Theses and Dissertations University of Iowa Year The Influence Of Smoking On Cytokines In The Gingival Crevicular Fluid In Patients With Periodontal Disease Keelen D. Tymkiw The University of Iowa This paper is posted at Iowa Research Online. http://ir.uiowa.edu/etd/26
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Theses and Dissertations
University of Iowa Year
The Influence Of Smoking On Cytokines
In The Gingival Crevicular Fluid In
Patients With Periodontal Disease
Keelen D. TymkiwThe University of Iowa
This paper is posted at Iowa Research Online.
http://ir.uiowa.edu/etd/26
THE INFLUENCE OF SMOKING ON CYTOKINES IN THE GINGIVAL
CREVICULAR FLUID IN PATIENTS WITH PERIODONTAL DISEASE
by
Keelen D. Tymkiw
A thesis submitted in partial fulfillment of the requirements for the Master of Science
degree in Oral Science in the Graduate College of The University of Iowa
May 2008
Thesis Supervisor: Dr. Janet Guthmiller
Graduate College The University of Iowa
Iowa City, Iowa
CERTIFICATE OF APPROVAL
_______________________
MASTER'S THESIS
_______________
This is to certify that the Master's thesis of
Keelen D. Tymkiw
has been approved by the Examining Committee for the thesis requirement for the Master of Science degree in Oral Science at the May 2008 graduation.
___________________________________ Georgia Johnson
___________________________________ Kim Brogden
___________________________________ Sophie Joly
___________________________________ Joseph Cavanaugh
ii
ACKNOWLEDGMENTS
Sincere thanks to Dr. Janet Guthmiller, Dr. Georgia Johnson and Dr. Kim A.
Brogden for their guidance, advice, and friendship. Gratitude to Dr. Sophie Joly for her
input and support; Dr. Joseph Cavanaugh for his statistical expertise. Thanks to my wife,
Jennifer, my son, Jakson, and my parents, for their patience, love, support and
encouragement.
iii
TABLE OF CONTENTS
LIST OF TABLES…………………………………………………………………….…..v
LIST OF FIGURES………………………………………………………………….…..vii
CHAPTER I. INTRODUCTION........................................................................................1
CHAPTER II. REVIEW OF THE LITERATURE.............................................................3 Gingival Crevicular Fluid..........................................................................3 Methods of Collection ...............................................................................3 Gingival Washing Methods.......................................................................3 Capillary Tubing or Micropipettes ............................................................4 Absorbent Filter Paper Strips ....................................................................5 Methods of Estimating Volume Collection Following use of Absorbent Filter Paper Strips ....................................................................5 GCF Contamination...................................................................................7 Sample Recovery.......................................................................................7 Protein Analysis.........................................................................................8 Western Blot ..............................................................................................9 ELISA........................................................................................................9 Multiplex Protein Analysis (Luminex®)...................................................9 Cytokine Concentrations .........................................................................10 Role of Cytokines in Periodontitis ..........................................................11 Role of Smoking in Periodontitis ............................................................12 Effects of Smoking on GCF Flow Rate and Cytokine Levels.................16
CHAPTER III. SIGNIFICANCE AND SPECIFIC AIMS...............................................32 Hypothesis ...............................................................................................33
CHAPTER IV. MATERIALS AND METHODS ............................................................34 Subject Population...................................................................................34 Site Selection ...........................................................................................34 Clinical Evaluation ..................................................................................35 Gingival Crevicular Fluid Collection ......................................................36 Preparation of GCF Fluid for Analysis ...................................................37 Statistical Analysis ..................................................................................38
CHAPTER V. RESULTS .................................................................................................42 Subject Demographics.............................................................................42 Site Characteristics ..................................................................................42
Clinical Parameters ..........................................................................42 Total Gingival Crevicular Fluid Volume .........................................42 Cytokines Detected in GCF..............................................................42
Chemokines: IL-8, IP-10, MCP-1, MIP-1, RANTES and Eotaxin ......................................................................................47 Regulators of T-cells and NK cells: IL-7, IL-15.......................50
CHAPTER VI. DISCUSSION..........................................................................................86 Gingival Crevicular Fluid Volume and Associated Disease Status ........86 Association of Cytokines and Periodontal Disease .................................87 GCF Cytokine Profiles in Smokers .........................................................91 GCF Variability .......................................................................................93 Future Directions .....................................................................................94 Conclusions .............................................................................................99
Table 2. Translation of Periotron® values to clinical conditions and gingival index with which they may be associated....................................................................30
Table 6. Demographic characteristics of the study population.........................................53
Table 7. Site characteristics of the study population. .......................................................54
Table 8. Mean volumes of GCF in all groups...................................................................55
Table 9. Th1 and Th2 cytokines: Intra-group comparisons: Healthy and diseased sites in smokers and non-smokers (Total/30s) ...................................................56
Table 10. Th1 and Th2 cytokines: Inter-group comparisons: Healthy controls vs. healthy and diseased sites in non-smokers (total/30s) .......................................57
Table 11. Th1 and Th2 cytokines: Inter-group comparisons: Healthy controls vs. healthy and diseased sites in smokers (total/30s) ..............................................58
Table 12. Th1 and Th2 cytokines: Inter-group comparisons: Healthy and diseased sites in smokers vs. non-smokers (total/30s) .....................................................59
Table 13. Th1 and Th2 cytokines: Pooled comparisons: Healthy vs. diseased sites in periodontitis subjects (total/30s)....................................................................60
Table 14. Th1 and Th2 cytokines: Pooled comparisons: Healthy controls vs. healthy and diseased sites in periodontitis subjects (total/30s)..........................61
Table 15. Pro-inflammatory cytokines: Intra-group comparisons: Healthy and diseased sites in smokers and non-smokers (Total/30s) ....................................62
Table 16. Pro-inflammatory cytokines: Inter-group comparisons: Healthy controls vs. healthy and diseased sites in non-smokers (Total/30s) ................................63
Table 17. Pro-inflammatory cytokines: Inter-group comparisons: Healthy controls vs. healthy and diseased sites in smokers (Total/30s)........................................64
Table 18. Pro-inflammatory cytokines: Inter-group comparisons: Healthy and diseased sites in smokers vs. non-smokers (Total/30s) .....................................65
Table 19. Pro-inflammatory cytokines: Pooled comparisons: Healthy vs. diseased sites in periodontitis subjects (Total/30s) ..........................................................66
Table 20. Pro-inflammatory cytokines: Pooled comparisons: Healthy controls vs. healthy and diseased sites in periodontitis subjects (Total/30s) ........................67
vi
Table 21. Chemokines: Intra-group comparisons: Healthy and diseased sites in smokers and non-smokers (Total/30s) ...............................................................68
Table 22. Chemokines: Inter-group comparisons: Healthy controls vs. healthy and diseased sites in non-smokers (Total/30s) .........................................................69
Table 23. Chemokines: Inter-group comparisons: Healthy controls vs. healthy and diseased sites in smokers (Total/30s).................................................................70
Table 24. Chemokines: Inter-group comparisons: Healthy and diseased sites in smokers vs. non-smokers (Total/30s) ................................................................71
Table 25. Chemokines: Pooled comparisons: Healthy vs. diseased sites in periodontitis subjects (Total/30s).......................................................................72
Table 26. Chemokines: Pooled comparisons: Healthy controls vs. healthy and diseased sites in periodontitis subjects (Total/30s)............................................73
Table 27. Regulators of T-cells and NK cells: Intra-group comparisons: Healthy and diseased sites in smokers and non-smokers (Total/30s)..............................74
Table 28. Regulators of T-cells and NK cells: Inter-group comparisons: Healthy controls vs. healthy and diseased sites in non-smokers (Total/30s) ..................75
Table 29. Regulators of T-cells and NK cells: Inter-group comparisons: Healthy controls vs. healthy and diseased sites in smokers (Total/30s)..........................76
Table 30. Regulators of T-cells and NK cells: Inter-group comparisons: Healthy and diseased sites in smokers vs. non-smokers (Total/30s)...............................77
Table 31. Regulators of T-cells and NK cells: Pooled comparisons: Healthy vs. diseased sites in periodontitis subjects (Total/30s)............................................78
Table 32. Regulators of T-cells and NK cells: Pooled comparisons: Healthy controls vs. healthy and diseased sites in periodontitis subjects (Total/30s)..........................................................................................................79
Table 33. P values of intra-group, inter-group and pooled comparisons of Th1 and Th2 cytokines, pro-inflammatory cytokines, chemokines and regulators of T-cells and NK cells. .....................................................................................80
Table 34. Comparison of studies on the effects of smoking on GCF cytokine/chemokine levels.................................................................................97
vii
LIST OF FIGURES
Figure 1. Collection of gingival crevicular fluid by means of filter paper strips (Periopaper®). Strip is inserted into gingival crevice and fluid is collected via capillary action. ............................................................................31
Figure 2. Means of Clinical Parameters: a) probing depth b) recession c) clinical attachment level d) gingival crevicular fluid .....................................................81
Figure 3. Th1 and Th2 cytokines: Median amounts (Total/30s): a) IL-2 b) INF-γ c) IL-3 d) IL-4 ........................................................................................................82
Figure 4. Pro-inflammatory cytokines: Median amounts (Total/30s): a) IL-1α b) IL-1β c) IL-6 d) IL-12(p40) e) GM-CSF ................................................................83
Figure 5. Chemokines: Median amounts (Total/30s): a) IL-8 b) IP-10 c) MCP-1 d) MIP-1 e) RANTES f) Eotaxin ...........................................................................84
Figure 6. Regulators of T-cells and NK cells: Median amounts (Total/30s): a) IL-7 b) IL-15 ..............................................................................................................85
1
CHAPTER I
INTRODUCTION
The role of cigarette smoking in the pathogenesis of periodontal disease has been
extensively studied and well documented over the past two decades. Cigarette smoking is
a significant risk factor in the pathogenesis of periodontal disease and is associated with
periodontal disease progression (Bergstrom and Preber 1994). Of equal importance in
the pathogenesis of the disease, is the fact that bacterial products stimulate
monocytes/macrophages and lymphocytes as well as resident cells (fibroblasts,
keratinocytes, and endothelial cells) to secrete pro-inflammatory and immuno-regulatory
cytokines.
