Analysis of host responses and

risk for disease progression

Sinem E. Sahingur & Robert E. Cohen
Periodontal diseases affect 530% of the adult popu-
lation, constituting one of the most common bacter-
ial infections in humans (203). The literature is
replete with studies compiled during the past 20
years that have investigated the etiology, pathogen-
esis, and treatment of periodontal diseases (56, 58,
80, 102, 199, 312). It has become evident that specic
bacteria are essential for the initiation and progres-
sion of periodontal diseases, and that tissue destruc-
tion as a consequence of periodontitis may result
from an imbalance in host protective and destructive
mechanisms initiated by an infectious process.
Although a signicant 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 identied that might be able to
detect individuals or populations with particular sus-
ceptibility or resistance to periodontitis.
In the broadest sense, risk is the possibility of suf-
fering a loss. In medicine, risk has been dened as
the probability of developing a particular disease or
experiencing a change in health status, given a
dened 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 multifac-
torial 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 dis-
continuous pattern of exacerbation and remission
characterized by disease-active and disease-inactive
sites (80, 199). Methods for the detection of period-
ontitis and the identication 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 period-
ontitis, they do not measure disease activity. Conse-
quently, 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 appro-
priate 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 identied 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,
57
Periodontology 2000, Vol. 34, 2004, 5783 Copyright
#
Blackwell Munksgaard 2004
Printed in Denmark. All rights reserved
PERIODONTOLOGY 2000
and sublingual as well as by numerous minor
glands (170). The basic secretory units of those
glands are acini, which comprise cell clusters secret-
ing 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 bicarbo-
nate 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 character-
istics of the major salivary glands are summarized in
Table 1.
Saliva can be assessed as whole (mixed), or as
gland-specic 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 sal-
iva 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-specic saliva can be collected
with or without gustatory stimulation. Unstimulated
whole saliva is composed of secretions from the par-
otid, submandibular, sublingual, and minor mucous
glands, as well as from gingival crevicular uid, des-
quamated epithelial cells, microorganisms, leuko-
cytes, food residue, and blood. Stimulated saliva
has been obtained in response to masticatory or
gustatory stimulation using a variety of methods,
including parafn 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 dur-
ing 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 (pros-
taglandins), immunodeciency (secretory IgA), and
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Sahingur & Cohen
58
tobacco usage (cotinine) have been assessed (171,
172, 309). In dentistry, salivary-based caries suscept-
ibility tests also have become relatively well estab-
lished (28, 172).
Proposed salivary diagnostic markers for peri-
odontal diseases have included serum and salivary
molecules such as immunoglobulins, enzymes,
consti-tuents of gingival crevice uid, bacterial com-
ponents or products, volatile compounds, and phe-
notypic 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 sali-
vary gland cells may not provide data of appropriate
specicity 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 leuko-
cytes, 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) investi-
gated 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 aminopepti-
dases was detected in saliva from patients with
chronic periodontitis, compared to healthy controls.
There were no signicant differences in enzyme activ-
ity between the chronic periodontitis and localized
aggressive periodontitis groups, but patients with
aggressive periodontitis exhibited greater amounts
of salivary butyrate esterase and cysteine aminopep-
tidase. Based on those data, the authors noted that a
signicant proportion of salivary enzymes were most
likely derived from the oral microora.
Gibbons & Etherden (87) compared the levels of
bronectin-degrading enzymes in whole saliva from
Table 2. Selected components of human mixed saliva
Electrolytes Organic
compounds
Proteins and
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. Selectedfunctions 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).
59
Host responses and risk for disease progression
periodontally healthy subjects. Unstimulated saliva
was collected immediately after awakening (i.e. lack
of recent oral hygiene measures), as well as immedi-
ately after toothbrushing, with substantially greater
enzyme activity detected in saliva obtained after
awakening. They concluded that oral hygiene mea-
sures might contribute to variations observed in the
levels of salivary proteases. Uitto et al. (285) investi-
gated collagenase activity in whole and gland-
specic salivas from subjects with or without period-
ontitis. 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 origi-
nated 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 inamed human gingiva, gingival crevicu-
lar uid, and saliva all were derived from polymor-
phonuclear 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 periodon-
tal diseases.
Greater amounts of immunoglobulin-degrading
enzymes have been reported in whole saliva from
patients with localized aggressive periodontitis, com-
paredtohealthy controls (94). These authors proposed
that production of enzymes by periodontal patho-
gens impart an ecologic advantage, enhance micro-
biological colonization, and may have a role in the
pathogenesis of aggressive periodontal diseases.
