Responses of human neutrophils to nicotine and/or Porphyromonas gingivalis.
ABSTRACT Tobacco smoking is considered a major modifiable risk factor for periodontal disease. Nicotine is the addictive ingredient in tobacco and has been shown to affect multiple cellular processes. Neutrophils are the first line of host defense and are critical cells in the maintenance of periodontal health through their role in the control of bacteria, but they can also contribute to the progression of periodontal disease by the production and release of reactive oxygen species (ROS). Virulence factors from periodontal pathogens, such as Porphyromonas gingivalis (Pg), stimulate the respiratory burst of neutrophils. The objective of this study is to explore the oxidative activity of neutrophils when stimulated with Pg, nicotine, or both.
Neutrophils were separated from buffy coats by the double dextran gradient method. The generation of ROS by neutrophils was determined using luminol-dependent chemiluminescence assays. The reaction was followed for 90 minutes, and the neutrophil activation was recorded as the total integrated energy output.
The Pg and Pg plus nicotine groups had a significantly higher active and peak chemiluminescence than the nicotine group (all with P <0.0001). The Pg and Pg with nicotine groups were not significantly different (P = 0.90).
In the presence of Pg, the nicotine did not further enhance the ROS release by the neutrophils, suggesting that the bacteria induced the maximum ROS release in this model system.
- [Show abstract] [Hide abstract]
ABSTRACT: Tobacco smoking is a significant risk factor for periodontal diseases. Nicotine, one of the most studied constituents in cigarette smoke, is thought to modify immune responses. Dendritic cells (DCs), which are key mediators between innate and adaptive immunity, stimulate naive T cells to differentiate to effector T-cell subsets that may be actively involved in the immunopathogenesis of periodontal diseases. In this study, we evaluated the effects of nicotine and lipopolysaccharide (LPS) from Porphyromonas gingivalis, alone and in combination, on the functions of human monocyte-derived DCs to elucidate the mechanism of tissue destruction of smoking-associated periodontal diseases. P. gingivalis LPS-stimulated DCs differentiated with nicotine (NiDCs) induced lower T-cell proliferation and human leukocyte antigen (HLA)-DR expression, but elevated expression of programmed cell death ligand 1. Additionally, NiDCs impaired interferon-γ production but maintained interleukin (IL)-5 and IL-10 production in co-cultured T cells. Furthermore, NiDCs produced lower levels of proinflammatory cytokines compared with DCs differentiated in the absence of nicotine. Interestingly, NiDCs preferentially produced the T helper 2 (Th2)-type chemokines macrophage chemotactic protein-1 and macrophage-derived chemokine. These results suggest that the presence of nicotine during differentiation of DCs modulates the immunoregulatory functions of P. gingivalis LPS-stimulated DCs.European Journal Of Oral Sciences 10/2012; 120(5):408-14. · 1.73 Impact Factor
Endotoxemia and thehost
systemic response during
Wahaidi VY, Kowolik MJ, Eckert GJ, Galli DM: Endotoxemia and the host systemic
response during experimental gingivitis. J Clin Periodontol 2011; 38: 412–417. doi:
Aim: To assess endotoxemia episodes and subsequent changes in serum inflammatory
biomarkers using the experimental gingivitis model.
Materials and Methods: Data from 50 healthy black and white adult males and
females were compared for serum concentrations of endotoxin, and serum biomarkers
[neutrophil oxidative activity, interleukin (IL)-1b, IL-6, IL-8, C-reactive protein
(CRP), and fibrinogen] at baseline, at 3 weeks of experimental gingivitis, and after
2 weeks of recovery. Means were compared using repeated measures analysis of
Results: Endotoxemia was reported in 56% of the serum samples at 3 weeks of
induced gingivitis. At 2 weeks of recovery, endotoxin levels decreased to levels similar
to those reported at baseline. Neutrophil oxidative activity increased significantly
following 3 weeks of gingivitis versus baseline (po0.05). In the endotoxin-negative
group this increase was associated with the black subjects whereas in the endotoxin-
positive group change in neutrophil activity was driven by the female subpopulation.
