University of Iowa

Iowa Research Online

Theses and Dissertations

2008

The influence of smoking on cytokines in the

gingival crevicular fluid in patients with periodontal
disease
Keelen D Tymkiw
University of Iowa

Copyright 2008 Keelen D Tymkiw

This thesis is available at Iowa Research Online: https://ir.uiowa.edu/etd/26

Recommended Citation
Tymkiw, Keelen D. "The influence of smoking on cytokines in the gingival crevicular fluid in patients with periodontal disease." MS
(Master of Science) thesis, University of Iowa, 2008.
https://doi.org/10.17077/etd.s6um7g51

Follow this and additional works at: https://ir.uiowa.edu/etd

Part of the Oral Biology and Oral Pathology Commons

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.

Thesis Committee: ___________________________________

Janet Guthmiller, Thesis Supervisor

___________________________________
Georgia Johnson

___________________________________
Kim Brogden

___________________________________
Sophie Joly

___________________________________
Joseph Cavanaugh
 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.

ii
 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
GCF Flow Rate.................................................................................16
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
Th1 and Th2 Cytokines: IL-2, IL-3, IL-4, INF-γ ......................43
Pro-inflammatory Cytokines: IL-1α, IL-1β, IL-6, IL-
12(p40), GM-CSF .....................................................................45

iii
 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

REFERENCES ................................................................................................................101

iv
 LIST OF TABLES

Table 1. Cytokine Properties.............................................................................................18

Table 2. Translation of Periotron® values to clinical conditions and gingival index

with which they may be associated....................................................................30

Table 3. Subject Groups....................................................................................................39

Table 4. Patient Inclusion Criteria ....................................................................................40

Table 5. Patient Exclusion Criteria ...................................................................................41

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

v
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

vi
 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

vii
 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

cytokines (IL-1α, IL-1β, IL-6, IL-12 (p40), GM-GSF), chemokines (IL-8, IP-10, MCP-1,

MIP-1, RANTES, Eotaxin) and regulators of T-cells and natural killer cells (IL-7 and IL-
 2

15). The remainder of this chapter will review the literature relative to gingival

crevicular fluid, smoking, and the relationship cytokines have in the pathogenesis of

chronic periodontitis.
 3

CHAPTER II

REVIEW OF THE LITERATURE

Gingival Crevicular Fluid

Gingival crevicular fluid (GCF) is a transudate that is released circumferentially

into the gingival sulcus of teeth. This transudate is a blood serum product and primarily

composed of inflammatory cells, most notably polymorphonuclear neutrophilic

leukocytes (PMN’s) and serum proteins (Cimasoni 1983). Additional contents include

bacteria, tissue breakdown products, enzymes, antibodies, complement, and numerous

inflammatory mediators (Armitage 1996). As GCF is produced by the periodontal

tissues, analysis of its constituents may provide an early indicator of tissue inflammatory

changes. In that regard, GCF provides a unique window for analysis of the periodontal

condition. The flow rate of GCF has been shown to increase up to 30-fold in periodontitis

compared to healthy sulci (Goodson 2003). Correspondingly, it has been shown that an

increase in GCF volume is associated with an increase in severity of inflammation (Loe

and Holm-Pedersen 1965; Oliver, Holm-Pederen et al. 1969).

Methods of Collection

It has been shown that the collection of GCF is simple, noninvasive, and

nontraumatic and allows for the evaluation of inflammatory status. The collection of

GCF can be accomplished using a variety of methods, each with distinct advantages and

disadvantages. The method selected is usually, based on the objective of the study.

There are three techniques which can be used to collect GCF including; gingival washing,

capillary tubing, and absorbent filter strip collection.

Gingival Washing Methods

In the gingival wash technique, the gingival crevice is perfused with a fixed

volume of an isotonic solution, such as Hanks' balanced salt solution. A customized

 4

acrylic stent can be employed to isolate the gingival tissues from the rest of the oral

cavity (Oppenheim 1970). The tissues are irrigated for 15 min, with the saline solution,

using a peristaltic pump, and the diluted GCF is removed (Skapski and Lehner 1976).

The fluid collected then represents a dilution of crevicular fluid and contains both cells

and soluble constituents such as plasma proteins (Griffiths 2003). This technique is

particularly valuable for harvesting cells from the gingival crevice (Griffiths 2003).

However, many disadvantages exist in relation to the complexity of this method. The

production of customized acrylic stents is technically demanding and GCF from

individual sites cannot be analyzed. Additionally, complete fluid recovery during the

aspiration and re-aspiration procedure is unpredictable. As a result, accurate

quantification of GCF volume or composition is not possible as the precise dilution factor

cannot be determined (Griffiths 2003).

Capillary Tubing or Micropipettes

In an alternative method of GCF collection, capillary tubes of known internal

diameter are inserted into the entrance of the gingival crevice following isolation and

drying of the site. GCF from the crevice migrates into the tube via capillary action. The

volume of fluid collected can accurately be determined by measuring the distance the

GCF has migrated in the known diameter of the tube (Sueda, Bang et al. 1969). This

technique is advantageous in that it allows for the analysis of an undiluted sample of

'native' GCF (Griffiths 2003). However, this technique is time consuming as an adequate

volume of GCF (≥0.1µl) must be collected (Griffiths 2003). The collection time for an

individual healthy site can exceed 30min and still may result in an inadequate volume for

analysis (Griffiths 2003). Due to the duration of time required for this technique, trauma

to the periodontal tissue must be questioned. Additional disadvantages of this methods

are difficulty in removing the complete sample from the tubing which can ultimately

affect the accuracy of the fluid volumes and concentrations (Griffiths 2003).
 5

Absorbent Filter Paper Strips

In this most commonly employed technique, the sample area is isolated, gently

dried with air, then an absorbable paper filter strip is placed into or laid outside of the

gingival sulcus (Figure 1). The extracrevicular variation of this method involves

overlaying the strip on the gingival crevice region in an attempt to minimize trauma. In

contrast, the intracrevicular method is the method used most frequently and the one

employed in the present study. In this technique the strip is inserted into the entrance of

the gingival crevice until minimum resistance is felt (Brill 1959). Collection time can

vary from 30-60 seconds. A shorter duration of collection (30 seconds) is used most

commonly to decrease the probability of blood contamination and to prevent the

collection of excessive volumes (>1.0µl) usually found in diseased sites, which are

unable to be measured with the Periotron 6000®. However, the minimal detection limit

(≥0.1µl) of the Periotron must also be reached which can be difficult to obtain in healthy

sites sampled for short durations. This technique is relatively simple and time efficient

and can be used to assay select sites with minimal tissue trauma (Griffiths 2003).

Methods of Estimating Volume Collection Following use

of Absorbent Filter Paper Strips

While the collection of GCF readily lends itself to comparative studies of various

conditions, the reliability of data is sometimes problematic because of the difficulty in

measuring and assaying the small volumes obtained in many cases and the variations in

collection protocols and processing methodology (Palmer, Wilson et al. 2005).

In early studies, GCF volumes were assessed simply by measuring the linear

distance the fluid migrated on the strip. A greater level of accuracy was achieved by

assessing the area of filter paper saturated by the GCF sample. Further accuracy was

achieved by staining the strips with ninhydrin which produced a purple color where GCF

had accumulated (Cimasoni 1983). These primitive methods had many inherent
 6

disadvantages including the inability to be employed chairside. This delayed the

measuring process and resulted in fluid evaporation and volume errors. Additionally,

staining of the strips for volumetric determination prevented further laboratory study of

the GCF components, effectively limiting the technique to volume determination

(Griffiths 2003).

An alternative historical approach, involved weighing the strips before and after

sample collection (Cimasoni and Giannopoulou 1988). This technique showed

reasonable accuracy but required a very sensitive scale to estimate the minute volumes of

fluid collected, especially that from a healthy sulcus. Similar to the first technique,

evaporation introduced potential volume errors (Griffiths 2003).

Today, electronic measuring devices (i.e. Periotron®), have introduced an

accurate and time efficient method of GCF volume measurements. This instrument

quantifies the volume of GCF or saliva collected on filter papers by measuring the

electrical capacitance of the wet paper strip (Tozum, Hatipoglu et al. 2004). A wet strip

results in increases in capacitance in proportion to the volume of fluid. This volume

correlates with disease status according to the manufactures guidelines (Table 2).

The electronic technique allows for immediate and rapid volume measurements

and has no effect on the GCF sample. Fluid evaporation is minimized as samples are

tested chairside (Tozum, Hatipoglu et al. 2004). Despite the advantages of this method of

analysis, limitations of the Periotron® exist. One of the limitations is the limited range of

volumes that can be measured. Volumes between 0.1-1.0µl can be measured, however,

measurements at the lower end of this range have been shown to have decreased accuracy

(Griffiths 2003). Machine calibration errors have been found to be only 3.2 +/- 7.5%,

however, standard deviations for volumes below 0.2µl were as high as 18.7% (Chapple,

Cross et al. 1995). While there are significant differences in volume readings between

different machines (p<0.0004) and between the same volume of different fluids

(p<0.00001), intramachine Periotron® calibrations are extremely reproducible and

 7

reliable (Chapple, Cross et al. 1995). Chapple concludes that the Periotron 6000 is a

reliable and convenient instrument for measuring fluid volumes greater than 0.2µl. For

optimum accuracy, the digital display should be re-set to zero after each sample is

measured and each machine should be calibrated with known volumes to produce an

individualized standard curve to enhance volume accuracy (Chapple, Cross et al. 1995;

Griffiths 2003).

GCF Contamination

The major sources of contamination of the GCF sample include blood, saliva, and

plaque (Griffiths 2003). Blood contamination is managed by discarding the sample and

not including it in the data analysis. Saliva contamination is minimized through gentle

air drying of the sample site and cotton roll isolation. Samples that are contaminated with

saliva are discarded. Studies utilizing alpha-amylase assays have confirmed that the

likelihood of a significant contribution of saliva in GCF samples is small (Griffiths,

Wilton et al. 1992). In contrast, plaque contamination of filter strips has been shown to

have a marked effect on GCF volume (Stoller, Karras et al. 1990). Experiments where

dental plaque was applied directly to filter paper strips showed that a large plaque mass

contained a considerable amount of fluid, which would obviously influence volume

determinations (Griffiths 2003). This has been supported by other studies which showed

that failure to remove plaque adequately from the site prior to sampling had a major

effect on the determined volume (D'Aoust and Landry 1994). Thus, it is essential to

carefully remove the supragingival plaque prior to GCF collection.

