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Curr Oral Health Rep (2014) 1:95–103
DOI 10.1007/s40496-014-0015-x

IMMUNOLOGY (G NUSSBAUM, SECTION EDITOR)

Neutrophil Dysfunction and Host Susceptibility to Periodontal
Inflammation: Current State of Knowledge
Corneliu Sima & Michael Glogauer

Published online: 25 March 2014
# Springer International Publishing AG 2014

Abstract Normal polymorphonuclear neutrophil (PMN)
function is critical for the maintenance of host-biofilm equilibrium and periodontal tissue homeostasis. Mounting evidence suggests that PMNs play important roles in the control
of commensal periodontal flora and initiation of resolution
following inflammation caused by accumulating subgingival
plaque. Quantitative and qualitative alterations of PMNs in
bone marrow, blood, periodontal tissues, and gingival crevicular fluid contribute to host-microbial dysbiosis and onset of
irreversible loss of clinical attachment around teeth. Recent
findings of specific PMN phenotypes associated with different
disease states bring us closer to understanding disease activity
and addressing chronic, non-resolved, periodontal inflammation to better monitor and predict patient-specific treatment
outcomes. The present review addresses the current state of
knowledge in PMN biology in the pathogenesis of periodontal
inflammation and the onset of periodontitis.
Keywords Neutrophil . Inflammation . Gingivitis .
Periodontitis . Host-biofilm . Innate immunity

Introduction
Polymorphonuclear neutrophils (PMNs) are the most abundant leukocytes in humans accounting for 50–70 % of all
circulating white blood cells. They are produced by the bone
marrow at a rate of approximately 109 cells/kg per day from
common myeloid precursors. A combination of stem cell
factors, interleukin (IL)-3, and granulocyte macrophage
C. Sima : M. Glogauer
Matrix Dynamics Group, University of Toronto, Toronto, ON,
Canada
C. Sima : M. Glogauer (*)
Faculty of Dentistry, Department of Periodontology, University of
Toronto, Room 221 Fitzgerald Building, 150 College Street, Toronto,
ON M5S 3E2, Canada
e-mail: michael.glogauer@utoronto.ca

colony-stimulating factor (GM-CSF) is required for commitment to the myeloid lineage [1]. Differentiated PMNs are
released into blood as band (incompletely matured) or segmented (mature) neutrophils after a 12-day maturation period.
Classically, it was thought that PMNs spend 6–8 h in the
circulation before they migrate into the tissues where they
can remain for days and then undergo apoptosis and be cleared
by macrophages. More recently, several studies have shown
that PMNs can circulate in the blood for up to 5 days [2, 3]. In
the extravascular compartment, PMNs participate in several
critical functions of the innate immune system including
phagocytosis and killing of microorganisms, extra-cellular
matrix degradation, and post-inflammatory restoration of tissue homeostasis. It is generally accepted that PMN recruitment and microbicidal function are essential for the maintenance of periodontal health. Research has shown that deviations from normal PMN activity, including altered
production, release, recruitment, impaired function, or
hyper-reactivity with the exception of some forms of
reduced superoxide production, are associated with disruption of periodontal tissue homeostasis that results in
tissue destruction and loss of clinical attachment [4•].
Furthermore, PMNs play important roles in periodontitis
pathogenesis through impaired apoptotic programs and
activation of adaptive immunity that leads to failure to
resolve inflammation and disease progression.

Neutrophil Physiology in the Periodontium
Neutrophil Recruitment
Several lines of evidence suggest that PMN recruitment to,
and function in the periodontal tissue are essential for maintaining homeostasis. It is estimated that 30,000 PMNs transit
through periodontal tissues every minute and that their presence in the gingival crevicular fluid (GCF) is physiological. In
fact, >90 % of GCF cells are PMNs and they form a barrier
between the junctional epithelium and the subgingival biofilm