Serum cytokines, CRP, and fibrinogen levels did not change during the study.
Conclusions: Experimental gingivitis was associated with endotoxemia and
hyperactivity of circulating neutrophils, but not with changes in systemic levels of
cytokines and acute-phase proteins. This may be attributed to the mild nature and the
short duration of the induced gingivitis.
Key words: acute-phase proteins; cytokines;
dental plaque; endotoxin; experimental
gingivitis; inflammatory response; neutrophils
Accepted for publication 15 January 2011
There is widespread interest in studying
the systemic impact of oral infection.
Gingivitis is universal, reversible, and
rapidly inducible. Thus, it provides an
appropriate model for investigation of
the host response to oral infection. It can
(EGM) (Lo ¨e et al. 1965).
Gingivitis is associated with thinning
of the sulcular epithelium and dilation of
the gingival vasculature (Zoellner et al.
2002), thus facilitating invasion of the
epithelial barrier by oral bacteria and/or
their products into the systemic circula-
tion. A distinctive bacterial product that
can be found in blood is endotoxin,
which is a cell wall component of
Gram-negative bacteria. It has been
shown previously that dental plaque
maturation in the EGM is accompanied
by a shift towards increasingly Gram-
negative bacteria (Theilade et al. 1966)
including periodontal pathogens of the
red and orange complex species (Salvi
et al. 2005). Indeed, it has been sug-
gested that an infected periodontium is a
major source of persistent endotoxemia,
even as a consequence of gentle masti-
cation (Geerts et al. 2002). Furthermore,
periodontal pathogens have also been
detected in atheromatous plaques and
in abdominal aortic aneurysms, further
supporting the occurrence of bactere-
(Haraszthy et al. 2000, Kurihara et al.
In addition to producing bacteremia/
endotoxemia, we hypothesized that den-
tal plaque accumulation may elicit a
host systemic inflammatory response to
the local dental plaque and/or bactere-
mia/endotoxemia. In this study potential
changes in levels of serum cytokines
and acute-phase proteins as well as
Vivian Y. Wahaidi1, Michael J.
Kowolik1, George J. Eckert2and
Dominique M. Galli3
1Department of Periodontics and Allied
Dental Programs, Indiana University School
of Dentistry, Indianapolis, IN, USA;2Division
of Biostatistics, Indiana University School of
Medicine, Indianapolis, IN, USA;
3Department of Oral Biology, Indiana
University School of Dentistry, Indianapolis,
Conflict of interest and source of
This study was supported by a research
grant provided by National Institutes of
Health (grant #R01 DEO1514501).
The authors declare that they have no
conflicts of interest related to this study.
J Clin Periodontol 2011; 38: 412–417 doi: 10.1111/j.1600-051X.2011.01710.x
r 2011 John Wiley & Sons A/S
neutrophil oxidative activity as a result
of gingivitis were assessed.
Peripheral blood neutrophils are the
primary cells of the acute inflammatory
response. Neutrophils can mediate tissue
destruction in inflammatory diseases
(Hansen 1995). Dental plaque accumula-
tion and periodontal disease elicit a per-
ipheral neutrophil response (Kowolik et
al. 2001, Matthews et al. 2007, Wahaidi
et al. 2009), which includes the release of
enzymes, both of which possess detri-
mental tissue-damaging effects.
In response to infection, inflamma-
tory cytokines promote a series of
events, at the local infection site and
systemically (Gabay & Kushner 1999).
Dental plaque accumulation in both
spontaneous and experimental gingivitis
induces changes in gingival crevicular
fluid cytokine levels (Ebersole et al.
1993, Offenbacher et al. 2010, Salvi et
al. 2010, Trombelli et al. 2010) with
potential for systemic dissemination
(Ebersole et al. 1999). Human experi-
mental endotoxemia is reported to
induce changes in levels of serum cyto-
kines (van Eijk et al. 2007, Dorresteijn
et al. 2010). Systemic effects of these
cytokines include production of acute
phase reactants (Baumann & Gauldie
1994), leukocyte recruitment (Yoshi-
mura et al. 1987), upregulation of
endothelial adhesion factors (Munro
1993), and induction of procoagulation
(Nachman et al. 1986).
