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Periodontitis in twins : smoking, microbiological and immunological aspects

Torres de Heens, G.L.

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Periodontitis in Twins


Smoking, microbiological and immunological aspects

Gaudy L. Torres de Heens

Gaudy L

. Torres de Heens

UITNODIGING

Voor het bijwonen van de openbare verdediging van het proefschrift van

Gaudy L. Torres de Heens

Receptie ter plaatse na afloop van de

promotie.

Paranimfen:

Sergio [email protected]: 06-28305362

Tony Begazo

[email protected]: 06-83240412



Gaudy Luzcar Torres de Heens

Copyright © 2009, Gaudy Luzcar Torres de Heens

Periodontitis in Twins. Smoking, microbiological and immunological aspects

ISBN 978-90-9025050-2

Printed by Ipskamp Drukkers BV, Enschede

Cover: "Induction Chromatique 54.” Carlos Cruz-Diez



ACADEMISCH PROEFSCHRIFT

ter verkrijgen van de graag van doctor

aan de Universiteit van Amsterdam

op gezag van de Rector Magnificus Prof. Dr. D.C. van den Boom

in het openbaar te verdedigen in de Agnietenkapel

op dinsdag 09 februari 2010, te 14.00

door

Gaudy Luzcar Torres de Heens

Geboren te Caracas, Venezuela

Promotiecommissie

Promotores: Prof. dr. U. van der Velden

Prof. dr. B. G. Loos

Overige leden: Prof. dr. F. Abbas

Prof. dr . W. Beertsen

Prof. dr. V. Everts

Prof. dr. A. J. van Winkelhoff

Dr. G. A. van der Weijden

Faculteit der Tandheelkunde

The research presented in this thesis was performed at the Department of

Periodontology of the Academic Centre for Dentistry Amsterdam (ACTA)

“Science is the art of the systematic observation.” Gaudy Torres de Heens

to my parents

to Patrick, Salvador en Aïda

to my patients

Contents

Chapter 1

Chapter 2

Chapter 3

Chapter 4

Chapter 5

Chapter 6

General Introduction

Effects of smoking on the ex vivo cytokine production in

periodontitis

Cigarette smoking enhances T cell activation and a Th2 immune

response; an aspect of the pathophysiology in periodontal disease

Monozygotic twins are discordant for chronic periodontitis.

Clinical and bacteriological findings

Monozygotic twins are discordant for chronic periodontitis.

White blood cell counts and cytokine production after ex vivo

stimulation.

Summary and Conclusions

Samenvatting

Resumen

Acknowledgements

9

23

43

59

83

105

115

125

141

9

Chapter 1

General Introduction

10

Introduction

The tissues that surround and support the teeth are collectively called the

periodontium (Socransky & Haffajee 1997). Their main functions are to support,

protect, and provide nourishment to the teeth. The periodontium consists of

cementum, alveolar bone, periodontal ligament, and gingiva. Microbial plaque

accumulating in the gingival crevice region induces an inflammatory response.

Gingivitis, the mildest form of periodontal inflammation, involves the marginal soft

tissue structures surrounding the teeth, and is characterized by redness, swelling and

bleeding of the gingiva. Gingivitis, one of the most common human diseases, affects

50-90% of adults worldwide and is readily reversible by simple, effective oral hygiene

(Pihlstrom et al. 2005).

In certain susceptible individuals, gingivitis can extend deeper into the tissues,

resulting in periodontitis, which involves the loss of supportive connective tissue and

alveolar bone (Albandar & Rams 2002). The most common form of periodontitis,

chronic periodontitis, has been reported to affect up to 30% of the adult population

with approximately 7-13% of adults affected with severe disease (Nares 2003). Some

loss of periodontal attachment and alveolar bone is to be expected in older persons,

but age alone in a healthy adult does not lead to a critical loss of periodontal support

(Burt 1994). Clinically, the disease is characterized by deepened periodontal pockets

as a result of loss of connective tissue attachment and bone loss in conjunction with

bleeding upon pocket probing due to the inflammation. With disease progression teeth

become mobile and may show migration and in some cases excessive recession of the

gums. If left untreated, teeth may eventually exfoliate. In humans, periodontitis is one

of the most important causes of tooth loss in adult age (Akhter et al. 2008, Ong 1998).

Treatment of periodontitis is generally good possible and should establish periodontal

health, prevent recurrence of disease, and preserve the dentition in state of health,

comfort, and function. This goal can be accomplished by various non-surgical and

surgical therapies, depending on the specific treatment objective (Pihlstrom et al.

2005).

With a new awareness that not all individuals were equally susceptible to

periodontal disease, scientists turned their attention to the “risk” for disease, i.e. the

probability that an individual will develop periodontitis (Van der Velden et al. 2006,

Williams 2008). In this respect, three types of variables are important (Beck 1994).

11

The first type are risk factors; i.e., characteristics that are thought to be aetiologic for

the disease of interest and that have shown to increase one’s odds for developing a

disease, e.g. a specific bacterium. The second type of variable involves background

characterisics that are not considered to be aetiologic, are immutable to change and

are often referred to as a risk determinant (e.g. age, gender and race). The third type of

variable are risk predictors. These are usually either biological markers that are

indicative of disease or disease progression (e.g. cytokine production or gene

polymorphism associated to periodontitis) and historical measures of the disease (e.g.

past evidence of periodontal disease) (Beck 1994).

Periodontitis is a multifactorial infectious disease. There is no doubt that the

interaction between host immune mechanisms and periodontal bacteria is fundamental

in the different manifestations of chronic inflammatory periodontal disease (Gemmell

& Seymour 1994b). The subgingival microflora in periodontitis can harbor hundreds

of bacterial species, but only a small number is considered etiologically important and

has been associated with progression of disease (Concensus report 1996). Due to the

episodic nature of periodontal disease and our lack of sensitive diagnostic clinical

tests that can detect disease activity, it is difficult to ascertain the causality of specific

pathogens in periodontitis (Ezzo & Cutler 2003). However, three putative periodontal

pathogens have been implicated as risk factor and risk predictors in periodontitis, i.e.

Aggregatibacter actinomycetemcomitans (Aa) (Fine et al. 2007, Van der Velden et al.

2006) as a risk factor and Porphyromonas gingivalis (Pg) and Tannerella forsythia

(Tf) as risk predictors (Ezzo & Cutler 2003, van Winkelhoff et al. 2002). Subgingival

periodontal pathogens are essential for the initiation and progression of the disease,

although it is the resulting host reaction that primarily mediates tissue damage in the

susceptible host (Gaffen & Hajishengallis 2008). In some individuals, neutrophils and

cell mediated immunity may limit the extent of attachment loss. However, in

susceptible people as determined by genetic and lifestyle factors, the clearance of the

periodontal pathogens by the host immune response seems to be unsatisfactory and

therefore disease progression may occur (Gemmell & Seymour 2004).

Our understanding of the pathogenesis of periodontitis has continued to evolve

as our understanding of the underlying mechanisms of the inflammatory immune

response has become more sophisticated. Tissue injury mediated by inflammation is a

consequence of the inability of the host to resolve the inflammation, not the initial

inflammation itself. This is an important distinction, because inflammation is

12

necessary to protect the host from infection, but persistent inflammation can also

cause disease and irreversible damage (Van Dyke & Serhan 2003). In this respect, the

ultimate outcome of periodontal disease in adults depends on patient susceptibility

which is modulated by the host immune response.

The host immune response in periodontitis

In response to an inflammatory trigger, like periodontal pathogens, two

distinct, yet intricately linked, immune responses occur: innate and adaptive. The

innate immune response is the first line of defense against invading microorganisms.

It acts through the recruitment of cells, activation of the complement system,

identification and removal of foreign substances, and activation of the adaptive

immune system. The main players in innate immunity are phagocytes such as

neutrophils, macrophages, and dendritic cells. These cells can discriminate between

pathogens and self by utilizing signals from the Toll-like receptors (TLRs). Toll-like

receptors (TLR) are signal molecules essential for the cellular response to bacterial

cell wall components (Folwaczny et al. 2004). In mammals there are at least 10

members of the TLR family that recognize specific components conserved among

microorganisms. Stimulation of immune cells by the binding of various bacterial

components (e.g. lipopolysaccharides, bacterial DNA) to TLRs causes an immediate

defensive response (Akira 2003, Mahanonda & Pichyangkul 2007). Upon activation

of TLRs, an intracellular signaling cascade is stimulated that leads to the activation of

transcription factors and the production of various cytokines (Graves 2008). These

chemical signals regulate the traffic of leukocytes and control the leukocyte response

(Van Dyke & Serhan 2003). The production of appropriate cytokines in response to

infection is necessary for the development of protective immunity (Seymour et al.

1996). It has been shown that these released mediators direct the subsequent

development of differential specific immune responses by eliciting subsets of T helper

(Th) cells (Kinane & Lappin 2001). Essentially, two types of Th cells develop from

the same CD4 + T cell precursor, termed Th1 and Th2 cells, which secrete different

patterns of cytokine (Gemmell et al. 2007).

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

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

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

many physiological responses (Seymour & Gemmell 2001a). Cytokines produced by

13

monocytes and other antigen presenting cells (APC) drive polarization of non-

committed T helper cells (Th) into either Th1 or Th2 (Kidd 2003). In particular,

interleukin (IL)-12 produced by monocytes, macrophages, neutrophils and dendritic

cells during innate immune responses, promotes naive T cells to differentiate into

Th1, while the anti-inflammatory IL-10 cytokine favors Th2 differentiation (Gemmell

& Seymour 2004). Th1 lymphocytes are characterized by the production of interferon

(IFN)- and IL-2, whereas Th2 cells produce mainly IL-4 and IL-13 (Mosmann &

Coffman 1989, Seder 1994). In periodontitis it is generally accepted that the stable

lesion is largely mediated by cells with a Th1 cytokine profile, while the progressive,

unstable lesion involves Th2-like cells (Gemmell & Seymour 2004). It is clear that the

immunoregulatory control of Th1/Th2 cytokine profiles is fundamental in determining

the ultimate outcome of periodontitis. Advances in knowledge of the pathogenesis of

periodontitis suggest that a group of disease modifiers, including smoking and

genotype, contribute strongly to individual patient differences in the susceptibility to

periodontitis (Kornman 2008). Indeed, many of the widespread systemic effects of

smoking may provide mechanisms for the increased susceptibility to periodontitis and

the poorer response to treatment.

Cigarette Smoking and periodontitis

Smoking is recognized as an important risk predictor in periodontitis (Palmer

et al. 2005). Various factors contribute to the deleterious periodontal effects of

smoking, including alterations in both microbial and host response factors (Johnson &

Hill 2004). Cigarette smoking affects the oral environment and ecology, the gingival

tissues, the vasculature, the inflammatory response and the homeostasis and healing

potential of periodontal connective tissues (Palmer et al. 2005). There are now a

number of studies that suggest a trend for smokers to harbour more or greater

numbers of potential periodontal pathogens than non-smokers without increasing the

amount of plaque (Haffajee & Socransky 2001, Kamma et al. 1999, Zambon et al.

1996a). Gingival blood flow is suppressed by smoking (Morozumi et al. 2004).

Indeed, Nair et al. (2003) have shown an increased gingival bleeding in subjects on a

successful quit-smoking programme, suggesting certain recovery of the inflammatory

response (Nair et al. 2003). Smoking may affect multiple functions of neutrophils and

may shift the net balance of neutrophil activities into the more destructive direction

(Palmer et al. 2005). It has also been shown that cigarette smoke results in reduced

14

concentration of immunoglobulin G (IgG) antibodies (Graswinckel et al. 2004).

Increased numbers of T-cells and elevated T-cell responsiveness in patients who

smoke may be one of several explanations why smoking increases the risk for

periodontitis (Loos et al. 2004). The majority of clinical trials show significantly

greater reductions in probing depths and bleeding on probing, and significantly

greater gain of clinical attachment following non-surgical and surgical treatments in

non-smokers compared with smokers (Heasman et al. 2006). Furthermore, after

periodontal therapy, smoker patients remain culture positive for periodontal

pathogens, which may contribute to the often observed unfavourable treatment results

in smoker periodontitis patients (Van der Velden et al. 2003). However, the

mechanism of how the different effects of smoking contributes to increase the severity

of periodontal breakdown in smoker periodontitis patients needs to be further

investigated.

Genetics in Periodontitis

Genetic variance, environmental exposures and life style factors are the key

determinants to phenotypic differences between individuals. Most diseases have a

genetic component in their etiology. However, the extent of the genetic contribution

to disease can and does vary greatly. Life style factors may diversely affect the

phenotypic expression of the genotype of different individuals (Hodge &

Michalowicz 2001). Therefore, differences in disease susceptibility may have, besides

a genetic and environmental component, a life style constituent. In addition, according

to the model proposed by Kinane and Hart (2003), the interaction between genetic,

environmental and life style factors is as important as these factors alone, and may be

pivotal to determine periodontitis (Kinane & Hart 2003).

Evidence for a genetic predisposition to periodontitis comes from three areas

of research: (1) population studies, (2) family studies, and (3) twin studies. In this

respect, probably the most powerful method to study genetic aspects of periodontal

disease is the twin model. Studying phenotypic characteristics of twins is a method of

differentiating variations due to environmental and genetic factors. Despite the twin

model being a powerful method of providing evidence of a genetic predisposition to

periodontitis, very few twin studies of chronic adult periodontitis have been

conducted, possibly, due to the enormous difficulty in gathering a homogeneous twin

population representative for the disease. Existent twin studies in periodontitis have

15

suggested a substantial role of genetic factors in the etiology (Corey et al. 1993,

Michalowicz et al. 1991, Michalowicz et al. 2000, Mucci et al. 2005). Nevertheless

the main limitation of these studies is the fact that subjects were selected based on

their twinship rather than their periodontal condition, resulting in populations with

mild periodontal breakdown.

Genetic polymorphisms in a candidate gene approach have been explored as

risk predictors for periodontitis. There is limited evidence that some polymorphisms

in the genes encoding interleukin (IL)-1, Fc gamma receptors, IL-10 and the vitamin

D receptor, may be associated with periodontitis in certain ethnic groups. However

relatively large variations in carriage rates of the Rare (R)-alleles among studies on

any polymorphism were observed (Loos et al. 2005). To date, genetic studies in

relation to periodontitis have revealed only one major disease gene, a functional

polymorphism for the cathepsin C gene, displaying decreased cathepsin C activity and

responsible for the occurrence of pre-pubertal periodontitis (Hart et al. 2000).

However till today there is no strong evidence for target genes and gene

polymorphisms that play a key role in the susceptibility to and severity of

periodontitis.

Aim and scope of this thesis

Periodontitis results from the inflammatory response to periodontopathic

bacteria. However the susceptibility of the patient determines the ultimate outcome of

the disease progress (Gemmell et al. 2002). Lifestyle factors, such as smoking, not

only modify the host response, but they also may diversely affect the phenotypic

expression of the genotype of different individuals, thereby being major determinants

of the enormous variation in susceptibility (Hodge & Michalowicz 2001). In the

genetically susceptible host, inappropriate immunological mechanisms can be

triggered by environmental factors. In the present thesis, we attempted to get more

insight in the susceptibility to periodontitis.

The main purpose of this thesis was to determine the contribution of the host

genetic make up in chronic adult periodontitis by means of the classical twin model.

Since smoking could be a confounding factor in the analysis of the twin population, it

was decided to explore the influence of smoking on the immune response in

periodontitis, in a non-twin population, previous to the start of the twin study. Stable

periodontal lesions are regarded to be predominantly Th1, whereas the active lesions

16

express a Th2 profile. In addition, the Th1/Th2 balance in periodontitis has been

investigated and currently periodontitis is considered as a Th2-type disease. We

hypothesized that the Th2 pattern in periodontitis may be accentuated by smoking,

accelerating disease progression and relapse in treated periodontitis patients. Since

monocytes are considered as orchestrating cells in the innate and adaptive immunity,

we firstly studied the monocytic cytokines secreted after ex vivo stimulation. We

hypothesized that in treated periodontitis patients the monocytic cytokines

representative for a shift towards Th2 immune response was increased in smokers

compared to non-smokers (Chapter 2).

The monocytic cytokine production gives only an indication of the ensuing

adaptive immune response. However, to investigate the actual Th1/Th2 balance, the

measurement of the lymphocytic cytokine production was needed. It was supposed

that the cytokine pattern to be found in the lymphocytic cytokine production should be

in line with the findings from monocytes. In this respect, if indeed smoking

potentiates the monocytic Th2 immune response in the studied periodontitis

population (i.e. lower IL-12 p40/IL-10 ratio in smokers) consistently, the lymphocyte

cytokine production should reflect and confirm this finding (i.e. higher IL-13 cytokine

production in smokers) (Chapter 3).

Previous twin studies on periodontal disease have suggested that in the

population approximately half of the variance in the clinical parameters of

periodontitis is attributed to genetic variance (Michalowicz et al. 1991, Michalowicz

et al. 2000). Contrary, the presence of periodontal pathogens has been shown not to be

under the genetic control (Michalowicz et al. 1999). In their twin study on the

presence of periodontal bacteria, Michalowicz et al. (1999) concluded that any effect

of the genetic make up on the presence of the periodontal microorganisms is not

apparent in adulthood. However, since their conclusions are founded on the clinical

and microbiological results of twins mildly affected by periodontal breakdown, they

warned that their results may not necessarily be extrapolated to more advanced

disease states. Thus, the question whether these findings would be also applicable for

moderate to severe periodontitis was an important one. Therefore we studied in

monozygotic (MZ) and dizygotic (DZ) twin pairs, selected on the basis of one sib of a

twin pair having moderate to severe chronic periodontitis, the contribution of genetics,

life style factors and periodontal pathogens to the clinical phenotype of the disease

(Chapter 4). In addition, we investigated the extent of concordance in number of

17

white blood cells and monocytic and lymphocytic cytokine secretion after ex vivo

stimulation among the previously studied twin population selected on the basis of one

sib of a twin pair suffering from moderate to severe chronic periodontitis (Chapter 5).

The main findings and conclusions of these studies are summarized in Chapter 6 and

future lines of investigations are suggested.

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treatment on the subgingival microflora. Journal of Clinical Periodontology

30, 603-610.

van Dyke, T. E. & Serhan, C. N. (2003) Resolution of inflammation: a new paradigm

for the pathogenesis of periodontal diseases. Journal of Dental Research 82,

82-90.

van Winkelhoff, A. J., Loos, B. G., van der Reijden, W. A. & van der Velden, U.

(2002) Porphyromonas gingivalis, Bacteroides forsythus and other putative

periodontal pathogens in subjects with and without periodontal destruction.


Williams, R. C. (2008) Understanding and managing periodontal diseases: a notable

past, a promising future. Journal of Periodontology 79, 1552-1559.

Zambon, J. J., Grossi, S. G., Machtei, E. E., Ho, A. W., Dunford, R. & Genco, R. J.

(1996) Cigarette smoking increases the risk for subgingival infection with

periodontal pathogens. Journal of Periodontology 67, 1050-1054.

22

23

Chapter 2

Effects of smoking on the ex vivo cytokine production in

periodontitis

G. L. Torres de Heens1, R. Kikkert2, L. A. Aarden2, U. van der Velden1

and B. G. Loos1

1Department of Periodontology, Academic Center for Dentistry Amsterdam, ACTA, The

Netherlands and 2Department of Immunopathology, Sanquin Research and Landsteiner Laboratory,

Academic Medical Center, University of Amsterdam, The Netherlands

Journal of Periodontal Research 2009; 44, 28-34

24

Abstract

Background and Objective: Smoking is associated with increased severity of

periodontitis. The underlying mechanisms of this phenomenon are not well

understood. The purpose of the present study was to compare the monocyte-derived T

cell directing (Th1/Th2) response and pro-inflammatory cytokine production in ex

vivo whole blood cell cultures (WBCC) of smoking and non-smoking chronic

periodontitis patients.

Material and Methods: Venous blood was collected from 29 periodontitis patients

(18 non-smokers and 11 smokers) receiving supportive periodontal treatment, and

diluted 10-fold for WBCC. WBCC were stimulated for 18 hours with Neisseria

meningitidis lipo-oligosaccharide (LOS) and Porphyromonas gingivalis sonic extract

(Pg-SE). The production of the T cell directing cytokines interleukin (IL)-12 p40 and

IL-10, as well as the pro-inflammatory cytokines IL-1 , IL-6 and IL-8, was measured

in the culture supernatants.

Results: After LOS stimulation of WBCC, smokers showed a lower IL-12 p40/IL-10

ratio than non-smokers (p<0.05). IL-1 production was significantly lower in smokers

as compared to non-smokers after stimulation with both LOS and Pg-SE (p<0.05). IL-

6 and IL-8 production was similar between both groups, for both LOS and Pg-SE.

Conclusion: A more pronounced Th2 response in smoking periodontitis patients may

be related to increased severity of the disease.

25

Introduction

Periodontitis is a chronic, multifactorial, infectious disease of the supporting

tissue of the teeth. Periodontitis patients suffer from gradual loss of tooth attachment

in the jaw bone leading to periodontal pockets, receding gums, loose teeth and

eventually tooth exfoliation (Kinane & Lappin 2001). The onset and progression of

periodontitis is due to an imbalance of the interaction between bacterial pathogens and

host immunity. Host immunity is greatly influenced by both genetic susceptibility and

environmental risk factors (Page et al. 1997). Although several specific

periodontopathogens have been implicated in the disease, two Gram-negative

bacteria, Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans have

the strongest association (van Winkelhoff et al. 2002). Cigarette smoking is

considered as one of the most important environmental risk factors in periodontitis

(Martinez-Canut et al. 1995) as more clinical attachment loss and bone loss have been

observed in smoking than in non-smoking patients (Grossi et al. 1995, Grossi et al.

1994). Moreover, smoking may be responsible for a less favorable outcome after

periodontal treatment and may more frequently cause disease progression despite a

strict periodontal maintenance care program (Darby et al. 2005).

Monocytes play a crucial role in orchestrating the host immune system. When

triggered by whole bacteria as well as bacterial components, monocytes produce

cytokines (monokines) which direct both innate and adaptive immunity (Seymour &

Gemmell 2001b). Monokines such as the pro-inflammatory interleukin (IL)-1 , IL-6,

IL-8, IL-12 and anti-inflammatory IL-10 have been shown to be part of the

inflammatory response in periodontitis and most likely may determine the host

susceptibility and thereby variation in periodontal destruction (Gemmell et al. 1997,

Gemmell et al. 2002, Niho et al. 1998, Seymour & Gemmell 2001a).

Cytokines derived mainly from dendritic cells, monocytes and macrophages,

play a pivotal role in directing lymphocytic differentiation of non-committed

precursors CD4+ T cells into either T-helper (Th)1 or Th2 cells (Kidd 2003). In

human infections, an imbalance of Th1 and Th2 cytokine production may be related

to disease progression (Kidd 2003). The role of T-helper cells in amplifying immune

responsiveness is well established. In periodontitis, the nature of the lymphocytic

infiltrate seems to be crucial in disease progression (Sigusch et al. 1998). A Th1

cytokine profile is the major mediator in the early/stable lesion, while the dominance

26

of B-cells/plasma cells in the advanced/progressive lesion would suggest a role for

Th2 cells. Therefore, it is likely that in those later stages, changes in cytokine profiles

that modulate the Th1/Th2 balance may affect the susceptibility to or the course of the

periodontal infection (Gemmell et al. 2002). Previous studies have shown that

periodontitis patients display a monocytic-cytokine profile which may favor a Th2

immune response. Indeed, periodontitis is currently regarded as a Th2-type disease;

therefore, the Th2-monocytic promoting phenotype may be an important risk factor

(Fokkema et al. 2002, Gemmell & Seymour 1994a).

Differences in monocytic cytokine production between smoking and non-

smoking periodontitis patients have been scarcely investigated. However, clinical data

demonstrate that, compared to non-smokers, patients who smoke show a more severe

disease and relapse during supportive periodontal treatment (SPT). In other

inflammatory diseases it has been shown that tobacco smoke may exacerbate disease

progression through “priming” of immune cells toward a Th2 phenotype (Byron et al.

1994, Cozen et al. 2004). In several inflammatory conditions, including periodontitis,

ex vivo stimulation assays of peripheral blood cells with LPS stimulant have been

used as a measure of the host immune capacity (Fokkema et al. 2003, Malave et al.

1989, Swaak et al. 1997, van der Pouw Kraan et al. 1997). Monocyte responsiveness,

both in isolated monocyte cell cultures and whole blood cell cultures (WBCC), is

regarded as a reliable measure for the in vivo situation (van den Heuvel et al. 1998).

The aim of the present study was to compare the ex vivo production of IL-1 ,

IL-6, IL-8, IL-10, and IL-12 p40 in whole blood cell cultures of smoking and non-

smoking periodontitis patients positive for Porphyromonas gingivalis (P. gingivalis),

after stimulation with lipooligosaccharide from Neisseria meningitidis (LOS) and a

sonic extract of P. gingivalis (SE-Pg).

Material and Methods

Patients

The study population consisted of patients referred to our clinic (Department

of Periodontology at the Academic Center for Dentistry Amsterdam [ACTA]) for the

treatment of periodontal disease. Patients were selected from a pool of 900

consecutive patients that were sampled for bacteriological investigation at the intake

before periodontal treatment. The selection criteria included: 1) Western European

27

Caucasian descent, 2) diagnosis of chronic adult periodontitis at intake, 3) receiving

SPT after non-surgical and surgical treatment had been completed, 4) age between 40

and 60 years, 5) presence of 20 permanent teeth, 6) periodontal bone loss of 1/3 of

the total root length at 2 teeth as visible on peri-apical radiographs, and 7)

subgingival presence of P. gingivalis. Exclusion criteria were: 1) presence of any

systemic condition that may affect the periodontal status, 2) pregnancy, 3) use of

antibiotics within the last 6 months preceding the study, and 4) use of any medicine

that may interfere with the periodontal health. This selection resulted in 48 potentially

eligible patients. Of the 48 patients, 29 volunteered to participate in the present study.

Subsequently, patients were classified into two groups according to the reported

smoking status: a) non-smokers: those who had never smoked or had ceased smoking

more than 10 years before entering the study, and b) smokers: current smokers who

had been smoking for at least 10 years with a consumption of 10 cigarettes/day (c/d).

The final study population included 18 non-smokers and 11 smokers. For each

subject, all teeth were radiographically examined for interproximal bone loss at the

mesial and distal aspects, using cemento-enamel junction (CEJ) of the tooth and the

bone crest as reference points. With the use of a translucent plastic ruler (Schei ruler

technique), the percentage of bone loss at the deepest proximal site of each tooth was

measured.

Approval for this study was obtained by the Medical Ethical Committee of the

Academic Medical Center of the University of Amsterdam. Participants were

informed both verbally and in writing about the purpose of the study, and provided

signed informed consent.

Stimuli

Lipo-oligosaccharide (LOS) as used previously (van der Pouw Kraan et al.

1995) was purified from Neisseria meningitidis, strain H44/76 (a kind gift from Dr. J.

Poolman, RIVM, Bilthoven, The Netherlands). P. gingivalis strain 381 was grown in

brain heart infusion broth enriched with hemin (5 mg/l) and menadione (1 mg/l) in an

anaerobic atmosphere (80% N2, 10% H2, 10% CO2) for 48 h at 37 °C during 48 h at

37 °C. The P. gingivalis bacteria were harvested in the log phase, pelleted by

centrifugation (8000 g), washed three times in phosphate-buffered saline (PBS), and

resuspended in PBS at a concentration of optical density (OD)690 =1, corresponding to

approximately 7 x 108 colony forming units per ml. Aliquots (500 l) of resuspended

28

bacteria were disrupted using a sonifier in a sonifying vessel on ice (Soniprep MSE

150, amplitude 18, 4 min, 5 s intervals).

