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Oral Microbiology Immunology 2002: 17: 100–107 Copyright C Blackwell Munksgaard 2002
Printed in Denmark. All rights reserved

ISSN 0902-0055

Inhibitory effects of whole and K. L. Wozniak1,3, A. Arribas2,3,

J. E. Leigh2,3, P. L. Fidel Jr1,3
1
Department of Microbiology, Immunology,

parotid saliva on
and Parasitology, Louisiana State University
Health Sciences Center, New Orleans, LA,
USA, 2Department of General Dentistry,
Louisiana State University School of

immunomodulators Dentistry, New Orleans, LA, USA, 3Center for

Excellence in Oral and Craniofacial Biology,
Louisiana State University School of
Dentistry, New Orleans, LA, USA

Wozniak KL, Arribas A, Leigh JE, Fidel PL Jr. Inhibitory effects of whole and
parotid saliva on immunomodulators.
Oral Microbiol Immunol 2002: 17: 100–107. C Blackwell Munksgaard, 2002.

Based on the presence of cytokines in whole saliva and their association with
resistance and susceptibility to infectious disease, the present study was designed
to evaluate the diagnostic potential of a large panel of cytokines and chemokines
in saliva. Despite the endogenous presence of Th1/Th2 and pro-inflammatory
cytokines and several chemokines in whole and parotid saliva of most individuals
tested, the detection of known concentrations of several recombinant cytokines
and chemokines was inhibited immediately following their addition to each type
Key words: chemokines; cytokines; oral
of saliva. In contrast, purified immunoglobulins were unaffected by either whole immunology; parotid saliva; saliva
or parotid saliva. Further studies revealed that the inhibition of immunoreactivity
involved sequestration of the majority of cytokines affected and degradation of Paul Fidel, Jr, Department of Microbiology,
chemokines. These results suggest that absolute concentrations of cytokines/ Immunology, and Parasitology, Louisiana
State University Health Sciences Center,
chemokines may not be fully detectable in saliva. Therefore, the diagnostic value 1901 Perdido St., New Orleans, LA 70112,
of any cytokine/chemokine is questionable and should be evaluated USA
independently as such. Accepted for publication August 20, 2001

Oral health is considered a mirror of
general health (7, 8, 9, 26, 28, 31),
whereby changes in oral health can be
indicative of changes in general health
status. As such, immunomodulators in
saliva can be used diagnostically in re-
lation to infection/disease. Accordingly,
antibodies in saliva have been used as
an indicator of infection status (e.g.
antibodies to HIV) (6, 9, 11, 13). This,
together with the ease of collection,
makes saliva a potentially effective bio-
logical fluid to be used as a diagnostic
tool and a predictor of overall health
status (9, 11, 18, 28, 31).
Cytokines have been used to predict
disease/infection status. For example, T
helper (Th)1-type cytokines are indica-
tive of protection against intracellular
pathogens and fungi, whereas Th2-type
cytokines are indicative of protection
against extracellular bacteria and para-
sitic infections (27, 32, 34, 37, 40). The
alternate cytokine profile usually infers
susceptibility to infection/disease (27,
32, 34, 37, 40). Therefore, cytokines and
chemokines in saliva have the potential
to be predictive of susceptibility/re-
sistance to infection or an indicator of
disease status.
Cytokines and chemokines have been
identified in whole saliva and/or oral
tissue by protein (25) or mRNA (10)
levels, or both. Cytokines that have been
detected in saliva include Th1/Th2-type
and pro-inflammatory cytokines (5, 10,
29). In fact, different salivary Th1/Th2
cytokine profiles have been reported for
HIV-infected persons with and without
oropharyngeal candidiasis, an oppor-
tunistic fungal infection (5, 25). To date,
a limited number of chemokines have
been identified only at the mRNA level
in oral tissues, including interleukin-8
(IL-8) and macrophage chemoattractant
proteinª1 (MCP)-1 (44).
Inasmuch as saliva has the potential to
be a diagnostic tool, the predictive value
may be limited by putative inhibitors of
immunoreactivity. This inhibition could
affect absolute concentration and/or de-
tection of immunomodulators, espe-
cially if a differential inhibition is evident
either with specific immunomodulators
or individuals. Currently, there have
been no reports of immunomodulator
inhibitors in saliva, although inhibitors
of cysteine proteinases have been de-
scribed (1–4, 14, 19–21, 30, 35, 39). In
contrast, the detection of both cytokines
and antibodies is inhibited in cervical
mucus, a common constituent of cerv-
ico-vaginal secretions (12).