An increase in serum levels of C-
reactive protein (CRP) and fibrinogen
occurs in response to infection including
periodontal infection (Kweider et al.
1993, Noack et al. 2001, Beck &
Offenbacher 2002, Mattila et al. 2005,
Paraskevas et al. 2008). CRP has pro-
thrombotic effects (Cermak et al. 1993)
and activates complement (Volanakis
1982). Fibrinogen increases blood visc-
osity (Lowe et al. 1997), promotes pla-
telet aggregation (Cook & Ubben 1990),
and stimulates smooth muscle prolifera-
tion (Ishida & Tanaka 1982).
derived endotoxin and the potential sub-
sequent host systemic response has not
been previously investigated in gingivi-
tis, including the EGM.
This substudy was part of a larger
investigation (subject N5128) that
examined the systemic response to den-
tal plaque accumulation, using the EGM
in a population of black and white
healthy adult male and female subjects
(Wahaidi et al. 2009). The results of this
study indicated that neutrophil activity
increased in black but not white subjects
in response to experimental gingivitis.
No gender differences were noted.
The aim of the present study was to
compare data from a subpopulation
(n550) of the original study (Wahaidi
et al. 2009), to determine if (1) dental
plaque accumulation and the ensuing
gingivitis results in endotoxemia, and
(2) whether the presence of endotoxin
would result in race and gender-asso-
ciated differences in a subsequent host
Materials and Methods
The original study has been described in
detail previously (Wahaidi et al. 2009).
The protocol was approved by the Insti-
tutional Review Board of Indiana Uni-
versity Purdue University Indianapolis/
Clarian Health (approval number 0405–
50). All participants provided written
informed consent for participation. One
week after all subjects had received a
professional oral prophylaxis and oral
hygiene (OH) instructions, they entered
the three study phases (Table 1). In the
‘‘control phase’’ (weeks 0–3) subjects
performed optimal OH practices. In the
‘‘experimental phase’’ (weeks 3–6),
subjects refrained from all OH measures
including the use of dentifrices, mou-
thrinses, dental floss, and gum. At the
end of the experimental phase, which
was also the start of the ‘‘recovery
phase’’ (weeks 6–9) subjects received
a professional oral prophylaxis and
resumed normal OH practices (Table
1). Subjects visited the clinic weekly.
During each visit, oral examinations
were conducted, dental plaque and gin-
givitis were assessed, and peripheral
blood samples were collected. Serum
was separated and stored for laboratory
analyses. The blood samples at week 6
were collected before the administration
of oral prophylaxis.
This sub-study analysed data received
from healthy adults (n550, whites5
34, blacks516, males522, females5
28) (mean age: 24.7, range: 18–30
years) during weeks 3 (end of the control
phase and beginning of the experimental
phase), 6 (end of the experimental phase
and beginning of the recovery phase),
and 8 (2 weeks of the recovery phase) of
the study. The subjects were in posses-
sion of a minimum of 20 natural teeth
and showed no signs of periodontal
o4mm) or gross dental caries. Exclu-
sion criteria included the current use of
tobacco products, any medication known
to affect the oral soft tissue or local/
systemic response, or antimicrobial pro-
ducts within 3 months preceding the
Oral examinations and assessments of
dental plaque and gingivitis were per-
formed by a single trained and cali-
brated examiner (weighted k for intra-
examiner reproducibility 40.7) using
the plaque index (PI) (Silness & Lo ¨e
1964) and gingival index (GI) (Lo ¨e &
Table1. Overview of the clinical procedures performed during the study
7 days before studyControl phase Experimental phaseRecovery phase
Week of study
Plaque and gingivitis assessment
Oral hygiene practices
Only data from weeks 3, 6, and 8 were analysed in this sub-study.
nSame day of study.
wSame day of study.
zThe blood sample at week 6 was collected before the administration of oral prophylaxis.
