Periodontics

New knowledge of the pathogenesis of periodontal diseaseAntonio Bascones, MD, DDS, PhDVJorge Gamonal, DDS, PhD /Maria Gomez, PhDVAugusto Silva, PhDVMiguel Angel Gonzalez, MD, DDS,

The aim ot this study was to evaiuate the relationship between tbe accumulation of interleukins IL-1 ß,TNF-t,, IL-8, and chemokine RANTES (Regulated upon Activation Normal T-cell Expressed and Secreted)in gingivai fluid and periodontal support tissues in patients with periodontitis. A review is also provided ofapoptotic processes as events of major importance, highlighting the presence of TUNEL cells and uitra-structural morphologic changes associated withceii apoptosis. There appears to be further evidence tosupport the important rcle of inflammation control, Gytokines may be considered as markers of the pro-gression and severity of periodontitis as well as indicators of an appropiate respcnse to treatment.However, further studies are needed to support and characterize this concept. (Ouintessence Int2004:35:706-716)

Periodontal diseases are a beterogeneous group ofdiseases characterized by inflammation and tbe

subsequent destruction of the tootb-supporting tissue.Today it is quite clear that periodontal diseases are ofan infectious nature and tbat tbe microorganisms pres-ent in the subgingival bacterial plaque are tbe primaryetiologic agents.'-^ Tbe destruction of tbe periodonfiumis associated with the presence of gram-negativeanaerobic bacteria localized in the subgingival region,and include typically Porphyromonas gingivalts (Pg),Prevotella intermedia (Pi), Actinobacillus actino-mycetemcomitans (Aa), and Bacteroides forsythus (Bf),

'Chair of Oral Medicine and Periodontology, Départirent of Stomatology,Sctiool o¡ Dentistry, University Compiutense of Madrid, Madrid, Spain,

^Professor, Department oí Periodontology, Schooi ot Dentistry, University oíSantiago of Ctiiie, Santiago, Ctiile,

^Doctor oí Biocherriistry Research, Bioctiemistry and Moiecuiar BioiogyDepartrrent, Sctiool of Medicine. Llniversity Complutense ol Madrid,Madrid, Spain,

'Doctor oí ßioicgical Researcii, Deparlrrent oí Immunoiogy, CentroInvestiaclones Bioiógicas, Madrid, Spain,

^Professor, Departrrent ot Stomatology, Schooi oí Dentistry, University oíGranada, Granada, Spam,

Reprint requests: Dr Antonio Bascones. Facultad de Odontclogia,Universidad Compiutense de Madrid, C/ Piaza Ramon y Cajal. s/n,E-28040-Madrid, Spain. E-mail: avances@arrakis,es

Tbese bacteria are considered to play a significant rolein tbe patbogenesis of periodontitis and tbe formationof the periodontal pocket, destruction of tbe connec-tive tissue, and résorption of the alveolar bone. Whileit is the bacterial infection that triggers the destructiveprocess, it is the host's immune response to the bacter-ial challenge that is responsible for the molecularprocesses leading to periodontal tissue destruction,''

Tbe bacteria colonizing the subgingival region mul-tiply and extend in an apical direction and, in tbeprocess, bring about loss of epithelial and connectivetissue attacbment. The bacteria may give rise to de-struction processes caused by both direct and indirectmecbanisms due to tbe activation of tbe host's im-munologie and inflammatory reactions,^-^

Although it is not possible to attribute the etiology ofperiodontal diseases to a specific bacterial agent, thereare a number of studies pointing to a group of bacteriabelieved to play a special role in the triggering and sub-sequent development of tbe disease,'" Tbere are over500 bacterial species capable of colonizing the subgingi-val region, but tbe number of these commonly impli-cated in tbe disease process is around 10 or 15 gram-negative anaerobes and spirochaetes,"'^ Tbedesignation of periodontal patbogen applies to thosebacteria that possess specific mechanisms to breakdown the host's defense systems and cause destructionof the periodontal tissues.

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Fig 1 Pathogenesis o( perio-dontai disease.

PMN iinfosB/plasmaticslnf!ammatQry j f •'"H1/TH2 maoroptiages

infiltrate / ^ ^

Inf !a m matory \ ^ ^ ^ ^ ^ ^exúdate A . Antibody oytoitmes _ ^

PGE2 MMPs chemo!<ines^

— Apoptosis ^ ^

Ciinicai destruction ^ ^ ^ ^ ^ .

^ ^ ^ ^ - Tooth !oss

Host sensibiüty —1

IL-lß!L-4

Gingivitis does not always progress to periodontitisalthough the latter is always preceded by gingivitis,"Yet, the proportion of gingivitis cases progressing toperiodontitis and the factors involved in this processare unknown.'^

The disease progression model points to a periodicor episodic phenomenon, with periods of quiescence,when neither periodontal destruction occurs nor peri-ods of exacerbation and destruction of the periodontalstructures. These are characterized histologically byacute inflammation, witb a significant increase in thenumber of neutrophils''-" (Fig 1),

The interaction between the pathogenic bacteriaand a bost's defense systems could lead to tbe devel-opment of a periodontal pocket, loss of connective tis-sue, and bone résorption.'" Once periodontitis is es-tablisbed, tbe inflammatory infiltration present iscomposed of different cell types, such as neutrophils, Tand B lymphocytes, and macrophages migrating to theperivascular connective tissue, as demonstrated by im-munologie studies.'''"-' Tbese cells produce variousspecific subtypes of cytokines tbat take part in tbe de-struction of periodontal connective tissue attachment.-'Tbe immune response is also regulated by tbe selec-tion and death of the immun ocompetent cells broughtabout by a programmed cell death mechanism, re-ferred to as apoptosis,^' and wbich could play a signifi-cant role in the pathogenesis of periodontitis." •' Thepresence of proinflammatory cytokines and chemo-kines, such as interleukin-lß (IL-lß), tumor necrosisfactor a (TNFc), interleukin-10 (IL-IO), interleukin-8{IL-8), and chemokine RANTES {Regulated uponActivation Normal T-cell Expressed and Secreted) S,in the gingival fluid is more marked in sites with pro-gressive loss of connective tissue attachment.Following treatment, there is a significant reduction inthe levels of IL-lß, TNF a, IL-10, IL-8, and RANTESin the gingival crevicular fluid, thus suggesting a rela-tionship between cytokine production and the occur-rence

The cytotoxicity of various bacterial cell compo-nents, such as short-chain carboxylic acids present inPorphyromonas gingivalis, Prevotella loescheii, andFusobacteriiim nucleatum give rise to apoptosis in Tcells^ä^* and keratinocytes.^" On the other hand,lipopolysaccharides (LPS) are common componentsof the cell wall of the gram-negative bacteria that willstimulate butyric acid, to induce apoptosis in tbemononuclear cells." In addition, A actinomycetem-comitrans leukotoxin can cause apoptosis in the peri-odontal tissue B lymphocytes.'