Sample Recovery

In addition to GCF volume determination, the composition of the GCF is also

frequently evaluated. In this case, it is necessary to recover the GCF sample from the

filter paper strips. Studies employing a centrifugal elution technique have shown greater

than 90% protein recovery from samples (Cimasoni and Giannopoulou 1988; Nakashima,
 8

Demeurisse et al. 1994; Gustafsson 1996). However, variances in the concentration of

the original protein sample, the filter strip material and the elution reagents, have shown

significant differences in the percentage of protein recovery from filter papers (Johnson

1999). Comparisons between filter strip materials (Periopaper® vs. Durapore®) showed

significant differences in recovery, favoring the Periopaper® (Johnson 1999). The values

obtained with the Durapore strips were too low for accurate measurements of small

volumes of fluid (Gustafsson 1996). Additionally, it has been shown that proteins at low

concentrations are less efficiently eluted from GCF collection papers than those at higher

concentrations (Johnson 1999). The elution reagents also affect recovery (Gustafsson

1996). The ideal elution solution is made of isotonic buffers at neutral pH without

detergents. This resulted in the best and most highly reproducible recovery (Gustafsson

1996). These methodological variances may explain the conflicting results of GCF

analysis due to potential protein entrapment or protein binding on the filter paper.

Protein Analysis

The analysis of GCF proteins has been extensively explored in the literature in an

attempt to understand the complexities of GCF as an indication of inflammation, disease

activity and ultimately for use as a diagnostic marker. Due to the site-specific nature of

collection, clinical parameters can be linked to the proteins at the site of sample

collection (Polson and Goodson 1985). However, due to limited volumes collected from

individual sample sites, often times only single analytes can be examined or samples

must be pooled resulting in the loss of site-specific information (Griffiths 2003). It may

be possible, however, with more sensitive techniques, to estimate multiple analytes from

a given sample.

There are numerous techniques that have been used for GCF protein analysis

including western blot, ELISA, and multiplex protein analysis.

 9

Western Blot

Western blotting, or immunoblotting, is a method used to identify and obtain size

information of a specific protein in a mixture of proteins. This technique requires that the

protein of interest is antigenic and reacts specifically with an antibody. The protein

mixture is first separated electrophoretically by SDS PAGE and then transferred to a

solid support, such as a nitrocellulose, polyvinylidene difluoride (PVDF), or a cationic

nylon membrane. The membrane is then exposed to the primary antibody, which binds to

the protein of interest. Subsequently, a secondary antibody, which is covalently attached

to an easily assayed enzyme or radiolabel, binds to the primary antibody.

Generally, either audioradiography, detection of a radioactive isotope, or

chemiluminescence, detection of light produced from a chemical reaction, is used to

visualize the presence of the protein of interest.

ELISA

Protein analysis of GCF has typically been evaluated by enzyme-linked

immunosorbent assays (ELISA). ELISA is a biochemical technique where an unknown

antigen is affixed to a surface, and then a specific enzyme-linked antibody is washed over

the surface to enable binding to the antigen and subsequent detection. This well-

developed assay requires significant sample volume, is labor intensive, and is limited to

single analytes, and thus, is not amenable to multiplex analysis. Therefore, the accurate

evaluation of multiple immune mediators has been problematic due to the lack of a

validated multiplex analysis methodology for protein expression.

Multiplex Protein Analysis (Luminex®)

Recently, several multiplex protein analysis techniques have been developed.

Flow cytometry-based systems are currently the most widely used multiplex biomarker

analysis technology. The Luminex® system is one of these and uses uniformly sized

color coded microspheres internally labeled with graded proportions of a red and a near
 10

infrared fluorophore (658 and 712 nm), providing the capacity to identify and classify

numerous discrete beads specific to a particular bioassay (Martins 2002). This is in

contrast to the BD CBA system that discriminates beads based upon fluorescence

intensity from a single fluorophore, which limits the multiplexing capacity (Cook, Stahl

et al. 2001). Beads of a single identity are covalently coupled to a specific capture

antibody for the analyte of interest. A second detection antibody is used to quantitate the

amount of analyte captured on the bead. This secondary antibody is either directly

conjugated to the phycoerythrin (PE) fluorophore or biotin, which is then reacted with

streptavidin-phycoerythrin. Lasers are used to excite the internal dyes that identify each

microsphere particle, and also any reporter dye captured during the assay. Numerous

readings are made on each bead set, further validating the results. This technique allows

multiplexing of up to 100 unique analytes within a single sample thus allowing a more

comprehensive and precise analysis of GCF constituents (Offenbacher, Barros et al.

2007).

The advantages of the multiplex cytokine assays over the standard ELISA assay

include smaller sample volumes required (~36% less), less labor intense and lower costs

relative to equivalent ELISAs (66% less) (Dupont, Wang et al. 2005). Luminex

technology has numerous inherent advantages over the other discussed techniques and is

a suitable alternative for GCF protein analysis.

Cytokine Concentrations

Levels of proteins can be reported as cytokine concentrations (pg/µl) or as a "total

amount" of a substance per unit sample time (pg/30 sec). The latter has been

recommended in studies that attempt to identify cytokines as markers for active disease

(Lamster, Oshrain et al. 1986; Chapple, Garner et al. 1999). This is due to a significant

relationship between increases in pocket depth and GCF volume. This has a direct effect
 11

on analyte concentration in association studies of periodontitis (Brock, Butterworth et al.

2004).

Role of Cytokines in Periodontitis

Periodontitis is initiated by specific bacteria. These bacteria are known to

stimulate the host response, which plays an important role in the recruitment of

leukocytes and the subsequent release of inflammatory mediators and cytokines such as

interleukin (IL)-1, IL-6, IL-8, and TNF-α. Such mediators are thought to play an

important role in the pathogenesis of the disease. Increased levels of these and other

cytokines are involved in periodontal tissue destruction (Genco 1992). In contrast to their

destructive role, cytokines also contribute to the immunopathology of a number of

diseases, and the production of appropriate cytokines is essential for the development of

protective immunity. Thus, cytokines have both protective and destructive roles which

can greatly influence the onset and progression of disease (Seymour and Gemmell 2001).

Cytokines are cell regulators that have a major influence on the production and

activation of different effector cells. T cells and macrophages are a major source of

cytokines, although they are produced by a wide range of cells that play important roles

in many physiological responses. Cytokines are low-molecular-weight proteins involved

in the initiation and effector stages of immunity and inflammation, in which they regulate

the amplitude and duration of the response. They are usually transiently produced,

extremely potent (generally acting at picomolar concentrations), and interact with

specific cell surface receptors (usually expressed in relatively low numbers) (Balkwill

and Burke 1989). Some cytokines are produced by a restricted type of cell, such as IL-2

produced by T cells, whereas others, including IL-1 and IL-6, are produced by many

different cell types. Target cells may also be restricted or diverse (Seymour and Gemmell

2001). Many cytokines are pleiotropic, having multiple activities on different target cells

and or overlapping cell regulatory activities (Seymour and Gemmell 2001). The response
 12

of a cell to a given cytokine depends on the local concentration, the cell type and other

cell regulators to which it is exposed. Cytokines interact in a network: first by inducing

each other; second by transmodulating cell surface receptors; and third by synergistic,

additive, or antagonistic interactions on cell function (Balkwill and Burke 1989).

The interrelationships of various cytokines and their correlation with periodontitis

have been explored through the biochemical analysis of GCF. Among many

inflammatory and immune mediators identified in GCF, cytokines have attracted

particular attention and are believed to be involved in both inflammation related

destruction and repair of the periodontal tissues. Increased levels of several cytokines

such as IL-1, IL-2, IL-6, IL-8 and TNF-α have been observed in the GCF of patients with

periodontal disease (Kamma, Giannopoulou et al. 2004). Certain cytokines have been

proposed as potentially useful diagnostic or prognostic markers for periodontal disease

activity and wound healing (Genco 1992; Champagne, Buchanan et al. 2003). These

include; IL-1β, IL-4, IL-6 and IL-8 which have been shown to function in concert with

other members of the cytokine network in order to regulate the cellular inflammatory

response in the periodontium. The cytokines assessed in the present study include: IL-1α,

IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12(p40), IL-12(p70), IL-13, IL-

15, IP-10, Eotaxin, IFN-γ, GM-CSF, MCP-1, MIP-1α, RANTES, and TNF-α. Table 1

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).

Table 1. Cytokine Properties

Cytokine Sources# Biologic Activity# Relationship to Periodontitis

Interleukin -1 • Produced by • Chemoattractant for leukocytes, stimulation • Significantly increased in the periodontal
monocytes, of T-helper cells (IL-2 secretion), tissues and gingival fluid from diseased
(IL-1) macrophages, proliferation of B cells, ↑ B-cell sites, compared with healthy sites (Masada,
neutrophils, responsiveness to IL-5, proliferation and Persson et al. 1990).
endothelial cells, activation of NK-cells and fibroblasts, • Can act on a large number of cells
fibroblasts, smooth thymocytes, glioblastoma cells. (fibroblasts, chondrocytes, bone cells,
muscle cells, • Enhances the metabolism of arachidonic neutrophils and lymphocytes) suggests that
keratinocytes, acid (prostacyclin and PGE2) in periodontal destruction and repair in
langerhans cells of the inflammatory cells (fibroblasts, synovial periodontitis may in part be associated with
skin, osteoclasts, cells, chondrocytes, endothelial cells, this cytokine (Orozco, Gemmell et al.
astrocytes, epithelial hepatocytes, and osteoclasts) 2006).
cells of the thymus
and the cornea, T- • Increased secretion of inflammatory • IL-1β up-regulates matrix
cells, B-cells, NK- proteins such as neutral proteases metalloproteinases and down-regulates
cells. (collagenase, elastase and plasminogen tissue inhibitors of metalloproteinase
activator). Antagonizes the effects of TGF- production (Ohshima, Otsuka et al. 1994).
• Production stimulated β on the extracellular matrix
by TNF-α, IFN-α, • Powerful and potent bone-resorbing
IFN-β and IFN- γ, • Promotion of wound healing (angiogenesis, cytokine (Shirodaria, Smith et al. 2000).
bacterial endotoxins, proliferation of fibroblasts, neutrophil • Plays a role in degrading the extracellular
viruses, mitogens, and chemotaxis). matrix in periodontitis (Schwartz,
antigens. Goultschin et al. 1997).
Interleukin -2 • Produced by T-cells, • Significant anti-tumor activity for a variety • Involved in B-cell activation and stimulates
and B-cells, AK cells of tumor cell types since it supports the macrophages, NK cells and T-cell
(IL-2) (lymphokine-activated proliferation and clonal expansion of T- proliferation. It is regarded as a pro-
killer cells) and NK- cells that specifically attack certain tumor inflammatory cytokine (Tew, Engel et al.
cells. types. 1989).