The degree of disruption of the bacteria was assessed by phase-contrast

microscopy and with Gram-staining by light microscopy. Sonicates were stored at 4

ºC until use. Before use, P. gingivalis sonicates were centrifuged (8000 g, 1 min) and

used in WBCC as described below.

A mouse monoclonal antibody raised against human CD3 (anti-CD3,

CLB-T3/4.E) was from CLB, Amsterdam, The Netherlands and has been previously

described (van Lier et al. 1987b).

Whole Blood Cell Cultures

Preliminary experiments were performed to determine the most optimal LOS

and Pg-SE concentration for the WBCC. Whole blood samples of periodontally

healthy donors were used for this purpose. From each subject, venous blood was

collected by venipuncture from the antecubital fossae in a sterile pyrogen-free blood

collection tube (Vacuette, Greiner, Alphen a/d Rijn, The Netherlands) containing

sodium heparine and diluted 10-fold in pyrogen-free Iscove’s modified Dulbecco’s

medium (IMDM, Bio Whittaker, Verviers, Belgium) supplemented with 0.1% fetal

calf serum (FCS, Bodinco, Alkmaar, The Netherlands), 100 IU/ml penicillin, 100

g/ml streptomycin (Gibco, Merelbeke, Belgium), and 15 IU/ml sodium-heparin (Leo

Pharmaceutical Products B.V., Weesp, The Netherlands). Diluted whole blood in 200

l flat-bottom microtitre culture plates (Nunc, Roskilde, Denmark) was stimulated

during 18 hours with different LOS concentrations (1000, 250, 62, 16 pg/ml) (van der

Pouw Kraan et al. 1995) or Pg-SE dilutions (1:100, 1:400, 1:1600, 1:64000), and

cytokine production of IL-1 , IL-6, IL-8, IL-10 and IL-12 p40 was measured. The

most optimal LOS and Pg-SE concentrations for WBCC stimulation were 1000 pg/ml

and 1:100 respectively (data not shown). Especially for IL-10, the capacity of both

stimulants alone to elicit cytokine at the indicated concentrations was still weak. To

increase IL-10 production we decided to combine monocyte stimulation with a T cell

stimulus. In the 18 hr incubation period anti-CD3 alone did not lead to measurable

cytokine production. However, when combined with monocyte stimuli such as LOS

or Pg-SE, anti-CD3 led to strongly increased production of cytokines such as IL-10

and IL-12 (data not shown).

29

From each subject of the final study population (29 subjects) diluted whole

blood as described previously was stimulated with LOS at a final concentration of

1000 pg/ml or with Pg-SE 1:100 dilution, in the presence of anti-CD3 at 1 g/ml.

Unstimulated diluted whole blood served as a negative control. Supernatants were

harvested and stored at -20 °C until cytokine measurements.

Venous blood of each participant was also collected in an EDTA (K3)-

containing tube (Becton Dickinson Vacutainer System Europe, Meylan, France) for

the determination of the total number of leukocytes, and leukocyte differentiation

(neutrophils, eosinophils, basophils, lymphocytes and monocytes), with standard

automated procedures (Cell-Dyn 4000, Hematology Analyzer, Abbott Laboratories,

Park, Illinois, USA.) operated in the clinical chemistry laboratory of the Slotervaart

Hospital, Amsterdam, The Netherlands.

Toll-like receptor-transfected human embryonic kidney 293 (HEK 293)

cell cultures

Human Embryonic Kidney 293 (HEK 293) cells stably transfected with CD14,

CD14-Toll-like receptor (TLR)2 or CD14-TLR4 were a kind gift from Drs. D.

Golenbock and E. Latz, Worcester, University of Massachusetts Medical School,

Division of Infectious Diseases, MA, USA. Transfected HEK cells were cultured in

IMDM supplemented with 5% heat-inactivated FCS, 100 U/ml penicillin, 100 l/ml

streptomycin, 50 M 2-mercaptoethanol (Sigma-Aldrich, Steinheim, Germany), and 5

g/ml puromycin (Sigma-Aldrich). For stimulation experiments, cells were seeded at

5 x 105 cells per well in 96-well flat-bottomed microtitre plates (Nunc, Roskilde,

Denmark), and stimulated the next day with the appropriate preparation. HEK 293-

CD14-TLR4 cells were stimulated in the presence of 5% human serum as described

elsewhere (25). After 16-20 h incubation, supernatants were harvested for

determination of IL-8 production as a marker for nuclear factor kappa B (NF- B)

activation, i.e. cell activation.

Assays for Cytokines

Cytokine levels of IL-1 , IL-6, IL-8, IL-10, and Il-12 p40 were measured in

the supernatants of WBCC using commercially available enzyme-linked

immunosorbent assay (ELISA) kits (PeliKine Compact™ human ELISA kits, CLB,

30

Amsterdam, The Netherlands) as previously described (van der Pouw Kraan et al.

1997). The plates were read in an ELISA-reader (Labsystems Multiskan Multisoft,

Helsinki, Finland) at 450 nm, with 540 nm as a reference. Cytokine production in

WBCC supernatants was adjusted per 106 monocytes.

Statistical Analysis

The SPSS package version 11.0 for Windows (Chicago, IL, USA) was used

for descriptive data, data analysis and box plot generation. Differences between

smokers and non-smokers were analyzed by Mann Whitney U-test. Differences in

number of subjects per group were tested by Fisher exact tests.

Results

Subject background characteristics age, gender and educational level are

presented in Table 1. Cigarette smoking consumption reported by current smokers

showed an average of 14 c/d during 35 years with a range of 10-20 cigarettes/day. The

levels of total white blood cells were significantly higher in smokers than non-

smokers (7.42 and 5.78 x109/l respectively, p= 0.001). This was mainly due to

increased numbers of neutrophils in smokers compared to their non-smoking

counterparts. Values for eosinophils, basophils, lymphocytes and monocytes did not

differ between the two groups (Table 1). Dental radiographic analysis of subjects at

the moment of intake is presented in Table 2. Smokers had, on average, 16.1 teeth

with 30% bone loss and 5.1 teeth with 50%, while non-smoking patients had 12.1

and 3.7 respectively (differences not significant).

31

Table 1. Characteristics of the study population (non-smoking and smoking periodontitis patients). Values are

means ± standard deviation or number (%) of subjects.

Non-smokers Smokers

(N = 18) ( N = 11 ) p-value

Age (years) 54 ± 5.3 51 ± 7.2 0.310

Gender

Female 11 (61%) 7 (64%) 1.000

Male 7 (39%) 4 (36%)

Education level

High school 6 (33%) 2 (18%) 0.671

> High school 12 (67%) 9 (82%)

Smoking habits

Number of cigarettes/day 0 14.2 ± 4.4

Number of years smoking 0 35 ± 6.9

Total WBC (109/l) 5.78 ± 1.36 7.42 ± 1.11 0.001

Neutrophils (109/l) 3.22 ± 1.11 4.31 ± 0.81 0.005

Eosinophils (109/l) 0.14 ± 0.12 0.19 ± 0.12 0.280

Basophils (109/l) 0.02 ± 0.07 0 ± 0 0.261

Lymphocytes (109/l) 1.90 ± 0.46 2.21 ± 0.77 0.079

Monocytes (109/l) 0.46 ± 0.13 0.50 ± 0.13 0.485

Table 2. Dental radiographic characteristics of the study population at intake (non-smoking and smoking

periodontitis patients). Values are means ± standard deviation.

Non-smokers Smokers

(N = 18) ( N = 11 ) p-value

Number of teeth 26.4 ± 2.7 26.2 ± 3.2 0.964

Teeth 30% bone loss 12.1 ± 7.5 16.1 ± 5.6 0.120

Teeth 50% bone loss 3.7 ± 3.3 5.1 ± 4.3 0.420

After stimulation of WBCC with LOS, supernatants were analyzed for the

cytokines IL-12 p40 and IL-10 (Figure 1, panels A and B). Regarding IL-12 p40 no

32

differences could be assessed between smokers and non-smokers. The median of IL-

10 for non-smokers was 14300 ng/106 monocytes, whereas in smokers the median

was 21300 ng/106 monocytes. However the values were not significantly different (p=

0.217). To explore the balance of Th1/Th2, we calculated the IL-12 p40/IL-10 ratio

(Fig. 1, panel C). Results showed a significant lower ratio in smokers when compared

to non-smokers (p= 0.022), which may indicate a more pronounced Th2 response in

this group. Production of IL-1 , IL-6 and IL-8 after LOS stimulation is presented in

Figure 1, panels D, E and F, respectively. The IL-1 production was significantly

higher in non-smokers than smokers (p= 0.012), whereas no differences were found

for IL-6 and IL-8 cytokine production between the two groups.

In addition to the LOS stimulation experiments, Pg-SE was similarly used to

explore the cytokine production of WBCC when challenged with a periodontal

pathogen. Results are displayed next to the LOS box plots in Figure 1. No difference

in IL-12 p40 production was found between smokers and non-smokers (panel A). The

levels of IL-10 and the calculated IL-12 p40/IL10 ratio showed no difference between

smokers and non-smokers (Fig. 1, panels B and C). Pro-inflammatory cytokine IL-1 ,

as it occurred after LOS stimulation, was significantly higher in non-smokers than

smokers (p= 0.015, Fig. 1, panel D), while IL-6 and IL-8 did not differ between the

groups (Fig. 1, panels E and F).

Smokers and non-smokers produced higher amounts of IL-8 after stimulation

with Pg-SE when compared to LOS (p< 0.001, Fig. 1, panel F) showing a clear

difference in the levels of cytokine production dependent on the stimulation used. It is

well known that Toll-like receptors (TLR) have the ability of transmitting LPS

signaling across the cell membrane. It has been previously shown that the activation

of either TLR2 or TLR4 may influence the TH1/TH2 balance. Therefore, in an effort to

explain the observed differences between LOS and Pg-SE, it was decided to test the

specificity of LOS and Pg-SE used in our experiments for both TLR2 and TLR4 on

transfected HEK 293 cells. After stimulation of the cells with either LOS or Pg-SE,

IL-8 production was measured in harvested supernatants. We observed that LOS

stimulated HEK-CD14-TLR4 but not HEK-CD14-TLR2 to produce IL-8. Inversely,

when Pg-SE was used, only HEK-CD14-TLR2 cells were able to produce IL-8 (one

graph representative of three experiments is shown, Figure 2). So in line with

33

expectations we observed that LOS signaled through TLR4, whereas Pg-SE

stimulated TLR2.

Figure 1. Boxplots for T cell directing monocytic cytokines IL-12 p40, IL-10, IL-12 p40/ IL-10 ratio

(panels A, B, C, respectively), and pro-inflammatory cytokines IL-1 , IL-6, IL-8 (panels D, E, F) in

ng/106 monocytes of whole blood cell cultures from non-smoking ( ) and smoking ( ) periodontitis

patients after 18 hrs stimulation with Neisseria meningitidis LOS (LOS) and Porphyromonas gingivalis

sonicate extract (Pg-SE), in the presence of anti-CD3 1 g/ml. *: p<0.05 and ***: p<0.001

A. D.IL-12 p40 80

LOS Pg-SE

60

40

20

0

B. IL-10

LOS Pg-SE

ng/1

06 mon

ocyt

es

60

40

20

0

50

30

10

Ratio IL-12 p40/IL-10

LOS Pg-SE

ratio

*

7

6

5

4

3

2

10

C.

IL-1ß

LOS Pg-SE

ng/1

06 mon

ocyt

es

25

20

15

10

5

0

**

IL-6E.

LOS Pg-SE

ng/

106 m

onoc

ytes

1200

1000

800

600

400

200

0

IL-8F.

LOS Pg-SE

ng/

106 m

onoc

ytes

4000350030002500200015001000

5000

***

**

*

ng/

106 m

onoc

ytes

34

Figure 2. IL-8 cytokine production of TLR2 and TLR4 in transfected HEK cell cultures induced by LOS

and Pg-SE. HEK 293 cell lines were stimulated with Neisseria meningitidis LOS (LOS) at

concentrations 0.01, 0.1, 1 and 10 pg/ml (panel A) and Porphyromonas gingivalis sonicate extract (Pg-

SE) at serial dilutions (panel B).

Discussion

A body of evidence indicates that both prevalence and severity of periodontal

disease is increased in smokers compared to non-smokers, and that smokers in SPT

show faster relapse and/or progression of disease (Bergstrom 2003, Bergstrom 2004,

Zambon et al. 1996b). However, to date, no comparative study of stimulated cytokine

profile in smoking and non-smoking periodontitis patients exists in the literature. The

present study constitutes a first attempt to explore the effects of smoking on the

monocytic cytokine profile in chronic adult periodontitis patients. In previous studies

a common and highly purified LPS from Escherichia coli (E. coli) has been mostly

used for WBCC stimulation purposes. However, the use of E. coli may be critised.

Firstly, in the in vivo situation and during infection, LPS is surely not the only

bacterial component interacting with immune cells. Secondly, E. coli is not a

periodontal pathogen. In order to overcome these problems to some extent, we

selected periodontitis patients who before treatment were subgingival positive for P.

gingivalis, a major periodontal pathogen, and used next to N. meningitidis LOS, a

sonic extract of P. gingivalis for WBCC stimulation. The addition of anti-CD3 in the

WBCC enhanced the monocytic cytokine production elicited by the single use of

either LOS or Pg-SE. Via stimulation of the T cell, anti-CD3 leads to co-stimulation

Pg-SE dilutions

TLR2TLR4

B

12 3 8 2 0.50

500

1000

1500

2000

IL-8

(pg/

ml)

LOS

TLR2TLR4

A

0.01 0.1 1 100

500

1000

1500

2000IL

-8 (p

g/m

l)

35

of TLR-triggered monocytes, possibly by upregulation of CD40-ligand (Kennedy et

al. 1996). It could be argued that a potential mechanism of co-stimulation would be

multivalent immunoglobulin (Ig) exposure to low-affinity Fc(gamma) receptors

(Fc R) or complement activation. However, to avoid such complications we made use

of an anti-CD3 monoclonal antibody of the IgE isotype which is incapable of

interacting with Fc R and with complement (Van Lier et al. 1987a).

In our experiments the decision of measuring IL-12 p40 and not IL-12 p70 is

supported by previous observations. Firstly, IL-12 p40 subunit can be produced in

large excess over the heterodimer IL-12 p70, favouring IL-12 p40 detection in the

supernatants as IL-12 p70 could be practically undetectable (Bucht et al. 1996, Venier

et al. 2007). Secondly, the production of IL-12 p40 and IL-12 p70 by LPS-IFN- -

stimulated macrophages is affected to the same degree by smokeless tobacco

suggesting that both cytokines may be similarly influenced by tobacco (Petro et al.

2002). Moreover, others have used the measurement of either p40 mRNA or p40 itself

as indicators of total IL-12 production in humans (Chougnet et al. 1996, Fulton et al.

1996). The Th1-promoting activity of IL-12 p40 has been shown and this cytokine is

considered as an indicator of Th1 differentiation (Giambartolomei et al. 2004,

Nakahara et al. 2005, Prebeck et al. 2001, Zaitseva et al. 1996).

Cytokine production has been usually studied in cultures of peripheral blood

mononuclear cells (PBMC). However the study of cytokine production in WBCC has

some advantages over separated cultures. Firstly, the whole blood culture system

reduces the likelihood of endotoxin contamination due to minimal handling of the

cells. Secondly, the risk of cellular activation due to the isolation procedures is

reduced. Thirdly, the WBCC system may represent more closely the natural

environment with the presence of various immunomodulating and pro- and anti-

inflammatory mediators in whole blood. Fourth, disturbances in the ratios of different

cell types due to purification procedures are avoided (Muller et al. 1998, van der

Pouw Kraan et al. 1997). Therefore, the integrity of the cellular interactions is

maintained as best possible, although the whole blood is diluted 1/10. It has been

extensively studied and shown in parallel cultures of whole blood and freshly isolated

monocytes as well as in kinetics that WBCC stimulated with LPS specifically reflect

the behaviour of the monocytes (Snijders et al. 1996, Snijders et al. 1998, van der

Pouw Kraan et al. 1995).

36

The present data show that smokers suffering from periodontitis have a lower

IL-12 p40/IL-10 ratio after LOS stimulation and lower IL-1 production after LOS

and Pg-SE stimulation than their non-smoking counterparts. Recently it was reported

that tobacco smoke leads to increase in cAMP levels (Du et al. 2005). In T cells

cAMP elevations lead to a strongly decreased IL-12 p40/IL-10 ratio (van der Pouw

Kraan et al. 1995). The lower IL-1 production is in agreement with in vitro

experiments that demonstrated that tobacco smoke can inhibit cytokine production by

peripheral blood monocytes, including production of IL-1 (Pabst et al. 1995).

Furthermore, it has been shown that IL-1 is involved in the up-regulation of interferon

gamma (IFN- ) production by Th1 cells and down-regulation of IL-4 production by

Th2 cells (O'Garra & Murphy 1994, Sandborg et al. 1995, Schmitz et al. 1993). Since

we found in smokers lower levels of IL-1 production and a lower IL-12 p40/IL-10

ratio it may be suggested that in periodontitis smoking influences the Th1/Th2

balance into a more pronounced Th2 profile. This may result in continuous polyclonal

B cell activation with less protective antibody production (Gemmell & Seymour

2004) which could explain partly the clinical finding of more severe periodontitis in

smokers.

The current study also showed that more IL-8 was produced in response to Pg-

SE compared to LOS stimulation, both in smoking and non-smoking periodontitis

patients. The finding that Pg-SE stimulates via TLR2, whereas LOS is recognised by

TLR4, might provide an explanation for the difference in IL-8 production. This is in

line with a body of evidence demonstrating striking differences in cytokine gene

transcription in TLR2 and TLR4 activation (Pulendran et al. 2001, Re & Strominger

2001). Alternatively, the difference in IL-8 levels might be caused by differences in

the cell types involved in IL-8 production. Since TLR2, but not TLR4, is substantially

expressed on the surface of human neutrophils, it is likely that Pg-SE activates both

monocytes and neutrophils, whereas after LOS stimulation only monocytes are

activated. (Kurt-Jones et al. 2002). It is clear that the role of TLRs in periodontal

pathogen recognition and subsequent cytokine production is not yet completely

understood, and that the use of different microbial stimuli may be an important

consideration for the interpretation of past and future data.

In summary, the results showed differences between smoking and non-

smoking periodontitis patients in ex vivo cell culture cytokine production. Our

37

findings suggest that the Th2 pattern in periodontitis may be accentuated by smoking.

This hypothesis is consistent with the contention that a strong innate immune response

is associated with a protective Th1 pattern, favoring inflammatory responses that

contain the infection with periodontal pathogens. Therefore, a more pronounced Th2

phenotype may accelerate disease progression and account for the relapse during SPT

as frequently observed in smoking periodontitis patients. Our findings may serve as

another step in revealing generalized inflammatory patterns in smoking periodontitis

patients.

References

Bergstrom, J. (2003) Tobacco smoking and risk for periodontal disease. Journal of

Clinical Periodontology 30, 107-113.

Bergstrom, J. (2004) Tobacco smoking and chronic destructive periodontal disease.

Odontology 92, 1-8.

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43

Chapter 3

Cigarette smoking enhances T cell activation and a Th2

immune response; an aspect of the pathophysiology

in periodontal disease

G. L. Torres de Heens1, U. van der Velden1 and B. G. Loos1

1Department of Periodontology, Academic Center for Dentistry Amsterdam, ACTA,

The Netherlands

Cytokine 2009; 47, 157-161

44

Abstract

Background and Objective: Smoking is a strong risk factor for periodontitis.

Treated patients who smoke show increased risk for further periodontal breakdown,

despite receiving maintenance care. Previous work indicated that such patients have a

monocytic cytokine response favoring Th2 activity. The purpose of the present study

was to investigate the T lymphocytic cytokine production representing Th1 and Th2

subpopulations in smokers and non-smokers.

Material and Methods: Venous blood was collected from 30 treated periodontitis

patients (12 smokers) and 24 healthy subjects (12 smokers). Whole blood cell cultures

were stimulated and interferon (IFN)- and interleukin (IL)-13 were measured in the

culture supernatants, representing type 1 and 2 Th subpopulations respectively.

Results: Unadjusted data showed that smokers had more lymphocytes, and higher

levels of IFN- and IL-13, irrespective of being periodontal patient. However in a

multivariate analysis, increased IFN- production was not significantly explained by

smoking, while higher IL-13 was strongly explained by smoking (21%, p<0.001).

Conclusion: We suggest that the increased Th activity and specifically an elevated

Th2 profile in smokers may constitute a risk for smoking patients which may induce

conversion of periodontal stability into progressive disease. This phenomenon may be

equally important in other conditions, where connective tissue and bone loss are

hallmarks of disease pathophysiology.

45

Introduction

Periodontitis is a chronic inflammatory disease of the supportive tissues of the

teeth characterized by loss of periodontal attachment and alveolar bone.

Approximately 10% of the population suffers from this condition and, if untreated, it

may result in tooth loss. The inflammation is precipitated by the subgingival bacterial

biofilm; a major pathogen associated with periodontitis is Porphyromonas gingivalis

(Pg) (Slots & Ting 1999, van Winkelhoff et al. 2002). An important role in the onset

and progression of periodontitis is played by the host immune response; susceptibility

for periodontitis is greatly influenced by genetic and life style risk factors (Loos et al.

2005, Page et al. 1997). Cigarette smoking is considered as one of the most important

life style risk factors for periodontitis (Bergstrom 2004); more clinical attachment loss

and more alveolar bone loss have been observed in smoking than in non-smoking

patients (Baharin et al. 2006, Grossi et al. 1995, Grossi et al. 1994, Xu et al. 2002).

Moreover, smoking may be responsible for a less favorable outcome after periodontal

treatment and is frequently associated with disease progression despite a strict

periodontal maintenance care program (Baharin et al. 2006, Bergstrom 2004,

Heasman et al. 2006). However exact mechanisms how smoking affects the host are

still unclear.

In any inflammatory response, cytokines produced by monocytes and other

antigen presenting cells (APC) drive polarization of non-committed T helper cells

(Th) into either Th1 or Th2 (Kidd 2003, Mosmann & Coffman 1989). In particular,

interleukin (IL)-12 produced by monocytes, macrophages, neutrophils and dendritic

cells during innate immune responses, promotes naive T cells to differentiate into

Th1, while the anti-inflammatory IL-10 cytokine favors Th2 differentiation (Kidd

2003). Th1 lymphocytes are characterized by the production of interferon (IFN)- and

IL-2, whereas Th2 cells produce mainly IL-13 and IL-4 (Kidd 2003). For the disease

periodontitis it is generally accepted that the stable lesion (as in treated patients in the

periodontal maintenance phase) is largely mediated by cells with a Th1 cytokine

profile, while the progressive, unstable lesion involves Th2-like cells (Gemmell &

Seymour 2004, Gemmell et al. 2007, Seymour et al. 1996). Th2 cells may induce

expansion of activated B cells resulting in local antibody production and the pro-

inflammatory IL-1, the latter inducing tissue destruction.

46

We hypothesize that smoking may convert stable periodontal lesions into

unstable progressive lesions, among other causes, by tipping the Th1/Th2 balance

towards a Th2 dominated host response. This may thus explain why smoking is a well

known risk factor for further periodontal destruction despite supportive therapy

(Baharin et al. 2006, Bergstrom 2004, Palmer et al. 2005). Some studies have

investigated the effect of cigarette smoking and related toxic metabolites on the

Th1/Th2 balance (Byron et al. 1994, Cheung et al. 1988, Cozen et al. 2004, Hagiwara

et al. 2001, Ouyang et al. 2000, Simhan et al. 2005). From these studies, a shift

towards a Th2 phenotype in smokers is most likely occurring. In our previous study

we observed in smoking periodontal maintenance patients a type 2 monocytic

response to lipopolysaccharide (LPS) compared to non-smokers (Torres de Heens et

al. 2009). However, the effect of smoking on the actual Th1/Th2 balance has not been

investigated.

Therefore the aim of the present study was to investigate whether T cells in

smoking periodontal patients who are in maintenance therapy, show a more

pronounced Th2 cytokine profile than similar non-smoker subjects. To confirm that

the observed effect on T cells is indeed related to smoking and not mainly due to

being a periodontal patient, we also recruited a periodontally healthy control group of

smokers and non-smokers. We measured the representative Th1 and Th2 cytokines

IFN- and IL-13 after T cell stimulation with a combination of anti-CD3/anti-CD28.

Materials and Methods Subjects

The study population consisted of patients and personnel from the Academic

Center for Dentistry Amsterdam (ACTA). The selection criteria included: 1) Western

European Caucasian descent, 2) age between 25 and 65 years and 3) being current

smoker (smoking for at least 10 years with a consumption of 10 cigarettes/day

[cig/day]) or being non-smoker (never smoker or ceased smoking more than 10 years

before entering the study). Periodontal patients were subjects attending the

periodontics clinic who had been treated for chronic periodontitis. These individuals

were in a 3-monthly maintenance program. Periodontally healthy controls were

recruited among subjects registered for restorative dental procedures or who visited

the dental school for regular dental check-ups or who worked as personnel in the

47

dental school. For both patients and controls exclusion criteria were: 1) pregnancy, 2)

presence of any other acute or chronic medical condition, including diabetes, viral,

fungal or bacterial infections, or any systemic condition that may affect the

periodontal status, 3) use of antibiotics within the last 6 months preceding the study,

and 4) use of any medicine that may interfere with the lymphocyte function, such as

non-steroidal anti-inflammatory drugs.

The Medical Ethical Committee of the Academic Medical Center of the

University of Amsterdam approved the study. Participants were informed both

verbally and in writing about the purpose of the study, and provided signed informed

consent.

Sample collection and whole blood cell cultures (WBCC)

From each subject, venous blood was collected by venipuncture from the

antecubital fossa in a sterile pyrogen-free blood collection tube containing sodium

heparine (Vacuette, Greiner, Alphen a/d Rijn, The Netherlands). Heparinized venous

blood was used and cultured at a final 1:10 dilution. Whole blood cell cultures

(WBCC) were performed in a 96-well flat bottom microtitre plate (Nunc, Roskilde,

Denmark). All cultures were carried out in endotoxin-free Iscove’s modified

Dulbecco’s medium (IMDM, BioWhittaker, Verviers, Belgium), supplemented with

penicillin (100 IU/ml), streptomycin (100 μg/ml) (Gibco, Merelbeke, Belgium), 0.1%

endotoxin-free fetal calf serum (FCS, Bodinco, Alkmaar, The Netherlands), and 15

U/ml sodium heparin (Leo Pharmaceutical Products B.V., Weesp, The Netherlands).

To stimulate T lymphocytes a combination of a mouse monoclonal antibody against

human CD3 (anti-CD3, CLB-T3/4.E, 1 μg/ml) and CD28 (anti-CD28 CLB-T3/4.E, 1

μg/ml) (both from Sanquin, Amsterdam, The Netherlands) was added to the aliquot of

200 μl of diluted blood, as previously described (Gerards et al. 2003). Cultures were

performed in duplicate and unstimulated diluted whole blood served as a negative

control. After 72 hours of incubation with anti-CD3/anti-CD28, supernatants were

harvested and stored at -20 °C until cytokine analysis was performed.