There are many bacteria able to induce tissue degen-eration," but it is likely that it is the host's mechanismsthat are responsible for damaging the connective tissue,tn this setting, loss of connective tissue may be tbe re-sult of tbe host's mecbanism of self-defense to preventthe progression of the lesion, by promoting the prolifer-ation of the junctional epithelium in an apical directionin order to avoid contact with a "toxic" root surface.'''-^'

It has been sbown tbat tbere is a significant rela-tionsbip between tbe severity of the disease and tbetotal amount of antibodies. Tbus, antibody titres couldbe a more accurate indicator of disease severity thantbe number of microorganisms present.''"

In view of the complexity of the bost's immunologieresponse phenomena, tbe progression from gingivitisto periodontitis and its rate of development cannot beexplained merely by tbe presence of a microbiota. Tbelatter, tbough necessary, cannot account for the con-nective tissue loss. Clinical studies have shown thatthe individual's inherent susceptibility is of great im-portance in determining tbe initiation and progressionof tbe disease. Understanding tbe factors that affectthis susceptibility may be crucial in the elucidation ofthe initiation and progression of periodontitis. Tbesefactors include tbe genetic makeup of the individualdetermining different cell responses. Environmentalrisk factors, bacteria, smoking, and stress, as well asdiseases (eg, diabetes), may alter the balance betweentbe bost and the putative microbiota and lead to dif-ferent clinical forms of periodontitis.

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Accordingly, the model of periodontitis progressionhas changed from the concept of continuous slow pro-gression to a pattern of discontinuous progression char-acterized by periods of exacerbation and remission.

BASIC IMMUNOLOGIC MECHANISMS

The manifestation of periodontitis depends on the inter-action between the host and environmental and micro-biologic factors, and it is probable that specific environ-mental and/or genetic factors determine the individual'ssusceptibility to disease. There are, however, specifichost defense mechanisms to the bacterial challenge inthe adaptive response of the immune system>^

Gingival crevicular fluid is a serous transudate thatcovers the sulcular and junctional epithelium. Thisfluid increases in cases of inflammation and containsmost of the serum components. In periodontitis, com-ponents of the serum are enriched with other sub-stances from the inflammatory cell infiltrate. Bacteriallipopolysaccharides (LPS) have a proinflammatory ef-fect on most of the periodontal tissue ceU types, suchas macrophages, lymphocytes, fibrohlasts, and os-teoblasts/osteoclasts.^^"^

The lipopolysaccharide-binding protein (LPS) andthe surrounding protein form a complex, which assistsin their binding to the specific CD14 receptor ex-pressed on monocytes. The activation of the CD14 re-ceptor produces the secretion of proinflammatory mol-ecules such as lL-1, TNFa, PGE2, and IL-6, which inturn will lead to the release of mediators such asplatelet-activating factor (PAF), bioactive amines{bradykinin and histamine), and prostaglandins.^''

The complement system is important in periodontaldisease, as is the role it plays in protecting against pe-riodontal pathogenic bacteria. Its activation is one ofthe host's earliest immune responses in the gingivalcrevicular space. * ActivaUon of C3 varies accordingto the severity of the periodontal inflammation andmay contribute to distinguishing between the differentforms oí periodontitis.^'' The bacteria related to peri-odontal disease exhibit different mechanisms to evadethe action of the complement system.

Some bacterial strains express polysaccharides ontheir surface that mask the molecules activating thecomplement system. In other words, the carbobydratestructures on the surface can determine the nature ofthe interaction between bacteria and the componentsof the complement system.''^''^ Certain periodontalpathogenic bacteria exbibit cell surface proteolytic ac-tivily tbat is capable of degrading specific componentsof the complement system, such as C3 and C5, therebypreventing opsonization."^ Bacteria such as Pg and Aapresent complex carbohydrate structures with intense

antigenic and immunogenic potency capable of mduc-ing a significant IgG2 response.'"-'*

Neutropbils, tbe main cell type present in tbe gingi-val sulcus, which make up the first line of defenseagainst periodontal pathogenic bacteria, constitute an-other level of the immune response.' •'' •5<'-'' Monocytesare phagocytic cells that represent the second line ofdefense and become activated wben infection is presentbut only if neutrophils are ineffective.'" The accretion ofbacterial plaque in bealthy individuals produces a sig-nificant increase in the number of polymorphonuclearcells (PMN), leading to the triggering of a systemic in-flammatory response. '' Active neutrophils in the gingi-val sulcus clear microorganisms through phagocytosis.However, some periodontal pathogenic bacteria sucb asPg and Aa, succeed in escaping the neutropbils, givingrise to a continuous flow of tbese phagocytes towardthe gingival crevicular space, causing continttous cellaccumulation and decay as a result of ineffectivephagocytosis.'''''^ Neutrophil degranulation entails therelease of endogenous proteases that, along with bacte-rial proteases, give rise to the destruction of the extra-cellular matrix. Thus, the normal activity of neutrophilsrepresents an essential component of the host's contri-bution to periodontal destruction** (Fig 2).

Systemic exposure to oral cavity pathogens seemsto occur in most patients, resulting in a specific anti-body response.'^ As a rule, the highest antibody titresare associated with the most severe forms of the dis-ease, as has been demonstrated for Pg in cbronicperiodontitis."

Elevated levels of antibodies against Aa ' *'' andPg5s,5û ¡.javg bggj.) described in localized aggressive andin generalized aggressive periodontitis. It bas beensuggested that tbe antibody against tbeir particularantigen is regulated by certain genes. Thus, it seemsreasonable to assume that the antibody responsemight be more protective for one individual than foranother. Therefore, some groups of patients migbt bemore prone to periodontitis because of their inabilityto elicit a response of bigh-affinity antibodies againsttbe dominant bacteriai antigens. '-*^

Antibody response to the specific characteristics ofbacterial virulence is apparently critical for the resolutionor halting the progression of the disease. There are manystudies showing a correlation between PMN phagocyticactivily and antibody affinity and fitres.^''**'^*'^

CHEMOKINES, CYTOKINES, AND PERIODONTITIS

Inflammatory processes give rise to macrophage activa-tion as well as leukocyte infiltration. The activated im-munocompetent cells produce and secrete cytokines.These activated host cells include monocytes.

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Fig Z Histologie rnfiammations in frozen sections ot gingiva from periodontitis patients (left), contiol biopsies of iiealthy gingiva ícentsr),and gingiva with periodontitis posttreatment (right) (original magnitication x20)

tnacrophages, lymphocytes, and fibroblasts."^*Cytokines are signaling proteins produced hy cell acfi-vation, acting in the local milieu as autocrine,paracrine, and sometimes, endocrine substances inorder to regulate cell function, ^ and thereby inducingand maintaining the periodontal inflammatory re-sponse.™' Several cytokines, including IL-lß, TNF-a,and IL-6, take part in this tissue destruction process asa response to the bacterial plaque.