18
Table 1. Continued

• Production stimulated • Induces the secretion of other soluble • IL-2 has been also implicated in the
by vitamin E. mediators, including TNF-α, TNF-β, and stimulation of osteoclast activity in bone
• Production inhibited IFN-γ. These effects may contribute to the resorption (Ries, Seeds et al. 1989).
by dexamethasone or antitumor activity of IL- as well as to its • There is evidence indicating that IL-2 may
Cyclosporin A. dose-related toxicity. 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 • Produced by T-cells, • Is a growth factor that establishes the link
keratinocytes, NK- between the immune system and the
(IL-3) cells, mast cells, hematopoietic system.
endothelial cells, and • Supports the proliferation and development
monocytes. of almost all types of hematopoietic
• Production inhibited progenitor cells.
by glucocorticoids or
CsA (Cyclosporin A).

19
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 • Produced by activated • Promotes the proliferation and • Potent downregulator of macrophage
T-cells (Th2) or (Th1), differentiation of activated B-cells, the function by inhibiting the secretion of IL-
(IL-4) Non-T/Non-B-cells of expression of MHC class 2 antigens, and of 1β, TNF-α and IL-6 (Kamma,
the lineage of mast low affinity IgE receptors in resting B-cells. Giannopoulou et al. 2004).
cells. • Enhances expression of MHC class 2 • Inhibits the secretion of prostaglandin
• Production stimulated antigens on B-cells. It can promote their (PGE2) by human monocytes which leads
by IL-2 and PAF capacity to respond to other B-cell stimuli to bone resorption (Shapira, van Dyke et al.
(platelet activating and to present antigens for T-cells. 1992).
factor). • Absence of IL-4 has been associated with
• Production inhibited periodontal disease activity and progression
by TGF-beta. (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).

20
Table 1. Continued

Interleukin -5 • Produced by T-cells • Specific hematopoietic growth factor that is

responsible for the growth and
(IL-5) 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 • Produced by • Pleiotropic cytokine influencing antigen- • Contributes to the terminal differentiation
monocytes, specific immune responses and of B-lymphocytes to plasma cells and
(IL-6) fibroblasts, and inflammatory reactions. stimulates secretion of immunoglobulin
endothelial cells. • B-cell differentiation factor in vivo and in (Ig)A and IgG (Fujihashi, Kono et al.
Macrophages, T-cells vitro and an activation factor for T-cells. 1993).
and B-lymphocytes, • Smoking causes a depression of IgG and
granulocytes, smooth • In the presence of IL-2, IL-6 induces the
differentiation of mature and immature T- possibly IgA production in serum (Quinn,
muscle cells, Zhang et al. 1996).
eosinophils, cells into cytotoxic T-cells.
chondrocytes, • IL-6 also induces the proliferation of • Induces bone resorption, both by itself and
osteoblasts, mast cells, thymocytes and probably plays a role in the in conjunction with other bone-resorbing
glial cells, and development of thymic T-cells. agents (Mundy 1991).
keratinocytes. • 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.

21
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 • Produced by • Chemotactic for all known types of • Powerful chemotactic functions for
monocytes, T- migratory immune cells. polymorphonuclear leukocytes but also for
(IL-8) lymphocytes, lymphocytes and macrophages (Kamma,
• Differs from all other cytokines in its
macrophages, ability to specifically activate neutrophil Giannopoulou et al. 2004).
fibroblasts, endothelial granulocytes. • Is a potent mediator of granulocyte
cells, keratinocytes, accumulation at the sites of inflammation
melanocytes, and • Inhibits histamine release from human
basophils induced by histamine releasing (Bickel 1993).
chondrocytes.
factors, CTAP-3 (connective tissue • Increased levels of IL-8 are found in the
• Production stimulated activating protein-3), and IL-3. GCF of inflamed sites (Tsai, Ho et al.
by IL-1, TNF-α. Antagonizes IgE production by human B- 1995).
cells.

22
Table 1. Continued

• Production inhibited • Inhibits the adhesion of leukocytes to • Powerful chemotactic functions for
by 5' lipoxygenase, activated endothelial cells and therefore polymorphonuclear leukocytes but also for
and vitamin D3. possesses anti-inflammatory activities. lymphocytes and macrophages (Kamma,
• Supports angiogenesis and may play a role Giannopoulou et al. 2004).
in disorders such as rheumatoid arthritis, • Is a potent mediator of granulocyte
tumor growth, and wound healing that accumulation at the sites of inflammation
critically depend on angiogenesis. (Bickel 1993).
• Increased levels of IL-8 are found in the
GCF of inflamed sites (Tsai, Ho et al.
1995).
Interleukin -10 • Produced by T-cells • Potent and specific T-cell chemoattractant.
(Th2 cells but not • Inhibits the synthesis of a number of
(IL-10) Th1 T-helper cells), cytokines such as IFN-γ, IL-2 and TNF-β in
B-cells, and Th1 T-helper subpopulations of T-cells but
keratinocytes. not of Th2 T-helper cells.
• Production is • This activity is antagonized by IL-4.
inhibited 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.

23
Table 1. Continued

Interleukin -12 • Produced by • Is a heterodimer comprised of p35 and p40 • Produced by proinflammatory infiltrates in
monocytes, subunits, which form the bioactive IL-12 periodontitis tissues.
(IL-12) macrophages, (p70). • High levels will contribute to the immune
neutrophils, B-cells • Principal mediator of the early innate reaction to Th1 type (Lamont and Adorini
and to a lesser extent immune response to intracellular microbes 1996).
by T-cells. and is a key inducer of cell-mediated • IL-12 is an inducer of IFN-γ production.
• Production immunity. IFN-γ itself can also activate IL-12
stimulated by IL-12, • Stimulates IFN-γ production by T cells and production (Lamont and Adorini 1996).
bacteria, bacterial natural killer cells and so promote Th1
products, and • LPS of periodontopathogens are also
responses. activators of IL-12 (Lamont and Adorini
parasites.
• Promotes Th1 development by stimulating 1996).
the production of IL-12 by macrophages • Significantly higher proportions found in
and the expression of functional IL-12 diseased sites (Lamont and Adorini 1996).
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.
Tumor Necrosis • Produced by • Inhibits anticoagulatory mechanisms and • Stimulates fibroblasts, including gingival
macrophages, promotes thrombotic processes and fibroblasts, to produce collagenase (Meikle,
Factor-Alpha
monocytes, therefore plays an important role in Atkinson et al. 1989) which is implicated in
(TNF-α) neutrophils, T-cells, pathological processes such as venous the tissue destruction of periodontal disease,
NK-cells, astrocytes, thromboses, arteriosclerosis, vasculitis, and and to stimulate bone resorption (Bertolini,
microglial cells, disseminated intravasal coagulation. Nedwin et al. 1986).
smooth muscle cells, • Potent chemoattractant for neutrophils and • Activates monocytes and stimulates the
and fibroblasts. also increases their adherence to the production of IL-1β and platelet activating
endothelium. factor (Erdemir, Duran et al. 2004).

24
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.
Interferon- • Produced by T-cells, B-
cells, natural killer cells
Gamma
activated by antigens, or
(IFN-γ) lymphocytes expressing
the surface antigens CD4
and CD8.
• Induces the synthesis of a number of • Monocyte stimulation by
chemoattractant cytokines, including IP-10, lipopolysaccharide enhances the production
JE, KC, in a cell-type and tissue-specific of TNF-α, which has also been shown to
manner. induce collagenase release and bone
• Inhibits the growth of endothelial cells in resorption in vivo (Meikle, Atkinson et al.
1989).
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.
• Antiviral and antiparasitic activities and • Studies have suggested an inhibitory effect
also inhibits the proliferation of a number of IFN-γ on RANKL-associated
of normal and transformed cells. osteoclastogenesis and bone remodeling in
• Synergizes with TNF-α and TNF-β in vitro and in vivo (Takayanagi, Ogasawara et
inhibiting the proliferation of various cell al. 2000).
types.
25
Table 1. Continued
• Production stimulated by • Main biological activity of IFN-γ appears
IL-2, bFGF, and EGF. to be immunomodulatory in contrast to the
• Production inhibited by other interferons which are mainly
1-alpha,25-Dihydroxy antiviral.
vitamin D3, • In T-helper cells IL-2 induces the synthesis
dexamethasone and CsA of IFN-γ and other cytokines. IFN-γ acts
(Cyclosporin A). 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).
26
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 • Produced by T-cells, • Important in the growth and development • Up-regulated in neutrophil-mediated
macrophages, of progenitors of granulocytes and pathology and is associated with
Macrophage
endothelial cells and macrophages. It stimulates myeloblasts and periodontal inflammation (Takematsu and
Colony fibroblasts. monoblasts and triggers irreversible Tagami 1990; Baqui, Meiller et al. 1999).
• Production stimulated differentiation of these cells. • Variety of effects on neutrophils potentially
Stimulating
by TNF-α, TNF-β, IL- • Strong chemoattractant for neutrophils. important in the pathogenesis of
Factor 1, IL-2 and IFN. periodontitis, including dose-dependent
• Enhances microbicidal activity, oxidative
metabolism, and phagocytotic activity of chemotaxis or inhibition of movement,
(GM-CSF)
neutrophils and macrophages. inhibition of apoptosis and priming for
increased phagocytic and respiratory burst
• Stimulates the proliferation precursors of activity (Fossati, Mazzucchelli et al. 1998).
neutrophils, eosinophils, and monocytes.
• Enhances phagocytotic activities of
neutrophil granulocytes and the
cytotoxicity of eosinophils.