Cytokines such as IL-1, IL-6, IL-8 and TNF-α are considered to be involved in
the host response of periodontal disease as mediators of tissue destruction. Increased
levels of these cytokines have been observed in the gingival crevicular fluid (GCF) of
patients with periodontal disease (Rossomando, Kennedy et al. 1990; Wilton, Bampton et
al. 1992; Geivelis, Turner et al. 1993). However, the levels of these cytokines have had
large inter- and intra-individual variations, suggesting that these parameters are
influenced by a multitude of other factors which have been poorly characterized to date.
In order to better understand the role of cytokines in the pathogenesis of periodontal
disease, our study utilized an extensive and highly quantitative assay to evaluate the
cytokine profile in periodontally diseased subjects and the influence that smoking may
have on cytokine response and concentration.
The overall purpose of this project was to evaluate the GCF cytokine profile in
chronic periodontitis subjects and the influence of cigarette smoking on the GCF
concentrations of Th1 and Th2 cytokines (IL-2, IL-12 (p70), IFN- γ), pro-inflammatory
reviews the sources, biologic activity and relationship to periodontitis for these cytokines.
Role of Smoking in Periodontitis
Smoking is recognized as the most important environmental risk factor in the
pathogenesis of chronic periodontitis. Risk calculations suggest that 40% of chronic
periodontitis cases may be attributed to smoking (Tomar and Asma 2000). Smokers are
four times (odds ratio of 4.0) as likely to have chronic periodontitis than non-smokers
(Tomar and Asma 2000). Smoking is associated with more attachment loss, bone loss
and tooth loss, but, paradoxically, less signs of inflammation. Extensive clinical trials
13
have also shown poorer response to non-surgical and surgical periodontal treatment in
smokers. Additionally, treatments for periodontal disease are likely to be more
efficacious in non-smokers than in smokers, with the response of previous smokers being
intermediate between these two groups (Bergstrom, Eliasson et al. 2000). Further
evidence for smoking as a risk factor for chronic periodontitis is strengthened by the
ability to demonstrate a dose–response (years of exposure) of tobacco products to the
severity of periodontal disease (Martinez-Canut, Lorca et al. 1995). The literature
strongly supports the observation that the longer or the greater the number of cigarettes a
patient smokes, the greater the severity of periodontal disease.
Tobacco smoking greatly affects the oral environment, including; the gingival
tissues and vasculature, the inflammatory response, the immune response and the
homeostasis and healing potential of the periodontal connective tissues. Clinically,
smokers commonly present with fibrotic gingiva, with limited gingival redness and
edema relative to disease severity; proportionally greater pocketing in anterior and
maxillary lingual sites; gingival recession at anterior sites; and a lack of association
between periodontal status and level of oral hygiene (Haber 1994).
Smoking has chronic effects, on the vasculature of the periodontal tissues, versus
acute vasoconstrictive effects following a smoking episode. The impairment of the
vasculature observed includes less gingival redness, less bleeding on probing and fewer
vessels histologically (Bergstrom, Eliasson et al. 2000).
While smoking is accepted as a strong modifying factor for periodontal diseases,
there is lack of consensus regarding its precise mechanisms. Smoking does not seem to
influence the subgingival colonization of some important periodontal pathogens, as most
studies suggest that smoking and non-smoking periodontitis patients largely exhibit the
same microflora (Preber, Bergstrom et al. 1992; Van der Velden, Varoufaki et al. 2003).
Smoking, affects many aspects of the host’s immune response, therefore it is probable
that this may be its primary role in contribution to the pathogenesis of the disease.
14
It is well known that neutrophils are critical immune cells in the maintenance of
periodontal health because of their multifaceted roles in the control of bacterial plaque.
They likely, however, also contribute to the progression of periodontitis in chronic
inflammatory responses. While data is conflicting, it is clear that smoking affects
multiple functions of neutrophils and may shift the net balance of neutrophil activities
into one of more destruction. While tobacco smoke exposure increases the number of
neutrophils found in the systemic circulation, the numbers of neutrophils entering the
gingival sulcus and oral cavity remain unaffected or even reduced (Pauletto, Liede et al.
2000). These findings imply that neutrophil transmigration across the periodontal
microvasculature is impeded in tobacco smokers (Palmer, Wilson et al. 2005). Seow
examined the effects of nicotine on neutrophil function at concentrations simulating those
found in oral tissues. The results showed a dose-dependent suppression of both
chemotaxis and phagocytosis (Seow, Thong et al. 1994). Additionally, it has long been
hypothesized that tobacco smoking increases the proteolytic activity of neutrophils.
While the tobacco-induced release of proteolytic enzymes from neutrophils has not been
demonstrated definitively in the periodontal tissues themselves, neutrophils are
considered to be a major source of the elastase and matrix metallo-proteases (MMPs)
associated with periodontal disease destruction (Persson, Bergstrom et al. 2003).
Furthermore, tobacco smoke and components are known to stimulate the release of such
enzymes from neutrophils in vivo and in vitro (Seow, Thong et al. 1994). Seow also
examined the effects of nicotine on neutrophil function at concentrations found in oral
tissues and showed an enhancement of neutrophil degranulation. As a result, the
literature supports the fact that tobacco may contribute to the progression of periodontal
disease, at least in part, through the release of proteases from periodontal neutrophils.
Following non-surgical periodontal therapy, most authors report greater probing
depth reductions in non-smokers as compared to smokers and show significantly greater
pocket depth reductions (0.9-1.1mm) in non-smokers compared with smokers at 1 and 3
15
months following non-surgical therapy (Preber and Bergstrom 1986; Preber, Linder et al.
1995; Grossi, Zambon et al. 1997; Renvert, Dahlen et al. 1998; Preshaw, Lauffart et al.
1999). Papantonopoulos (1999) showed that significantly more smokers (42.8%) than
non-smokers (11.5%) required additional treatment at reevaluation, following non-
surgical therapy (Papantonopoulos 1999). Long term studies involving both surgical and
non-surgical therapy show similar results, with non-smokers showing greater probing
depth reduction and gain of attachment level (Kaldahl, Johnson et al. 1996; Renvert,
Dahlen et al. 1998; Preshaw, Lauffart et al. 1999). In a radiographic study, Meinberg et
al. (2001) reported significantly more bone loss at 12 months following non-surgical
therapy in smokers compared with non-smokers (Meinberg, Canarsky-Handley et al.
2001).
The differences in clinical responses following non-surgical therapy in smokers
versus non-smokers has also been observed following surgical treatment (Preber and
Bergstrom 1990). Tonetti et al. (1995) reported a significant difference in clinical
attachment gains following guided tissue regeneration of intrabony defects for smokers
and non-smokers of 2.1mm and 5.2mm respectively (Tonetti, Pini-Prato et al. 1995).
They also concluded that higher plaque levels were seen consistently in smokers
compared with non-smokers which can also influence clinical outcomes (Tonetti, Pini-
Prato et al. 1995).
The majority of studies investigating the effects of smoking cessation on
periodontal disease acknowledge the benefits of smoking cessation counseling and
conclude that smoking cessation may result in long-term benefits to the periodontium
(Ramseier 2005). Further, the implementation of population-based smoking cessation
programs may have a significant impact on the prevalence and progression of periodontal
diseases (Susin, Oppermann et al. 2004). Bolin et al. (1993) reported results from a 10-
year radiographic follow-up study of alveolar bone loss which found that the progression
of bone loss was significantly retarded in those who had quit smoking during the study
16
compared with continual smokers (Bolin, Eklund et al. 1993). Preshaw et al. (2005)
showed similar radiographic results over 12 months with former smokers as well as a
significant reduction in probing depths and a higher incidence of probing depth
reductions of 2 and 3 mm in the former smokers as compared to current smokers
(Preshaw and Heasman 2005).
Effects of Smoking on GCF Flow Rate and Cytokine
Levels
GCF Flow Rate
Smoking has been shown to decrease the resting GCF flow rate (Persson,
Bergstrom et al. 1999). Additionally, in a study examining subjects in a smoking
cessation program, Morozumi et al. (2004) showed that GCF flow was greater at 5 days
following smoking cessation (Morozumi, Kubota et al. 2004). These findings are thought
to be related to the effects of smoking on gingival blood flow and the inflammatory
response (Palmer, Wilson et al. 2005).