Arginase is an arginine-depleting enzyme belong-
ing 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 period-
ontitis patients and 15 systemically and periodon-
tally healthy controls. Patients with periodontitis
had signicantly greater amounts of arginase, as well
as greater pocket depths, loss of clinical attachment,
plaque index and gingival index. However, there was
no statistically signicant 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 4. Saliva collection methods
Saliva Method
Mixed Draining/spitting method: The subject is asked to accumulate saliva in the floor of the mouth
and then spit into a pre-weighed or graduated test tube (11, 190).
Mixed Suction method: Saliva is continuously aspirated from the floor of the mouth into a suitable
collection vessel (11, 190).
Mixed Swab (absorbent) method: A pre-weighed swab, cotton roll, or gauze sponge is placed in the
mouth at the orifices of the major glands and is removed for reweighing at the end of the
collection period (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/
sublingual
Since submandibular and sublingual secretions enter the oral cavity via a common duct system, it
is difficult to collect individual salivas separately. For combined submandibular-sublingual saliva,
custom-made collectors can befabricated (214). Those devices contain a central chamber for the
collection of submandibular saliva and one or two side chambers for the collection of sublingual
saliva (214).
Submandibular/
sublingual
Alternatively, Fox et al. (74) have described a simpler method for the collection of submandibular-
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.
Minor glands Minor gland secretions can be collected by micropipette, absorbent filter paper or strips from the
inner surface of lips, palate, or buccal mucosa and quantitated by weight differences or using a
Periotron
1
device (249).
60
Sahingur & Cohen
Zambon et al. (313) found reduced amounts of leu-
cine, valine, cysteine aminopeptidases, caprylate
esterase lipase, trypsin, b-galactosidase, b-glucuro-
nidase, and b-glucosidase in whole saliva from
chronic periodontitis patients after periodontal ther-
apy. 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 micro-
organisms and the effectiveness of periodontal ther-
apy could be monitored by changes in the amount of
specic 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 signicantly 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 statisti-
cally signicant.
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 acti-
vity 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 signicantly higher
in the study group than in the control group at base-
line. After the initial treatment phase at 8 months, all
three proteases were signicantly reduced, but the
concentration of glycosidases remained elevated.
Uponcompletionof periodontal therapy at 20months,
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 reect the severity of periodontal disease,
and that salivary elastase may have diagnostic poten-
tial for assessment of periodontal inammation and
the response to periodontal therapy.
Ingman et al. (124) also examined proteolytic
activity in patients with chronic periodontitis, loca-
lized 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 signicantly 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 inves-
tigators (125) determined that gelatinase from saliva
and GCF was similar to gelatinase isolated from poly-
morphonuclear leukocytes and broblasts. They sug-
gested 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 Makela et al. (168), who found
that the concentration of matrix metalloproteinase-9
(MMP-9 or 92 kDa gelatinase) was signicantly 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) concentra-
tion 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
Table 5. Systemic conditions associated with neutro-
phil disorders and periodontal diseases
Disease Reference
Diabetes mellitus (13, 41, 60)
Chediak-Higashi Syndrome (108, 181)
Chronic Granulomatous Disease (39, 44, 141)
Cyclic neutropenia (42, 244)
Leukocyte Adhesion Deficiency (36, 180)
Down's Syndrome (4, 181, 238)
Papillon-Lefevre Syndrome (86, 165)
Agranulocytosis (22, 119)
Crohn's disease (71, 131)
Actin Dysfunction Syndrome (32)
Congenital neutropenia (62, 81, 213)
Glycogen Storage Disease (110)
Systemic Lupus Erythematosis (34, 196)
Lazy Leukocyte Syndrome (81, 182)
Myeloperoxidase Deficiency (53, 159)
Ulcerative Colitis (27, 271)
Hyperimmunoglobulin E
(Job's Syndrome)
(39, 228)
Familial Benign Chronic Neutropenia (61)
61
Host responses and risk for disease progression
consisted of procollagenase, while mainly active col-
lagenase was present in whole saliva from patients
with periodontal diseases. Signicant reciprocal
changes of TIMP-1 and collagenase levels, that is,
increase in TIMP-1 concentration and decrease in
collagenase activity, were observed after initial peri-
odontal therapy.
How may systemic conditions affect
salivary enzymes?