IL-1 is a proinflammatory cytokitie that contributesto bone résorption hy stimulating release of PGE2 andpromoting metalloproteinase release that denatitratesextracelltilar matrix proteins."^'' The predominantform of IL-1 in periodontal tissues is IL-lß producedmainly by the macrophages.'"^ IL-1 is increased in in-flamed gingival tissue in patients with periodontitis.""IL-1 is also detected in the gingival crevicular fluid inareas affected by periodontitis.^''* Periodontal destruc-tion and disease profession are associated with ele-vated cytokine levels."

TNF-a is another cytokine produced mainly bymacrophages in response to such antigens as LPS. Itexhibits a wide variety of biologic effects related to thedestruction of periodontal tissue.""*

The biologic effects of IL-1 are associated withrecruiting inflammatory cel!s, facilitating neutrophildegranulation, increasing PGE2 and MMP produc-tion, inhibiting collagen synthesis, and activating Tand B cells.'«

In addition, TNF-a is involved in apoptotic pro-grammed cell death, bone résorption, MMP secretion,and intercellular adhesion molecule (ICAM) expres-sion, while promoting IL-6 production." IL-6 hasbeen identified in patients with periodontitis**' and a

number of acfions, proliferation of B cells and T cellsas well as bone résorption, are attributed to this cy-tokine in periodontifis.*'

IL-10 modulates the expression of cytokines ofmyeloid origin resulting in major functional outcomesin the triggering and maintenance of the immune re-sponse. These could consist of an anti-inflammatoryand immunosuppressive action in experimental mod-g[g32,83 similar to certain regulatory effects on the im-mune response of periodontitis.^"'*^ IL-10 might beproduced by type 2 T helper (Th2) lymphocytes andbe responsible for the inhibition of the cytokine pro-duction of the type 1 T helper (Thl) cells.^' This ac-tion of interfering with cytokine synthesis is due to itsinhibitory eñects on monocytes and macrophages.^

The presence of IL-lß (95%) and IL-8 (100%) isdetected in patients with periodontitis, in comparisonwith heahhy subjects. * The total amount of IL-lß andIL-8 decreases noticeably after completion of perio-dontal treatment and after the inflammatory process iseradicated.*^

Chemokines are cytokines with low molecularweights that attract and activate different suhpopula-tions of lymphocytes, acting as mediators in variouspathologic situations, including inflammation.*'"^'Chemokines represent a large family of proteinsgrouped in four subfamilies. One of the chemokinesubtypes is "CXC" and another "CC," being differenfi-ated by the initial two cysteine amino acids, whichmay he either adjoining (CC) or separated by anotheramino acid (CXC). On the other hand, the C familyhas a single N-terminal cysteine residue and in theCX3 C family, these cysteines are separated hy threeamino acids.^'''

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Cytokine receptors arc expressed selectively in lym-pbocytes and bave such activities as the destruction ofthe gingival connective tissue, loss of periodontal liga-ment insertion, and bone résorption. Accretion of bac-terial plaque is followed by leukocyte infiltration oftbe gingival connective tissue, in a perivascular local-ization below tbe dentogingival junction in its initialstages. Leukocyte extravasation takes place due to anactivation of the endothelial cells. Recruitment ofleukocjites into the gingival connective tissue followsthereafter. There is a constant flow of neutrophils fromthe blood vessels of the gingival plexus toward thejunctional epithelium and finally to reach the gingivalcrevical or periodontal pocket region. Accordingly,amongst the host's defense mechanisms, two immuno-logie axes are described. One is localized in tbe ep-itbelial tissue and formed of complement-antibody-neutrophils, and the other is sited in the connectivetissue and composed of macrophagcs-lymphocyte-cy-tokines,'' It is therefore of special interest to identifythe different cytokines that take part selectively in theattraction of the cells forming the infiltrate present inthe host's response to bacterial accumulation.

RANTES is a member of the family of CC chemo-kines that activates the monocytes, eosinophils, andbasopbils, induces chemotaxis, and releases otber me-diators.««'' Higb levels of RANTES are found in tbegingival crevicular fluid in patients with periodontitisin comparison witb bealtby subjects."^ It could be sug-gested tbat in periodontal disease, migration and accu-mulation of monocytes and macropbages at sites of in-flammation could be associated witb the release ofchemokines such as RANTES,

INFLAMMATORY INFILTRATION

The inflammatory infiltration of periodontitis is com-posed of mononuclear cells, mainly mononuclearphagocytes and lymphocytes.'^ T lymphocytes pre-dominate in the nonprogressive stable periodontal le-sion in wbich CD4-H cells predominate over the CD8-I-type.™ The proportion of B lymphocytes and plasmaceUs increases as periodontitis progresses. - ^

It has been sbown that macrophages, lymphocytes,and local cells, such as fibroblasts and vascular en-dothelial cells, synthesize and secrete various cy-tokines in response to the the stimulus of bacterialproducts. There are marked differences in tbe degreeof inflammation and cytokine secretion in patientswitb periodontitis when compared witb bealthy indi-viduals. Thus, an increase in T and B cells andmacropbages bas been observed in tbe biopsy leuko-cyte infiltration seen in patients with periodontitis incomparison with healthy individuals^ (Fig 3).

RANTES is a chemoattractant for the Th 1 (helper)but not the Th 2 ceils (suppressor). For these reasons,RANTES may play a major role in the regulation ofthe local immune reactions by controlling the balancebetween tbe proinflammatory cells and anti-inflamma-tory T cells, Tbe presence of T cells and macropbagesin connective tissue biopsies in patients witb peri-odontitis would imply a possible involvement ofRANTES in the tissue destruction mechanisms associ-ated with periodontitis.^^

APOPTOSIS AND PERIODONTITIS

Programmed cell death is a normal physiologicprocess that contributes to maintaining tissue home-ostasis. Cells die due to apoptosis during morphogene-sis or synapto gene sis, during tissue replacement or aspart of the immune response.

