0.9 3.0 ± 0.3 6.5 ± 1.3* 6.6 ± 1.3* 2.5 ± 0.7 2.9 ± 0.7 6.5 ± 1.3*
Gingival crevicular fluid (µl) 1.6 ± 1.0 1.3 ± 0.9 2.3 ± 0.8 1.9 ± 0.9# 1.1 ± 0.7 1.5 ± 1.0† 2.1 ± 0.08†‡
Open in a new tab

NH=healthy sites in periodontitis non-smoking subjects; SH=healthy sites in periodontitis smoking subjects; ND=diseased sites in periodontitis non-smoking subjects; SD=diseased sites in periodontitis smoking subjects; Con=healthy control; H (pooled) =healthy sites in smoking and nonsmoking periodontitis subjects; D (pooled) =diseased sites in smoking and nonsmoking periodontitis subjects.

*

Significantly different from healthy control subjects (Con).

†

Significantly different from healthy controls (Con).

‡

Significantly different from healthy sites in the diseased subjects (H (pooled)).

#

Significantly different from diseased sites in the nonsmoking periodontitis subjects (ND).

Subjects had not received periodontal therapy for four months preceding their participation in the study. Smokers were enrolled if they regularly smoked ≥20 cigarettes per day, and non-smokers were characterized as not having smoked one hundred or more cigarettes in their lifetime. Periodontally healthy subjects included nonsmokers with CAL and PD ≤ 3 mm and bleeding on probing (BOP) at ≤10% of sites. To participate in the study, all subjects were in good general health. Subjects were excluded from participating if they were pregnant or had a history of diabetes or intake of medication, such as antibiotics and anti-inflammatory agents, due to their possible effects on the microbial flora and/or the immune or inflammatory response, for six months prior to the study.

Subject selection and data collection were performed in the Department of Periodontics at the College of Dentistry, University of Iowa. Written informed consent was obtained from each subject, and the study protocol was approved by the University of Iowa Institutional Review Board.

Site selection

Two diseased (PD and CAL ≥5 mm with BOP) and two healthy sites (PD and CAL ≤3mm with no BOP) were identified in each of the 40 periodontitis subjects and two healthy sites were identified in the 12 periodontally healthy control subjects. The diseased and healthy sites in the 20 smokers were identified as SD and SH, respectively. In the 20 nonsmokers, the diseased and healthy sites were designated as ND and NH, respectively (Table 2). Four healthy sites were sampled from the twenty periodontally healthy, non-smoking individuals and classified as (Con).

GCF collection

GCF collection took place at a subsequent visit following the initial examination. Prior to GCF sampling, the individual tooth site was isolated with cotton rolls, supragingival plaque was carefully removed and the site was gently air-dried with the air syringe. A paper strip (Periopaper, Amityville, NY, USA) was inserted into the crevice 1–2 mm for 30s. The GCF volume was determined based on measurements made using a Periotron 8000 (Oraflow Inc., Plainview, NY, USA) as recently described (Thunell et al., 2010). In cases of visible contamination with blood, the strips were discarded and new sites sampled. Strips from each subject were placed into labeled tubes containing 300 µl 0.01 M PBS, pH 7.2 and protease inhibitor (Complete Mini, protease inhibitor cocktail tablets, Roche Applied Science, IN, USA). After shaking for 20 min, the strips were removed and the eluates centrifuged for 5 min at 5,800 × g to remove plaque and cellular elements. The samples were frozen at −80° C until further analysis.

Cytokine and chemokine analysis

Th1 cytokines (IL-2, IL-12(p70), and IFN-γ), Th2 cytokines (IL-3, IL-4, IL-5, IL-10, and IL-13), proinflammatory cytokines (IL-1α, IL-1β, IL-6, GM-CSF, TNF-α, and IL-12(p40)), chemokines (IL-8, IP-10, MCP-1, MIP-1α, RANTES and Eotaxin), and regulators of T and natural killer cell activation and proliferation (IL-7 and IL-15) in GCF (pg/30s) were determined as previously described (Thunell 2010) using a commercial multiplexed fluorescent bead-based immunoassay (Millipore, Billerica, MA) in the Luminex 100 IS Instrument (Luminex®, Austin, TX). Each sample was assayed in duplicate. Briefly, 50 µl of 0.01 M PBS, pH 7.2 containing GCF samples were incubated with anti-human multi-cytokine beads at 4° C for 18 h. Unbound material was removed by filtration. Anti-human multi-cytokine biotin reporter was added, and reactions were incubated at room temperature for 1.5 h in the dark. Streptavidin–phycoerythrin then was added, and the plates were incubated at room temperature for an additional 30 min. Stop solution was added, and the plates were read in a plate reader (Model 100 IS, Luminex, Austin, TX). Cytokine quantities in each sample were extrapolated based on standards utilizing Beadview software (Millipore, Billerica, MA).

Statistical analysis

Analysis of normality was conducted, and nonparametric approaches were used based on the distribution of the data. Total cytokine amounts (pg/30s) were analyzed and reported for each cytokine. Cytokine and GCF volumes were analyzed using the Mann-Whitney test to compare cytokine and GCF levels. This analysis was completed for inter-group and pooled comparisons. Inter-group comparisons consisted of comparing the healthy controls (Con) to both healthy and diseased sites in smoking and non-smoking diseased subjects (SH, SD, NH, ND). Additionally, smokers and non-smokers were compared in relation to healthy and diseased sites (SH vs NH; SD vs ND). Pooled comparisons consisted of comparing the healthy controls (Con) to both healthy and diseased sites in pooled smoker and non-smoker periodontitis subjects (H pooled, D pooled). Intra-group and pooled comparisons for matched-paired groups were completed using the Wilcoxon matched-pairs signed-rank test. Intra-group matched-paired comparisons consisted of comparing healthy and diseased sites in smokers and in non-smokers (SH vs SD; NH vs ND). Pooled matched-paired comparisons consisted of comparing healthy and diseased sites in diseased subjects (H pooled vs D pooled). In each case the level of significance was set at p<0.05 (Table 3).

Table 3.

P values of intra-group, inter-group and pooled comparisons of pro-inflammatory cytokines.

