Theses and Dissertations

University of Iowa Year

The Inuence Of Smoking On Cytokines In The Gingival Crevicular Fluid In Patients With Periodontal Disease
Keelen D. Tymkiw
The University of Iowa

This paper is posted at Iowa Research Online. http://ir.uiowa.edu/etd/26

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.

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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- ......................43 Pro-inflammatory Cytokines: IL-1, IL-1, IL-6, IL12(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

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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

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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- c) IL-3 d) IL-4 ........................................................................................................82 Figure 4. Pro-inflammatory cytokines: Median amounts (Total/30s): a) IL-1 b) IL1 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

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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- 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- ), pro-inflammatory cytokines (IL-1, IL-1, 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-

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 (PMNs) 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

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 (0.1l) 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).

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 (>1.0l) usually found in diseased sites, which are unable to be measured with the Periotron 6000. However, the minimal detection limit (0.1l) 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

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 0.1-1.0l 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 0.2l 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

reliable (Chapple, Cross et al. 1995). Chapple concludes that the Periotron 6000 is a reliable and convenient instrument for measuring fluid volumes greater than 0.2l. 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,

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.

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 welldeveloped 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

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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/l) 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

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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-. 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

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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- 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-1, 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-1, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12(p40), IL-12(p70), IL-13, IL15, IP-10, Eotaxin, IFN-, GM-CSF, MCP-1, MIP-1, RANTES, and TNF-. 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

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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 doseresponse (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 hosts immune response, therefore it is probable that this may be its primary role in contribution to the pathogenesis of the disease.

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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

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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 nonsurgical 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, PiniPrato 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 10year 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-1. This is in agreement with the observations of Bostrm et al. (2000), who analyzed GCF levels of IL-1 and its receptor antagonist IL-1ra with respect to smoking in patients with moderate to severe periodontal disease. IL-1 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-1, IL-2, interferon (IFN)- and TNF- (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- 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- in a smaller group of smokers (Bostrom, Linder et al. 1999). Bostrom et al. (2000) then compared levels of IL-1 and IL-1ra and found no significant differences (Bostrom, Linder et al. 2000). However, Rawlinson et al. (2003) found levels of IL-1 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-1 in GCF of smokers was approximately half that found in non-smokers (Petropoulos, McKay et al. 2004).

Table 1. Cytokine Properties

Cytokine Interleukin -1 (IL-1)

Sources# Produced by monocytes, macrophages, neutrophils, endothelial cells, fibroblasts, smooth muscle cells, keratinocytes, langerhans cells of the skin, osteoclasts, astrocytes, epithelial cells of the thymus and the cornea, Tcells, B-cells, NKcells. Production stimulated by TNF-, IFN-, IFN- and IFN- , bacterial endotoxins, viruses, mitogens, and antigens. Produced by T-cells, and B-cells, AK cells (lymphokine-activated killer cells) and NKcells.

Biologic Activity# Chemoattractant for leukocytes, stimulation of T-helper cells (IL-2 secretion), proliferation of B cells, B-cell responsiveness to IL-5, proliferation and activation of NK-cells and fibroblasts, thymocytes, glioblastoma cells. Enhances the metabolism of arachidonic acid (prostacyclin and PGE2) in inflammatory cells (fibroblasts, synovial cells, chondrocytes, endothelial cells, hepatocytes, and osteoclasts) Increased secretion of inflammatory proteins such as neutral proteases (collagenase, elastase and plasminogen activator). Antagonizes the effects of TGF on the extracellular matrix Promotion of wound healing (angiogenesis, proliferation of fibroblasts, neutrophil chemotaxis). Significant anti-tumor activity for a variety of tumor cell types since it supports the proliferation and clonal expansion of Tcells that specifically attack certain tumor types.

Relationship to Periodontitis Significantly increased in the periodontal tissues and gingival fluid from diseased sites, compared with healthy sites (Masada, Persson et al. 1990). Can act on a large number of cells (fibroblasts, chondrocytes, bone cells, neutrophils and lymphocytes) suggests that periodontal destruction and repair in periodontitis may in part be associated with this cytokine (Orozco, Gemmell et al. 2006). IL-1 up-regulates matrix metalloproteinases and down-regulates tissue inhibitors of metalloproteinase production (Ohshima, Otsuka et al. 1994). Powerful and potent bone-resorbing cytokine (Shirodaria, Smith et al. 2000). Plays a role in degrading the extracellular matrix in periodontitis (Schwartz, Goultschin et al. 1997). Involved in B-cell activation and stimulates macrophages, NK cells and T-cell proliferation. It is regarded as a proinflammatory cytokine (Tew, Engel et al. 1989).

Interleukin -2 (IL-2)

18

Table 1. Continued

Production stimulated by vitamin E. Production inhibited by dexamethasone or Cyclosporin A.

Induces the secretion of other soluble mediators, including TNF-, TNF-, and IFN-. These effects may contribute to the antitumor activity of IL- as well as to its dose-related toxicity.

IL-2 has been also implicated in the stimulation of osteoclast activity in bone resorption (Ries, Seeds et al. 1989). There is evidence indicating that IL-2 may also be a relevant factor in the pathogenesis of periodontal disease. Lymphocytes cultured from the chronically inflamed periodontal tissues of patients with alveolar bone loss produced IL-2 (Seymour, Cole et al. 1985). The levels of IL-2 in the sera of periodontitis patients are elevated when compared to those of normal subjects (McFarlane and Meikle 1991). Due to its biological properties, IL-2 has been suggested to be a useful marker of pathologic inflammatory activity in systemic diseases (John, Turner et al. 1998) and periodontal conditions (McFarlane and Meikle 1991).

Interleukin -3 (IL-3)

Produced by T-cells, keratinocytes, NKcells, mast cells, endothelial cells, and monocytes. Production inhibited by glucocorticoids or CsA (Cyclosporin A).

Is a growth factor that establishes the link between the immune system and the hematopoietic system. Supports the proliferation and development of almost all types of hematopoietic progenitor cells.