The term apoptosis describes a cell-deatb process inwbicb tbe cell actively participates. It is a cefl deathbeneficial for tbe survival of the organism.^' When thecell undergoes apoptosis, it exhibits a series of mor-phologic changes in various structures. There is loss ofasymmetry and adhesion in the cell membrane, cyto-plasm, and nucleus condensation and breaking up oftbe DNA into internucleosomal fragments. The finalstate is cell fragmentation into the so-called "apoptoticbodies," which are eliminated by the phagocytic cellswithout producing inflammation, thus avoiding dam-age to neighboring celis and surrounding tissue.^'-""Conversely, during necrosis, a cellular inflammatoryreaction is triggered as a result of tbe release of tbe cy-toplasmatic contents of tbe cells, including largeamounts of proteolytic enzymes,'''

Apoptosis is a highly controlled process that affectsvarious components at the onset, amplificafion, andcompletion of tbe destructive process. During apopto-sis, caspases (cytosolic proteases) become acti-vated,"' •"'•* and chromafin condensation as well as theactivation of specific endonucleases breaking up tbeDNA into internucleosomal fragments occur. " In ad-dition, tbe apoptotic stimulus may activate differentmechanisms, which include death cell receptor lig-ands, such as tbe Fas/Fas ligand complexes,'"^ andthese stimuli induce molecules directly or indirectlyinvolved in apoptosis, such as p53,"" Members of thefamily of Bcl-2 proteins have also been shown to playa regulating role in the apoptotic process (Fig 5), Thistype of protein, belonging to the antiapoptotic pro-teins group, can prevent or hmit cell deatb induced bydifferent stimuli,'""'™ Therefore, detection oí apoptosisin inflamed gingival tissues in patients with periodon-titis with pockets more tban 5 mm deep and attacb-ment tissue loss of over 3 mm could partly explain the

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Fig 3 iníiammatory infiltrate by immunohistochemislry on frozen sections of gingiva of patients with periodontitis Detection ot T ceils (anti-CD3, -CD4, -CD8¡, B ceils (anti-CD19¡, monocyte/macrophages (anti-CDI lo), and CCR5 ctiemokine receptor (onginai magnification x20).

mechanisms associated with tbe destruction of tbetooth support tissues.

The most appropriate metbod for identifying apopto-sis is dependent on the type of cells and tissue to bestudied,""'" Thus, light and electron microscopy pro-vide the tools for detecting tbe cbanges occurring duringapoptosis in the ceU membrane, cytoplasm, and nucleus,

Apoptosis is accompanied by the activation of a se-ries of cysteine-type proteases, named caspases, be-cause of tbeir catabolic properties. Detecting somemembers of tbis protease family may be indicative ofthe occurrence of apoptosis- Caspases are syntbesizedas inactive proenzymes, wbich are then processed inthe cells undergoing apoptosis, Caspase 3 has been in-volved as a key protease activated during the develop-ment of apoptosis,I'^"5 Active caspase 3 is present inthose cells tbat are undergoing apoptosis; the caspaseis split by proteolysis and activates other caspases.

Tbe induction of apoptosis in the host's cells pro-voked by certain pathogens, or by their products, is aphenomenon involved in tbe patbogenesis of infectiousdiseases.'^-"^ On the otber band, bacterial pbagocyto-sis or exposure to different bacterial components sucbas LPS, may delay apoptosis of the PMNs,""'"

DNA fragmentation is a process tbat takes placedue to tbe activation of tbe endonucleases during tbeapoptotic process. These nucleases break chromatininto smali fragments. The plasma membrane is the site

where redistribution of tbe pbospbatidyiserine pbos-pholipid residues takes place, wbich move from theinner cell layer to the outside of the membrane. In tbeplasma membrane, tbere are receptors involved in tberegulation of apoptosis, sucb as Fas (CD95), Fas lig-and (Fas L) can induce apoptosis in cells expressingtbe Fas receptor; this mechanism is important in Tlymphocytes and NK cells apoptosis,"'^

Apoptosis may therefore be an important pbenome-non in the regulation of the inflammatory responseagainst chronic bacterial accumulation, affecting bothtbe increase in cellularity and tbe extent of tbe inflam-matory inflltration. The number oí active caspase 3positive cells in the biopsies of subjects with periodon-titis is comparable with tbat obtained using tbeTUNEL technique"« (Fig 4), Tbe presence of morpbo-logic changes in the cell associated with apoptosis in-dicate that apoptosis mechanisms may be involved inthe patbogenesis of periodontitis.^' However, tbe smallnumber of positive cells for tbese apoptosis markerssuffer a signiflcant loss of affinity to tissue, suggestingtbat the tissue destruction taking place in tbis condi-tion, may occur after a long time of evolution,

Neutrophils are the cells initially responding to thepathogenic action of the bacteria, which comprise theplaque; they play an especially important role in thepathogenesis of periodontal disease,'^'''^' These cellspredominate in the ¡unctiona! epithelium and the gin-

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0 *^ '- îmmFig 4 TÚNEL and immunohistDc!iemica!

detection in frozen tissue biopsy sectionstrom patients with periodontitis, TUNEL-pos-itive ce!is (white arrow) and negative oelis(arrow) are observed in ciironio intlamma-tcny cei!s in a gingivai section of a patientwith periodontitis (left) Eiectrcn microscopyol the suicuiar epit!ie!ium of a ce!i undergo-ing apcptosis and shewing chfcmatin con-densation (originai magniticaticn X3,OOO)(rigfit).

•ft*» ' V t " ' / • • i •

•

b

M

cK ' • • , ' . '

Fig 5 Charactenzaticn of inflammatory in-¡i!trate by immunochemistry on secticns ofgingnfa of periodontitis, (a) Bci-2: lamüy ofproteins invo!ved in the reguiaticn of apop-tosis (arrow): (b) CASPASA-3; protease acti-vated during the development of apoptosis(arrow): (c) Fas: receptor invoived in thereguiation of apoptcsis (arrow); (d) Fas L: in-duce apoptosis in celis expressing the Fasreceptor (arrow).

gival sulcus, representing a leukocyte wall between thebacterial plaque and tbe periodontal connective tissue.Tbis interaction is crucial for understanding tissue de-struction in periodontitis.

Circulating neutropbils bave a sbort life span, andtbe loss of their functions, such as adhesion andpbagocytosis, is closely related to the occurrence ofapoptosis in these cells.' ^ Inhibition or delay of neu-trophil apoptosis in the inflammatory tissues is regu-lated by different cytokines'""'' or by factors secretedduring cell immune response such as LPS.'^'

One must consider the presence of apoptotic cellsin periodontal tissue again, as their presence poses thequestion of wbetber tbe induction mechanisms involvetbe Fas L apoptosis receptor, a member of tbe familyof TNF-a cytokines tbat, on binding to tbe Fas recep-tor, produce apoptosis. It may be considered, tbere-fore, that the Fas/Fas L complex is involved in gingi-

val tissue apoptosis. Fas and Fas L positive cells areobserved in periodontal tissue biopsies wbile they arenot detected in biopsies from healthy subjects, thus,tbis finding confirms tbe involvement of apoptoticmechanisms in periodontitis."^ Since certain apopto-sis activators require the presence of p53 protein, itsinvolvement in the apoptosis present in periodontitiscan be assumed."^ This p53 protein is a tumor-sup-pressant, and when activated, it induces genes relatedto the cell regulation cycle, DNA repair mechanism,and apoptosis induction. p53 protein is present in nor-mal tissues and cells; due to its short halfiiie, it maynot be detectable in healthy tissues.'^

The scientific studies described above support tbeconcept of apoptosis playing a role in tbe pathogenesisof periodontitis.'i^'i^^ Nevertheless, further studies arerequired to improve our knowledge of the control of in-flammation in the pathogenesis of periodontal disease.