Based on the effects of cervical mu-
cus on immunomodulators and the fact
that saliva contains similar large mucin-
like proteins, we asked whether or not
saliva has any inhibitory effects on im-
munomodulators that ultimately could
affect detection and/or function. To this
end, the effects of saliva on a large
panel of cytokines and chemokines
were evaluated to elucidate its diagnos-
tic potential.
Materials and methods
Human saliva samples
Twenty-five normal healthy volunteers
were enrolled in accordance with the
stated guidelines of the Institutional Re-
view Board of Louisiana State Univer-
sity Health Sciences Center. Partici-
pants included 10 females and 15 males,
with ages ranging from 20 to 45 years.
Exclusion criteria included any form of
oral pathology, oral infections, peri-
odontitis or gingivitis. Once informed
consent was obtained from each sub-
ject, saliva was collected from each indi-
vidual at approximately the same time
of day (late afternoon). For this, sub-
jects were asked to expectorate 10 ml of
unstimulated whole saliva into a 50-ml
sterile plastic centrifuge tube. Following
collection of whole saliva, 10 ml of pa-
rotid saliva was collected. For this, the
Lashley collection device (22, 23) was
affixed to the duct of the parotid gland,
and parotid saliva production was
stimulated by application of lemon
juice to the tongue. Following collec-
tion, whole and parotid saliva was clari-
fied by centrifugation for 5 min at 800 g
at 4æC. The supernatants were collected
and sterile filtered using a 45-mm low-
protein binding filter. The filtered saliva
was then aliquoted into 1-ml volumes
and frozen at ª80æC until use.
Protein assay
For each saliva sample, the total protein
content was determined using the BCA
protein assay (Pierce, Rockford, IL) with
bovine serum albumin as the standard
(Sigma, St. Louis, MO) as previously de-
scribed (38). The protein was quantified
using an automated plate reader at 495
nm (Ceres 900, Bio-tek, Wisnooski, VT).
The protein concentrations were ex-
pressed as mg/ml. Protein concen-
trations in whole and parotid saliva were
similar, with no significant difference
[whole (mean ∫ standard error of the
Immunomodulator inhibitors in saliva
mean (SEM)), 1.485 ∫ 0.2 mg/ml vs. pa-
rotid, 1.328 ∫ 0.16 mg/ml].
ELISAs
For chemokine and cytokine ELISAs,
Opt EIA kits (BD Pharmingen, San Di-
ego, CA) were used according to manu-
facturer’s instructions. Briefly, each
plate was coated with a 1 : 250 dilution
of coating antibody in coating buffer
(0.1 M Na HCO3, pH 8.2) at 50 ml/well
and incubated at 4æC overnight. After
washing with phosphate-buffered saline
(PBS)-Tween buffer (1 ¿ PBS π 0.05%
Tween 20), the nonspecific binding sites
were blocked with 10% fetal bovine
serum (FBS) in PBS for 1 h at 21æC
(room temperature). Following
blocking, the plates were washed and
the samples (undiluted) and standards
were added at 50 ml/well. Standards were
prepared using purified recombinant
chemokines or cytokines. Each stan-
dard curve was prepared beginning at
2000 pg/ml and serially diluted 1 : 2 in
PBS. After a 2-h incubation at 21æC, the
plates were washed followed by the ad-
dition of 50 ml/well of secondary anti-
body plus horseradish peroxidase en-
zyme (HRP), both diluted 1 : 250 into
blocking buffer for 1 h at 21æC. Follow-
ing a final washing step, o-phenyline di-
amine dihydrochloride (OPD) (Sigma)
was added at 50 ml/well. After incuba-
tion in the dark (for 15–30 min) at 21æC,
plates were read at 450 nm in the auto-
mated plate reader (Ceres 900, Bio-tek).
The sensitivity of the cytokine and
chemokine assays in our laboratory was
approximately 2–10 pg/ml. Optical den-
sities corresponding to concentrations
below these levels were considered un-
detectable. The coefficient of variance
for samples tested multiple times in the
ELISAs was ⬍7%. Concentrations
were determined by extrapolation from
a standard curve and expressed as pg/
ml or pg/mg total protein.
The procedure for the antibody
ELISAs was identical to that for the
chemokine/cytokine ELISAs, except for
