Analysis of host responses and risk for disease progression

Sinem E. Sahingur &Robert E. Cohen

Periodontal diseases affect 5±30% of the adult population, constituting one of the most common bacterial infections in humans (203). The literature is replete with studies compiled during the past 20 years that have investigated the etiology, pathogenesis, and treatment of periodontal diseases (56, 58, 80, 102, 199, 312). It has become evident that speci®c bacteria are essential for the initiation and progression of periodontal diseases, and that tissue destruction as a consequence of periodontitis may result from an imbalance in host protective and destructive mechanisms initiated by an infectious process. Although a signi®cant subset of the population appears to be susceptible to periodontitis, there also are individuals who seem to be more resistant to the most severe forms of disease (81, 151, 153, 234). It thus has been hypothesized that host factors or responses could be identi®ed that might be able to detect individuals or populations with particular susceptibility or resistance to periodontitis.

In the broadest sense, risk is the possibility of suffering a loss. In medicine, risk has been de®ned as the probability of developing a particular disease or experiencing a change in health status, given a de®ned condition (68). Any genetic, environmental, or behavioral characteristics of an individual that increase the probability of disease outcome are called risk factors (212). Periodontal diseases are multifactorial conditions that are affected by both genetic and environmental factors (81). In general, risk factors for periodontitis include smoking history (89, 96, 97, 103, 316), genetic predisposition (113, 158, 178), age (96), gender (97), stress (85, 186), and diet (193, 194), as well as medical conditions such as diabetes mellitus (70, 96, 152, 235, 251, 275), osteoporosis (226, 299), Down's syndrome (4, 82, 135), and acquired immunode®-ciency syndrome (135, 154, 315).

The natural history of periodontitis follows a discontinuous pattern of exacerbation and remission characterized by disease-active and disease-inactive sites (80, 199). Methods for the detection of periodontitis and the identi®cation of patients at risk for progressive disease are under active investigation (220). Although clinical parameters such as probing depth, attachment level, bleeding on probing, plaque index, and radiographic assessment of alveolar bone loss provide information on the severity of periodontitis, they do not measure disease activity. Consequently, microbiological testing (257, 312), analysis of the host response (150), and genetic analyses (143) have been proposed in an effort to monitor and identify patients at increased risk for periodontitis. Ideally, development and application of rapid and simple diagnostic tests based on host salivary or immune factors may facilitate early detection of patients at risk for periodontal diseases, allow appropriate intervention, decrease the need for more aggressive treatment, and improve the response to periodontal therapy. Salivary-based tests for caries susceptibility currently exist, but are not employed on a routine basis in clinical practice. Although many studies have identi®ed salivary and host factors and have attempted to determine their relationships to periodontal diseases, the applicability of diagnostic tests ± and the potential utility of risk assessment based upon analysis of salivary factors and host response ± await further development.

Saliva as a diagnostic fluid in periodontal diseases

What is saliva, where is it formed, and what types of human saliva exist?

Saliva is the ¯uid that bathes the hard and soft tissues of the oral cavity. Human saliva is produced by three major glands ± the parotid, submandibular, and sublingual ± as well as by numerous minor glands (170). The basic secretory units of those glands are acini, which comprise cell clusters secreting water, electrolytes, proteins, and glycoproteins, and whose products ¯ow into collecting ducts (283). That ¯uid is further altered within the ducts. For example, much of the sodium is actively reabsorbed, and additional quantities of potassium and bicarbonate ions are secreted (170, 303). Small collecting ducts within salivary glands then lead to larger ducts, eventually forming a terminal duct discharging into the oral cavity. Some of the morphologic characteristics of the major salivary glands are summarized in Table 1.

Saliva can be assessed as whole (mixed), or as gland-speci®c saliva. The composition of saliva is affected by many factors, such as the originating gland, diet, use of pharmacological agents, and the systemic or oral health of the patient (170, 173, 303). Parotid saliva is a serous, watery secretion whereas the submandibular glands produce a mixed serous and mucous ¯uid; a predominantly mucous ¯uid is derived from the sublingual glands. Some of the components commonly found in mixed human saliva are noted in Table 2. The function of saliva and its components have been extensively reviewed (65, 133, 149, 161, 170, 270), and some of their functional properties are outlined in Table 3.

What techniques are used to collect and analyze saliva?

Both whole and gland-speci®c saliva can be collected with or without gustatory stimulation. Unstimulated whole saliva is composed of secretions from the parotid, submandibular, sublingual, and minor mucous glands, as well as from gingival crevicular ¯uid, desquamated epithelial cells, microorganisms, leukocytes, food residue, and blood. Stimulated saliva has been obtained in response to masticatory or gustatory stimulation using a variety of methods, including paraf®n wax, rubber bands, gum base, and citric acid (74, 170, 263). Some of the more common techniques (190) for obtaining whole-and duct-collected saliva are described in Table 4.

Since saliva can be collected more easily and less invasively compared to blood, interest in saliva as a diagnostic ¯uid has received increased attention during the past two decades. Indeed, saliva has been used as a diagnostic ¯uid in medicine, where clinical problems such as digitalis toxicity (calcium and potassium measurement), affective disorders (prostaglandins), immunode®ciency (secretory IgA), and

Table 1. Summary of morphologic characteristics of the major salivary glands

Parotid Submandibular Sublingual

Gland type PositionSizeMain excretory duct Interlobular ducts Striated ducts Intercalated ducts

Terminal secretory units

Stroma/interstitial tissue

Compound acinar Mandibular ramus Largest Stensen's duct; opens opposite maxillary 2nd molar

All lined with pseudostratified columnar epithelium Single layer cuboidal to columnar cells

Single layer flat to cuboidal; ducts are long, narrow, and branching Serous acini

Connective tissue septa, lobulated

Compound tubuloacinar Beneath mandible IntermediateWharton's duct; opens on either side of frenulum of tongue Same as parotid Same as parotid, but with more abundant and longer ducts Same as parotid, but ducts are shorter and fewerSerous acini predominate; fewer mucous acini with serous demilunes Same as parotid Compound tubuloacinarFloor of mouth SmallestBartholin's duct; opens into or beside Wharton's duct; minor ducts open into oral cavity on sublingual fold Same as parotid Same as parotid but ducts are rare Same as parotid, but ducts are rare Mucous acini with demilunes predominate; some serous acini Same as parotid

Modified from (304).

Electrolytes Organic Proteins and compounds macromolecules

Ammonia	Amino acids	Aggregins
Bicarbonate	Creatinine	Albumin
Calcium	Fatty acids	Blood group substances
Chloride	Glucose	Cystatins
Fluoride	Lactic acid	Enzymes
Hypothiocyanate	Lipids	Fibronectin
Iodine	Sialic acid	Fucose-rich glycoprotein
Magnesium	Urea	Growth factors
Nitrate	Uric acid	Gustin
Nitrite		Histatins
Phosphates		Immunoglobulins
Potassium		Kallikrein
Sodium		Lactoferrin
Sulfate		Mucin glycoproteins
Thiocyanate		Proline-rich proteins
		Ribonucleases
		Secretory component
		Serum proteins
		Tyrosine-rich proteins

Adapted from (43, 73, 74, 134).

Table 3. Selected functions of salivary components Antimicrobial activity

Buffering capacity Clearance and adherence of microbial flora Food preparation and digestion Formation of intraoral pellicles Heterotypic complexing with other salivary

components Lubrication and protection of soft and hard tissue

interfaces Pellicle formation Post-translational molecular processing Remineralization Speech Taste Tissue coating Utilization as a microbial metabolic substrate Water balance

Adapted from (43, 134, 170).

tobacco usage (cotinine) have been assessed (171, 172, 309). In dentistry, salivary-based caries susceptibility tests also have become relatively well established (28, 172).

Proposed salivary diagnostic markers for periodontal diseases have included serum and salivary molecules such as immunoglobulins, enzymes, consti-tuents of gingival crevice ¯uid, bacterial components or products, volatile compounds, and phenotypic markers such as epithelial keratins (134, 171). The use of saliva for periodontal diagnosis also has been recently reviewed (134). In general, factors that are native to saliva and derived from products of salivary gland cells may not provide data of appropriate speci®city for use as periodontal disease markers (134, 171, 172, 308). Consequently, considerable research efforts have been directed to analysis of salivary enzymes and immunoglobulins.

Which salivary enzymes are derived from oral microorganisms and polymorphonuclear leukocytes?

Salivary enzymes can be produced by salivary glands, oral microorganisms, polymorphonuclear leukocytes, oral epithelial cells, or be derived from gingival crevice ¯uid (171). Consequently, attempts have been made to correlate enzymatic activity in human saliva with periodontal status. Studies also have assessed changes in salivary enzyme activity in response to periodontal therapy.