Salivary enzyme proles 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 collage-
nase 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 con-
trol 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 con-
trol 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 inammation 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, sys-
temically 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. Indepen-
dent 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
reect and directly participate in periodontitis asso-
ciated 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, demon-
strating a signicantly decreased amount of salivary
proteolytic activity and MMP-8 levels in current smo-
kers than in former smokers. Consequently, the
authors suggested that care should be taken in inter-
preting results suggesting that salivary proteinases
might be potential diagnostic markers for periodon-
tal 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, parafn-stimulated sal-
iva or oral rinse samples were assayed for elastase
activity, and neutrophils were quantitated by stain-
ing 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 signicant changes. Analysis of
elastase in stimulated whole saliva also showed that
smokers had signicantly 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 period-
ontal status in smokers.
Additional salivary host-derived non-immune fac-
tors include lactoferrin, lysozyme, sialoperoxidase,
histatin, and amylase. Although many pathogens
have capsules or other cell wall protective mechan-
isms that resist lysozyme, it induces cell lysis and
death by hydrolyzing the specic bonds in bacterial
cell surface (109). Indeed, Markkanen et al. (177)
reported lower lysozyme concentration in mixed sal-
iva in periodontitis patients compared to healthy
controls. Smith et al. (254) reported increased perox-
idase activity at the onset of gingival inammation
that declined after initiation of oral hygiene. Over et
al. (208) demonstrated increased myeloperoxidase
activity in patients with aggressive and chronic per-
iodontitis, compared to controls, with the highest
activity levels found in the aggressive periodontitis
group. They concluded that increased MPO activity
was due to increased inltration and degranulation
of polymorphonuclear leukocytes. In contrast, Saxen
et al. (239) demonstrated decreased MPO activity in
62
Sahingur & Cohen
localized aggressive periodontitis patients, and pos-
tulated that reduced peroxidase-mediated host
defense mechanisms could be characteristic of that
disease. Guven et al. (100) measured salivary perox-
idase 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 inammation in such patients.
Jalil et al. (126) studied the relationships among
thiocyanate, lysozyme, lactoferrin, plaque accumula-
tion and gingivitis in resting and stimulated whole
saliva of 94 children 1214 years old. An inverse rela-
tionship was observed between salivary thiocyanate
the amounts of plaque and gingival inammation.
Lactoferrin and lysozyme concentrations in stimu-
lated 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 inammatory
mediator as well. Platelet-activating factor affects
vascular permeability, smooth muscle contraction,
and inammatory cell stimulation (221, 258, 298).
In one study, Garito et al. (78) found that salivary
platelet-activating factor occurred in greater concen-
trations in the presence of periodontal inammation.
More specically, 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 period-
ontal therapy. Following scaling and root planing,
they found a signicant reduction in salivary plate-
let-activating factor and polymorphonuclear leuko-
cytes that were concurrent with improvements in
probing depths and bleeding on probing.
Kojima et al. (142) studied the presence of low mole-
cular 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 gin-
gival crevice uid and saliva from periodontitis and
healthy patients, four dominant low-molecular-mass
(814 kDa) proteins were observed that were not found
in serum, and were less pronounced in saliva from
edentulous and newborn subjects. These proteins were
identied by immunoblotting as members of the S100
family of calcium-binding proteins. The authors
hypothesized that one protein, MRP14 (S100A9), may
have a dened role in the gingival sulcus and could
serve as a potential periodontal disease marker. How-
ever, the practical utility of that protein, or its expres-
sion following treatment, awaits further investigation.
Aurer et al. (12) studied the potential role of nitric
oxide in the development of periodontitis by analyz-
ing salivary NO
2

concentrations in 25 subjects with

generalized aggressive periodontitis, 25 with chronic
periodontitis, and in 25 periodontally healthy sub-
jects. Individuals with periodontitis had signicantly
less salivary NO
2

than healthy subjects. Subjects

with aggressive periodontitis had lower NO
2

con-
centrations than those with chronic periodontitis.
In addition, parotid saliva contained less NO
2

than
sublingual or whole salivas. It was concluded that
local NO
2

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 per-
iodontally healthy controls. Although they found
similar quantities of Gc in parotid saliva from
patients in both groups, Gc in whole saliva was sig-
nicantly higher in periodontitis patients. In that
study, Gingival Index scores also were positively cor-
related positively with Gc.
Cystatins are potent inhibitors of cysteine pepti-
dases that depend on the highly reactive thiol group
of a cysteine residue at the catalytic site for their
activity (15, 16, 1820, 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 signicantly higher in whole and
parotid salivas of patients with chronic periodontitis,
compared to controls, but that the concentration of
63
Host responses and risk for disease progression
cystatin S was lower. Baron et al. (16) also found that
the salivary concentration of patients with periodon-
titis 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 period-
ontitis and control groups. Interestingly, Lie et al.