19

Table 1. Continued

Supports the differentiation of early nonlineage-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. Promotes the proliferation and differentiation of activated B-cells, the expression of MHC class 2 antigens, and of low affinity IgE receptors in resting B-cells. Enhances expression of MHC class 2 antigens on B-cells. It can promote their capacity to respond to other B-cell stimuli and to present antigens for T-cells. Potent downregulator of macrophage function by inhibiting the secretion of IL1, TNF- and IL-6 (Kamma, Giannopoulou et al. 2004). Inhibits the secretion of prostaglandin (PGE2) by human monocytes which leads to bone resorption (Shapira, van Dyke et al. 1992). Absence of IL-4 has been associated with periodontal disease activity and progression (Shapira, van Dyke et al. 1992). Clinical importance in the treatment of inflammatory diseases since it inhibits the production of inflammatory cytokines such as IL-1, IL-6 and TNF- by monocytes and of TNF by T-cells (Kamma, Giannopoulou et al. 2004).

Interleukin -4 (IL-4) Produced by activated T-cells (Th2) or (Th1), Non-T/Non-B-cells of the lineage of mast cells. Production stimulated by IL-2 and PAF (platelet activating factor). Production inhibited by TGF-beta.

20

Table 1. Continued
Interleukin -5 (IL-5) Interleukin -6 (IL-6) Produced by monocytes, fibroblasts, and endothelial cells. Macrophages, T-cells and B-lymphocytes, granulocytes, smooth muscle cells, eosinophils, chondrocytes, osteoblasts, mast cells, glial cells, and keratinocytes.

Produced by T-cells

Specific hematopoietic growth factor that is responsible for the growth and differentiation of eosinophils. Promotes the generation of cytotoxic Tcells from thymocytes. Induces the proliferation of pre-activated Bcells and their differentiation. Stimulates the production of IgM and IgA. Pleiotropic cytokine influencing antigenspecific immune responses and inflammatory reactions. B-cell differentiation factor in vivo and in vitro and an activation factor for T-cells. In the presence of IL-2, IL-6 induces the differentiation of mature and immature Tcells into cytotoxic T-cells. IL-6 also induces the proliferation of thymocytes and probably plays a role in the development of thymic T-cells. IL-6 is capable of inducing the final maturation of B-cells into immunoglobulinsecreting plasma cells if the cells have been pre-activated by IL-4. In B-cells IL-6 stimulates the secretion of antibodies. Contributes to the terminal differentiation of B-lymphocytes to plasma cells and stimulates secretion of immunoglobulin (Ig)A and IgG (Fujihashi, Kono et al. 1993). Smoking causes a depression of IgG and possibly IgA production in serum (Quinn, Zhang et al. 1996). Induces bone resorption, both by itself and in conjunction with other bone-resorbing agents (Mundy 1991).

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

Interleukin -8 (IL-8)

22

Table 1. Continued

Production inhibited by 5' lipoxygenase, and vitamin D3.

Inhibits the adhesion of leukocytes to activated endothelial cells and therefore possesses anti-inflammatory activities. Supports angiogenesis and may play a role in disorders such as rheumatoid arthritis, tumor growth, and wound healing that critically depend on angiogenesis.

Powerful chemotactic functions for polymorphonuclear leukocytes but also for lymphocytes and macrophages (Kamma, Giannopoulou et al. 2004). Is a potent mediator of granulocyte accumulation at the sites of inflammation (Bickel 1993). Increased levels of IL-8 are found in the GCF of inflamed sites (Tsai, Ho et al. 1995).

Interleukin -10 (IL-10)

Produced by T-cells (Th2 cells but not Th1 T-helper cells), B-cells, and keratinocytes. Production is inhibited by IL-4.

Potent and specific T-cell chemoattractant. Inhibits the synthesis of a number of cytokines such as IFN-, IL-2 and TNF- in Th1 T-helper subpopulations of T-cells but not of Th2 T-helper cells. This activity is antagonized by IL-4. In the human system, IL-10 is produced by, and down-regulates the function of, Th1 and Th2 cells. In macrophages stimulated by bacterial lipopolysaccharides, IL-10 inhibits the synthesis of IL-1, IL-6 and TNF-. In human monocytes IFN- 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 (IL-12)

Produced by monocytes, macrophages, neutrophils, B-cells and to a lesser extent by T-cells. Production stimulated by IL-12, bacteria, bacterial products, and parasites.

Is a heterodimer comprised of p35 and p40 subunits, which form the bioactive IL-12 (p70). Principal mediator of the early innate immune response to intracellular microbes and is a key inducer of cell-mediated immunity. Stimulates IFN- production by T cells and natural killer cells and so promote Th1 responses. Promotes Th1 development by stimulating the production of IL-12 by macrophages and the expression of functional IL-12 receptors on T cells. The importance of IL-12 is not limited to initiating an immune response, but may contribute to maintaining immunity because Th1 responses, smooth muscle cells, and fibroblasts also secrete TNF. Inhibits anticoagulatory mechanisms and promotes thrombotic processes and therefore plays an important role in pathological processes such as venous thromboses, arteriosclerosis, vasculitis, and disseminated intravasal coagulation. Potent chemoattractant for neutrophils and also increases their adherence to the endothelium.

Produced by proinflammatory infiltrates in periodontitis tissues. High levels will contribute to the immune reaction to Th1 type (Lamont and Adorini 1996). IL-12 is an inducer of IFN- production. IFN- itself can also activate IL-12 production (Lamont and Adorini 1996). LPS of periodontopathogens are also activators of IL-12 (Lamont and Adorini 1996). Significantly higher proportions found in diseased sites (Lamont and Adorini 1996).

Tumor Necrosis Factor-Alpha (TNF-)

Produced by macrophages, monocytes, neutrophils, T-cells, NK-cells, astrocytes, microglial cells, smooth muscle cells, and fibroblasts.