Endotoxemia and the systemic response
r 2011 John Wiley & Sons A/S
Silness 1963), respectively. Measure-
ments were made on six sites per tooth
mesiolingual, lingual, distolingual) on
all teeth present, with the exception of
Serum endotoxin levels were assayed
using a Limulus Amebocyte Lysate
commercial kit (Charles River Labora-
tories, Charleston, SC, USA). The
(Charles River Laboratories), a kinetic
colorimetric assay, was used for quanti-
fication of endotoxin levels. All samples
were processed in duplicate.
Neutophils were isolated by a density
gradient centrifugation method (Wahai-
di et al. 2009). Total neutrophil activity
was measured via luminol-enhanced
chemiluminescence using a 1251 Bio-
Orbit Luminometer (Bio-Orbit, Turku,
Finland) for 90min. (Wahaidi et al.
2009) and expressed in millivoltage/
min. Samples were processed in tripli-
Serum cytokine levels were assayed
using commercial immunoassay kits. The
LINCO plext multiplex immunoassays
kit (Linco Research Inc., St. Charles,
MO, USA) was used for quantification
of interleukin (IL)-1b, IL-6, and IL-8.
Samples were processed in duplicate.
Plasma fibrinogen levels were mea-
sured using an automated clot detection
system in the Clarian Health Labora-
tories. A commercial kit, the Activet
CRP ELISA (Diagnostic
Laboratories Inc., Webster, TX, USA),
was used for quantification of CRP.
Samples were processed in duplicate.
Means and 95% confidence intervals
were calculated. Comparisons between
means of the endpoints of the three
different phases were performed using
repeated measures analysis of variance,
using the log-transformed data due to
the skewed distributions of the measure-
ments. The level of statistical signifi-
cance was set at 0.05.
No measurable endotoxin was detected
in weeks 4 and 5 of the experimental
phase. The percentage of endotoxin-
positive subjects at week 6 was overall
56% (whites: 59%, blacks: 50%, males:
50%; and females: 61%). In the endo-
endotoxin levels (EU/ml) were signifi-
cantly higher at week 6 versus week 3
(po0.0001) (Table 2) with a mean of
0.72EU/ml for whites, 0.78EU/ml for
0.81EU/ml for females. These levels
decreased significantly at week 8 to
levels similar to those reported at base-
Plaque and gingivitis
PI and GI increased significantly at
week 6 versus week 3 (po0.0001) in
all subjects as reported previously
(Wahaidi et al. 2009). This increase
was independent of endotoxin status
(Table 2). PI and GI decreased signifi-
cantly at week 8 versus week 6
(po0.0001), although week 8 PI values
were still significantly higher than base-
Total chemiluminescence increased sig-
nificantly during the experimental phase
with a p value of o0.001 in the endo-
toxin-negative group and 0.009 in the
endotoxin-positive group (Table 2).
This change was dependent on race in
the endotoxin-negative group with an
increase of 5.88 in blacks (po0.001)
but only 2.95 in whites (p50.051) (Fig.
1). In the endotoxin-positive group gen-
der influenced overall neutrophil activ-
ity with a significant increase of 5.77 in
females (po0.001) versus a decrease in
activity by 1.94 in males (p50.131)
(Fig. 1). Neutrophil activity for blacks
(p50.038) and females (p50.006)
returned to baseline levels during the
During the course of the study, no
statistically significant changes were
observed in levels of the analysed cyto-
kines (p40.05). Moreover, there were
no significant differences in the fre-
quency (defined as a measurable con-
detection of cytokines between endotox-
in-positive and endotoxin-negative sub-
jects (p40.05) (data not shown). High
individual variability in the pattern of
cytokine profiles was observed (data not
Levels of serum CRP and fibrinogen
did not change significantly during the
study course for any of the groups
To our knowledge, this is the first report
that directly links gingivitis to endotox-
emia. In this study, levels of systemic
endotoxin increased significantly fol-
lowing 21 days of dental plaque accu-
mulation in the majority of subjects,
with no differences between individuals
Table2. Mean, 95% confidence intervals, and significance for study outcomes over the course of the study
Total neutrophil activity
8.39nn(5.88, 10.9) 5.54
0.15 (0.13, 0.18) 2.12nnn(2.00, 2.24)
0.46 (0.42, 0.49) 1.22nnn(1.16, 1.28)
5.47 (4.20, 6.74) 9.44nnn(7.21, 11.7)
8.51 (6.28, 10.8)
0.14 (0.12, 0.16)
0.41 (0.38, 0.44)
5.77 (3.81, 7.74)
Comparisons between means of the control and experimental phase, and the experimental and recovery phase within each group were performed using
repeated measures ANOVA.