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preventing its apical migration [5]. IL-8 and intercellular
adhesion molecule 1 (ICAM-1) gradients in the junctional
epithelium mediate PMN migration to the gingival sulcus
[6]. In gingival post-capillary venules, activated endothelial
cells increase the expression of chemokines (macrophage
inflammatory protein 2-alpha, MIP2-α or CXCL2 and IL-8
or CXCL8), selectins (P and -E selectin or CD62P and
CD62E), and intercellular adhesion molecules or ICAMs for
capture, rolling, and attachment of circulating PMNs. Activated PMNs increase surface expression of chemokine receptors
(CXCR1 or CD181, CXCR2 or CD182), selectin ligands (Pselectin glycoprotein ligand 1 or PSGL-1 or CD162 and
SLewx containing glycoproteins), and β2-integrins (lymphocyte function-associated antigen 1 [LFA-1] or CD11a/CD18).
The role of commensal periodontal bacteria in PMN recruitment is incompletely understood. Recent evidence suggests
that PMN migration into the healthy periodontium is mediated
by selective CXCL2 chemokine upregulation in response to
non-pathogenic periodontal bacteria [7]. These observations
support the current paradigm that chemoattractants can be
functionally divided into “intermediate”, found at the blood
tissue interface (CXCL8, LTB4) and generally produced by
endothelial cells, and “end-target” chemoattractants, found in
the immediate vicinity of bacteria (formylmethionyl-leucylphenylalanine; fMLP, C5a). It appears that PMNs preferentially respond to the latter group [8•]. It is therefore possible
that specific recruitment mechanisms result in different PMN
phenotypes in the tissue and that sequential chemokine engagement can also mediate recruitment. Prior to transmigration into gingival tissues, PMNs undergo rolling mediated
by P-selectin-PSGL-1 interactions followed by slow
rolling and attachment mediated by LFA-1-ICAM-1 interactions. Using a mouse model of acute gingivitis, we have
shown that the leukocyte rolling rate 2 h after proinflammatory stimulation with tumor necrosis factor-α shows a
dose-response relationship with the stimulus [9]. Several
physiological inhibitors of PMN integrins have been described. Galectin-1, Lipoxin A4, and Resolvin D series
interfere with integrin activation by altered transcription,
decreased expression of endothelial ligands, and decreased upregulation. Pentraxin 3 (PTX-3), growth differentiation factor 15, and developmental endothelial locus
1 (del-1) interfere with the interaction between integrins
by antagonism of receptor engagement, interception of
inside-out signaling, and antagonism of integrin engagement, respectively [10]. At the disease level, Lipoxin A4
was shown to prevent bone loss and mediate bone regeneration in a rabbit model of ligature-induced periodontitis
[11], while del-1 was shown to inhibit PMN infiltration
and IL-17-mediated alveolar bone loss in aging mice.
Further, del-1-deficient mice developed spontaneous
periodontitis characterized by PMN infiltration and IL17 expression [12].

Curr Oral Health Rep (2014) 1:95–103

Bacterial Killing
It is currently generally believed that periodontitis is a
polymicrobial inflammatory disease initiated by bacteria,
and that several bacteria are associated with disease; however,
which organisms actually initiate it remains unknown. PMNs
play key roles in the control of periodontal biofilm composition by the killing and clearance of pathogens. PMNs can
eliminate pathogens by phagocytosis and intracellular killing
through oxidative and proteolytic means, and by extracellular
mechanisms such as degranulation and release of neutrophil
extracellular traps (NETs). Phagocytosis is exponentially enhanced by opsonization through IgG and complement protein
C3b. Following recognition of opsonized pathogens, the PMN
forms phagocytic cups and then phagosomes, which ultimately fuse with lysosomes to form digestive vacuoles
(phagolysosomes) where oxidative and proteolytic antimicrobial molecules are released into an acidic milieu. These include lactoferrin, lysozyme, β2-microglobulin, MMP 2 and 9
(collagenase and gelatinase), histaminase, heparinase,
sialidase, superoxide, hypochlorous acid (HOCl), and
peroxynitrate (ONOO-). Although GCF PMN can phagocytose bacteria in the gingival crevice, this may not be the
predominant mechanism of protection in this particular niche.
When exposed to pathogens, PMN employ efficient oxidative
means to kill phagocytosed bacteria mainly through production of reactive oxygen species (ROS) and derivatives (HOCl,
ONOO-). However, ROS generation by PMN may not be
critical for maintenance of periodontal health because patients
with chronic granulomatous disease having PMNs deficient in
producing ROS do not have increased susceptibility to periodontitis [13, 14]. Interestingly, it has been shown that patients
with a hyperactive ROS response are more susceptible to
periodontitis [15, 16]. Degranulation may be a more powerful
mechanism for control of compositional biofilm changes [5].
The relationship between crevicular PMN and commensal
periodontal bacteria is poorly defined. It is believed that
similar to intestinal bacteria, oral bacteria associated with
periodontal health induce immune tolerance and prevent the
host immune system from being activated [17]. It is also likely
that non-invading commensal bacteria in the gingival sulcus
maintain the continuous influx of PMN that contribute to
control of subgingival biofilm composition. The shift from
in-offensive to pathogenic subgingival biofilms remains poorly understood. Recent evidence suggests that some periodontal bacteria associated with disease such as Porphyromonas
gingivalis can influence the pathogenicity of subgingival
biofilms by disrupting the host-microbial homeostasis. On
the one hand, P. gingivalis can stimulate proinflammatory
cytokine production by PMNs and alter their apoptosis
through upregulation of Triggering Receptor Expressed on
Myeloid cells 1 (TREM-1) [18]. On the other hand, it can
trigger changes in the composition and amount of commensal