Watanabe et al. (301) found positive relationships between salivary protease activity and calculus index, as well as between protease activity and periodontal pocket depth. Later, Nakamura & Slots (189) investigated enzyme activity in whole saliva and parotid saliva in 10 periodontally healthy individuals, 10 patients with chronic periodontitis, and four patients with localized aggressive periodontitis. Increased activity for alkaline phosphatase, esterase, b-glucuro-nidase, a-and b-glucosidase, and other aminopeptidases was detected in saliva from patients with chronic periodontitis, compared to healthy controls. There were no signi®cant differences in enzyme activity between the chronic periodontitis and localized aggressive periodontitis groups, but patients with aggressive periodontitis exhibited greater amounts of salivary butyrate esterase and cysteine aminopeptidase. Based on those data, the authors noted that a signi®cant proportion of salivary enzymes were most likely derived from the oral micro¯ora.

Gibbons & Etherden (87) compared the levels of ®bronectin-degrading enzymes in whole saliva from

Table 4. Saliva collection methods

Saliva Method

Mixed Suction method: Saliva is continuously aspirated from the floor of the mouth into a suitable collection vessel (11, 190).

Parotid Modified Carlson-Crittenden device (246). The device has two chambers, an inner and an outer chamber. The inner chamber is placed over the orifice of the parotid duct and attached to tubing that carries saliva to the collection vessel. The outer chamber is connected to a vacuum squeeze bulb. The bulb is compressed and the collector is then placed over the duct opening.

Submandibular/	Alternatively, Fox et al. (74) have described a simpler method for the collection of submandibular-
sublingual	sublingual. After blocking the parotid saliva secretion by placing a gauze pad at the orifice of the
	parotid ducts, saliva can be collected from the floor of the mouth with a micropipette.

periodontally healthy subjects. Unstimulated saliva was collected immediately after awakening (i.e. lack of recent oral hygiene measures), as well as immediately after toothbrushing, with substantially greater enzyme activity detected in saliva obtained after awakening. They concluded that oral hygiene measures might contribute to variations observed in the levels of salivary proteases. Uitto et al. (285) investigated collagenase activity in whole and gland-speci®c salivas from subjects with or without periodontitis. Mammalian collagenase was detected in whole saliva from all subjects, but not in parotid, sublingual or submandibular saliva. Periodontitis patients demonstrated greater collagenase activity compared to controls and, in those patients, signi®-cant reductions in collagenase were reported after periodontal therapy. The authors concluded that most of the collagenase in the saliva samples originated from polymorphonuclear leukocytes entering the oral cavity via the gingival sulcus. Further studies by Sorsa et al. (261) supported the previous ®ndings and demonstrated that collagenases isolated from extracts of in¯amed human gingiva, gingival crevicular ¯uid, and saliva all were derived from polymorphonuclear leukocytes and not from ®broblasts. Those studies collectively reinforced the potential utility of assessing saliva for components of tissue destruction that might be associated with periodontal diseases.

Greater amounts of immunoglobulin-degrading enzymes have been reported in whole saliva from patients with localized aggressive periodontitis, compared to healthy controls (94). These authors proposed that production of enzymes by periodontal pathogens impart an ecologic advantage, enhance microbiological colonization, and may have a role in the pathogenesis of aggressive periodontal diseases.

Arginase is an arginine-depleting enzyme belonging to the l-arginine/nitric oxide pathway. Ozmeric et al. (209) examined the possible role of salivary arginase in the pathogenesis of periodontal disease using whole saliva samples from 20 chronic periodontitis patients and 15 systemically and periodontally healthy controls. Patients with periodontitis had signi®cantly greater amounts of arginase, as well as greater pocket depths, loss of clinical attachment, plaque index and gingival index. However, there was no statistically signi®cant correlation between the biochemical and periodontal variables within the patient groups.

A variety of studies have investigated the effects of periodontal therapy on salivary enzyme activity.

Table 5. Systemic conditions associated with neutrophil disorders and periodontal diseases

Disease Reference

Chediak-Higashi Syndrome (108, 181)

Cyclic neutropenia (42, 244)

Down's Syndrome (4, 181, 238)

Agranulocytosis (22, 119)

Actin Dysfunction Syndrome (32)

Glycogen Storage Disease (110)

Lazy Leukocyte Syndrome (81, 182)

Ulcerative Colitis (27, 271)

Familial Benign Chronic Neutropenia (61)

Zambon et al. (313) found reduced amounts of leucine, valine, cysteine aminopeptidases, caprylate esterase lipase, trypsin, b-galactosidase, b-glucuro-nidase, and b-glucosidase in whole saliva from chronic periodontitis patients after periodontal therapy. The proportions of subgingival black-pigmented Bacteroides and motile organisms also decreased in those patients. The investigators hypothesized that these salivary enzymes were derived from oral microorganisms and the effectiveness of periodontal therapy could be monitored by changes in the amount of speci®c bacterial enzymes in whole saliva. Similarly, salivary collagenase and gelatinases were evaluated in patients having chronic periodontitis, localized aggressive periodontitis, and in periodontally healthy controls before and after periodontal treatment (77). Collagenase activity was signi®cantly elevated in patients with periodontitis compared to controls. Moreover, periodontal therapy resulted in reduced amounts of active collagenase and elastase. Although clinical parameters such as gingival index, plaque index, and pocket depth positively correlated with enzyme activity, the relationships were not statistically signi®cant.

In a 20-month longitudinal study, Nieminen et al. (192) assessed enzyme activity in whole saliva of proteases and glycosidases using a study group of 24 adults with advanced periodontitis, compared to a control group of 25 subjects with healthy periodontal tissues. Clinical parameters and levels of enzyme activity were assessed at baseline and following different stages of periodontal therapy. The mean values of the proteolytic enzymatic activity and the activity of two glycosidases in whole saliva were signi®cantly higher in the study group than in the control group at baseline. After the initial treatment phase at 8 months, all three proteases were signi®cantly reduced, but the concentration of glycosidases remained elevated. Upon completion of periodontal therapy at 20 months, the activity of both the proteases and glycosidases approximated the values of the healthy group. The authors postulated that the enzyme activity in whole saliva may re¯ect the severity of periodontal disease, and that salivary elastase may have diagnostic potential for assessment of periodontal in¯ammation and the response to periodontal therapy.

Ingman et al. (124) also examined proteolytic activity in patients with chronic periodontitis, localized aggressive periodontitis, and periodontal health. Chronic periodontitis patients had greater protease, collagenase and elastase-like activity in comparison with localized aggressive periodontitis patients and the healthy controls. Scaling and root planing signi®cantly reduced protease and elastase activities in the chronic periodontitis group, but not in patients with aggressive periodontitis. The authors concluded that salivary elastase activity might have the potential to be a simple and accurate indicator of treatment effectiveness. Further work by those investigators (125) determined that gelatinase from saliva and GCF was similar to gelatinase isolated from polymorphonuclear leukocytes and ®broblasts. They suggested that multiple forms of gelatinase present in saliva may be involved in tissue destruction. Salivary gelatinase levels in relation to periodontal status was further evaluated by MaÈkelaÈ et al. (168), who found that the concentration of matrix metalloproteinase-9 (MMP-9 or 92 kDa gelatinase) was signi®cantly higher in whole saliva of periodontitis patients compared with healthy subjects, and that periodontal treatment resulted in reduced amounts of those enzymes.

Hayakawa et al. (116) reported that total TIMP-1 (tissue inhibitor of metalloproteinases-1) concentration in whole saliva of periodontally diseased subjects was clearly lower than that of clinically healthy subjects. They also found that most of the total collagenase in whole saliva of healthy subjects consisted of procollagenase, while mainly active collagenase was present in whole saliva from patients with periodontal diseases. Signi®cant reciprocal changes of TIMP-1 and collagenase levels, that is, increase in TIMP-1 concentration and decrease in collagenase activity, were observed after initial periodontal therapy.

How may systemic conditions affect salivary enzymes?

Salivary enzyme pro®les also have been investigated in patients with systemic conditions that typically are associated with periodontal pathology. Halinen et al. (107) reported that there was greater salivary collagenase activity in children with Down's syndrome, compared to controls. They postulated that MMP activity derived from polymorphonuclear leukocytes or cytokine-activated ®broblasts may, in part, be responsible for early periodontal tissue and alveolar bone destruction sometimes observed in patients with that condition. Collin et al. (47) studied the salivary levels and activities of the MMP-8 and MMP-9 in 45 type 2 diabetic patients and in 77 control subjects. They found that the major MMPs in the type 2 diabetic patients' saliva were MMP-8 and MMP-9. Salivary MMP levels and activities in type 2 diabetic patients were similar to those in the control group. However, multiple regression analysis revealed that gingival bleeding, pocket depths and HbA1c were associated with increased MMP-8 levels. They postulated that advanced periodontitis in type 2 diabetes may be related to elevated salivary MMP-8, and measurement of that enzyme might be useful in monitoring periodontal disease in diabetics.