Cytokine Levels
It is well known that tobacco components modify the production of cytokines or
inflammatory mediators; however their effects on various proteins are variable and
contradictory throughout the literature. Giannopoulou et al. (2003) showed associations
between smoking and total amounts of GCF IL-4, IL- 6 and IL-8. In the smoking group
they showed an increase in IL-6 and IL-8 but a decrease in IL-4 and no association with
the levels of IL-1β. This is in agreement with the observations of Boström et al. (2000),
who analyzed GCF levels of IL-1β and its receptor antagonist IL-1ra with respect to
smoking in patients with moderate to severe periodontal disease. IL-1β was detected in
almost all GCF samples, but smoking showed no association with GCF levels of this
17
cytokine nor with those of IL-1ra. It has been suggested or reported that cigarette smoke
contains potent inhibitors of specific cytokine production, including IL-1β, IL-2,
interferon (IFN)-γ and TNF-α (Ouyang, Virasch et al. 2000). However, when the
influence of smoking is examined in regards to IL-6 content of GCF in patients with
moderate to severe forms of periodontal disease, no statistically significant differences
are observed between smokers and non-smokers (Bostrom, Linder et al. 1999) in contrast
to the findings by Giannopoulou et al. (2003) a slight increase was observed. Elevated
concentrations of IL-6 were also observed in the plasma of smokers by Tappia (Tappia,
Troughton et al. 1995). Bostrom et al. (1998) also showed higher levels of TNF-α in GCF
in smokers and former smokers compared with non-smokers, with comparable levels of
moderate/severe periodontitis (Bostrom, Linder et al. 1998). In a follow-up study of a
comparable group of subjects using similar protocols they confirmed the presence of
higher levels of TNF-α in a smaller group of smokers (Bostrom, Linder et al. 1999).
Bostrom et al. (2000) then compared levels of IL-1β and IL-1ra and found no significant
differences (Bostrom, Linder et al. 2000). However, Rawlinson et al. (2003) found levels
of IL-1β and IL-1ra to be significantly lower in GCF from diseased sites in smokers
compared with non-smokers (Rawlinson, Grummitt et al. 2003). Petropoulos et al. (2004)
showed that the concentration of IL-1α in GCF of smokers was approximately half that
found in non-smokers (Petropoulos, McKay et al. 2004).
18
Table 1. Cytokine Properties
Cytokine Sources#
Biologic Activity#
Relationship to Periodontitis
Interleukin -1
(IL-1)
• Produced by
monocytes,
macrophages,
neutrophils,
endothelial cells,
fibroblasts, smooth
muscle cells,
keratinocytes,
langerhans cells of the
skin, osteoclasts,
astrocytes, epithelial
cells of the thymus
and the cornea, T-
cells, B-cells, NK-
cells.
• Production stimulated
by TNF-α, IFN-α,
IFN-β and IFN- γ,
bacterial endotoxins,
viruses, mitogens, and
antigens.
• Chemoattractant for leukocytes, stimulation
of T-helper cells (IL-2 secretion),
proliferation of B cells, ↑ B-cell
responsiveness to IL-5, proliferation and
activation of NK-cells and fibroblasts,
thymocytes, glioblastoma cells.
• Enhances the metabolism of arachidonic
acid (prostacyclin and PGE2) in
inflammatory cells (fibroblasts, synovial
cells, chondrocytes, endothelial cells,
hepatocytes, and osteoclasts)
• Increased secretion of inflammatory
proteins such as neutral proteases
(collagenase, elastase and plasminogen
activator). Antagonizes the effects of TGF-
β on the extracellular matrix
• Promotion of wound healing (angiogenesis,
proliferation of fibroblasts, neutrophil
chemotaxis).
• Significantly increased in the periodontal
tissues and gingival fluid from diseased
sites, compared with healthy sites (Masada,
Persson et al. 1990).
• Can act on a large number of cells
(fibroblasts, chondrocytes, bone cells,
neutrophils and lymphocytes) suggests that
periodontal destruction and repair in
periodontitis may in part be associated with
this cytokine (Orozco, Gemmell et al.
2006).
• IL-1β up-regulates matrix
metalloproteinases and down-regulates
tissue inhibitors of metalloproteinase
production (Ohshima, Otsuka et al. 1994).
• Powerful and potent bone-resorbing
cytokine (Shirodaria, Smith et al. 2000).
• Plays a role in degrading the extracellular
matrix in periodontitis (Schwartz,
Goultschin et al. 1997).
Interleukin -2
(IL-2)
• Produced by T-cells,
and B-cells, AK cells
(lymphokine-activated
killer cells) and NK-
cells.
• Significant anti-tumor activity for a variety
of tumor cell types since it supports the
proliferation and clonal expansion of T-
cells that specifically attack certain tumor
types.
• Involved in B-cell activation and stimulates
macrophages, NK cells and T-cell
proliferation. It is regarded as a pro-
inflammatory cytokine (Tew, Engel et al.
1989).
19
Table 1. Continued
• Production stimulated
by vitamin E.
• Production inhibited
by dexamethasone or
Cyclosporin A.
• Induces the secretion of other soluble
mediators, including TNF-α, TNF-β, and
IFN-γ. These effects may contribute to the
antitumor activity of IL- as well as to its
dose-related toxicity.
• IL-2 has been also implicated in the
stimulation of osteoclast activity in bone
resorption (Ries, Seeds et al. 1989).
• There is evidence indicating that IL-2 may
also be a relevant factor in the pathogenesis
of periodontal disease. Lymphocytes
cultured from the chronically inflamed
periodontal tissues of patients with alveolar
bone loss produced IL-2 (Seymour, Cole et
al. 1985).
• The levels of IL-2 in the sera of
periodontitis patients are elevated when
compared to those of normal subjects
(McFarlane and Meikle 1991).
• Due to its biological properties, IL-2 has
been suggested to be a useful marker of
pathologic inflammatory activity in
systemic diseases (John, Turner et al. 1998)
and periodontal conditions (McFarlane and
Meikle 1991).
Interleukin -3
(IL-3)
• Produced by T-cells,
keratinocytes, NK-
cells, mast cells,
endothelial cells, and
monocytes.
• Production inhibited
by glucocorticoids or
CsA (Cyclosporin A).
• Is a growth factor that establishes the link
between the immune system and the
hematopoietic system.
• Supports the proliferation and development
of almost all types of hematopoietic
progenitor cells.
20
Table 1. Continued
• Supports the differentiation of early non-
lineage-committed hematopoietic
progenitor cells into colonies of
granulocytes, macrophages, erythroid cells,
megakaryocytes, and mast cells
• Chemoattractant for eosinophils
• Induces the proliferation of mast cells and
macrophages.
• Significantly enhances the secretion of
other cytokines including IL-1, IL-6 and
TNF.
• In vitro IL-3 also stimulates the
proliferation of keratinocytes.
Interleukin -4
(IL-4)
• Produced by activated
T-cells (Th2) or (Th1),
Non-T/Non-B-cells of
the lineage of mast
cells.
• Production stimulated
by IL-2 and PAF
(platelet activating
factor).
• Production inhibited
by TGF-beta.
• Promotes the proliferation and
differentiation of activated B-cells, the
expression of MHC class 2 antigens, and of
low affinity IgE receptors in resting B-cells.
• Enhances expression of MHC class 2
antigens on B-cells. It can promote their
capacity to respond to other B-cell stimuli
and to present antigens for T-cells.
• Potent downregulator of macrophage
function by inhibiting the secretion of IL-
1β, TNF-α and IL-6 (Kamma,
Giannopoulou et al. 2004).
• Inhibits the secretion of prostaglandin
(PGE2) by human monocytes which leads
to bone resorption (Shapira, van Dyke et al.
1992).
• Absence of IL-4 has been associated with
periodontal disease activity and progression
(Shapira, van Dyke et al. 1992).
• Clinical importance in the treatment of
inflammatory diseases since it inhibits the
production of inflammatory cytokines such
as IL-1, IL-6 and TNF-α by monocytes and
of TNF by T-cells (Kamma, Giannopoulou
et al. 2004).
21
Table 1. Continued
Interleukin -5
(IL-5)
• Produced by T-cells • Specific hematopoietic growth factor that is
responsible for the growth and
differentiation of eosinophils.
• Promotes the generation of cytotoxic T-
cells from thymocytes.
• Induces the proliferation of pre-activated B-
cells and their differentiation.
• Stimulates the production of IgM and IgA.
Interleukin -6
(IL-6)
• Produced by
monocytes,
fibroblasts, and
endothelial cells.
Macrophages, T-cells
and B-lymphocytes,
granulocytes, smooth
muscle cells,
eosinophils,
chondrocytes,
osteoblasts, mast cells,
glial cells, and
keratinocytes.
• Pleiotropic cytokine influencing antigen-
specific immune responses and
inflammatory reactions.
• B-cell differentiation factor in vivo and in
vitro and an activation factor for T-cells.
• In the presence of IL-2, IL-6 induces the
differentiation of mature and immature T-
cells into cytotoxic T-cells.
• IL-6 also induces the proliferation of
thymocytes and probably plays a role in the
development of thymic T-cells.