27
Table 1. Continued
RANTES • Produced by T-cells.
• Production stimulated by
TNF-α and IL-1α.
Eotaxin
Interferon- • Produced by monocytes,
keratinocytes, and
Inducible
fibroblasts, neutrophils.
Protein-10 • Production stimulated by
(IP-10) IFN-γ, TNF-α, and LPS.
• Production inhibited by
IL-10 and IL-4.
• Chemotactic for T-cells, human eosinophils • Plays an important role in the host response
and basophils and plays an active role in by recruiting inflammatory cells into the
recruiting leukocytes into inflammatory foci of active inflammation and by inducing
sites. the release of other cell mediators
• Increases the adherence of monocytes to (Gamonal, Acevedo et al. 2000).
endothelial cells and selectively supports • Important mediators of the host response in
the migration of monocytes and T- chronic adult periodontitis (Emingil, Atilla
lymphocytes expressing the cell surface et al. 2004).
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.
• 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
• 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.
28
Table 1. Continued

Monocyte • Produced by • Chemotactic for monocytes but not

monocytes, vascular neutrophils.
Chemotactic
endothelial cells, • Elevated levels of MCP-1 are observed in
Proteins-1 smooth muscle cells, macrophage-rich atherosclerotic plaques.
glomerular mesangial
(MCP-1) cell, osteoblastic cells, • Regulates the expression of cell surface
and human pulmonary antigens (CD11c, CD11b) and the
type 2 like epithelial expression of cytokines (IL-1, IL-6). MCP-
cells. 1 is a potent activator of human basophils,
inducing degranulation and the release of
• Production stimulated histamines.
by peripheral blood
mononuclear • Induces the proliferation and activation of
leukocytes, killer cells known as CHAK (CC-
phytohemagglutinin Chemokine-activated killer).
(PHA), bacterial
lipopolysaccharides,
and IL-1.
Macrophage • Produced by • Caused local inflammatory responses in
macrophages vivo, and induces superoxide production by
inflammatory
neutrophils in vitro.
protein-1

(MIP-1)

Source: # Information adapted and revised from: http://www.copewithcytokines.de/

29
 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

1 of 2 calibrated examiners: probing depths (PD), recession (REC), clinical attachment

levels (CAL), bleeding on probing (BOP), and plaque. All measurements were taken

prior to collection of gingival crevicular fluid.

In order to minimize potential technique errors and inter-examiner variability,

both examiners were standardized by probing sites from four patients, which was

repeated twice. Probing was completed using standardized color coded probes.

Recession measurements were made by measuring the level of the free gingival margin

from the CEJ. Clinical attachment levels were calculated by adding the probing depth

value to the recession value. Bleeding on probing was assigned to a site based on the

presence or absence of bleeding following PD measurements.

 36

Gingival Crevicular Fluid Collection

The sampling of GCF is inherently technique sensitive. In order to minimize

potential technique errors and inter-examiner variability, two calibrated examiners

completed all sampling in this study. At the beginning of the study, both examiners were

standardized by sampling sites from four patients, which was repeated twice. In addition,

the sampling time was standardized, and the insertion of the Periopaper® into the

Periotron® was consistently done in a single direction for all samples. The Periotron

8000® was calibrated prior to insertion of each sample by cleaning the device with an

alcohol gauze and placing a dry unused Periopaper® strip into the machine and the

output reading was zeroed. Sampling techniques published in the literature were

evaluated and the most widely utilized protocol was used in this study.

GCF sampling was performed following completion of the periodontal

assessment. The selected sites were isolated by cotton rolls, rinsed gently with water and

dried with a gentle air spray directed perpendicular to the gingival margin (Griffiths

2003). A saliva ejector was used to avoid salivary contamination of the samples. Gentle

removal of supragingival plaque was completed utilizing dry gauze, and a sterile filter

paper strip (Periopaper®, Amityville, NY, USA) was gently inserted into the entrance of

the selected site until the first sign of resistance was felt. The strip was held in place for

30 seconds (Figure 3). Attempts were made to avoid the insertion of the strips to the full

depth of the pocket, to minimize the risk of contaminating the GCF with blood. Strips

contaminated with blood were discarded and an alternative site was sampled. The GCF

volume was determined immediately using a Periotron 8000 (Oraflow Inc., Plainview,

NY, USA), which was calibrated using known volumes of 0.01 M sodium phosphate

buffer, pH 7.2 containing 140 mM NaCl (0.01 M PBS, pH 7.2) and protease inhibitor.

Strips from each subject were placed in a labeled tube containing 300 µl 0.01 M PBS, pH

7.2 and protease inhibitor. The samples were transported on ice to the laboratory and

then stored at -80◦ C until further analysis.

 37

Preparation of GCF Fluid for Analysis

GCF samples were kept separate for each sample site. GCF samples were eluted

(extracted) from paper strips by the following technique: each Periopaper® was taken

from the freezer, and the orange (non-sampling end) was removed. The strip was then

placed back into the vial with 300µl of PBS and protease inhibitor and vortexed for ten

seconds and eluted at 4◦C on a rocker for twenty minutes. The strips were removed and

the eluates were centrifuged for 5 minutes at 5800g to remove plaque and cellular

elements.

The concentrations of cytokines and chemokines (pg/mL) were determined using

a commercial multiplexed fluorescent bead-based immunoassay (Kit 48-011, Millipore,

Billerica, MA), the Luminex 100 IS Instrument (Luminex, Austin, TX). This multiplex

kit detects 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 (IL1-α, 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).

To determine the concentrations of cytokines, the manufactures instructions were

followed. 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 hours. Unbound material was removed by

filtration. 25 µl of anti-human multi-cytokine biotin reporter was added, and reactions

were incubated at room temperature for 1.5 hours in the dark. 25 µl of streptavidin–

phycoerythrin was then added, and the plates were incubated at room temperature for an

additional 30 minutes. 25 µl of stop solution was added, and the plates were read in the

plate reader (model 100 IS, Luminex, Austin, TX). Total amounts of cytokines in each

sample were extrapolated from those of standards by use of Beadview software

(Millipore, Billerica, MA).

 38

Statistical Analysis

In some subjects, the concentrations of cytokines were below the detectable

capacity of the assay. Rather than using zero, 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 above the detectable capacity of the assay, they were

simply diluted and rerun again.

Analysis of normality was conducted and nonparametric approaches were used

based on the distribution of the data. Total cytokine volumes (total/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 to both healthy and diseased sites in smoking and non-

smoking diseased subjects. Additionally, smokers and non-smokers were compared in

relation to healthy and diseased sites. Pooled comparisons consisted of comparing the

healthy controls to both healthy and diseased sites in diseased subjects. 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. Pooled matched-

paired comparisons consisted of comparing healthy and diseased sites in diseased

subjects. In each case the level of significance was set at p≤0.05.

 39

Table 3. Subject Groups

Subject Group Abbreviation

Healthy Control HC

Smoking subjects: healthy sites SH

Smoking subjects: diseased sites SD

Non-smoking subjects: healthy sites NH

Non-smoking subjects: diseased sites ND

Diseased sites in periodontitis subjects DP

Healthy sites in periodontitis subjects HP

 40

Table 4. Patient Inclusion Criteria

• Age range 40-75 years

• Good general health
• Diagnosis of Generalized Advanced Chronic Periodontitis (>1/3 the
dentition, CAL ≥5mm, BOP)
• No periodontal therapy in last 4 months
• Smoking Status
o Smokers were enrolled if they regularly smoke ≥20 cigarettes
per day
o Non-smokers were classified as not having smoked 100 or
more cigarettes in their lifetime.
 41

Table 5. Patient Exclusion Criteria

• Pregnant
• Gingival overgrowth
• Diabetes
• Systemic antibiotics in last 6 months
• Premedication required
• Regular use of anti-inflammatory medications in last 6 months
• Immunosuppression
 42

CHAPTER V

RESULTS

Subject Demographics

The study included 52 subjects, 20 smokers, 20 non-smokers and 12 healthy

controls (HC) (Table 6). The study population included 30 females and 22 males. The

average age was 55 ± 9.6 years. All subjects were Caucasian with the exception of one

Asian and one Latino subject.

Site Characteristics

Clinical Parameters

Diseased sites from both smokers and non-smokers demonstrated statistically

significant deeper (p<0.05) probing depths, recession, and more CAL (Figure 2)

compared to sites evaluated in HC (Table 7).

Total Gingival Crevicular Fluid Volume

Total GCF volumes collected for 30 seconds ranged from 0.17 to 3.28 µl.

Volumes of GCF were significantly higher for both HP and DP (pooled) sites in

periodontitis subjects (p=0.0202 and <0.0001, respectively) compared to healthy controls.

When the pooled sites were compared, volumes for DP sites were significantly higher (p=

0.0001) compared to the HP sites. GCF volumes for SD sites were significantly lower

(p=0.0300) compared to ND sites. No significant differences were found between SH

and NH sites (p=0.2790). GCF volumes are presented in Table 8.

Cytokines Detected in GCF

17 out of the panel of 22 cytokines were found at detectable levels in the GCF.

These included: IL-2 and IFN-γ (Th1 cytokines); IL-3 and IL-4 (Th2 cytokines); IL1-α,

IL1-β, IL-6, GM-CSF, and IL-12(p40) (pro-inflammatory cytokines); CXCL8/IL8,

 43

CXCL10/IP-10, CCL2/MCP-1, CCL3/MIP-1α, CCL5/RANTES, and CCL11/eotaxin

(chemokines); and IL-7 and IL-15 (regulators of T and natural killer cell activation and

proliferation). The following cytokines were below the detectable limits of the multiplex

assay utilized in this study: (IL-5, IL-10, IL-12(p70), IL-13, and TNF-α). Therefore, they

were not included in the analysis.

Th1 and Th2 Cytokines: IL-2, IL-3, IL-4, IFN-γγ

Th1 and Th2 cytokines were present but generally were found in low

concentrations (ranging from 0.0 to 349.2 pg/30 sec) for all subjects in all groups. The

amounts of IL-2 (12.2 to 77.4 pg/30 sec), IFN-γ (0.0 to 152.4 pg/30 sec), and IL-4 (0.6 to

123.0 pg/30 sec) were lower than that for IL-3 (22.2 to 349.2 pg/30 sec). The GCF from

the healthy control group exhibited the least of each of these 4 cytokines (Figure 3a-d).

Th1 and Th2 Cytokines: Intra-Group Comparisons

Non-smokers: healthy sites vs. diseased sites

Comparison of Th1 and Th2 cytokine amounts in NH and ND sites showed

significantly higher amounts of IFN-γ (p<0.01) in ND sites. No significant differences in

amounts of IL-2, IL-3 or IL-4 were found between NH and ND sites (Table 9).

Smokers: healthy sites vs. diseased sites

Comparison of Th1 and Th2 cytokine amounts in SH and SD sites showed

significantly greater amounts of IFN-γ in SD sites (p<0.05). No significant differences in

amounts of IL-2, IL-3 or IL-4 were found between SH and SD sites (Table 9).

Th1 and Th2 Cytokines: Inter-Group Comparisons

Healthy controls vs. healthy sites in non-smokers

Comparison of Th1 and Th2 cytokine amounts in HC and NH sites showed no

significant differences in amounts of IL-2, IL-3, IL-4, and IFN-γ (Table 10).
 44

Healthy controls vs. diseased sites in non-smokers

Comparison of Th1 and Th2 cytokine amounts in HC and ND sites showed

significantly greater amounts of IL-2 (p<0.005), IL-3 (p<0.0005), IL-4 (p<0.001), and

IFN-γ (p<0.05) in ND sites (Table 10).