Venous blood of all participants was also collected in an EDTA (K3)-

containing tube (Becton Dickinson Vacutainer System, Meylan, France) for the

determination of the total number of leukocytes, and leukocyte differentiation

(neutrophils, lymphocytes, monocytes, eosinophils and basophils), with standard

automated procedures (Cell-Dyn 4000, Hematology Analyzer, Abbott Laboratories,

48

Park, IL, U.S.A.) operated in the clinical chemistry laboratory of the neighboring

hospital (Slotervaart, Amsterdam, The Netherlands).

Cytokine assays

Supernatants of WBCC were analyzed for levels of IFN- and IL-13 using

commercially available ELISA kits (PeliKine Compact™ human ELISA kits,

Sanquin) as described previously (van der Pouw-Kraan et al. 1993).

Statistical analysis

Data analyses were performed with the SPSS 15.0 package (SPSS Inc.,

Chicago, IL, USA). Means, standard deviations and frequency distributions for all

characteristics of the study population were calculated. Possible differences in these

background parameters between 4 groups of subjects (smokers and non-smokers for

both periodontal patients and controls) were analyzed with one-way ANOVA (post

hoc testing with Bonferroni correction) or X2-test, where appropriate. The outcome

variables IFN- and IL-13 in culture supernatants were compared between the 4

groups of subjects with non-parametric Kruskal-Wallis tests (post hoc testing with

Bonferroni correction). For further explorative analysis, IFN- and IL-13 values were

first log-transformed since they were not normally distributed (Kolmogorov-Smirnov

goodness-of-fit test p<0.05). A multivariate analysis (backward stepwise linear

regression with p 0.10 to enter and p 0.05 to leave) was performed to identify factors

explaining the observed variation in the IFN- and IL-13 values. For these latter

analyses predictor variables entered were smoking, periodontal patients, age, gender

and number of lymphocytes. p values <0.05 were considered statistically significant.

Results

Subject background characteristics are presented in Table 1. Cigarette

smoking consumption reported by current smokers showed a mean of 14.8 cig/day

during 33.9 years for those with periodontal disease and 17.6 cig/day on average

during 20.6 years for controls. The levels of total peripheral leukocytes were

significantly different among the 4 groups, with smokers having higher levels than

non-smokers. Also the numbers of lymphocytes and monocytes were significantly

different among the 4 groups, showing the same trends as total leukocytes (Table 1).

49

Table 1. Characteristics of the study population (smoking and non-smoking periodontal patients and controls).

Patients Controls

Smokers Non-smokers Smokers Non-smokers

(N = 12 ) (N = 18) (N = 12) ( N = 12 )

Age (years) 53.6 ± 5.8 50.3 ± 3.9 40.4 ± 12.2 39.6 ± 11.1

Gender

Female 7 (64%) 11 (61%) 7 (58%) 7 (58%)

Male 4 (36%) 7 (39%) 5 (42%) 5 (42%)

Smoking habits

Number of cigarettes/day 14.8 ± 5.8 0 17.6 ± 5.8 0

Number of years smoking 33.9 ± 6.7 0 20.6 ± 11.6 0

Total Leukocytes (109/L) 1 7.30 ± 0.84 6.12 ± 1.42 8.61 ± 2.10 2 6.55 ± 1.79

Neutrophils (109/L) 4.30 ± 0.76 3.48 ± 1.20 4.87 ± 1.87 3.91 ± 1.72

Lymphocytes (109/L) 3 2.29 ± 0.32 1.96 ± 0.49 2.76 ± 0.54 4 1.90 ± 0.69

Monocytes (109/L) 5 0.50 ± 0.17 0.47 ± 0.13 0.73 ± 0.26 6 0.54 ± 0.14

Eosinophils (109/L) 0.17 ± 0.11 0.14 ± 0.12 0.21 ± 0.13 0.17 ± 0.13

Basophils (109/L) 0.08 ± 0.02 0.02 ± 0.07 0.03 ± 0.04 0.01 ± 0.03

Values are means ± standard deviation or number (%) of subjects. 1 overall ANOVA p=0.001 2 value is higher than for non-smoking patients (p=0.001) and than for non-smoking controls (p=0.016) 3 overall ANOVA p<0.001 4 value is higher than for non-smoking patients (p=0.001) and than for non-smoking controls (p=0.001) 5 overall ANOVA p=0.002 6 value is higher than for smoking patients (p=0.016) and than for non-smoking patients (p=0.002)

WBCC were incubated with a combination of anti-CD3/anti-CD28 and

supernatants were analyzed for IFN- and IL-13 cytokine production, both indicators

for the direction of the Th response (data presented in Fig 1). Overall a significant

difference between the 4 groups was observed for IFN- (p=0.012). In Fig. 1A it can

be seen that smokers in general had higher levels of IFN- than non-smokers

irrespective of periodontal disease. Post-hoc testing showed that smokers with

periodontal disease produced higher levels than non-smoking controls. Similarly,

among the 4 groups, there was an overall difference for IL-13 production in

50

supernatants of WBCC (p=0.001); smokers produced more IL-13 irrespective of

periodontal disease, and their levels were higher in post hoc testing than in non-

smokers with periodontal disease.

Figure 1. Boxplots for T cell cytokines IFN- (panel A) and IL-13 (panel B), in supernatants from

whole blood cell cultures from smoking and non-smoking subjects with and without periodontal

disease after 72 hrs stimulation with a combination of anti-CD3/anti-CD28 mouse monoclonal

antibody. Overall p values from non-parametric testing (Kruskal-Wallis) in heading; post hoc, adjusted

for multiple testing: * p<0.05, ** p<0.01.

51

To further explore which factors explained significantly the observed variation

in IFN- and IL-13 levels in the WBCC, we applied a linear regression analysis. For

IFN- a significant final model (p<0.001) was obtained, where all included

explanatory variables were retained (Table 2). From this analysis we could infer that

female gender and older age were associated with lower levels of IFN- ; these

variables explained together 25.4% of the variation. Also higher numbers of

lymphocytes and being a periodontal patient correlated positively with IFN- levels in

the supernatants. Smoking contributed not significantly to higher levels of IFN-

(p=0.080); adjusted mean values of IFN- for smokers and non-smokers were

estimated to be 97.7 and 67.8 x 103 pg/ml respectively (Table 3). IL-13 as

independent variable in the regression model yielded the same results as above, since

IL-13 was not retained (p=0.236), suggesting that the IFN- lymphocyte

subpopulation behaved independent of IL-13.

In the final regression model exploring the IL-13 levels which are indicative of

a type 2 T cell reactivity (overall p<0.001, Table 2), smoking was the strongest

explanatory variable (p<0.001); the adjusted correlation coefficient for smoking with

IL-13 was 0.457, explaining 20.9% of the variation. The adjusted mean values for

smokers and non-smokers for IL-13 were estimated to be 3.49 and 1.59 x 103 pg/ml

respectively (Table 3). In addition to smoking, also periodontal patient was retained

(p=0.025, negatively correlated, explaining only 7.3%), while age, number of

lymphocytes and gender were not of influence on the observed variation in the study

population (Table 2). Interestingly, levels of IL-13 were not associated with INF- , as

IFN- levels were not included in the final model. This suggests that an IL-13

producing subpopulation of T lymphocytes behaves independently from the IFN-

subpopulation.

52

Table 2. Results of the multivariate analyses for IFN- and IL-13 values in the supernatants of whole blood

cell cultures, using IFN- and IL-13 as dependent variable and age, gender, periodontal disease, number

(no.) of lymphocytes and smoking status as predictor variables (N=54). In the model exploring IFN- , IL-

13 levels were also entered; in the model exploring IL-13, IFN- levels were also entered.

IFN- 1 IL-13 2

(95% CI)

Explainedvariance p-value (95% CI) Explained

variance p-value

Smoking 3 0.158 (-0.020, 0.336) 5.2% 0.080 0.457 (0.166, 0.518) 20.9% <0.001

Periodontal patient4 0.295 (0.012, 0.396) 8.7% 0.038 -0.270 (-0.377, -0.026) 7.3% 0.025

Age -0.315 (-0.190, -0.001) 9.9% 0.029 n.r. - -

No. of lymphocytes 0.297 (0.020, 0.316) 8.8% 0.027 n.r. - -

Gender5 -0.394 (-0.435, -0.114) 15.5% 0.001 n.r. - -

n.r. = not retained in final model 1 final model: p<0.001, where IL-13 was not retained. 2 final model: p<0.001, where IFN- was not retained. 3 in statistical analysis “non-smoker”= 0, “smoker”=1. 4 in statistical analysis “not a periodontal patient”=0, “being a periodontal patient”=1. 5 in statistical analysis “female”=0, “male”=1.

Table 3. Adjusted mean values (95% confidence interval) for levels of IFN- and IL-13 in

supernatants from the ANCOVA model for all smokers and non-smokers, and Padj-values.

Smokers Non-smokers

(N = 24) (N = 30) Padj-value

IFN- 97.7 (73.1 - 130.3) 67.8 (52.6 - 87.5) 0.080

IL-13 3.49 (2.58 - 4.72) 1.59 (1.21 - 2.08) <0.001

Values are x 103 pg/ml

53

Discussion

This study was conducted to investigate the influence of cigarette smoking on

the type of Th cell response in periodontitis patients who are in maintenance phase.

We added a control group of smokers and non-smokers to support that the

observations in smokers are not due to the disease process itself. In a previous study

we found that in the same type of smoking patients (receiving supportive periodontal

care), T cell directing cytokines derived from monocytic cells showed a more

pronounced type 2 profile than in non-smoking patients (Torres de Heens et al. 2009).

In the present study we stimulated lymphocytes from smoking and non-smoking

periodontal patients and similarly from control subjects to study whether indeed

lymphocytes from smokers are more inclined to a type 2 phenotype. The overall

results show more lymphocytes in blood of smokers and more lymphocytic cytokine

production in smokers. We suggest that the increased IFN- production in the present

study is multifactorial, due to increased numbers of lymphocytes, periodontal status

and strongly influenced by female gender and age, and not significantly by smoking.

For IL-13 levels however, smoking was a strong explanatory variable and being a

periodontal patient a minor explanatory variable, while it was not explained by a

higher number of lymphocytes, age and gender. We suggest from these data that

smoking stimulates the proliferation of a pronounced Th2-type cell subpopulation.

Important to note is that both IFN- and IL-13 productions were independent

of each other, indicative of two independent Th subpopulations. Moreover, the higher

IL-13 levels are not due the disease process itself, as similar findings were in both the

periodontal as well as the control study groups (Fig. 1); on the contrary, from the

multivariate analysis on IL-13 levels, periodontal patient status is inversely correlated

with IL-13. This latter observation and the results of the IFN- multivariate analysis

reflect that the controls and the treated periodontal patients showed a good periodontal

health; IFN- was positively associated with being a periodontal patient, suggestive

for the previously described Th1-associated periodontal stability (Gemmell &

Seymour 2004, Gemmell et al. 2007, Seymour et al. 1996).

Other studies have also investigated the effect of cigarette smoking and related

toxic metabolites on the Th1/Th2 balance (Byron et al. 1994, Cheung et al. 1988,

Cozen et al. 2004, Hagiwara et al. 2001, Ouyang et al. 2000, Simhan et al. 2005). For

example, after ex vivo mitogen stimulation of peripheral blood mononuclear cells

54

(PBMC) from healthy individuals it appeared that smoking was associated with an

increase in IL-13 and IL-4 production in supernatant of cell cultures (Byron et al.

1994, Cozen et al. 2004), whereas IFN- did not differ between smokers and non-

smokers (Cozen et al. 2004). Furthermore, smoking was associated with a lower

number of Th1 cytokine-secreting cells in bronchoalveolar lavage fluid (BALF) in

smokers compared to non-smokers (Hagiwara et al. 2001). Also, cigarette smoke

extracts suppressed the production of IFN- from isolated human PBMC and mouse

spleen cells (Cheung et al. 1988, Ouyang et al. 2000). Thus, smoking seems an

important factor in the Th1/Th2 immunoregulation, promoting a shift towards a Th2

phenotype (Byron et al. 1994, Cozen et al. 2004, Hagiwara et al. 2001). Notably, in a

previous study we found in smoking periodontal maintenance patients a type 2

monocytic response to lipopolysaccharide (LPS) compared to non-smokers (Torres de

Heens et al. 2009); in the current study in a newly recruited study population we

observed that indeed smoking was a strong factor explaining increased levels of IL-

13, the marker cytokine for Th2. It is thus highly likely that the action of cigarette

smoking is related primarily to the activation of Th2 rather than Th1 lymphocytes,

although smoking is associated with increased numbers of lymphocytes.

Previous observations in the literature have shown increased lymphocyte

reactivity in smokers (Loos et al. 2004, Silverman et al. 1975). T cells play a

fundamental role in periodontal disease (Gemmell & Seymour 2004, Gemmell et al.

2007). It has been suggested that susceptible individuals for periodontitis may have a

predominant Th2 type response, with a stimulatory effect on B-cells, which in turn

contribute to tissue breakdown, most likely through release of IL-1 from activated B-

cells (Aoyagi et al. 1995, Bartova et al. 2000, Gemmell & Seymour 2004, Gemmell et

al. 2007, Manhart et al. 1994). In addition, early publications have shown a bias

towards Th2 cytokine production in periodontitis after challenge of immune cells with

two major periodontal pathogens (Jotwani et al. 2003, Mahanonda et al. 2006). A Th1

type cell mediation in periodontal disease may reflect periodontal stability or health.

With the current study we add new information that smoking is a factor promoting a

Th2 type lymphocytic phenotype. This may make smoking periodontal patients in

maintenance care vulnerable for renewed disease progression and may explain the

frequently observed increase of relapse into active periodontitis (Baharin et al. 2006,

Bergstrom 2004, Heasman et al. 2006).

55

This is the first report on the effect of smoking on the actual cytokine

production by lymphocytes in periodontal patients and normal subjects. Our study

provides evidence for earlier speculations that smoking appears to be associated with

increased levels of Th2 cytokines (Byron et al. 1994, Cozen et al. 2004, Torres de

Heens et al. 2009). The present results suggest that cigarette smoking can prime

lymphocytes toward a more pronounced Th2 phenotype, which in turn may

exacerbate or increase severity in periodontitis or may induce recurrence of disease in

treated subjects, possibly resulting in a hyperactivity of B cells in the periodontal

tissues. This phenomenon may be equally important in other conditions where

connective tissue and bone loss are hallmarks of disease pathophysiology.

Acknowledgements

This work was financially supported by the Dental Research Institute (IOT) of

our institution. The authors thank Prof. Dr. L. A. Aarden, Sanquin, Amsterdam, The

Netherlands, for his useful comments.

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59

Chapter 4

Monozygotic twins are discordant for chronic periodontitis

Clinical and bacteriological findings



The Netherlands

Journal of Clinical Periodontology; accepted for publication and in press 2010

60

Abstract

Objectives: The aim of this study was to assess, in monozygotic (MZ) and dizygotic

(DZ) twin pairs of which the proband of the twin pair is suffering from moderate to

severe chronic periodontitis, the contribution of genetics, periodontal pathogens and

life style factors to the clinical phenotype.

Material and Methods: For this study 18 adult twin pairs were selected on the basis

of interproximal attachment loss 5 mm in 2 non-adjacent teeth in 1 twin member.

The study included 10 MZ and 8 DZ twin pairs, in which the periodontal condition,

presence of periodontal pathogens, educational level, smoking behavior and Body

Mass Index was evaluated.

Results: Both MZ and DZ twins were discordant regarding attachment loss and

alveolar bone loss. Discordance was greater in DZ compared to MZ twins. In MZ

twins the discordance could not be explained by education, smoking, Body Mass

Index and periodontal pathogens. In DZ twins 45.6% of the discordance could be

explained by more pack-years of the probands.

Conclusion: The results confirm a possible role of genetic factors in periodontitis.

However, the magnitude of the genetic effects on disease severity may have

previously been overestimated.

61

Introduction

Periodontitis is initiated by microbial plaque, which accumulates at the gingival

crevice region and at present Aggregatibacter actinomycetemcomitans and

Porphyromonas gingivalis are recognized as main periodontal pathogens (Fine et al.

2007, Van der Velden et al. 2006, van Winkelhoff et al. 2002). Although bacteria are

essential for inducing an inflammatory response in the periodontal tissues, they are

insufficient to cause periodontitis as sole etiological factor (Page et al. 1997). Life style

factors, like smoking are also believed to be important for the severity as well as

treatment of periodontitis (Palmer et al. 2005). Moreover, there is now consensus that

genetic factors play a role in the susceptibility and severity to periodontitis (Loos et al.

2005, Tonetti & Claffey 2005).

There are a limited number of family studies on periodontitis, but collectively

their results suggest that periodontitis aggregates in families (Loos et al. 2008).

Although family studies might give a first impression of familial aggregation, they can

not distinguish between the influence of genetic and shared environmental effects as an

explanation for the familial clustering of periodontitis. In this respect twin studies are

especially useful. For chronic periodontitis relatively few twin studies have been

carried out, but the results suggest a substantial role of genetic factors in the etiology

(Corey et al. 1993, Michalowicz et al. 1991a, Michalowicz et al. 2000, Mucci et al.

2005). Nevertheless the latter studies have limitations; the results of Corey et al. (1993)

and Mucci et al. (2005) are based on self reported evidence of periodontal disease and

the subjects in the studies of Michalowicz et al. (1991a,b, 2000) were mildly affected

by periodontal breakdown. Interestingly, results of another twin study of Michalowicz

et al. (1999) suggested that the presence of periodontal bacteria in subgingival plaque

was not determined by host genetic factors. An earlier family study on periodontitis

indicated that the main periodontal pathogens A. actinomycetemcomitans and P.

gingivalis, can be transmitted between parents and their children (Petit et al. 1993).

Therefore, it is plausible that susceptibility or resistance to periodontitis, as it was

proposed for other infectious diseases, may be dependent on genetically controlled

differences in immune responses after pathogen exposure; thus the disease is not

exclusively restricted to the exposure itself, but rather by the mechanisms of the host

elicited in response to the exposure (Baker et al. 2000). This concept combines both

environment, life style and genetics as contributing factors to the risk of multifactorial

diseases like periodontitis.

62

Susceptibility to periodontitis may increase due to the experienced life style

factors. The effect of smoking in the development of chronic periodontitis is well

documented. There is strong evidence that smoking contributes to a higher prevalence

and severity of periodontitis (Albandar & Rams 2002, Baharin et al. 2006, Tomar &

Asma 2000). Moreover, a twin study showed that the nicotine dependence is

influenced for 75% by genetic factors, which provides evidence for a substantial

impact of genetic factors on the smoking behavior (Vink et al. 2005). Another factor

which may promote the progression of periodontitis is overweight. (Ylostalo et al.

2008) found in a large epidemiological study among non-diabetic, non smoking adults

a significant relationship between body weight and periodontitis. For Body Mass Index

(BMI) there is overwhelming evidence that variation in the population is influenced by

genotype. Results from twin studies suggest that genetic factors explain 50 to 90% of

the variance in BMI (Maes et al. 1997, Schousboe et al. 2003).

Furthermore, it has been suggested that measures of socioeconomic status

including education are fairly good indicators for periodontitis. Groups with low

education are at a higher risk of having periodontitis (Drury et al. 1999) and twin

studies suggest a moderate heritability for education (Silventoinen et al. 2000). It has

also been suggested that common genetic factors may affect educational attainment

and body weight (Silventoinen et al. 2004). Therefore, in order to avoid

oversimplification, a number of factors have to be considered for the understanding of

the complexity of multifactorial diseases like periodontitis.

We hypothesized that with regard to periodontitis monozygotic (MZ) twin pairs

would have a comparable periodontal phenotype whereas dizygotic twin pairs may

differ to some extent. Therefore, the aim of the present study was to assess, in MZ and

dizygotic (DZ) twin pairs selected on the basis of one sib of a twin pair having

moderate to severe chronic periodontitis, the contribution of genetics, life style factors

and periodontal pathogens to the clinical phenotype of the disease.

63


Subjects

Twin pairs were obtained as follows. A first set of twins was recruited through

the identification of patients with moderate to severe periodontal breakdown, who were

part of a twin pair, and who were referred to various periodontal clinics across the

Netherlands for the treatment of periodontitis (including patients referred to the clinic

of the Department of Periodontology at the Academic Center for Dentistry Amsterdam

[ACTA]). Another set of twins was recruited with the aid of the Dutch Association of

Twins. Possible eligible subjects from this latter set of twins, underwent a preliminary

periodontal clinical examination (screening for suitability) to determine whether their

periodontal status met the inclusion criteria of our study.

The selection criteria for the twin subjects with moderate to severe periodontal

breakdown included: 1) Caucasian descent, 2) age between 25 and 65 years, 3)

diagnosis of chronic periodontitis in one member of the twin pair defined by the

presence of interproximal attachment loss 5 mm at 2 non-adjacent teeth. Exclusion

criteria were: 1) presence of any systemic condition that may affect the periodontal

status, 2) pregnancy, and 3) use of antibiotics within the last 6 months preceding the

study. The periodontal condition of the co-twin was not part of the selection procedure

as the apparent phenotype of the co-twin is part of the results of the present study. The

patient recruitment resulted in 25 potentially eligible pairs of twins. Of the 25 twin

pairs, 18 pairs (36 subjects) volunteered to participate in the present study. Common

reasons for refusal of participation were lack of agreement of both subjects of the twin

pair to participate or distant household location making transportation to the research

venue difficult.

The study population consisted of 18 reared-together twin pairs and prior to

the clinical examination a verbal and a written informed consent were obtained from

all twins. This study was approved by the Medical Ethical Committee of the

Academic Medical Center of the University of Amsterdam. Data from the twin

subjects were obtained in the following order: 1) microbiological samples from

buccal mucous membranes and tongue, 2) venous blood, 3) full-mouth periapical

radiographs, 4) periodontal examination: attachment loss (AL), probing pocket depth

(PPD), bleeding on probing (BOP) and plaque index (PI); and 5) microbiological

samples of supragingival and subgingival plaque.

64

Clinical examination

The clinical examination was carried out at the interproximal sites of all teeth

from buccal and lingual aspects. The following assessments were performed: PI

according to (Silness & Löe 1964); BOP recorded as 0 = no bleeding, 1 = point

bleeding within 30 seconds, 2 = immediate and overt bleeding; PPD, recorded in mm

(measurements were rounded off to the nearest mm marking) and AL, again in whole

mm, using the cemento-enamel junction as a reference. All clinical assessments were

performed using a periodontal probe (PQW, Hu-Friedy, Chicago, IL., USA).

Radiographic examination

All participants underwent a full-mouth radiographic survey consisting of 14

periapical and 2 bitewing radiographs using the long-cone paralleling technique with

a Heliodent MD digital device, setting of 70 kV, 7mA (Sirona Dental Systems,

Bensheim, Germany). Images were obtained using the Emago/Advanced 5.2

program (Exan Academic Inc., Port Coqvitlam, BC, Canada) and printed on

photographic paper (Drystar DT 1 B, dry medical film, 25 x 30) using the Agfa

Drystar 4500 printer (Agfa, Mortsel, Belgium). All teeth were radiographically

examined for interproximal bone loss at the mesial and distal sites, using cemento-

enamel junction (CEJ) of the tooth and the bone crest as reference points. Using the

Schei ruler technique, the percentage of bone loss at the deepest interproximal site of

each tooth was measured (Schei 1959).

Microbiological procedures

Prior to any clinical measurement, samples for microbiological analysis were

obtained from the buccal mucous membranes: right and left buccal mucosa, and

dorsum of the tongue (from the vallate papillae to the tip of the tongue). The mucous

membranes were sampled with a sterile swab and were immediately suspended in

Reduced Transport Fluid (RTF). After the clinical examination, 4 sites (1 from each

quadrant) were chosen for bacterial sampling according to the following criteria and

in the following order: 1) the deepest pocket with the greatest amount of AL and

BOP; 2) if no AL was found, the deepest pocket which showed BOP; 3) if only

shallow healthy pockets were present, samples were taken mesially from the first

permanent molars. The selected sites were isolated with cotton rolls and

supragingival plaque samples were taken with a sterile Gracey curette. Subsequently,

the remaining supragingival plaque was removed and subgingival plaque samples

were obtained by inserting 1 sterile paper point per pocket during 10 s. Both pooled

65

supragingival and pooled subgingival plaque samples were suspended in RTF. All

microbial samples were transported to the laboratory and processed within 24 h.

The presence and proportions of A. actinomycetemcomitans were determined

by means of TSBV plates and that of P. gingivalis, Prevotella intermedia,

Fusobacterium nucleatum, Parvimonas micra, Tannerella forsythia and

Campylobacter rectus by means of blood agar plates. These isolates were all purified

and identified to species level as described by Van Winkelhoff et al. (2002).

Zygosity testing

First information about zygosity was obtained during the questionnaire-based

medical history by asking the participant if he/she was part of a MZ or DZ twin.

Aware of the possibility of zygosity misclassification solely by self-report (with an

agreement between zygosity diagnoses from questionnaire and DNA data of 97%),

and to verify the obtained verbal report, DNA testing from each member of the twin

pair was done (Middeldorp et al. 2006, Reed et al. 2005, Rietveld et al. 2000).

Genomic DNA from all twin pairs (38 subjects) was extracted from EDTA venous

blood samples with a commercially available DNA purification kit according to the

manufacturer's instructions (Puregene DNA isolation kit, Gentra Systems,

Minneapolis, MN, USA). Thereafter, zygosity was assesssed by the department of

paternity testing (Sanquin Diagnostic Services, Sanquin, Amsterdam, The

Netherlands) by testing 17 autosomal short tandem repeats (STR) loci. The PCR

amplification was performed using the fluorescent STR multiplex system

PowerPlex16 (Promega, Madison, WI, USA) and the AmpFISTR™PCR

Amplification kit (Applied Biosystems, Foster City, CA, USA). The PCR products

were separated by capillary electrophoresis on an ABI PRISM 310 Genetic Analyzer

(Applied Biosystems). Data analysis was performed using the GeneScan Analysis

and Genotyper software (Applied Biosystems) and further statistical analysis was

performed using the Kinship program (Brenner 1997).

Life style characteristics

Data on life style characteristics were obtained by means of a self-

administered questionnaire. For assessment of the education level, the reported

highest education level was used and classified according to 3 categories: 1.

secondary education low level, 2. secondary education high level and 3. higher

education. In the questionnaire, current and former smokers were asked to estimate

their daily number of cigarettes usually smoked and the number of years they had

66

smoked. Pack-years were calculated on the basis of 20 cigarettes per pack (Grossi et

al., 1994). The BMI (kg/m2) was calculated on the basis of the self reported height

and weight.

Data Analysis

Twins were considered both as individuals and as members of a pair

depending on the analysis. After both subjects of each twin pair were clinically

examined, members of each twin pair were classified as either the proband or co-twin.

The term proband is used to define the sib showing the greatest mean AL, and the

remaining brother/sister is termed the co-twin.

Descriptive statistics and data analysis were performed with statistical

software from SPSS (version 14.0 for Windows, Chicago, IL, USA). First the data

were analysed whether they showed normal distributions (Kolmogorov-Smirnov

goodness-of-fit test p<0.05). For comparisons between probands and co-twins

irrespective of zygosity, paired t-tests and Wilcoxon matched-pairs signed ranks tests

were used when appropriate. A repeated measures ANOVA was employed for

comparisons between MZ probands and MZ co-twins versus DZ probands and DZ

co-twins followed by paired t-tests to assess difference between probands and co-

twins. In case of non-normal distributions, differences between MZ twins and DZ

twins were tested by means of the Mann-Whitney U test followed by Wilcoxon

matched-pairs signed ranks tests for comparisons between probands and co-twins

within each twin type. In DZ twins, a multivariate analysis (backward stepwise linear

regression with p 0.10 to enter and p 0.05 to leave) was performed to identify

factors explaining the observed variation in periodontal breakdown between DZ

probands and co-twins. The predictor variables entered were smoking, education,

BMI and periodontal pathogens subgingivally. p values <0.05 were considered

statistically significant.