Stimulates fibroblasts, including gingival fibroblasts, to produce collagenase (Meikle, Atkinson et al. 1989) which is implicated in the tissue destruction of periodontal disease, and to stimulate bone resorption (Bertolini, Nedwin et al. 1986). Activates monocytes and stimulates the production of IL-1 and platelet activating factor (Erdemir, Duran et al. 2004).

24

Table 1. Continued

Production stimulated by interferons, IL-2, GMCSF, SP, Bradykinin, Immune complexes, inhibitors of cyclooxygenase and platelet activating factor. Production inhibited by IL-6, TGF-, vitamin D3, prostaglandin E2, dexamethasone, CsA (Cyclosporin A), and antagonists of platelet activating factor.

Induces the synthesis of a number of chemoattractant cytokines, including IP-10, JE, KC, in a cell-type and tissue-specific manner. Inhibits the growth of endothelial cells in vitro and is a potent promoter of angiogenesis in vivo. Growth factor for normal human diploid fibroblasts and promotes the synthesis of collagenase and prostaglandin E2 in fibroblasts. In resting macrophages TNF induces the synthesis of IL-1 and prostaglandin E2. Stimulates phagocytosis and the synthesis of superoxide dismutase in macrophages. Activates osteoclasts and thus induces bone resorption. Stimulates the expression of class 1 and II HLA and differentiation antigens, and the production of IL-1, colony stimulating factors, IFN-, arachidonic acid metabolism. Stimulates the biosynthesis of collagenases in endothelial cells and synovial cells. Antiviral and antiparasitic activities and also inhibits the proliferation of a number of normal and transformed cells. Synergizes with TNF- and TNF- in inhibiting the proliferation of various cell types.

Monocyte stimulation by lipopolysaccharide enhances the production of TNF-, which has also been shown to induce collagenase release and bone resorption in vivo (Meikle, Atkinson et al.
1989).

InterferonGamma (IFN-) Produced by T-cells, Bcells, natural killer cells activated by antigens, or lymphocytes expressing the surface antigens CD4 and CD8.

Studies have suggested an inhibitory effect of IFN- on RANKL-associated osteoclastogenesis and bone remodeling in vitro and in vivo (Takayanagi, Ogasawara et al. 2000).

25

Table 1. Continued

Production stimulated by IL-2, bFGF, and EGF. Production inhibited by 1-alpha,25-Dihydroxy vitamin D3, dexamethasone and CsA (Cyclosporin A).

Main biological activity of IFN- appears to be immunomodulatory in contrast to the other interferons which are mainly antiviral. In T-helper cells IL-2 induces the synthesis of IFN- and other cytokines. IFN- acts synergistically with IL-1 and IL-2 and appears to be required for the expression of IL-2 receptors on the cell surface of Tlymphocytes. IFN- is a modulator of T-cell growth and functional differentiation. It is a growthpromoting factor for T-lymphocytes and potentiates the response of these cells to mitogens or growth factors. IFN- inhibits the growth of B-cells induced by IL-4. IFN-gamma and Anti-Ig costimulate the proliferation of human Bcells. 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- can positively modulate the expression of proresorptive 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-+ 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- expression significantly reduces the severity of periodontal bone loss in mice after mounting a microbial challenge (Baker, Dixon et al. 1999). IFN- 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- 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- induces the secretion of TNF- and the transcription of genes encoding G-CSF and M-CSF. In macrophages IFN- stimulates the release of reactive oxygen species. IFN- is involved also in processes of bone growth and inhibits bone resorption probably by partial inhibition of the formation of osteoclasts. IFN- 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. Important in the growth and development of progenitors of granulocytes and macrophages. It stimulates myeloblasts and monoblasts and triggers irreversible differentiation of these cells. Strong chemoattractant for neutrophils. Enhances microbicidal activity, oxidative metabolism, and phagocytotic activity of neutrophils and macrophages. Stimulates the proliferation precursors of neutrophils, eosinophils, and monocytes. Enhances phagocytotic activities of neutrophil granulocytes and the cytotoxicity of eosinophils. Up-regulated in neutrophil-mediated pathology and is associated with periodontal inflammation (Takematsu and Tagami 1990; Baqui, Meiller et al. 1999). Variety of effects on neutrophils potentially important in the pathogenesis of periodontitis, including dose-dependent chemotaxis or inhibition of movement, inhibition of apoptosis and priming for increased phagocytic and respiratory burst activity (Fossati, Mazzucchelli et al. 1998).

Granulocyte Macrophage Colony Stimulating Factor (GM-CSF)

Produced by T-cells, macrophages, endothelial cells and fibroblasts. Production stimulated by TNF-, TNF-, IL1, IL-2 and IFN.

27

Table 1. Continued
RANTES

Produced by T-cells. Production stimulated by TNF- and IL-1.

Chemotactic for T-cells, human eosinophils and basophils and plays an active role in recruiting leukocytes into inflammatory sites. Increases the adherence of monocytes to endothelial cells and selectively supports the migration of monocytes and Tlymphocytes expressing the cell surface markers CD4 and UCHL1. Activates human basophils from some select basophil donors and causes the release of histamines. Expressed by human synovial fibroblasts and may participate, therefore, in the ongoing inflammatory process in rheumatoid arthritis. 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.

Plays an important role in the host response by recruiting inflammatory cells into the foci of active inflammation and by inducing the release of other cell mediators (Gamonal, Acevedo et al. 2000). Important mediators of the host response in chronic adult periodontitis (Emingil, Atilla et al. 2004).

Eotaxin

InterferonInducible Protein-10 (IP-10)

Produced by monocytes, keratinocytes, and fibroblasts, neutrophils. Production stimulated by IFN-, TNF-, and LPS. Production inhibited by IL-10 and IL-4.