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CONCLUSION

The local immune response in periodontal tissueagainst bacterial antigens is characterized by an in-flammatory infiltration exhibiting the clustering ofleukocytes and the subsequent release and accumula-tion of IL-lß, TNF-a, and IL-8.

Cbemokines such as RANTES have been detectedin the gingival fluid of patients with periodontifis, incomparison with healthy subjects. The pattern of thereleased cytokines matches that of a proinflammatoryimmune response, with higher levels of these media-tors being measured in pockets more than 5 mm deepand at sites of attachment tissue loss larger than 2 to 3mm. On the other hand, periodontal treatment bringsabout a considerable reduction in the levels of IL-lß,IL-8, and RANTES.

Another important aspect in ascertaining the cellu-lar and molecular bases of periodontal infection is thestudy of tbe inflammatory infiltrafion, composed of Tand B lymphocytes and macrophages, at sites with pe-riodontifis. T ceüs are found at places with stable dis-ease, wbereas B cells are sbown at sites exbibiting pro-gression of tissular destruction, in comparison witbareas with no attacbment tissue loss.

Botb tbe cytokine levels and tbe characteristics oftbe inflammatory infiltration change 2 months after pe-riodontal treatment, the situation returning to that ob-served in heaitby subjects. This finding shows that theinfiltrate and the cytokine levels bear a close relation tothe local inflammatory features . A fundamental aspectof this series of phenomena is the presence of apopto-sis, which is eventually a crucial event in periodontitis.The presence of positive TUNEL cells and the ultra-structural morphologic changes are associated with cellapoptosis. Bcl-2, p53. Fas, Fas L, and active caspase 3have been detected in the apoptotic infiltrate.

In summary, cytokines may be considered as mark-ers of tbe progression and severity of periodontitis, aswell as indicators of an appropriate response to treat-ment, ahbougb further studies are needed to supportthis concept.

REFERENCES

1. EUison SA, Oral bacteria in periodontal disease. J Dent Res1970;59(suppl 2):198-202.

2. Genco RJ, Evans RT, Ellison S. Dental research in microbi-ology with emphasis on periodontal disease. ] Am DentAssoc 1969;78:1016-1026.

3. Socransky SS. Microbiology of periodontal dis ease-Pre sentstatus and future considerations. J Periodontol 1977;48:550-S54.

4. Page RC. Kornman KS. The pathogenesis of human perio-dontitis: An introduction. Periodontology 2000 1997;14:9-11.

5. Genco RJ, Mashimo P. Krygier G, Eilison S. Antibody-medi-ated effects on the periodontium. | Periodontol 1974;45:330-337.

6. Hausmann E. Potential pathways for bone résorption inhuman periodontal disease. J Periodontol 1974;45:338-343.

7 Horton JE, Leinkin S, Oppenheim J]. Human lymphoproiif-erative reactions to saiiva and dentai plaque deposits: An invitro correlation with periodontai disease. J Periodontol1972:43:522-527

8. Nisengard RJ. Immediate hypersensitivity and periodontaldisease. J Periodontoi 1974;45;344-347.

9. Page RC, Schroeder HE, Current oí host immune responsein periodontal disease. J Periodontol 1981;52:477-491.

10. Clarif W, Loe H. Mechanism of initiation and progression ofperiodontal disease. Periodontoiogy 2000 1993;2:72-82.

11. Haffajee AD, Socransltj' SS, Smithe JY, Dihart S. Relation ofbaseline microbial parameters to future periodontal attach-ment ioss. J CHn Periodontol 1991;18:744-750.

12. Moore WEC, Moore LH, Ranney RR, Smibert RM,Burmeister JA, Schenkein HA, The microflora of periodon-tal sites showing active destructive progression. J CiinPeriodontol 1991;18:729-739

13. Loe H, Morrison E. Epidemioiogy of periodontai disease. In:Genco RJ, Goidman HM, Cohen DW (eds). ContemporaryPeriodontics. St Louis: Mosby, 1990:106-.116.

14. Lindhe J, Haffajee AD, Socransky S. Progression of diseasein adult subjects in the absence of periodontal therapy. JChn Periodontoi 1983;10:433-442.

15. Haffajee A, Socransiiy S, Goodson J. Clinical parameters aspredictors of destructive periodonta! disease activity. | ClinPeriodontol 1983;10:257-265.

16. Beciter W, Berg L, Becker BE, Untreated periodontal dis-ease: A longitudinal study. J Periodontoi 1979;5:234-243,

17 Goodson JM, Tanner ACR, Haffajee AD, Sombenger GC,Socransity SS. Patterns of progression and regression of ad-vanced destructive periodontai disease, J Clin Periodontol1982;9:472-481.

18. Page R, Schoeder H. Periodontitis in Man and OtherAnimals. A Comparative Review. New York: S Kanger,1982:5-76.

19. Miyasaki KT. The neutrophil: Mechanisms of controllingperiodontal bacteria. J Periodontoi 1991;62:761-774.

20. Seymour GJ, Gemmeil E, Reinhardt RA, Eastcott J,Taubman MA, Inmuno pathogène sis of chronic infiamma-tory periodontal disease: Ceiiular and moiecular mecha-nisms. J Periodontal Res 1993 ;28:478-486.

21. Tonetti M, Imboden M, Geber L, Lang N. Compartmentali-zation of inflammatory cell phenotypes in normal gingivaand peri-implant keratinized mucosa. J Clin Periodontol1995:22:735-742.

22. Cohen J, Duke R, Apoptosis and programmed cell death inimmunity. Annu Rev Immunol 1992;10:267-293.

23. Sawa T, Nishimura F, Ohyama H, Takahashi K, TakashibaS, Murayama Y. In vitro induction of activation-induced celldeath in lymphocytes from chronic periodontal lesions byexogenous Fas ligand. Infect Immun 1999;67:1450-1454.

Quintessenoe International 713

• Bascones et al

24. Koulouri O, Lappin DF, Radvar M, Iíinane DR Cell divi-sion, synthetic capacity, and apoptosis analysed by in situhybridisation and immunohistocheniistry. ] Clin Periodontolt999;26:552-559.

25. Masada M, Persson R, Kenney J, Lee S, Page R, Allison A.Measurement of interleukin-lo and -ß in gingival crevitularfluid: Implieation for the pathogenesis of periodcntal dis-ease. I Periodontal Res 1990;25;t56-163.