(162) found that smoking decreased the amount of
salivary cystatin in patients with gingivitis. Collec-
tively, those studies suggest that cystatin may be
an inammatory 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 inhi-
bitor 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 con-
trols (30, 122).
Other studies have assessed the relationship
between certain salivary ions, such as Ca
2
(205),
increased number of inammatory cells (49, 224),
and volatile sulfur compounds (145) to determine
whether a relationship existed between periodontal
disease and those salivary components. However,
specic associations were not found and the useful-
ness of the latter salivary components as potential
diagnostic markers remains doubtful. Similarly, the
presence of free amino acids in saliva did not signif-
icantly 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 popula-
tions. 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-specic uids. Indeed, the literature suggests
that analysis of host-derived salivary enzymes, such
as collagenase, elastase, and gelatinase, may hold sig-
nicant promise for periodontal diagnosis (77, 151).
Moreover, host inammatory mediators that predo-
minate 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 analy-
sis. 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 poten-
tial diagnostic markers for periodontal disease.
One obstacle associated with many studies report-
ing associations between potential salivary markers
and periodontal diseases is relatively small study
populations. Considerable variations in the concen-
trations 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 specic marker,
deviations from which might be considered abnor-
mal. 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 regard-
ing a particular individual's susceptibility to period-
ontitis 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 infec-
tion 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 microo-
rganisms or other foreign macromolecules. This
system includes physical barriers, phagocytic cells,
eosinophils, natural killer (NK) cells, and various
64
Sahingur & Cohen
blood-borne molecules. Specic immunity, however,
is induced upon exposure to foreign substances,
recognizes distinct macromolecules, and increases
in magnitude and capability with each successive
antigenic exposure. Specic immunity can be char-
acterized as being either cell-mediated (via T lym-
phocytes) 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 frac-
tion (9, 279). Immunoglobulins are heterogeneously
distributed in various biological uids and on the
surfaceof somelymphocytesubsets. Humanimmuno-
globulin molecules can be divided into ve distinct
classes or isotypes basedondifferences inserologic
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 signicant host patho-
logy (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 polymorphonuc-
lear leukocytes successfully eliminate the pathogens
and their byproducts via phagocytosis and inter-
cellular killing mechanisms, the clinical result may
be limited to gingivitis. However, if those mechan-
isms are evaded and inammation continues in host
tissues, then a transition from gingivitis to period-
ontitis 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 sufcient 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 per-
iodontal infection, the potential application of anti-
body 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 disadvan-
tage associated with whole-cell titers is the possibility
of nonspecic cross-reactivity with other organisms
that share common epitopes. Consequently, the pur-
ied antigenic preparations or targeted epitopes
unique for a particular pathogen generally are super-
ior to whole-cell lysates, and improve the sensitivity
and the specicity of the antibody-based diagnostic
assays (120, 219, 223, 253). Antibodies to bacteria and
other antigens typically are measured in serum, sal-
iva, and gingival crevicular uid. (Diagnostic applica-
tions involving gingival crevicular uid components
are reviewed elsewhere in this volume (9)).
Individuals with periodontitis often have elevated
antibody titers to periodontal pathogens, as com-
pared 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 demon-
strate 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,
signicant uctuations in the amount of antibodies
specic for periodontal pathogens were observed in
some subjects. The authors hypothesized that such
variation might reect changes in the infectious
process.
Elevated concentrations of IgG, IgM, and IgA class
antibodies to Actinobacillus actinomycetemcomitans
have been reported in aggressive and chronic period-
ontitis 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 signicant correlation
between antibody avidity and clinical parameters of
65
Host responses and risk for disease progression
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 general-
ized aggressive periodontitis patients. Positive treat-
ment 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 anti-
gens, their isotype, and subclass were measured in
four groups of patients (219) including subjects posi-
tive for P. gingivalis (with or without periodontitis)
and negative for P. gingivalis (with or without period-
ontitis). There were no differences in IgG1, IgG3 and
IgM concentrations, but the amount of IgG2 in sub-
jects without periodontitis or P. gingivalis seroreac-
tivity was signicantly higher than in patients
positive for P. gingivalis. For patients in the latter
group, the IgG4 and IgA concentrations also were
signicantly lower. Seropositivity to IgG3 was almost
exclusively seen in patients without periodontitis.