28

Table 1. Continued
Monocyte Chemotactic Proteins-1 (MCP-1)

Produced by monocytes, vascular endothelial cells, smooth muscle cells, glomerular mesangial cell, osteoblastic cells, and human pulmonary type 2 like epithelial cells. Production stimulated by peripheral blood mononuclear leukocytes, phytohemagglutinin (PHA), bacterial lipopolysaccharides, and IL-1. Produced by macrophages

Chemotactic for monocytes but not neutrophils. Elevated levels of MCP-1 are observed in macrophage-rich atherosclerotic plaques. Regulates the expression of cell surface antigens (CD11c, CD11b) and the expression of cytokines (IL-1, IL-6). MCP1 is a potent activator of human basophils, inducing degranulation and the release of histamines. Induces the proliferation and activation of killer cells known as CHAK (CCChemokine-activated killer).

Macrophage inflammatory protein-1 (MIP-1)

Caused local inflammatory responses in vivo, and induces superoxide production by neutrophils in vitro.

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 020 2140 4180 81200

Level of Gingival Inflammation healthy mild moderate severe

Gingival Index 0 1 2 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- 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-), Th2 cytokines (IL-3, IL-4, IL-5, IL-10, and IL13), pro-inflammatory cytokines (IL-1, IL-1, IL-6, GM-CSF, TNF-, 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 nonsmokers 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 l 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 300l of PBS and protease inhibitor and vortexed for ten seconds and eluted at 4C 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-), Th2 cytokines (IL-3, IL-4, IL-5, IL-10, and IL-13), pro-inflammatory cytokines (IL1-, IL-1, IL-6, GM-CSF, TNF-, 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). To determine the concentrations of cytokines, the manufactures instructions were followed. 50 l of 0.01 M PBS, pH 7.2 containing GCF samples were incubated with anti-human multi-cytokine beads at 4C for 18 hours. Unbound material was removed by filtration. 25 l of anti-human multi-cytokine biotin reporter was added, and reactions were incubated at room temperature for 1.5 hours in the dark. 25 l of streptavidin phycoerythrin was then added, and the plates were incubated at room temperature for an additional 30 minutes. 25 l 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 nonsmoking 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 matchedpaired comparisons consisted of comparing healthy and diseased sites in diseased subjects. In each case the level of significance was set at p0.05.

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 l. 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- (Th1 cytokines); IL-3 and IL-4 (Th2 cytokines); IL1-, IL1-, IL-6, GM-CSF, and IL-12(p40) (pro-inflammatory cytokines); CXCL8/IL8,

43

CXCL10/IP-10, CCL2/MCP-1, CCL3/MIP-1, 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-). Therefore, they were not included in the analysis. Th1 and Th2 Cytokines: IL-2, IL-3, IL-4, IFN- 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- (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- (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- 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- (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- (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- (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- 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- (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- (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- (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- (p=0.0024), IL-3 (p= 0.0007) and IL-4 (p=0.0461) compared to healthy controls (Table 14). Pro-inflammatory Cytokines: IL-1, IL-1, 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-1 were the highest (ranging from 41.5 to 32580.0 pg/30 sec), followed by IL-1 (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-1 (p<0.0001), IL-1 (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-1 in SD sites (p<0.0001). No significant differences in amounts of IL-1, 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-1 (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-1 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-1 (p<0.0001), IL-1 (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-1 (p<0.01) in SH sites. No significant differences in amounts of IL-1, 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-1 (p<0.005) and IL-1 (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-1, IL-1, 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 IL1 and GM-CSF. SD sites showed significantly less IL-1 (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-1 (p=0.0001), IL-1 (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-1 were significantly higher (p=0.0012) compared to healthy controls (Table 13). DP sites showed significantly greater amounts of IL-1 (p<0.0001), IL-1 (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), IP10 (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 IL15 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-, IL-1, IL-1, IL2, 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-1 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-1, 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, IL12(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 Characteristics Mean age (years) Gender Female Male 10 2 8 12 12 8 51.2 5.2 51.2 7.4 61.2 9.7 Healthy Control Smokers Non-smokers

Table 7. Site characteristics of the study population.

Clinical characteristics Probing depth (mm) Recession (mm) CAL (mm) BOP (% positive) HC 2.3 0.6 0.1 0.4 2.5 0.7 0.0 0.0 HP 2.7 0.5 0.3 0.6 2.9 0.7 0.0 0.0 DP 5.7 0.8 0.8 1.0 6.5 1.3 100 0.0 NH 2.6 0.6 0.3 0.7 2.9 0.9 0.0 0.0 SH 2.8 0.4 0.3 0.4 3.0 0.3 0.0 0.0 ND 5.8 0.9 0.7 0.9 6.5 1.3 100 0.0 SD 5.6 0.5 1.0 1.1 6.6 1.3 100 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 HC HP DP NH SH ND SD

Mean GCF Volume SD (l)

1.1 0.7 1.5 1.0 2.1 0.8 1.6 1.0 1.3 0.9 2.3 0.8 1.9 0.9

p value
NS <0.05 (HP vs. HC) <0.001 (DP vs. HC) NS NS NS <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
Median Th1 cytokines IL-2 IFN- Th2 cytokines IL-3 IL-4 75.5 14.8 78.6 32.9 35.0 38.2 88.5 14.2 24.0 14.0 26.6 21.4 8.9 18.0 26.5 23.2 Mean SD Median

ND
Mean SD p value Median

SH
Mean SD Median

SD
Mean SD p value

27.2 31.8

1.7 31.8

NS <0.01

26.7 13.4

28.3 19.2

10.8 16.0

23.1 15.2

27.3 24.7

10.2 23.3

NS <0.05

91.5 36.2

48.9 40.2

NS NS

68.1 16.3

74.5 50.0

46.9 47.2

83.0 16.4

85.9 49.9

37.9 47.4

NS 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
Median Th1 cytokines IL-2 IFN- Th2 cytokines IL-3 IL-4 55.3 12.4 56.2 28.0 30.7 35.7 72.0 14.3 21.3 12.6 24.2 15.5 9.3 11.1 23.1 15.2 Mean SD Median