26. Matsuki Y, Yamamotg T, Hara K, Localization of inter-leukin-1 (IL-1) mRNA-expressing macrophages in humaninflamed gingiva and lL-1 activity girigival crevicular fluid. JPeriodontal Res 1993;28:35-42.

27. Tsai C, Ho Y, Chen C. Levels of interleukin-lß and inter-leukin-8 in gingival crevicular fluids in aduit periodontitis. JPeriodontoi 1995;66:852-859.

28. Kurita-Ochiai T, Fukushima K, Ochiai K. Butyric acid in-duced apoptosis of murine thymocytes, splenic T ceiis, andhuman Jurkat T celis. Infect Immun 1997;65:35-41.

29. Geatch DR, Harris JI, Heasman PA, Tayior JJ. In vitro stud-ies of lymphoeyte apoptosis induced by the periodontalpathogen Porphyromonas gingivalis. J Periodontai Res1999;34:70-78.

30. Sorkin BC, Niederman R. Short chain carbosylic acids de-crease human gingivai iteratinocyte proiiferation and in-crease apoptosis and necrosis. J Clin Periodontol 1998;25:3n-3t5.

31. Kurita-Ochiai T, Pukushima K, Ochiai K. Lipopolysaccharidestimulates butyric acid-induced apoptosis in iiuman periph-eral biood mononuciear celis. Infect Immun I999;67;22-29.

32. Ohguchi M, Ishisald A, Koide M, Koseki T, Yamamoto K,Noguchi T. Actinobaciilus actinomycetemeomitans toxis in-duces both celi cycle arrest in the G2/M phase and apopto-sis. Infect Immun I998;65:5980-5987.

33. Briifedal-Hansen H. Role of cytokines and inflammatorymediators in tissue destruction. J Periodontai Res 1993;28:500-510.

34. Genco RJ. Host responses in periodontal disease: Currentconcepts. J Periodontoi I992;63:338-355.

35. Tonetti MS. Etiology and pathogenesis. In: Lang NP,Karring T (eds). Proceedings of the 1st European Workshopon Peri odontology. Beriin: Quintessence, 1993:54-89.

36. Williams R. Periodontal disease. New Engl Med 1990-322-373-382.

37. Page RD. The role of inflammatory mediators in tbe patho-genesis of periodontal disease. ] Periodontal Res 1991;2fi-230-242.

38. Offenbacher S. Periodontai diseases: Pathogenesis. AnnPeriodontoi 1996;l:82I-878.

39. Seymour GJ. Importance of the host response in the peri-odontium. J Clin Periodontoi 1991; 18:421-426.

40. Wheeler TT, McArthur WP, Magnusson I. et al. Modeiingthe relationship between clinical, microbiologie, and im-munoiogic parameters and aiveolar bone levels in an eiderlypopulation. J Periodontol 1994;65:65-78.

41. Kinane DF, Lindhe J. Pathogenesis of periodontitis. In:Lindhe J (ed]. Clinieal Periodontology and ImpiantDentistry, ed 3. Copenhagen: Munksgaard, 1997:189-225.

42. Wilson M. Bioiogical activities of lipopolysaccharides fromorai bacteria and their relevance to the pathogenesis ofchronic periodontitis. Sei Prog 1995;78:19-34.

43. Takashiba S, Van Dyke TE, Shapira L, Amar S. Lipopoly-saccharide-lnducible ad sai icy late-sensitive nuelear factor(s) on human tumor necrosis factor alpba promoter. InfectImmun 1995;63:1529-1534.

44. Bone RC. The pathogenesis of sepsis. Ann InternMed 1991 ;115:457-469,

45. Schifferle RE, Cben PB, Davem LB, Aguirre A, Genco R],Levine MJ. Modification of expérimentai Porphyromonasgingivaiis murine infection by immunization with a poiysac-charide -protein coniugate. Orai Microbiol Immunol1993:8:266-271.

46. Hart TC, Shapira L, Van Dyke TE. Neutrophil defects asrisk factors of periodontai diseases. J Periodontol 1994;65:521-529.

47 Gemmell E, Polak B, Reinhardt RA, Eccieston J, SeymourGJ. Antibody responses of Porphyromonas gingivalis infectedgingivitis and periodontitis subjects. Oral Dis 1995;l:63-69.

48. Haffajee AD, Socransky SS, Taubman MA, Sioson ¡, SmithDJ. Patterns of antibody response in subjects with periodon-titis. Oral Microbiol Immunol 1995;10:129-137.

49. Lopatin DE, Blaciíbum E. Avidity and titer of immunoglobu-iin G subciasses to Porpbyromonas gingivalis in aduit perio-dontitis patients. Oral Microbiol Immunol 1992;7:332-337.

50. Travis J, Pike R, lmamura T, Potcmpa J, The roie of prote-oiytic enzymes in the development of pulmonary emphy-sema and periodontal disease. Am J Respir Crit Care Med1994;150:S143-S146.

51. Madianos PN, Papanou PN, Sandros J. Porphyromonas gin-givaiis infection of oral epitheiium inhibits neutrophiltransepithelial migration. Infect Immun 1997;65:3983-3990.

52. Sigusch B, Erick S, Pfister W, Klinger G, Gloekmann E.Altered ehemotactic behavior of crevicuiar PMNs in differentforms of periodontitis. J Clin Periodontol 2001;28:162-167.

53. Del Fabbro M, Francetti L, Pizzoni L, Weinstein RL. Con-genitai neutrophil defects and periodontal diseases. MinervaStomatol 2000:49:293-311.

54. Kowoiik MJ, Dowsett AS. Rodriguez J, De La Rosa RM,Eckert GJ, Systemic neutropbil response result from dentalplaque accumulation. J Periodontol 2001;72:146-151.

55. Eick S, Pfister W, Sigusch B, Sträube E. Phagocytosis of pe-riodontopathogenic bacteria by crevicular granulocytes isdepressed in progressive periodontitis. Infection 2000;28:301-304.

56. Engstrom PE, Larsson A, Norhagen G, et al. Specificity andlevels of oral and systemic antibodies to Actinobaciilusactinomyeetemcomitans. J Clin Periodontoi 1993:20:746-751.

57. Lopatin DE, LaBelle D, Lee SW. Measurement of relativeavidity of antibodies reactive with Porphyromonas(Bacteroides) gingivalis in the sera of subjects having adultperiodontitis. J Periodontal Res 1991;26:167-175.

58. Meghji S, Henderson B, Kirby A, Newman HN, Wilson M.Serum antibody response to surface-associated materialfrom periodontopathogenie bacteria. FEMS Immunol MedMicrobiol 1995;10:101-108.

59. Ling TY, Sims TJ, Chen HA, et al. Titer and subciass distrib-ution of serum lgG antibody reactive with Actinobaciilusactinomycetemeomitans in iocalized juvenile periodontitis, JClin Immunol 1993;13:101-U2.