• IL-6 is capable of inducing the final
maturation of B-cells into immunoglobulin-
secreting plasma cells if the cells have been
pre-activated by IL-4.
• In B-cells IL-6 stimulates the secretion of
antibodies.
• Contributes to the terminal differentiation
of B-lymphocytes to plasma cells and
stimulates secretion of immunoglobulin
(Ig)A and IgG (Fujihashi, Kono et al.
1993).
• Smoking causes a depression of IgG and
possibly IgA production in serum (Quinn,
Zhang et al. 1996).
• Induces bone resorption, both by itself and
in conjunction with other bone-resorbing
agents (Mundy 1991).
22
Table 1. Continued
• Production induced by
IL-1, bacterial
endotoxins, TNF,
PDGF, and Oncostatin
M. In fibroblasts the
synthesis of IL-6 is
stimulated by IFN-β,
TNF-α, PDGF, and
viral infections. IL-6
can also stimulate or
inhibits its own
synthesis, depending
upon the cell type. In
epithelial, endothelial,
and fibroblastic cells
secretion of I-L6 is
induced by IL-17.
• Production inhibited
by Glucocorticoids,
IL-4, and TGF-beta.
Interleukin -8
(IL-8)
• Produced by
monocytes, T-
lymphocytes,
macrophages,
fibroblasts, endothelial
cells, keratinocytes,
melanocytes, and
chondrocytes.
• Production stimulated
by IL-1, TNF-α.
• Chemotactic for all known types of
migratory immune cells.
• Differs from all other cytokines in its
ability to specifically activate neutrophil
granulocytes.
• Inhibits histamine release from human
basophils induced by histamine releasing
factors, CTAP-3 (connective tissue
activating protein-3), and IL-3.
Antagonizes IgE production by human B-
cells.
• Powerful chemotactic functions for
polymorphonuclear leukocytes but also for
lymphocytes and macrophages (Kamma,
Giannopoulou et al. 2004).
• Is a potent mediator of granulocyte
accumulation at the sites of inflammation
(Bickel 1993).
• Increased levels of IL-8 are found in the
GCF of inflamed sites (Tsai, Ho et al.
1995).
23
Table 1. Continued
• Production inhibited
by 5' lipoxygenase,
and vitamin D3.
• Inhibits the adhesion of leukocytes to
activated endothelial cells and therefore
possesses anti-inflammatory activities.
• Supports angiogenesis and may play a role
in disorders such as rheumatoid arthritis,
tumor growth, and wound healing that
critically depend on angiogenesis.
• Powerful chemotactic functions for
polymorphonuclear leukocytes but also for
lymphocytes and macrophages (Kamma,
Giannopoulou et al. 2004).
• Is a potent mediator of granulocyte
accumulation at the sites of inflammation
(Bickel 1993).
• Increased levels of IL-8 are found in the
GCF of inflamed sites (Tsai, Ho et al.
1995).
Interleukin -10
(IL-10)
• Produced by T-cells
(Th2 cells but not
Th1 T-helper cells),
B-cells, and
keratinocytes.
• Production is
inhibited by IL-4.
• Potent and specific T-cell chemoattractant.
• Inhibits the synthesis of a number of
cytokines such as IFN-γ, IL-2 and TNF-β in
Th1 T-helper subpopulations of T-cells but
not of Th2 T-helper cells.
• This activity is antagonized by IL-4.
• In the human system, IL-10 is produced by,
and down-regulates the function of, Th1
and Th2 cells.
• In macrophages stimulated by bacterial
lipopolysaccharides, IL-10 inhibits the
synthesis of IL-1, IL-6 and TNF-α.
• In human monocytes IFN-γ and IL-10
antagonize each other's production and
function. IL-10 has been shown also to be a
physiologic antagonist of IL-12.
• IL-10 acts as a costimulator of the
proliferation of mast cells (in the presence
of IL-3 and/or IL-4) and peripheral
lymphocytes.
24
Table 1. Continued
Interleukin -12
(IL-12)
• Produced by
monocytes,
macrophages,
neutrophils, B-cells
and to a lesser extent
by T-cells.
• Production
stimulated by IL-12,
bacteria, bacterial
products, and
parasites.
• Is a heterodimer comprised of p35 and p40
subunits, which form the bioactive IL-12
(p70).
• Principal mediator of the early innate
immune response to intracellular microbes
and is a key inducer of cell-mediated
immunity.
• Stimulates IFN-γ production by T cells and
natural killer cells and so promote Th1
responses.
• Promotes Th1 development by stimulating
the production of IL-12 by macrophages
and the expression of functional IL-12
receptors on T cells.
• The importance of IL-12 is not limited to
initiating an immune response, but may
contribute to maintaining immunity
because Th1 responses, smooth muscle
cells, and fibroblasts also secrete TNF.
• Produced by proinflammatory infiltrates in
periodontitis tissues.
• High levels will contribute to the immune
reaction to Th1 type (Lamont and Adorini
1996).
• IL-12 is an inducer of IFN-γ production.
IFN-γ itself can also activate IL-12
production (Lamont and Adorini 1996).
• LPS of periodontopathogens are also
activators of IL-12 (Lamont and Adorini
1996).
• Significantly higher proportions found in
diseased sites (Lamont and Adorini 1996).
Tumor Necrosis
Factor-Alpha
(TNF-α)
• Produced by
macrophages,
monocytes,
neutrophils, T-cells,
NK-cells, astrocytes,
microglial cells,
smooth muscle cells,
and fibroblasts.
• Inhibits anticoagulatory mechanisms and
promotes thrombotic processes and
therefore plays an important role in
pathological processes such as venous
thromboses, arteriosclerosis, vasculitis, and
disseminated intravasal coagulation.
• Potent chemoattractant for neutrophils and
also increases their adherence to the
endothelium.
• Stimulates fibroblasts, including gingival
fibroblasts, to produce collagenase (Meikle,
Atkinson et al. 1989) which is implicated in
the tissue destruction of periodontal disease,
and to stimulate bone resorption (Bertolini,
Nedwin et al. 1986).
• Activates monocytes and stimulates the
production of IL-1β and platelet activating
factor (Erdemir, Duran et al. 2004).
25
Table 1. Continued
• Production stimulated by
interferons, IL-2, GM-
CSF, SP, Bradykinin,
Immune complexes,
inhibitors of
cyclooxygenase and
platelet activating factor.
• Production inhibited by
IL-6, TGF-β, vitamin
D3, prostaglandin E2,
dexamethasone, CsA
(Cyclosporin A), and
antagonists of platelet
activating factor.
• Induces the synthesis of a number of
chemoattractant cytokines, including IP-10,
JE, KC, in a cell-type and tissue-specific
manner.
• Inhibits the growth of endothelial cells in
vitro and is a potent promoter of
angiogenesis in vivo.
• Growth factor for normal human diploid
fibroblasts and promotes the synthesis of
collagenase and prostaglandin E2 in
fibroblasts.
• In resting macrophages TNF induces the
synthesis of IL-1 and prostaglandin E2.
• Stimulates phagocytosis and the synthesis
of superoxide dismutase in macrophages.
• Activates osteoclasts and thus induces bone
resorption.
• Stimulates the expression of class 1 and II
HLA and differentiation antigens, and the
production of IL-1, colony stimulating
factors, IFN-γ, arachidonic acid
metabolism.
• Stimulates the biosynthesis of collagenases
in endothelial cells and synovial cells.
• Monocyte stimulation by
lipopolysaccharide enhances the production
of TNF-α, which has also been shown to
induce collagenase release and bone
resorption in vivo (Meikle, Atkinson et al.
1989).
Interferon-
Gamma
(IFN-γ)
• Produced by T-cells, B-
cells, natural killer cells
activated by antigens, or
lymphocytes expressing
the surface antigens CD4
and CD8.
• Antiviral and antiparasitic activities and
also inhibits the proliferation of a number
of normal and transformed cells.
• Synergizes with TNF-α and TNF-β in
inhibiting the proliferation of various cell
types.
• Studies have suggested an inhibitory effect
of IFN-γ on RANKL-associated
osteoclastogenesis and bone remodeling in
vitro and in vivo (Takayanagi, Ogasawara et
al. 2000).
26
Table 1. Continued
• Production stimulated by
IL-2, bFGF, and EGF.
• Production inhibited by
1-alpha,25-Dihydroxy
vitamin D3,
dexamethasone and CsA
(Cyclosporin A).
• Main biological activity of IFN-γ appears
to be immunomodulatory in contrast to the
other interferons which are mainly
antiviral.
• In T-helper cells IL-2 induces the synthesis
of IFN-γ and other cytokines. IFN-γ acts
synergistically with IL-1 and IL-2 and
appears to be required for the expression of
IL-2 receptors on the cell surface of T-
lymphocytes.
• IFN-γ is a modulator of T-cell growth and
functional differentiation. It is a growth-
promoting factor for T-lymphocytes and
potentiates the response of these cells to
mitogens or growth factors.
• IFN-γ inhibits the growth of B-cells
induced by IL-4. IFN-gamma and Anti-Ig
costimulate the proliferation of human B-
cells. IFN-gamma also inhibits the
production of IgG1 and IgE elicited by IL-4
in B-cells stimulated by bacterial
lipopolysaccharides.