Healthy controls vs. healthy sites in smokers

Comparison of Th1 and Th2 cytokine amounts in HC and SH sites showed no

significant difference in amounts of IL-2, IL-3, IL-4, and IFN-γ (Table 11).

Healthy controls vs. diseased sites in smokers

Comparison of Th1 and Th2 cytokine amounts in HC and SD sites showed

significantly higher amounts of IL-3 (p<0.05) in SD sites. No significant differences in

the amounts of IL-2, IL-4 and IFN-γ were found between HC and SD sites (Table 11).

Healthy sites in non-smokers vs. healthy sites in smokers

Comparison of Th1 and Th2 cytokine amounts in NH and SH sites showed no

significant differences in levels of IL-2, IL-3, IL-4, and IFN-γ (Table 12).

Diseased sites in non-smokers vs. diseased sites in smokers

Comparison of ND and SD sites showed no significance differences in amounts of

IL-2, IL-3 and IL-4 and IFN- γ (Table 12).

Th1 and Th2 Cytokines: Pooled Comparisons

Healthy sites in all periodontitis subjects vs. diseased sites in all periodontitis subjects

Comparison of Th1 and Th2 cytokine amounts in HP and DP sites showed no

significant differences in levels of IL-2, IL-3 and IL-4. DP sites however demonstrated

significantly higher amounts of IFN-γ (p=0.0008) than HP sites (Table 13).

 45

Healthy sites & diseased sites in periodontitis subjects vs. healthy control

Total cytokine amounts were compared between healthy controls and HP and

healthy controls and DP sites. No significant differences were found between HP sites

and healthy controls. DP sites showed significantly greater amounts of IL-2 (p=0.0212),

IFN- γ (p=0.0024), IL-3 (p= 0.0007) and IL-4 (p=0.0461) compared to healthy controls

(Table 14).

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

Pro-inflammatory cytokines were present and ranged from 0.0 to 32580.0 pg/30

sec. The levels of IL-1α were the highest (ranging from 41.5 to 32580.0 pg/30 sec),

followed by IL-1β (0.0 to 1662.0 pg/30 sec), IL-6 (10.2 to 1632.0 pg/30 sec), IL-12(p40)

(7.2 to 436.2 pg/30 sec), and GM-CSF (0.6 to 130.8 pg/30 sec) (Figure 4a-e).

Pro-inflammatory Cytokines: Intra-Group Comparisons

Non-smokers: healthy sites vs. diseased sites

Comparison of pro-inflammatory cytokine amounts in NH and ND sites showed

significantly higher amounts of IL-1α (p<0.0001), IL-1β (p<0.005), IL-12(p40) (p<0.01)

and GM-CSF (p<0.05) in ND sites. No significant differences in amounts of IL-6 were

found between NH and ND sites (Table 15).

Smokers: healthy sites vs. diseased sites

Comparison of pro-inflammatory cytokine amounts in SH and SD sites showed

significantly higher levels of IL-1β in SD sites (p<0.0001). No significant differences in

amounts of IL-1α, IL-6, IL-12(p40) and GM-CSF were found between SH and SD sites

(Table 15).
 46

Pro-inflammatory Cytokines: Inter-Group Comparisons

Healthy controls vs. healthy sites in non-smokers

Comparison of pro-inflammatory cytokine amounts in HC and NH sites showed

significantly higher quantities of IL-1β (p<0.01), IL-6 (p<0.001) and IL-12(p40) (p<0.05)

in NH sites. No significant differences were found for IL-1α and GM-CSF between HC

and NH sites (Table 16).

Healthy controls vs. diseased sites in non-smokers

Comparison of pro-inflammatory cytokine amounts in HC and ND sites showed

significantly more IL-1α (p<0.0001), IL-1β (p<0.0001), IL-6 (p<0.0001) and IL-12(p40)

(p<0.0001) in ND sites. No significant differences in amounts of GM-CSF were found

between HC and ND sites (Table 16).

Healthy controls vs. healthy sites in smokers

Comparison of pro-inflammatory cytokine amounts in HC and SH sites showed

significantly higher levels of IL-1β (p<0.01) in SH sites. No significant differences in

amounts of IL-1α, IL-6, IL-12(p40) and GM-CSF were found between HC and SH sites

(Table 17).

Healthy controls vs. diseased sites in smokers

Comparison of pro-inflammatory cytokine amounts in HC and SD sites showed

significantly higher levels of IL-1α (p<0.005) and IL-1β (p<0.0001) in SD sites. No

significant differences in amounts of IL-6, IL-12(p40) and GM-CSF were found between

HC and SD sites (Table 17).

 47

Healthy sites in non-smokers vs. healthy sites in smokers

Comparison of pro-inflammatory cytokine amounts in NH and SH sites showed

no significant difference in levels of IL-1α, IL-1β, GM-CSF (Table 16). SH sites showed

significantly less IL-6 (p<0.0001) and IL-12 (p40) (p=0.0145) than NH sites (Table 18).

Diseased sites in non-smokers vs. diseased sites in smokers

Comparison of ND and SD sites showed no significant differences in levels of IL-

1β and GM-CSF. SD sites showed significantly less IL-1α (p=0.0479), IL-6 (p<0.0001)

and IL-12 (p40) (p<0.0001) than ND sites (Table 18).

Pro-Inflammatory Cytokines: Pooled Comparisons

Healthy sites in all periodontitis subjects vs. diseased sites in all periodontitis subjects

Comparison of pro-inflammatory cytokines in HP and DP sites showed no

significant differences in levels of GM-CSF (Table 15). DP sites showed significantly

elevated IL-1α (p=0.0001), IL-1β (p=0.0003), IL-6 (p=0.0280), IL-12 (p40) (p=0.0017)

than HP sites (Table 19).

Healthy sites & diseased sites in periodontitis subjects vs. healthy control

Total cytokine amounts were compared between healthy controls and HP and

healthy controls and DP sites. No significant differences were observed in total amounts

of GM-CSF. In HP sites total amounts of IL-1β were significantly higher (p=0.0012)

compared to healthy controls (Table 13). DP sites showed significantly greater amounts

of IL-1α (p<0.0001), IL-1β (p<0.0001), IL-6 (p=0.0113) and IL-12 (p40) (p<0.0001) than

healthy controls (Table 20).

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

Several chemokines were present in the GCF ranging from 0.6 to 24306.0 pg/30

sec. The amounts of IL-8 (21.2 to 24306.0 pg/30 sec), IP-10 (36.0 to 3672.0 pg/30 sec),
 48

RANTES (0.6 to 1098.0 pg/30 sec) and MIP-1 (18.6 to 573.6 pg/30 sec) were more than

that found for Eotaxin (12.0 to 392.4 pg/30 sec), and MCP-1 (1.2 to 111.6 pg/30 sec)

(Figure 5a-f).

Chemokines: Intra-Group Comparisons

Non-smokers: healthy sites vs. diseased sites

Comparison of chemokines in NH and ND sites showed significantly elevated

MCP-1 (p<0.05) and Eotaxin (p<0.05) in ND sites. No significant differences in IL-8,

IP-10, MIP-1 and RANTES were found between NH and ND sites (Table 21).

Smokers: healthy sites vs. diseased sites

Comparison of chemokine amounts in SH and SD sites showed no significant

differences in amounts of IL-8, IP-10, MCP-1, MIP-1, RANTES and Eotaxin (Table 21).

Chemokines: Inter-Group Comparisons

Healthy controls vs. healthy sites in non-smokers

Comparison of chemokine amounts in HC and NH sites showed significantly

higher volumes of IL-8 (p<0.0001), MIP-1 (p<0.01) and RANTES (p<0.05) in NH sites.

In contrast, IP-10 (p<0.05) showed significantly lower levels in NH compared to HC

sites. No significant differences in MCP-1 and Eotaxin quantities were found between

HC and NH sites (Table 22).

Healthy controls vs. diseased sites in non-smokers

Comparison of chemokine amounts in HC and ND sites showed significantly

higher volumes of IL-8 (p<0.0001), MCP-1 (p<0.0001), MIP-1 (p<0.0001), RANTES

(p<0.0001) and Eotaxin (p<0.005) in ND sites. In contrast, IP-10 (p<0.05) was

significantly less in ND sites compared to HC sites (Table 22).

 49

Healthy controls vs. healthy sites in smokers

Comparison of chemokine amounts in HC and SH sites showed significantly

lower volumes of IP-10 (p<0.0001) and MCP-1 (p<0.01) in SH sites. No significant

differences in amounts of IL-8, MIP-1, RANTES and Eotaxin were found between HC

and SH sites (Table 23).

Healthy controls vs. diseased sites in smokers

Comparison of chemokine amounts in HC and SD sites showed significantly

lower volumes of IP-10 (p<0.0001) in SD sites. No significant differences in amounts of

IL-8, MCP-1, MIP-1, RANTES and Eotaxin were found between HC and SD sites (Table

23).

Healthy sites in non-smokers vs. healthy sites in smokers

Comparison of chemokine amounts in NH and SH sites showed no significant

difference in levels of Eotaxin. SH sites showed a significantly less IL-8 (p=0.0013), IP-

10 (p=0.0342), MCP-1 (p<0.0001), MIP-1a (p=0.0160) and RANTES (p=0.0042) (Table

24).

Diseased sites in non-smokers vs. diseased sites in smokers

Comparison of ND and SD sites showed significantly less amounts of IL-8

(p<0.0001), IP-10 (p=0.0148), MCP-1 (p<0.0001), MIP-1 (p=0.0023) and RANTES

(p=0.0016) in SD sites (Table 24).

Chemokines: Pooled Comparisons

Healthy sites in all periodontitis subjects vs. diseased sites in all periodontitis subjects

Comparison of chemokine amounts in HP and DP sites showed no significant

difference in levels of IL-8, IP-10 and Eotaxin. DP sites showed significantly greater

amounts of MCP-1 (p=0.0137), MIP-1 (p=0.0168) and RANTES (p=0.0391) (Table 25).
 50

Healthy sites & diseased sites in periodontitis subjects vs. healthy control

Total cytokine amounts were compared between healthy controls and HP and

healthy controls and DP sites. No significant differences were observed in total amounts

of Eotaxin. In HP sites total amounts of IL-8 were significantly higher (p=0.0046)

compared to healthy controls. IP-10 was significantly decreased in both HP sites

(p=0.0001) and DP sites (p=0.0002) compared to healthy controls. DP sites showed

significantly more IL-8 (p=0.0001), MIP-1 (p=0.0009) and RANTES (p=0.0055) than

healthy controls (Table 26).