Patients harboring HIV frequently demonstrate pronounced gingival in¯ammation or attachment loss. Mellanen et al. (179) investigated the presence of ®broblast-type matrix (MMP-1) and neutrophil (MMP-8) collagenases, stromelysin-1 (MMP-3), and myeloperoxidase in saliva of HIV-positive patients at different phases of HIV-infection. HIV-negative, systemically healthy, age-matched patients served as controls. The activity of interstitial collagenase was increased in saliva from different phases of HIV-infected patients compared to the controls. Independent of the phase of HIV-infection, saliva samples contained pro-and active forms of MMP-1, -3 and -8. Saliva samples from healthy controls were found to contain minimal immunoreactivity for MMP-1 or MMP-8, but considerable amounts of MMP-3 were detected. Increased amounts of myeloperoxidase in HIV-patients' saliva relative to controls also were reported. The authors concluded that the increased amounts of MMPs and myeloperoxidase might re¯ect and directly participate in periodontitis associated with HIV-infection.

Smoking represents one of the major risk factors for periodontal diseases (81, 135, 137). Liede et al. (163) investigated the associations between smoking, periodontal status, and salivary proteases, demonstrating a signi®cantly decreased amount of salivary proteolytic activity and MMP-8 levels in current smokers than in former smokers. Consequently, the authors suggested that care should be taken in interpreting results suggesting that salivary proteinases might be potential diagnostic markers for periodontal disease activity. In another study, Pauletto et al. (215) studied the differences in elastase levels between smokers and non-smokers in patients with periodontitis. In that study, paraf®n-stimulated saliva or oral rinse samples were assayed for elastase activity, and neutrophils were quantitated by staining the cells in oral rinse smears. In non-smokers, periodontitis patients exhibited elevated numbers of neutrophils compared to healthy subjects, while the smokers showed no signi®cant changes. Analysis of elastase in stimulated whole saliva also showed that smokers had signi®cantly lower oral elastase levels than former smokers in both advanced and moderate chronic periodontitis groups. The authors concluded that cigarette smoking leads to lowered elastase and neutrophil levels in the oral cavity. Consequently, they also noted that an oral neutrophil elastase assay may not be applicable for measurement of periodontal status in smokers.

Additional salivary host-derived non-immune factors include lactoferrin, lysozyme, sialoperoxidase, histatin, and amylase. Although many pathogens have capsules or other cell wall protective mechanisms that resist lysozyme, it induces cell lysis and death by hydrolyzing the speci®c bonds in bacterial cell surface (109). Indeed, Markkanen et al. (177) reported lower lysozyme concentration in mixed saliva in periodontitis patients compared to healthy controls. Smith et al. (254) reported increased peroxidase activity at the onset of gingival in¯ammation that declined after initiation of oral hygiene. Over et al. (208) demonstrated increased myeloperoxidase activity in patients with aggressive and chronic periodontitis, compared to controls, with the highest activity levels found in the aggressive periodontitis group. They concluded that increased MPO activity was due to increased in®ltration and degranulation of polymorphonuclear leukocytes. In contrast, SaxeÂn et al. (239) demonstrated decreased MPO activity in localized aggressive periodontitis patients, and postulated that reduced peroxidase-mediated host defense mechanisms could be characteristic of that disease. Gu

Èven et al. (100) measured salivary peroxidase activity in whole saliva from 10 insulin-depen-dent diabetes mellitus patients and 10 healthy controls and reported increased peroxidase activity in the diabetic group. The authors concluded that salivary peroxidase activity might serve as a marker for gingival in¯ammation in such patients.

Jalil et al. (126) studied the relationships among thiocyanate, lysozyme, lactoferrin, plaque accumulation and gingivitis in resting and stimulated whole saliva of 94 children 12±14 years old. An inverse relationship was observed between salivary thiocyanate the amounts of plaque and gingival in¯ammation. Lactoferrin and lysozyme concentrations in stimulated saliva also were directly related to the amounts of plaque and gingivitis. The authors speculated that the origin of those enzymes might have been due to contributions from gingival crevicular ¯uid.

Are there other salivary components that may be potentially useful to assess periodontitis risk?

In addition to enzymes and other products derived from cells of the immune system, numerous other proteins also occur in saliva. One such phospholipid component, platelet-activating factor, originally was described as a mediator of platelet stimulation, but currently is recognized to be a potent in¯ammatory mediator as well. Platelet-activating factor affects vascular permeability, smooth muscle contraction, and in¯ammatory cell stimulation (221, 258, 298). In one study, Garito et al. (78) found that salivary platelet-activating factor occurred in greater concentrations in the presence of periodontal in¯ammation. More speci®cally, they found a higher percentage of pocket depths greater than 4 mm, more sites that bled upon probing, and greater numbers of salivary polymorphonuclear leukocytes as salivary platelet-activating factor levels increased. The authors hypothesized that salivary platelet-activating factor originated from polymorphonuclear leukocytes. Similarly, Rasch et al. (225) measured the amount of salivary platelet-activating factor in 15 patients with chronic periodontitis before and after periodontal therapy. Following scaling and root planing, they found a signi®cant reduction in salivary plate-let-activating factor and polymorphonuclear leukocytes that were concurrent with improvements in probing depths and bleeding on probing.

Kojima et al. (142) studied the presence of low molecular weight proteins found in gingival crevice ¯uid, but not in serum, in chronic periodontitis patients. Gingival crevice ¯uid, serum, and whole saliva were collected from healthy and periodontitis patients, as well as from edentulous and newborn subjects. In gingival crevice ¯uid and saliva from periodontitis and healthy patients, four dominant low-molecular-mass (8±14 kDa) proteins were observed that were not found in serum, and were less pronounced in saliva from edentulous and newborn subjects. These proteins were identi®ed by immunoblotting as members of the S100 family of calcium-binding proteins. The authors hypothesized that one protein, MRP14 (S100A9), may have a de®ned role in the gingival sulcus and could serve as a potential periodontal disease marker. However, the practical utility of that protein, or its expression following treatment, awaits further investigation.

Aurer et al. (12) studied the potential role of nitric oxide in the development of periodontitis by analyzing salivary NO2^{^±}concentrations in 25 subjects with generalized aggressive periodontitis, 25 with chronic periodontitis, and in 25 periodontally healthy subjects. Individuals with periodontitis had signi®cantly less salivary NO2^{^±}than healthy subjects. Subjects with aggressive periodontitis had lower NO2^{^±}concentrations than those with chronic periodontitis. In addition, parotid saliva contained less NO2^{^±}than sublingual or whole salivas. It was concluded that local NO2^{^±}production is decreased in patients with periodontitis; this effect was more pronounced in patients with aggressive periodontal diseases.

Gc, a vitamin D-binding protein, is a membrane component of monocytes, B-lymphocytes, and T-lymphocytes involved in signal transduction (218, 217). Krayer et al. (146) compared the amount of salivary Gc in patients with periodontitis and in periodontally healthy controls. Although they found similar quantities of Gc in parotid saliva from patients in both groups, Gc in whole saliva was sig-ni®cantly higher in periodontitis patients. In that study, Gingival Index scores also were positively correlated positively with Gc.

Cystatins are potent inhibitors of cysteine peptidases that depend on the highly reactive thiol group of a cysteine residue at the catalytic site for their activity (15, 16, 18±20, 45). They are present in a variety of ¯uids and tissues, including saliva. Hens-kens et al. (117) reported that the concentration of cystatin C (as well as the amount of amylase and total protein) was signi®cantly higher in whole and parotid salivas of patients with chronic periodontitis, compared to controls, but that the concentration of cystatin S was lower. Baron et al. (16) also found that the salivary concentration of patients with periodontitis was depleted. On the other hand, Aguirre et al. (3) noted considerable variability in cystatin levels and were not able to demonstrate differences in the amount and activity of salivary cystatins from periodontitis and control groups. Interestingly, Lie et al. (162) found that smoking decreased the amount of salivary cystatin in patients with gingivitis. Collectively, those studies suggest that cystatin may be an in¯ammatory marker, but the variability in cystatin concentration in periodontitis patients, as well as the apparent sensitivity to smoking status, likely decreases the potential utility of this proteinase inhibitor as a risk or disease status marker.

Both epidermal growth factor and vascular endothelial growth factor have been shown to be involved in wound healing. The concentrations of both those factors have been shown to be greater in patients with periodontitis, compared with controls (30, 122).