714 Volume 35, Number 9, 2004

• Bascones et al

60. Takahashi J, Saito A, lNakagawa T, Yamada S, Ishihara K,Okuda K. Dynamics of serum Immunoglohulin G avidity forPorphyromonas gingivalis in adult periodontitis. J Perio-dontoi 1998:69:367-373.

61. Mooney J, Adonogianaiii E. Kinane DF. Relative avidity ofserum antihodies to putative periodontopathogens in perio-dontal disease. J Periodontal Res 1993;28:444-450.

62. Kinane DF, Mooney J, MacFarlane TW, McDonald M.Local and systemic antibody response to putative periodon-topathogens in patients with chronic periodontitis:Correlation with clinical indices. Oral Microhiol Immunol1993:8:65-68,

63. Mooney ], KJnane DF. Humoral immune responses toForphyromonas gingivalis and Actinobacillus actino-mycetemcomitans In adult periodontitis and rapidly progres-sive periodontitis. Oral Microhiol Immunoi 1994;9:321-326.

64. Sjostrom K, Darveau R, Page R, Whitney C, Engel D.Opsonic antihody activity against Actinobacillus actino-mycetemc omitan s in patients with rapidly progressive perio-dontitis. Infect Immun 1992;60:4819-4825 {erratum 1993:61:1167).

65. Sjostrom K, Ou J, Whitney C, et al. Effect of treatment ontiter, function and antigen recognition of serum antibodiesto Actinobacillus acti no mycetemcomitans in patients withrapidiy progressive periodontitis. Infect Immun 1994:62:145-151.

66. Underwood K. Sjostrom K, Darveau R, et al. Serum anti-hody opsonic activity against Actinohacillus actino-mycetemc omit ans in human periodontai diseases. J InfectDis 199j; 168:1436-1448.

67 Wiiton JM, Hurst TJ, Sterne JA. Eievated opsonic forPorphyromonas (Bacteroides) gingivaiis in serum from pa-tients with a history of destructive periodontal disease. Acase control study. J Ciin Periodontoi 1993:20:563-569.

68. Vilcek J. Le J. Immunology of cytokines: An introduction.In: Thomson A ¡ed]. The Cytoliine Handbook. London:Harcourt Brace. 1996,

69. Meikle M, Heath ], Reynolds J. Reievance to the pathogene-sis of periodontai disease and a new hypothesis. J OralPathol 1986:15:239-250.

70. Dewhhît F, Stashenko P, Mole J, Tsurumachi T Purificationand partiai sequence of human osteociast-activating factor:Identify with inter)eukin-lß. J Immunol 1985:135:2562-2568.

71. Graves DT, Cochran DL. Clinical, radiographie and bio-chemical assessment of IL-1/TNF-aifa antagonist inhibitionof bone loss in experimental periodontitis, J Clin Perio-dontoi 2C]02;29:137-143.

72. Matsuki Y, Yamamoto T, Hara K. Interleukin-I mRNA-ex-pressing macrophages in human chronically inflamed gingi-val tissues. Am J Fathol 1991:138:1299-1305.

73. Matsuki Y, Yamamoto T, Hara K. Detection of inflammatorycytokine messenger RNA (mRNA)-expressing cells inhuman inflamed gingiva by combined in situ hybridizationand immunohistochemistry. Immunoi 1992:76:42-47.

74. Honing ], Rordorf-Adam C, Siegmund C, Wiedermann W,Erard F, Interieukin-i beta (IL-lß] concentration in gingivaltissue from periodontitis patients and healthy control suh-jects. J Periodontal Res 1989:24:362-367

75. Tokoro Y, Yamamoto T, Hara K. IL-lß mRNA as the pre-dominant inflammatory cytokine transcript: Correlationwith inflammatory cell infiltration into human gingiva. JOral Pathol Med 1996;26:225-231.

76, Reinhardt R, Masada MF, Kaldahl WB, ct al. Gingival fiuidIL-1 and IL-6 levéis in retractory periodontitis. J ClinFeriodontol 1993:20:225-231.

77 Stashencko P, Fukiyoshi P, Obernesser M, Prostak L,Hatfajee A, Socransky S. Levels of Interleukin-la in tissuefrom sites of active periodontal disease. J Clin Periodontoi1991;18:548-554.

78. Meikle M, Atkinson S, Ward R, Murphy G, Reynoids J.Gingival flbrobiast degrade type 1 coliagen films when stim-ulated with tumor necrosis factor and interleukin 1.Evidence that breakdown is mediated by metailopro-teinases. J Periodontai Res 1989:24:207-213.

79. Butler L. Layman N, Sbarp J, Bedade A. Interieukin-1-in-duced pathophysiology. Ciin Immunoi lmmunopathol1989:53:400-406.

80. Takahashi S, Takahashi K, Takashiba S, et al. Assessment ofinterleukin-6 in the pathogenesis of periodontal disease. JPeriodontoi 1994:65:147-153.

81. Ishimi Y, Miyaura C, Jin CH, et al. IL-6 is produced by os-teoblasts and induces bone résorption. J Immunol 1990;145:3297-3303.

82. Rosemhaun J, Angelí E. Paradoxical eflects of IL-10 in en-dotoxin-induced uveitis. J Immunol 1995:155:4090-4094.

83. Tumpey T, Einer V, Chen S, Oakes J. Lauscb R, Interleukin-10 treatment can suppress stromal keratitis induced by her-pes simplex virus type 1. J Immunoi 1994:153:2258-2265.

84. Stein S, Hendrix C. Intericukin-lO promotes anti-coilagcnantibody production in gingivai mononuclcar cells [abstract1122]. J Dent Res 1996;75(special issue):158.

85. Yamamoto M, Fujihashi K, Hiroi T, McGee J, Van Dyke T,Kiyono H. Molecular and cellular mechanism for periodon-tai diseases: Role of Thl and Th2 type cytokines in induc-tion of mucosal infiammation. J Feriodontal Res 1997;32:115-119.

86. Florentino D, Bond M, Mosmann T. Two types of mousehelper T celi. IV. Th2 ciones secrete a factor that inhibits cy-tokine production by Thl clones. J Exp Med 1989:170:2081-2095.

87. Florentino D, Zlotnik A, Mosmann T, O'Garra H, lL-10 in-hihits cytokine production by activated macrophages, JImmunol 1991; 147:3815-3822.

88. Gamonal J, Jorge O, Silva A. Interleuquina 1& e inter-ieuquina 8 en pacientes aduitos con periodontitis destruc-tiva: efectos del tratamiento periodontal. Av Perio-doncia 1999:11:183-193.