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

The levels of IL-7 and IL-15 in GCF ranged from 21.2 to 196.8 pg/30 sec and IL-

15 ranged from 10.9 to 48.8 pg/30 sec, respectively (Figure 6a, b).

Regulators of T-cells and NK cells: Intra-Group Comparisons

Non-smokers: healthy sites vs. diseased sites

Comparison of cytokine amounts in NH and ND sites showed no significant

differences in amounts of IL-7 and IL-15 (Table 27).

Smokers: healthy sites vs. diseased sites

Comparison of cytokine levels in SH and SD sites showed no significant

differences in amounts of IL-7 and IL-15 (Table 27).

Regulators of T-cells and NK cells: Inter-Group Comparisons

Healthy controls vs. healthy sites in non-smokers

Comparison of cytokine amounts in HC and NH sites showed significantly higher

volumes of IL-15 (p<0.05) in NH sites. No significant differences in levels of IL-7 were

found between HC and NH sites (Table 28).

 51

Healthy controls vs. diseased sites in non-smokers

Comparison of cytokine amounts in HC and ND sites showed significantly higher

volumes of IL-7 (p<0.05) and IL-15 (p<0.0005) in ND sites (Table 28).

Healthy controls vs. healthy sites in smokers

Comparison of cytokine levels in HC and SH sites showed no significant

differences in amounts of IL-7 and IL-15 (Table 29).

Healthy controls vs. diseased sites in smokers

Comparison of cytokine amounts in HC and SD sites showed no significant

differences in amounts of IL-7 and IL-15 (Table 29).

Healthy sites in non-smokers vs. healthy sites in smokers

Comparison of cytokine levels in NH and SH sites showed no significant

difference in levels of IL-7 and IL-15 (Table 30).

Diseased sites in non-smokers vs. diseased sites in smokers

Comparison of cytokine amounts in ND and SD sites showed a significant

decrease in IL-7 (p=0.0056) and IL-15 (p=0.0331) in SD sites (Table 30).

Regulators of T-cells and NK cells: Pooled Comparisons

Healthy sites in all periodontitis subjects vs. diseased sites

in all periodontitis subjects

Comparison of cytokine amounts in HP and DP sites showed no significant

difference in levels of IL-7. DP sites showed significantly more IL-15 (p=0.0373) than

HP sites (Table 31).

 52

Healthy sites & diseased sites in periodontitis subjects vs. healthy control

Total cytokine amounts were compared between healthy controls and HP and

healthy controls and DP sites. No significant differences were observed in levels of IL-7.

DP sites showed significantly greater amounts of IL-15 (p=0.0013) than healthy controls

(Table 32).

In summary, periodontitis subjects had significantly elevated GCF volumes and

cytokine and chemokine profiles when compared to healthy controls. When comparing

diseased sites in periodontitis subjects to healthy controls, diseased sites showed a

significant increase in various chemokines and cytokines such as IFN-γ, IL-1α, IL-1β, IL-

2, IL-3, IL-6, IL-8, IL-12 (p40), IL-15, MIP-1 and RANTES. Healthy sites in

periodontitis subjects also showed an increase in IL-1β and IL-8 when compared to

healthy controls (Table 33).

Smoking appeared to have an inhibitory effect on the production of GCF and

several pro-inflammatory cytokines, chemokines and regulators of T-cells and NK-cells,

however, little influence was observed on Th1/Th2 cytokines. Smokers showed a

significant decrease in IL-1α, IL-6, IL-7, IL-8, IL-12(p40), IL-15, IP-10, MCP-1, MIP-1

and RANTES. Of interest, was the novel finding of decreased concentrations of IL-7, IL-

12(p40), IL-15, IP-10, MCP-1 and MIP-1 within smokers, which have not been reported

in relation to smoking and periodontitis in the literature to date (Table 33).

 53

Table 6. Demographic characteristics of the study population.

Demographic Healthy Control Smokers Non-smokers

Characteristics

Mean age (years) 51.2 ± 5.2 51.2 ± 7.4 61.2 ± 9.7

Gender

Female 10 8 12

Male 2 12 8
Table 7. Site characteristics of the study population.

Clinical characteristics HC HP DP NH SH ND SD

Probing depth (mm) 2.3 ± 0.6 2.7 ± 0.5 5.7 ± 0.8 2.6 ± 0.6 2.8 ± 0.4 5.8 ± 0.9 5.6 ± 0.5

Recession (mm) 0.1 ± 0.4 0.3 ± 0.6 0.8 ± 1.0 0.3 ± 0.7 0.3 ± 0.4 0.7 ± 0.9 1.0 ± 1.1

CAL (mm) 2.5 ± 0.7 2.9 ± 0.7 6.5 ± 1.3 2.9 ± 0.9 3.0 ± 0.3 6.5 ± 1.3 6.6 ± 1.3

BOP (% positive) 0.0 ± 0.0 0.0 ± 0.0 100 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 100 ± 0.0 100 ± 0.0

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.

54
 55

Table 8. Mean volumes of GCF in all groups.

Group Mean GCF Volume ± SD (µl) p value

HC 1.1 ± 0.7 NS

HP 1.5 ± 1.0 <0.05 (HP vs. HC)

DP 2.1 ± 0.8 <0.001 (DP vs. HC)

NH 1.6 ± 1.0 NS

SH 1.3 ± 0.9 NS

ND 2.3 ± 0.8 NS

SD 1.9 ± 0.9 <0.05 (SD vs. ND)

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.
Table 9. Th1 and Th2 cytokines: Intra-group comparisons: Healthy and diseased sites in smokers and non-smokers (pg/30s)

NH ND SH SD

Median Mean SD Median Mean SD p value Median Mean SD Median Mean SD p value

Th1
cytokines

IL-2 24.0 26.6 8.9 26.5 27.2 1.7 NS 26.7 28.3 10.8 23.1 27.3 10.2 NS

IFN- γ 14.0 21.4 18.0 23.2 31.8 31.8 <0.01 13.4 19.2 16.0 15.2 24.7 23.3 <0.05

Th2
cytokines

IL-3 75.5 78.6 35.0 88.5 91.5 48.9 NS 68.1 74.5 46.9 83.0 85.9 37.9 NS

IL-4 14.8 32.9 38.2 14.2 36.2 40.2 NS 16.3 50.0 47.2 16.4 49.9 47.4 NS

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.

56
Table 10. Th1 and Th2 cytokines: Inter-group comparisons: Healthy controls vs. healthy and diseased sites in non-smokers (pg/30s)

HC NH HC ND

Median Mean SD Median Mean SD p value Median Mean SD Median Mean SD p value

Th1
cytokines

IL-2 21.3 24.2 9.3 23.1 26.6 10.3 NS 23.1 24.2 9.3 27.7 28.6 8.9 <0.005

IFN- γ 12.6 15.5 11.1 15.2 21.4 18.9 NS 12.6 15.5 11.1 25.5 33.2 29.8 <0.0005

Th2
cytokines

IL-3 55.3 56.2 30.7 72.0 78.6 51.1 NS 55.3 56.2 30.7 95.4 99.3 66.2 <0.001

IL-4 12.4 28.0 35.7 14.3 32.9 38.7 NS 12.4 28.0 35.7 15.9 41.9 43.0 <0.05

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.

57
Table 11. Th1 and Th2 cytokines: Inter-group comparisons: Healthy controls vs. healthy and diseased sites in smokers (pg/30s)

HC SH HC SD

Median Mean SD Median Mean SD p value Median Mean SD Median Mean SD p value

Th1
cytokines

IL-2 21.3 24.2 9.3 25.2 27.9 12.1 NS 21.3 24.2 9.3 22.9 27.0 11.0 NS

IFN- γ 12.6 15.5 11.1 13.6 18.6 16.4 NS 12.6 15.5 11.1 15.0 24.2 23.2 NS

Th2
cytokines

IL-3 55.3 56.2 30.7 70.2 72.7 58.3 NS 55.3 56.2 30.7 74.4 83.8 50.4 <0.05

IL-4 12.4 28.0 35.7 15.2 46.9 47.2 NS 12.4 28.0 35.7 13.6 48.0 46.9 NS

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.

58
Table 12. Th1 and Th2 cytokines: Inter-group comparisons: Healthy and diseased sites in smokers vs. non-smokers (pg/30s)

NH SH ND SD

Median Mean SD Median Mean SD p value Median Mean SD Median Mean SD p value

Th1
cytokines

IL-2 23.1 26.6 10.3 25.2 27.9 12.1 NS 27.7 28.6 9.0 22.9 27.0 11.0 NS

IFN- γ 15.2 21.4 18.9 13.6 18.6 16.4 NS 25.5 33.2 29.8 15.0 24.2 23.2 NS

Th2
cytokines

IL-3 72.0 78.6 51.1 70.2 72.7 58.3 NS 95.4 99.3 66.2 74.4 83.8 50.4 NS

IL-4 14.3 32.9 38.7 15.2 46.9 47.2 NS 15.9 41.9 43.1 13.6 48.0 46.1 NS

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.

59
 60

Table 13. Th1 and Th2 cytokines: Pooled comparisons: Healthy vs. diseased sites in
periodontitis subjects (pg/30s)

HP DP

Median Mean SD Median Mean SD p value

Th1
cytokines

IL-2 24.2 27.3 11.2 24.4 27.9 9.9 NS

IFN- γ 13.6 20.1 17.6 22.4 29.2 27.4 <0.001

Th2
cytokines

IL-3 70.2 75.5 54.7 83.4 92.5 60.0 NS

IL-4 14.6 40.3 43.6 15.4 44.6 44.6 NS

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.
Table 14. Th1 and Th2 cytokines: Pooled comparisons: Healthy controls vs. healthy and diseased sites in periodontitis subjects
(pg/30s)

HC HP HC DP

Median Mean SD Median Mean SD p value Median Mean SD Median Mean SD p value

Th1
cytokines

IL-2 21.3 24.2 9.3 24.2 27.3 11.2 NS 21.3 24.2 9.3 24.4 27.9 9.9 <0.05

IFN- γ 12.6 15.5 11.1 13.6 20.1 17.6 NS 12.6 15.5 11.1 22.4 29.2 27.4 <0.01

Th2
cytokines

IL-3 55.3 56.2 30.7 70.2 75.5 54.7 NS 55.3 56.2 30.7 83.4 92.5 60.0 <0.001

IL-4 12.4 30.1 35.7 14.6 40.3 43.6 NS 12.4 30.1 35.7 15.4 44.6 44.6 <0.05

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.