Collectively, those ndings suggested that the pre-
sence of IgG3 might reect a lack of susceptibility to
periodontitis, while a relative deciency of IgG4 may
be indicative of periodontal health and lack of infec-
tion. Although those results appeared promising, the
practical utility was compromised due to the rela-
tively small sample size of the population.
Celenligil & Ebersole (37) investigated the speci-
city of serum antibodies to dened oral microorgan-
isms 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 sero-
types c and a, while antibody levels to A. actinomy-
cetemcomitans serotype b were signicantly 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 con-
cluded that signicant and considerable variation
existed in the systemic antibody responses to period-
ontal pathogens in patients from both countries.
More recently, serum IgG responses to six period-
ontal 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 popu-
lation, prior disease and subsequent attachment loss
both were associated with elevated serum IgG anti-
body 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 ele-
vated serum IgG antibody to P. gingivalis may be
reective of progressive, destructive periodontal dis-
ease, and the presence of such antibodies may be
considered a risk factor for disease progression in
these populations. Nevertheless, the possible inu-
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 periodonti-
tis patients, and 5 sites with clinically progressive reces-
sion (273). They found no signicant 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 signicantly 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 func-
tions 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 difculty 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,
66
Sahingur & Cohen
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-
nicantly elevated IgG2 responses compared to other
patients. The mean IgG4 level of maintenance
patients was signicantly higher compared to the
other groups, and a statistically signicant positive
correlation between IgG2 levels and changes in bone
levels also was observed. Since maintenance patients
with high IgG2 and low IgG4 concentrations demon-
strated greater bone loss than those with low IgG2
and high IgG4, the authors suggested that a pro-
longed IgG2 response following periodontal therapy
might indicate recurrent or persistent periodontal
destruction.
Beickler et al. (24) assessed specic 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 scal-
ing 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
signicantly 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 associa-
tion was statistically insignicant. 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. actino-
mycetemcomitans might have a protective effect
against periodontal disease progression in patients
harboring those microorganisms.
Antibody titers and avidity to P. gingivalis, A. acti-
nomycetemcomitans, 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 mainte-
nance procedures (15 smokers and 16 non-smokers)
(185). Decreased titers to A. actinomycetemcomitans,
P. intermedia and T. denticola were found in main-
tenance 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 period-
ontal treatment.
More recent evidence suggests that inammatory
mediators or bacteremia secondary to chronic peri-
odontal inammation may affect systemic health by
increasing the risk of developing cardiovascular dis-
ease, association with respiratory disorders, or deli-
vering low birth weight infants (58, 128, 240, 241).
Dasanayake et al. (57) determined IgG concentra-
tions specic for dened 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-specic maternal serum IgG
in the low birth weight group compared to the nor-
mal birth-weight group. Women with higher levels of
P. gingivalis-specic 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 pre-
term 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 identied 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 sali-
vary 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 tis-
sue 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 patho-
gens. A number of studies have investigated the rela-
tionship of both specic 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
67
Host responses and risk for disease progression
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 inammation. Reiff
et al. (227) examined serum and salivary concentra-
tions 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 period-
ontitis 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, com-
pared to non-diabetic periodontitis patients and per-
iodontally 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 periodonti-
tis 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 immunosup-
pressed group.
Barr-Agholme et al. (17) investigated the salivary
concentration of immunoglobulins sIgA, IgM, and
IgG and albumin in 20 patients with Down's syn-
drome, compared to 19 healthy controls. They found
that results from analysis of sIgA, IgM, total IgG, and
albumin did not differ signicantly 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 distribu-
tion 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 periodon-
titis 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 dis-
ease severity and decreased sodium, calcium, and
magnesium concentrations. On the other hand, smo-
kers responded favorably to treatment, resulting in
the elimination of the differences in salivary compo-
sition. 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 differentiat-
ing 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 period-
ontitis. Salivary IgG antibody to A. actinomycetemco-
mitans was signicantly increased in 55% of
individuals with untreated localized aggressive peri-
odontitis, and in 28% of treated subjects with that
disease. Furthermore, 28% of the patients with
chronic periodontitis had signicantly elevated
salivary IgG antibody levels to A. actinomycetemco-
mitans subtype Y4. In that study, however, IgA con-
centrations did not show appreciable difference
among study groups. Schenk et al. (242) measured
salivary IgA specic 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. actinomyce-
temcomitans, and Eubacteriun saburreum anti-
bodies. 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 sam-
ples 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 signicantly lower concentration and rate of secre-
tion of total salivary IgA in the experimental group.