Other studies have assessed the relationship between certain salivary ions, such as Ca^{2^‡}(205), increased number of in¯ammatory cells (49, 224), and volatile sulfur compounds (145) to determine whether a relationship existed between periodontal disease and those salivary components. However, speci®c associations were not found and the usefulness of the latter salivary components as potential diagnostic markers remains doubtful. Similarly, the presence of free amino acids in saliva did not significantly correlate with disease status (268, 269).

Summary: how useful are salivary analyses for identifying patients at increased risk for the progression of periodontitis?

Saliva can be collected more easily, in larger amounts, and with less patient discomfort, relative to gingival crevicular ¯uid. Salivary diagnostic tests also may be applicable for screening large populations. However, the utility of saliva as a diagnostic ¯uid may be compromised due to its complex origin and derivation from a variety of sources, such as salivary glands, serum, gingival crevice ¯uid, oral microorganisms, sloughed oral epithelial cells, and foreign substances such as food and oral hygiene products. Moreover, the salivary secretion rate has a diluting effect that can prevent detection of a potentially discriminating diagnostic factor (283). Nevertheless, analysis of whole salivary components may be better suited to capture a greater variety of potential diagnostic markers or risk factors than gland-speci®c ¯uids. Indeed, the literature suggests that analysis of host-derived salivary enzymes, such as collagenase, elastase, and gelatinase, may hold sig-ni®cant promise for periodontal diagnosis (77, 151). Moreover, host in¯ammatory mediators that predominate in gingival crevice ¯uid, such as arachidonic acid metabolites and cytokines, as well as enzymes such as aspartate aminotransferase and b-glucuroni-dase, also are potentially amenable to salivary analysis. Conversely, studies of the potential diagnostic value of other salivary factors, such as ions, volatile components, and non-enzyme/non-immunoglobulin (glyco-) proteins, is less conclusive, and controlled, longitudinal studies will be necessary to more fully elucidate the importance of those molecules as potential diagnostic markers for periodontal disease.

One obstacle associated with many studies reporting associations between potential salivary markers and periodontal diseases is relatively small study populations. Considerable variations in the concentrations of salivary components also are routinely observed in individuals and populations, among healthy patients as well as those with periodontal diseases. Consequently, few well-accepted normal values have been proposed for any speci®c marker, deviations from which might be considered abnormal. Moreover, most authors have used cross-sec-tional designs to analyze salivary components of interest that have arisen following the development of periodontal diseases. Although such factors may represent markers having potential diagnostic value, they may not necessarily provide information regarding a particular individual's susceptibility to periodontitis prior to the onset of periodontal breakdown.

Salivary and serum immunoglobulins as potential diagnostic markers for periodontitis

How does an immune response protect the host?

The immune system protects the host against infection and prevents the initiation and dissemination of malignant tumors (63, 300). Healthy individuals defend themselves against antigenic stimuli via innate or acquired immunity. Innate immunity is present prior to exposure to infectious microorganisms or other foreign macromolecules. This system includes physical barriers, phagocytic cells, eosinophils, natural killer (NK) cells, and various blood-borne molecules. Speci®c immunity, however, is induced upon exposure to foreign substances, recognizes distinct macromolecules, and increases in magnitude and capability with each successive antigenic exposure. Speci®c immunity can be characterized as being either cell-mediated (via T lymphocytes) or humoral (mediated by antibodies). Antibodies belong in the third fastest migrating group of serum globulins, the gamma globulins. The term immunoglobulin (Ig) refers to the immu-nity-conferring portion of the gamma globulin fraction (9, 279). Immunoglobulins are heterogeneously distributed in various biological ¯uids and on the surface of some lymphocyte subsets. Human immunoglobulin molecules can be divided into ®ve distinct classes ± or isotypes ± based on differences in serologic and chemical properties. Those include IgG, IgA, IgM, IgD, and IgE. In addition, IgG and IgA can be further subdivided based on minor differences in the constant heavy chains into a number of subclasses (IgG1, IgG2, IgG3, and IgG4; IgA1 and IgA2).

Immune responses to pathogens ideally should accomplish their objectives with minimal injury to normal host tissues. However, prolonged, exuberant or inappropriate immune responses often are capable of inducing tissue injury and signi®cant host pathology (80, 138, 143, 201, 260). Following periodontal infection, the initial response typically is recruitment and migration of polymorphonuclear leukocytes to the site of periodontal infection. If polymorphonuclear leukocytes successfully eliminate the pathogens and their byproducts via phagocytosis and intercellular killing mechanisms, the clinical result may be limited to gingivitis. However, if those mechanisms are evaded and in¯ammation continues in host tissues, then a transition from gingivitis to periodontitis is facilitated. The production of antibodies by plasma cells at this stage can limit the infection, but if the action of polymorphonuclear leukocytes and antibodies are not suf®cient for bacterial clearance then antigens and antigenic byproducts can result in macrophages and T-lymphocyte activation.

Is analysis of serum antibody levels to putative periodontal pathogens useful in identifying patients who are at an increased risk for the progression of periodontitis?

Due to the relationship between antibodies and periodontal infection, the potential application of antibody titers for periodontal diagnostic purposes has received considerable research attention. The methods used to determine antibody titers include enzyme-linked immunosorbent assays (ELISA) and immunoblotting techniques. The primary disadvantage associated with whole-cell titers is the possibility of nonspeci®c cross-reactivity with other organisms that share common epitopes. Consequently, the pur-i®ed antigenic preparations or targeted epitopes unique for a particular pathogen generally are superior to whole-cell lysates, and improve the sensitivity and the speci®city of the antibody-based diagnostic assays (120, 219, 223, 253). Antibodies to bacteria and other antigens typically are measured in serum, saliva, and gingival crevicular ¯uid. (Diagnostic applications involving gingival crevicular ¯uid components are reviewed elsewhere in this volume (9)).

Individuals with periodontitis often have elevated antibody titers to periodontal pathogens, as compared to periodontally healthy controls (66, 139). However, some studies could not demonstrate an increase in antibody concentration over baseline prior to the onset of disease (24, 104, 273). In one study, (66), 22 patients were monitored for up to 5 years, and found that two-thirds of the patients had elevated serum antibody to the same bacteria during multiple phases of disease. In another longitudinal investigation (274), 51 subjects were monitored bimonthly for 5 years. That study failed to demonstrate a relationship between disease progression and changes in serum antibody titer to periodontal pathogens. While most serum antibody levels remained relatively constant throughout the study, signi®cant ¯uctuations in the amount of antibodies speci®c for periodontal pathogens were observed in some subjects. The authors hypothesized that such variation might re¯ect changes in the infectious process.

Elevated concentrations of IgG, IgM, and IgA class antibodies to Actinobacillus actinomycetemcomitans have been reported in aggressive and chronic periodontitis patients, compared to healthy controls (67). However, those results are in contrast to another study (98) that demonstrated decreased amounts of anti-A. actinomycetemcomitans antibody in patients with severe aggressive periodontitis. In the latter study, the authors proposed that decreased antibody levels might be useful as a clinical indicator for increased disease susceptibility.

Tolo et al. (280) suggested that serum antibodies to Porphyromonas gingivalis determinants detected prior to the onset of bone loss might be predictive for disease progression. In another study, O'Dell & Ebersole (198) demonstrated a signi®cant correlation between antibody avidity and clinical parameters of disease severity in patients with chronic periodontitis or localized aggressive periodontitis. Similarly, Chen et al. (40) reported an increase in antibody avidity to

P. gingivalis after nonsurgical treatment of generalized aggressive periodontitis patients. Positive treatment effects on IgG antibody concentration and avidity to A. actinomycetemcomitans have also been demonstrated (164), as well as a decrease in antibody levels to P. gingivalis following periodontal therapy (121). In the latter study, the authors suggested that the changes in serum IgG levels to P. gingivalis were related to bacterial suppression.

Antibodies to immunodominant P. gingivalis antigens, their isotype, and subclass were measured in four groups of patients (219) including subjects positive for P. gingivalis (with or without periodontitis) and negative for P. gingivalis (with or without periodontitis). There were no differences in IgG1, IgG3 and IgM concentrations, but the amount of IgG2 in subjects without periodontitis or P. gingivalis seroreactivity was signi®cantly higher than in patients positive for P. gingivalis. For patients in the latter group, the IgG4 and IgA concentrations also were signi®cantly lower. Seropositivity to IgG3 was almost exclusively seen in patients without periodontitis. Collectively, those ®ndings suggested that the presence of IgG3 might re¯ect a lack of susceptibility to periodontitis, while a relative de®ciency of IgG4 may be indicative of periodontal health and lack of infection. Although those results appeared promising, the practical utility was compromised due to the relatively small sample size of the population.