types of antibodies and antibody di-
lutions. To coat, 20 mg/ml anti-IgA
(monoclonal a-chain specific) and 5 mg/
ml polyclonal anti-IgG (Sigma) were di-
luted in coating buffer. For standards,
purified antibodies (reagent grade hu-
man IgA and IgG) were used (Sigma)
beginning at 40 000 ng/ml IgA and 625
ng/ml IgG. Each standard was serially
diluted 1 : 2 in PBS, while samples were
diluted 1 : 100. For detection, HRP con-
101
jugated-anti-IgA (monomeric a-chain
specific) was diluted to 125 ng/ml and
HRP conjugated-anti-IgG (polyclonal)
(Sigma) was diluted to 530 ng/ml in
blocking buffer. Following incubation
and washing, substrate (OPD) was
added to each well, and the plates were
read at 450 nm. Concentrations were ex-
trapolated from the standard curves,
and final concentrations were expressed
as ng/ml or ng/mg protein.
Stability of exogenous chemokines and
cytokines
To sterile-filtered whole pooled saliva
samples (n Ω 5), 1000 pg/ml of purified
recombinant chemokines (IL-8,
RANTES, and MCP-1) and cytokines
[IL-12, interferon gamma (IFN-g), IL-
4-4, and IL-10] were added in a cocktail.
The sample was separated into aliquots
and incubated for 48 h in sterile, closed
containers ª80, 4, 21 or 37æC. An ad-
ditional portion of the pooled sample
was set aside and similarly treated with
exogenous chemokines and cytokines
and tested immediately (no incubation).
Each sample was evaluated in the
chemokine/cytokine ELISAs undiluted.
Effect of whole and parotid saliva on
exogenous chemokines, cytokines and
antibodies
To all available whole and parotid sal-
iva samples, 1000 pg/ml of purified re-
combinant chemokines (IL-8, RANT-
ES, and MCP-1) and cytokines (Th1,
Th2, and pro-inflammatory) was added
to each sample in a cocktail. For exoge-
nous antibodies, 100 mg/ml purified IgA
and 25 mg/ml purified IgG (Sigma) were
added to saliva samples. Each sample
was tested immediately in ELISAs,
either undiluted (chemokines and cyto-
kines) or at a 1 : 100 dilution (anti-
bodies). Endogenous levels of each im-
munomodulator were also evaluated in
parallel for each saliva sample. Controls
included the addition of chemokines,
cytokines and antibodies to PBS to de-
termine the actual concentration of
each immunomodulator added.
Solubilization of large proteins by
bromelain
Bromelain (Sigma) was prepared at 4
mg/ml dissolved in Tyrode’s salt solu-
tion (Sigma). To clarified saliva, an
equal volume of bromelain or Tyrode’s
salt solution (vehicle control) was added
102 Wozniak et al.
for 15 min at room temperature. Fol-
lowing treatment, the saliva was sterile
filtered, aliquoted, and frozen at ª80æC
until use.
Western blots
Western blots were conducted as per
standard protocol (16). Briefly, saliva
samples were first prepared for electro-
phoresis along with the positive con-
trols, purified IgA and IgG (5000 ng
each), with sample buffer (Laemmeli
sample buffer, Bio-Rad, Hercules, CA)
and b-mercaptoethanol. Following boil-
ing for 15 min, 20 ml of each sample was
added to the wells of a 12% polyacryl-
amide gel along with 20 ml of prestained
broad-range molecular weight standard
(Bio-Rad). Electrophoresis was carried
out at 150 V for 1 h in tris-glycine run-
ning buffer. The samples and standards
from the SDS-PAGE were then trans-
ferred to nitrocellulose paper at 25 V
overnight in tris-glycine buffer with
20% methanol. Nonspecific protein-
binding sites on the nitrocellulose paper
were blocked by overnight incubation
with 10% goat serum in tris-buffered sa-
line (TBS) with 0.05% Tween at room
temperature on an orbital shaker. Fol-
lowing this, HRP-conjugated-anti-hu-
man IgA (polyclonal) was added at 250
ng/ml and anti-human IgG (polyclonal)
was added at 1600 ng/ml in blocking
buffer for 1 h at 21æC and washed 4
times for 30 min each with blocking
buffer. Finally, OPD substrate (Sigma)
was added and the bands visualized
after 15 min.
Statistics
The unpaired Student’s t-test was used to
analyze data in all exogenous immuno-
modulator experiments and bromelain
studies. Correlation statistics of re-