67

Results

Results of the zygosity testing by means of DNA analysis showed that one

twin pair classified as DZ by questionnaire was typed to be MZ. Thus, the final study

sample consisted of 10 MZ twin pairs (6 female and 4 male) and 8 DZ twin pairs (7

same-sexed pairs: 6 female and 1 male, and 1 opposite-sexed pair). The aid of the

Dutch Association of Twins resulted in 6 MZ twin pairs and the contribution of the

periodontal clinics included 4 MZ and 8 DZ twin pairs.

Descriptive characteristics of the study population regarding demographic

and life style data, clinical parameters and oral microbiological parameters are

presented in Table 1 for probands and co-twins, irrespective of zygosity. The mean

age was 48.2 years and close to 75% of the participants were females. The majority

of the subjects had completed the high level of secondary education or higher

education. In this respect no difference could be assessed between probands and co-

twins.

With regard to smoking, a minority of subjects were never smokers i.e. 2 and

5 out of the 18 probands and co-twins respectively. The probands included more

current or former smokers compared to the co-twins. They also showed a higher

number of pack-years although this failed to reach the level of statistical

significance. The mean BMI was of normal weight and comparable between

probands co-twins.

The number of teeth ranged between 12-32 in the probands and between 17-

29 in the co-twins. Comparing probands and co-twins for their periodontal condition,

analysis showed significant higher values for probing pocket depth, attachment loss,

number and percentage of teeth with attachment loss 5 mm, percentage of teeth

with 30% and with 50% bone loss in the probands compared to the co-twins

(Table 1). Regarding the oral presence of periodontal bacteria the results showed that

P. gingivalis was more prevalent in probands than in their co-twins (p=0.03). Few

subjects harbored A. actinomycetemcomitans and C. rectus, whereas F. nucleatum

was present in all twins (Table 1).

68

Table 1. Demographic, lifestyle, clinical and laboratory data in probands and co-twins of MZ and DZ

twins combined.

Parameters

Probands

(N= 18)

Co-twins

(N= 18)

p-value

Age

48.2 ± 12.0

48.2 ± 12.0

nd

Gender (female) 14 13 nd

Education

Secondary education

low level

high level

Higher education

4

7

7

2

7

9

0.19

Smoking status

Never smokers

Former smokers

Current smokers

2

9

7

5

9

4

0.04

Pack-years 12.2 ± 10.8 6.9 ± 7.2 0.08

Body MassIindex (kg/cm2)

Clinical parameters

23.8 ± 2.5

23.6 ± 2.5

0.77

No. of teeth 23.8 ± 5.2 25.4 ± 3.1 0.17

Plaque Index 0.9 ± 0.6 0.9 ± 0.3 0.98

Bleeding on probing 0.8 ± 0.6 0.8 ± 0.4 0.90

Probing pocket depth 3.4 ± 0.9 2.8 ± 0.5 0.02

Attachment loss (AL)

# of teeth AL 5 mm

3.0 ± 1.4

9.1 ± 6.0

1.4 ± 0.6

1.8 ± 2.2

< 0.001

< 0.001

% teeth AL 5 mm

% of teeth 30% bone loss

39.0 ± 24.9

59.4 ± 39.4

2.7 ± 7.1

15.7 ± 17.4

< 0.001

< 0.001

% of teeth 50% bone loss 14.4 ± 14.0 2.7 ± 0.1 0.006

Bacteriological parameters: culture positive at subject level

A. actinomycetemcomitans 1 3 0.16

P. gingivalis 9 3 0.03

P. intermedia 8 8 1.00

T. forsythia 14 12 0.48

P. micra 15 14 0.66

F. nucleatum 18 18 1.0

C. rectus 2 1 0.32

69

Descriptive characteristics of periodontal data for MZ and DZ sibs separately,

are presented in Table 2. It can be seen that the DZ probands showed the most severe

periodontal condition in terms of attachment loss and bone loss. Both within MZ

twins and within DZ twins, analysis showed that the probands had a worse

periodontal condition compared to their co-twins. The differences between probands

and co-twins were smaller in the MZ twins compared to the DZ twins. The

periodontal condition of the MZ co-twins was very similar to that of the DZ co-

twins. Both co-twin groups were suffering from periodontitis to a lesser extent since

they showed either no teeth with attachment loss 5 mm or only a few.

Age and lifestyle characteristics of MZ and DZ twins are presented separately

in Table 3. The mean age of the MZ and DZ twins was comparable i.e. 49.5 and 48

years respectively. No differences could be assessed between MZ sibs as well as DZ

sibs for education level. However, the difference between probands and co-twins was

significantly smaller in MZ twins compared to DZ twins. MZ sibs had the same

education, 9 out of the 10, whereas in the DZ sibs this was 5 out of 8. Furthermore,

in 2 DZ twins the probands had the lowest education level whereas their co-twins

had the highest.

Evaluation of smoking behavior showed that in MZ twins 8 probands and 7

co-twins were smokers or former smokers, whereas in the DZ twins the numbers

were 8 and 6 respectively (Table 3). The probands and co-twins of the MZ twins

showed a comparable amount of pack-years (mean difference 0.9 ± 7.6) ranging

between 0.5-28 and 2-22 pack-years respectively. In the DZ twins the probands

smoked significantly more than their co-twins, 18.7 versus 5.6 pack-years

respectively (mean difference 13.1 ± 12.9). The difference in pack-years between

probands and co-twins was significantly smaller in MZ twins compared to DZ twins

(Table 3). Analysis of the 4 groups showed that the probands of the DZ twins had the

highest amount of pack-years. To further explore which factors explained

significantly the observed difference in periodontal breakdown between DZ

probands and co-twins a linear regression analysis was performed. For the number of

teeth with AL 5 mm a final model was obtained in which only the number of pack

years was retained as significant (p = 0.004) explaining 45.6% of the variation.

Eighteen of the 20 MZ sibs were of normal weight i.e. the BMI was below 25

kg/m2. One MZ proband and 1 non-related co-twin were overweight: BMI values of

26.6 and 29.5 kg/m2 respectively. In the DZ twins, 6 sibs, 4 probands and 2 co-twins,

were overweight, BMI values ranging between 25.2-29.4 and 25.4-27.8 kg/m2

70

respectively. The difference in BMI between the MZ sibs (1.1 ± 2.2 kg/m2) was

significantly smaller than between the DZ sibs (1.9 ± 3.2 kg/m2) (Table 3).

Tabl

e 2.

Per

iodo

ntal

cha

ract

eris

tics (

mea

n va

lues

± S

D) i

n m

onoz

ygot

ic (M

Z) a

nd d

izyg

otic

(DZ)

twin

s

MZ

(N=

10 p

airs

)

D

Z (N

= 8

pairs

)

Clin

ical

par

amet

ers

Pr

oban

d

C

o-tw

in

p-

valu

e

Prob

and

Co-

twin

p-

valu

e

p-

valu

e*

dMZ

vers

us d

DZ

# of

teet

h Pl

aque

Inde

x

B

leed

ing

on p

robi

ng

Prob

ing

pock

et d

epth

A

ttach

men

t los

s (A

L)

# of

teet

h A

L 5

mm

%

teet

h A

L 5

mm

%

teet

h 3

0% b

one

loss

%

teet

h 5

0% b

one

loss

24

.7 ±

4.1

1.1

± 0.

5

1.0

± 0.

5

3.4

± 0.

7

2.3

± 1.

3

7.2

± 5.

2

30.3

± 2

2.1

41

.7 ±

29.

3

8

.4 ±

9.7

25

.0 ±

3.5

0.9

± 0.

4

0.9

± 0.

5

2.9

± 0.

5

1.6

± 0.

8

2.2

± 2.

4

9.2

± 9.

7

15.6

± 1

7.7

3.2

± 8

.9

0.

85

0.

21

0.

39

0.

09

0.

04

0.

005

0.00

5 0.

006

0.2

0

22

.8 ±

6.5

0.6

± 0.

4

0.5

± 0.

4

3.4

± 1.

1

3.

5 ±

1.2*

*

11.

2 ±

6.4*

*

50.

0 ±

25.1

**

8

1.5

± 40

.1**

21.

7 ±

15.5

**

26

.0 ±

2.7

0.9

± 0.

2

0.6

± 0.

3

2.7

± 0.

3

1.2

± 0.

4

1.4

± 2.

1

5.4

± 7.

8

15.7

± 1

8.1

2.1

± 4

.0

0.

07

0.

015

0.52

0.12

<0.0

001

0.

001

0.00

1 0.

001

0

.01

0.

20

0.

05

0.

30

0.

59

0.

01

0.

08

0.

03

0.

01

0.

05

* p-

valu

es in

dica

te w

heth

er th

e di

ffer

ence

s (d)

bet

wee

n M

Z tw

ins a

re si

gnifi

cant

ly d

iffer

ent f

rom

thos

e of

DZ

twin

s.

**

val

ues o

f DZ

prob

ands

are

sign

ifica

ntly

hig

her c

ompa

red

to M

Z pr

oban

ds a

nd M

Z co

-twin

s p<

0.01

Tabl

e 3.

Age

and

life

styl

e ch

arac

teris

tics e

duca

tion,

smok

ing

and

Bod

y M

ass I

ndex

(BM

I) o

f mon

ozyg

otic

(MZ)

and

diz

ygot

ic (D

Z) tw

ins.

Num

ber o

f sub

ject

s and

mea

n va

lues

(SD

) for

pac

k-ye

ars a

re p

rese

nted

.

MZ

(N=

10 p

airs

)

D

Z (N

= 8

pairs

)

Life

styl

e ch

arac

teris

tics

Pr

oban

d

C

o-tw

in

p-

valu

e

Prob

and

Co-

twin

p-va

lue

p-

valu

e*

dMZ

vers

us d

DZ

Age

49.5

± 1

3.6

48.0

± 1

0.3

0.

26

Educ

atio

n

Sec

onda

ry e

duca

tion

low

leve

l

hi

gh le

vel

H

ighe

r edu

catio

n Sm

okin

g st

atus

Nev

er sm

oker

For

mer

smok

er

C

urre

nt sm

oker

Sm

okin

g

Pac

k-ye

ars

Bod

y M

ass I

ndex

(kg/

m2 )

1 4 5 2 5 3

7.1

± 9.

8

22.8

± 1

.9

1 5 4 3 4 3 8.

0 ±

8.8

23

.9 ±

2.4

0.

19

0.

56

0.

87

0.

94

3 3 2 0 4 4

18.7

± 8

.8**

25.0

± 2

.7

1 2 5 2 5 1 5.

6 ±

4.9

23

.1 ±

2.6

0.

10

0.

06

0.

02

0.

12

0.

03

0.

12

0.

01

0.

03

*

p-va

lues

indi

cate

whe

ther

the

diff

eren

ces (

d) b

etw

een

MZ

twin

s are

sign

ifica

ntly

diff

eren

t fro

m th

ose

of D

Z tw

ins.

**

val

ues o

f DZ

prob

ands

are

sign

ifica

ntly

hig

her c

ompa

red

to M

Z pr

oban

ds a

nd M

Z co

-twin

s p<

0.05

Tabl

e 4.

Pre

vale

nce

of p

erio

dont

al p

atho

gens

on

a su

bjec

t lev

el a

nd a

t 4 o

ral s

ites i

n m

onoz

ygot

ic (M

Z) a

nd d

izyg

otic

(DZ)

twin

s. a)

Num

ber o

f pos

itive

subj

ects

and

m

ean

prop

ortio

ns in

pos

itive

subj

ects

(in

pare

nthe

sis)

are

pre

sent

ed, b

) Num

ber o

f pos

itive

subj

ects

.

M

Z

(N=

10 p

airs

)

DZ

(N=

8 pa

irs)

Bac

terio

logi

cal

para

met

ers

Pr

oban

d N

(%)

Co-

twin

N

(%)

Prob

and

N (%

)

Co-

twin

N

(%)

a)

Per

site

Su

bgin

giva

l

pl

aque

Su

prag

ingi

val

pla

que

Tong

ue

Muc

osa

b) A

ll si

tes

A.

act

inom

ycet

emco

mita

ns

P. g

ingi

valis

P.

inte

rmed

ia

T. f

orsy

thia

P.

mic

ra

F. n

ucle

atum

C

. rec

tus

A. a

ctin

omyc

etem

com

itans

P. g

ingi

valis

P.

inte

rmed

ia

T. fo

rsyt

hia

P. m

icra

F.

nuc

leat

um

T. fo

rsyt

hia

P. m

icra

F.

nuc

leat

um

P. in

term

edia

P.

mic

ra

F. n

ucle

atum

A.

act

inom

ycet

emco

mita

ns

P. g

ingi

valis

P.

inte

rmed

ia

T. fo

rsyt

hia

P. m

icra

F.

nuc

leat

um

C. r

ectu

s

0

5

(18

.5)

5

(3.2

)

9

(3

.6)

9

(5.1

)

10

(3

.9)

0

0

4

(1.4

)

2

(0

.4)

6

(0.4

)

5

(0

.5)

9

(1.8

)

2

(0

.5)

1

(2.0

)

8

(0

.3)

1

(0.2

)

0

3

(0

.6)

0

5

7

9

10

10

0

2

(4.0

)

2

(14

.8)

3

(0.5

)

7

(1

.7)

9

(3.6

)

10

(3

.8)

0

1

(0.0

1)

2

(1.2

)

2

(5

.5)

1

(0.3

)

2

(2

.0)

9

(1.8

)

0

2

(0

.2)

8

(1.0

)

0

1

(0

.3)

4

(0.8

)

2

2

3

7

9

10

0

1

(2

.0)

4 (

16.1

)

2

(1

.3)

5

(5.1

)

5

(10

.0)

8

(8.7

)

2

(12

.5)

1

(0.0

1)

3

(3.4

)

0

3

(2

.0)

3

(1.0

)

7

(1

.0)

1

(0

.04)

1

(1

.3)

6

(1.8

)

0

1

(0

.01)

5

(0

.2)

1

4

2

5

5

8

2

1

(0

.9)

1 (5

6.3)

4

(1

.4)

5

(3.2

) 5

(7

.5)

8

(5.1

) 1

(3

.0)

1 (

0.01

) 1

(0.

8)

2 (

0.7)

2

(2.

0)

4 (

1.3)

8

(2.

3)

1 (

0.01

) 1

(0.

1)

5 (

1.2)

1

(0.

06)

0 6 (

0.8)

1 1 5 5 5 8 1

No

sign

ifica

nt d

iffer

ence

s wer

e fo

und

74

In Table 4 the prevalence of the periodontal pathogens at the various oral sites is

presented for MZ and DZ twins separately. It can be seen that the highest prevalence of

periodontal pathogens was found in the subgingival plaque followed by the supragingival

plaque. For all oral sites no significant differences were found between the probands and co-

twins of both the MZ as well as the DZ twins. Also no significant differences could be

assessed between the intra-pair discrepancies of MZ and DZ twins. Although the prevalence

values of the periodontal pathogens were the highest in the MZ probands compared to the

other 3 groups this failed to reach the level of significance. Nevertheless P. gingivalis was

both in MZ and DZ probands present subgingivally in half of the subjects, whereas for the

co-twins this was 2 out of 10 and 1 out of 8 for MZ and DZ respectively. Further analysis of

the subgingival presence of P. gingivalis in DZ twins revealed that in the only case that the

co-twin was P. gingivalis positive, the proband twin was positive as well. For the MZ twins

it was found that in 1 twin pair both sibs were positive, in 4 twin pairs only the probands

were positive and in 1 twin pair only the co-twin was P. gingivalis positive.

Analysis of the periodontal condition of MZ twins with regard to subgingival

presence of P. gingivalis showed no difference between P. gingivalis positive and negative

subjects. However, in DZ twins, when P. gingivalis positive and negative subjects were

compared it was found that P. gingivalis positive subjects had more attachment loss (mean

attachment loss (mm): 3.7 ± 1.6 vs 1.7 ± 0.9, p=0.007), a higher percentage of teeth with AL

5 mm (57.0 ± 33.5 vs 14.2 ± 14.3, p=0.003) and a higher percentage of teeth with bone

loss 30% (86.7 ± 55.7 vs 31.3 ± 28.7, p= 0.01).

Discussion

Historically, most studies on the heritability of periodontitis concentrate on

segregation analysis of nuclear families selected on the basis of probands suffering from

Juvenile Periodontitis/ Early Onset Periodontitis (EOP). The results of these studies all

suggested a substantial role for genetics in the development of EOP (Loos et al. 2008). The

most powerful tool to study the heritability of periodontitis is the twin model. Only one study

has evaluated the periodontal condition in terms of probing depths in juvenile MZ and DZ

twins and found no evidence that pocket depth was an inherited characteristic (Ciancio et al.

1969). This finding is most likely due to the inherent difficulties in finding the appropriate

young twins suffering from severe periodontitis, which at that age has a very low prevalence.

75

For the current study it was anticipated that for chronic periodontitis twin studies may be

easier to perform since the prevalence of the disease is approximately 10% (Albandar et al.

1999, Page & Eke 2007). Unfortunately, it appeared that also for chronic periodontitis it was

extremely difficult to recruit moderate to severe periodontitis patients having a MZ or DZ sib

as a primary selection criterion. Nevertheless a major effort was made to obtain these

patients from private periodontal clinics, the clinic of the department of periodontology of

ACTA and with the aid of the Dutch Association of Twins.

To date results of twin studies of chronic periodontitis show converging results

suggesting a substantial role of genetics in this condition (Corey et al. 1993, Michalowicz et

al. 1991b, Mucci et al. 2005). Nevertheless, these studies have some limitations. The results

of the study of Corey et al. (1993) and Mucci et al. (2005) were based on questionnaires and

not on clinical measurements. The studies of Michalowicz et al. (1991a,b, 2000) included

study populations with relatively minor periodontal destruction. Up to now all twin studies

have been based on subjects selected because of their twinship and not on the presence of

moderate to severe periodontitis. Interestingly, a case report of a 40 year old MZ twin

presented a proband who suffered from localized moderate to severe alveolar bone loss

around several premolars and molars whereas her twin sister had shallow pockets and

essentially normal bone architecture (McDaniel et al. 1999). One of the present authors

(UvdV) also came across such a case in his practice. Therefore, the aim of the present study

was to initiate a twin study in which twins were selected on the basis of a proband with

moderate to severe periodontitis. Consequently, the patient selection for this study was based

on the presence of interproximal attachment loss 5 mm at 2 non-adjacent teeth.

Surprisingly, the plaque and bleeding scores were relatively low. This phenomenon was

mainly due to the 12 twin pairs of which the proband was referred to the periodontal clinics.

The subjects of these twins had mean plaque and bleeding score scores of 0.7 and 0.6

respectively, whereas the plaque and bleeding scores of the 6 twin pairs recruited with the

aid of the Dutch Association of twins amounted to 1.26 and 1.23 respectively (p-values <

0.001). Most likely, the lower plaque and bleeding scores of the referral twins is caused by

previous treatment in the practice of the general practitioner before referral, resulting in

improved oral hygiene and reduced inflammation at shallow pockets in the probands.

The most important result of this study is the finding that MZ twins appeared to be

discordant with regard to mean attachment loss, number and percentage of teeth with AL 5

mm and percentage of teeth with bone loss 30 %. This finding is in agreement with the

results of Tabrizi et al. (2007) who found in monozygotic twins discordant for coronary hart

76

disease that the twin patient with coronary hart disease was also discordant for periodontal

breakdown. Although the number of twins included in the present study was rather small, the

statistical power for the assessed differences was at or above 80%. These discrepancies can

obviously not be explained by basic variations in genetic make-up, neither could it be

explained by the prevalence of periodontal pathogens nor by the life style factors education,

smoking and BMI, factors that are all known to be related to destructive periodontal disease

(Grossi et al. 1994, Tomar & Asma 2000, van Winkelhoff et al. 2002, Ylostalo et al. 2008).

As a matter of fact these life style factors were concordant and in agreement with the

literature: there is consistent evidence from twin studies that genetic factors play a role in

educational attainment (Silventoinen et al. 2000, Silventoinen et al. 2004), smoking (Munafo

& Johnstone 2008, Vink et al. 2005) and BMI (Maes et al. 1997, Schousboe et al. 2003).

Since MZ twins are discordant for the amount of periodontal breakdown, it is not surprising

that the DZ twins are discordant as well. It must be noted that DZ sibs differed to a greater

extent from each other than the MZ sibs, confirming that the genetic component does play a

role. The DZ probands showed the worst periodontal condition compared DZ co-twins, MZ

probands and MZ co-twins. This finding is in line with the studied life style factors, because

this group included only 2 subjects with higher education, showed the highest percentage of

smokers with the highest number of pack-years, and 4 out of 8 subjects were overweight. In

the study population all subjects had attachment loss to some extent; the least in one MZ co-

twin having at 3 interproximal sites 2 mm attachment loss and the most in one DZ proband

having at 9 teeth (40%) 9 mm or more interproximal attachment loss. Results showed that for

all parameters of periodontal breakdown used, all individual probands had more periodontal

breakdown than their co-twins. The finding that MZ twins are discordant for the amount of

periodontal breakdown could imply that the influence of genetics in the development of

chronic periodontitis may have been overestimated although it may still play a significant

role.

The subgingival microbiological profile of the probands of the present study

population, consisting of subjects with moderate to severe periodontitis and having a mean

age of 48 years, is in agreement with the prevalence of periodontal pathogens in periodontitis

patients of that age (van Winkelhoff et al. 2002). The prevalence of periodontal pathogens on

the mucous membranes seems somewhat lower than expected (Van der Velden et al. 2006).

The additional sampling of the mucous membranes did not give extra information compared

to supra- and subgingival sampling only. In the present study no statistical significant

influence of the microbial flora could be assessed. However, it must be realized that the

77

number of twins in the present study is small and the statistics did not include corrections for

multiple comparisons. Therefore, from one point of view the results on the basis of p-values

0.01 should be interpreted with care. However, on the other hand the small number of

twins may have been also responsible for the many non significant differences, e.g. the

subgingival presence of P. gingivalis. In the MZ twin group, 5 out of the 10 probands were

positive for P. gingivalis, whereas 2 out of the 10 co-twins were positive for this bacterium.

A larger study population of MZ twins could have shown that P. gingivalis plays a

significant role in the etiology of periodontitis.

At present the discordant MZ twin model is regarded as the best option to study the

etiology of a disease (Vaag & Poulsen 2007). Discordance between MZ twins regarding

diseases has been reported for a number of disorders. For example, it has been found that the

discordance of MZ twins for a complex disease like rheumatoid arthritis may amount to about

85% (Silman et al. 1993). Because MZ twins start life with identical genomes, within twin

pair differences reflect exposure to an individual-specific environment and life style which

may ultimately act through genetic or epigenetic modifications of gene expression (den

Braber et al. 2008). Epigenetic mechanisms result in heritable modifications of the DNA,

resulting in variation of expression of genes independent of basic DNA code. (Petronis 2001)

suggested that epigenetic mis-regulation of genes is more consistent with features of complex

diseases than the DNA sequence. Discordance of MZ twins is usually explained by the

differential effect of environmental and life style factors. At present both aging, smoking and

environmental factors like nutrition have been shown to be involved in epigenetic changes

(Fraga et al. 2005, Zochbauer-Muller et al. 2001, Kauwell 2008) possibly explaining

discordance in MZ twins. Epigenetic changes may be important for controlling the immune

and inflammatory responses and thus for controlling periodontitis (Wilson 2008). DNA

modifications by environmental and life style factors (epigenetics) could explain the

discordance in the periodontal condition of the MZ twins in the present study.

In conclusion, since in the present study MZ sibs are discordant regarding the

amount of periodontal breakdown, the role of genetics in the development of chronic

periodontitis may have been overestimated although it clearly plays a role. In addition,

differences in the periodontal condition of the MZ sibs could not be explained by differences

in the microbial flora nor by the life style factors education, smoking and BMI. Furthermore,

the factors that play an important role in the development of chronic periodontitis have yet to

be determined. To this end, studies including large numbers of MZ twins selected for the

presence of moderate to severe periodontitis are needed.

78

Acknowledgements

The authors are grateful to Mark Timmerman for his assistance in the periodontal

clinical examination of the twin population. The authors wish to thank A. Smid and J. Smid,

chairmen of the Dutch Association of Twins and the following periodontists for their help in

the recruitment of the twins: P.G.G.L. van der Avoort, D.J. Bossers, R.A. Driessen, S.J.

Fokkema, P.C. van Gils, H. Hamming, J.W. Hutter, G. Maffei, G.N.Th. de Quincey, J.

Steinfort, R.W.R. Steures, A. Varoufaki. The authors thank Prof. dr. D.I. Boomsma,

Department of Biological Psychology, Vrije Universiteit, Amsterdam, The Netherlands, for

her useful comments.

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83

Chapter 5

Monozygotic twins are discordant for chronic periodontitis

White blood cell counts and cytokine production after ex vivo stimulation



The Netherlands

Journal of Clinical Periodontology; accepted for publication and in press 2010

84

Abstract

Objectives: The aim of this study was to investigate the extent of concordance in number of

leukocytes and their cytokine secretion after ex vivo stimulation in a twin population

discordant for the amount of periodontal breakdown.

Material and Methods: Venous blood was collected from 18 adult twin pairs (10

monozygotic and 8 dizygotic twins). Each twin pair consisted of a diseased twin (proband)

and his/her co-twin. In venous blood, leukocytes were counted. The cytokines interleukin

(IL)-1 , IL-6, IL-8, IL-10 and IL-12p40 were assessed after stimulation of monocytic cells,

while IL-13 and interferon (IFN)- were determined after lymphocytic stimulation.

Results: In the study population as a whole probands showed higher total numbers of

leukocytes and lower IL-12p40 levels compared to their co-twins. In monozygotic twins no

difference was found in leukocyte counts, but probands secreted more IL-6 than their co-

twins; an opposite trend was found for IL-12p40.

Conclusion: The results suggest that the observed discordance in periodontal breakdown in

the studied monozygotic twin population may be related to relatively high levels of IL-6 and

low levels of IL-12p40 secretion after ex vivo stimulation of whole blood cell cultures. This

cytokine secretion profile may be regarded as a risk indicator of periodontitis.

85

Introduction

Periodontitis is a chronic, multifactorial, infectious disease of the supporting tissues of

the teeth characterized by gradual loss of periodontal attachment and alveolar bone (Kinane &

Lappin 2001). Periodontitis is initiated by microbial plaque, which accumulates in the

gingival crevice region and induces an inflammatory response. Although bacteria are essential

for the induction of the inflammatory response, they are insufficient to cause the disease (Page

et al. 1997). In conjunction with the bacterial challenge, the host immune response plays an

important role in the onset and progression of periodontitis (Ebersole & Taubman 1994). In

fact, variability in host response may be a component of a genetic predisposition to

periodontitis (Michalowicz et al. 1991). It is possible that genetically determined differences

in immune regulation or in homeostatic bone remodeling are also important to the outcome of

periodontal disease (Baker et al. 2000, Kornman et al. 1997).