Celenligil & Ebersole (37) investigated the speci®-city of serum antibodies to de®ned oral microorganisms in a Turkish population having aggressive periodontitis. In that study, IgG antibodies to 14 oral microorganisms were determined by ELISA in 89 localized aggressive periodontitis patients, 86 patients with generalized aggressive periodontitis, and 94 periodontally healthy controls. All localized aggressive periodontitis patients exhibited elevated antibody levels to A. actinomycetemcomitans serotypes c and a, while antibody levels to A. actinomycetemcomitans serotype bwere signi®cantly higher in localized aggressive patients from the United States. In addition, 87% and 77% of the Turkish patients with localized aggressive periodontitis had elevated antibody responses to P. gingivalis and E. corrodens, respectively, which was not observed in the corresponding U.S. patients. The authors concluded that signi®cant and considerable variation existed in the systemic antibody responses to periodontal pathogens in patients from both countries.

More recently, serum IgG responses to six periodontal pathogens were evaluated in 23 Asian, 48 Afri-can-American, and 37 Hispanic subjects (50). The concentration of anti-P. gingivalis IgG was higher in African-Americans, and anti-B. forsythus IgG was diminished in Hispanics. For the entire study population, prior disease and subsequent attachment loss both were associated with elevated serum IgG antibody to P. gingivalis. Increasing pocket depth, attachment level, gingival erythema, and age also were positively correlated with serum IgG antibody to P. gingivalis, but not with antibodies to ®ve other subgingival species. The authors suggested that elevated serum IgG antibody to P. gingivalis may be re¯ective of progressive, destructive periodontal disease, and the presence of such antibodies may be considered a risk factor for disease progression in these populations. Nevertheless, the possible in¯u-ence of various environmental and socioeconomic factors on serum IgG antibody levels in the study groups also was noted.

Another study investigated the presence of serum antibodies to subgingival species in 16 healthy subjects, 21 patients with gingivitis, 11 early chronic periodontitis patients, and 5 sites with clinically progressive recession (273). They found no signi®cant differences in serum antibody levels among the healthy, gingivitis, and initial periodontitis subjects. In this case, it was concluded that serum antibody levels might not be sensitive risk markers for initial periodontitis.

The associations among multiple humoral and cell-mediated host immunologic risk factors also have been studied. (272). These authors studied 68 patients with aggressive periodontitis, 51 patients suspected of having aggressive periodontitis, 43 with chronic periodontitis, and 36 periodontally healthy subjects. Patients with aggressive periodontitis were associated with signi®cantly elevated antibodies to

A. actinomycetemcomitans, P. gingivalis, T. denticola, and F. nucleatum, compared with healthy controls. Nevertheless, there were considerable variations among members of all groups. Moreover, aggressive periodontitis patients showed wider intradiagnostic group variations in certain host defensive cell functions than the other groups. The authors concluded that the association of host immunologic risk factors in aggressive periodontitis patients might be more complex than previously thought, and emphasized the dif®culty of attributing the pathogenesis of aggressive periodontal disease on the basis of a single host risk factor.

Sakai et al. (233) investigated the relationships among serum IgG subclass, antibody concentration, and alveolar bone loss in 20 treated and maintained periodontal maintenance patients, in 30 untreated patients, and in 19 periodontally healthy subjects. They found that both treated and untreated patient groups had detectable levels of IgG1, IgG2, and IgG4. Members of the untreated group also exhibited sig-ni®cantly elevated IgG2 responses compared to other patients. The mean IgG4 level of maintenance patients was signi®cantly higher compared to the other groups, and a statistically signi®cant positive correlation between IgG2 levels and changes in bone levels also was observed. Since maintenance patients with high IgG2 and low IgG4 concentrations demonstrated greater bone loss than those with low IgG2 and high IgG4, the authors suggested that a prolonged IgG2 response following periodontal therapy might indicate recurrent or persistent periodontal destruction.

Beickler et al. (24) assessed speci®c serum IgA, IgG and IgG reactivities against the 110-kDa protein of A. actinomycetemcomitans in 34 adult patients with untreated periodontitis. Patients were randomly assigned to receive full-mouth scaling alone or scaling with an adjunctive antimicrobial therapy, with each treatment regimen followed by periodontal maintenance. The authors found that in patients harboring A. actinomycetemcomitans, IgG4 antibody reactivity to the 110-kDa protein was associated with signi®cantly higher tooth survival rates, as well as the number of teeth associated with attachment loss of 2 mm or greater. Although the same trend was noted for IgG3 and IgG2 antibody reactivities, the association was statistically insigni®cant. Moreover, no association was observed between clinical treatment outcome and IgA, IgG, or IgG1 antibody reactivities. The authors concluded that systemic IgG4 antibody reactivity against the 110-kDa protein of A. actinomycetemcomitans might have a protective effect against periodontal disease progression in patients harboring those microorganisms.

Antibody titers and avidity to P. gingivalis, A. actinomycetemcomitans, P. intermedia, T. denticola and

B. forsythus) was assessed in a population of 34 untreated patients (17 smokers and 17 non-smokers), and in 31 patients undergoing periodontal maintenance procedures (15 smokers and 16 non-smokers) (185). Decreased titers to A. actinomycetemcomitans,

P. intermedia and T. denticola were found in maintenance patient who were smokers, but not in the untreated patients. Avidity to P. gingivalis also was lower in maintenance patients who were smokers, but not in untreated patients. The authors concluded that these results were consistent with interruption of immune maturation in smokers following periodontal treatment.

More recent evidence suggests that in¯ammatory mediators or bacteremia secondary to chronic periodontal in¯ammation may affect systemic health by increasing the risk of developing cardiovascular disease, association with respiratory disorders, or delivering low birth weight infants (58, 128, 240, 241). Dasanayake et al. (57) determined IgG concentrations speci®c for de®ned periodontal pathogens in maternal serum and correlated those ®ndings to infant birth weight while controlling for other known birth weight risk factors. The authors reported higher amounts of P. gingivalis-speci®c maternal serum IgG in the low birth weight group compared to the normal birth-weight group. Women with higher levels of

P. gingivalis-speci®c IgG also had a higher odds ratio of having low birth-weight infants. This study opens a door for the development of periodontal pathogen-esis-based tools to monitor the risk groups for preterm delivery.

Is analysis of salivary antibody levels to putative periodontal pathogens useful in identifying patients who are at an increased risk for the progression of periodontitis?

Immunoglobulins were identi®ed in human saliva more than 40 years ago (69). Secretory IgA (sIgA) is the predominant salivary immunoglobulin, derived primarily from plasma cells associated with the salivary glands. sIgA was ®rst demonstrated by Tomasi & Ziegelbaum in 1963 (281), and further characterized through other studies describing molecular isotypes, structure, function, origin, and immunization routes effective for the induction of salivary immune responses (1, 93, 99). sIgA appears to contribute to the immune response in the oral cavity by reducing the adherence of bacterial cells to hard and soft tissue surfaces. Conversely, salivary IgG and IgM are mainly derived from serum via gingival crevicular ¯uid and are usually present in comparatively lower concentrations (134, 255).

Individuals with periodontal diseases typically demonstrate salivary antibodies, as well as systemic immune responses, to putative periodontal pathogens. A number of studies have investigated the relationship of both speci®c and total salivary immunoglobulin to disease status. For example, Sandholm et al. (236) reported increased amounts of IgA, IgG and IgM in whole saliva from 21 patients with localized aggressive periodontitis, compared to 27 periodontally healthy siblings and 17 controls. In contrast, Bokor et al. (29) reported that IgA levels in mixed unstimulated saliva were lower in subjects with more pronounced gingival in¯ammation. Reiff et al. (227) examined serum and salivary concentrations of IgG and IgA in periodontitis patients before and after initial therapy, and found a reduction in both IgG and IgA after treatment. Basu et al. (21) evaluated IgG and IgA in whole saliva from 12 periodontitis patients and found increased amounts of IgG and decreased IgA concentrations before therapy, compared to post-treatment levels.

Further studies have explored the relationship between salivary and systemic immunoglobulins in patients with other oral or systemic conditions. For example, increased concentrations of salivary IgG and IgA were found in periodontitis patients with non-insulin-and insulin-dependent diabetes, compared to non-diabetic periodontitis patients and periodontally healthy controls (7, 276). Harding et al. (112) reported decreased amounts of monomeric IgA and IgG, but elevated sIgA in saliva of patients with necrotizing ulcerative gingivitis. Myint et al. (187) measured IgA concentration in parotid saliva from HIV-positive and HIV-negative individuals with healthy gingiva, chronic gingivitis, chronic marginal periodontitis, and necrotizing ulcerative periodontitis and found that the IgA concentration was elevated in all patients harboring HIV. Kirstila et al. (136) analyzed total IgA, IgG, IgM, anti-Streptococcus mutans IgA, IgG, and IgM antibodies and lysozyme, lactoferrin, salivary peroxidase, myeloperoxidase, hypothiocyanite, thiocyanate, and agglutinins in whole saliva of patients who were on Ig-replacement therapy. The results showed no notable differences between the healthy group and the immunosuppressed group.