Wahaidi et al.
r 2011 John Wiley & Sons A/S
of different race/gender. As anticipated,
resolution of gingivitis following treat-
ment resulted in the reduction of endo-
toxin to baseline levels.
Only endotoxemia was evaluated in
this study, not bacteremia. Endotoxemia
is a surrogate marker for Gram-negative
comprise a significant portion of the
mature subgingival plaque flora (Thei-
lade et al. 1966). Whereas endotoxemia
occurred at a high frequency in a very
short period of time in the present
study, only low levels of bacteremia in
individuals with periodontal disease and
reported (Kinane et al. 2005, Forner
et al. 2006). This may be due to the
challenges associated with blood culture
Endotoxemia, at sufficient levels, is a
potent immune and inflammatory stimu-
lator (Guthrie et al. 1984, Lindemann et
al. 1988), and may ultimately, in the
long run, result in multiple organ system
failure (Brandtzaeg et al. 1989). With a
mean of 0.74EU/ml, the amount of
serum endotoxin detected in this study
was relatively low. This may be attrib-
uted to the low severity of gingivitis as a
periodontal infection. Indeed, it has
been shown that levels of endotoxemia
are positively associated with the sever-
ity of periodontal disease (Geerts et al.
2002). Nonetheless, although mild, as a
chronic insult gingivitis may cause per-
sistent delivery of endotoxin into the
systemic circulation. In fact, a systemic
endotoxin level 450pg/ml (equivalent
to 0.5 EU) in individuals with chronic
infections has been identified as a risk
factor for early atherogenesis (Wieder-
mann et al. 1999).
Endotoxin is known to stimulate neu-
trophil activity (Guthrie et al. 1984,
Fittschen et al. 1988), although the
effect of endotoxin on neutrophils varies
depending on whether endotoxin is free
or bound. Persistent neutrophil hyperac-
tivity plays a role in tissue destruction
(Di Filippo et al. 2007, Wittkowski et al.
2007). In this study, a systemic periph-
eral neutrophil response was noted fol-
lowing 3 weeks of dental plaque
accumulation. We previously reported
a heightened neutrophil response in
blacks versus whites following 3 weeks
of experimental gingivitis in healthy
adults (Wahaidi et al. 2009). Results of
the current study showed that the neu-
trophil response following dental plaque
accumulation in blacks was independent
of endotoxin status. In contrast, an asso-
ciation between endotoxin and neutro-
phil activity was observed as it relates to
showed a significant increase in neutro-
phil activity in week 6 that dropped back
to baseline levels during the recovery
phase. In contrast, neutrophil activity for
males decreased in the experimental
phase, although the change was not
significant. In accordance with our find-
ings, gender disparity has been reported
in the acute neutrophil inflammatory
response. In a rat model an increase in
phagocytic activity in circulating neu-
trophils was noted in female but not
male animals in response to an endotox-
in challenge (Spitzer 1999). Further-
more, van Eijk et al. (2007) reported
a more pronounced
response in females in a human experi-
dimorphism in the innate inflammatory
response including neutrophil biology
has been associated with the effect of
sex hormones (Miyagi et al. 1992, Mol-
loy et al. 2003).
In the present study, experimental
gingivitis and the subsequent endotox-
emia were not associated with changes
in serum levels of cytokines and acute-
phase proteins. However, high indivi-
dual variability in the pattern of cyto-
agreement, earlier studies demonstrated
no association between clinical para-
meters of periodontitis and serum levels
of cytokines including IL-1b and IL-6,
and a high variability of cytokine pro-
files (Chen et al. 1997, Gorska et al.
2003). Likewise, the literature supports
a gingival crevicular fluid cytokine
experimental gingivitis (Deinzer at al.