Although no differences were detected in the con-
centration or secretion of P. gingivalis-reactive IgA
between groups, the specic fraction of P. gingivalis-
reactive 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 specic IgA in human saliva.
68
Sahingur & Cohen
Collectively, those studies suggest that detectable
amounts of antibodies specic for periodontal
pathogens are indicative of an infectious process,
the presence of periodontal disease, or both. Unfor-
tunately, data describing the temporal relationship
between antibody response and infection are sparse.
Consequently, longitudinal studies in high-risk sub-
jects are required to determine whether the analysis
of salivary antibodies may have clinical utility as a
predictor of the risk of infection or disease. More-
over, other host factors or conditions may inuence
immunoglobulin production. For example, Granade
et al. (92) found that signicant differences in sali-
vary 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 immuno-
globulin 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 specic
for putative periodontopathic organisms would
appear to have considerable potential for periodontal
diagnosis. Although sIgA is the predominant immu-
noglobulin in saliva, it seems to be related less to
periodontal inammation than IgG, which is derived
primarily from gingival crevice uid. In any event,
the data indicate that identication of specic
immunoglobulins directed to periodontal bacterial
components may be more specic and provide more
useful information than quantitation of nonspecic
immunoglobulin isotypes.
Longitudinal studies investigating the concentra-
tion, specicity, 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 ther-
apy, variations in treatment modalities and the dif-
ferences 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, antibo-
dies 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. Specic determinants
unique for a particular bacterium may decrease
cross-reactivity, but limiting the focus to specic
antigens may cause an underestimation of the host
response. Nevertheless, it has been suggested that
puried antigen preparations may lead to improved
sensitivity and specicity of antibody-based diagnos-
tics (201).
During the past 20 years, extensive information has
become available describing the specicity and con-
centrations of serum antibodies to putative period-
ontal 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 dened
due to the lack of sufciently robust data. Well-
designed longitudinal investigations and controlled
clinical treatment studies for relatively long time per-
iods are required to more fully explore the applic-
ability 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-dened tests to clinicians that will
help to improve patient management and the devel-
opment 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 consti-
tute the primary mammalian phagocytic cell system
for host defense against exogenous agents. Some of
69
Host responses and risk for disease progression
their functions include ingestion of foreign organ-
isms, microbial processing via secretion of lytic
enzymes, release of inammatory mediators impor-
tant in the initiation and regulation of immune and
inammatory 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 5070% 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). Fol-
lowing differentiation from bone marrow progenitor
cells, neutrophils circulate in blood for relatively
short time periods (t
1/2
67 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 inammatory event secondary to cell
injury or infection, neutrophils in the marginated
pool exit the circulation by adhering to the endothe-
lium and projecting pseudopods between endothe-
lial cells via diapedesis or transendothelial migration
(14, 54, 64, 183). Major superfamilies of adhesion
molecules participating in polymorphonuclear leu-
kocyteendothelial 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 che-
moattractants for polymorphonuclear leukocytes
include N-formylmethionyl peptides (fMLP), C5a,
leukotriene B
4
, 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 chemoat-
tractants and specic 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 neutro-
phil cell surfaces, thereby amplifying that process.
The mechanisms through which neutrophils migrate
are thought to involve a change in conguration
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 parti-
culate matter through specic interactions between
the polymorphonuclear leukocyte membrane and
one or more components on the target particle sur-
face (169). Efcient phagocytosis often requires that
the particle be coated with one or more host serum
proteins through opsonization, which requires inter-
action with IgG antibodies and cleavage fragments of
complement component C3 (C3b and 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 neutro-
phils include the primary (azurophil) and secondary
(specic) granules, which contain enzymatic and
non-enzymatic proteins that are bactericidal or bac-
teriostatic 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, gener-
ates superoxide, which is converted to hydrogen per-
oxide 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, neutro-
phils undergo lysis or programmed cell death (apop-
tosis) after the completion of their primary protective
function. Abnormalities in any of these steps might
result in a delayed or less efcient 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 inam-
mation, typically making up about 90% of the cells in
the gingival sulcus (54, 210). Studies have demon-
strated that neutrophils have a protective role by
serving as a barrier between the periodontal patho-
gens and the periodontal tissues (157, 183, 232). Neu-
trophils function by altering bacterial colonization
and growth, or through modulating bactericidal pro-
cesses. Some of the mechanisms through which
70
Sahingur & Cohen
neutrophils respond to periodontal bacteria have
been extensively reviewed (79, 115, 141, 183).