gression analysis were performed on all
endogenous immunomodulator detec-
tions in whole and parotid saliva. Sig-
nificant differences were defined as a
confidence level at which P was ⬍0.05,
using a two-tailed test. All statistics were
evaluated using GraphPad software
(GraphPad Software, San Diego, CA).
Results
Endogenous levels of chemokines shown
by ELISA
To evaluate endogenous chemokines in
matched whole and parotid saliva, each
saliva sample was quantified for
RANTES, IL-8, and MCP-1 by com- Table 1. Endogenous cytokines in saliva
mercial ELISA and normalized to total Cytokine Median concentration,
protein. The individual and median pg/mg proteina (range)
concentrations illustrated in Fig. 1 show
that all chemokines measured were de- Parotid saliva Whole saliva
tectable in both parotid and whole sal- IL-12 15.6 16.3
iva. RANTES was detected in 55 and (0–483) (0–254)
81% of parotid and whole saliva IFN-g 15.3 8.6
samples, respectively, while IL-8 and (0–655) (0–229)
IL-4 13.7 15.2
MCP-1 were detected in 95 and 100% of
(0–160) (0–2929)
both parotid and whole saliva samples, IL-10 0 0
respectively. Of all chemokines tested, (0–216) (0–100)
MCP-1 was found at the highest con- TGF-b 0 47
centrations in both parotid and whole (0–2.2) (0–255)
saliva, followed by IL-8 and RANTES. IL-1a 65.1 362
(0–200) (0–673)
IL-6 45.4 91.8
Endogenous levels of cytokines shown by (26.2–172) (34.8–1306)
ELISA TNF-a 35.0 85.6
(13.1–92.9) (27.2–1074)
Cytokines evaluated included Th1-type
a
cytokines (IL-12 and IFN-g), Th2-type Saliva concentration determined for 25 sub-
cytokines [IL-4, IL-10, and trans- jects with all available samples tested for
each cytokine.
forming graph factor-beta (TGF-b)],
and pro-inflammatory cytokines [IL-
1a, tumor necrosis factor-alpha (TNF-
a), and IL-6]. The individual and me-
dian concentrations are summarized in tration in whole saliva, followed by IL-
Table 1. For the Th1-type cytokines, IL- 6, TNF-a, and TGF-b. All other cyto-
12 was detected in 93 and 87% and kines measured in whole and parotid
IFN-g in 70 and 65% of parotid and saliva were detected at lower concen-
whole saliva samples, respectively. For trations (below 100 pg/mg protein).
the Th2-type cytokines, IL-4 and IL-10 In correlation analyses, there was a
were detected in 50 and 10%, respec- significant correlation in amount of
tively, of both parotid and whole saliva IFN-g between matched parotid and
samples, while TGF-b was detected in whole saliva (R Ω 0.6018, P Ω 0.0006)
10 and 90% of parotid and whole saliva, (data not shown). None of the chemo-
respectively. For the pro-inflammatory kines and no other cytokines showed a
cytokines, IL-1a was detected in 91% of significant correlation between the
both parotid and whole samples, while amounts detected in the matched
IL-6 and TNF-a were each detected in samples. Additionally, the only signifi-
100% of both parotid and whole saliva cant differences between concentrations
samples. Of all cytokines measured, IL- in whole and parotid saliva were for IL-
1a was detected at the highest concen- 1a (P Ω 0.029), TNF-a (P Ω 0.022), and
TGF-b (P Ω 0.0003).
Stability of chemokines and cytokines in
saliva
To assess the stability of cytokines and
chemokines in saliva, a preliminary ex-
periment using a small number of
pooled whole saliva from individuals
(n Ω 5) was performed. The poded
sample was separated into aliquots
spiked with 1000 pg/ml of recombinant
chemokines (MCP-1, IL-8, and RANT-
Fig. 1. Endogenous chemokines in saliva. ES) and cytokines (IL-12, IFN-g, IL-4,
Whole and parotid saliva was collected from
and IL-10) and assayed immediately or
25 subjects, centrifuged, and sterile filtered.
All available samples were assayed for
after a 48-h incubation at room tem-
RANTES, IL-8, and MCP-1 by ELISA. Re- perature, chemokines was detected at
sults are expressed as pg/mg protein in saliva. ª80, 4, 21 or 37æC. Each cytokine
Individual and median concentrations of showed ⬍400 pg/ml with no differences
each chemokine are shown. detected between samples stored under