89. Bacon KB, Schall TJ. Chemokines as mediators of allergicinflammation. Int Arch Ailergy Immunol 1996;1G9:97-1O9.

90. Howard OM, Ben-Baruch A, Oppenheim J). Chemokines.Progress toward identifying molecular targets for therapeu-tic agents. Trends Biotechnoi 1996:14:46-51.

91. Ward SG, Bacon K, Westwick J, Chemokines and T lym-phocytes: More than attraction. Immunity 1998:9:1-11.

92. Baggiolini M. Chemokines and leukocyte traffic. Nature1998:392:565-568.

93. Murphy PM. Chemokine receptors. Structure, function, androle in microbiai pathogenesis. Cytoidne Growth Fact Rev1996:7:47-64.

94. Baggioiini M, Dahinden CA, Chemokines in allergic inflam-mation. Immunol Today 1994;15:127-133.

95. Schroeder HE. Pathogenesis of inflammatory periodontaldisease. A summary of current work. Lab Invest 1976:34:235-249.

Quintessence Inter national 715

• Bascones et ai

96, Zappa U, Reinking-Zappa M, Graf H, Espelatid M, Cellpopulatiorts and episodic periodontal attachment loss inhumans, J Clin Periodontol 1991;18:508-515,

97, Gamonal ], Acevedo A, Bascones A, Jorge O, Silva A.Charac ter iza t ion of cellular infiltrate de tec t ion ofchemokine receptor CCR5 and interleukin 8 and RANTESchemokines in adult periodontitis, J Periodont Res 2000;35:1-10,

98, Siveke |T, Hamanti A. T helper 1 and T helper 2 cells re-spond differentially to chemokines, ) Immunol 1998;160:550-554,

99, Kerr J, Wyllie A, Curric A. Apoptosis: A basic biologic phe-nomenon with wide-ranging implications in tissue kinetics,Br) Cancer 1972;26;239-257.

100, Abrams ), White K, Fessier L, Steller H, Programmed celldeath during Drosophila embryogenesis. Development1993;117:29-43,

101, Robertson A, Thomson ), Morphology of programmed celldeath in the ventral nerve cord of Caenorhabditis eleganslarvae, ) Embryol Exp Morphol 1982;67:89-100,

102, Majno G, Joris J, Apoptosis. oncosis and necrosis. Anoverview of cell death. Am J Pathol 1995;146:3-16.

103, Thornberry NA, Caspases: Key mediators of apoptosis,Chem Biol 1998;5:97-103,

104, Salvesen GS, Dixit VM. Caspase activation: The induced-proximity model. Proc Nati Acad Sei USA 1999;96:10,964-10,967,

105, Nagata S, Apoptotic DNA fragmentation, Exp Cell Res2000;255:12-18,

106, Nagata S, Fas l igand-induced apoptos i s , Ann ReuGenet 1999;33:29-55,

107, Sionov RV, Haupt Y. The cellular response to p53: The deci-sion between life and death, Oncogenc 1999; 18:6,145-6,157,

108, Reed JC, Bcl-2 family prote ins Oncogene 1998;15:3,225-3,236.

109, Ekert PG, Silke J, Vatt?: DL, Caspase inhibitors. Cell DeathDiffer 1999;6:1081-10S6,

110, Schwartz ZL. Osbome B, Metbods in Cell Biology, Vol 46:Cell death. London: Academic Press, 1995:XV-XVIII,

111, McCarthy N, Evan G. Cellular and molecular proceduresin developmental biology, Metbods for detecting and quan-tifying apoptosis, London: Academic Press, 1998: 259-278,

112, Nicholson D, Ali A, Thornberry NA, et al. Identificationand inhibition of the ICE/CED-3 protease necessary formammalian apoptosis. Nature 1995;376:37-43.

113, Patel T, Gores G, Katifmann S. The role of proteases dur-ing apoptosis, FASEB J 1996;10:587-597,

114, Chen Y, Zychlinsky A, Apoptosis induced by bacterialpathogens, Microb Pathog 1994; 17:203-212,

115, Mangan D, Taichman N, Lally E, Wahl S. Lethal eÖects ofActinobacillus actinomycetemcomitans leucotoAis onhuman T lymphocytes. Infect Immun 1991;59:3267-3272,

116, Zychlinsky A, Prévost M, Sansonetti P, Sbigella flexneri in-duces apoptos i s in infected macrophages . Nature1992;358:167-169,

117 Baran J, Guzik K, Hryniewicz W, Ernst M, Piad H, PryjmaJ,Apoptosis of monocytes and prolonged survival of granulo-cytes as a result of phagocytosis of bacteria. Infect Immun1996;64:4242-4248,

118, Colotta F, Re F, Polentarutti N, Sozzani S, Mantovani A,Modulation of granulocytc survival and programmed celldea th by cytokines and bacter ia l p roduc t s . Blood1992;80:2012-2020,

119, Gamonal J, Bascones A, Acevedo A. Blanco E, Silva A,Apoptosis in cbronic adult periodontitis analyzed by in situDNA breaks, electron microscopy, and immunohistochem-istry, J Periodontol 2001;72:517-525,

120, Daniel M, Van Dyke T, Alterations in phagocyte functionand pe r iodon ta l infection, J Pe r iodon to l 1996;67;1070-1076,

121, Tonetti M, Gerber L, Lang N, Vascular adhesion moleculesand initial development of inflammation in clinicallyhealthy human keratinized mucosa around teeth and os-seointegrated implant, J Periodontal Res 1994;29:386-392,

122, Dransfield I. Stocks S, Haslett C, Regulation of cell adhe-sion molecule expression and function associated withneutrophil apoptosis. Blood 1995;85:3264-3273,

123, Dibbert B, Weber M, Nikolaizik WH, et al, Cytokine-medi-ated Bax deficiency and consequent delayed neutrophilapoptosis: A general mechanism to accumulate effectorcells in inflammation, Proc Nati Aead Sei USA 1999;96:13330-13335,

124, Cox G, Gauldie J, Jordana M, Bronchial epithelial cell-de-rived cytokines {G-CSF and GM-CSF] promote the sur-vival of peripheral blood neutrophils in vitro. Am J RespirCell Mol Biol 1992;7:507-513,

125, Ichinose Y, Hara N, Ohta M, et al. Recombinant gratiulo-cyte colony-stimulating factor and lipopolysaccharidemaintain the phenotype of and Superoxide anión genera-tion by neutrophils. Infect Immun 1990;58:1647-1652.

126, Gamonal J, Bascones A, Jorge O, Silva A. ChemokineRATES in gingival crevicular fluid of adult patients withperiodontitis, J Clin Periodontol 2001;27:675-681,

127, Ko L, Prives C, P53: Puzzle and paradigm. Genes Dev1996;10:1054-1072.

128, Ellis S, Tucci M, Serio F, Johson R, Factors for progressionof per iodontal diseases, J Oral Pathol Med 1998;27:101-105,

716 Volume 35, Number 9. 2004