Curr Oral Health Rep (2014) 1:95–103

bacteria ultimately leading to alveolar bone loss [19•]. Therefore, it seems that the persistence of PMNs inside periodontal
tissues combined with ineffective bacterial killing through
non-oxidative means contributes to disease activity and tissue
breakdown in periodontitis.

Neutrophil Extracellular Traps
NETs are extracellular fibers extruded actively by the PMN as
a type of biological “spider’s web.” NETs are made up of
granule and nuclear constituents that entrap and kill bacteria in
the extracellular space [20, 21]. Only about 30 % of transiently
resident PMN release NETs with some evidence that only
viable cells can produce them. The current hypothesis of
NET formation states that the dying neutrophil is characterized by nuclear swelling, chromatin degradation, and extracellular extrusion of large strands of de-condensed nuclear or
mitochondrial DNA that carry with them proteins from the
cytosol, granules, and histones from the nuclei [22]. NETs
entrap bacteria but they do not appear to be bactericidal.
Incubation of entrapped Staphylococcus aureus bacteria and
Candida albicans blastospores with DNAse did not result in
the killing of these pathogens [23]. The presence of NETs is
significantly increased in areas of gingivitis compared with
healthy gingival tissues.
Initiation of NET formation is dependent on ROS and
patients with chronic granulomatous disease cannot form
NETs [24]. Upon ROS release, peptidyl arginine deiminase4 is activated. The latter is known to hypercitrullinate the
condensed nuclear chromatin promoting chromatin decondensation. PMN elastase translocates to the nucleus where
it digests nucleosomal histones assisting chromatin unfolding
[25]. Ultimately, the space between inner and outer nuclear
membranes enlarges, forming distinct vesicles that fuse with
granule membranes resulting in the release of elastase,
myeloperoxidase, and LL-37, and co-localization with nuclear
chromatin. Disintegration of the nuclear envelope allows for
the DNA/histone complex mixed with granular contents to fill
the cytoplasmic space. The final stage involves rupture of the
PMN cell membrane and extrusion of the DNA/histone/
cathelicidin antimicrobial peptide mix through changes in
the actin cytoskeleton and the microtubular complex [21].
Although the role of NETs in pathogenesis of periodontitis
is at an early stage of research, the increased DNAse activity
in gingival crevicular fluid during 21-day experimental gingivitis would suggest that removal of biofilm or NET-derived
DNA is necessary for inflammation resolution and restoration
of health. Therefore, it appears that NET formation in periodontal tissues may not be critical for control of biofilm
composition because patients with chronic granulomatous
disease do not have increased susceptibility to periodontitis,
but NET persistence at sites of periodontal inflammation