different temperatures and those spiked
and tested immediately.
Effects of saliva on exogenous
chemokines and cytokines
In the light of the immediate effects
of whole saliva on concentrations of
exogenous cytokines, a formal evalu-
ation of the effects of parotid and
whole saliva on cytokines and chemo-
kines was conducted. For this, a
cocktail of recombinant chemokines
(MCP-1, RANTES, and IL-8) or
cytokines (IL-12, IFN-g, IL-4, IL-10,
TGF-b, IL-1a, IL-6, and TNF-a)
were added to each saliva sample or
PBS (control) at 1000 pg/ml, and
samples were assayed for each cyto-
kine/chemokine immediately by ELI-
SA. The results in Fig. 2 show that,
compared to the concentration de-
tected in the spiked sample of PBS,
the chemokines IL-8 and MCP-1 were
detected at significantly lower concen-
trations (P ⬍ 0.05) in parotid saliva
samples, while all chemokines tested
were detected at significantly lower
concentrations in whole saliva
samples (P ⬍ 0.002). For all chemo-
kines, concentrations in whole saliva
were significantly lower than in pa-
rotid saliva (P ⬍ 0.02).
Th1/Th2 cytokines were reduced to
Fig. 2. Effects of saliva on exogenous chemokines. PBS and whole and parotid saliva (n Ω 20)
were spiked with 1000 pg/ml of each chemokine (RANTES, IL-8, and MCP-1) in a cocktail.
Results are expressed as pg/ml detected of each chemokine in PBS, parotid saliva, and whole
saliva (with endogenous amounts subtracted). The percentages shown indicate the percent
reduction compared to the PBS spike. Asterisks (*) represent significant reductions compared
to PBS spike (P ⬍ 0.05). Mean concentrations and standard error of the mean (SEM) are
shown for each chemokine.
Immunomodulator inhibitors in saliva
an average of 75% in whole and 20% in
parotid saliva, with cytokines in whole
saliva again showing the largest reduc-
tion (P ⬍ 0.005) (Fig. 3A). For pro-in-
flammatory cytokines, little to no re-
duction was seen for IL-1a in either pa-
rotid or whole saliva, while IL-6 and
TNF-a were reduced to approximately
50% in both parotid and whole saliva
(Fig. 3B). For all cytokines except IL-
1a and IL-6, inhibition was significantly
greater in whole than in parotid saliva
(P ⬍ 0.03).
Effects of bromelain treatment on
endogenous immunomodulators
As concentrations of cytokines and
chemokines were reduced in saliva, a
study was conducted to determine
whether the chemokines and cytokines
were present but masked from detection
or, alternatively, degraded in the saliva
sample. For this, all available whole sal-
iva samples were treated with brome-
lain, whereby the bromelain-dependent
solubilization of large proteins would
be expected to enhance the detection of
any masked or sequestered cytokine.
The results in Fig. 4 show that the
amounts of endogenous Th1/Th2 cyto-
kines and IL-6 increased significantly
(P ⬍ 0.04) following treatment of saliva
with bromelain compared to the vehicle
103
control, whereas concentrations of
TGF-b (Fig.4A), IL-1a, and TNF-a
(Fig.4B), and the chemokines IL-8,
MCP-1, and RANTES (Fig.4C) were
unchanged.
Effects of saliva on exogenous
immunoglobulins
A design similar to that for exogenous
chemokines and cytokines was applied
to immunoglobulins. For this, purified
human IgA and IgG were added to
whole and parotid saliva at 100 and 25
mg/ml, respectively, and assayed im-
mediately by ELISA. The results shown
in Fig. 5 indicate that concentrations of
exogenous immunoglobulins in either
parotid or whole saliva were not differ-
ent than that detected in PBS.
Western blotting of immunoglobulins in
saliva
To determine whether saliva affects the
structure but not the immunoreactivity
of immunoglobulins, purified IgG and
IgA were added to saliva and evaluated
immediately by Western blotting and
compared to immunoglobulins in PBS.
The results shown in Fig. 6 indicate that
saliva had no discernable effect on puri-
fied IgA and IgG compared to that in
PBS. In contrast, saliva treated with
bromelain showed significant degrada-
tion of endogenous immunoglobulins.
Saliva treated with the vehicle control
(Tyrode’s salt solution) showed no
degradation (data not shown).
Discussion
The detection of immunomodulators in
saliva has implications for infectious
diseases. Antibodies can be an indicator
of infection and/or convalescence. A lo-
cal Th1/Th2 cytokine profile may indi-
cate resistance or susceptibility to infec-
tion. Pro-inflammatory cytokines may
be indicative of acute inflammation,
while chemokines are indicative of T-
cell, macrophage, and/or PMN mi-
gration to areas of infection. The po-
tential of saliva as a tool to diagnose
health status is dependent upon several
factors such as detectability and sta-
bility of the named substances in saliva.
Accordingly, we have shown that immu-
nomodulators such as chemokines,
cytokines, and immunoglobulins can be
104 Wozniak et al.