Barr-Agholme et al. (17) investigated the salivary concentration of immunoglobulins sIgA, IgM, and IgG and albumin in 20 patients with Down's syndrome, compared to 19 healthy controls. They found that results from analysis of sIgA, IgM, total IgG, and albumin did not differ signi®cantly between the two groups. However, the proportion of IgG1 was signi®-cantly higher in the Down's syndrome groups. The authors concluded that an alteration in the distribution of IgG subclasses occurred in the saliva of patients with Down's syndrome.

Zuabi et al. (317) evaluated the effect of smoking on periodontal status and the composition of whole saliva in subjects with established chronic periodontitis before and after periodontal therapy. They found that subjects with periodontitis had elevated concentrations of salivary electrolytes and proteins compared to controls. Smokers exhibited greater disease severity and decreased sodium, calcium, and magnesium concentrations. On the other hand, smokers responded favorably to treatment, resulting in the elimination of the differences in salivary composition. Mansheim et al. (174) evaluated salivary IgA concentration to P. gingivalis in patients with localized and generalized aggressive periodontitis and found no difference when compared to controls. Nevertheless, Sandholm et al. (237) have suggested that salivary antibody may be useful for differentiating among periodontal diseases. They reported an increased concentration of whole salivary IgG in 34% of patients with moderate chronic periodontitis, and in 57% of patients with severe chronic periodontitis. Salivary IgG antibody to A. actinomycetemcomitans was signi®cantly increased in 55% of individuals with untreated localized aggressive periodontitis, and in 28% of treated subjects with that disease. Furthermore, 28% of the patients with chronic periodontitis had signi®cantly elevated salivary IgG antibody levels to A. actinomycetemcomitans subtype Y4. In that study, however, IgA concentrations did not show appreciable difference among study groups. Schenk et al. (242) measured salivary IgA speci®c for a number of periodontal pathogens in a model of experimental gingivitis and demonstrated that individuals with low bleeding scores had elevated anti-S. mutans, A. actinomycetemcomitans, and Eubacteriun saburreum antibodies. Nieminen et al. (191) reported that the concentration of salivary IgA and IgG antibody to

A. actinomycetemcomitans was correlated signi®-cantly with serum antibody levels in patients with severe periodontitis. They concluded that saliva samples could be used to assess the serum antibody response to infection with Actinobacillus in severe chronic periodontitis patients.

Hagewald et al. (106) evaluated total and P. gingi-valis-reactive salivary IgA in generalized aggressive periodontitis patients, compared to age-and gender-matched periodontally healthy controls. They found a signi®cantly lower concentration and rate of secretion of total salivary IgA in the experimental group. Although no differences were detected in the concentration or secretion of P. gingivalis-reactive IgA between groups, the speci®c fraction of P. gingivalisreactive IgA within the total IgA fraction was signi®-cantly higher in patients with periodontitis. The authors concluded that P. gingivalis selectively activates IgA lymphocyte clones and induces a switch in the fraction of speci®c IgA in human saliva.

Collectively, those studies suggest that detectable amounts of antibodies speci®c for periodontal pathogens are indicative of an infectious process, the presence of periodontal disease, or both. Unfortunately, data describing the temporal relationship between antibody response and infection are sparse. Consequently, longitudinal studies in high-risk subjects are required to determine whether the analysis of salivary antibodies may have clinical utility as a predictor of the risk of infection or disease. Moreover, other host factors or conditions may in¯uence immunoglobulin production. For example, Granade et al. (92) found that signi®cant differences in salivary IgG concentration were associated with HIV status, gender, edentulousness, gingival bleeding, and time elapsed since a subject's most recent meal. The authors suggested that these factors could affect diagnostic methods that are based on the immunoglobulin concentration in oral ¯uids.

Summary: what potential value do serum and salivary antibody levels to putative periodontal pathogens have in identifying patients who are at an increased risk for the progression of periodontitis?

Detection and measurement of antibodies speci®c for putative periodontopathic organisms would appear to have considerable potential for periodontal diagnosis. Although sIgA is the predominant immunoglobulin in saliva, it seems to be related less to periodontal in¯ammation than IgG, which is derived primarily from gingival crevice ¯uid. In any event, the data indicate that identi®cation of speci®c immunoglobulins directed to periodontal bacterial components may be more speci®c and provide more useful information than quantitation of nonspeci®c immunoglobulin isotypes.

Longitudinal studies investigating the concentration, speci®city, and avidity of antibodies relative to periodontal status also support the concept that characterization of immunoglobulins may hold some promise as a marker of disease activity at the level of the subject, as well as for individual diseased sites. While the literature generally suggests that serum antibody levels decrease following periodontal therapy, variations in treatment modalities and the differences in sampling times may introduce confounding factors that may adversely affect the reliability of such tests.

The utility of immunoglobulins as periodontal diagnostics may be further complicated due to the presence of commensal microorganisms that also may initiate an immune response. Moreover, antibodies detected in the oral cavity may be related to nonoral bacteria, and serum antibodies interacting with oral bacteria might exhibit some cross-reactivity with nonoral bacteria (201). Whether the use of whole bacterial cells or particular antigens isolated from those cells is more appropriate to assess the quality and quantity of humoral immune responses also awaits further study. Speci®c determinants unique for a particular bacterium may decrease cross-reactivity, but limiting the focus to speci®c antigens may cause an underestimation of the host response. Nevertheless, it has been suggested that puri®ed antigen preparations may lead to improved sensitivity and speci®city of antibody-based diagnostics (201).

During the past 20 years, extensive information has become available describing the speci®city and concentrations of serum antibodies to putative periodontal pathogens. However, many of those studies have been derived from cross-sectional data and are equivocal regarding the ability of serum antibody to identify disease activity or susceptibility (98, 233, 253, 272).

At present, the clinical usefulness of various types of immunologic markers cannot be clearly de®ned due to the lack of suf®ciently robust data. Well-designed longitudinal investigations and controlled clinical treatment studies for relatively long time periods are required to more fully explore the applicability of salivary and immunologically based tests for periodontitis, and their use generally has been limited to research environments. Although such tests are not yet generally available to clinicians, future work will require adaptation of those research tools to clinical situations, ideally providing simple, accurate, well-de®ned tests to clinicians that will help to improve patient management and the development of individualized treatment strategies.

Neutrophil defects as risk factors for periodontal disease

What is the role of polymorphonuclear leukocytes in host defense?

Polymorphonuclear leukocytes and mononuclear phagocytes (monocytes and macrophages) are non-lymphoid cells of myeloid origin. Those cells constitute the primary mammalian phagocytic cell system for host defense against exogenous agents. Some of their functions include ingestion of foreign organisms, microbial processing via secretion of lytic enzymes, release of in¯ammatory mediators important in the initiation and regulation of immune and in¯ammatory responses, and facilitation of tissue repair and remodeling (35, 91, 147, 169, 183, 286, 296, 297, 302).

Neutrophils are the primary phagocytic cells found in the peripheral circulation, comprising 50±70% of the total white blood cell population (14). As a result, neutrophils are considered to be the ®rst line of defense against extracellular bacteria (115, 183). Following differentiation from bone marrow progenitor cells, neutrophils circulate in blood for relatively short time periods (t^1/²ˆ 6±7 h) (127). About one-half to two-thirds of the peripheral blood neutrophils are located in marginated pools loosely associated with the vascular endothelium (127, 183). Upon initiation of an in¯ammatory event secondary to cell injury or infection, neutrophils in the marginated pool exit the circulation by adhering to the endothelium and projecting pseudopods between endothelial cells via diapedesis or transendothelial migration (14, 54, 64, 183). Major superfamilies of adhesion molecules participating in polymorphonuclear leu-kocyte±endothelial interactions during diapedesis include integrins, members of immunoglobulin supergene family (ICAMs) and selectins (64, 136, 183).