NH
Mean SD p value Median

HC
Mean SD Median

ND
Mean SD p value

26.6 21.4

10.3 18.9

NS NS

23.1 12.6

24.2 15.5

9.3 11.1

27.7 25.5

28.6 33.2

8.9 29.8

<0.005 <0.0005

78.6 32.9

51.1 38.7

NS NS

55.3 12.4

56.2 28.0

30.7 35.7

95.4 15.9

99.3 41.9

66.2 43.0

<0.001 <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
Median Th1 cytokines IL-2 IFN- Th2 cytokines IL-3 IL-4 55.3 12.4 56.2 28.0 30.7 35.7 70.2 15.2 21.3 12.6 24.2 15.5 9.3 11.1 25.2 13.6 Mean SD Median

SH
Mean SD p value Median

HC
Mean SD Median

SD
Mean SD p value

27.9 18.6

12.1 16.4

NS NS

21.3 12.6

24.2 15.5

9.3 11.1

22.9 15.0

27.0 24.2

11.0 23.2

NS NS

72.7 46.9

58.3 47.2

NS NS

55.3 12.4

56.2 28.0

30.7 35.7

74.4 13.6

83.8 48.0

50.4 46.9

<0.05 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
Median Th1 cytokines IL-2 IFN- Th2 cytokines IL-3 IL-4 72.0 14.3 78.6 32.9 51.1 38.7 70.2 15.2 23.1 15.2 26.6 21.4 10.3 18.9 25.2 13.6 Mean SD Median

SH
Mean SD p value Median

ND
Mean SD Median

SD
Mean SD p value

27.9 18.6

12.1 16.4

NS NS

27.7 25.5

28.6 33.2

9.0 29.8

22.9 15.0

27.0 24.2

11.0 23.2

NS NS

72.7 46.9

58.3 47.2

NS NS

95.4 15.9

99.3 41.9

66.2 43.1

74.4 13.6

83.8 48.0

50.4 46.1

NS 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 Median
Th1 cytokines

DP SD Median Mean SD p value

Mean

IL-2 IFN-
Th2 cytokines

24.2 13.6

27.3 20.1

11.2 17.6

24.4 22.4

27.9 29.2

9.9 27.4

NS <0.001

IL-3 IL-4

70.2 14.6

75.5 40.3

54.7 43.6

83.4 15.4

92.5 44.6

60.0 44.6

NS 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
Median Th1 cytokines IL-2 IFN- Th2 cytokines IL-3 IL-4 55.3 12.4 56.2 30.1 30.7 35.7 70.2 14.6 21.3 12.6 24.2 15.5 9.3 11.1 24.2 13.6 Mean SD Median

HP
Mean SD p value Median

HC
Mean SD Median

DP
Mean SD p value

27.3 20.1

11.2 17.6

NS NS

21.3 12.6

24.2 15.5

9.3 11.1

24.4 22.4

27.9 29.2

9.9 27.4

<0.05 <0.01

75.5 40.3

54.7 43.6

NS NS

55.3 12.4

56.2 30.1

30.7 35.7

83.4 15.4

92.5 44.6

60.0 44.6

<0.001 <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
Median
Proinflammatory cytokines

ND
SD Median Mean SD p value Median

SH
Mean SD Median

SD
Mean SD p value

Mean

IL-1

858.6 22.3 109.2 109.1 23.2

1121.0 45.6 148.1 104.1 25.1

1230.5 57.8 102.7 39.3 23.4

1765.5 112.0 165.9 147.1 23.9

2407.8 161.6 220.0 151.8 29.5

2444.7 157.7 171.1 77.1 25.3

<0.0001 <0.005 NS <0.01 <0.05

857.6 17.5 49.3 78.9 23.1

907.8 42.6 69.6 77.2 28.7

570.0 63.0 47.3 30.2 20.9

1102.8 69.9 60.4 80.6 22.6

1442.7 199.9 81.5 94.6 27.8

1092.8 314.4 55.5 40.6 20.6

NS <0.0001 NS NS NS

IL-1

IL-6

IL-12(p40)

GM-CSF

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
Median
Proinflammatory cytokines

NH
SD Median Mean SD p value Median

HC
Mean SD Median

ND
Mean SD p value

Mean

IL-1

612.0 0.0 68.7 75.0 23.8

778.4 14.1 78.4 76.5 25.8

624.3 34.7 44.3 58.7 19.9

786.0 14.3 106.2 99.0 23.2

1121.0 45.6 148.7 104.1 25.1

1438.0 84.0 143.7 57.3 25.3

NS <0.01 <0.001 <0.05 NS

612.0 0.0 68.7 75.0 23.8

778.4 14.1 78.4 76.5 25.8

624.3 34.7 44.3 58.7 19.9

1647.0 44.7 114.0 135.3 28.4

2762.4 134.7 213.2 156.4 33.2

4393.8 165.4 276.3 90.7 28.6

<0.0001 <0.0001 <0.0001 <0.0001 NS

IL-1

IL-6

IL-12(p40)

GM-CSF

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
Median
Proinflammatory cytokines

SH
SD Median Mean SD p value Median

HC
Mean SD Median

SD
Mean SD p value

Mean

IL-1

612.0 0.0 68.7 75.0 23.8

778.4 14.1 78.4 76.5 25.8

624.3 34.7 44.3 58.7 19.9

738.0 16.9 54.8 82.2 24.3

870.7 39.4 68.7 73.4 27.5

636.3 68.0 60.4 36.0 22.1

NS <0.01 NS NS NS

612.0 0.0 68.7 75.0 23.8

778.4 14.1 78.4 76.5 25.8

624.3 34.7 44.3 58.7 19.9

1176.0 38.6 57.4 87.0 23.2

1606.2 119.0 80.0 92.3 27.4

1463.4 161.0 59.5 51.0 21.0

<0.005 <0.0001 NS NS NS

IL-1

IL-6

IL-12(p40)

GM-CSF

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
Median
Proinflammatory cytokines