• Regulates the expression of MHC class 2
genes and is the only interferon that
stimulates the expression of these proteins.
• Stimulates the expression of IgA antigens
on the cell surface, the expression of CD4
in T-helper cells, and the expression of
high-affinity receptors for IgG in myeloid
cell lines, neutrophils, and monocytes.
• There is also evidence that IFN-γ can
positively modulate the expression of pro-
resorptive factors in periodontal
microorganism-specific periodontal CD4+
Th1 cells, which can further mediate
osteoclastogenesis associated with alveolar
bone loss in vivo (Takayanagi, Ogasawara
et al. 2000).
• It has also been shown that IFN-γ+ Th1
cells are strongly associated with enhanced
alveolar bone loss during periodontal
infections (Valverde, Kawai et al. 2004).
• There is strong evidence suggesting that
deficient IFN-γ expression significantly
reduces the severity of periodontal bone
loss in mice after mounting a microbial
challenge (Baker, Dixon et al. 1999).
• IFN-γ can up-regulate the expression of
major histocompatibility complex (MHC)
class II and other accessory molecules on
the antigen-presenting cells, which may
further recruit other signaling molecules
and/or immune effectors associated with
bone remodeling (Ellis and Beaman 2004).
• A fine balance of IFN-γ under various
inflammatory conditions (for instance,
periodontal diseases) may directly or
indirectly modulate Th1 cells for
osteoclastogenesis (Ellis and Beaman
2004).
27
Table 1. Continued
• In monocytes and macrophages IFN-γ
induces the secretion of TNF-α and the
transcription of genes encoding G-CSF and
M-CSF.
• In macrophages IFN-γ stimulates the
release of reactive oxygen species.
• IFN-γ is involved also in processes of bone
growth and inhibits bone resorption
probably by partial inhibition of the
formation of osteoclasts.
• IFN-γ inhibits the proliferation of
endothelial cells and the synthesis of
collagens by myofibroblasts. It thus
functions as an inhibitor of capillary growth
mediated by myofibroblasts and fibroblast
growth factors and PDGF.
Granulocyte
Macrophage
Colony
Stimulating
Factor
(GM-CSF)
• Produced by T-cells,
macrophages,
endothelial cells and
fibroblasts.
• Production stimulated
by TNF-α, TNF-β, IL-
1, IL-2 and IFN.
• Important in the growth and development
of progenitors of granulocytes and
macrophages. It stimulates myeloblasts and
monoblasts and triggers irreversible
differentiation of these cells.
• Strong chemoattractant for neutrophils.
• Enhances microbicidal activity, oxidative
metabolism, and phagocytotic activity of
neutrophils and macrophages.
• Stimulates the proliferation precursors of
neutrophils, eosinophils, and monocytes.
• Enhances phagocytotic activities of
neutrophil granulocytes and the
cytotoxicity of eosinophils.
• Up-regulated in neutrophil-mediated
pathology and is associated with
periodontal inflammation (Takematsu and
Tagami 1990; Baqui, Meiller et al. 1999).
• Variety of effects on neutrophils potentially
important in the pathogenesis of
periodontitis, including dose-dependent
chemotaxis or inhibition of movement,
inhibition of apoptosis and priming for
increased phagocytic and respiratory burst
activity (Fossati, Mazzucchelli et al. 1998).
28
Table 1. Continued
RANTES
• Produced by T-cells.
• Production stimulated by
TNF-α and IL-1α.
• Chemotactic for T-cells, human eosinophils
and basophils and plays an active role in
recruiting leukocytes into inflammatory
sites.
• Increases the adherence of monocytes to
endothelial cells and selectively supports
the migration of monocytes and T-
lymphocytes expressing the cell surface
markers CD4 and UCHL1.
• Activates human basophils from some
select basophil donors and causes the
release of histamines.
• Expressed by human synovial fibroblasts
and may participate, therefore, in the
ongoing inflammatory process in
rheumatoid arthritis.
• Plays an important role in the host response
by recruiting inflammatory cells into the
foci of active inflammation and by inducing
the release of other cell mediators
(Gamonal, Acevedo et al. 2000).
• Important mediators of the host response in
chronic adult periodontitis (Emingil, Atilla
et al. 2004).
Eotaxin
• Belongs to the platelet factor-4 family of
chemokines.
• Potent stimulator of eosinophils in vitro.
• Does not possess suppressive activity
against immature subsets of myeloid
progenitors
Interferon-
Inducible
Protein-10
(IP-10)
• Produced by monocytes,
keratinocytes, and
fibroblasts, neutrophils.
• Production stimulated by
IFN-γ, TNF-α, and LPS.
• Production inhibited by
IL-10 and IL-4.
• Thought to play an important role in
hypersensitivity reactions of the delayed
type.
• Thought to play a role in regulation of the
growth of immature hematopoietic
progenitor cells.
• Potent endogenous inhibitor of
angiogenesis.
29
Table 1. Continued
Monocyte
Chemotactic
Proteins-1
(MCP-1)
• Produced by
monocytes, vascular
endothelial cells,
smooth muscle cells,
glomerular mesangial
cell, osteoblastic cells,
and human pulmonary
type 2 like epithelial
cells.
• Production stimulated
by peripheral blood
mononuclear
leukocytes,
phytohemagglutinin
(PHA), bacterial
lipopolysaccharides,
and IL-1.
• Chemotactic for monocytes but not
neutrophils.
• Elevated levels of MCP-1 are observed in
macrophage-rich atherosclerotic plaques.
• Regulates the expression of cell surface
antigens (CD11c, CD11b) and the
expression of cytokines (IL-1, IL-6). MCP-
1 is a potent activator of human basophils,
inducing degranulation and the release of
histamines.
• Induces the proliferation and activation of
killer cells known as CHAK (CC-
Chemokine-activated killer).
Macrophage
inflammatory
protein-1
(MIP-1)
• Produced by
macrophages
• Caused local inflammatory responses in
vivo, and induces superoxide production by
neutrophils in vitro.
Source: # Information adapted and revised from: http://www.copewithcytokines.de/
30
Table 2. Translation of Periotron® values to clinical conditions and gingival index with which they may be associated.
Periotron® Reading Level of Gingival Inflammation Gingival Index
0–20 healthy 0
21–40 mild 1
41–80 moderate 2
81–200 severe 3
Source: Griffiths, G. S., J. M. Wilton, et al. (1992). "Contamination of
human gingival crevicular fluid by plaque and saliva." Arch Oral Biol
37(7): 559-64.
31
Figure 1. Collection of gingival crevicular fluid by means of filter paper strips (Periopaper®). Strip is inserted into gingival crevice and fluid is collected via capillary action.
32
CHAPTER III
SIGNIFICANCE AND SPECIFIC AIMS
The role of cigarette smoking in the pathogenesis of periodontal disease has been
extensively studied and well documented over the past two decades. Cigarette smoking is
a significant risk factor in the pathogenesis of periodontal disease, and is also associated
with disease progression (Bergstrom and Preber 1994). Of equal importance in the
pathogenesis of the disease is the fact that bacterial products stimulate
monocytes/macrophages and lymphocytes as well as resident cells (fibroblasts,
endothelial cells) to secrete pro-inflammatory and immuno-regulatory cytokines.
Cytokines such as IL-1, IL-6, IL-8, and TNF-α are considered to be involved in
the host response of periodontal disease as mediators of tissue destruction. Increased
levels of these cytokines have been observed in GCF of patients with periodontal disease
(Rossomando, Kennedy et al. 1990; Wilton, Bampton et al. 1992; Geivelis, Turner et al.
1993). These associations however have large inter- and intra-individual variations
which suggest that these parameters are influenced by a multitude of other factors which,
so far, have been poorly quantified. In order to further establish their diagnostic value, an
extensive pro-inflammatory cytokine profile which is highly quantitative, was evaluated
in periodontally diseased subjects exposed to the environmental risk factor, smoking.
The objective of this study was to employ a highly quantitative protein assay to
evaluate a unique and comprehensive panel of gingival crevicular fluid (GCF) Th1
cytokines (IL-2, IL-12(p70), and IFN-γ), Th2 cytokines (IL-3, IL-4, IL-5, IL-10, and IL-
13), pro-inflammatory cytokines (IL-1α, IL-1β, IL-6, GM-CSF, TNF-α, and IL-12(p40)),
chemokines (IL-8, IP-10, MCP-1, MIP-1, RANTES and Eotaxin), and regulators of T
and natural killer cell activation and proliferation (IL-7 and IL-15), in chronic
periodontitis subjects and the influence of cigarette smoking on these mediators.
33
Hypothesis
The hypothesis of this study was that the GCF cytokine profiles of smokers are
significantly different from diseased nonsmokers. Furthermore, we expected that the
GCF cytokine profiles are significantly different in healthy versus diseased sites in the
diseased populations. Additionally, we expected cytokine profiles to significantly differ
between the diseased populations and the non-smoking healthy population.
The specific aims of this study were:
1) To determine the differences in GCF cytokine production between smokers and non-
smokers in sites with periodontal disease.
2) To measure cytokine levels in GCF from healthy sites within the diseased populations
and compare these to diseased sites.
3) To compare GCF cytokine production in the diseased populations with a periodontally
healthy non-smoking population.