Intra-Group
Comparisons		 Inter-Group
Comparisons		 Pooled
Comparisons
NH/ND SH/SD Con/NH Con/ND Con/SH Con/SD NH/SH ND/SD H pooled)/
D (pooled)		 Con/
H (pooled)		 Con/
D (pooled)
IL-1α <0.0001* 0.0897 0.2425 <0.0001* 0.3986 <0.005* 0.5972 <0.05* <0.001* 0.2408 <0.001*
IL-1β <0.005* <0.0001* <0.01* <0.0001* <0.01* <0.0001* 0.8749 0.2862 <0.001* <0.01* <0.001*
IL-6 0.0728 0.2024 <0.001* <0.0001* 0.0767 0.6141 <0.001* <0.001* <0.05* 0.4365 <0.05*
IL-12(p40) <0.01* 0.1231 <0.05* <0.0001* 0.8974 0.1365 <0.05* <0.001* <0.01* 0.1708 <0.001*
GM-CSF <0.05* 0.4954 0.7371 0.2630 0.7902 0.7453 0.5183 0.3848 0.2580 0.9792 0.3813
Open in a new tab

NH=healthy sites in periodontitis non-smoking subjects; SH=healthy sites in periodontitis smoking subjects; ND=diseased sites in periodontitis non-smoking subjects; SD=diseased sites in periodontitis smoking subjects; Con=healthy control; H (pooled) =healthy sites in periodontitis subjects; D (pooled)=diseased sites in periodontitis subjects.

*

P values ≤ 0.05 were considered significant.

In some subjects, the concentrations of cytokines were beyond the detectable capacity of the assay, which was 2.3 to 5,000 pg/ml. For those chemokines and cytokines whose concentrations were below the detectable limit of the immunoassay, a value of 1.3 pg/ml was assigned by subtracting 1.0 from 2.3 pg/ml, the lowest value of the standard curve. When the concentrations of cytokines were higher the detectable capacity of the assay, the samples were diluted and rerun.

Results

Site characteristics among subjects

Diseased sites within the smoking and non-smoking periodontitis groups (SD, ND, D (pooled) had significantly (p < 0.05) higher values for PD, REC and CAL compared to the sites in the healthy controls (Con) (Table 2). GCF volumes were significantly higher in both healthy and diseased sites in periodontitis subjects compared to the healthy controls. (D pooled vs Con; H (pooled) vs Con, p = 0.0366 and < 0.001, respectively). GCF volumes in diseased sites of the smoking and non-smoking periodontitis subjects were also significantly higher (p = 0.0005) than healthy sites (D pooled vs H pooled). Diseased sites within smokers (SD) showed significantly lower (p = 0.0121) GCF volumes than diseased sites in the non-smoking population (ND).

Chemokine and Cytokine Concentrations

Seventeen of the 22 chemokines and cytokines assayed were detected in the GCF, including IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-6, IL-7, IL-8, IL-12(p40), IL-15, IP-10, IFN-γ, GM-CSF, MCP-1, MIP-1, RANTES and Eotaxin. The quantities of IL-5, IL-10, IL-12(p70), IL-13, and TNF-α were below the detectable limits of the multiplex assay. Therefore, they were excluded from further analysis.

Pro-inflammatory cytokines: IL-1α, IL-1β, IL-6, IL-12(p40), GM-CSF

The quantities of pro-inflammatory cytokines ranged from 0.0 to 32,580.0 pg/30s. The ranges for IL-1α were the broadest (ranging from 41.5 to 32,580.0 pg/30s), followed by IL-1β (0.0 to 1,662.0 pg/30s), IL-6 (10.2 to 1,632.0 pg/30s), IL-12(p40) (7.2 to 436.2 pg/30s), and GM-CSF (0.6 to 130.8 pg/30s) (Figure 1a–e). The significant differences among the chemokine and cytokine values are presented in Table 3.

Figure 1.

Figure 1

Open in a new tab

Pro-inflammatory cytokines: Median amounts (pg/30s): a) IL-1α b) IL-1β c) IL-6 d) IL-12(p40) e) GM-CSF. HC=healthy control; H (pooled) =healthy sites in periodontitis subjects; D (pooled) =diseased sites in periodontitis subjects; NH=healthy sites in periodontitis non-smoking subjects; SH=healthy sites in periodontitis smoking subjects; ND=diseased sites in periodontitis non-smoking subjects; SD=diseased sites in periodontitis smoking subjects. * p≤0.05, ** p≤0.01, *** p≤0.005, + p≤0.001, ++ p≤0.0005, +++ p≤0.0001.

Intra-group comparisons

Comparison of healthy and diseased sites in non-smokers (NH vs. ND) showed significantly higher amounts of IL-1α, IL-1β, IL-12(p40) and GM-CSF in diseased sites (Figure 1a,b,d,e, Table 3). Diseased sites in the smoking population (SD) also showed significantly higher quantities of IL-1β than healthy sites (SH) (Figure 1b).

Inter-group comparisons

Healthy sites in non-smokers with periodontitis (NH) showed significantly higher amounts of IL-1β, IL-6 and IL-12(p40) than the healthy controls (Con) (Figure 1b–d, Table 3) Diseased sites in the non-smoking population (ND) had significantly more IL-1α, IL-1β, IL-6 and IL-12(p40) than the healthy controls (Con) (Figure 1a–d, Table 3). Healthy sites in smokers (SH) showed significantly higher quantities of IL-1β than in healthy controls (Con) (Figure 1b, Table 3). Diseased sites in smokers (SD) showed significantly higher values for IL-1α and IL-1β than in the healthy controls (Con) (Figure 1a,b, Table 3). Comparison of healthy sites in smokers and non-smokers (SH vs. NH) showed significantly less IL-6 and IL-12 (p40) in the smokers (Figure 1c,d, Table 3). Additionally, diseased sites in smokers (SD) showed significantly less IL-1α, IL-6 and IL-12 (p40) than non-smokers (ND) (Figure 1a,c,d, Table 3).

Pooled comparisons

Diseased sites in pooled periodontitis subjects (D pooled) demonstrated significantly higher amounts of IL-1α, IL-1β, IL-6, IL-12 (p40) than healthy sites in periodontitis subjects (H pooled) (Figure 1a–d, Table 3). When compared to healthy controls (Con), diseased sites in periodontitis subjects (D pooled) showed significantly greater quantities of IL-1α, IL-1β, IL-6 and IL-12 (p40) (Figure 1a–d, Table 3). Healthy sites in periodontitis subjects (H pooled) also showed significantly higher IL-1β levels than that in the healthy controls (Con) (Figure 1b, Table 3).

Chemokines: IL-8, IP-10, MCP-1, MIP-1, RANTES and Eotaxin

Several chemokines were present in the GCF ranging in amounts from 0.6 to 24,306.0 pg/30s. IL-8 was found in the highest amounts (ranging from 21.2 to 24,306.0 pg/30s). The remaining chemokines had the following ranges: IP-10 (36.0 to 3,672.0 pg/30s); MIP-1 (18.6 to 573.6 pg/30s); Eotaxin (12.0 to 392.4 pg/30s); RANTES (0.6 to 1,098.0 pg/30s); and MCP-1 (1.2 to 111.6 pg/30s). The significant differences among the chemokine and cytokine values are listed Table 4.

Table 4.

P values of intra-group, inter-group and pooled comparisons of chemokines.