In the innate and adaptive immunity, monocytes play an orchestrating role. When

triggered by bacteria, monocytes produce cytokines which direct both immune responses

(Seymour & Gemmell 2001). Furthermore, cytokines derived mainly from dendritic cells,

monocytes and macrophages, play a pivotal role in directing lymphocytic differentiation of

non-committed precursors CD4+ T cells into either T helper type 1 (Th1) or Th2 cells.

Previous studies have shown that periodontitis patients display a monocytic-cytokine profile

which may favor a Th2 immune response (Fokkema et al. 2002, Gemmell & Seymour 1994).

Interestingly, the monocytic directional Th2 response is even more pronounced in smokers

(Torres de Heens et al. 2009). It is likely that changes in cytokine profiles that modulate the

Th1/Th2 balance may affect the susceptibility to or the course of the periodontal infection

(Gemmell et al. 2002). Studies in infectious diseases other than periodontitis provide

convincing evidence that host genetic factors are important in determining who will succumb

to the pathogen and who will not (Davies & Grange 2001, Lama & Planelles 2007).

Susceptibility or resistance to many infectious diseases is dependent on genetically controlled

differences in inflammatory responses, cytokine secretion, or T-cell recruitment after

exposure to the pathogen (Gervais et al. 1984, Skamene 1994, Stevenson & Tam 1993).

Moreover, twin studies have confirmed a genetic component for cytokine production

(Grainger et al. 1999, Reuss et al. 2002).

For chronic periodontitis relatively few twin studies have been carried out, but the

results suggest a substantial role of genetic factors in the etiology (Corey et al. 1993,

Michalowicz et al. 1991, Michalowicz et al. 2000, Mucci et al. 2005). Nevertheless these

studies have limitations. The results of Corey et al. (1993) and Mucci et al. (2005) are based

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on self reported evidence of periodontal disease. Subjects in the studies of Michalowicz et al.

(1991a, b, 2000) were selected based on their twin ship rather than their periodontal condition,

resulting in a population with mild periodontal breakdown. Therefore a twin study was

initiated that studied in monozygotic (MZ) and dizygotic (DZ) twin pairs selected on the basis

of one sib of a twin pair (the proband) suffering from moderate to severe chronic

periodontitis, the contribution of genetics, life style factors and periodontal pathogens to the

clinical phenotype of the disease (Torres de Heens et al. 'in press'). The clinical results

showed that the MZ twins were discordant regarding attachment loss and bone loss. The

discordance was greater in DZ compared to MZ twins. In MZ twins the discordance could not

be explained by education, smoking, Body Mass Index or presence of periodontal pathogens.

In DZ twins the discordance could be explained by more cigarette smoking of the probands.

The aim of the present study was to investigate the extent of concordance in number of white

blood cells and monocytic and lymphocytic cytokine secretion after ex vivo stimulation

among the previously studied twin population selected on the basis of one sib of a twin pair

suffering from moderate to severe chronic periodontitis


Subjects

Twin subjects were recruited as previously described (Torres de Heens et al. 'in

press'). In short, subjects were recruited among patients referred to various periodontal clinics

across the Netherlands for the treatment of periodontitis, and from members of the Dutch

Association of Twins whose periodontal status met the inclusion criteria of our study. The

selection criteria included: 1) Caucasian descent, 2) age between 25 and 65 years, 3) diagnosis

of chronic periodontitis in one member of the twin pair defined by the presence of

interproximal attachment loss 5mm in 2 non-adjacent teeth. Exclusion criteria were: 1)

presence of any systemic condition that may affect the periodontal status, 2) pregnancy, and

3) use of antibiotics within the last 6 months preceding the study. Approval for this study was

obtained by the Medical Ethical Committee of the Academic Medical Center of the University

of Amsterdam.

The study population consisted of 18 reared-together twin pairs and prior to the

clinical examination a verbal and written informed consent was obtained from all twins. The

clinical examination was carried out at the interproximal sites of all teeth from buccal and

lingual aspects. The following assessments were performed: Plaque Index according to

87

(Silness & Löe 1964); bleeding on probing recorded as 0 = no bleeding, 1 = point bleeding

within 30 seconds, 2 = immediate and overt bleeding; probing pocket depth, recorded in mm

(measurements were rounded off to the nearest mm marking) and AL, again in whole mm,

using the cemento-enamel junction as a reference. All clinical assessments were performed

using a Hu-Friedy® PQW probe (Chicago, Illinois, USA). In addition, the participants

underwent a full-mouth radiographic survey on which all teeth were examined for

interproximal bone loss at the mesial and distal sites, using cemento-enamel junction (CEJ) of

the tooth and the bone crest as reference points. By means of the Schei ruler technique, the

percentage of bone loss at the deepest interproximal site of each tooth was measured (Schei et

al. 1959). Reported smoking habits of the twins were recorded in pack-year according to three

groups: (1) non-smokers: subjects who had never smoked, (2) former smokers: subjects that

stopped smoking before entering the study, and (3) smokers: subjects who were current

smokers. Zygosity was assessed by the department of paternity testing (Sanquin Diagnostic

Services, Sanquin, Amsterdam, The Netherlands) by testing 17 autosomal short tandem

repeats (STR) loci as previously described (Torres de Heens et al. 'in press')

Venous blood collection and differential cell counts

From each subject, venous blood was collected by venipuncture from the antecubital

fossa in sterile pyrogen-free blood collection tubes. For whole blood cell cultures (WBCC)

sodium heparine tubes were used (Vacuette, Greiner, Alphen a/d Rijn, The Netherlands). For

differential blood cell counts EDTA (K3)-containing tubes (Becton Dickinson Vacutainer

System Europe, Meylan, France) were used and the cell counts (neutrophils, eosinophils,

basophils, lymphocytes and monocytes) carried out in the clinical chemistry laboratory of the

Slotervaart Hospital Amsterdam, The Netherlands, using standard automated procedures

(Cell-Dyn 4000, Hematology Analyzer, Abbott Laboratories, Park, Illinois, U.S.A.).

Preparation of stimuli

Three stimuli for WBCC were used in the study:

(1) Lipo-oligosaccharide (LOS) was purified from Neisseria meningitides strain

H44/76 (a kind gift from Dr. J. Poolman, Rijksinstituut voor Volksgezondheid en Milieu,

Bilthoven, The Netherlands (van der Pouw Kraan et al. 1995).

(2) Porphyromonas gingivalis (Pg) strain 381 was grown in brain heart infusion broth

enriched with hemin (5 mg/l) and menadione (1 mg/l) in an anaerobic atmosphere (80% N2,

10% H2, 10% CO2) for 48 h at 37 °C. Bacteria were harvested in the log phase, pelleted by

88

centrifugation (8000 g), washed three times in PBS, and resuspended in PBS at an optical

density of 1 at 690 nm, corresponding approximately to 7 x 108 CFU/ml. Aliquots (500 l) of

resuspended bacteria were disrupted using a sonifier in a sonicating vessel on ice (Soniprep

MSE 150, York, United Kingdom; amplitude 18, 4 min, 5 sec intervals). The degree of

disruption of the bacteria was assessed by phase-contrast microscopy and with Gram-staining

by light microscopy. Sonicates were stored at 4 ºC until use. Before use, P. gingivalis

sonicates were centrifuged (8000 x g, 1 min) and used in WBCC as described below.

(3) Mouse monoclonal antibodies raised against human CD3 (anti-CD3, CLB-T3/4.E)

and CD28 (anti-CD28 CLB-T3/4.E) were obtained from Sanquin, Amsterdam, The

Netherlands (van Lier et al. 1987).

Whole Blood Cell Cultures

WBCC were performed in 96-well flat bottom microtitre plate (Nunc, Roskilde,

Denmark). Heparinized venous whole blood was diluted 1/10 with Iscove’s modified

Dulbecco’s medium (IMDM, Bio Whittaker, Verviers, Belgium), supplemented with

penicillin (100 IU/ml), streptomycin (100 μg/ml) (Gibco, Merelbeke, Belgium), 0.1%

endotoxin-free fetal calf serum (FCS, Bodinco, Alkmaar, The Netherlands), and 15 U/ml

sodium heparin (Leo Pharmaceutical Products B.V., Weesp, The Netherlands).

Two hundred l of the diluted blood was stimulated during 18 hours with LOS at a

final concentration of 1000 pg/ml or with Pg sonic extract (Pg-SE) 1:100 dilution, in the

presence of anti-CD3 at 1 g/ml. LOS and Pg-SE concentrations used have been previously

shown to be the most optimal concentration for these stimulation assays (Torres de Heens et

al. 2009). Unstimulated diluted whole blood served as a negative control. Supernatants were

harvested and stored at -20 °C until cytokine measurements (Gerards et al. 2003).

To stimulate T lymphocytes a combination of a mouse monoclonal antibody against

human CD3 and CD28 was added to the 200 μl aliquot of diluted whole blood, as previously

described (Gerards et al. 2003). Cultures were performed in duplicate and unstimulated

diluted whole blood served as a negative control. After 72 hours of incubation with anti-

CD3/anti-CD28, supernatants were harvested and stored at -20 °C until cytokine analysis was

performed.

Assays for cytokines

Cytokine levels of IL-1 , IL-6, IL-8, IL-10, IL-12p40, IL-13 and IFN- were

measured in the supernatants of WBCC using commercially available enzyme-linked

89

immunosorbent assay (ELISA) kits (PeliKine Compact™ human ELISA kits, Sanquin,

Amsterdam, The Netherlands) as previous described (van der Pouw Kraan et al. 1997). The

plates were read in an ELISA reader (Labsystems Multiskan Multisoft, Helsinki, Finland) at

450 nm, with 540 nm as a reference. Cytokine production of IL-1 , IL-6, IL-8 and IL-12p40

was adjusted for the number of monocytes and neutrophils, IL-10 only for the number of

monocytes and IL-13 and IFN- for the number of lymphocytes.

Data Analysis

Twins were considered both as individuals and as members of a pair depending on the

analysis. Members of each twin pair were classified as either the proband or co-twin. The

term proband is used to define the sib showing the greatest mean attachment loss (AL), and

the remaining brother/sister is termed the co-twin.

Descriptive statistics and data analysis were performed with statistical software from

SPSS (version 14.0 for Windows, Chicago, IL, USA). First the data were analyzed whether

they showed normal distribution (Kolmogorov-Smirnov goodness-of-fit test p<0.05). For

comparisons between probands and co-twins irrespective of zygosity, paired t-tests and

Wilcoxon matched-pairs signed ranks tests were used when appropriate. A repeated measures

ANOVA was employed for comparisons between MZ probands and MZ co-twins versus DZ

probands and DZ co-twins, followed by paired t-tests to assess a difference between probands

and co-twins. In case of non-normal distributions, differences between MZ twins and DZ

twins were tested by means of the Mann-Whitney U test followed by Wilcoxon matched-pair

signed ranks tests for comparisons between probands and co-twins within each twin type. The

significance was set at p<0.05.

90

Results

Descriptive characteristics of the twin population including demographic, life style,

clinical and microbiological data have been reported before (Torres de Heens et al. 'in press').

In brief, the final study sample consisted of 10 MZ twin pairs (6 female and 4 male) and 8 DZ

twin pairs (7 same-sexed pairs: 6 female and 1 male, and 1 opposite-sexed pair). The mean

age was 48.2 years and the probands included more current or former smokers than their co-

twins. Probands showed more attachment- and bone loss than their co-twins which was highly

significant (Table 1). White blood cell counts revealed that the total number of leukocytes and

lymphocytes were significantly higher in probands than in their co-twins.

Table 1. Background clinical parameters and White blood cell (WBC) data (mean values ± SD) in probands and co-twins of MZ and DZ twins combined. Parameters

Probands (N= 18)

Co-twins (N= 18)

p-value

Clinical parameters

No. of teeth 23.8 ± 5.2 25.4 ± 3.1 0.17 Plaque Index 0.9 ± 0.6 0.9 ± 0.3 0.98 Bleeding on probing 0.8 ± 0.6 0.8 ± 0.4 0.90 Probing pocket depth 3.4 ± 0.9 2.8 ± 0.5 0.02 Attachment loss (AL) 3.0 ± 1.4 1.4 ± 0.6 < 0.001 % of teeth 30% bone loss 59.4 ± 39.4 15.7 ± 17.4 < 0.001 Smoking status

Non- or former smoker 12 14 0.31 Current smoker 6 4

Total Leukocytes (109/L) 7.41 ± 2.84 6.13 ± 1.74 0.02 Monocytes 0.53 ± 0.14 0.50 ± 0.12 0.48 Lymphocytes 2.50 ± 0.77 2.11 ± 0.61 0.04 Neutrophils 4.16 ± 2.15 3.39 ± 1.27 0.09 Basophils 0.02 ± 0.04 0.01 ± 0.03 0.41 Eosinophils 0.13 ± 0.10 0.16 ± 0.07 0.26

Table 2 presents data on cytokine secretion in the supernatants of WBCC from all the

probands and their co-twins, after stimulation with LOS, Pg and anti-CD3/CD28. Probands

showed significantly lower amounts of IL-12p40 after LOS stimulation. With regard to the

other cytokines measured after LOS stimulation, no differences were found between the

probands and their co-twins. After Pg stimulation no differences in cytokine values could be

91

assessed although the IL-12p40 values tended to be higher in the co-twins, but they did not

reach significance (p = 0.07). IL-12p40/IL 10 ratios were calculated, but both for LOS and Pg

stimulation no differences between probands and their co-twins were found. After anti-

CD3/CD28 stimulation no significant differences in IFN- and IL-13 values were found

between probands and their co-twins.

Table 2. Cytokine secretion (picograms/ml x 102): IL-1 , IL-6, IL-8, IL-12p40 adjusted for the number of monocytes and neutrophils together, IL-10 adjusted for the number of monocytes and IFN- and IL-13 adjusted for the number of lymphocytes, after LOS, Pg and anti CD3/CD28 stimulation of whole blood in monozygotic (MZ) and dizygotic (DZ) twins. Values represent mean ± SD.

Parameters

Probands (N= 18)

Co-twins (N= 18)

p-value

Cytokine production after LOS stimulation

IL-1 1.00 ± 0.74 1.03 ± 0.59 0.19 IL-6 6.60 ± 0.48 5.51 ± 3.82 0.10 IL-8 25.63 ± 14.44 32.06 ± 36.03 0.91 IL-10 1.64 ± 20.77 1.58 ± 2.44 0.52 IL-12p40 0.24 ± 0.16 0.34 ± 0.31 < 0.001 IL-12p40/IL10 0.01 ± 0.03 0.02 ± 0.02 0.26 Cytokine production after Pg stimulation

IL-1 1.24 ± 0.77 1.26 ± 0.83 0.61 IL-6 6.57 ± 2.81 7.06 ± 4.49 0.77 IL-8 62.56 ± 38.96 59.10 ± 38.75 0.91 IL-10 0.88 ± 1.11 1.02 ± 1.43 0.83 IL-12p40 0.29 ± 0.26 0.38 ± 0.31 0.07 IL-12p40/IL10 0.03 ± 0.06 0.02 ± 0.02 0.61 Cytokine production after CD3/CD28 stimulation

IFN- 219.88 ± 28.42 216.10 ± 17.05 0.65 IL-13 13.78 ± 12.72 14.03 ± 8.53 0.40

Probands of DZ twins had the most severe attachment loss and the highest percentage

of teeth with 30% bone loss. In addition, there was an absence of concordance for both

these measures in both the MZ and DZ twins (Table 3). No differences in white blood cell

counts were found between the MZ probands and their co-twins. Significantly higher

leukocyte and lymphocyte counts were found in the DZ probands compared to their co-twins.

These values were also higher than those found in the MZ probands and their co-twins. In

addition, the difference in the lymphocyte counts between MZ probands and their co-twins

was smaller than the difference between the DZ twin pairs.

Tabl

e 3.

Bac

kgro

und

clin

ical

par

amet

ers a

nd w

hite

blo

od c

ell d

ata

(mea

n va

lues

± S

D) i

n m

onoz

ygot

ic (M

Z) a

nd d

izyg

otic

(DZ)

twin

s.

M

Z (N

= 10

pai

rs)

D

Z (N

= 8

pairs

)

Para

met

ers

Pr

oban

d

C

o-tw

in

p-

valu

e

Prob

and

Co-

twin

p-

valu

e

p-

valu

e*

dMZ

vers

us d

DZ

Clin

ical

par

amet

ers

#

of t

eeth

24.7

± 4

.1

25.

0 ±

3.5

0.

85

22.

8 ±

6.5

2

6.0

± 2.

7

0.07

0.

20

P

laqu

e in

dex

1

.1 ±

0.5

0.9

± 0

.4

0.21

0.6

± 0.

4

0.9

± 0.

2 0.

015

0.05

Ble

edin

g on

pro

bing

1.0

± 0

.5

0

.9 ±

0.5

0.

39

0.

5 ±

0.4

0.6

± 0.

3

0.52

0.

30

P

robi

ng p

ocke

t dep

th

3

.4 ±

0.7

2.9

± 0

.5

0.09

3.4

± 1

.1

2.

7 ±

0.3

0.12

0.

59

A

ttach

men

t los

s (A

L)

2

.3 ±

1.3

1.6

± 0

.8

0.04

3.5

± 1.

2 **

1.2

± 0.

4

<0.0

001

0.01

% te

eth

30%

bon

e lo

ss

41

.7 ±

29.

3

15.

6 ±

17.7

0.

006

81.

5 ±

40.1

**

15.

7 ±

18.1

0.

001

0.01

Sm

okin

g st

atus

N

on- o

r for

mer

smok

er

7 7

1.00

5

7 0.

16

0.27

Cur

rent

smok

er

3 3

3

1

Tota

l Leu

cocy

tes (

109 /L

) 6

.29

± 1.

54

5.8

5 ±

1.58

0.

39

8.8

0 ±

3.54

**

6.4

8 ±

1.95

0.

02

0.13

Mon

ocyt

es

0.4

9 ±

0.13

0

.48

± 0.

10

0.92

0

.56

± 0.

13

0.5

2 ±

0.15

0.

35

0.43

Lym

phoc

ytes

2

.09

± 0.

60

2.0

7 ±

0.57

0.

89

3.0

1 ±

0.70

**

2.1

6 ±

3.01

0.

02

0.02

Neu

troph

ils

3.5

9 ±

1.11

3

.12

± 1.

12

0.31

4

.87

± 2.

93

3.7

3 ±

1.43

0.

18

0.77

Bas

ophi

ls

0.0

1 ±

0.03

0

.02

± 0.

42

0.56

0

.03

± 0.

05

0 ±

0 0.

08

0.83

Eos

inop

hils

0

.09

± 0.

06

0.1

0 ±

0.06

0.

45

0.1

6 ±

0.11

0

.17

± 0.

06

0.43

0.

72

* si

gnifi

cant

p-v

alue

s ind

icat

e th

at th

e di

ffer

ence

s (d)

bet

wee

n M

Z tw

ins a

re si

gnifi

cant

ly d

iffer

ent f

rom

thos

e of

DZ

twin

s. **

val

ues o

f DZ

prob

ands

sign

ifica

ntly

hig

her c

ompa

red

to M

Z pr

oban

ds a

nd c

o-tw

ins p

< 0.

01

93

In Table 4 the cytokine secretion in the supernatants of WBCC is presented for

probands and their co-twins of the MZ and DZ twins. After LOS stimulation IL-6 levels in MZ

twins were higher for the probands than for their co-twins. The values of the other cytokines

were not significantly different between probands and their co-twins, both in MZ and DZ

twins. The MZ probands as well as their co-twins had significantly higher values of IL-6 and

IL-12p40 after LOS stimulation than the DZ probands and their co-twins. After Pg stimulation,

the IL-12p40 values of the DZ twins were lower for the probands than for their co-twins. For

the other cytokines after Pg stimulation no significant differences between probands and their

co-twins were found both in MZ and DZ twins. The MZ probands as well as their co-twins had

significantly higher values of IL-12p40 after Pg stimulation compared to DZ probands and

their co-twins. With regard to the IL-12p40/IL-10 ratios both in MZ and DZ twins no

differences were found between probands and their co-twins. Also after lymphocytic

stimulation with anti-CD3/CD28 no significant differences between probands and their co-

twins in MZ and DZ twins were found for IFN- and IL-13 values.

Tabl

e 4.

Cyt

okin

e se

cret

ion

(pic

ogra

ms/

ml x

102 ):

IL-1

, IL-

6, IL

-8, I

L-12

p40

adju

sted

for t

he n

umbe

r of m

onoc

ytes

and

neu

troph

ils

toge

ther

, IL-

10 a

djus

ted

for t

he n

umbe

r of m

onoc

ytes

and

IFN

- a

nd IL

-13

adju

sted

for t

he n

umbe

r of l

ymph

ocyt

es, a

fter L

OS,

Pg

and

anti

CD

3/C

D28

stim

ulat

ion

of w

hole

blo

od in

mon

ozyg

otic

(MZ)

and

diz

ygot

ic (D

Z) tw

ins.

Val

ues r

epre

sent

mea

n ±

SD.

MZ

(N=

10 p

airs

)

D

Z (N

= 8

pairs

)

Cyt

okin

e pr

oduc

tion

Pr

oban

d

C

o-tw

in

p-

valu

e

Prob

and

Co-

twin

p-

valu

e

p-

valu

e*

dMZ

vers

us

dDZ

afte

r LO

S st

imul

atio

n

IL-1

1.

08 ±

0.7

8

1

.19

± 0.

30

0.22

0

.90

± 0.

72

0.8

4 ±

0.37

0.

57

0.75

IL

-6

8.52

± 5

.67*

*

6

.33

± 4.

65**

0.

02

4.2

1 ±

1.85

4

.48

± 2.

34

0.91

0.

08

IL-8

27.

15 ±

14.

95

4

0.30

± 4

6.75

0.4

9

23

.73

± 14

.55

2

1.76

± 1

1.16

0.

81

0.49

IL

-10

0.71

± 0

.77

1.1

1 ±

2.31

0.

93

2.8

1 ±

2.64

2

.18

± 2.

62

0.26

0.

58

IL-1

2p40

0.

29 ±

0.1

7**

0.4

6 ±

0.37

**

0.11

0

.18

± 0.

13

0.1

9 ±

0.10

0.

43

0.61

IL

-12p

40/IL

10

0.01

± 0

.01

1.7

8 ±

1.84

0.

56

0.0

2 ±

0.05

0

.01

± 0.

02

0.28

0.

81

afte

r Pg

stim

ulat

ion

IL

-1

1.40

± 0

.88

1.5

2 ±

0.96

0.

33

1.0

4 ±

0.61

0

.92

± 0.

50

0.89

0.

86

IL-6

7.

09 ±

1.7

9

8.

31 ±

5.4

10.

83

5

.90

± 3.

77

5.

51 ±

2.4

90.

860.

96

IL-8

63.

35 ±

43.

45

6

6.39

± 0

.40

0.88

61

.59

± 35

.45

4

9.10

± 3

7.38

0.

78

0.50

IL

-10

0.42

± 0

.48

0.7

8 ±

1.07

0.

32

1.4

5 ±

1.43

1

.33

± 1.

82

0.51

0.

24

IL-1

2p40

0.

37 ±

0.2

2***

0

.45

± 0.

37**

* 0.

78

0.1

8 ±

0.27

0

.29

± 0.

20

0.04

0.

06

IL-1

2p40

/IL10

0.

04 ±

0.0

8

0

.02

± 0.

01

0.44

0.

003

± 0.

006

0.0

2 ±

0.03

0.

19

0.11

af

ter C

D3/

CD

28 st

imul

atio

n

IF

N-

3

00.3

7 ±

364.

33

2

26.9

3 ±

143.

71

0.64

11

9.27

± 6

8.23

202.

56 ±

208

.90

0.33

0.

26

IL-1

3

14.

80 ±

12.

06

12.8

9 ±

7.65

0.

76

12.

50 ±

14.

24

1

5.45

± 9

.86

0.27

0.

27

*

sign

ifica

nt p

-val

ues i

ndic

ate

that

the

diff

eren

ces (

d) b

etw

een

MZ

twin

s are

sign

ifica

ntly

diff

eren

t fro

m th

ose

of D

Z tw

ins.

**

valu

es o

f MZ

prob

ands

and

co-

twin

s sig

nific

antly

hig

her c

ompa

red

to D

Z pr

oban

ds a

nd c

o-tw

ins p

< 0.

01

***

valu

es o

f MZ

prob

ands

and

co-

twin

s sig

nific

antly

hig

her c

ompa

red

to D

Z pr

oban

ds p

< 0.

01

95

Discussion

Periodontitis is considered to be a complex disease of which the phenotype is

determined by the genetic make-up, the environmental influences and the life style of the

affected individual (Loos et al. 2008). In the previous clinical analysis of our twin study

the results showed that the MZ probands suffered from more severe periodontitis than

their co-twins (Torres de Heens et al. 'in press'). This discrepancy between the MZ twins

could not be explained by the studied life style and environmental factors, such as

education, smoking and periodontal pathogens. Since the MZ probands and co-twins

were significantly discordant for the amount of periodontal breakdown, it was not

surprising that the DZ twins were also discordant. Analysis showed that the difference in

periodontal condition between the DZ twin pairs differed to a greater extent than the

differences between the MZ twin pairs, suggesting that some genetic component is at

play (Torres de Heens et al. 'in press').

In order to investigate whether monocytic and lymphocytic cytokine secretion

after ex vivo stimulation could explain the observed discordance in periodontal

breakdown of MZ and DZ twins, LOS from N. meningitidis and a sonicate of P.

gingivalis were used (Torres de Heens et al. 2009). In stimulation studies often LPS from

Escherichia coli has been used, however this may be criticized since in the in vivo

situation and during infection, E. coli LPS is surely not the only bacterial component

interacting with immune cells (Fokkema et al. 2002). Secondly, E. coli is not a

periodontal pathogen. In order to overcome these problems to some extent, a sonic

extract of P. gingivalis, a major periodontal pathogen which signals through Toll-like

receptor 2 (TLR2), was used. Nevertheless, it must be realized that 50% of the probands

and 81% of the co-twins were culture negative for P. gingivalis (Torres de Heens et al. 'in

press'). Therefore, in addidion to the Pg sonic extract, a generic stimulant (N.

meningitidis LOS) which signals through TLR4 was used for WBCC stimulation. In our

results, the amount of cytokine measured in the supernatants stimulated with LOS or Pg

was adjusted for to the number of monocytes and/or neutrophils where appropriate. It

should be noted that on a cell basis the production of IL-1 , IL-6, IL-8 and IL-12p40

from neutrophils is much less than that from monocytes, which are the principal

producers of all these cytokines (Moore et al. 1993, Snijders et al. 1996, van der Pouw

96

Kraan et al. 1995, Wang et al. 1994). Nevertheless, there may be a significant

contribution of neutrophils to the overall cytokine production during inflammation due to

the quantitative predominance of these cells over the monocytes in the peripheral blood

and at sites of acute inflammation. For these reasons it was decided to consider both

monocytes and neutrophils as the main producing cells for the mentioned cytokine set.

Levels of IL-10 were only adjusted for the number of monocytes, since neutrophils do

not secrete this cytokine. IFN- and IL-13 production were corrected for lymphocyte cell

counts because we selectively stimulated those cells with anti- CD3 and CD28 (Gerards

et al. 2003, Yamada-Ohnishi et al. 2004).