61
Table 15. Pro-inflammatory cytokines: Intra-group comparisons: Healthy and diseased sites in smokers and non-smokers (pg/30s)

NH ND SH SD

Median Mean SD Median Mean SD p value Median Mean SD Median Mean SD p value

Pro-
inflammatory
cytokines

IL-1α 858.6 1121.0 1230.5 1765.5 2407.8 2444.7 <0.0001 857.6 907.8 570.0 1102.8 1442.7 1092.8 NS

IL-1β 22.3 45.6 57.8 112.0 161.6 157.7 <0.005 17.5 42.6 63.0 69.9 199.9 314.4 <0.0001

IL-6 109.2 148.1 102.7 165.9 220.0 171.1 NS 49.3 69.6 47.3 60.4 81.5 55.5 NS

IL-12(p40) 109.1 104.1 39.3 147.1 151.8 77.1 <0.01 78.9 77.2 30.2 80.6 94.6 40.6 NS

GM-CSF 23.2 25.1 23.4 23.9 29.5 25.3 <0.05 23.1 28.7 20.9 22.6 27.8 20.6 NS

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.

62
Table 16. Pro-inflammatory cytokines: Inter-group comparisons: Healthy controls vs. healthy and diseased sites in non-smokers
(pg/30s)

HC NH HC ND

Median Mean SD Median Mean SD p value Median Mean SD Median Mean SD p value
Pro-
inflammatory
cytokines

IL-1α 612.0 778.4 624.3 786.0 1121.0 1438.0 NS 612.0 778.4 624.3 1647.0 2762.4 4393.8 <0.0001

IL-1β 0.0 14.1 34.7 14.3 45.6 84.0 <0.01 0.0 14.1 34.7 44.7 134.7 165.4 <0.0001

IL-6 68.7 78.4 44.3 106.2 148.7 143.7 <0.001 68.7 78.4 44.3 114.0 213.2 276.3 <0.0001

IL-12(p40) 75.0 76.5 58.7 99.0 104.1 57.3 <0.05 75.0 76.5 58.7 135.3 156.4 90.7 <0.0001

GM-CSF 23.8 25.8 19.9 23.2 25.1 25.3 NS 23.8 25.8 19.9 28.4 33.2 28.6 NS

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.

63
Table 17. Pro-inflammatory cytokines: Inter-group comparisons: Healthy controls vs. healthy and diseased sites in smokers (pg/30s)

HC SH HC SD

Median Mean SD Median Mean SD p value Median Mean SD Median Mean SD p value
Pro-
inflammatory
cytokines

IL-1α 612.0 778.4 624.3 738.0 870.7 636.3 NS 612.0 778.4 624.3 1176.0 1606.2 1463.4 <0.005

IL-1β 0.0 14.1 34.7 16.9 39.4 68.0 <0.01 0.0 14.1 34.7 38.6 119.0 161.0 <0.0001

IL-6 68.7 78.4 44.3 54.8 68.7 60.4 NS 68.7 78.4 44.3 57.4 80.0 59.5 NS

IL-12(p40) 75.0 76.5 58.7 82.2 73.4 36.0 NS 75.0 76.5 58.7 87.0 92.3 51.0 NS

GM-CSF 23.8 25.8 19.9 24.3 27.5 22.1 NS 23.8 25.8 19.9 23.2 27.4 21.0 NS

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.

64
Table 18. Pro-inflammatory cytokines: Inter-group comparisons: Healthy and diseased sites in smokers vs. non-smokers (pg/30s)

NH SH ND SD

Median Mean SD Median Mean SD p value Median Mean SD Median Mean SD p value
Pro-
inflammatory
cytokines

IL-1α 786.0 1121.0 1438.0 738.0 870.7 636.2 NS 1647.0 2762.4 4393.8 1176.0 1606.2 1463.4 <0.05

IL-1β 14.3 45.6 84.1 16.9 39.4 68.0 NS 69.9 199.9 314.4 44.7 134.7 165.4 NS

IL-6 106.2 148.7 143.7 54.8 68.7 60.4 <0.001 114.0 213.2 276.3 57.4 80.0 59.5 <0.001

IL-12(p40) 99.0 104.1 57.3 82.2 73.4 36.0 <0.05 135.3 156.4 90.7 87.0 92.3 51.0 <0.001

GM-CSF 23.2 25.1 25.3 24.3 27.5 22.1 NS 28.4 33.2 28.6 23.2 27.4 21.0 NS

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.

65
 66

Table 19. Pro-inflammatory cytokines: Pooled comparisons: Healthy vs. diseased sites in
periodontitis subjects (pg/30s)

HP DP

Median Mean SD Median Mean SD p value

Pro-inflammatory
cytokines

IL-1α 756.0 989.9 1094.2 1533.0 2253.7 3462.4 <0.001

IL-1β 15.6 42.4 75.6 66.9 171.2 260.5 <0.001

IL-6 89.7 106.8 114.9 103.8 154.6 219.9 <0.05

IL-12(p40) 84.0 88.0 49.5 112.5 128.2 82.0 <0.01

GM-CSF 24.3 26.4 23.6 26.6 30.6 25.6 NS

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.
Table 20. Pro-inflammatory cytokines: Pooled comparisons: Healthy controls vs. healthy and diseased sites in periodontitis subjects
(pg/30s)

HC HP HC DP

Median Mean SD Median Mean SD p value Median Mean SD Median Mean SD p value
Pro-
inflammatory
cytokines

IL-1α 612.0 778.4 624.3 756.0 989.9 1094.2 NS 612.0 778.4 624.3 1533.0 2253.7 3462.4 <0.001

IL-1β 0.0 14.1 34.7 15.6 42.4 75.6 <0.01 0.0 14.1 34.7 66.9 171.2 260.5 <0.001

IL-6 68.7 78.7 44.3 89.7 106.8 114.9 NS 68.7 78.7 44.3 103.8 154.6 219.9 <0.05

IL-12(p40) 75.0 76.5 58.7 84.0 88.0 49.5 NS 75.0 76.5 58.7 112.5 128.2 82.0 <0.001

GM-CSF 23.8 25.8 19.9 24.3 26.4 23.6 NS 23.8 25.8 19.9 26.6 30.6 25.6 NS

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.

67
Table 21. Chemokines: Intra-group comparisons: Healthy and diseased sites in smokers and non-smokers (pg/30s)

NH ND SH SD

Median Mean SD Median Mean SD p value Median Mean SD Median Mean SD p value

Chemokines

IL-8 1408.2 1352.3 740.3 1512.0 2004.1 1851.4 NS 574.8 734.5 548.4 580.4 728.2 502.6 NS

IP-10 377.56 470.1 535.5 320.6 525.3 591.2 NS 203.9 286.5 232.4 248.2 277.0 205.6 NS

MCP-1 15.8 16.9 8.6 98.7 25.7 20.5 <0.05 10.2 9.4 4.8 10.7 11.3 4.5 NS

MIP-1 245.6 217.0 98.0 258.5 250.7 9.6 NS 148.2 161.7 76.4 167.1 180.5 88.2 NS

RANTES 21.9 33.5 44.0 30.2 76.1 89.8 NS 17.6 18.5 10.8 17.4 22.4 17.9 NS

Eotaxin 34.6 83.5 79.3 66.6 98.7 77.4 <0.05 50.1 108.3 90.3 32.8 104.6 91.7 NS

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.

68
Table 22. Chemokines: Inter-group comparisons: Healthy controls vs. healthy and diseased sites in non-smokers (pg/30s)

HC NH HC ND

Median Mean SD Median Mean SD P value Median Mean SD Median Mean SD P value

Chemokines

IL-8 452.4 607.0 460.1 1092.0 1352.3 944.4 <0.0001 452.4 607.0 460.1 1656.0 2461.1 4399.2 <0.0001

IP-10 495.9 769.6 799.6 367.5 470.1 585.5 <0.05 495.9 769.6 799.6 320.7 527.3 663.65 <0.05

MCP-1 12.9 12.8 8.4 14.6 16.9 10.7 NS 12.9 12.8 8.4 20.8 24.6 20.2 <0.0001

MIP-1 147.3 161.6 76.0 237.9 217.0 103.1 <0.01 147.3 161.6 76.0 240.9 255.3 115.3 <0.0001

RANTES 17.2 18.3 10.5 19.5 33.5 61.8 <0.05 17.2 18.3 10.5 23.6 80.4 177.8 <0.0001

Eotaxin 39.2 70.1 70.1 36.9 83.5 79.2 NS 39.2 70.1 70.1 107.7 112.2 84.2 <0.005

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.

69
Table 23. Chemokines: Inter-group comparisons: Healthy controls vs. healthy and diseased sites in smokers (pg/30s)

HC SH HC SD

Median Mean SD Median Mean SD p value Median Mean SD Median Mean SD p value

Chemokines

IL-8 452.4 607.0 460.1 564.6 745.7 677.8 NS 452.4 607.0 460.1 594.0 810.3 705.7 NS

IP-10 495.9 769.6 799.6 170.1 278.3 258.9 <0.0001 495.9 769.6 799.6 205.2 274.9 261.9 <0.0001

MCP-1 12.9 12.8 8.4 9.1 8.9 7.5 <0.01 12.9 12.8 8.4 11.1 11.1 6.8 NS

MIP-1 147.3 161.6 76.0 145.2 163.8 81.0 NS 147.3 161.6 76.0 173.7 182.8 87.9 NS

RANTES 17.2 18.3 10.5 16.7 18.2 12.7 NS 17.2 18.3 10.5 17.2 22.1 18.4 NS

Eotaxin 39.2 70.1 70.1 40.7 104.6 90.0 NS 39.2 70.1 70.1 31.6 102.7 90.0 NS

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.

70
Table 24. Chemokines: Inter-group comparisons: Healthy and diseased sites in smokers vs. non-smokers (pg/30s)

NH SH ND SD

Median Mean SD Median Mean SD p value Median Mean SD Median Mean SD p value

Chemokines

IL-8 1092.0 1352.3 944.4 564.6 745.7 677.8 <0.01 1656.0 2461.1 4399.2 594.0 810.3 705.7 <0.001

IP-10 367.5 470.1 585.5 170.1 278.3 258.9 <0.05 320.7 527.3 663.7 205.2 274.8 261.9 <0.05

MCP-1 14.6 16.9 10.7 9.1 8.9 7.5 <0.001 20.8 24.6 20.2 11.1 11.1 6.8 <0.0001

MIP-1 237.9 217.0 103.1 145.2 163.8 81.0 <0.05 240.9 255.3 115.3 173.7 182.8 87.9 <0.001

RANTES 19.5 33.5 61.8 16.7 18.2 12.7 <0.01 23.6 80.4 177.8 17.2 22.1 18.4 <0.01

Eotaxin 36.9 83.5 79.2 40.7 104.6 90.0 NS 107.7 112.2 84.2 31.6 102.6 90.0 NS

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.