Intra-Group
Comparisons		 Inter-Group
Comparisons		 Pooled
Comparisons
NH/ND SH/SD Con/NH Con/ND Con/SH Con/SD NH/SH ND/SD H pooled)/
D (pooled)		 Con/
H (pooled)		 Con/
D (pooled)
IL-8 0.1769 0.9854 <0.0001* <0.0001* 0.3364 0.3856 <0.01* <0.001* 0.2734 <0.01* <0.001*
IP-10 0.7562 0.8983 <0.05* <0.05* <0.0001* <0.0001* <0.05* <0.05* 0.8666 <0.001* <0.0005*
MCP-1 <0.05* 0.1536 0.0703 <0.0001* <0.01* 0.3054 <0.001* <0.0001* <0.01* 0.5524 0.0649
MIP-1 0.1054 0.0826 <0.01* <0.0001* 0.8359 0.2213 <0.05* <0.001* <0.05* 0.0959 <0.001*
RANTES 0.0583 0.3683 <0.05* <0.0001* 0.4793 0.5190 <0.01* <0.01* <0.05* 0.3726 <0.01*
Eotaxin <0.05* 0.0897 0.9465 <0.005* 0.5525 0.9719 0.5336 0.0820 0.4639 0.7495 0.0656
Open in a new tab

NH=healthy sites in periodontitis non-smoking subjects; SH=healthy sites in periodontitis smoking subjects; ND=diseased sites in periodontitis non-smoking subjects; SD=diseased sites in periodontitis smoking subjects; Con=healthy control; H (pooled) =healthy sites in periodontitis subjects; D (pooled)=diseased sites in periodontitis subjects.

*

P values ≤ 0.05 were considered significant.

Intra-group comparisons

Diseased sites in non-smokers (ND) had significantly higher amounts of MCP-1 and Eotaxin than healthy sites (NH) (Figure 2c,f, Table 4). In smokers, there were no significant differences in any of the chemokines between diseased sites (SD) when compared to healthy sites (SH) (Figure 2a–f, Table 4).

Figure 2.

Figure 2

Open in a new tab

Chemokines: Median amounts (pg/30s): a) IL-8 b) IP-10 c) MCP-1 d) MIP-1 e) RANTES f) Eotaxin. HC=healthy control; H (pooled) =healthy sites in periodontitis subjects; D (pooled) =diseased sites in periodontitis subjects; NH=healthy sites in periodontitis non-smoking subjects; SH=healthy sites in periodontitis smoking subjects; ND=diseased sites in periodontitis non-smoking subjects; SD=diseased sites in periodontitis smoking subjects. * p≤0.05, ** p≤0.01, *** p≤0.005, + p≤0.001, ++ p≤0.0005, +++ p≤0.0001.

Inter-group comparisons

Comparison of chemokine amounts in healthy sites of non-smokers (NH) compared to the healthy controls (Con) showed significantly higher values for IL-8, MIP-1 and RANTES in NH sites (Figure 2a,d,e Table 4). In contrast, IP-10 showed significantly lower amounts in the healthy (NH) and diseased (ND) sites of non-smokers than in the healthy controls (Con) (Figure 2b, Table 4). Comparison of chemokine amounts in diseased sites of non-smokers (ND) compared to the healthy controls (Con) showed significantly higher quantities of IL-8, MCP-1, MIP-1, RANTES, and Eotaxin in ND sites (Figure 2a,c,d,e,f, Table 4).

Healthy (SH) and diseased (SD) sites in smokers showed significantly less IP-10 than the healthy controls (Con) (Figure 2b, Table 4). Healthy sites in smokers also had lower amounts of MCP-1 than the healthy controls (Con) (Figure 2c, Table 4). Healthy (SH) and diseased (SD) sites in the smoking population showed significantly less IL-8, IP-10, MCP-1, MIP-1α and RANTES than healthy (NH) and diseased sites (ND) of non-smokers with periodontitis (Figure 2a–e, Table 4).

Pooled comparisons

Diseased sites in pooled periodontitis subjects (D pooled) showed significantly greater amounts of MCP-1, MIP-1 and RANTES than healthy sites (H) (Figure 2c–e, Table 4). In healthy (H) sites of periodontitis subjects, amounts of IL-8 were significantly higher than in the healthy controls (Con) (Figure 2a, Table 4). IP-10 was significantly decreased in both H (pooled) sites and D (pooled) sites compared to healthy controls (Con) (Figure 2b, Table 4). Diseased (D) sites in periodontitis subjects had significantly greater levels of IL-8, MIP-1 and RANTES than healthy controls (Con) (Figure 2a,d,e, Table 4).

Th1 and Th2 cytokines: IL-2, IFN-γ, IL-3, and IL-4

Th1 and Th2 cytokines were present but generally were found in low amounts (ranging from 0.0 to 349.2 pg/30s) for all subjects in all groups. The ranges for these cytokines were as follows: IL-2 (12.2 to 77.4 pg/30s); IFN-γ (0.0 to 152.4 pg/30s);IL-4 (0.6 to 123.0 pg/30s) and IL-3 (22.2 to 349.2 pg/30s). The GCF from the healthy control group exhibited the lowest values of each of these 4 cytokines (Figure 3a–d). The significant differences among the chemokine and cytokine responses are shown in Table 5.

Figure 3.

Figure 3

Open in a new tab

Th1 and Th2 cytokines: Median amounts (pg/30s): a) IL-2 b) IFN-γ c) IL-3 d) IL-4. HC=healthy control; H (pooled) =healthy sites in periodontitis subjects; D (pooled) =diseased sites in periodontitis subjects; NH=healthy sites in periodontitis non-smoking subjects; SH=healthy sites in periodontitis smoking subjects; ND=diseased sites in periodontitis non-smoking subjects; SD=diseased sites in periodontitis smoking subjects. * p≤0.05, ** p≤0.01, *** p≤0.005, + p≤0.001, ++ p≤0.0005, +++ p≤0.0001.

Table 5.

P values of intra-group, inter-group and pooled comparisons of Th1 and Th2 cytokines and regulators of T and NK cells.

Intra-Group
Comparisons		 Inter-Group
Comparisons		 Pooled
Comparisons
NH/ND SH/SD Con/NH Con/ND Con/SH Con/SD NH/SH ND/SD H pooled)/
D (pooled)		 Con/
H (pooled)		 Con/
D (pooled)
IL-2 0.6226 0.4900 0.3307 <0.005* 0.0859 0.3418 0.6380 0.1658 0.7718 0.1116 <0.05*
IFN-γ <0.01* <0.05* 0.2133 <0.0005* 0.7486 0.1314 0.4331 0.0802 <0.001* 0.3687 <0.01*
IL-3 0.4091 0.2935 0.5214 <0.001* 0.305 <0.05* 0.4955 0.3447 0.1650 0.1220 <0.001*
IL-4 0.8906 0.6215 0.5214 <0.05* 0.3645 0.2124 0.6672 0.6845 0.7377 0.3612 <0.05*
IL-7 0.7841 0.4304 0.6034 <0.05* 0.8451 0.6893 0.4682 <0.01* 0.7933 0.8629 0.3039
IL-15 0.0897 0.2024 p<0.05* <0.0005* 0.6362 0.0877 0.0784 <0.05* <0.05* 0.1157 <0.01*
Open in a new tab

NH=healthy sites in periodontitis non-smoking subjects; SH=healthy sites in periodontitis smoking subjects; ND=diseased sites in periodontitis non-smoking subjects; SD=diseased sites in periodontitis smoking subjects; Con=healthy control; H (pooled) =healthy sites in periodontitis subjects; D (pooled)=diseased sites in periodontitis subjects.