Discordance of periodontal breakdown in MZ twins offers the unique possibility

to disentangle the etiology of the disease. Therefore the number of leukocytes and their

cytokine production after ex vivo stimulation were investigated. In the study population

as a whole, probands showed higher total numbers of leukocytes compared to their co-

twins. This discrepancy could almost completely be explained by the DZ probands

having the worst periodontal condition, compared to DZ co-twins, MZ probands and MZ

co-twins. The higher number of leukocytes in these subjects is in agreement with

previous studies which showed that periodontitis patients have higher numbers of

leukocytes compared to controls (Christan et al. 2002, Fredriksson et al. 1998, Kweider et

al. 1993, Loos et al. 2000). Nevertheless, the MZ probands who suffered also from

moderate to severe periodontitis, showed comparable numbers of leukocytes to their own

co-twins as well as to the DZ co-twins both having a far better periodontal condition.

This finding may be explained by a lower degree of severity of periodontitis in the MZ

probands as compared to the DZ probands. Possibly the severity of disease of the MZ

probands was not severe enough to cause a significant increase in the number of

leukocytes.

Leukocytes, when triggered by whole bacteria as well as bacterial components,

produce cytokines which direct both innate and adaptive immunity (Seymour & Gemmell

2001). Cytokines such as the pro-inflammatory interleukin IL-1 , IL-6, IL-8, IL-12 and

anti-inflammatory IL-10 have been shown to be part of the inflammatory response in

periodontitis and may determine the host susceptibility and thereby variation in

periodontal destruction (Gemmell et al. 1997, Gemmell et al. 2002, Niho et al. 1998,

97

Seymour & Gemmell 2001). In the twin population as a whole, probands and co-twins

showed no differences in cytokine secretion, except for a lower level of IL-12p40 in the

probands after LOS stimulation and to a lesser extent after Pg stimulation. In addition, in

the probands, a trend towards higher levels of IL-6 after LOS stimulation can be seen (p-

value 0.10). The IL-6 data of the MZ twins show that MZ probands secrete higher levels

of this cytokine than their co-twins. IL-6 is a pleiotropic cytokine and plays a major role

in bone remodeling, neuro-endocrine homeostasis, haemopoiesis and immune-

inflammatory response regulation. In particular, it plays a pivotal role in acute phase

responses and in balancing the pro-inflammatory/anti-inflammatory pathways. As

reviewed (Ershler & Keller 2000), it is suggested that elevated expression of IL-6 may

contribute to (generalized) autoimmune diseases, such as rheumatoid arthritis. Since after

stimulation the cells of the MZ probands secreted more IL-6 than their co-twins and the

MZ probands have more periodontal breakdown an association may be suggested

between hyper responsive cells secreting IL-6 and the risk for periodontal breakdown.

Since the MZ twins have identical DNA sequence, other phenomena might play a role;

epigenetic modifications of DNA have recently been observed (Barros & Offenbacher

2009). Whether the higher secretion level is due to epigenetic mechanisms or other than

the studied environmental and life style factors education, smoking, BMI and periodontal

pathogens, cannot be deduced on the basis of the present data.

The lower levels of IL-12p40 as found in the probands compared to their co-twins

in the study population as a whole, is indicative for a Th2 response. This together with

the greater periodontal breakdown found in the probands, is in line with the concept that

considers periodontitis as a Th2-type disease. In general it is assumed that susceptible

individuals may have a predominant Th2 type response, which contributes to tissue

breakdown (Aoyagi et al. 1995, Bartova et al. 2000, Gemmell et al. 2007, Manhart et al.

1994, Torres de Heens et al. 2009). However in the present study this is neither supported

by a lower IL-12p40/IL-10 ratio, which would strongly indicate a type 2 response, nor by

the elevated IL-13 values of the probands compared to their co-twins in both MZ and DZ

twins. The lack of supporting evidence may be due to the finding that the subjects in this

twin population showed relatively low plaque and bleeding scores, suggestive for

previous periodontal treatment in general practice before referral to the periodontal

98

clinics. This supposition is supported by the finding that the twins recruited via

periodontal clinics have lower plaque and bleeding scores than the non referred twins

recruited with the aid of the Dutch Association of Twins. The low bleeding scores, that

were almost identical for probands and co-twins in both MZ and DZ twin groups, are

indicative for relatively little inflammation in the periodontal pockets. Therefore, the Th2

profile of subjects with active disease may have changed into a Th1 profile associated

with periodontal stability (Gemmell & Seymour 2004, Gemmell et al. 2007, Seymour et

al. 1996). On the other hand the lack of supporting data of the present study towards a

type 2 response is in line with the recent understanding that the Th1/Th2 paradigm cannot

accurately describe periodontal disease independent of the involvement of the novel Th17

subset (Gaffen & Hajishengallis 2008). Unfortunately, IL-17 measurements were not

included in the present study and therefore no real suggestions can be made.

Nevertheless, in the study population as a whole, monocytes of the probands, when

stimulated ex vivo, secreted less IL-12p40 than the co-twins. Also, in the small number of

MZ twins a trend may be seen towards lower levels of IL-12p40 after LOS stimulation in

the probands. If 16 instead of the present 10 MZ twins would have been included in this

study, a statistical significant difference could have been assessed suggesting an

association between the level of IL-12p40 secretion and disease risk. On the other hand, it

must be realized that the number of twins in the present study is small and the statistics

did not include corrections for multiple comparisons. Therefore the results on the basis of

p-values 0.01 should be interpreted with care. However, the small number of twins may

have been also responsible for the many non significant differences.

In the search of etiological factors for diseases in general the twin model has been

used for decades and with time, epidemiologic studies included increasing numbers of

twins. Traditionally, MZ and DZ twins were recruited from databases or twin registers,

irrespective of disease status. However, during the last decade studies are focusing on

MZ twins that are discordant for the disease in question. The discordant twin design,

which may have small numbers of twins, allows the investigation of between twin

differences that are specifically due to influences of environmental and life style risk

factors (Martin et al. 1997). Because MZ twins start life with identical genomes, within

twin pair differences reflect exposure to an individual-specific environment which may

99

ultimately act through genetic or epigenetic modifications of gene expression (den Braber

et al. 2008). For example for the development of distinct Th cell lineages, the initial

instructions are received by the naive CD4+ T cells from the antigen-presenting cells. The

instruction are converted by responding T cells into changes in the abundance,

interactions and locations of transcription factors, which in turn lead to changes in gene

expression. As suggested before (Wilson et al. 2009), more precise gene expression is

achieved through epigenetic processes which facilitate heritable and stable programs of

gene expression. Such mechanisms may well have been the cause for the observed

differences in IL-6 and to a lesser extent IL-12p40 in the MZ twins.

In conclusion, the results of the present study suggest that the observed

discordance in periodontal breakdown in the studied twin population may be related to

relatively high levels of IL-6 and low levels of IL-12p40 secretion after ex vivo

stimulation of WBCCs. This cytokine secretion profile may be regarded as a risk

indicator of periodontitis.

Acknowledgements

The authors are grateful to Mark Timmerman for his assistance in the periodontal

clinical examination of the twin population. The authors wish to thank A. Smid and J.

Smid, chairmen of the Dutch Association of Twins and the following periodontists for

their help in the recruitment of the twins: P.G.G.L. van der Avoort, D.J. Bossers, R.A.

Driessen, S.J. Fokkema, P.C. van Gils, H. Hamming, J.W. Hutter, G. Maffei, G.N.Th. de

Quincey, J. Steinfort, R.W.R. Steures, A. Varoufaki. The authors thank Prof. dr. D.I.

Boomsma, Department of Biological Psychology, Vrije Universiteit, Amsterdam, The

Netherlands, for her useful comments.

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Chapter 6

Summary and Discussion

106

Summary and Discussion

Periodontitis is a complex, multifactorial, chronic disease that affects the

supporting tissues of the teeth and is characterized by inflammation and destruction of

connective tissues and alveolar bone (Pihlstrom et al. 2005). Disease expression involves

intricate interactions of the bacterial biofilm with the host immune inflammatory

response and subsequent alterations in the bone and connective tissue homeostasis

(Preshaw 2008). Certain individuals appear to be more susceptible to periodontitis (Loos

et al. 2008), and this increased susceptibility is largely determined by the immune

inflammatory response that develops in the periodontal tissues following chronic

exposure to bacterial plaque (Preshaw 2008). The immune inflammatory response against

bacterial plaque can thus be viewed as a 'two-edged sword'. That is, the response is

protective by intent, and provides antibodies and polymorphonuclear neutrophils that are

responsible for the control of the bacterial infection. However, the inflammatory response

in susceptible individuals, results in the local production of excessive quantities of

destructive enzymes and inflammatory mediators that result in the tissue destruction

which is observed clinically (Preshaw 2008). It is paradoxical that the inflammatory

response to the bacterial challenge is primarily responsible for the breakdown of the

periodontal hard and soft tissues. Although the bacterial challenge initiates inflammation

in the tissues, genetic factors and life style factors, such as smoking, modulate the

inflammatory response and determine the resulting disease progression and severity that

is seen clinically as bone and attachment loss (Kornman 2008, Loos et al. 2008, Page et

al. 1997, Palmer et al. 2005, Pihlstrom et al. 2005, Preshaw 2008).

The present thesis was intended initially to examine the extent to which genetic

variation determines phenotypic variation of moderate to severe chronic periodontitis. A

better understanding of complex diseases like periodontitis involves the evaluation of the

interaction between genetic factors and the life style and bacterial influences. Twin

studies have been a valuable source of information about the genetic basis of

multifactorial traits. To date results of twin studies of chronic periodontitis show

converging results suggesting a substantial role of genetics in this condition (Corey et al.

1993, Michalowicz, 1994, Michalowicz et al. 1991a, Michalowicz et al. 1991b,

Michalowicz et al. 2000, Michalowicz et al. 1999, Mucci et al. 2005). However, these

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studies have some limitations as they based their results on periodontal observations by

questionnaires and not on clinical measurements (Corey et al. 1993, Mucci et al. 2005)

and included study populations with relatively minor periodontal destruction

(Michalowicz et al. 1991a, Michalowicz et al. 1991b, Michalowicz et al. 2000). Up to

now all twin studies selected twins because of their twinship and not on the presence of

moderate to severe periodontitis. Thus it may still be questioned to what extent genetics

contribute to the development of moderate to severe chronic periodontitis. As prelude to

investigating this issue, and motivated by the fact that the interplay between the host

genetic make up and smoking as main lifestyle factor is of pivotal importance, it was

decided to explore first the effect of smoking on the innate and adaptive immune

response in periodontitis. This approach would help us to perhaps explain phenotypic

variation among identical twins.

Studies have shown that on average 50% of the patients suffering from

periodontitis are smokers or former smokers (Bergstrom 1989, Tonetti 1998, van der

Weijden et al. 2001, Xu et al. 2002). This proportion is high compared to the overall adult

Dutch population of which approximately 27% are smokers (Tabakspreventie 2008).

Most periodontists would consider that smokers are the most challenging patients to

manage in the periodontal treatment. Smokers tend to have more advanced periodontal

disease than non-smokers and less favorable outcomes following periodontal treatment

(Johnson & Hill 2004, Kinane & Chestnutt 2000, Palmer et al. 2005, Tonetti 1998). In

addition, smoking has major effects on the host immune response (Graswinckel et al.

2004, Kinane and Chestnutt 2000, Loos et al. 2004, Palmer et al. 2005).

T cells play an important immunoregulatory role in the pathogenesis of

periodontal diseases (Gemmell et al. 2007, Kinane & Lappin 2001, Seymour et al. 1996).

Evidence supporting the concept that periodontitis is a Th2-type disease comes from both

histological analysis of inflamed periodontal tissues and ex vivo cytokine production in

periodontitis patients compared to healthy controls (Gemmell et al. 2007). However it

was not yet investigated whether this Th2 profile in periodontitis would be potentiated by

smoking, while at least half of the patients smoke. This presumption set the basis for our

first study. In Chapter 2, we investigated the monocytic derived T cell directing

(Th1/Th2) response and pro-inflammatory cytokine production in ex vivo whole blood

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cell cultures (WBCC) of smoking and non-smoking chronic periodontal patients. Venous

blood was collected from 29 patients (18 non-smokers and 11 smokers) receiving

supportive periodontal treatment (SPT), and diluted 10-fold for WBCC. WBCC were

stimulated for 18 hours with Neisseria meningitidis lipooligosaccharide (LOS) and

Porphyromonas gingivalis sonic extract (Pg-SE). The production of the T cell directing

cytokines interleukin IL-12p40 and IL-10, as well as the pro-inflammatory cytokines IL-

1 , IL-6 and IL-8, was measured in the culture supernatants. Based on previous studies it

is clear that monocytes/macrophages are the source for these cytokines (van der Pouw

Kraan et al. 1997, van der Pouw Kraan et al. 1995). Our data shows that smoking

periodontal patients have a lower IL-12p40/IL-10 ratio after LOS stimulation and lower

IL-1 production after LOS and Pg-SE stimulation than their non-smoking counterparts.

These findings were suggestive of a more pronounce Th2 immune response in smoking

periodontal patients.

Although our findings provided evidence that smoking patients display a more

pronounced Th2 type monocytic response, we felt it needed confirmation that indeed the

T cells express such a cytokine pattern. In Chapter 3, we investigated the T lymphocytic

cytokine production representing Th1 and Th2 subpopulations in smoking and non-

smoking patients and healthy controls. WBCC were stimulated with specific T cell

stimulants and IFN- and IL-13 were measured in the culture supernatants, representing

type 1 and 2 Th subpopulations respectively. Our results showed that smokers had more

lymphocytes, and higher levels of IFN- and IL-13, irrespective of being a periodontal

patient. However, in a multivariate analysis we found that the increased IFN-

production was not significantly explained by smoking, while higher IL-13 was strongly

explained by smoking. The secretion of IFN- and IL-13 was independent of each other

which demonstrate that they indeed were secreted by two distinct T cell populations (i.e.

Th1 and Th2 type). We suggested that the increased Th activity and specifically an

elevated Th2 profile in smokers may constitute a risk for smoking patients, which may

induce conversion of periodontal stability into progressive disease. This study allowed us

to confirm our previous finding on the monocytic cytokine profile in smokers. The

observed Th2 profile measurable in the monocytic response in the smoking periodontitis

patients is consistent with the cytokine profile produced by the lymphocytes.

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Our results showed differences between smoking and non-smoking periodontal

patients in ex vivo cell culture cytokine production. IL-12 and IL-10 are major cytokines

of the innate immunity with main effects on the ensuing adaptive immune response

(Seymour & Gemmell 2001). High IL-12 levels will contribute to the Th1 type immune

reaction, as it is a strong inducer of IFN- production (Tsai et al. 2005). In contrast, IL-10

reduces the secretion of IFN- and has a potential role in diminishing IFN- -mediated

responses and thereby Th1 type of immunity (Lappin et al. 2001). Furthermore, IL-1 is

involved in the up-regulation of IFN- production by Th1 cells, down-regulation of IL-4

production by Th2 cells (Sandborg et al. 1995, Schmitz et al. 1993) and can be inhibited

by smoking (Pabst et al. 1995). Taken together, the lower IL-12p40/IL-10 ratio and the

lower IL-1 production in the studied group of smokers were indicative of a stronger Th2

response (Chapter 2), which was confirmed by higher IL-13 levels produced by a Th2

cell population in smoking subjects (Chapter 3).

It is well known that there is a shift from a predominantly T cell to B cell lesion

in the progression from gingivitis to periodontitis. It is interesting to speculate that a shift

from cell-mediated immunity (Th1) to humoral immunity (Th2) occurs during the

development of periodontal disease (Kinane & Lappin 2001). It is apparent that in

gingivitis T cells probably exceed cells of the B cell lineage, and when this progresses

into periodontitis, B cells then predominate (Kinane & Lappin 2001). The dominance of

B-cells/plasma cells in the advanced/ progressive lesion would suggest a role for Th2

cells. Th1 and Th2 cells induce B cell proliferation, but Th2 cells are generally more

efficient than Th1 in this capacity (Rothermel et al. 1991). Furthermore, activated B cells

have the potential to contribute to the amplification or maintenance of the ongoing

polarized immune response (Harris et al. 2000). Therefore, it is plausible that a more

pronounced Th2 response in smoking subjects may increases the B cell proliferation,

inducing a stronger humoral response, which in turn may reinforce the existing Th2

response, thereby increasing the risk for recurrent periodontal destruction in the smoking

patient.

B cells have the capacity to make pleitropic cytokines such as IL-6 and tumor

necrosis factor (TNF)- , which regulate diverse aspects of bone resorption and formation

in inflammatory diseases (Harris et al. 2000). These cytokines may induce bone

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resorption directly and indirectly by affecting the production of the essential osteoclast

differentiation factors (Boyce et al. 2005, Gemmell et al. 1997). Therefore, it may be

suggested that these bone resorbing cytokines produced by increased activated B cells

may contribute to the periodontal breakdown in the smokers. In addition, the pronounced

B cell stimulation and activation may contribute to higher production of autoantibodies. It

is known that autoimmune mechanisms may contribute to periodontal disease

pathogenesis (Rajapakse & Dolby 2004). A recent study suggested the involvement of

serum autoantibodies directed to extracellular matrix components in the pathogenesis of

chronic periodontitis (De-Gennaro et al. 2006). In addition, a local production of

autoantibodies to autoantigen in granulomatous tissues housed within the periodontal

lesion has been described (Rajapakse & Dolby 2004). Therefore, autoimmune

mechanisms triggered by the increase B cell activation may contribute to the periodontal

breakdown in the smoking subjects. The previous studies on the influence of smoking on

aspects of the immune response in the periodontal disease provided important

background to further study the severity of periodontal breakdown observed in the

periodontal patient. The aim of these studies was to provide a useful framework for the

data analysis of our twin population.

In order determine the relative contribution of genetic, environmental and life

style factors; such as smoking, in the etiology of moderate to severe chronic periodontitis,

the classic twin method was used. In Chapter 4, monozygotic (MZ) and dizygotic (DZ)

twins reared together, were recruited to assess the contribution of genetics, periodontal

pathogens and life style factors to the clinical phenotype. Important for this study was

that the adult twin pairs were selected on the basis of at least one of the 2 twin members

having interproximal attachment loss 5 mm in 2 non-adjacent teeth. The study

included 10 MZ and 8 DZ twin pairs, in which the periodontal condition, presence of

periodontal pathogens, educational level, smoking behavior and Body Mass Index (BMI)

was evaluated. The most important result of this study is the finding that MZ twins

appeared to be rather discordant with regard to mean attachment loss, number and

percentage of teeth with AL 5 mm and percentage of teeth with bone loss 30%. By

selection the MZ probands suffered from moderate to severe periodontitis, whereas the

MZ co-twins were not selected on the presence of periodontitis and showed periodontal

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breakdown to a much lesser extent. This finding was surprising since on the basis of

previous research it was supposed that genetics contribute to 50% of the severity of

periodontitis (Michalowicz et al. 2000) and thus less large differences were to be

expected in the MZ twins. Analysis of our twin data showed that the lack of concordance

could not be explained by periodontal bacteria, smoking and BMI, factors that are all

known to be related to destructive periodontal disease. However, it must be realized that

the number of twins in the present study was small. This small number may have been

also responsible for the many non significant differences, e.g. the subgingival presence of

P. gingivalis. In the MZ twin group, 5 out of the 10 probands were positive for P.

gingivalis, whereas 2 out of the 10 co-twins were positive for this bacterium. A larger

study population of MZ twins could have shown that P. gingivalis plays a significant role

in the etiology of periodontitis. Smoking as an explanatory variable could not be

evaluated in the MZ twins since these genetically identical pairs showed also a similar

smoking behaviour. Interestingly, this was in contrast to smoking behaviour found within

the DZ twins where no equal smoking habits between pairs were noted. In the DZ twins

45.6% of the variation in terms of periodontal breakdown could be explained by

smoking. Within MZ twin pairs the discordance regarding periodontal breakdown was

smaller than within DZ twin. This was expected as this phenomenon has been previously

shown in large-scale twin data.

Since immune response mechanisms have been shown to play an important role

in the periodontal breakdown, the analysis of the cytokine production in this twin

population constituted an interesting parameter to be explored as a possible explanation

for the discrepancy in periodontal phenotype between the MZ and DZ twin pairs. In

Chapter 5, we investigated the extent of concordance in number of leukocytes and their

cytokine secretion after ex vivo stimulation in the previously studied twin population.

Probands of both MZ and DZ twin sets pooled together showed higher total numbers of

leukocytes and lower IL-12p40 levels compared to their co-twins. The higher number of

leukocytes is similar to findings in the literature where it has been shown that with

increasing severity of periodontal disease, leukocyte numbers increase (Loos 2005). It is

well known that lower IL-12p40 level favors the Th2 immunity and decreases the

stimulation of the Th1 type immune response. Our finding of increased alveolar bone loss

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together with the lower IL-12p40 production in the total group of probands compared to

the group of co-twins is consistent with the characteristic Th2 type response in

periodontitis. Furthermore, MZ probands secreted more IL-6 than their co-twins. This

observation is in line with previous results showing a dose response in systemic levels of

IL-6: increased severity leads to higher IL-6 secretion (Loos et al. 2000). IL-6 is a

multifunctional cytokine, of which biological activities include B-lymphocyte

differentiation, T-lymphocyte proliferation and stimulation of immunoglobulin secretion

by B-lymphocytes (Hirano et al. 1990). Of particular significance is the ability of IL-6 to

induce bone resorption, both by itself and in conjunction with other bone-resorbing

agents (Ishimi et al. 1990). Therefore, it may be concluded that within our twin

population, the higher IL-6 production in MZ probands seems to be associated to the

increased bone loss found in this group. The results of our twin study suggested that the

observed lack of strong concordance of periodontal destruction in the studied MZ twin

population can not be explained by differences in numbers of leukocytes, but that low

levels of IL-12p40 and high levels of IL-6 secretion after ex vivo stimulation may be

regarded as risk indicators for the severity of periodontitis.

Based on our results from the twin population, we suggest that the role of genetics

in periodontitis may have been overestimated. The host genetic make-up, the traditional

lifestyle factors and the immune responses after stimulation of cells could not explain the

lack of straight forward concordance in periodontal destruction, especially in MZ twins,

indicative that other factors play an important role in the extent and severity of

periodontal disease. We have several suggestions. For example, nutritional factors and

dietary supplementation have been associated with the inflammatory response and

disease severity in periodontal disease (Amaliya et al. 2007, Rosenstein et al. 2003,

Staudte et al. 2005). A reduced-calorie diet dampens the inflammatory response and

reduces active periodontal breakdown associated with an acute microbial challenge

(Branch-Mays et al. 2008). In addition, fish oil dietary supplementation may have

potential benefits as a host modulatory agent in the prevention and/or adjunctive

management of periodontitis (Bendyk et al. 2009). Another explanation for discordance

of periodontal breakdown among twins could be diversity of concomitant infectious

agents in the periodontal tissues. Herpesvirus infections may initiate or accelerate

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periodontal breakdown via their ability to stimulate cytokine release from host cells. The

ensuing inflammation might impair host defense mechanisms, resulting in less defensive

capacities against the resident periodontopathic bacteria (Contreras et al. 2000, Slots

2007).

Furthermore, although genetics modulate the inflammatory response, there is

increasing evidence that epigenetic mechanisms are critical for regulating the

inflammatory response (Offenbacher et al. 2008). Epigenetic events act through the

remodeling of the chromatin structure due to DNA methylation and histone acetylation

which can selectively activate or inactivate genes and determine their expression. In

general, increased DNA methylation in the promoter region of genes, causes gene

silencing (Franco et al. 2008). The epigenetic process, by inducing a change in cytokine

profile, may subsequently influence the pathogenesis and determine the outcome of many

infectious diseases (Gomez et al. 2009). For example, preliminary findings suggested that

the gene for IL-6, a cytokine involved in the final differentiation of B-cells into

immunoglobulin-secreting cells, undergoes a decrease in methylation in periodontal

disease tissues compared to control samples. These preliminary findings suggest that the

IL-6 gene may be preferentially upregulated in expression in periodontal disease

(unpublished data, Offenbacher et al. 2008). This suggestion is in line with the finding in

our twin study that in stimulated whole blood cell cultures, MZ probands suffering from

moderate to severe periodontitis secreted higher levels of IL-6 than their co-twins with

minor periodontal breakdown. It has been shown also that infection can lead to host

epigenetic modification of an imprinted gene (Bobetsis et al. 2007). Thus previous

infectious diseases, and/or viral infectious in the proband of a MZ twin pair could have

triggered genetic variation and as a consequence developing more severe periodontal

disease. Unfortunately, there is scarce information of epigenetic events during

periodontitits. Hopefully, future research will help us understand, for example, how

systemic exposures, like smoking, may alter global epigenetic patterns to affect the

expression of periodontitis (Barros & Offenbacher 2009). Taken together, the above

mentioned suggestions may potentially influence the disease expression and therefore

could explain the lack of clear concordance in periodontal condition of the MZ twins in

the present study.

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The identification of individuals with increased susceptibility to periodontitis

remains a great challenge in the dental practice. Tailored prevention and treatment

strategies of the subject at higher risk for periodontitis are needed. Hopefully, studies on

larger twin populations involving the assessment of other potential factors which may

influence the host immune response may shed light on the pathways by which some

individuals develop periodontitis and will provide knowledge on how to prevent

effectively the disease onset and progression. It can be envisaged that in periodontitis, the

interaction between genetic and epigenetic mechanisms influenced by life style- and

enveronmental factors provides an interesting line of research in our field.

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Samenvatting en Discussie

Parodontitis is een complexe, multifactoriele, chronische aandoening van de

steunweefsels van de gebitselementen (het parodontium), en wordt gekenmerkt door

ontsteking en afbraak bindweefsel en kaakbot (Pihlstrom et al. 2005). Het verloop van de

aandoening wordt bepaald door de interactie tussen de bacteriën in de biofilm op het

tandoppervlak en het afweersysteem van de gastheer resulterend in een verstoring van de

bindweefsel en kaakbot homeostasis (Preshaw 2008). Sommige individuen blijken

vatbaarder te zijn voor parodontitis dan anderen (Loos et al. 2008). Deze verhoogde

vatbaarheid wordt grotendeels veroorzaakt door de immunologische ontstekingsreactie

van de gastheer op de bacteriële tandplaque (Preshaw 2008). Deze immune respons op de

bacteriële plaque kan gezien worden als een twee snijdend zwaard. Dat wil zeggen dat de

respons in principe beschermend is en leidt tot de productie van antistoffen en migratie

van polymorphonucleare leukocyten (PMNs) die verantwoordelijk zijn voor de

verdediging tegen de bacteriële infectie. De ontstekingsreactie bij personen die

vatbaarheid zijn voor parodontitis gaat echter gepaard met excessieve productie van

destructieve enzymen en ontstekingsmediatoren resulterend in parodontale afbraak

(Preshaw 2008). Het is eigenlijk paradoxaal dat de in principe beschermende

ontstekingsreactie tevens oorzaak is van de afbraak van de harde en zachte weefsels van

het parodontium. Hoewel bacteriën de ontstekingsreactie in de weefsels initiëren, blijken

genetische en life style factoren, zoals roken, de ontstekingsreactie te beïnvloeden

waardoor deze bepalend zijn voor de ernst en progressie van de parodontitis (Kornman

2008, Loos et al. 2008, Page et al. 1997, Palmer et al. 2005, Pihlstrom et al. 2005,

Preshaw 2008).