Fig. 4. Effects of bromelain solubilization on

endogenous chemokines and cytokines.
Whole saliva samples (n Ω 15) were treated
with 1 : 1 volumes of either vehicle control
Fig. 3. Effects of saliva on exogenous cytokines. PBS and whole and parotid saliva (n Ω 20) (Tyrode’s salts) or bromelain. Results are ex-
were spiked with 1000 pg/ml of each cytokine (Th1, Th2, or pro-inflammatory) in cocktails. pressed as pg/ml (A) Th1/Th2 cytokines,
Results are expressed as pg/ml detected of (A) Th1/Th2 cytokines and (B) pro-inflammatory (B) pro-inflammatory cytokines, and (C)
cytokines in PBS, parotid saliva, and whole saliva (with endogenous amounts subtracted). chemokines. Asterisks (*) indicate significant
The percentages shown indicate the per cent reduction compared to the PBS spike. Asterisks increases in cytokine/chemokine detection.
(*) represent significant reductions compared to the PBS spike (P ⬍ 0.05). Mean concen- Mean concentrations and standard error of
trations and standard error of the mean (SEM) are shown for each cytokine. the mean (SEM) are shown for each chemo-
kine and cytokine.

detected in saliva by standard ELISA kines were found in relatively low

and are stable in storage, but may be
negatively affected quickly by salivary
components.
Chemokines were found in both pa-
rotid and whole saliva. Of those evalu-
ated, MCP-1 (chemotactic for macro-
phages and T cells) was found at the
highest concentration, followed by IL-8
(chemotactic for PMNs) and RANTES
(chemotactic for T cells), with IL-8 and
MCP-1 detected in 95% of samples. No
significant differences were observed be-
tween the amounts detected in parotid
and in whole saliva. This is the first re-
port of the detection of chemokines in
saliva, although mRNA of chemokines
in oral tissues would have predicted
their presence (44).
Of the cytokines tested, Th-type cyto-
amounts compared to the other cyto-
kines. Despite this, based on concen-
tration, the overall profile in the cohort
was a mix of Th1 and Th2 cytokines
(Th0), similar to our previous finding in
healthy HIV-negative persons (25). In
contrast, median concentrations of pro-
inflammatory cytokines (IL-1a, IL-6,
and TNF-a) were generally higher than
those of Th1 and Th2-type cytokines,
with the vast majority of samples (97%)
showing detectable amounts of pro-in- Fig. 5. Effects of saliva on exogenous im-
flammatory cytokines. Additionally, the munoglobulins. PBS and whole and parotid
saliva samples (n Ω 15) were spiked with 100
majority of the cytokines tested were
mg/ml IgA and 25 mg/ml IgG. Results are ex-
detected at similar levels in whole and pressed as ng/ml IgA and IgG in PBS, pa-
in parotid saliva with no positive corre- rotid saliva, and whole saliva (with endoge-
lation in matched samples. The excep- nous amounts subtracted). Mean concen-
tions were TGF-b, found only in whole trations and standard error of the mean
saliva, IL-1a, found at significantly (SEM) are shown for each immunoglobulin.