*

P values ≤ 0.05 were considered significant.

Intra-group comparisons

Comparison of healthy and diseased sites within smokers (SH vs. SD) and non-smokers (NH vs. ND) showed significantly higher amounts of IFN-γ in the diseased sites of both groups (Figure 3b, Table 5).

Inter-group comparisons

Diseased sites in non-smokers (ND) showed significantly greater amounts of IL-2, IFN-γ IL-3, and IL-4 than in the healthy controls (Con) (Figure 3a–d, Table 5). Diseased sites in the smoking population (SD) showed significantly higher amounts of IL-3 than in the healthy controls (Con) (Figure 3c, Table 5). Comparisons of healthy (NH vs. SH) and diseased (ND vs. SD) sites between smokers and non-smokers showed no significant differences in levels of any Th1 or Th2 cytokine (Figure 3a–d, Table 5).

Pooled comparisons

Diseased sites in pooled periodontitis subjects (D pooled) which included smokers and nonsmokers demonstrated significantly higher amounts of IFN-γ than in healthy sites (H pooled) (Figure 3b, Table 3). Additionally, D (pooled) sites also showed significantly greater amounts of IL-2, IFN- γ, IL-3 and IL-4 compared to healthy controls (Con) (Figure 3a–d, Table 3).

Regulators of T-cells and NK cells: IL-7, IL-15

The values for IL-7 in GCF ranged from 21.2 to 196.8 pg/30s, and IL-15 ranged from 10.9 to 48.8 pg/30s (Figure 4a, b). The significant differences among the chemokine and cytokine responses are found in Table 3.

Figure 4.

Figure 4

Open in a new tab

Regulators of T-cells and NK cells: Median amounts (pg/30s): a) IL-7 b) IL-15 HC=healthy control, H (pooled)=healthy sites in periodontitis subjects, D (pooled)=diseased sites in periodontitis subjects, NH=healthy sites in periodontitis non-smoking subjects, SH=healthy sites in periodontitis smoking subjects, ND=diseased sites in periodontitis non-smoking subjects, SD=diseased sites in periodontitis smoking subjects. * p≤0.05, ** p≤0.01, *** p≤0.005, + p≤0.001, ++ p≤0.0005, +++ p≤0.0001.

Intra-group comparisons

No significant differences in amounts of IL-7 or IL-15 were found when comparing healthy and diseased sites within smokers (SH vs. SD) and non-smokers (NH vs. ND) (Figure 4a,b, Table 3).

Inter-group comparisons

Healthy (NH) and diseased (ND) sites in non-smokers with periodontitis showed significantly higher quantities of IL-15 than healthy controls (Con) (Figure 4a,b, Table 3). Diseased sites in non-smokers (ND) also showed significantly higher IL-7 than healthy controls (Con) (Figure 4a, Table 3). Comparison of cytokine amounts in diseased sites of smokers (SD) and non-smokers (ND) showed a significant decrease in IL-7 and IL-15 in smokers (Figure 4a, b, Table 3).

Pooled comparisons

Comparison of healthy (H pooled) and diseased (D pooled) sites in pooled periodontitis subjects showed significantly higher levels of IL-15 in diseased sites (Figure 4b, Table 3). Diseased sites in periodontitis subjects (D pooled) also showed significantly greater amounts of IL-15 when compared to healthy controls (Con) (Figure 4b, Table 3).

Discussion

The use of the multiplex assay in this study made it possible to evaluate the levels of a comprehensive panel of cytokines and chemokines in the GCF of periodontally diseased subjects and healthy controls and the influence of smoking on these parameters. Several previous studies have evaluated GCF biomarkers in using enzyme-linked immunosorbent assays (ELISA’s), but a limitation is that this technology can evaluate a limited number of mediators in each sample. The most important finding of the present study was that smokers with periodontitis, as compared to nonsmoking periodontitis subjects, exhibited a decrease in several pro-inflammatory cytokines, chemokines and certain regulators of T-cells and NK-cells. Consistent with this, there was a higher frequency of differences in biomarker levels between healthy and diseased sites within nonsmokers than in smokers. Overall, periodontitis sites (nonsmokers and smokers pooled) and diseased sites in nonsmokers had significantly elevated cytokine and chemokine profiles as compared to healthy control subjects.

The Impact of Disease on Biomarker Expression

Within pooled periodontitis subjects, diseased sites as compared to healthy sites contained elevated amounts of several chemokines and pro-inflammatory cytokines, including IL-1α, IL-1β, IL-6, IL-12 (p40), MCP-1, MIP-1α and RANTES. IFN-γ, a Th1 cytokine, and IL-15, a lymphocyte growth factor, were also elevated in these sites. All of these biomarkers as well as IL-2 IL-3, IL-4 (Th1/Th2 cytokines) and IL-8 were increased in diseased sites in periodontitis subjects relative to healthy controls.

Numerous studies have shown increased GCF amounts of pro-inflammatory cytokines in periodontitis, including IL-1 (Teles et al., 2010, Tsai et al., 1995), IL-6 (Geivelis et al., 1993), and IFN-γ (Dutzan et al., 2009). Interestingly, healthy sites in pooled periodontitis subjects exhibited increased IL-1β and IL-8 when compared to healthy control subjects. This pattern has also been reported by Teles and coworkers (Teles et al., 2010), and Engebretson et al (Engebretson et al., 2002), where subjects with severe disease had higher levels of IL-1β in shallow sites compared to subjects with less severe disease. It has been suggested that this may reflect an overall patient factor such as genotype that influences host response to bacterial challenge or a pre-clinical stage of inflammation.