The protective function of neutrophils in period-
ontal diseases has been supported through clinical
investigations of patients with primary and second-
ary immunodeciencies (83, 115, 135, 290). Neutro-
phil 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 sur-
face adherence protein LFA-1 (5). In addition, indi-
viduals whose quantity and function of neutrophils
are reduced due to systemic diseases such as dia-
betes mellitus (41, 95, 202), Down's syndrome (238,
248), and Papillon-Lefevre syndrome (291) also exhi-
bit increased periodontal destruction. Some systemic
conditions that are characterized in part by neutro-
phil defects, and are associated with periodontal dis-
eases, are noted in Table 5.
Additional support for the benecial role of neu-
trophils in periodontal diseases has been derived
from data suggesting that many periodontal patho-
gens possess virulence factors that either kill neutro-
phils or result in diminished neutrophil function
(222). The leukotoxin produced by A. actinomycetem-
comitans. (77, 111, 243) and proteases produced by
P. gingivalis (52, 130) are examples of factors that are
believed to destroy neutrophils or affect their func-
tion. Moreover, susceptibility of neutrophils to these
virulence factors may vary between individuals. poly-
morphonuclear leukocytes from periodontally
healthy individuals also may be more susceptible
to A. actinomycetemcomitans leukocidin than neu-
trophils from patients with localized aggressive per-
iodontitis (132, 294). Thus, identication of
neutrophil susceptibility factors potentially may pro-
vide 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 per-
iodontitis frequently possess neutrophil-response
dysfunctions, including chemotactic defects (55,
287, 293, 295), phagocytotic defects (292), altered
superoxide generation (292), and altered leukotriene
B
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 specic 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
characterizedbyrecurrentcutaneous, periodontal, res-
piratory or soft tissue infections (139). Although the
precise neutrophil defect in each disease may be dif-
ferent, severe periodontitis typically is associated with
those conditions. Altered expression of neutrophil
adhesion molecules could inuence PMN migration
and lead to inappropriate release of reactive oxygen
species and tissue injury. Congenital leukocyte adher-
ence deciency (LAD) (31, 36) is an autosomal reces-
sive disorder of leukocyte function. Such patients lack
surface adhesive glycoprotein receptors of the Mac-1,
LFA-1 and p150,95 (CD11/18) group. Recurrent infec-
tions, delayed wound healing, impaired suppuration,
as well as aggressive periodontal disease (generalized
prepubertal periodontitis) are commonly associated
with LAD (139). Resting polymorphonuclear leuko-
cytes 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 aggres-
sive periodontitis also exhibit decreased shedding of
l-selectin after fMLP stimulation, increased basal pro-
duction of H
2
O
2
by polymorphonuclear leukocytes,
decreased polymorphonuclear leukocyte oxidative
burst, increased plasma IL-8 levels, and decreased
plasma l-selectin levels. However, those abnormal-
ities were not found in patients with chronic period-
ontitis 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-Lefevre syn-
drome (86, 165), actin dysfunction syndrome (32),
Crohn's disease (131), and aggressive periodontal dis-
eases (140, 207, 282, 293, 295, 311). Upon stimulation
with a chemotactic factor, morphologic changes and
directedneutrophil movement result fromreorganiza-
tion of plasma membrane-associated structures (25,
51, 204206), with actin serving as one of the major
components of the microlamentous 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
71
Host responses and risk for disease progression
weredetectedbetweenthetwogroups, andthedatadid
not show a correlation between the kinetics of actin
polymerization-depolymerization and the abnormal
chemotactic response observed in polymorphonuc-
lear leukocytes from patients with localized aggressive
periodontitis. The authors concluded that the associa-
ted chemotactic defect may be mediated by signaling
events that are independent of cytoskeletal integrity.
Nevertheless, substantial evidence has associated
impaired PMN chemotaxis with aggressive period-
ontal disease (54, 115, 292). Moreover, approximately
75% of African-Americans with localized aggressive
periodontitis demonstrate chemotactic defects that
makethemsusceptibletoperiodontal 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 alsobeenshowntobepresent in
diminished quantities on neutrophils from patients
withlocalizedaggressive 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 inammatory 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 particu-
lar 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 identication of such
genetic markers that would predict susceptibility for
certain periodontal diseases.