34
CHAPTER IV
MATERIALS AND METHODS
Subject Population
Fifty-two subjects, including forty periodontally diseased subjects of which
twenty were smokers and twenty were non-smokers, and twelve periodontally healthy
non-smokers, participated in this study. Diseased subjects were between 40-75 years of
age with good general health and had a diagnosis of generalized advanced chronic
periodontitis with greater than thirty percent of sites with a clinical attachment level
(CAL) and probing depth (PD) greater than or equal to 5mm and bleeding on probing
BOP (Armitage 1996). Subjects had not received periodontal therapy for four months
preceding their participation in the study. Smokers were classified and enrolled if they
regularly smoked ≥20 cigarettes per day (Grossi, Zambon et al. 1997). Non-smokers
were classified as not having smoked one hundred or more cigarettes in their lifetime.
Healthy subjects were classified as non-smokers and free of periodontal disease with
CAL and PD ≤3mm and BOP ≤10% (Table 4). Subjects were excluded from
participating if they were pregnant, manifested gingival hypertrophy, diabetic or intake of
medication, such as antibiotics and anti-inflammatory agents, which may effect microbial
flora, the immune system or the inflammatory response for six months prior to
participation in the study (Table 5).
Subject selection and data collection were performed in the Department of
Periodontics at the University of Iowa College of Dentistry. Informed consent was
obtained from each subject and in accordance with guidelines established by the
University of Iowa Institutional Review Board (ID# 200603706).
Site Selection
A total of four sites were sampled from each of the twenty smokers. Sample sites
were located in different sextants of the oral cavity. Alternative sites were selected and
35
sampled in instances when the sampled site did not meet the established criteria. Two
samples were selected from diseased sites (PD and CAL ≥5mm with BOP) based on sites
with deepest probing depths, accessibility and avoidance of salivary contamination and
were classified as (SD). Another two samples were taken from healthy sites (PD and
CAL ≤3mm with no BOP) and were classified as (SH). An additional four samples were
taken from each of the twenty non-smoking participants. For participants who had
periodontitis but did not smoke, two samples were taken from diseased sites and
classified as (ND), and two samples were taken from healthy sites classified as (NH).
Four healthy sites were sampled from the twenty periodontally healthy non-smoking
individuals and classified as (HC). Healthy and diseased sites in all non-smoking and
smoking periodontitis subjects were classified as (HP) and (DP), respectively (Table 3).
Clinical Evaluation
All subjects eligible for participation were screened to assess level of disease by a
review of radiographs and a clinical evaluation. For sites selected to be sampled and
categorized as either healthy or diseased the following measurements were completed by
HC=healthy control, HP=healthy sites in periodontitis subjects, DP=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.
80
Table 33. P values of intra-group, inter-group and pooled comparisons of Th1 and Th2 cytokines, pro-inflammatory cytokines, chemokines and regulators of T-cells and NK cells.
The volume of GCF has been shown to be associated with the status of
periodontal disease. GCF is an inflammatory transudate of serum origin (Tollefsen and
Saltvedt 1980). Goodson showed that the flow rate of GCF can increase up to 30-fold in
periodontitis sites compared to healthy sulci (Goodson 2003). Other studies have also
shown an increase in GCF volume with an increase in severity of inflammation (Loe and
Holm-Pedersen 1965; Oliver, Holm-Pederen et al. 1969). Our study found the mean
GCF volume of all sites to be 1.6µl with a range of 0.17-3.28µl. Similar to other studies,
the volume of GCF in diseased sites was significantly higher than that found in healthy
sites of the periodontitis subjects. Interestingly, GCF volumes in both the healthy and
diseased sites of the periodontitis subjects were statistically greater than sites sampled in
the healthy control subjects. The general increase in the GCF volume in all sites in
diseased subjects suggests a physiological reactive mechanism that plays a special role in
the homeostasis of the periodontium. Alternatively, this increase in GCF could also
reflect the inflammatory and tissue breakdown process. In the present study, diseased
sites in smoking subjects demonstrated significantly less GCF volumes when compared
to diseased sites in non-smokers. This has been reported in other studies and has been
explained by the effects of smoking on gingival vasculature and subsequent decrease in
GCF production (Morozumi, Kubota et al. 2004). Studies have shown that smokers have
a lower resting GCF flow rate however after smoking cessation the GCF volumes
increased to those comparable with nonsmokers (Persson, Bergstrom et al. 1999;
Morozumi, Kubota et al. 2004).
87
Association of Cytokines and Periodontal Disease
Cytokines such as IL-1, IL-6, IL-8 and TNF-α have been reported to play an
important role in the host response of periodontal disease as mediators of tissue
destruction. This group represents inflammatory cytokines which are induced during the
course of an inflammatory response (Okada and Murakami 1998). These cytokines are
also prominent regulators of normal tissue homeostasis and can therefore also be detected
in healthy gingival tissues (Okada, Murakami et al. 1996). Increased levels of these
cytokines have been observed in the GCF of patients with periodontal disease
(Rossomando, Kennedy et al. 1990; Wilton, Bampton et al. 1992; Geivelis, Turner et al.
1993). Our study also found significant increases in levels of these and other chemokines
and cytokines within diseased sites of periodontitis subjects, which correspond with
reports in the literature. The levels of TNF-α could not be compared in our study due to
values falling below the detectable limit of the assay.
The total amount of numerous chemokines and pro-inflammatory cytokines was
increased (pg/30s) in the diseased sites of periodontitis subjects relative to the sites
sampled from the healthy controls. These included IFN-γ, IL-1α, IL-1β, IL-2, IL-3, IL-4,
IL-6, IL-8, IL-12 (p40), IL-15, MIP-1 and RANTES. Healthy sites of periodontitis
subjects also showed an increase in IL-1β and IL-8 relative to sites sampled from the
healthy controls. The increase of these two cytokines may represent a pre-clinical
initiation of the inflammatory process and may be potential indicators of future
periodontal breakdown. When comparing diseased sites to healthy sites in periodontitis
subjects, an increase of IFN-γ, IL-1α, IL-1β, IL-6, IL-12 (p40), IL-15, MCP-1, MIP-1 and
RANTES was observed in diseased sites suggesting a stimulated inflammatory and
immunological host response. The results of our study correspond favorably with other
reports in the literature showing an increase in specific pro-inflammatory cytokines (IL-1,
IL-6 and IL-8) found within diseased sites of periodontitis subjects.
88
Orozco showed that IL-1β can act on a large number of cells (fibroblasts,
chondrocytes, bone cells, neutrophils and lymphocytes) suggesting that both periodontal
destruction and repair is likely associated with this cytokine (Orozco et al. 2006). IL-1β
is a potent bone-resorbing cytokine in that it affects the differentiation and activation of
osteoclasts (Shirodaria, Smith et al. 2000). It also plays a role in degrading the
extracellular matrix in periodontitis by up-regulating matrix metalloproteinases and
down-regulating tissue inhibitors of metalloproteinase production (Ohshima, Otsuka et al.
1994; Schwartz, Goultschin et al. 1997).
IL-6 is linked to periodontitis through its action on the terminal differentiation of
B-lymphocytes to plasma cells and stimulating the secretion of immunoglobulin IgA and
IgG (Fujihashi, Kono et al. 1993). In addition, it is believed to play an important role in
the local regulation of bone turnover (Ishimi, Miyaura et al. 1990).
Because of its pro-inflammatory and neutrophil chemotactic properties, IL-8 is
also thought to play a significant role in the pathogenesis of periodontitis (Kamma,
Giannopoulou et al. 2004). It is likely that locally secreted IL-8 induces neutrophil
extravasation at the site of inflammation and that the numerous neutrophils present in the
lamina propria and the epithelium of inflamed gingiva is directed there by IL-8 (Okada
and Murakami 1998). Continuous and excessive IL-8-mediated chemotactic and
activation effects on neutrophils in the inflamed gingiva may contribute to local tissue
destruction of the periodontal tissues (Okada and Murakami 1998).
While not thought to be directly involved in the host response of periodontal
disease, various cytokines can act indirectly, enhancing or suppressing other tissue
destructive mediators. Our study found an increase of various Th1 and Th2 cytokines
(IFN-γ, IL-2, IL-3, IL-4), chemokines (MCP-1, MIP-1 and RANTES), and regulators of
T-cells and natural killer cells (IL-15) within diseased sites. To our knowledge, this is
the first report to identify an increase of these mediators in the GCF from periodontally
diseased sites.
89
Gemmell and Seymour proposed that the stable periodontal lesion is mediated
principally by cells within the Th1 cytokine profile (IFN-γ, IL-2), while the progressive
lesion involves Th2 cells which secrete cytokines (IL-3, IL-4) mainly acting on B-cells
(Gemmell and Seymour 1994). Immunoglobulin is indirectly produced from the B-cell
population and may produce protective antibodies and eliminate pathogenic organisms.
Alternatively, non-protective antibodies and/or IL-1β may be produced resulting in tissue
breakdown. Ebersole and Taubman proposed an opposing theory whereby Th1 cells are
prominent in diseased sites and Th2 cells are protective rather than destructive (Taubman,
Stoufi et al. 1984). Our study is consistent with numerous investigations reporting both
Th1 and Th2-type cytokines in diseased periodontal sites. It is reasonable to speculate
that both Th1 and Th2 cytokines are involved in the pathogenesis of periodontitis.