Although IL-12 (p40) was elevated in periodontitis subjects, IL-12(p70) was below the detection level of the assay. IL-12 is a heterodimeric cytokine made up of a 40 kDa (p40) subunit and a 35 kDa (p35) subunit that comprise the bioactive form, IL-12(p70), which induces Th1 cell-mediated immunity. The IL-12 p40 subunit is shared by IL-23, another heterodimeric cytokine that has biological activities similar to, yet distinct from, IL-12 (Belladonna et al., 2002). High amounts of the p40 component inhibit binding of the active IL-12(p70) to its receptor and therefore inhibit IL-12 bioactivity (Gately et al., 1998). Reduced levels of the bioactive form of IL-12 in GCF of periodontitis subjects have been noted by others, with a trend towards decreasing amounts from gingivitis to periodontitis (Orozco et al., 2006). A relative reduction in bioactive IL-12 could favor the predominant Th2 response observed in periodontitis. IL-15 is produced by lipopolysaccharide stimulated fibroblasts, keratinocytes, macrophages and endothelial cells and, similar to the current study where it was increased in periodontitis sites, it has also been reported to be elevated in gingivitis sites (Buduneli et al., 2003). Other investigators, however, found that it was lower in inflamed as compared to healthy tissues (Johnson and Serio, 2007).

Of the chemokines evaluated, IL-8, RANTES, MCP-1 and MIP-1α were elevated in diseased sites compared to healthy sites in pooled periodontitis subjects (smokers and non-smokers combined). This is consistent with other studies showing higher GCF IL-8 (Tsai et al., 1995, Teles et al., 2010, Pradeep et al., 2009), RANTES and/or MCP-1 levels in patients with aggressive or chronic periodontitis as compared to healthy controls (Emingil et al., 2004, Kurtis et al., 2005, Pradeep et al., 2009). MIP-1α has not been evaluated in GCF, although it has been detected in diseased gingival tissues (Gemmell et al., 2001). In non-periodontal infections, MIP-1α and MCP-1 are consistent with monocyte infiltration that occurs during infection (Maurer and von Stebut, 2004, Deshmane et al., 2009). IP-10 was the only marker that was reduced in periodontitis (in both healthy and diseased sites) when compared to healthy controls. This reduction in diseased sites consistently occurred within smokers and nonsmokers, as well as between periodontitis subjects and healthy controls. This unique finding deserves further investigation. IP-10 induction is likely important in recruiting lymphocytes, cells prominent in periodontal lesions and likely participants in the adaptive immune response (Taubman and Kawai, 2001). The balance among the various types of chemokines and their receptors determines the inflammatory and immune cell profile of the lesion and is proposed to play a role in the balance between disease activity and stability (Gemmell et al., 2001).

The Impact of Smoking on Biomarker Expression

In this study, smokers exhibited a decrease in several pro-inflammatory cytokines (IL-1α, IL-6, IL-12(p40)), chemokines (IL-8, IP-10, MCP-1, MIP-1α and RANTES) and regulators of T-cells and NK cells (IL-7 and IL-15) in diseased sites as compared to diseased sites in nonsmokers. To our knowledge, a significant decrease in IL-6, IL-7, IL-12(p40), IL-15, IP-10, MCP-1, MIP-1α and RANTES within smokers has not been previously reported. In fact, the majority of these biomarkers have not been evaluated before in smokers. In contrast to the pro-inflammatory cytokines and chemokines, there were no apparent inhibitory effects of smoking on Th1 and Th2 cytokines.

Smoking’s inhibition of certain pro-inflammatory cytokines in GCF is supported by other studies that have shown depressed IL-1α (Petropoulos et al., 2004) and IL-1β (Rawlinson et al., 2003) in the GCF of smokers with periodontitis compared to nonsmokers with periodontitis. In contrast, other studies have shown higher GCF levels of IL-1β, IL-6 or TNF-α in smokers (Zhong et al., 2007, Bostrom et al., 1998, Bostrom et al., 1999) or no effect of smoking on these mediators (Bostrom et al., 2000, Kamma et al., 2004, Erdemir et al., 2004). Likewise, in vitro studies of the effects of nicotine or smoke on IL-1β release by various cell types have yielded conflicting outcomes (Kamer et al., 2006, Wendell and Stein, 2001, Johnson and Organ, 1997, Payne et al., 1996, Bernzweig et al., 1998, de Heens et al., 2009, Ryder et al., 2002). The majority of studies, however, support a depressive impact of smoking on IL-1β. These findings underscore how variables such as the model system, the stimulant (nicotine or smoke), GCF collection techniques and assays used for detection of biomarker expression contribute to the differences reported in these studies.

Regardless, we observed greater amounts of IL-1α, IL-1β and IL-3 within diseased sites of smokers when compared to periodontally healthy controls. This relative increase in IL-1β in diseased smokers is consistent with previous research that found greater total amounts of IL-1β in smokers with “early-onset” periodontitis as compared to healthy, nonsmoking control subjects (Kamma et al., 2004). Even though production of pro-inflammatory biomarkers is depressed in smokers, these mediators still occur at concentrations capable of pathogenesis.

IL-8 has been extensively evaluated in GCF in smokers. However, the effects of smoking vary depending on the type of periodontal disease (gingivitis, aggressive or chronic periodontitis)(Giannopoulou et al., 2003a, Giannopoulou et al., 2003b, Kamma et al., 2004). Our results demonstrate a reduction of IL-8, MIP-1α, MCP and RANTES in smokers. The observed reduction in chemokines could contribute to the phenomena of impaired neutrophil chemotaxis and migration in the periodontium in spite of the presence of leukocytosis (Palmer et al., 2005).

Recently, others have found that exposure to nicotine suppresses the innate immune response to infection by reducing the activity of antimicrobial peptides (Radek et al., 2010). It is possible that a similar phenomenon may occur with the production of chemokines and cytokines in smokers. Such a situation would help explain the lower amount of chemokines and cytokines expressed in smokers.

Similar to other studies, smokers in this study had lower GCF volume compared to nonsmokers (Apatzidou et al., 2005). One might question if this was the primary reason that smokers showed decreased levels of several of the biomarkers. The data, however, was also analyzed based on concentration, which takes into consideration GCF volume, and similar findings were noted. In another study in which smokers and nonsmokers had similar GCF volumes, the smokers had lower IL-1α concentrations (Petropoulos et al., 2004). Finally, Cesar-Neto and coworkers reported that both gingival tissue levels mRNA and protein expression of IL-1α, IL-10, TNF- α were depressed in smokers with periodontitis as compared to nonsmokers with periodontitis (Cesar-Neto et al., 2007), further supporting a depressed host response in smokers.

There is generally good correlation between multiplex assays and ELISA’s for most cytokines in biologic samples; however the multiplex assays may either yield higher or lower quantitative values, depending on the cytokine being measured (Elshal and McCoy, 2006). Therefore, trends in cytokine expression are similar when comparing the types of assay, although actual cytokine values may not be directly comparable. Evidence suggests that these differences are minimized by the use of similar capture and reporter antibodies, diluents and serum blockers. The inability to detect TNF-α in the present study may, in part, be related to these variables. Other investigators have also noted that TNF-α was frequently undetectable in GCF (Erdemir et al., 2004); furthermore, differences in GCF sampling techniques contribute to inter-study variation in cytokine values.