Fig. 6. Western blot of salivary immunoglobulins. PBS (control) and whole saliva (n Ω 3) were spiked with 5000 ng/ml (A) IgA or (B) IgG (lanes
1 and 2) and detected with appropriate anti-IgA or IgG antibodies. Lane 3: whole saliva untreated; lane 4: whole saliva treated with bromelain.
The figure shows a representative saliva sample of three tested.
higher concentrations in whole saliva,
TNF-a, found at significantly higher
concentrations in whole saliva, and
IFN-g, which showed a positive corre-
lation in matched samples. Overall, the
lack of differences between concen-
trations of cytokines in whole and pa-
rotid saliva suggests that, in most cases,
the parotid gland is a contributor to
cytokines in whole saliva. The lack of
specific correlations of immunomodula-
tors between whole and parotid saliva
in matched samples suggests that con-
tributions to saliva by the parotid gland
can vary from subject to subject. It is
interesting to note that similar levels of
cytokines and chemokines were found
in parotid and whole saliva, especially
considering that whole saliva contains
contributions from many glands and
from buccal epithelial cells and gingival
crevicular fluid. Of the three cytokines
that showed increased concentrations in
whole saliva relative to parotid saliva,
two were pro-inflammatory cytokines
that are known to be produced by buc-
cal epithelial cells (41). Where similar
concentrations were found in the two
types of saliva, perhaps the glandular
contributions of the chemokines and
cytokines become diluted in whole sal-
Immunomodulator inhibitors in saliva
iva, thereby reducing the concentration.
Formal studies designed to evaluate
putative inhibitory effects of whole and
parotid saliva on exogenous chemo-
kines and cytokines were conducted im-
mediately following the addition of the
cytokine/chemokine cocktail, based on
preliminary stability studies showing
early inhibitory effects. As indicated by
the preliminary results, compared to
spiked PBS samples, the detection of
Th1/Th2 cytokines and chemokines was
clearly reduced/inhibited following con-
tact with both parotid (20–40%) and
whole (⬃75%) saliva. This reduction in
detection is similar to that observed for
cytokines in cervical mucus (12). Cervi-
cal mucus also contains substantial mu-
cins capable of cytokine and antibody
sequestration and/or degradation,
which limits detection by ELISA (12).
In contrast, a smaller inhibition was ob-
served with pro-inflammatory cyto-
kines, without any difference between
parotid and whole saliva. Interestingly,
no inhibition of any kind was found for
IL-1a, consistent with it being at the
highest concentration for any cytokine
tested in either type of saliva. This sug-
gests that saliva can affect cytokines
and chemokines differentially. In the
105
case of IL-1a, the lack of inhibition
could be caused by several factors.
First, since IL-1a is found in signifi-
cantly higher amounts in whole saliva
and is produced by buccal epithelial
cells (41), it may be continually replaced
if inhibited to some level. Secondly, IL-
1a may be resistant to an existing sali-
vary inhibitor(s), suggesting some speci-
ficity of the inhibition in saliva. Size
should not be considered a factor as all
the cytokines are ⬃15–25 kDa.
The inhibition of chemokines and
cytokines in parotid saliva indicates
that components from the parotid
gland, at least in part, contribute to the
inhibition. However, the greater inhi-
bition by whole than by parotid saliva
suggests that the inhibitory compo-
nent(s) is more concentrated in whole
saliva. If the putative inhibitor(s) is a
protein, as expected, this difference in
inhibition is not attributable to salivary
protein concentration, since the protein
content was virtually identical in the
two types of saliva. Alternatively,
whole, but not parotid, saliva contains
mucins similar to that in cervical mucus
(12). Thus, mucins or mucin-like mol-
ecules in whole saliva are expected to
play some role in the cytokine/chemo-
106 Wozniak et al.
kine inhibition. Confirmation will re-
quire submandibular saliva that is al-
most completely composed of mucin.
In contrast to salivary effects on
chemokines and cytokines, immuno-
globulin detection is not affected by
either type of saliva. This may be ex-
plained by the difference in size of im-
munoglobulins compared to cytokines/
chemokines (150 vs. 8–25 kDa, respec-
tively). Alternatively, immunoglobulins
utilized were purified proteins, while the