Altered or over-production of cytokines derived from Th1 and Th2 cells may be
responsible for periodontal destruction through humoral and/or cellular exaggerated
immune responses (Okada and Murakami 1998).
IFN-γ is an example of a Th1 cytokine which has been shown to modulate the
expression of pro-resorptive factors in periodontal microorganism-specific periodontal
CD4+ Th1 cells. This can further mediate osteoclastogenesis associated with alveolar
bone loss in vivo (Takayanagi, Ogasawara et al. 2000). It has also been shown that IFN-γ
and Th1 cells are strongly associated with enhanced alveolar bone loss during periodontal
infections (Valverde, Kawai et al. 2004). IFN-γ can also up-regulate the expression of
major histocompatibility complex (MHC) class II and other accessory molecules on the
antigen-presenting cells, which may further recruit other signaling molecules and/or
immune effectors associated with bone remodeling (Ellis and Beaman 2004).
There is evidence indicating that IL-2 plays a primary role in the pathogenesis of
periodontal disease (McFarlane and Meikle 1991). IL-2 is a Th1 cytokine involved in B-
cell activation and stimulating macrophages, natural killer cells and T-cell proliferation,
which mediate the cellular immune response (Tew, Engel et al. 1989). IL-2 has been
90
implicated in the stimulation of osteoclast activity in bone resorption (Ries, Seeds et al.
1989). Localized IL-2 production has been shown from lymphocytes cultured from
chronically inflamed periodontal tissues of patients with alveolar bone loss produced IL-2
(Seymour, Cole et al. 1985). Correspondingly, systemic IL-2 has been shown to be
elevated in the sera of periodontitis patients when compared to those of normal subjects
(McFarlane and Meikle 1991). Due to its biological properties, IL-2 has been suggested
to be a useful marker of pathologic inflammatory activity in systemic diseases (John,
Turner et al. 1998) and periodontal conditions (McFarlane and Meikle 1991).
IL-3 is a Th2 cytokine that has been shown to induce the proliferation of mast
cells and macrophages and causes the synthesis of histamines by mast cells and
phagocytosis in macrophages. It also significantly enhances the secretion of other pro-
inflammatory cytokines such as IL-1, IL-6 and TNF-α. The stimulatory effects of IL-3
on macrophages, mast cells and pro-inflammatory cytokines may explain its contributing
role in the pathogenesis of periodontitis.
IL-4 may play a role in inhibiting periodontitis as a potent down regulator of
macrophage function by inhibiting the secretion of IL-1β, tumor necrosis factor-α (TNF-
α) and IL-6 (Kamma, Giannopoulou et al. 2004). It is a Th2 cytokine and is known to
inhibit the secretion of prostaglandin E2 by human monocytes which leads to bone
resorption (Shapira, van Dyke et al. 1992). Localized absence of IL-4 in diseased
periodontal tissues has been associated with periodontal disease activity and progression
(Shapira, van Dyke et al. 1992). An increase in this cytokine likely demonstrates a
compensatory reaction in an attempt to balance the pro-inflammatory response.
Our study showed an increase in other chemokines in diseased sites including:
MCP-1, MIP-1 and RANTES. Chemokines are a family of structurally related
glycoproteins with potent leukocyte activation and/or chemotactic activity. Monocyte
chemotactic protein-1 (MCP-1) is chemotactic for monocytes and is known to regulate
the expression of pro-inflammatory cytokines such as IL-1 and IL-6. MCP-1 is also a
91
potent activator of human basophils, inducing degranulation and the release of
histamines, thus likely contributing to inflammatory responses seen in periodontitis.
Macrophage inflammatory protein-1 (MIP-1) is known to cause local
inflammatory responses in vivo, and induces superoxide production by neutrophils in
vitro.
RANTES plays an important role in the host response by recruiting inflammatory
cells into the foci of active inflammation and by inducing the release of other cell
mediators (Gamonal, Acevedo et al. 2000). RANTES has also been shown to be an
important mediator of the host response in chronic adult periodontitis (Emingil, Atilla et
al. 2004).
Our study also showed an increase in IL-15 within diseased sites. IL-15 is a
regulator of T-cells and natural killer cells (NK). It specifically increases the antitumor
activities of these cells and the production of CD4+ lymphocytes. An increase within the
GCF of diseased sites of periodontitis subjects represents a heightened host response.
Interestingly, Interferon-Inducible Protein-10 (IP-10) was the only cytokine that
was less prevalent (in both healthy and diseased sites) when compared to healthy
controls. IP-10 is a chemokine that is thought to play an important role in delayed type
hypersensitivity reactions. It is also thought to regulate the growth of immature
hematopoietic progenitor cells and is a potent endogenous inhibitor of angiogenesis. Our
study showed a decrease in IP-10 within diseased sites which may suggest a
compensatory mechanism to increase angiogenesis as part of the inflammatory host
response.
GCF Cytokine Profiles in Smokers
Smoking’s potent inhibition of the activity and amounts of chemokines and pro-
inflammatory cytokines, is supported throughout the literature (Rawlinson, Dalati et al.
2000; Kamma, Giannopoulou et al. 2004; Petropoulos, McKay et al. 2004). We saw a
92
decrease in various pro-inflammatory cytokines (IL-1α, IL-6, IL-12(p40)), chemokines
(IL-8, IP-10, MCP-1, MIP-1 and RANTES) and regulators of T-cells and NK cells (IL-7
and IL-15) in diseased sites in smokers as compared to diseased sites in nonsmokers
within our study. In contrast there were no apparent inhibitory effects of smoking on Th1
and Th2 cytokines.
The decrease in IL-1α is consistent with Petropoulos et al. (2004) findings (Table
32) whereby the concentration of IL-1α in GCF of smokers was approximately half that
found in non-smokers (Petropoulos, McKay et al. 2004). Kamma et al. 2004 showed a
statistically significant decrease in IL-8 in smokers with aggressive periodontitis. A
decrease in IL-8 was also observed in our study with a chronic periodontitis subject
population. Our study showed no difference in levels of IL-4 levels between smokers
and non-smokers which is also consistent with the findings of Kamma et al 2004.
To our knowledge, the findings of a significant decrease in IL-6, IL-7, IL-
12(p40), IL-15, IP-10, MCP-1, MIP-1 and RANTES within smokers, in the present study
have not been previously reported. This decrease in pro-inflammatory cytokines,
chemokines and regulators of T-cells and NK cells is expected, as nicotine induces an
immunosuppressed state. Smokers display suppressed migration and chemotaxis of
neutrophils which may be explained by the decrease in chemokines such as IL-8, IP-10,
MCP-1, MIP-1 and RANTES. In our study, smokers also showed a decrease in
regulators of T-cells and NK cells such as IL-7 and IL-15 which may explain a reduction
in CD4+ lymphocytes found in smokers (Loos, Roos et al. 2004). A decrease in pro-
inflammatory cytokines found in this study may be explained through the reduction in IL-
7 which is known to induce the synthesis of IL-1, IL-6 and GM-CSF in activated human
T-cells.
Some studies have reported increased cytokine amounts in smokers. We found
greater amounts of IL-1α, IL-1β and IL-3 within diseased sites of smokers when
compared to healthy controls. However, diseased sites of non-smokers also displayed
93
similar increases when compared to healthy controls, which questions the true effect of
smoking on these cytokines. The relative increase in IL-1β observed in diseased smokers
is consistent with earlier reports by Kamma et al. (2004) who also reported greater total
volumes of IL-1β in smokers. Our study also showed greater levels of IL-3 within
diseased sites of smokers compared to healthy controls, which to our knowledge has not
been previously reported. IL-3 is known to stimulate the production of IL-1 which was
observed in our study. Bostrom et al. (1998, 1999) showed higher levels of TNF-α in
GCF in smokers and former smokers compared with non-smokers, with comparable
levels of moderate/severe periodontitis (Bostrom, Linder et al. 1998; Bostrom, Linder et
al. 1999). This relationship could not be confirmed within the present study due to the
fact that total amounts of TNF-α fell below the detectable limit of the assay. Also in
contrast to the present study, Giannopoulou et al. 2003 showed an increase in total
amounts of IL-6 and IL-8 in GCF of smoking subjects in an experimental gingivitis
model. Differences in the results found in our study may again be explained by variances
in study design methodology and analysis. Giannopoulou et al. pooled 2 strips sampled
for 15s each, versus our protocol where we used a single strip per site and held the strip
in place for 60s. Bostrom et al. used an aspiration method for GCF sampling where
complete fluid recovery is known to be unpredictable. They also reported the
concentration of cytokines (pg/µl) which is greatly affected by differences in GCF
volumes, versus total cytokine volumes (total/30s) which were reported in the present
study.
GCF Variability
Variations in GCF parameters (including volume, cytokine concentration and total
amounts of cytokines) are widespread throughout the periodontal literature. Jin et al
(2000) suggested that this variability might be indicative of the episodic nature of
periodontal disease progression, the various stages of inflammation, disease severity,
94
shifts in host-bacterial interactions, or the presence of certain putative periodontal
pathogens. Other explanations for GCF cytokine variability in studies may reflect the
complex multifactorial nature of the disease and differences in sampling techniques and
assays used for analysis.