The multiplex immunoassay (Luminex®) employed in this study enabled the evaluation of a comprehensive chemokine and cytokine profile, in both smoking and non-smoking periodontitis subjects. This is the most comprehensive investigation to date of the impact of smoking and periodontitis on GCF biomarkers. A key finding was that smokers with periodontitis, as compared to nonsmoking periodontitis subjects, exhibited a decrease in several pro-inflammatory cytokines, chemokines and certain regulators of T-cells and NK-cells. Compared to healthy controls, GCF in subjects with periodontitis contained significantly higher amounts of the majority of biomarkers evaluated. The nonsmokers mainly accounted for these findings; compared to the smokers, they had a higher frequency of elevated biomarker quantities in diseased sites as compared to healthy controls. They also demonstrated more intra-group differences between healthy and diseased sites.

Smoking inhibited the expression of several biomarkers, including pro-inflammatory cytokines (IL-1α, IL-6, IL-12 (p40)), chemokines (IL-8, MCP-1, MIP-1α, RANTES) and regulators of T-cells and NK cells (IL-7, IL-15). The Th1/Th2 cytokines were the only group of cytokines not decreased by smoking. A reduction of pro-inflammatory cytokines is consistent with lower levels of clinical inflammation in smokers. Interestingly, levels of IL-1α and IL-1β at diseased sites in smokers were still elevated relative to periodontally healthy controls, underscoring the centrality of pro-inflammatory cytokines in the pathogenesis of periodontal diseases. Chemokine levels in smokers with periodontitis were not significantly different from the healthy controls, whereas nonsmokers with periodontitis had elevated chemokines relative to both smokers and the healthy controls. This suggests a major role for a diminished chemokine response in the pathogenesis of periodontitis in smokers. An inability to recruit inflammatory and immune cells could lead to an ineffective defense against periodontal pathogens and increased susceptibility to tissue destruction.

With the progression of our understanding of the pathogenesis of periodontal diseases, we are better able to identify potential diagnostic biochemical marker(s) that could be used to predict disease status and/or disease progression. Several studies have evaluated various cytokines and inflammatory mediators, intracellular and extracellular host enzymes, and byproducts of tissue breakdown as potential markers of periodontal diseases. The multi-bead array assay used in this study facilitated characterization of a broad biomarker profile in smokers and nonsmokers with periodontitis. Future studies assaying these or additional cytokines using this methodology would improve our understanding of the role of the host response in periodontitis and smoking’s impact in the inflammatory disease process.

Clinical Relevance

Scientific rationale for the study

The impact of smoking on the host response in periodontitis is not fully understood.

Principal findings

Smokers with periodontitis, as compared to nonsmokers with periodontitis, had decreased GCF quantities of several pro-inflammatory cytokines, chemokines and key regulators of T cell function. Within smokers and nonsmokers with periodontitis, GCF from diseased sites exhibited higher levels of mediators than healthy sites.

Practical application

Smoking suppresses the inflammatory and immune response which may contribute to increased periodontal disease susceptibility in smokers.

Acknowledgements

This work was supported by funds from R01 DE13334 (JMG) and R01 DE014390 (KAB) from the National Institute of Dental and Craniofacial Research, National Institutes of Health.

Footnotes

Conflict of interest and source of funding statement: The authors declare that they have no conflict of interests.