SH
SD Median Mean SD p value Median

ND
Mean SD Median

SD
Mean SD p value

Mean

IL-1

786.0 14.3 106.2 99.0 23.2

1121.0 45.6 148.7 104.1 25.1

1438.0 84.1 143.7 57.3 25.3

738.0 16.9 54.8 82.2 24.3

870.7 39.4 68.7 73.4 27.5

636.2 68.0 60.4 36.0 22.1

NS NS <0.001 <0.05 NS

1647.0 69.9 114.0 135.3 28.4

2762.4 199.9 213.2 156.4 33.2

4393.8 314.4 276.3 90.7 28.6

1176.0 44.7 57.4 87.0 23.2

1606.2 134.7 80.0 92.3 27.4

1463.4 165.4 59.5 51.0 21.0

<0.05 NS <0.001 <0.001 NS

IL-1

IL-6

IL-12(p40)

GM-CSF

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 Median
Pro-inflammatory cytokines
IL-1

DP SD Median Mean SD p value

Mean

756.0 15.6 89.7 84.0 24.3

989.9 42.4 106.8 88.0 26.4

1094.2 75.6 114.9 49.5 23.6

1533.0 66.9 103.8 112.5 26.6

2253.7 171.2 154.6 128.2 30.6

3462.4 260.5 219.9 82.0 25.6

<0.001 <0.001 <0.05 <0.01 NS

IL-1

IL-6

IL-12(p40)

GM-CSF

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
Median
Proinflammatory cytokines

HP
SD Median Mean SD p value Median

HC
Mean SD Median

DP
Mean SD p value

Mean

IL-1

612.0 0.0 68.7 75.0 23.8

778.4 14.1 78.7 76.5 25.8

624.3 34.7 44.3 58.7 19.9

756.0 15.6 89.7 84.0 24.3

989.9 42.4 106.8 88.0 26.4

1094.2 75.6 114.9 49.5 23.6

NS <0.01 NS NS NS

612.0 0.0 68.7 75.0 23.8

778.4 14.1 78.7 76.5 25.8

624.3 34.7 44.3 58.7 19.9

1533.0 66.9 103.8 112.5 26.6

2253.7 171.2 154.6 128.2 30.6

3462.4 260.5 219.9 82.0 25.6

<0.001 <0.001 <0.05 <0.001 NS

IL-1

IL-6

IL-12(p40)

GM-CSF

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
Median Mean SD Median

ND
Mean SD p value Median

SH
Mean SD Median

SD
Mean SD p value

Chemokines

IL-8 IP-10 MCP-1 MIP-1 RANTES Eotaxin

1408.2 377.56 15.8 245.6 21.9 34.6

1352.3 470.1 16.9 217.0 33.5 83.5

740.3 535.5 8.6 98.0 44.0 79.3

1512.0 320.6 98.7 258.5 30.2 66.6

2004.1 525.3 25.7 250.7 76.1 98.7

1851.4 591.2 20.5 9.6 89.8 77.4

NS NS <0.05 NS NS <0.05

574.8 203.9 10.2 148.2 17.6 50.1

734.5 286.5 9.4 161.7 18.5 108.3

548.4 232.4 4.8 76.4 10.8 90.3

580.4 248.2 10.7 167.1 17.4 32.8

728.2 277.0 11.3 180.5 22.4 104.6

502.6 205.6 4.5 88.2 17.9 91.7

NS NS NS NS NS 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
Median Mean SD Median

NH
Mean SD P value Median

HC
Mean SD Median

ND
Mean SD P value

Chemokines

IL-8 IP-10 MCP-1 MIP-1 RANTES Eotaxin

452.4 495.9 12.9 147.3 17.2 39.2

607.0 769.6 12.8 161.6 18.3 70.1

460.1 799.6 8.4 76.0 10.5 70.1

1092.0 367.5 14.6 237.9 19.5 36.9

1352.3 470.1 16.9 217.0 33.5 83.5

944.4 585.5 10.7 103.1 61.8 79.2

<0.0001 <0.05 NS <0.01 <0.05 NS

452.4 495.9 12.9 147.3 17.2 39.2

607.0 769.6 12.8 161.6 18.3 70.1

460.1 799.6 8.4 76.0 10.5 70.1

1656.0 320.7 20.8 240.9 23.6 107.7

2461.1 527.3 24.6 255.3 80.4 112.2

4399.2 663.65 20.2 115.3 177.8 84.2

<0.0001 <0.05 <0.0001 <0.0001 <0.0001 <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
Median Mean SD Median

SH
Mean SD p value Median

HC
Mean SD Median

SD
Mean SD p value

Chemokines

IL-8 IP-10 MCP-1 MIP-1 RANTES Eotaxin

452.4 495.9 12.9 147.3 17.2 39.2

607.0 769.6 12.8 161.6 18.3 70.1

460.1 799.6 8.4 76.0 10.5 70.1

564.6 170.1 9.1 145.2 16.7 40.7

745.7 278.3 8.9 163.8 18.2 104.6

677.8 258.9 7.5 81.0 12.7 90.0

NS <0.0001 <0.01 NS NS NS

452.4 495.9 12.9 147.3 17.2 39.2

607.0 769.6 12.8 161.6 18.3 70.1

460.1 799.6 8.4 76.0 10.5 70.1

594.0 205.2 11.1 173.7 17.2 31.6

810.3 274.9 11.1 182.8 22.1 102.7

705.7 261.9 6.8 87.9 18.4 90.0

NS <0.0001 NS NS NS 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
Median Mean SD Median

SH
Mean SD p value Median

ND
Mean SD Median

SD
Mean SD p value

Chemokines

IL-8 IP-10 MCP-1 MIP-1 RANTES Eotaxin

1092.0 367.5 14.6 237.9 19.5 36.9

1352.3 470.1 16.9 217.0 33.5 83.5

944.4 585.5 10.7 103.1 61.8 79.2

564.6 170.1 9.1 145.2 16.7 40.7

745.7 278.3 8.9 163.8 18.2 104.6

677.8 258.9 7.5 81.0 12.7 90.0

<0.01 <0.05 <0.001 <0.05 <0.01 NS

1656.0 320.7 20.8 240.9 23.6 107.7

2461.1 527.3 24.6 255.3 80.4 112.2

4399.2 663.7 20.2 115.3 177.8 84.2

594.0 205.2 11.1 173.7 17.2 31.6

810.3 274.8 11.1 182.8 22.1 102.6

705.7 261.9 6.8 87.9 18.4 90.0

<0.001 <0.05 <0.0001 <0.001 <0.01 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 Median
Chemokines