Het oorspronkelijke doel van het huidige proefschrift was om te onderzoeken in

welke mate genetische variaties bepalend zijn voor de fenotypische variatie van matige

tot ernstige chronisch parodontitis. Evaluatie van de interactie tussen genetische factoren

aan de ene kant en de invloed van lifestyle factoren en bacteriën aan de andere kant, zijn

van groot belang om een beter begrip te krijgen van de etiologie van chronische ziekten

zoals parodontitis. Bij het onderzoek naar de genetische achtergrond van multifactoriële

aandoeningen vormen tweelingen onderzoek een belangrijke bron van informatie. Op dit

moment lijken de resultaten van het beschikbare tweelingen onderzoek in de richting

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wijzen dat de erfelijke aanleg een belangrijke component is de etiologie van parodontitis

(Corey et al. 1993, Michalowicz 1994, Michalowicz et al. 1991a, Michalowicz et al.

1991b, Michalowicz et al. 2000, Michalowicz et al. 1999, Mucci et al. 2005). Echter,

deze bevindingen dienen voorzichtig geïnterpreteerd te worden aangezien de resultaten

gebaseerd zijn op parodontale bevindingen die verkregen zijn uit onderzoek met behulp

van enquêtes en niet op basis van klinische gegevens (Corey et al. 1993, Mucci et al.

2005) of bij patiënten populaties die niet op de aanwezigheid van parodontitis zijn

geselecteerd (Michalowicz et al. 1991a, Michalowicz et al. 1991b, Michalowicz et al.

2000). Tot nu toe zijn bij al het tweelingen onderzoek, de tweelingen niet geselecteerd op

basis van de aanwezigheid van matige tot ernstige parodontitis maar enkel en alleen op

basis van hun tweelingen status. Als gevolg hiervan kan men zich dus nog steeds

afvragen in welke mate de genetische aanleg bijdraagt tot het ontstaan van matige tot

ernstige parodontitis. Omdat bij parodontitis de wisselwerking tussen de genetische

aanleg voor de aandoening en de life style factor roken zo belangrijk is werd

vooruitlopend op eigen onderzoek bij tweelingen eerst het effect van roken op de

aangeboren en verworven immuunrespons bij parodontitis onderzocht. Deze benadering

zou kunnen helpen om fenotypische variaties tussen ééneiige tweelingen te verklaren.

Onderzoek heeft aangetoond dat gemiddeld 50% van de parodontitis patiënten

rokers zijn (Bergstrom 1989, Tonetti 1998, van der Weijden et al. 2001, Xu et al. 2002).

Dit percentage is hoog vergeleken met het gemiddelde van de Nederlandse bevolking

waarvan ongeveer 27% rookt (Tabakspreventie 2008). In het algemeen worden rokers als

de moeilijkst te behandelen patiënten beschouwd. Rokers hebben vaak ernstiger

parodontitis dan niet-rokers en reageren minder goed op de behandeling van parodontitis

dan niet-rokers (Johnson & Hill 2004, Kinane & Chestnutt 2000, Palmer et al. 2005,

Tonetti 1998). Bovendien heeft roken een grote invloed op het afweersysteem

(Graswinckel et al. 2004, Kinane and Chestnutt, 2000, Loos et al. 2004, Palmer et al.

2005).

Bij de pathogenese van parodontitis spelen T-cellen een belangrijke

immunoregulerende rol (Gemmell et al. 2007, Kinane & Lappin 2001, Seymour et al.

1996). Aanwijzingen op basis van histologische analyse van ontstoken parodontaal

weefsel en ex vivo cytokine productie van parodontitis patiënten en gezonde controles,

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ondersteunen het concept dat parodontitis een Th2-type aandoening is (Gemmell et al.

2007). Echter, er was nog niet onderzocht of roken de Th2 respons bij parodontitis zou

kunnen versterken, dit terwijl toch minstens de helft van de parodontitis patiënten rokers

zijn. Deze veronderstelling vormde de basis voor onze eerste studie. In Hoofdstuk 2, werd

de T cell sturende respons van de monocyten (Th1/Th2) en de pro-inflammatoire

cytokine produktie in ex vivo volbloed cel kweken van rokende en niet rokende

parodontitis patiënten onderzocht. Bij 29 patiënten (18 niet-rokers en 11 rokers) die in de

nazorg zaten werd veneus bloed afgenomen waarna dit vervolgens 10 keer werd verdund

voor volbloed kweken. De volbloed cel kweken werden gedurende 18 uur gestimuleerd

met zowel Neisseria meningitidis lipooligosaccharide (LOS) als een sonisch extract van

Porphyromonas gingivalis (Pg-SE). Vervolgens werd in het supernatant de productie van

zowel de T cel sturende cytokines interleukine IL-12p40 en IL-10, als de pro-

inflammatoire cytokines IL-1 , IL-6 and IL-8 bepaald. Eerdere studies hebben

aangetoond dat monocyten/macrofagen de belangrijkste bron zijn van de genoemde

cytokinen (van der Pouw Kraan et al. 1997, van der Pouw Kraan et al. 1995). Uit onze

gegevens blijkt dat parodontitis patiënten die roken hebben een lagere IL-12p40/IL-10

ratio na LOS stimulatie en een lagere IL-1 produktie na LOS en Pg-SE stimulatie

hebben dan hun niet-rokende tegenhangers. Deze resultaten suggereerden een versterkte

Th2 immuun respons bij rokende parodontitis patiënten.

Ondanks dat onze bevindingen een verhoogde Th2 respons na monocyten

stimulatie bij rokende parodontitis patiënten suggereerden, was een verdere bevestiging

dat T cellen een dergelijk cytokine patroon tot expressie brengen gewenst. Daarom werd

vervolgens in Hoofdstuk 3, de cytokine produktie van de Th1 en Th2 subpopulaties

bestudeerd in rokers en niet-rokers met en zonder parodontitis. Volbloed cel kweken

werden gestimuleerd met specifieke T cel stimulatoren waarna IFN- en IL-13 werden

gemeten representatief voor respectievelijk de Th1 en Th2 respons. De resultaten lieten

zien dat rokers meer lymfocyten hadden, en een hogere produktie van IFN- en IL-13 dan

niet rokers, ongeacht het feit of ze parodontitis hadden. In a multivariabele analyse werd

echter gevonden dat de toegenomen IFN- productie niet verklaard werd door roken

terwijl de toegenomen IL-13 productie wel sterk verklaard werd door roken. De IFN- en

IL-13 produktie was onafhankelijk van elkaar wat aantoont dat ze door twee

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verschillende T cel populaties werden geproduceerd (d.w.z. Th1 and Th2 type). Op basis

hiervan kan worden verondersteld dat de verhoogde Th activiteit en in het bijzonder het

versterkte Th2 profiel bij de rokers een risico factor vormt voor patiënten die roken. Bij

hen zou door het roken een stabiele situatie kunnen worden omgezet in een progressieve

laesie. Door middel van dit onderzoek konden wij onze eerdere bevindingen met

betrekking tot het cytokine profiel van monocyten bevestigen. Het Th2 profiel van

monocyten van parodontitis patiënten die roken komt overeen met de cytokine produktie

door de lymfocyten.

Onze resultaten hebben verschillen laten zien tussen rokende en niet rokende

parodontitis patiënten voor wat betreft de ex vivo volbloed cel kweek cytokine productie.

IL-12 en IL-10 zijn belangrijke cytokinen bij de aangeboren immuniteit die ook een groot

effect hebben op de verworven immuniteit (Seymour & Gemmell 2001). IL-12 expressie

speelt een belangrijke rol in het bepalen van het Th1 respons door de inductie van IFN-

secretie (Tsai et al. 2005). IL-10 daarentegen, verlaagt de productie van IFN- waardoor

de Th1 respons wordt onderdrukt (Lappin et al. 2001). Verder is IL-1 betrokken bij een

verhoogde IFN- productie door Th1 cellen, een verlaagde IL-4 productie door Th2

cellen (Sandborg et al. 1995, Schmitz et al. 1993) en wordt geremd door roken (Pabst et

al. 1995). Samenvattend lijkt de lagere IL-12p40/IL-10 ratio en lagere IL-1 productie bij

de onderzochte groep rokers indicatief te zijn voor een versterkte Th2 response

(Hoofdstuk 2), dit werd bevestigd door een hogere IL-13 productie door de Th2 cellen

van rokers (Hoofdstuk 3).

Het wordt algemeen aangenomen dat er een shift optreedt van een voornamelijk

T cel naar B cel laesie bij de overgang van gingivitis naar parodontitis. Men zou kunnen

veronderstellen dat tijdens het ontstaan van parodontitis er een verandering van cellulaire

immuniteit (Th1) naar de humorale immuniteit (Th2) optreedt (Kinane & Lappin 2001).

Het blijkt dat bij gingivitis meer T- dan B cellen worden aangetroffen terwijl bij

parodontitis de B cellen overheersen (Kinane & Lappin 2001). De dominantie van B-

cellen/plasma cellen bij de gevorderde/progressieve laesie suggereert een rol voor de Th2

cellen bij parodontitis. Zowel Th1 en Th2 cellen induceren B cel proliferatie, echter de

Th2 cellen zijn hierin meer efficiënt dan de Th1 cellen (Rothermel et al. 1991).

Daarnaast, kunnen geactiveerde B cellen bijdragen tot het uitbreiden en handhaven van

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een dergelijke immune respons (Harris et al. 2000). Het is daarom plausibel dat een

versterkte Th2 respons bij rokers kan leiden tot een verhoogde B cell proliferatie,

waardoor een versterkte humorale respons wordt geïnduceerd, dat op zijn beurt de

bestaande Th2 respons versterkt met als gevolg een toegenomen risico op steeds

terugkerende parodontale afbraak bij rokers.

B cellen hebben de mogelijkheid om multifunctionele cytokinen zoals IL-6 en

tumor necrosis factor (TNF)- te kunnen aanmaken, die de diverse aspecten van

botresorptie en het ontstaan van ontstekingen reguleren (Harris et al. 2000). Deze

cytokinen kunnen botafbraak direct induceren of indirect door de productie aan te tasten

van factoren die essentieel zijn voor de osteoclast differentiatie (Boyce et al. 2005,

Gemmell et al. 1997). Daarom zou men kunnen veronderstellen dat deze bot resorberende

cytokinen die geproduceerd worden door verhoogd geactiveerde B cellen kunnen

bijdragen tot parodontale afbraak bij rokers. Bovendien zou de verstrekte B cel stimulatie

ook kunnen bijdragen tot een verhoogde antilichaamproductie. Het is ook bekend dat

autoimmunologische mechanismen zouden kunnen bijdragen in de pathogenese van

parodontitis (Rajapakse & Dolby 2004). Een recent onderzoek liet zien dat

autoantilichamen tegen de extracellulaire matrix betrokken zijn bij de pathogenese van

parodontitis (De-Gennaro et al. 2006). Daarnaast is de lokale productie van

autoantilichamen tegen autoantigenen in granulatie weefsel van de parodontale laesie

beschreven (Rajapakse & Dolby 2004). Met andere woorden autoimmunologische

mechanismen opgewekt door de verhoogde B cel activatie zouden kunnen bijdragen aan

de parodontale afbraak bij rokers. Voorgaande studies over de invloed van roken op de

immuun respons bij parodontitis gaven belangrijke achtergrond informatie om verder

onderzoek te doen naar de oorzaak van de ernst van de parodontale afbraak bij

parodontitis patiënten. Het doel hiervan was om een kader te scheppen voor de data

analyse van het tweelingenonderzoek.

De klassieke tweeling onderzoek werd gebruikt voor het bepalen van de

bijdrage van genetische-, omgevings- en life style factoren zoals roken, in de etiologie

van matige tot ernstige chronisch parodontitis. Voor het onderzoek in Hoofdstuk 4

werden zowel eeneiige (MZ), als twee-eiige (DZ) tweelingen die samen waren

opgegroeid gerekruteerd om de bijdrage te onderzoeken van de genetica, parodontale

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pathogenen en life style factoren op het klinische fenotype. Voor deze studie was het

uiterst belangrijk dat de tweelingen werden geselecteerd op basis van het feit dat bij ten

minste één van de twee, de probandus, 5 mm of meer approximaal aanhechtingsverlies

bij twee niet aan elkaar grenzende elementen aanwezig was. Uiteindelijk kon bij 10 MZ

en 8 DZ tweeling paren de parodontale toestand, de aanwezigheid van parodontaal

pathogene bacteriën, het opleiding niveau, roken en Body Mass Index (BMI) worden

onderzocht. De belangrijkste bevinding van deze studie is het feit dat MZ tweelingen

behoorlijk discordant waren met betrekking tot het aanhechtings verlies, het aantal en

percentage elementen met AL 5 mm en het percentage elementen met botafbraak van

30%. De MZ probandi vertoonden de matige tot ernstige parodontitis op basis waarvan

zij geselecteerd waren, terwijl de MZ co-tweelingen die niet op parodontitis waren

geselecteerd, altijd veel minder ernstige parodontitis vertoonden. Dit was een verrassend

resultaat omdat op basis van eerder onderzoek verondersteld was dat erfelijkheid voor

50% bijdraagt aan de ernst van de parodontitis (Michalowicz et al. 2000) en er dus

minder grote verschillen te verwachten waren bij de MZ tweelingen. Analyse van ons

tweelingen materiaal liet zien dat het gebrek van concordantie niet te verklaren was door

de aanwezigheid van de parodontale pathogene bacteriën, roken en BMI, factoren die een

rol spelen bij de etiologie van parodontitis. Men moet zich echter wel realiseren dat het

aantal tweelingen in het onderhavige onderzoek klein is. Dit geringe aantal zou

verantwoordelijk kunnen zijn voor de vele niet significante verschillen zoals bijvoorbeeld

de subgingivale aanwezigheid van P. gingivalis. Bij de MZ tweelingen waren 5 van de

10 probandi positief voor P. gingivalis, terwijl slechts 2 van de 10 co-tweelingen positief

waren voor dit organisme. Een grotere onderzoekspopulatie zou hebben kunnen aantonen

dat P. gingivalis wel degelijk een significante rol speelt in de etiologie van parodontitis.

Roken, als verklarende variabele kon niet in de MZ groep geanalyseerd worden gezien

hun identieke rookgedrag. Dit was wel mogelijk bij de DZ tweelingen omdat hierbij wel

een verschillend rookgedrag aanwezig was tussen probandi en co-tweelingen. Bij de DZ

tweelingen kon 45.6% van verschil in de parodontale afbraak worden verklaard door

roken. Bij de MZ tweelingen was de discordantie in parodontale afbraak kleiner dan bij

de DZ tweelingen. Deze bevinding is in overeenstemming met de literatuur.

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Aangezien immunologische reacties een belangrijke rol spelen bij de

parodontale afbraak, was het interessant om de cytokine productie van de leukocyten van

deze tweeling populatie te onderzoeken om een mogelijke verklaring te vinden voor de

discrepantie in het parodontale fenotype tussen de MZ en DZ tweelingparen. In

Hoofdstuk 5, werd bij deze tweeling populatie de mate van concordantie in aantal

leukocyten en hun cytokine productie na ex vivo stimulatie onderzocht. Wanneer de data

van MZ en DZ tweelingen worden gecombineerd blijkt dat probandi een hoger aantal

leukocyten hadden en minder IL-12p40 productie vertoonden dan hun co-tweelingen. Dit

verhoogde aantal leukocyten is vergelijkbaar met eerdere bevindingen in de literatuur

waarbij een ernstiger vorm van parodontitis ook is gerelateerd aan een verhoogd aantal

leukocyten bij deze patiënten (Loos 2005). Het is ook bekend dat een lagere IL-12p40

productie de Th2 immuniteit bevordert en de stimulatie van de Th1 immuun respons

vermindert. De grotere mate van botafbraak bij MZ en DZ probandi in vergelijking met

hun co-tweelingen en de lagere IL-12p40 productie is in overeenstemming met de

karakteristieke Th2 respons bij parodontitis. Bovendien produceerden MZ probandi meer

IL-6 dan hun co-tweelingen. Dit beeld is in overeenstemming met eerder onderzoek

waaruit blijkt dat met toenemende ernst van de parodontitis de IL-6 productie ook

toeneemt (Loos et al. 2000). IL-6 is een multifactorieel cytokine met o.a. als functie B-

lymfocyten differentiatie, T-lymfocyten proliferatie en stimulatie van immunoglobuline

productie door B-lymfocyten (Hirano et al. 1990). Een belangrijk aspect van IL-6 is dat

het botresorptie kan induceren zowel zelfstandig als in combinatie met andere bot

resorberende stoffen (Ishimi et al. 1990). Dit leidt tot de conclusie dat binnen onze

tweeling populatie, de hogere IL-6 productie door de MZ probandi geassocieerd lijkt te

zijn met de sterkere bot afbraak in deze groep. De resultaten suggereren dat het gebrek

aan een sterke concordantie in parodontale afbraak bij de MZ tweelingen niet verklaard

kan worden door verschillen in het aantal leukocyten, maar dat de lagere productie van

IL-12p40 en hogere IL-6 productie na ex vivo stimulatie beschouwd zou kunnen worden

als risico factoren voor de ernst van parodontitis.

Op basis van de resultaten bij onze tweelingen populatie komen wij tot de

veronderstelling dat de rol van genetische factoren bij parodontitis wel eens overschat

zouden kunnen zijn. De genetische aanleg van de gastheer, de klassieke lifestyle factoren

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en de immuunrespons na stimulatie van de afweer cellen konden niet het gebrek in

concordantie van de parodontale afbraak, speciaal bij MZ tweelingen verklaren. Dit

impliceert dat andere factoren een belangrijke rol spelen en de ernst uitgebreidheid van

de ziekte bepalen. Er zijn verschillende mogelijkheden. Bijvoorbeeld, voeding en dieet

supplementen zijn geassocieerd met de ontstekingsreactie en ernst van parodontitis

(Amaliya et al. 2007, Rosenstein et al. 2003, Staudte et al. 2005). Een calorie-arm diet

vertraagt de ontstekingsreactie en vermindert actieve parodontale afbraak bij een

experimenteel geïnduceerde parodontitis (Branch-Mays et al. 2008). Daarnaast, zou

suppletie met visolie een positief effect hebben op het afweer systeem en daardoor een rol

kunnen spelen in de preventie en/of ondersteuning bij behandeling van parodontitis

(Bendyk et al. 2009). Een andere mogelijke verklaring van de gevonden discordantie in

de parodontale afbraak bij tweelingen zouden virale infecties kunnen zijn. Herpes virus

infecties zou de parodontale afbraak kunnen initiëren of versnellen door stimulatie van

gastheer cellen om cytokinen uit te scheiden. De hieruit voortvloeiende ontsteking zou de

afweer mechanismen van de gastheer kunnen verstoren met als resultaat een minder

effectieve respons tegen de aanwezige paropathogene bacteriën (Contreras et al. 2000,

Slots 2007).

Gedurende de laatste jaren zijn er in toenemende mate aanwijzingen dat, alhoewel

genetische factoren de immunologische afweer bepalen, epigenitische mechanismen

buitengewoon belangrijk zijn voor de regulatie van de ontstekingsreactie (Offenbacher et

al. 2008). Epigenetische veranderingen worden veroorzaakt door een remodeling van de

chromatine structuur ten gevolge van DNA methylering en histone acetylering waardoor

genen selectief kunnen worden geactiveerd en gedeactiveerd en hun expressie wordt

bepaald. In het algemeen veroorzaakt een toegenomen DNA methylering in de promotor

regio van genen een verminderde expressie van dat gen (Franco et al. 2008). Doordat het

epigenetische proces een verandering in cytokine profiel kan induceren, beïnvloedt het

daardoor de pathogenese en uiteindelijk resultaat van vele infectieziekten (Gomez et al.

2009). Preliminaire data suggereren bijvoorbeeld dat het gen voor IL-6, een cytokine dat

betrokken is bij de uiteindelijke differentiatie van B-cellen in immunoglobuline

producerende cellen, in parodontitis weefsel een verminderde methylation vertoont ten op

zichtte van controles. Deze preliminaire bevindingen suggereren dat bij parodontitis de

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expressie van het IL-6 gen is geopreguleerd (ongepubliceerde gegevens, Offenbacher et

al. 2008). Deze suggestie is in overeenstemming met de bevindingen in onze tweelingen

studie waarbij gevonden werd dat na stimulatie van volboed kweken, de MZ probandi

met matige tot ernstige parodontitis meer IL-6 produceerden dan hun co-tweelingen met

geringe parodontale afbraak. Daarnaast is ook aangetoond dat infectie kan leiden tot

epigenetische veranderingen van een imprinted gen van de gastheer (Bobetsis et al.

2007). Het zou dus zo kunnen zijn dat vroeger opgelopen infectie ziekten en/of virale

infecties bij de MZ proband heeft geresulteerd in een genetische variatie met als

consequentie het ontstaan van een ernstiger vorm van parodontitis. Helaas is slechts

schaarse informatie beschikbaar over epigenetische veranderingen bij parodontitis.

Hopelijk zal toekomstig onderzoek ons helpen te begrijpen hoe bijvoorbeeld de

blootstelling aan systemische factoren zoals roken, het gehele epigenetische patroon

verandert en de neiging om parodontitis te ontwikkelen beïnvloedt. Al met al kunnen

bovengenoemde veronderstellingen de mate van expressie van ziekten beïnvloeden en

kunnen dus ook het gebrek aan duidelijke concordantie in de parodontale toestand van de

MZ tweelingen van het onderhavige onderzoek verklaren.

Het identificeren van personen die verhoogd vatbaar zijn voor parodontitis blijft

een grote uitdaging voor de tandheelkundige praktijk. Preventie en behandelstrategieën

op maat voor personen met een verhoogd risico zijn hoogst nodig. Hopelijk zullen studies

met grotere aantallen tweelingen gericht op andere mogelijke factoren die

immunologische afweer van de gastheer beïnvloeden meer licht werpen op de vraag

waarom sommige personen parodontitis ontwikkelen en andere niet. Dit zou tevens

kunnen bijdragen tot een effectieve preventie van het ontstaan en progressie van

parodontitis. Bij de zoektocht naar de oorzaken van parodontitis lijkt onderzoek naar de

interactie tussen genetische en epigenetische mechanismen enerzijds en de omgevings- en

life stylefactoren anderzijds een aantrekkelijk gebied van onderzoek.

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125

Resumen y Discusión

Periodontitis es una enfermedad compleja, multifactorial y de caracter crónico, la

cual afecta a los tejidos de soporte de los dientes y que a su vez se caracteriza por la

inflamación y la destrucción del tejido conectivo y hueso alveolar (Pihlstrom et al. 2005).

La manifestación de la enfermedad conlleva interacciones intrincadas entre la placa

bacteriana y la respuesta inflamatoria por parte del sistema immune del huésped (Preshaw

2008). Ciertos individuos muestran mayor susceptibilidad a la enfermedad que otros

(Loos et al. 2008), lo cual aparentemente es determinado por la respuesta

inmunoinflamatoria que se genera en los tejidos periodontales como respuesta a la

exposición crónica a la placa bacteriana (Preshaw 2008). La respuesta

inmunoinflamatoria por combatir la placa bacteriana puede, por ende, ser considerada de

“doble filo”. Esto quiere decir que aunque la respuesta es protectiva por naturaleza y que

provee de anticuerpos y neutrófilos polimorfos, que son responsables para el control de la

infeccion bacteriana, en aquellos sujetos de alta susceptibilidad, resulta en una

producción local de cantidades excesivas de enzimas destructoras y de substancias

mediadoras de la inflamación que conducen a la destrucción periodontal que se aprecia

clínicamente (Preshaw 2008). Resulta paradójico el hecho de que la respuesta

inflamatoria frente al ataque bacteriano es también responsable de la destrucción de los

tejidos periodontales blandos y duros. Aún cuando el asalto bacteriano inicia la

inflamación en los tejidos, los factores genéticos y el estilo de vida, tal y como el uso del

cigarrillo, pueden modular la respuesta inflamatoria y así determinar la progresión y el

grado de severidad de la enfermedad, lo cual se traduce clínicamente como pérdida de

hueso alveolar e inserción (Kornman 2008, Loos et al. 2008, Page et al. 1997, Palmer et

al. 2005, Pihlstrom et al. 2005, Preshaw 2008).

La presente tesis fue diseñada inicialmente para examinar en qué medida la

variación genética determina la variación del fenotipo de la periodontitis crónica

moderada a severa. Una mejor comprensión de enfermedades de etiología compleja como

es el caso de la periodontitis, amerita la evaluación de la interacción entre los factores

genéticos y de estilo de vida, conjuntamente con las influencias bacteriana. Estudios en

gemelos han representado una herramienta valiosa que aporta información acerca de la

base genética de rasgos multifactoriales. Hasta el presente, resultados de estudios en

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gemelos relacionados a la periodontitis crónica muestran resultados convergentes, los

cuales sugieren el importante rol de la genética en esta enfermedad (Corey et al. 1993,

Michalowicz 1994, Michalowicz et al. 1991a, Michalowicz et al. 1991b, Michalowicz et

al. 2000, Michalowicz et al. 1999, Mucci et al. 2005). Sin embargo, los mismos tienen

algunas limitaciones debido a que sus resultados estan fundamentados en observaciones

obtenidas a través de cuestionarios mas no provenientes de mediciones clínicas (Corey et

al. 1993, Mucci et al. 2005), y además, las poblaciones estudiadas presentan escasa

destrucción periodontal (Michalowicz et al 1991a, Michalowicz et al. 1991b,

Michalowicz et al. 2000). Hasta el presente, las investigaciones en gemelos han

seleccionado los sujetos tomando como criterio el hecho de ser gemelos y no por la

presencia de periodontitis moderada a severa. Por ende, sigue siendo cuestionado hasta

qué medida los factores genéticos contribuyen al desarrollo de la periodontitis crónica

moderada a severa. Como preludio a investigar este tema, y motivado por el hecho de que

la interacción entre la configuración genética y el cigarrillo sería de gran importancia, nos

decantamos a explorar como punto de partida el efecto del fumar cigarrillo sobre el

sistema inmune, tanto innato como adquirido, de sujetos afectados con periodontitis. Este

enfoque nos ayudaría potencialmente a explicar la variación fenotípica dentro de los

gemelos idénticos.

Estudios han demostrado que en promedio un 50% de los pacientes que sufren de

periodontitis son fumadores o ex-fumadores (Bergstrom 1989, Tonetti 1998, van der

Weijden et al. 2001, Xu et al. 2002). Esta proporción es alta al ser comparada con el

promedio de la población holandesa para dicho parámetro siendo aproximadamente un

27% de fumadores (Prevención del Tabaco Stivoro 2008). La mayoría de los

periodoncistas consideraría a los fumadores como el grupo de pacientes de mayor

demanda en cuanto al tratamiento (Johnson & Hill 2004, Kinane & Chestnutt 2000,

Palmer et al. 2005, Tonetti 1998). Además, el fumar cigarrillo tiene grandes efectos en la

respuesta immune del huésped (Graswinckel et al. 2004, Kinane & Chesnutt 2000, Loos

et al. 2004, Palmer et al. 2005).

Los linfocitos T son muy importantes en la regulación inmunitaria dentro de la

patogénesis de las enfermedades periodontales (Gemmell et al. 2007, Kinane & Lappin

2001, Seymour et al. 1996). Existe evidencia tanto histológica como proveniente del

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análisis de las citokinas producidas por el tejido periodontal inflamatorio, que considera a

la periodontitis como una enfermedad de tipo Th2 (T-helper-2, es decir, caracterizada por

la proliferación de linfocitos T de ayuda del tipo 2) (Gemmell et al 2007). Sin embargo,

aún no se ha investigado si este perfil Th2 en la enfermedad estaría potenciado por el

cigarrillo, aspecto de interés debido al alto número de fumadores dentro de los pacientes.