References

  1. Apatzidou DA, Riggio MP, Kinane DF. Impact of smoking on the clinical, microbiological and immunological parameters of adult patients with periodontitis. Journal of Clinical Periodontology. 2005;32:973–983. doi: 10.1111/j.1600-051X.2005.00788.x. [DOI] [PubMed] [Google Scholar]
  2. Armitage GC. Periodontal diseases: diagnosis. Annals of Periodontolology. 1996;1:37–215. doi: 10.1902/annals.1996.1.1.37. [DOI] [PubMed] [Google Scholar]
  3. Barbour SE, Nakashima K, Zhang JB, Tangada S, Hahn CL, Schenkein HA, Tew JG. Tobacco and smoking: environmental factors that modify the host response (immune system) and have an impact on periodontal health. Critical Reviews in Oral Biology and Medicine. 1997;8:437–460. doi: 10.1177/10454411970080040501. [DOI] [PubMed] [Google Scholar]
  4. Belladonna ML, Renauld JC, Bianchi R, Vacca C, Fallarino F, Orabona C, Fioretti MC, Grohmann U, Puccetti P. IL-23 and IL-12 have overlapping, but distinct, effects on murine dendritic cells. J Immunol. 2002;168:5448–5454. doi: 10.4049/jimmunol.168.11.5448. [DOI] [PubMed] [Google Scholar]
  5. Bergstrom J, Preber H. Tobacco use as a risk factor. Journal of Periodontology. 1994;65:545–550. doi: 10.1902/jop.1994.65.5s.545. [DOI] [PubMed] [Google Scholar]
  6. Bernzweig E, Payne JB, Reinhardt RA, Dyer JK, Patil KD. Nicotine and smokeless tobacco effects on gingival and peripheral blood mononuclear cells. Journal of Clinical Periodontology. 1998;25:246–252. doi: 10.1111/j.1600-051x.1998.tb02435.x. [DOI] [PubMed] [Google Scholar]
  7. Bostrom L, Linder LE, Bergstrom J. Clinical expression of TNF-alpha in smoking-associated periodontal disease. Journal of Clinical Periodontology. 1998;25:767–773. doi: 10.1111/j.1600-051x.1998.tb02368.x. [DOI] [PubMed] [Google Scholar]
  8. Bostrom L, Linder LE, Bergstrom J. Smoking and cervicular fluid levels of IL-6 and TNF-alpha in periodontal disease. Journal of Clinical Periodontology. 1999;26:352–357. doi: 10.1034/j.1600-051x.1999.260604.x. [DOI] [PubMed] [Google Scholar]
  9. Bostrom L, Linder LE, Bergstrom J. Smoking and GCF levels of IL-1beta and IL-1ra in periodontal disease. Journal of Clinical Periodontology. 2000;27:250–255. doi: 10.1034/j.1600-051x.2000.027004250.x. [DOI] [PubMed] [Google Scholar]
  10. Buduneli E, Genel F, Atilla G, Kutukculer N. Evaluation of p53, bcl-2, and interleukin-15 levels in gingival crevicular fluid of cyclosporin A-treated patients. Journal of Periodontology. 2003;74:506–511. doi: 10.1902/jop.2003.74.4.506. [DOI] [PubMed] [Google Scholar]
  11. Cesar-Neto JB, Duarte PM, de Oliveira MC, Tambeli CH, Sallum EA, Nociti FH., Jr Smoking modulates interleukin-6:interleukin-10 and RANKL:osteoprotegerin ratios in the periodontal tissues. Journal of Periodontal Research. 2007;42:184–191. doi: 10.1111/j.1600-0765.2006.00934.x. [DOI] [PubMed] [Google Scholar]
  12. Cimasoni G. Crevicular fluid updated. Monographs in Oral Science. 1983;12:III–VII. 1–152. [PubMed] [Google Scholar]
  13. de Heens GL, Kikkert R, Aarden LA, van der Velden U, Loos BG. Effects of smoking on the ex vivo cytokine production in periodontitis. Journal of Periodontal Research. 2009;44:28–34. doi: 10.1111/j.1600-0765.2007.01047.x. [DOI] [PubMed] [Google Scholar]
  14. Deshmane SL, Kremlev S, Amini S, Sawaya BE. Monocyte chemoattractant protein-1 (MCP-1): an overview. Journal of Interferon and Cytokine Research. 2009;29:313–326. doi: 10.1089/jir.2008.0027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dutzan N, Vernal R, Hernandez M, Dezerega A, Rivera O, Silva N, Aguillon JC, Puente J, Pozo P, Gamonal J. Levels of interferon-gamma and transcription factor T-bet in progressive periodontal lesions in patients with chronic periodontitis. Journal of Periodontology. 2009;80:290–296. doi: 10.1902/jop.2009.080287. [DOI] [PubMed] [Google Scholar]
  16. Eggert FM, McLeod MH, Flowerdew G. Effects of smoking and treatment status on periodontal bacteria: evidence that smoking influences control of periodontal bacteria at the mucosal surface of the gingival crevice. Journal of Periodontology. 2001;72:1210–1220. doi: 10.1902/jop.2000.72.9.1210. [DOI] [PubMed] [Google Scholar]
  17. Elshal MF, McCoy JP. Multiplex bead array assays: performance evaluation and comparison of sensitivity to ELISA. Methods. 2006;38:317–323. doi: 10.1016/j.ymeth.2005.11.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Emingil G, Atilla G, Huseyinov A. Gingival crevicular fluid monocyte chemoattractant protein-1 and RANTES levels in patients with generalized aggressive periodontitis. Journal of Clinical Periodontology. 2004;31:829–834. doi: 10.1111/j.1600-051X.2004.00584.x. [DOI] [PubMed] [Google Scholar]
  19. Engebretson SP, Grbic JT, Singer R, Lamster IB. GCF IL-1beta profiles in periodontal disease. Journal of Clinical Periodontology. 2002;29:48–53. doi: 10.1034/j.1600-051x.2002.290108.x. [DOI] [PubMed] [Google Scholar]
  20. Erdemir EO, Duran I, Haliloglu S. Effects of smoking on clinical parameters and the gingival crevicular fluid levels of IL-6 and TNF-alpha in patients with chronic periodontitis. Journal of Clinical Periodontology. 2004;31:99–104. doi: 10.1111/j.0303-6979.2004.00454.x. [DOI] [PubMed] [Google Scholar]
  21. Gately MK, Renzetti LM, Magram J, Stern AS, Adorini L, Gubler U, Presky DH. The interleukin-12/interleukin-12-receptor system: role in normal and pathologic immune responses. Annual Review in Immunology. 1998;16:495–521. doi: 10.1146/annurev.immunol.16.1.495. [DOI] [PubMed] [Google Scholar]
  22. Geivelis M, Turner DW, Pederson ED, Lamberts BL. Measurements of interleukin-6 in gingival crevicular fluid from adults with destructive periodontal disease. Journal of Periodontology. 1993;64:980–983. doi: 10.1902/jop.1993.64.10.980. [DOI] [PubMed] [Google Scholar]
  23. Gemmell E, Carter CL, Seymour GJ. Chemokines in human periodontal disease tissues. Clinical and Experimental Immunology. 2001;125:134–141. doi: 10.1046/j.1365-2249.2001.01511.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Genco RJ. Host responses in periodontal diseases: current concepts. Journal of Periodontology. 1992;63:338–355. doi: 10.1902/jop.1992.63.4s.338. [DOI] [PubMed] [Google Scholar]
  25. Giannopoulou C, Cappuyns I, Mombelli A. Effect of smoking on gingival crevicular fluid cytokine profile during experimental gingivitis. Journal of Clinical Periodontology. 2003a;30:996–1002. doi: 10.1034/j.1600-051x.2003.00416.x. [DOI] [PubMed] [Google Scholar]
  26. Giannopoulou C, Kamma JJ, Mombelli A. Effect of inflammation, smoking and stress on gingival crevicular fluid cytokine level. Journal of Clinical Periodontology. 2003b;30:145–153. doi: 10.1034/j.1600-051x.2003.300201.x. [DOI] [PubMed] [Google Scholar]
  27. Goodson JM. Gingival crevice fluid flow. Periodontology 2000. 2003;31:43–54. doi: 10.1034/j.1600-0757.2003.03104.x. [DOI] [PubMed] [Google Scholar]
  28. Haffajee AD, Socransky SS. Relationship of cigarette smoking to the subgingival microbiota. Journal of Clinical Periodontology. 2001;28:377–388. doi: 10.1034/j.1600-051x.2001.028005377.x. [DOI] [PubMed] [Google Scholar]