2007, Andriankaja et al. 2009, Salvi
et al. 2010, Trombelli et al. 2010), but
related changes in serum levels of cyto-
kines have not been detected (Andrian-
kaja et al. 2009, Trombelli et al. 2010).
The inability to detect systemic cyto-
kines may be explained by the mild
nature of gingivitis (Trombelli et al.
2010) and the low level of the resultant
endotoxemia. In contrast, changes in
cytokine levels have been observed in
induced endotoxemia (van Eijk et al.
2007, Dorresteijn et al. 2010) where
serum endotoxin levels were signifi-
cantly higher when compared with the
Furthermore, it has been proposed
that circulating levels of cytokines
depend upon multiple factors including
not just the type of stimulant but also the
duration of exposure to the stimulant (Di
Padova et al. 1991). Hence, failure to
detect significant changes in serum
inflammatory markers may also be
attributed to the short duration of the
observed endotoxemia. It is important to
note that increases in serum endotoxin
levels were reported at week 6 of this
study, but not at week 5. In fact, it is
very likely that an extension of the
experimental phase by an additional
week would have resulted in an increase
Fig.1. Mean (? 95% confidence interval) peripheral blood neutrophil total activity (chemiluminescence) by race and gender at weeks 3
(baseline), 6 (experimental phase), and 8 (recovery phase). Comparisons between means of the control, experimental and recovery phase
within each group were performed using repeated measures analysis of variance.nnnpo0.001;nnpo0.01;npo0.05.
Endotoxemia and the systemic response
r 2011 John Wiley & Sons A/S
in number of endotoxin-positive sub-
jects and possibly endotoxin concentra-
tion. Instead, the short time-frame
used in our study was probably insuffi-
cient to induce prolonged endotoxemia
and an ensuing systemic response. Such
a finding may be of importance in
understand the temporal aspect of the
potential systemic response to endotox-
emia following the development of
It should be noted that, although well-
controlled, the conditions of the EGM
differ from those of spontaneous gingi-
vitis (Deinzer et al. 2007). The EGM
involves meticulous OH measures, fol-
lowed by complete neglect of OH for 21
days. In comparison, during sponta-
neous gingivitis dental plaque, in some
areas, accumulates for a longer duration
and some OH is practiced, which results
in transient bacteremia (Silver et al.
1977). It is conceivable that endotoxin
levels would have been much higher in
the present study had the blood samples
been collected immediately after the
subjects received dental prophylaxis in
week 6. These discrepancies between
the EGM and spontaneous gingivitis
may affect the nature and level of the
resultant bacteremias/endotoxemias and
the systemic host response.
Although the results from this study
did not show a definitive host systemic
response to dental plaque accumulation,
there were findings of potential clinical
relevance. The development of gingivi-
tis was the origin of systemic endotox-
emia and hyperactivity of circulating
neutrophils, the latter being associated
with gender in endotoxin-positive sub-
jects. The increase in neutrophil activity
observed in this study could be linked to
the local oral infection (i.e. gingivitis),
to the circulating bacteria/endotoxin, or
to both. As a chronic insult, spontaneous
gingivitis may cause persistent delivery
of endotoxin into the systemic circula-
tion. In the long-term, this ‘‘little and
often’’ endotoxemia and inflammatory
response may cumulatively cause detri-
mental systemic effects. The disparity in
the race/gender systemic neutrophil
response, in the endotoxin-positive and
endotoxin-negative groups, is worthy of
This study was supported by NIH grant
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Dominique M. Galli
Department of Oral Biology
Indiana University School of Dentistry
1121 West Michigan Street
Scientific rationale for study: Endo-
response may play a central role in
mediating the oral-systemic disease
and heightened neutrophil oxidative
Practical implications: Gingivitis is a
potential source of endotoxemia and
neutrophil activation. In the long-run,
persistent endotoxemia and neutro-
phil hyperactivity may be of clinical
significance. In this study, endotoxe-
mia and neutrophil activity in endo-
toxin-positive subjects decreased to
baseline levels following a profes-
sional oral prophylaxis and practicing
Endotoxemia and the systemic response
r 2011 John Wiley & Sons A/S