71
 72

Table 25. Chemokines: Pooled comparisons: Healthy vs. diseased sites in periodontitis
subjects (pg/30s)

HP DP

Median Mean SD Median Mean SD p value

Chemokines

IL-8 822.0 1034.5 866.0 1200.0 1734.8 3412.7 NS

IP-10 268.8 369.6 452.9 276.9 416.2 538.8 NS

MCP-1 12.1 12.7 10.0 14.3 18.7 17.1 <0.01

MIP-1 178.5 189.2 95.5 231.0 223.4 109.8 <0.05

RANTES 17.3 25.5 44.0 19.4 54.7 136.2 <0.05

Eotaxin 37.8 94.5 85.2 66.6 108.0 86.5 NS

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.
Table 26. Chemokines: Pooled comparisons: Healthy controls vs. healthy and diseased sites in periodontitis subjects (pg/30s)

HC HP HC DP

Median Mean SD Median Mean SD p value Median Mean SD Median Mean SD p value

Chemokines

IL-8 452.4 607.0 460.1 822.0 1034.5 866.0 <0.01 452.4 607.0 460.1 1200.0 1734.8 3412.7 <0.001

IP-10 495.9 769.6 799.6 268.8 369.6 452.9 <0.001 495.9 769.6 799.6 276.9 416.2 538.8 <0.0005

MCP-1 12.9 12.8 8.4 12.1 12.7 10.0 NS 12.9 12.8 8.4 14.3 18.7 17.1 NS

MIP-1 147.3 161.6 76.0 178.5 189.2 95.5 NS 147.3 161.6 76.0 231.0 223.4 109.8 <0.001

RANTES 17.2 18.3 10.5 17.3 25.5 44.0 NS 17.2 18.3 10.5 19.4 54.7 136.2 <0.01

Eotaxin 39.2 70.1 70.1 37.8 94.5 85.2 NS 39.2 70.1 70.1 66.6 108.0 86.5 NS

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.

73
Table 27. Regulators of T-cells and NK cells: Intra-group comparisons: Healthy and diseased sites in smokers and non-smokers
(pg/30s)

NH ND SH SD

Median Mean SD Median Mean SD p value Median Mean SD Median Mean SD p value

Regulators
of
T-cells
and NK
cells

IL-7 54.0 61.5 29.0 57.5 66.7 30.4 NS 51.6 53.4 17.3 51.3 50.9 17.6 NS

IL-15 19.2 21.7 5.8 21.5 23.6 7.7 NS 17.9 20.0 5.7 19.3 21.1 5.8 NS

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.

74
Table 28. Regulators of T-cells and NK cells: Inter-group comparisons: Healthy controls vs. healthy and diseased sites in non-smokers
(pg/30s)

HC NH HC ND

Median Mean SD Median Mean SD p value Median Mean SD Median Mean SD p value

Regulators
of
T-cells
and NK
cells

IL-7 50.2 57.9 32.4 52.5 61.5 31.2 NS 50.2 57.9 32.4 65.7 69.7 34.4 <0.05

IL-15 18.4 18.6 4.3 20.9 21.7 6.4 <0.05 18.4 18.6 4.3 22.7 25.0 8.4 <0.0005

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.

75
Table 29. Regulators of T-cells and NK cells: Inter-group comparisons: Healthy controls vs. healthy and diseased sites in smokers
(pg/30s)

HC SH HC SD

Median Mean SD Median Mean SD p value Median Mean SD Median Mean SD p value

Regulators
of
T-cells
and NK
cells

IL-7 50.2 57.9 32.4 47.9 53.2 21.2 NS 50.2 57.9 32.4 51.5 51.7 20.0 NS

IL-15 18.4 18.6 4.3 18.2 19.9 6.8 NS 18.4 18.6 4.3 19.3 21.5 6.9 NS

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.

76
Table 30. Regulators of T-cells and NK cells: Inter-group comparisons: Healthy and diseased sites in smokers vs. non-smokers
(pg/30s)

NH SH ND SD

Median Mean SD Median Mean SD p value Median Mean SD Median Mean SD p value

Regulators
of T-cells
and NK
cells

IL-7 52.5 61.5 31.2 47.9 53.1 21.2 NS 65.7 69.7 34.4 51.5 51.7 20.0 <0.01

IL-15 20.9 21.7 6.4 18.2 19.9 6.9 NS 22.7 25.0 8.4 19.3 21.5 6.9 <0.05

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.

77
 78

Table 31. Regulators of T-cells and NK cells: Pooled comparisons: Healthy vs. diseased
sites in periodontitis subjects (pg/30s)

HP DP

Median Mean SD Median Mean SD p value

Regulators
of T-cells
and NK cells

IL-7 48.3 57.2 26.6 57.6 61.8 30.2 NS

IL-15 18.9 20.8 6.6 20.9 23.5 8.0 <0.05

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.
Table 32. Regulators of T-cells and NK cells: Pooled comparisons: Healthy controls vs. healthy and diseased sites in periodontitis
subjects (pg/30s)

HC HP HC DP

Median Mean SD Median Mean SD p value Median Mean SD Median Mean SD p value

Regulators
of T-cells
and NK
cells

IL-7 50.2 57.9 32.4 48.3 57.2 26.6 NS 50.2 57.9 32.4 57.6 61.8 30.2 NS

IL-15 18.4 18.6 4.3 18.9 20.8 6.6 NS 18.4 18.6 4.3 20.9 23.5 8.0 <0.01

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.

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.

NH/ND SH/SD HC/NH HC/ND HC/SH HC/SD NH/SH ND/SD HP/DP HC/HP HC/DP
Th1
cytokines
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
Th2
cytokines
IL-3 0.4091 0.2935 0.5214 <0.001 0.3052 <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
Pro-
inflammatory
cytokines
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
Chemokines
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
Regulators of T-
cells and NK
cells
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 <0.05 <0.0005 0.6362 0.0877 0.0784 <0.05 <0.05 0.1157 <0.01
■ P values considered significant (p ≤ 0.05)
Note: 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

80
smoking subjects.
 81

Figure 2. Means of Clinical Parameters: a) probing depth b) recession c) clinical attachment level d)
gingival crevicular fluid

Note: 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.

* p≤0.05, ** p≤0.01, *** p≤0.005, + p≤0.001, ++ p≤0.0005, +++ p≤0.0001

 82

Figure 3. Th1 and Th2 cytokines: Median amounts (pg/30s): a) IL-2 b) IFN-γ c) IL-3 d) IL-4

Note: 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.

* p≤0.05, ** p≤0.01, *** p≤0.005, + p≤0.001, ++ p≤0.0005, +++ p≤0.0001

 83

Figure 4. Pro-inflammatory cytokines: Median amounts (pg/30s): a) IL-1α b) IL-1β c) IL-6 d) IL-12(p40)
e) GM-CSF

Note: 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.

* p≤0.05, ** p≤0.01, *** p≤0.005, + p≤0.001, ++ p≤0.0005, +++ p≤0.0001

 84

Figure 5. Chemokines: Median amounts (pg/30s): a) IL-8 b) IP-10 c) MCP-1 d) MIP-1 e) RANTES f)
Eotaxin

Note: 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.

* p≤0.05, ** p≤0.01, *** p≤0.005, + p≤0.001, ++ p≤0.0005, +++ p≤0.0001

 85

Figure 6. Regulators of T-cells and NK cells: Median amounts (pg/30s): a) IL-7 b) IL-15

Note: 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.

* p≤0.05, ** p≤0.01, *** p≤0.005, + p≤0.001, ++ p≤0.0005, +++ p≤0.0001

 86

CHAPTER VI

DISCUSSION

Gingival Crevicular Fluid Volume and Associated

Disease Status

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.

Table 34. Comparison of studies on the effects of smoking on GCF cytokine/chemokine levels.

Study Smokers Concentration Total volume

NS S NS S
H D H D H D H D
Bostrom et al. (1999) ↑ TNF-α * 12 (7.3-18.3) 61.0 (42-177)
(Bostrom, Linder et al. (pg/ml) (pg/ml)
1999)
IL-6 (no diff) 10 (0-28) (pg/ml) 5.0 (0-30.5) (pg/ml)

Bostrom et al. (2000) IL-1β (no diff) 61.50 (34.50- 60.5 (24.75-93.50)
(Bostrom, Linder et al. 113.75) (pg/ml) (pg/ml)
2000)
IL-1ra (no diff) 59.62 (40.28-71.89) 57.61 (46.92-
(pg/ml) 110.06) (pg/ml)
Rawlinson et al. ↓IL-1β * 393.8 73.1 (61.0) (pg/µl ) 2714.5 24.5 (29.2) (pg/µl) 0.27 (0.40) 1.06 (0.34) 0.32 (0.42) 1.39 (0.22)
(2003) (867.1)(pg/µl ) (4416.2)(pg/ul) (µl) (µl ) (µl ) (µl )
(Rawlinson, Grummitt
et al. 2003) 3.2x105 5.8x105 (9.7)
↓IL-1ra * 3.2x105 (2.3) 0.19x105 (0.07)
(2.3)(pg/µl) (pg/µl) (pg/ul) (pg/µl)
Petropoulos et al. ↓IL-1α * 3.29 (2.02) (pg/µl ) 1.59 (1.13) (pg/µl)
(2004)(Petropoulos,
McKay et al. 2004)
Erdemir et al. (2004) TNF-α (no diff) 0.51 (0.81) (pg/µl) 1.07 (1.32) (pg/µl)
(Erdemir, Duran et al.
2004)
IL-6 (no diff) 0.57 (0.75) (pg/µl) 0.32 (0.38) (pg/µl)

* Statistically significant (p≤0.05)

97
Table 34. Continued

Kamma et al. (2004) ↑IL-1β * 7.85 61.37 17.97 62.37

(Kamma, (pg/30s) (pg/30s) (pg/30s) (pg/30s)
Giannopoulou et al.
2004) 12.07 1.90 10.33 3.08
IL-4 (no diff)
(pg/30s) (pg/30s) (pg/30s) (pg/30s)

0.89 5.04 1.70 6.04

↑IL-6 * (pg/30s) (pg/30s) (pg/30s) (pg/30s)

24.00 72.96 20.43 68.30

↓IL-8 * (pg/30s) (pg/30s) (pg/30s) (pg/30s)

* Statistically significant (p≤0.05)

98
 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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