  29. Johnson GK, Organ CC. Prostaglandin E2 and interleukin-1 concentrations in nicotine-exposed oral keratinocyte cultures. Journal of Periodontal Research. 1997;32:447–454. doi: 10.1111/j.1600-0765.1997.tb00557.x. [DOI] [PubMed] [Google Scholar]
  30. Johnson RB, Serio FG. The contribution of interleukin-13 and -15 to the cytokine network within normal and diseased gingiva. Journal of Periodontology. 2007;78:691–695. doi: 10.1902/jop.2007.060204. [DOI] [PubMed] [Google Scholar]
  31. Kamer AR, El-Ghorab N, Marzec N, Margarone JE, 3rd, Dziak R. Nicotine induced proliferation and cytokine release in osteoblastic cells. International Journal of Molecular Medicine. 2006;17:121–127. [PubMed] [Google Scholar]
  32. Kamma JJ, Giannopoulou C, Vasdekis VG, Mombelli A. Cytokine profile in gingival crevicular fluid of aggressive periodontitis: influence of smoking and stress. Journal of Clinical Periodontology. 2004;31:894–902. doi: 10.1111/j.1600-051X.2004.00585.x. [DOI] [PubMed] [Google Scholar]
  33. Kinane DF, Chestnutt IG. Smoking and Periodontal Disease. Critical Reviews in Oral Biology and Medicine. 2000;11:356–365. doi: 10.1177/10454411000110030501. [DOI] [PubMed] [Google Scholar]
  34. Kurtis B, Tuter G, Serdar M, Akdemir P, Uygur C, Firatli E, Bal B. Gingival crevicular fluid levels of monocyte chemoattractant protein-1 and tumor necrosis factor-alpha in patients with chronic and aggressive periodontitis. Journal of Periodontology. 2005;76:1849–1855. doi: 10.1902/jop.2005.76.11.1849. [DOI] [PubMed] [Google Scholar]
  35. Loe H, Holm-Pedersen P. Absence and Presence of Fluid from Normal and Inflamed Gingivae. Periodontics. 1965;149:171–177. [PubMed] [Google Scholar]
  36. Maurer M, von Stebut E. Macrophage inflammatory protein-1. International Journal of Biochemistry and Cell Biology. 2004;36:1882–1886. doi: 10.1016/j.biocel.2003.10.019. [DOI] [PubMed] [Google Scholar]
  37. Oliver RC, Holm-Pederen P, Loe H. The correlation between clinical scoring, exudate measurements and microscopic evaluation of inflammation in the gingiva. Journal of Periodontology. 1969;40:201–209. doi: 10.1902/jop.1969.40.4.201. [DOI] [PubMed] [Google Scholar]
  38. Orozco A, Gemmell E, Bickel M, Seymour GJ. Interleukin-1beta, interleukin-12 and interleukin-18 levels in gingival fluid and serum of patients with gingivitis and periodontitis. Oral Microbiology and Immunology. 2006;21:256–260. doi: 10.1111/j.1399-302X.2006.00292.x. [DOI] [PubMed] [Google Scholar]
  39. Palmer RM, Wilson RF, Hasan AS, Scott DA. Mechanisms of action of environmental factors--tobacco smoking. Journal of Clinical Periodontology. 2005;32 Suppl 6:180–195. doi: 10.1111/j.1600-051X.2005.00786.x. [DOI] [PubMed] [Google Scholar]
  40. 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 monocytes. Journal of Periodontal Research. 1996;31:99–104. doi: 10.1111/j.1600-0765.1996.tb00470.x. [DOI] [PubMed] [Google Scholar]
  41. Petropoulos G, McKay IJ, Hughes FJ. The association between neutrophil numbers and interleukin-1alpha concentrations in gingival crevicular fluid of smokers and non-smokers with periodontal disease. Journal of Clinical Periodontology. 2004;31:390–395. doi: 10.1111/j.1600-051x.2004.00489.x. [DOI] [PubMed] [Google Scholar]
  42. Pradeep AR, Daisy H, Hadge P, Garg G, Thorat M. Correlation of gingival crevicular fluid interleukin-18 and monocyte chemoattractant protein-1 levels in periodontal health and disease. Journal of Periodontology. 2009;80:1454–1461. doi: 10.1902/jop.2009.090117. [DOI] [PubMed] [Google Scholar]
  43. Radek KA, Elias PM, Taupenot L, Mahata SK, O'Connor DT, Gallo RL. Neuroendocrine nicotinic receptor activation increases susceptibility to bacterial infections by suppressing antimicrobial peptide production. Cell and Host Microbe. 2010;7:277–289. doi: 10.1016/j.chom.2010.03.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Rawlinson A, Grummitt JM, Walsh TF, Ian Douglas CW. Interleukin 1 and receptor antagonist levels in gingival crevicular fluid in heavy smokers versus non-smokers. Journal of Clinical Periodontology. 2003;30:42–48. doi: 10.1034/j.1600-051x.2003.300107.x. [DOI] [PubMed] [Google Scholar]
  45. Ryder MI. The influence of smoking on host responses in periodontal infections. Periodontology 2000. 2007;43:267–277. doi: 10.1111/j.1600-0757.2006.00163.x. [DOI] [PubMed] [Google Scholar]
  46. Ryder MI, Saghizadeh M, Ding Y, Nguyen N, Soskolne A. Effects of tobacco smoke on the secretion of interleukin-1beta, tumor necrosis factor-alpha, and transforming growth factor-beta from peripheral blood mononuclear cells. Oral Microbiology and Immunology. 2002;17:331–336. doi: 10.1034/j.1399-302x.2002.170601.x. [DOI] [PubMed] [Google Scholar]
  47. Taubman MA, Kawai T. Involvement of T-lymphocytes in periodontal disease and in direct and indirect induction of bone resorption. Critical Reviews in Oral Biology and Medicine. 2001;12:125–135. doi: 10.1177/10454411010120020301. [DOI] [PubMed] [Google Scholar]
  48. Teles R, Sakellari D, Teles F, Konstantinidis A, Kent R, Socransky S, Haffajee A. Relationships among gingival crevicular fluid biomarkers, clinical parameters of periodontal disease, and the subgingival microbiota. Journal of Periodontology. 2010;81:89–98. doi: 10.1902/jop.2009.090397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Thunell DH, Tymkiw KD, Johnson GK, Joly S, Burnell KK, Cavanaugh JE, Brogden KA, Guthmiller JM. A multiplex immunoassay demonstrates reductions in gingival crevicular fluid cytokines following initial periodontal therapy. Journal of Periodontal Research. 2010;45:148–152. doi: 10.1111/j.1600-0765.2009.01204.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Tsai CC, Ho YP, Chen CC. Levels of interleukin-1 beta and interleukin-8 in gingival crevicular fluids in adult periodontitis. Journal of Periodontology. 1995;66:852–859. doi: 10.1902/jop.1995.66.10.852. [DOI] [PubMed] [Google Scholar]
  51. Umeda M, Chen C, Bakker I, Contreras A, Morrison JL, Slots J. Risk indicators for harboring periodontal pathogens. Journal of Periodontology. 1998;69:1111–1118. doi: 10.1902/jop.1998.69.10.1111. [DOI] [PubMed] [Google Scholar]
  52. van Winkelhoff AJ, Bosch-Tijhof CJ, Winkel EG, van der Reijden WA. Smoking affects the subgingival microflora in periodontitis. Journal of Periodontology. 2001;72:666–671. doi: 10.1902/jop.2001.72.5.666. [DOI] [PubMed] [Google Scholar]
  53. Wendell KJ, Stein SH. Regulation of cytokine production in human gingival fibroblasts following treatment with nicotine and lipopolysaccharide. Journal of Periodontology. 2001;72:1038–1044. doi: 10.1902/jop.2001.72.8.1038. [DOI] [PubMed] [Google Scholar]
  54. Zambon JJ, Grossi SG, Machtei EE, Ho AW, Dunford R, Genco RJ. Cigarette smoking increases the risk for subgingival infection with periodontal pathogens. Journal of Periodontology. 1996;67:1050–1054. doi: 10.1902/jop.1996.67.10s.1050. [DOI] [PubMed] [Google Scholar]
  55. Zhong Y, Slade GD, Beck JD, Offenbacher S. Gingival crevicular fluid interleukin-1beta, prostaglandin E2 and periodontal status in a community population. Journal of Clinical Periodontology. 2007;34:285–293. doi: 10.1111/j.1600-051X.2007.01057.x. [DOI] [PubMed] [Google Scholar]

RESOURCES