Although a 30-second sampling time is standard protocol throughout the literature
and considered adequate to sample diseased sites, using a 60 second sampling time could
be beneficial for sampling healthy sites to minimize the inflation of cytokine
concentration as a result of low GCF volumes typically collected from these sites. In the
present study and other studies, attempts were made to reduce inter and intra-examiner
variability and systematic errors. Therefore, variations in GCF cytokine values are likely
not exclusively due to technical sampling errors but also due to biological differences and
reflect the variability seen among subjects.
Mathur et al (1996) found that GCF cytokine amounts were highly variable at
healthy sites, as evidenced by large standard deviations. This was apparent in the present
study as well. Mathur attributed this, in part, to the relatively large error in estimating
fluid volumes at sites with low Periotron® readings. He showed that when total amounts
were used, cytokine levels (pg/30sec) at diseased sites were greater than those at healthy
sites. However, the inverse was true when he used cytokine concentrations. These
findings were similar to those by Lamster et al (1986) when levels of neutrophil enzymes
were evaluated. Chappie et al (1995) found that measurement error was greater for
samples with small (<0.2 µl) fluid volumes. Mathur et al (1996) further concluded that
reporting total amounts of cytokines is probably more valid or reliable than reporting
concentrations at sites with small GCF volumes.
Future Directions
This study utilized a highly quantitative assay to evaluate a panel of cytokines
seen in GCF, in order to further establish the diagnostic value of cytokines in
95
periodontitis and further explore their usefulness as a measure of disease activity.
Twenty-two cytokines were tested using multiplex protein analysis. The cytokines tested
in this study were based on previous reports of cytokines in periodontal diseases and
those available, as part of a kit from the manufacturer. Our study confirmed the reports
of increased levels of IL-1, IL-6 and IL-8 in periodontal disease. We also found an
increase in the following cytokines within diseased sites: IFN-γ, IL-2, IL-3, IL-4, IL-12
(p40), IL-15, MIP-1 and RANTES. To our knowledge, these have not been previously
reported or studied in the periodontal literature. Future studies may benefit by testing
other cytokines using this methodology to further expand this profile, in an attempt to
better understand the periodontal disease process.
With the progression of our understanding of the mechanics of periodontal
disease, 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 different cytokines and inflammatory mediators, intracellular and extracellular
host enzymes, and byproducts of tissue breakdown as potential markers of periodontal
diseases. The discovery of a marker(s) that could predict the shift from gingivitis to
periodontitis, or diagnose periodontitis at an early stage, would increase our ability to
manage periodontitis and to effectively design treatment plans for high risk patients
including appropriate mechanical and/or chemical interventions and earlier and/or more
aggressive intervention. So far, "there are insufficient data to determine the role of
proposed host-based diagnostic tests in treatment planning, and monitoring the effect of
periodontal therapy in patients with periodontitis"(Armitage 1996).
While cytokine profiling of GCF is a common method of protein analysis, other
more invasive methods of cytokine assessment such as evaluation of cytokine levels in
plasma samples and tissue biopsies could improve our understanding of the disease
process. Plasma samples reflect general concentrations of cytokines, but would not
reflect the differences between different periodontal sites as periodontal disease is
96
typically a site specific disease. Cytokines could also be extracted from tissue biopsies
and levels measured by ELISA.
Future studies should focus on reducing the inherent limitations found in this
study. The subject population should be more strictly defined to chronic periodontitis
subjects by a review of past records, to enhance the probability of exclusion of aggressive
periodontitis subjects. Smoking history and status should also be more strictly defined
and grouped to reduce the influence of variances of nicotine exposure levels on both GCF
volumes and cytokine amounts. Additionally, the relatively small sample size and
limited number of sites sampled within this study should be increased to expand the
statistical detection of additional cytokine relationships. An experimental gingivitis
model could also be employed, consisting of the sampling of GCF at different time
points, to determine the effects of various stages of inflammation on chemokine and
cytokine amounts and profiles.
97
Table 34. Comparison of studies on the effects of smoking on GCF cytokine/chemokine levels.
* Statistically significant (p≤0.05)
Concentration Total volume
NS S NS S
Study Smokers
H D H D H D H D
Bostrom et al. (1999)
(Bostrom, Linder et al.
1999)
↑ TNF-α *
IL-6 (no diff)
12 (7.3-18.3)
(pg/ml)
10 (0-28) (pg/ml)
61.0 (42-177)
(pg/ml)
5.0 (0-30.5) (pg/ml)
Bostrom et al. (2000)
(Bostrom, Linder et al.
2000)
IL-1β (no diff)
IL-1ra (no diff)
61.50 (34.50-
113.75) (pg/ml)
59.62 (40.28-71.89)
(pg/ml)
60.5 (24.75-93.50)
(pg/ml)
57.61 (46.92-
110.06) (pg/ml)
Rawlinson et al.
(2003)
(Rawlinson, Grummitt
et al. 2003)
↓IL-1β *
↓IL-1ra *
393.8
(867.1)(pg/µl )
3.2x105
(2.3)(pg/µl)
73.1 (61.0) (pg/µl )
3.2x105 (2.3)
(pg/µl)
2714.5
(4416.2)(pg/ul)
5.8x105 (9.7)
(pg/ul)
24.5 (29.2) (pg/µl)
0.19x105 (0.07)
(pg/µl)
0.27 (0.40)
(µl)
1.06 (0.34)
(µl )
0.32 (0.42)
(µl )
1.39 (0.22)
(µl )
Petropoulos et al.
(2004)(Petropoulos,
McKay et al. 2004)
↓IL-1α * 3.29 (2.02) (pg/µl ) 1.59 (1.13) (pg/µl)
Erdemir et al. (2004)
(Erdemir, Duran et al.
2004)
TNF-α (no diff)
IL-6 (no diff)
0.51 (0.81) (pg/µl)
0.57 (0.75) (pg/µl)
1.07 (1.32) (pg/µl)
0.32 (0.38) (pg/µl)
98
Table 34. Continued
* Statistically significant (p≤0.05)
Kamma et al. (2004)
(Kamma,
Giannopoulou et al.
2004)
↑IL-1β *
IL-4 (no diff)
↑IL-6 *
↓IL-8 *
7.85
(pg/30s)
12.07
(pg/30s)
0.89
(pg/30s)
24.00
(pg/30s)
61.37
(pg/30s)
1.90
(pg/30s)
5.04
(pg/30s)
72.96
(pg/30s)
17.97
(pg/30s)
10.33
(pg/30s)
1.70
(pg/30s)
20.43
(pg/30s)
62.37
(pg/30s)
3.08
(pg/30s)
6.04
(pg/30s)
68.30
(pg/30s)
99
Conclusions
The purpose of this study was to employ a quantitative assay to measure a broad
panel of cytokines in diseased and healthy sites in subjects with periodontal disease who
smoked, who did not smoke and to compare to each other and healthy controls. GCF
volumes were also evaluated and compared.
1. The GCF volumes were significantly increased in both healthy and
diseased sites of periodontitis subjects when compared to healthy
control subjects. Additionally, volumes in diseased sites were
significantly higher compared to healthy sites in periodontitis
subjects. Diseased sites in smokers showed significantly lower total
GCF volumes compared to diseased sites in non-smokers. GCF
volumes in healthy sites between smokers and non-smokers were not
significantly different.
2. Diseased sites in periodontitis subjects showed significantly greater
levels of several chemokines and cytokines including IFN-γ, IL-1α,
IL-1β, IL-2, IL-3, IL-4, IL-6, IL-8, IL-12(p40), IL-15, MIP-1 and
RANTES when compared to healthy controls. Healthy sites in
periodontitis subjects showed an increase in IL-1β and IL-8 when
compared to healthy controls. Interestingly, IP-10 was the only
cytokine that was less prevalent (in both healthy and diseased sites)
when compared to healthy controls.
3. Smoking appears to have a potent inhibitory effect on chemokine and
cytokine production. Smokers had significantly less IL-1α, IL-6, IL-
7, IL-8, IL-12(p40), IL-15, IP-10, MCP-1, MIP-1 and RANTES in
100
diseased sites compared to diseased sites in non-smokers. This
supports the concept that smoking induces an immunosuppressed
state, thus inhibiting an individual’s ability to combat bacterial
infection found in periodontitis. Novel cytokines such as IL-7, IL-
12(p40), IL-15, IP-10, MCP-1, MIP-1, have not been reported in
relation to smoking and periodontitis in the literature to date.
4. The multiplex immunoassay (Luminex®) employed in this study has
enabled the formation of a comprehensive chemokine and cytokine
profile, in both smoking and non-smoking periodontitis subjects.
This profile can be further expanded using the methodologies
employed in this study, in hopes of reducing the large degree of
variability inherent in GCF cytokine analysis.
5. The current study has suggested a GCF chemokine and cytokine
profile for periodontally diseased subjects, including smokers and
non-smokers and healthy controls. This profile increases our
understanding of the multifactorial nature of the disease by showing
the pleiotropic roles of chemokines and cytokines in the host
response. Future studies should focus on exploring how this panel of
chemokines and cytokines could be used in the diagnosis, prognosis
or in the treatment of periodontal disease.
101
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