The investigation of chemotactic defects in neutro-
phils frompatients with aggressive periodontitis led to
further investigations to identify possible signal trans-
duction anomalies associated with neutrophil chemo-
taxis (2, 55). For example, guanine nucleotide binding
proteins (G proteins), a family of homologous regula-
tory proteins that transfer information from cell
receptor to cell effector systems, are involved in recep-
tor transduction such as photoreception and the mod-
ulation 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 cal-
cium release from intracellular sites and the activa-
tion 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 mechan-
ism through which G proteins may regulate phos-
pholipase C activation in neutrophils is still not
thoroughly established. However, studies have inves-
tigated 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 immobiliza-
tion (2, 55), reduction of protein kinase C activity
(148), increased levels of diacylglycerol and reduced
diacylglycerol kinase activity (284).
Stimulatedneutrophils alsoactively generate oxyge-
nated derivatives of arachidonic acid such as 5-HETE,
12-HETE, and 5,12 di-HETE, and LTB
4
, all of which
can modulate cell function (183). The former com-
pound and their hydroperoxy precursors (HPETEs), as
well as LTB
4
, have been shown to alter calcium uxes
andtoinduce a variety of neutrophil responses suchas
degranulation and chemotaxis (88, 188). A depressed
LTB
4
chemotactic response (200) and impaired lipo-
oxygenase 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 comple-
tely dene any deciency as a disease marker.
Disorders associated with neutrophil phagocytosis
defects include diabetes mellitus and actin dysfunc-
tion 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, com-
pared 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). Evi-
72
Sahingur & Cohen
dence strongly suggests that variations among indi-
viduals 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 gin-
gival 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 dis-
ease, 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 predo-
minantly IgG2-mediated response to A. actinomyce-
temcomitans 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 elimina-
tion of bacteria by neutrophils.
Smoking is generally accepted to be a major risk
factor in the development and progression of period-
ontal disease (81). Accordingly, the inuence 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, Mariggio
et al. (176) demonstrated increased apoptosis of
polymorphonuclear leukocytes obtained from gingi-
val crevice uid of smokers with chronic periodonti-
tis compared to those isolated from periodontally
healthy controls, and proposed that smoking may
be associated with an inefcient host response.
Another study (230) found increased H
2
O
2
produc-
tion during the neutrophil oxidative burst in cells
exposed to smoke in vitro. Numabe et al. (197) inves-
tigated the effect of smoking, and passive smoking,
on the phagocytic function of salivary polymorpho-
nuclear leukocytes and found increased phagocytic
activity of salivary polymorphonuclear leukocytes.
In contrast, no signicant differences were found
in l-selectin and CD18 expression on neutrophils
isolated from smokers and non-smokers (231).
Although acquired neutrophil deciencies repre-
sent an attractive mechanism through which exo-
genous factors may alter the host's susceptibility to
periodontitis, many reports have been based on
in vitro observations or relatively small clinical inves-
tigations. As a result, more denitive studies are
required to establish the mechanisms by which those
factors inuence periodontal diseases, and whether
measurement of altered neutrophil function may be
useful to assess disease risk or treatment efcacy.
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 dis-
ease pathogenesis is well accepted. Considerable evi-
dence 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. Identication of genetic
components also has fostered additional research on
potential genetic markers that may correlate with
periodontal disease occurrence. In contrast to stu-
dies of acquired neutrophil defects, there are com-
pelling data linking some intrinsic neutrophil defects
with the risk of periodontitis. Although acquired neu-
trophil deciencies represent a very attractive
hypothesis regarding a host's susceptibility to peri-
odontitis, they generally are based on in vitro observa-
tions 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 direc-
ted 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, inuenced by
genetics as well as by the environment (81). The
recognition that individuals are not equally suscep-
tible 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 measure-
ments of pocket depth, attachment level, or bone
loss relate only to previous disease and current
health status, and may not be useful to identify indi-
viduals who may be at risk for future disease.
73
Host responses and risk for disease progression
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-specic uids.
However, considerable variations exist in the amount
of salivary components among individuals and popu-
lations, and few well-accepted normal values have
been established for any specic marker. Although
the identication of some host-derived salivary
enzymes such as collagenase, elastase, and gelati-
nase may be signicantly 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 par-
ticular 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 pre-
dictive 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 there-
fore 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 mea-
surement of antibodies specic for putative period-
ontopathic organisms may prove to be quite useful,
particularly for early identication of aggressive
periodontal diseases (e.g. through identication 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 com-
mercialization of tests based on neutrophil defects
may be of signicant 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 susceptibil-
ity. Well-designed longitudinal investigations also
will be required to more fully explore the applicabil-
ity of salivary and immunologically based tests for
periodontitis if they are to become clinically useful
for most patients and clinicians.
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Host responses and risk for disease progression