DP SD Median Mean SD p value

Mean

IL-8 IP-10 MCP-1 MIP-1 RANTES Eotaxin

822.0 268.8 12.1 178.5 17.3 37.8

1034.5 369.6 12.7 189.2 25.5 94.5

866.0 452.9 10.0 95.5 44.0 85.2

1200.0 276.9 14.3 231.0 19.4 66.6

1734.8 416.2 18.7 223.4 54.7 108.0

3412.7 538.8 17.1 109.8 136.2 86.5

NS NS <0.01 <0.05 <0.05 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
Median Chemokines Mean SD Median

HP
Mean SD p value Median

HC
Mean SD Median

DP
Mean SD p value

IL-8 IP-10 MCP-1 MIP-1 RANTES Eotaxin

452.4 495.9 12.9 147.3 17.2 39.2

607.0 769.6 12.8 161.6 18.3 70.1

460.1 799.6 8.4 76.0 10.5 70.1

822.0 268.8 12.1 178.5 17.3 37.8

1034.5 369.6 12.7 189.2 25.5 94.5

866.0 452.9 10.0 95.5 44.0 85.2

<0.01 <0.001 NS NS NS NS

452.4 495.9 12.9 147.3 17.2 39.2

607.0 769.6 12.8 161.6 18.3 70.1

460.1 799.6 8.4 76.0 10.5 70.1

1200.0 276.9 14.3 231.0 19.4 66.6

1734.8 416.2 18.7 223.4 54.7 108.0

3412.7 538.8 17.1 109.8 136.2 86.5

<0.001 <0.0005 NS <0.001 <0.01 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
Median Mean SD Median

ND
Mean SD p value Median

SH
Mean SD Median

SD
Mean SD p value

Regulators of T-cells and NK cells IL-7 IL-15 54.0 19.2 61.5 21.7 29.0 5.8 57.5 21.5 66.7 23.6 30.4 7.7 NS NS 51.6 17.9 53.4 20.0 17.3 5.7 51.3 19.3 50.9 21.1 17.6 5.8 NS 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
Median Mean SD Median

NH
Mean SD p value Median

HC
Mean SD Median

ND
Mean SD p value

Regulators of T-cells and NK cells IL-7 IL-15 50.2 18.4 57.9 18.6 32.4 4.3 52.5 20.9 61.5 21.7 31.2 6.4 NS <0.05 50.2 18.4 57.9 18.6 32.4 4.3 65.7 22.7 69.7 25.0 34.4 8.4 <0.05 <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
Median Mean SD Median

SH
Mean SD p value Median

HC
Mean SD Median

SD
Mean SD p value

Regulators of T-cells and NK cells IL-7 IL-15 50.2 18.4 57.9 18.6 32.4 4.3 47.9 18.2 53.2 19.9 21.2 6.8 NS NS 50.2 18.4 57.9 18.6 32.4 4.3 51.5 19.3 51.7 21.5 20.0 6.9 NS 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
Median Mean SD Median

SH
Mean SD p value Median

ND
Mean SD Median

SD
Mean SD p value

Regulators of T-cells and NK cells IL-7 IL-15 52.5 20.9 61.5 21.7 31.2 6.4 47.9 18.2 53.1 19.9 21.2 6.9 NS NS 65.7 22.7 69.7 25.0 34.4 8.4 51.5 19.3 51.7 21.5 20.0 6.9 <0.01 <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 Median
Regulators of T-cells and NK cells IL-7 IL-15 48.3 18.9 57.2 20.8 26.6 6.6 57.6 20.9

DP SD Median Mean SD p value

Mean

61.8 23.5

30.2 8.0

NS <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
Median Regulators of T-cells and NK cells IL-7 IL-15 50.2 18.4 57.9 18.6 32.4 4.3 48.3 18.9 Mean SD Median

HP
Mean SD p value Median

HC
Mean SD Median

DP
Mean SD p value

57.2 20.8

26.6 6.6

NS NS

50.2 18.4

57.9 18.6

32.4 4.3

57.6 20.9

61.8 23.5

30.2 8.0

NS <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
Th1 cytokines IL-2 IFN- Th2 cytokines IL-3 IL-4 Proinflammatory cytokines IL-1 IL-1 IL-6 IL-12(p40) GM-CSF Chemokines IL-8 IP-10 MCP-1 MIP-1 RANTES Eotaxin Regulators of Tcells and NK cells IL-7 IL-15

SH/SD

HC/NH

HC/ND

HC/SH

HC/SD

NH/SH

ND/SD

HP/DP

HC/HP

HC/DP

0.6226 <0.01

0.4900 <0.05

0.3307 0.2133

<0.005 <0.0005

0.0859 0.7486

0.3418 0.1314

0.6380 0.4331

0.1658 0.0802

0.7718 <0.001

0.1116 0.3687

<0.05 <0.01

0.4091 0.8906

0.2935 0.6215

0.5214 0.5214

<0.001 <0.05

0.3052 0.3645

<0.05 0.2124

0.4955 0.6672

0.3447 0.6845

0.1650 0.7377

0.1220 0.3612

<0.001 <0.05

<0.0001 <0.005 0.0728 <0.01 <0.05 0.1769 0.7562 <0.05 0.1054 0.0583 <0.05

0.0897 <0.0001 0.2024 0.1231 0.4954 0.9854 0.8983 0.1536 0.0826 0.3683 0.0897

0.2425 <0.01 <0.001 <0.05 0.7371 <0.0001 <0.05 0.0703 <0.01 <0.05 0.9465

<0.0001 <0.0001 <0.0001 <0.0001 0.2630 <0.0001 <0.05 <0.0001 <0.0001 <0.0001 <0.005