After diapedesis, polymorphonuclear leukocytes migrate toward the target particle that is to be ingested. The chemical substances capable of attracting neutrophils to the site of stimuli are referred to as chemoattractants or chemotaxins, and the directed movement of neutrophils towards these agents is referred as chemotaxis. Some of the chemoattractants for polymorphonuclear leukocytes include N-formylmethionyl peptides (fMLP), C5a, leukotriene B4, platelet activating factors, interleu-kin-8 (IL-8) and coagulation cascade products (e.g. ®brinopeptide B) (175, 204, 216). Chemotaxis is initiated through the interaction between chemoattractants and speci®c receptors on the cell surface. Following receptor activation, neutrophils alter their shape, becoming morphologically oriented toward the chemoattractant gradient and translocate in a curvilinear fashion without discrete pauses, rather than moving along surfaces (175, 204, 205, 216). The number of receptors also increases on neutrophil cell surfaces, thereby amplifying that process. The mechanisms through which neutrophils migrate are thought to involve a change in con®guration of actin, myosin, and microtubules within the cell cytoplasm; those substances also are important in neutrophil degranulation (25, 205, 206, 264, 267).

A key property of polymorphonuclear leukocytes includes their ability to recognize and ingest particulate matter through speci®c interactions between the polymorphonuclear leukocyte membrane and one or more components on the target particle surface (169). Ef®cient phagocytosis often requires that the particle be coated with one or more host serum proteins through opsonization, which requires interaction with IgG antibodies and cleavage fragments of complement component C3 (C3band iC3b

) (25, 169). Following particle recognition and uptake, a number of events associated with the bactericidal properties of neutrophils are initiated, including degranulation and respiratory burst (183). Two major classes of cytoplasmic granules present in neutrophils include the primary (azurophil) and secondary (speci®c) granules, which contain enzymatic and non-enzymatic proteins that are bactericidal or bacteriostatic for many organisms. Degranulation into phagocytic vacuoles leads to intracellular killing with minimal host injury, but extracellular degranulation may result in damage to host tissues.

In concert with degranulation, neutrophils increase oxygen consumption and generate toxic oxygen metabolites (respiratory burst) that facilitate bacterial killing and protease inactivation (183). NAPH oxidase, a plasma membrane enzyme, generates superoxide, which is converted to hydrogen peroxide by superoxide dismutase (26). Additional toxic oxygen metabolites such as hypochlorous acid and aldehydes also can be generated through interaction with myeloperoxidase. (5, 183, 262) Finally, neutrophils undergo lysis or programmed cell death (apoptosis) after the completion of their primary protective function. Abnormalities in any of these steps might result in a delayed or less ef®cient host response to bacterial challenge (6, 54, 160, 252, 288).

How may polymorphonuclear leukocytes affect the course of periodontal disease?

polymorphonuclear leukocytes are the predominant cell type found in early stages of periodontal in¯am-mation, typically making up about 90% of the cells in the gingival sulcus (54, 210). Studies have demonstrated that neutrophils have a protective role by serving as a barrier between the periodontal pathogens and the periodontal tissues (157, 183, 232). Neutrophils function by altering bacterial colonization and growth, or through modulating bactericidal processes. Some of the mechanisms through which neutrophils respond to periodontal bacteria have been extensively reviewed (79, 115, 141, 183).

The protective function of neutrophils in periodontal diseases has been supported through clinical investigations of patients with primary and secondary immunode®ciencies (83, 115, 135, 290). Neutrophil disorders associated with severe forms of periodontal destruction include cyclic neutropenia (42), Chediak-Higashi syndrome, (108), drug-induced agranulocytosis (22), abnormalities in the neutrophil glycoprotein receptor CR3 (277), and surface adherence protein LFA-1 (5). In addition, individuals whose quantity and function of neutrophils are reduced due to systemic diseases such as diabetes mellitus (41, 95, 202), Down's syndrome (238, 248), and Papillon-LefeÁvre syndrome (291) also exhibit increased periodontal destruction. Some systemic conditions that are characterized in part by neutrophil defects, and are associated with periodontal diseases, are noted in Table 5.

Additional support for the bene®cial role of neutrophils in periodontal diseases has been derived from data suggesting that many periodontal pathogens possess virulence factors that either kill neutrophils or result in diminished neutrophil function (222). The leukotoxin produced by A. actinomycetemcomitans. (77, 111, 243) and proteases produced by

P. gingivalis (52, 130) are examples of factors that are believed to destroy neutrophils or affect their function. Moreover, susceptibility of neutrophils to these virulence factors may vary between individuals. polymorphonuclear leukocytes from periodontally healthy individuals also may be more susceptible to A. actinomycetemcomitans leukocidin than neutrophils from patients with localized aggressive periodontitis (132, 294). Thus, identi®cation of neutrophil susceptibility factors potentially may provide valuable information regarding future disease risk, and may assist in the prevention or treatment of periodontitis.

Studies of individuals with aggressive periodontal diseases have also supported the relationship of functionally intact neutrophils to periodontal health. For instance, patients with localized aggressive periodontitis frequently possess neutrophil-response dysfunctions, including chemotactic defects (55, 287, 293, 295), phagocytotic defects (292), altered superoxide generation (292), and altered leukotriene 4 generation (200). Consequently, it is quite likely that some individuals with defective neutrophils may be more susceptible to various forms of periodontal disease, particularly those that may compromise the host responses to speci®c pathogens. Thus, the detection of neutrophil-based susceptibility markers may provide a risk marker for disease, particularly in cases of aggressive periodontal diseases.

What are some of the intrinsic neutrophil defects that might serve as markers for increased susceptibility to periodontitis?

In general, disorders of neutrophil function have been characterized by recurrent cutaneous, periodontal, respiratory or soft tissue infections (139). Although the precise neutrophil defect in each disease may be different, severe periodontitis typically is associated with those conditions. Altered expression of neutrophil adhesion molecules could in¯uence PMN migration and lead to inappropriate release of reactive oxygen species and tissue injury. Congenital leukocyte adherence de®ciency (LAD) (31, 36) is an autosomal recessive disorder of leukocyte function. Such patients lack surface adhesive glycoprotein receptors of the Mac-1, LFA-1 and p150,95 (CD11/18) group. Recurrent infections, delayed wound healing, impaired suppuration, as well as aggressive periodontal disease (generalized prepubertal periodontitis) are commonly associated with LAD (139). Resting polymorphonuclear leukocytes from patients with periodontal diseases have been shown to be associated with reduced levels of the adhesion molecules l-selectin, Lewis x, and sialyl-Lewis x (76). Some patients with generalized aggressive periodontitis also exhibit decreased shedding of l-selectin after fMLP stimulation, increased basal production of H2O2 by polymorphonuclear leukocytes, decreased polymorphonuclear leukocyte oxidative burst, increased plasma IL-8 levels, and decreased plasma l-selectin levels. However, those abnormalities were not found in patients with chronic periodontitis or localized aggressive periodontitis (167).

Disorders of cell motility and chemotaxis also have been described in certain diseases, including Chediak-Higashi syndrome (108), diabetes mellitus (60), Down's syndrome (310), Papillon-LefeÁvre syndrome (86, 165), actin dysfunction syndrome (32), Crohn's disease (131), and aggressive periodontal diseases (140, 207, 282, 293, 295, 311). Upon stimulation with a chemotactic factor, morphologic changes and directed neutrophil movement result from reorganization of plasma membrane-associated structures (25, 51, 204±206), with actin serving as one of the major components of the micro®lamentous cytoskeleton. In one investigation, cytoskeletal actin reorganization was investigated in peripheral blood neutrophils from 14 localized aggressive periodontitis patients and 12 periodontally healthy controls (38). No differences were detected between the two groups, and the data did not show a correlation between the kinetics of actin polymerization-depolymerization and the abnormal chemotactic response observed in polymorphonuclear leukocytes from patients with localized aggressive periodontitis. The authors concluded that the associated chemotactic defect may be mediated by signaling events that are independent of cytoskeletal integrity.

Nevertheless, substantial evidence has associated impaired PMN chemotaxis with aggressive periodontal disease (54, 115, 292). Moreover, approximately 75% of African-Americans with localized aggressive periodontitis demonstrate chemotactic defects that make them susceptible to periodontal destruction (54). Several receptors for chemotactic agents, such as fMLP (58, 59, 295), C5a (287, 295), and IL-8 (287), are present in diminished quantities in patients with localized aggressive periodontitis. Expression of GP110, a glycoprotein found on polymorphonuclear leukocyte surface, has also been shown to be present in diminished quantities on neutrophils from patients with localized aggressive periodontitis (288). However, the precise role of GP110 remains unclear.

The neutrophil chemotactic defects in aggressive periodontitis patients are present in cells isolated from peripheral blood as well as from lesion sites, and persist even after elimination of the pathogens and the resolution of the in¯ammatory lesion (287). The mode of inheritance of the defects has been reported to be autosomal dominant (114). A recent study (101) reported a higher frequency of a particular genotype in localized aggressive periodontitis, resulting in a conformational change in the fMLP receptor that may explain impaired chemotaxis observed in that patient population. It is likely that future research will focus on identi®cation of such genetic markers that would predict susceptibility for certain periodontal diseases.