chemokines/cytokines were recombin-
ant proteins. Thus, post-transcriptional
modifications/glycosylations on endog-
enous proteins may render them resis-
tant to inhibition. If so, one may expect
that the lack of glycosylations on re-
combinant cytokines and chemokines
would render them sensitive to inhi-
bition by other biological fluids. This is
not the case, however, as select recom-
binant cytokines were not affected by
plasma (data not shown). Despite this,
we cannot rule out the possibility that
the inhibition is restricted to recombin-
ant proteins, and that endogenous cyto-
kines/chemokines are not inhibited by
saliva. This is unlikely, however, based
on the differential inhibition observed
for various recombinant cytokines in
saliva. Moreover, detection of purified
immunoglobulins was affected by cervi-
cal mucus (12).
The inhibition of recombinant
chemokines and cytokines by saliva
prompted the question of whether a
putative inhibition of endogenous cyto-
kines and chemokines involved some
form of sequestering or masking of the
chemokines and cytokines from detec-
tion or irreversible degradation. Brome-
lain is an enzyme derived from pine-
apple that degrades large proteins and
solublizes mucins (17, 33). Specifically,
it cleaves proteins similar to papain, a
nonspecific endopeptidase (15, 17, 24,
33, 42, 43). Unlike papain, however,
which can be inhibited by cystatins in
saliva (39), bromelain is not affected by
cystatins (36). Based on the fact that
addition of bromelain significantly in-
creased the detectable amount of sev-
eral endogenous cytokines in whole sal-
iva, a sequestering of some cytokines by
mucin-like proteins is suggested. How-
ever, in the case of chemokines and
other select cytokines in saliva, brome-
lain treatment did not appreciably
change the detectable levels. This could
indicate either that these molecules are
degraded rather than sequestered or
that they are sequestered by a molecule
resistant to bromelain. Noteworthy is
the lack of effects of bromelain on
smaller endogenous molecules (chemo-
kines and cytokines) compared to anti-
bodies that are clearly affected. Perhaps
cleavage sites in the smaller chemokines
and cytokines are not as accessible due
to tighter tertiary structure, or perhaps
they do not contain the appropriate
amino acid sequences for enzymatic
cleavage by bromelain. Yet another
possibility is that cytokines and chemo-
kines are affected, but immunoreactiv-
ity remains intact. This is in fact the
case with immunoglobulins treated with
bromelain (data not shown).
The final and most important con-
sideration is the potential of cytokines
and chemokines in saliva to be used as
a diagnostic tool to indicate oral health
status, and to predict susceptibility to
oral or extra-oral infections. The differ-
ential sequestering and/or degradation
of chemokines, Th1/Th2 cytokines, and
pro-inflammatory cytokines by both
whole and parotid saliva limits the ab-
lilty to detect absolute concentrations
of cytokines and chemokines. Pre-
viously it was suggested that cytokines
detected in whole saliva from patients
with HIV may be indicative of disease
status (i.e. oral candidiasis) (25). These
results should be carefully interpreted
based on present results showing sig-
nificant inhibition or altered detection
of Th1/Th2 cytokines in saliva. How-
ever, in the case of these cytokines, sal-
iva may be treated such that true con-
centrations of at least some cytokines
and chemokines can be detected and
evaluated reliably in different groups of
individuals. Additionally, the present
study shows that bromelain could be
used to treat saliva as it had no demon-
strable effect on endogenous chemo-
kines and cytokines. Use of saliva to
measure antibodies diagnostically
should not present problems, as these
molecules are not affected by saliva,
and, as stated, can be detected equally
in bromelain-treated samples despite
degradation. However, use of saliva as
a diagnostic tool for chemokines and
some other select cytokines (i.e. TNF-
a, and TGF-b) is not encouraged be-
cause of the potential for degradation.
Thus, the choice of cytokines and
chemokines will be important in deter-
mining the diagnostic value. Overall,
while saliva has potential as a diagnos-
tic tool to indicate health status relative
to medically important conditions and
infectious disease, the value of each im-
munomodulator for this purpose will
need to be evaluated independently.
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