Esta presunción dió origen a nuestro primer estudio. En el Capítulo 2, hemos investigado

la probable caracterización del tipo de linfocitos Th y por ende la respuesta establecida

(Th1/Th2) a predecir a través de las citokinas generadas por los monocitos en

experimentos ex vivo, en cultivos celulares en sangre completa (CCSC) de pacientes

quienes han padecido periodontitis crónica, fumadores versus no-fumadores. Para tal fin

se extrajo sangre venosa de 29 pacientes (18 no-fumadores y 11 fumadores), quienes para

ese momento recibían tratamiento periodontal de mantenimiento, y se diluyó a razón de

diez. Dichos cultivos fueron estimulados durante 18 horas con lipooligosacárido

proveniente de Neisseria meningitidis (LOS) y extracto sonificado de Porphyromonas

gingivalis (Pg-SE). La producción de las citokinas que diferencian los linfocitos T

inmaduros bien sea en Th1 o Th2 fue medida. Es conocido que la interleukina (IL)-12p40

dirige la diferenciación de linfocitos inmaduros hacia el tipo 1, mientras que la IL-10 al

tipo 2. En nuestros experimentos, se extendió la evaluación de las citokinas inflamatorias

hacia la determinación de la IL-1 , IL-6 y la IL-8, cuya producción se midió en los

supernatantes provenientes de los cultivos en estimulación. Estudios previos han dejado

claro que los monocitos/macrófagos representan una fuente de las citadas citokinas (van

der Pouw Kraan et al. 1997, van der Pouw Kraan et al. 1995). Nuestros resultados

muestran que los pacientes afectados con periodontitis y fumadores tienen una

proporción IL-12p40/IL-10, posterior a la estimulacion con LOS y Pg-SE, inferior a la

que presentan sus contrarios no-fumadores, sugiriendo así una respuesta inmunne de tipo

2 más acentuada en los pacientes fumadores.

Aunque nuestros resultados ofrecen evidencia de que los pacientes fumadores con

periodontitis tienen una respuesta Th2 más pronunciada (a saber por las citokinas

generadas por los monocitos) nos vimos intrigados a confirmar este hallazgo en las

citokinas producidas por los propios linfocitos en cuestión. En el Capítulo 3, hemos

investigado la producción de citokinas por parte de los linfocitos, característica para las

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respuestas de tipo 1 o 2, Th1 o Th2, en un grupo de pacientes con afección periodontal y

otros periodontalmente sanos, ambos a su vez fumadores y no-fumadores. En los CCSC

fueron estimulados específicamente los linfocitos T y la producción de interferón-gamma

(IFN- ) y IL-13 (típicas citokinas para evaluar la respuesta Th1 y Th2, respectivamente)

fueron medidad en los supernatantes. Nuestros resultados muestran un mayor número de

linfocitos en los fumadores, y altos niveles de IFN- y IL-13, independiente del hecho de

que el sujeto padezca o no de periodontitis. Sin embargo, en un posterior análisis de

multivariabilidad se ha encontrado que la alta producción de IFN- no era explicanle de

forma significativa por el hecho de ser fumador, mientras que las altas cantidades de IL-

13 sí fueron fuertemente explicables por esta variable. La secreción de IFN- y de IL-13

fueron independientes la una de la otra, lo cual demuestra que se trata de dos

subpoblaciones celulares completamente independientes. Por lo tanto, sugerimos que este

aumento en la actividad de las celular Th, y más específicamente en las células Th2 en

los fumadores, constituye un riesgo para los pacientes fumadores, lo cual puede conducir

una situación periodontal inestable y de progesion en la destrucción. Este estudio nos ha

permitido confirmar nuestro previo hallazgo acerca del perfil de citokina generado por los

monocitos en los pacientes fumadores. Este perfil Th2 medido en la respuesta monocítica

de los pacientes fumadores es consistente con la respuesta generada por los linfocitos de

este grupo de pacientes.

Nuestros resultados dejan testimonio de las diferencias entre pacientes afectados

periodontalmente fumadores y no-fumadores con respecto a la producción de inmuno

mediadores en los cultivos sanguíneos de estimulación. Las citokinas IL-12 e IL-10

desempeñan un papel central en la inmunidad innata con importantes efectos en la

subsecuente inmunidad adquirida (Seymour & Gemmell 2001). Altos niveles de IL-12

contribuirían a una respuesta tipo 1 Th1, ya que ésta es inducida por la producción de

IFN- (Tsai et al. 2005). Al contrario, IL-10 reduce la secreción de IFN- y tiene un rol

potencial en la disminución de las respuestas mediadas por IFN- y por consiguiente en

las respuestas Th1 (Lappin et al. 2001). Además, IL-1 está involucrada en el aumento en

la producción de IFN- por parte de las celulas Th1 y a su vez de la disminución en la

producción de IL-4 por parte de las celulas Th2 (Sandborg et al. 1995 Schmitz et al.

1993), lo cual que puede ser inhibido por el efecto del cigarrillo (Pabst et al. 1995). En

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conjunto, la reducción en la proporción IL-12p40/IL-10 y la disminución en la

producción de IL-1 observada en nuestro grupo de fumadores fue indicativo de una

respuesta Th2 más pronunciada (Capítulo 2), lo cual fue confirmado posteriormente por

un alto nivel de IL-13 producido por las celulas Th2 en los sujetos fumadores (Capítulo

3).

Es bien conocido que existe un cambio de una población celular

predominantemente formada por linfocitos T a una de linfocitos B en la progresión de las

lesiones de gingivitis a periodontitis. Resulta interesante especular que un cambio de una

respuesta mediada por el infiltrado celular (Th1) hacia una respuesta humoral (Th2)

ocurre durante el desarrollo de la enfermedad periodontal (Kinane & Lappin 2001).

Parece evidente que en la gingivitis las células T probablemente exceden a las células B,

mientras que en el sucesiva inflamación del tejido, conduciendo a la periodontitis, las

celulas B sin duda son las predominantes (Kinane & Lappin 2001). El predominio de

células B/células plasma en las lesiones periodontales avanzadas/progresivas sería

indicativo del importante rol que las células Th2 desempeñan en estas lesiones. Las

células Th1 y Th2 inducen la proliferación de las células B, pero las células Th2 son

generalmente más eficientes que las Th1 en esta capacidad (Rothermel et al.1991).

Además, las células B tienen la particularidad de contribuir a la ampliación y

mantenimiento de la polarización establecida (Harris et al. 2000). Por consiguiente, es

plausible que una respuesta Th2 más pronunciada en los individuos fumadores pudiera

incrementar la proliferación de las celulas B, induciendo a su vez una más marcada

respuesta humoral, que reforzaría la respuesta Th2 existente, por lo cual se aumentaría el

riesgo de recurrencia de la destrucción periodontal en el paciente fumador.

Las células B tienen la capacidad de producir cytokinas con múltiples funciones,

tal es el caso de IL-6 y el factor tumor necrosis alfa, las cuales regulan diversos aspectos

en la absorción y aposición ósea en los procesos inflamatorios (Harris et al. 2000). Estas

citokinas inducen absorción ósea directa e indirectamente al afectar la producción de

factores de diferenciación osteoclástica (Boyce et al. 2005, Gemmell et al. 1997). Por

consiguiente, se puede sugerir que estas citokinas de reabsorción ósea producidas por las

células B activadas, pueden contribuir a la destrucción periodontal observada en los

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pacientes fumadores. Además, la pronunciada estimulación de células B y su activación

podría contribuir a la patogénesis de la enfermedad periodontal (Rajapakse & Dolby

2004). Un estudio reciente sugirió que el involucramiento de los anticuerpos en el serum

serían capaces de dirigir componentes del matrix extracelular durante la patogénesis de la

periodontitis crónica (De-Gennaro et al. 2006). Además, la producción local de

anticuerpos hacia autoantígenos en los tejidos granulomatosos embebidos dentro de las

lesiones periodontales han sido anteriormente descritos (Rajapakse & Dolby 2004). Por

ello, los mecanismos autoinmunes provocados por un aumento en la activación de las

células B pudiera contribuir a la destrucción periodontal en los sujetos fumadores. Los

estudios anteriores acerca de la influencia del cigarrillo sobre la respuesta inmune en

periodontitis proveen un importante marco de referencia para subsiguientes estudios que

pretendan aclarar asuntos relacionados a la severidad de la destrucción periodontal

observada en los pacientes. El propósito de estos sendos estudios fue el de aportar un

marco de referencia válido que aplicar, en este específico aspecto, en el análisis de la

población de gemelos.

Con la finalidad de determinar la contribución relativa de los genes, el medio-

ambiente y factores en el estilo de vida (tal como el fumar cigarrillo) en la etiología de la

periodontitis crónica moderada, se utilizó el método de estudios en gemelos. En el

Capítulo 4, gemelos monozigotos (MZ) y dizigotos (DZ) criados juntos fueron

seleccionados para evaluar la contribución de los genes, los patógenos periodontales y los

factores de estilo de vida, con el fenotipo clínico. De recalcar en este proceso es el hecho

de que los pares de gemelos fueron seleccionados en base de que al menos uno de cada

par presentara pérdida de inserción periodontal interproximal de 5 mm en 2 dientes no

adjacentes, este sujeto de cada par se denominó gemelo control, y su hermano/a sería el

co-gemelo/a. El estudio incluyó 10 MZ y 8 DZ pares de gemelos, en los cuales fue

evaluado la condición periodontal, presencia de patógenos, nivel de formación

profesional, hábitos del uso de cigarrillo y el indice índice de masa corporal (IMC). El

resultado más importante de este estudio es la discordancia encontrada en los gemelos

MZ en relación al promedio de pérdida de inserción periodontal, número de dientes con

pérdida de inserción 5 mm y el porcentaje de dientes con 30% de hueso alveolar. Los

gemelos control del grupo de MZ padecían de periodontitis crónica moderada a severa,

131

mientras que sus co-gemelos (quienes no fueron seleccionados en función de la

enfermedad periodontal) presentaron un grado de la enfermedad muy inferior. Este

resultado fue sorpresivo al considerar previas investigaciones de similar naturaleza en las

cuales se había encontrado un 50% de contribución genética al fenotipo de la enfermedad

periodontal (Michalowicz et al. 2000), razón por la cual una discrepancia menor dentro

de los MZ era esperada. Esta discrepancia no pudo ser explicada por factores como

presencia de bacteria periodontopatogénica, uso del cigarrillo o el IMC. Sin embargo, es

preciso puntualizar que la cantidad de participantes fue limitada, lo cual pudo haber

imposibilitado el cálculo de diferencias estadísticas que sí estarían presentes al

incrementar el número de participantes (por ejemplo en el caso de la presencia

subgingival de P. gingivalis). En el grupo de MZ, 5 de los 10 sujetos control resultaron

positivos para P. gingivalis, en contraposición a 2 de sus co-gemelos. Es muy factible que

una muestra con más participantes pudiera haber arrojado resultados estadísticamente

significativos a este respecto. La variable uso de cigarrrillo no puede ser explicativa de

alguna diferencia observada en el grupo (en cuanto a afección periodontal) ya que ambos

control y sus co-gemelos de los MZ fumaban aproximadamente la misma cantidad de

cigarrillos diarios. Este aspecto en los DZ sí difería, explicando así un 45.6% de la

diferencia de la destrucción periodontal en los DZ. En cuanto a la discrepancia en la

enfermedad periodontal, esta fue mayor entre los DZ que en los MZ, lo cual es

consistente con previos reportes en grupos de similitud y muestras de mayor escala.

La respuesta inmune ha sido considerada como muy relevante dentro de la

etiopatogenesis de la enfermedad periodontal, razón por la cual el análisis de este aspecto

constituiría un enfoque interesante para explicar la discrepancia encontrada en el grupo

de gemelos idénticos (MZ). En el Capítulo 5, se investigó el nivel de similitud en la

expresión de determinadas citokinas entre gemelos controles y sus co-gemelos para

ambos grupos (MZ y DZ). Cuando se realizó el análisis de estos mediadores de la

inflamación en los gemelos controles tanto de los MZ como de los DZ juntos se observó

un alto número de la cantidad total de leucocitos y de producción en la citokina IL-12p40

en comparación con el de los co-gemelos juntos. Los altos números en leucocitos

coinciden con previos hallazgos, que relacionan estos incrementos con un aumento de la

severidad de la enfermedad (Loos 2005). Es bien sabido que bajos niveles de IL-12p40

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favorece a la respuesta immune de tipo 2 (Th2), lo que a su vez suprime el desarrollo de

la respuesta de tipo 1 (Th1). Nuestros resultados han mostrado un aumento de la pérdida

de inserción periodontal, concomitante con baja producción de IL-12p40 en los gemelos

control, al ser comparados con sus co-gemelos, lo cual deja en evidencia la respuesta Th2

característica en la periodontitis. Asímismo, los gemelos controles MZ segregaron

mayores cantidades de IL-6 que sus co-gemelos, lo cual es congruente con observaciones

previas que han relacionado altos niveles de esta citokina con aumento en la severidad de

la enfermedad (Loos et al. 2000). IL-6 es una citokina multifuncional, con actividades

biológicas tales como diferenciación de los linfocitos B, proliferación de los linfocitos T

y estimulación de la secreción de inmunoglobulinas por parte de estos linfocitos (Hirano

et al. 1990). De especial importancia es la habilidad de IL-6 de inducir reabsorción ósea,

tanto por sí misma como cuando se conjuga con otros agentes de reabsorción ósea (Ishimi

et al. 1990). Por consiguiente, se llegó a la conclusión de que la alta producción de IL-6

encontrada en el grupo de gemelos control MZ parece estar asociado a un aumento de la

severidad de la destrucción periodontal exhibida en este grupo. La marcada discrepancia

en la destrucción periodontal del grupo de gemelos idénticos (MZ) no pudo ser asociada a

diferencias en cantidad de leucocitos, sin embargo, es interesante el hecho de que la baja

producción de IL-12p40 y los altos niveles de IL-6 en este grupo pudieran ser

considerados como indicadores de riesgo para la severidad de la periodontitis.

Basado en nuestros resultados provenientes del estudio en gemelos, sugerimos

que el rol de los genes en la enfermedad periodontal puede haber sido sobreestimada.

Factores genéticos, en conjunto con factores relacionados al estilo de vida, así como la

respuesta inmune generada después de la estimulación celular, no pudieron explicar la

carencia de concordancia en la manifestacion clínica de la periodontitis en el grupo de

gemelos idénticos (MZ), lo cual fuertemente apunta a que otros factores interactuan para

determinar el fenotipo periodontal. Tenemos algunas sugerencias para explicar dicho

hallazgo. Es el caso de los factores nutricionales y de suplementación nutricional, los

cuales en el pasado ya han sido relacionados con la respuesta inflamatoria y la severidad

de la enfermedad (Amaliya et al. 2007, Rosenstein et al. 2003, Staudte et al. 2005). Una

dieta baja en calorías puede suprimir la respuesta inflamatoria y reducir la destrucción

periodontal asociada a la respuesta frente a los periodontopatógenos (Branch-Mays et al.

133

2008). Adicionalmente, la suplementación nutricional con aceite de pescado podría

ofrecer efectos positivos en la prevención y tratamiento de la enfermedad periodontal

(Bendyk et al. 2009). Otra posible razón para explicar la marcada discordancia observada

en los gemelos idénticos podría ser la presencia, dentro de los tejidos periodontales, de

agentes provenientes de infecciones concomitantes. Por ejemplo, infecciones por herpes

virus podrían iniciar o acelerar la destruccción periodontal ya que estimulan la

producción de citokinas, lo cual podria interferir con la respuesta inmune del huésped

frente a los patógenos periodontales lo cual disminuiría las defensas contra las bacterias

que causan la periodontitis (Contreras et al. 2000, Slots 2007).

Por otra parte, aunque los genes modulan la respuesta inflamatoria, está en

ascenso la convicción de que los factores epigenéticos son críticos en la regulación de la

respuesta inflamatoria (Offenbacher et al. 2008). Los eventos epigenéticos actúan a través

de la remodelación de la estructura de la cromatina como resultado de la metilación del

ADN o metilación-acetilación de histonas del ADN, lo cual puede selectivamente activar

o inactivar los genes y así mismo determinar su expresión. En general, podrían causar un

aumento en la metilación del ADN en las zonas del promotor del gen, silenciándolo de

este modo (Franco et al. 2008). Los procesos epigenéticos, a través de la inducción de

cambios en la producción de citokinas, pueden influenciar la patogénesis y determinar el

progreso de muchas enfermedades infecciosas (Gómez et al. 2009). Por ejemplo,

resultados preliminares han sugerido que el gen encargado de la síntesis de IL-6 (citokina

involucrada en la diferenciación definitiva de las células B en células activas productoras

de anticuerpos) sufre una disminución en su metilación en los tejidos de pacientes con

periodontitis cuando ésto se compara a sujetos sanos. Lo cual sugiere que el gen para IL-

6 podría estar sobreestimulado en la enfermedad periodontal (resultados por publicar,

Offenbacher et al. 2008). Esta idea va en línea con nuestros resultados en donde los

gemelos controles del grupo de gemelos idénticos segregaron concentraciones de IL-6

superiores a sus contrapartes y a su vez mostraron mayor destrucción periodontal.

También se ha mostrado que procesos infectivos pueden conducir a cambios en los genes

impresos (Bobetsis et al. 2007). Por consiguiente, infecciones previas sufridas por el

gemelo control pudieran haber generado una variación genética lo cual a su vez pudiera

haber conducido a un aumento de la severidad de la periodontitis en estos sujetos.

134

Desafortunadamente existe muy escasa información acerca de los cambios epigenéticos

asociados a la periodontitis. Esperamos que investigaciones futuras nos puedan ayudar a

entender la forma en la cual la exposición a factores sistémicos, como el cigarrillo,

pudieran alterar los patrones epigenéticos y así afectar la expresión de la enfermedad

periodontal (Barros & Offenbacher 2009). En resumen, las sugerencias mencionadas

anteriormente podrían potencialmente influenciar la manifestación de la periodontitis por

lo cual ofrecen una respuesta escasa concordancia en los parámetros estudiados en los

gemelos idénticos.

La identificación de sujetos con alta susceptibilidad para desarrollar la

enfermedad periodontal sigue siendo un gran reto en la atención odontológica. Es

necesario una prevención a la medida, así como el diseño y aplicación de estrategias de

tratamiento para este grupo de alto riesgo. Al respecto, estudios en gemelos en muestras

de mayor escala, donde además de los indicadores clásicos de la enfermedad, también se

evalúen otros factores de influencia, contribuirían a aclarar los procesos hasta hoy poco

conocidos acerca de la etiología y progreso de la periodontitis. Basados en nuestros

estudios y en algunos otros publicados recientemente podemos concluir y además

especular que el análisis de la interacción de los factores genéticos y de los mecanismos

epigenéticos, influenciados a su vez por los factores medioambientales, constituyen

inequívocamente una novedosa e interesante línea de investigación dentro de nuestro

campo.

135

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Acknowledgements, Dankwoord, Agradecimientos

The best and most difficult moments of my doctoral dissertation journey have

been shared with many people. I wish to thank a number of them, who generously shared

their knowledge and time with me and made it possible.

I would like to express my gratitude to Prof. dr. Ubele van der Velden, whose

expertise, understanding, and patience, added considerably to my graduate experience.

His wisdom, flexibility, genuine caring, concern, and faith in me during the dissertation

process enabled me to attend to life while also earning my Ph.D. He’s been motivating,

encouraging, and enlightening. He has never judged nor pushed when he knew I needed

to juggle priorities. Dear Ubele, you provided me with direction, support and became

more a mentor and a friend, than a professor. Even after your retirement, you have

remained a supporter and provided insight and direction right up to the end. I am forever

grateful. Thank you!

I also want to thank Prof. dr. Bruno Loos for his support during these years. I

especially appreciate his good eye for detail. He participated actively in many steps of

this dissertation. Dear Bruno, I admire your enthusiasm in life and your passion for work.

Thank you for the insightful discussions and for your guidance.

I am thankful to Prof. dr. Wouter Beertsen. Dear Wouter, thank you for your

friendship and kindness that embraced me from the very beginning. I have enjoyed your

joyful way of living and working.

I am grateful to the remaining members of my dissertation committee, Prof. dr.

Frank Abbas, Prof. dr. Vincent Everts, Prof. dr. Arie-Jan van Winkelhoff, and Dr. Fridus

van der Weijden. I thank you for your willingness to review my thesis.

142

I would especially like to take the opportunity to thank the volunteers of the

experiments, especially the twins, and all the colleagues of the various periodontal

practices across the Netherlands for their efforts in recruiting patients.

Dear Sergio my “paranimfo”, my “ángel de la guarda at paro”, thank you for your

patience and kindness, for always having time for a question and for being truly

committed in helping me with End Note… I wish you success in your dissertation!

Dear Tony, my paranimf, thanks for sharing with me thoughts, achievements,

fears and joy. I am happy we can still count on each other!

My gratitude to Marja Laine for her kind support and guidance during my early

lab works at the VU and to Robert Kikkert for his collaboration and incisive comments

on my first publication.

Dear Mark, thank you for helping me in the clinical assessment of the twins, for

sharing with me your knowledge and critical eye for making nice and appealing

presentations (in dutch!)… I really learnt from you!

I am very grateful to Prof. dr. Lucien Aarden. Dear Lucien, thank you for

embracing me in your team, allowing me to run most of the experiments under your

guidance. Also thanks to my colleagues at the department of Immunopathology at

Sanquin Research Institute (colleagues at the CLB). The atmosphere was always friendly

and welcoming!

It has been a great privilege to spend these years working at the department of

Periodontology (nowadays section Periodontology… I still have to get used to it!). I

enjoyed that time of my life and keep nice memories of all of you. Dear “TPOers”, thank

you for cheering me up and making me laugh. Dear Elena Alvarez, many thanks for your

helping hand on the “Spanish section” of my book and I wish you luck with you research

project as well!

143

Dear Margriet (paro-rose), thank you for your smile and interest in my well

being… especially during my pregnancies. I miss you!

Dear Sandra, I remember your empathy from the very beginning… you are such a

nice human being, genuine and transparent. I’m glad you have that special connection

with Venezuela. Dear Ingrid, I will miss your positive energy and your tremendous

enthusiasm in life!

Many thanks to Spiros for the pleasant work atmosphere during these years, I will

miss you saying: …”I think it’s a good time to go home”…. My thanks to Elena Nicu for

the good moments we shared at ACTA and VU. My thanks to Gaia for the nice talks!

Siempre has sido una persona especial y valoro nuestra amistad.

Special thanks to Arjen van Wijk, for his assistance in the statistical analysis of

the twin data.

Financial support made my doctoral studies possible. I would like to thank the

IOT for its continuing support.

Dear colleagues at Dental Udding, specially Aletta and Wendy, thank you for de

gezelligheid during all these years. Dear Aletta, it has been a pleasure to witness your

achievements, very inspiring!

Thanks to all my close friends, who make me a rich, rich person… you know who

you are!

My gratitude to Mr. Carlos Cruz-Diez (Venezuelan painter and kinetic artist). I

am so honoured to have such a beautiful master piece on the cover of my book. Thank

you for all the gifts you have gave us through so many years, for creating new visual and

colourful sensations. Thank you for reacting so enthusiastic when I asked you permission

for using your serigraphy on my book. You are a pioneer in bringing Venezuelan art into

144

the international art scene. I admire you profoundly and feel very proud of being

Venezuelan as well! Como Ud. bien ha puntualizado: “El arte tiene que estar en la calle,

en las fábricas, no colgado de una pared”, creo que de algún modo mi portada contribuye

a esta filosofía!

The seeds of my interest in periodontology were planted as an undergraduate

student at the Universidad Central de Venezuela, my alma mater. I feel lucky that my

first patient ever had periodontitis and allowed me to learn so much from her. I am proud

you made it Drills and very happy I took part of it! Querida Drills, gracias por ser la

mejor paciente que un odontólogo puede desear y por demostrar que la enfermedad se

puede superar!

My dear parents, you mean so much to me!... thank you for making me feel so

proud just by being your daughter. Both of you have instilled many admirable qualities in

me and given me a good foundation with which to meet life. You’ve taught me about

hard work and self-respect, about persistence and about how to be independent. Thank

you for teaching me to love learning and for making my education one of your top

priorities. Thank you for taking care of me during all those years, even from a distance, I

know you care!... I know you always will! Huanito (dad) thank you for being a source of

inspiration, thank you for sharing with me your wisdom in life… thank you for giving me

freedom and most importantly for showing me that freedom equals responsibility… I

couldn´t agree more! Dear Mami (mom), you have such a personality! I´m so proud of

you, your attitude in life, your strength, resilience and warmth. Thank you for teaching

me to read at a very early age… thank you for your love, dedication and trust. Thank you

for loving me just the way I am!

Mis queridos padres… los amo y admiro profundamente. Si hay alguno aquí que

merece honores esos son Uds. Gracias por regalarme tanta vida, tanto amor, tanta

dedicación. Toda una vida no bastará para retribuirles los bienes recibidos. Estoy

bendecida por tenerlos y poderlos disfrutar. Como siempre Huani ha dicho: tus logros

nadie podra arrebatarlos! Aquí esta otro Huanito y Mami!

145

I would like to thank my dear sister, Maguy, for being a role model to me. Gracias

Evi por siempre estar allí, preparada para escucharme con amor y paciencia. Gracias por

darme el gusto de ser la hermana de tan brillante actuario. Also thanks to my brother,

Enrique, for his commitment to excellence in life and work. Querido Ergu, tu constancia

en los estudios y trabajo ha servido como marco de referencia en mi camino.

Special thanks to my family in Venezuela, for their support even though they are

still wondering what I was doing and above all why. Mi abuelita bella, mi Lucci, mil

gracias por tus oraciones y buenos deseos, han sido una lucecita en mi camino. Gracias a

toda mi hermosa familia en Venezuela, siempre los llevo en mi corazón y los recordaré

muy especialmente el día de la defensa de mi tesis!

My dearests, my Salvador, my Aïda, when you're old enough to read, I hope

you're as proud of your “mami” as she is of you both. Mommy thanks you little treasures

for providing a constant source of happiness. Mi Buggy, mi negrita, thank you for giving

me the opportunity to share your lives with me… you are “my little piece of heaven”.

Parenting is hard, hard work, but when combined with a dissertation it becomes a real

challenge. Fortunately I like hard work and have the best team!... Thank you “mis niños”

for having the patience to let me try to be the best mother for you. Thank you for letting

me be in your lives, allowing me to learn and grow together. Parenting is definitely the

most rewarding and colorful journey a human soul may go through… I am the luckiest

for enjoying this gift!

My dear Patrick (Neni), my love, my confident, my friend… you have been

supportive and patient in every way, I give you my love and gratitude. You went through

every excruciating step and mood change with me. Through your eyes I’ve seen myself

as a capable, intelligent woman who could do anything once I made up my mind,

reminding me why I fell in love with you in the first place. Thank you so much. A

lifetime with you will always be too short. Te amo!

146

Thank you God for your blessings, for the light I have been able to see and for

being there for me all the time! Also, thanks to you, my guide, I know you are proud of

me…

My heartfelt thanks to all others whose names I did not mention, but who

contributed in any form towards the successful completion of the dissertation. Thank you

all!

Documents

UvA-DARE (Digital Academic Repository) Periodontitis in ...smoking may provide mechanisms for the incr eased susceptibility to periodontitis and the poorer response to treatment. Cigarette