0.3986 <0.01 0.0767 0.8974 0.7902 0.3364 <0.0001 <0.01 0.8359 0.4793 0.5525

<0.005 <0.0001 0.6141 0.1365 0.7453 0.3856 <0.0001 0.3054 0.2213 0.5190 0.9719

0.5972 0.8749 <0.001 <0.05 0.5183 <0.01 <0.05 <0.001 <0.05 <0.01 0.5336

<0.05 0.2862 <0.001 <0.001 0.3848 <0.001 <0.05 <0.0001 <0.001 <0.01 0.0820

<0.001 <0.001 <0.05 <0.01 0.2580 0.2734 0.8666 <0.01 <0.05 <0.05 0.4639

0.2408 <0.01 0.4365 0.1708 0.9792 <0.01 <0.001 0.5524 0.0959 0.3726 0.7495

<0.001 <0.001 <0.05 <0.001 0.3813 <0.001 <0.0005 0.0649 <0.001 <0.01 0.0656

P values considered significant (p 0.05)

0.7841 0.0897

0.4304 0.2024

0.6034 <0.05

<0.05 <0.0005

0.8451 0.6362

0.6893 0.0877

0.4682 0.0784

<0.01 <0.05

0.7933 <0.05

0.8629 0.1157

0.3039 <0.01

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.

80

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. * p0.05, ** p0.01, *** p0.005, + p0.001, ++ p0.0005, +++ p0.0001

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Figure 3. Th1 and Th2 cytokines: Median amounts (pg/30s): a) IL-2 b) IFN- 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. * p0.05, ** p0.01, *** p0.005, + p0.001, ++ p0.0005, +++ p0.0001

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Figure 4. Pro-inflammatory cytokines: Median amounts (pg/30s): a) IL-1 b) IL-1 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. * p0.05, ** p0.01, *** p0.005, + p0.001, ++ p0.0005, +++ p0.0001

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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. * p0.05, ** p0.01, *** p0.005, + p0.001, ++ p0.0005, +++ p0.0001

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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. * p0.05, ** p0.01, *** p0.005, + p0.001, ++ p0.0005, +++ p0.0001

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 1.6l with a range of 0.17-3.28l. 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- 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- 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-, IL-1, IL-1, 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-1 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-, IL-1, IL-1, 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.

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Orozco showed that IL-1 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-1 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-, 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-, 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-1 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- 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- and Th1 cells are strongly associated with enhanced alveolar bone loss during periodontal infections (Valverde, Kawai et al. 2004). IFN- 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 Bcell 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 proinflammatory cytokines such as IL-1, IL-6 and TNF-. 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-1, tumor necrosis factor- (TNF) 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 Smokings potent inhibition of the activity and amounts of chemokines and proinflammatory 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-1, 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-1 is consistent with Petropoulos et al. (2004) findings (Table 32) whereby the concentration of IL-1 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, IL12(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 proinflammatory cytokines found in this study may be explained through the reduction in IL7 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-1, IL-1 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-1 observed in diseased smokers is consistent with earlier reports by Kamma et al. (2004) who also reported greater total volumes of IL-1 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- 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- 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/l) 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 l) 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-, 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 NS H
Bostrom et al. (1999) (Bostrom, Linder et al. 1999) IL-6 (no diff) Bostrom et al. (2000) (Bostrom, Linder et al. 2000) IL-1ra (no diff) Rawlinson et al. (2003) (Rawlinson, Grummitt et al. 2003) IL-1 * 393.8 (867.1)(pg/l ) 3.2x105 (2.3)(pg/l) IL-1 (no diff) TNF- *

Concentration S D
12 (7.3-18.3) (pg/ml) 10 (0-28) (pg/ml) 61.50 (34.50113.75) (pg/ml) 59.62 (40.28-71.89) (pg/ml) 73.1 (61.0) (pg/l ) 2714.5 (4416.2)(pg/ul) 5.8x105 (9.7) (pg/ul)

Total volume NS D
61.0 (42-177) (pg/ml) 5.0 (0-30.5) (pg/ml) 60.5 (24.75-93.50) (pg/ml) 57.61 (46.92110.06) (pg/ml) 24.5 (29.2) (pg/l) 0.27 (0.40) (l) 1.06 (0.34) (l ) 0.32 (0.42) (l ) 1.39 (0.22) (l )

S D H D

IL-1ra * IL-1 *

3.2x105 (2.3) (pg/l) 3.29 (2.02) (pg/l )

0.19x105 (0.07) (pg/l) 1.59 (1.13) (pg/l)

Petropoulos et al. (2004)(Petropoulos, McKay et al. 2004) Erdemir et al. (2004) (Erdemir, Duran et al. 2004)

TNF- (no diff)

0.51 (0.81) (pg/l)

1.07 (1.32) (pg/l)

IL-6 (no diff)

0.57 (0.75) (pg/l)

0.32 (0.38) (pg/l)

* Statistically significant (p0.05) 97

Table 34. Continued

Kamma et al. (2004) (Kamma, Giannopoulou et al. 2004) IL-1 * 7.85 (pg/30s) 12.07 (pg/30s) 0.89 (pg/30s) 24.00 (pg/30s) 61.37 (pg/30s) 1.90 (pg/30s) 5.04 (pg/30s) 72.96 (pg/30s) 17.97 (pg/30s) 10.33 (pg/30s) 1.70 (pg/30s) 20.43 (pg/30s) 62.37 (pg/30s) 3.08 (pg/30s) 6.04 (pg/30s) 68.30 (pg/30s)

IL-4 (no diff)

IL-6 *

IL-8 *

* Statistically significant (p0.05)

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-, IL-1, IL-1, 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-1 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-1, IL-6, IL7, 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 individuals ability to combat bacterial infection found in periodontitis. Novel cytokines such as IL-7, IL12(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.

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