The investigation of chemotactic defects in neutrophils from patients with aggressive periodontitis led to further investigations to identify possible signal transduction anomalies associated with neutrophil chemotaxis (2, 55). For example, guanine nucleotide binding proteins (G proteins), a family of homologous regulatory proteins that transfer information from cell receptor to cell effector systems, are involved in receptor transduction such as photoreception and the modulation of the adenylate cyclase system (183, 204, 205). Neutrophil signal transduction also is mediated via G-proteins (183, 204, 205), with activation thought to result primarily from the combined processes of calcium release from intracellular sites and the activation of protein kinase C (204). Both events occur as a result of generation of two intracellular second messengers, inositol triphosphate and diacylglycerol. The formation of those messengers occurs during the breakdown of 1-phosphatidyl-d-myoinositol 4,5-biphosphate by phospholipase C. The mechanism through which G proteins may regulate phospholipase C activation in neutrophils is still not thoroughly established. However, studies have investigated possible signal transduction defects in neutrophils from patients with localized aggressive periodontitis neutrophils, and have demonstrated changes in the G-protein pathway that may be responsible for reduced chemotaxis (54). Those changes include reduction in calcium immobilization (2, 55), reduction of protein kinase C activity (148), increased levels of diacylglycerol and reduced diacylglycerol kinase activity (284).

Stimulated neutrophils also actively generate oxygenated derivatives of arachidonic acid such as 5-HETE, 12-HETE, and 5,12 di-HETE, and LTB4, all of which can modulate cell function (183). The former compound and their hydroperoxy precursors (HPETEs), as well as LTB4, have been shown to alter calcium ¯uxes and to induce a variety of neutrophil responses such as degranulation and chemotaxis (88, 188). A depressed LTB4 chemotactic response (200) and impaired lipooxygenase activity as determined by reduced levels of 15-HETE (195) have been associated with aggressive periodontitis. However, controlled clinical trials with larger patient numbers are needed to more completely de®ne any de®ciency as a disease marker.

Disorders associated with neutrophil phagocytosis defects include diabetes mellitus and actin dysfunction syndrome (140). Defects in phagocytosis and bacterial killing are also associated with neutrophils from localized aggressive periodontitis patients (41, 132, 307), and Loesche et al. (166) reported reduced oxidative function in gingival crevicular neutrophils from such patients. In addition, downregulation of immunoglobulin G type II (Fcg) receptor expression and impaired phagocytosis also has been associated with gingival crevicular ¯uid polymorphonuclear leukocytes from chronic periodontitis patients, compared to periodontally healthy controls (48, 184, 265).

What are some of the acquired neutrophil defects that might serve as markers for increased susceptibility to periodontitis?

Periodontal infections are believed to occur as a result of an imbalance between pathogenic bacteria and host immune responses (80, 81, 183, 199). Evidence strongly suggests that variations among individuals exist in the severity and rate of progression of periodontitis that cannot be solely explained by the presence and amount of bacteria (81). Neutrophils are the ®rst cells that encounter bacteria in the gingival sulcus; consequently, neutrophil defects should be detrimental for the host and lead to increased susceptibility to periodontal destruction (115). In addition to inherited neutrophil defects that might represent possible risk markers for periodontal disease, neutrophil function can also be altered by environmental factors such as stress (85), as well as bacterial virulence factors such as lipopolysaccharide (169, 247), leukotoxin (72, 111, 305), and proteinases (118, 266), all of which are associated with increased susceptibility to periodontal destruction.

Wilson & Hamilton (306) have reported a predominantly IgG2-mediated response to A. actinomycetemcomitans lipopolysaccharide in patients with localized aggressive periodontitis. Since IgG2 has weak opsonization complement activation activities, the authors proposed that the dominance of IgG2 may represent a possible risk marker for periodontal destruction by limiting the recognition and elimination of bacteria by neutrophils.

Smoking is generally accepted to be a major risk factor in the development and progression of periodontal disease (81). Accordingly, the in¯uence of smoke or smoke products on the normal functions of the host response, especially on neutrophil functions, may explain the deleterious effect on the periodontium of smoking. In particular, MariggioÁ et al. (176) demonstrated increased apoptosis of polymorphonuclear leukocytes obtained from gingival crevice ¯uid of smokers with chronic periodontitis compared to those isolated from periodontally healthy controls, and proposed that smoking may be associated with an inef®cient host response. Another study (230) found increased H2O2 production during the neutrophil oxidative burst in cells exposed to smoke in vitro. Numabe et al. (197) investigated the effect of smoking, and passive smoking, on the phagocytic function of salivary polymorphonuclear leukocytes and found increased phagocytic activity of salivary polymorphonuclear leukocytes. In contrast, no signi®cant differences were found in l-selectin and CD18 expression on neutrophils isolated from smokers and non-smokers (231).

Although acquired neutrophil de®ciencies represent an attractive mechanism through which exogenous factors may alter the host's susceptibility to periodontitis, many reports have been based on in vitro observations or relatively small clinical investigations. As a result, more de®nitive studies are required to establish the mechanisms by which those factors in¯uence periodontal diseases, and whether measurement of altered neutrophil function may be useful to assess disease risk or treatment ef®cacy.

Summary: how useful are neutrophil function tests for identifying patients at increased risk for the progression of periodontitis?

The protective role of neutrophils in periodontal disease pathogenesis is well accepted. Considerable evidence has associated a variety of inherited or acquired neutrophil function defects with the development of systemic disease, systemic disease accompanied by periodontal diseases, and with periodontal disease alone. Recent advances in molecular biology have facilitated the elucidation of molecular mechanisms associated with those defects. Identi®cation of genetic components also has fostered additional research on potential genetic markers that may correlate with periodontal disease occurrence. In contrast to studies of acquired neutrophil defects, there are compelling data linking some intrinsic neutrophil defects with the risk of periodontitis. Although acquired neutrophil de®ciencies represent a very attractive hypothesis regarding a host's susceptibility to periodontitis, they generally are based on in vitro observations or relatively small clinical studies. Consequently, the usefulness of acquired neutrophil defects as disease susceptibility markers remains tenuous. However, it is likely that future research will be directed to identifying the molecular mechanisms of those defects and locating potential genetic markers that may be associated with disease susceptibility.

Summary and conclusions: what host factors hold the most promise for assessing the risk of periodontitis?

Periodontal diseases are multifactorial, in¯uenced by genetics as well as by the environment (81). The recognition that individuals are not equally susceptible to periodontitis has fostered investigations to assess potential diagnostic tools for management of periodontitis. The diagnosis of periodontal disease currently relies on subjective criteria founded on clinical and radiographic evaluation, but measurements of pocket depth, attachment level, or bone loss relate only to previous disease and current health status, and may not be useful to identify individuals who may be at risk for future disease.

Whole saliva serves as a reservoir for host-derived products (e.g. salivary gland components, gingival crevice ¯uid, host enzymes) as well as exogenous components (e.g. oral microorganisms and microbial products). Consequently, analysis of whole saliva may hold more promise than gland-speci®c ¯uids. However, considerable variations exist in the amount of salivary components among individuals and populations, and few well-accepted normal values have been established for any speci®c marker. Although the identi®cation of some host-derived salivary enzymes such as collagenase, elastase, and gelatinase may be signi®cantly correlated with existing periodontitis, their ability to predict future disease remains unclear. Those factors may yet represent markers with potential diagnostic value, but may not necessarily provide information regarding a particular individual's susceptibility to periodontitis prior to the onset of periodontal breakdown. In any event, additional controlled, longitudinal studies will be necessary to more fully elucidate the utility of salivary components as potential diagnostic or predictive markers.

Tests based on characteristics of the host's immune response currently are used for diagnosis of some infectious diseases (8, 23, 46, 278). It therefore has been postulated that similar diagnostic methods might be useful not only as predictors of periodontal disease activity, but also for diagnosis and treatment (211, 223). Indeed, detection and measurement of antibodies speci®c for putative periodontopathic organisms may prove to be quite useful, particularly for early identi®cation of aggressive periodontal diseases (e.g. through identi®cation of

A. actinomycetemcomitans or related antigens).

Finally, there are compelling data linking intrinsic neutrophil defects with the risk of periodontitis, as well as systemic disease. Data also exist that link intrinsic neutrophil defects with an increased risk of periodontitis. Further development and the commercialization of tests based on neutrophil defects may be of signi®cant value to patients and clinicians. Such tests also might be useful to detect underlying systemic conditions in situations where the initial presentation is manifested as periodontal disease. In any case, future studies likely will be directed to identifying the molecular mechanisms and genetic markers associated with disease onset or susceptibility. Well-designed longitudinal investigations also will be required to more fully explore the applicability of salivary and immunologically based tests for periodontitis if they are to become clinically useful for most patients and clinicians.

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