97

may be associated with failure of resolution and onset
of periodontitis.
Resolution of Inflammation
Resolution of inflammation is a tightly regulated active process that follows successful removal of non-self. It consists of
switching off proinflammatory pathways and clearing local
tissue debris ultimately leading to complete restoration of
homeostasis. Essential signals from front-line PMNs are needed to initiate resolution. Inside the tissues, PMNs coordinate a
lipid mediator switch from proinflammatory (prostaglandins,
leukotrienes) to pro-resolving arachidonate and omega-3 fatty
acid derivatives (lipoxins, resolving, protectins, and
maresins). It is the lipoxins that provide important signals to
switch from inflammatory states characterized by PMN infiltration and activation to resolution and return to tissue homeostasis [26••]. Lipoxin A4 is known to inhibit PMN migration
and stimulate non-inflammatory recruitment of monocytes
and apoptotic PMN phagocytosis by macrophages [27••].
Once it has accomplished its mission, the PMN triggers selfdestroying mechanisms in a non-inflammatory manner initiating apoptosis through sequential activation of caspases 8, 9,
7, and 3, and activation of calpains and ubiquitin-proteasome
complexes [28, 29]. This results in destruction of essential
cytoplasmic proteins including the ubiquitously present actin,
and chromatin inter-nucleosomal degradation in the nucleus.
Aged or dying PMN simultaneously expose
phosphatidycholine (PC), phosphatidylethanolamine (PE),
and phosphatidylserine (PS), oxidized phospholipids, and carbohydrates including fucose and N-acetyl-glucosamine on
their surface [30]. Several pattern recognition molecules
(PRM) bind some of the apoptotic cell surface markers and
act as bridging molecules to enhance phagocytosis of apoptotic PMNs by macrophages. PRM that have been correlated
with apoptotic cells include thrombospondin 1, C1q,
mannose-binding lectin, and surfactant proteins (SP-A, SPD) with affinity for carbohydrates, and pentraxins CRP, PTX3,
and serum amyloid (SAP) that probably bind chromatin released from dying cells. Specific scavenger receptors including scavenger receptors A, B, CD36, and CD68, αvβ3
integrin, PS receptor, and complement receptors (CR1, CR3,
CR4) will bind to these molecules activating macrophages and
dendritic cells to phagocytose and clear dying PMNs from the
tissue [31]. Some non-professional phagocytes such as
mesangial and epithelial cells may also help in the clearance
of apoptotic PMNs. This physiological “start of the end”
allows the PMNs to die silently while sending “find me” and
“eat me” signals to be cleared without releasing intracellular
components that are highly toxic for host tissues. Nevertheless, a late secondary apoptotic phenotype of PMN can be
induced by persistence and increase in proinflammatory lipid
mediators such as LTB4. This late apoptosis characterized by

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selective leakage of intracellular content, particularly DNA
fragments, is distinct from primary necrosis or NET formation
but it may lead to secondary necrosis in the absence of efficient
resolving mechanisms [32]. The local and newly recruited
monocytes become M2-type (anti-inflammatory) macrophages
inside the tissue and phagocytose apoptotic PMN and other
debris. Ultimately, macrophages drain via lymphatic vessels
into the circulation to be cleared in the spleen or undergo
apoptosis locally in a process called efferocytosis [33–35].

Neutrophil Pathology in Periodontal Diseases
Neutropenia
Defective PMN production in the bone marrow results in
neutropenia characterized by reduction of the absolute circulating PMN numbers to <1,500 cells/μL. Control of endogenous microbiota is significantly impaired at absolute counts of
<500 cells/μL and an inability to mount an inflammatory
response is seen at counts of <200 cells/μL [34]. Congenital
and acquired (autoimmune, HIV-, and cancer therapyassociated) neutropenia increases the incidence and severity
of periodontitis in both primary and permanent dentitions [36].
It is considered that an absolute PMN count of <1000 PMN/μL
increases the risk of gingivitis [37•]. Pathophysiology of neutropenia can be classified into altered bone marrow stem cell
development, altered release from the bone marrow, altered
distribution of circulating and marginating PMN pools, and
decreased survival of circulating PMN. Benign chronic neutropenia characterized by prolonged noncyclic neutropenia is
associated with hyperplastic, edematous, fiery-red gingiva, and
in some cases with periodontitis [38, 39]. Cyclic neutropenia
characterized by periodic oscillations in production and release
of mature PMN including a 21-day cycle with severe neutropenia persisting for 3–10 days is consistently associated with
gingivitis and periodontitis [40]. Treatment includes human
recombinant granulocyte colony stimulating factor (hrGCSF) three times per week with successful increase in PMN
numbers. Congenital neutropenia (Kostman syndrome) is an
inherited hematological disorder characterized by an arrest of
PMN hematopoiesis at promyelocyte/myelocyte stage and
absolute counts of <2000 cells/μL. Mutations in the ELANE
gene coding for PMN elastase have been associated with
periodontitis in patients with severe congenital neutropenia
[41]. Gingivitis and severe periodontitis are common complications and despite some temporary improvement in PMN
counts with granulocyte colony stimulating factor (G-CSF)
treatment, patients with congenital neutropenia tend to have
persistent gingivitis. Other conditions characterized by neutropenia and associated with periodontitis are Felty syndrome (an
uncommon complication of rheumatoid arthritis), lazy leukocyte syndrome manifested by qualitative and quantitative

Curr Oral Health Rep (2014) 1:95–103

PMN defects, and agranulocytosis manifested by a decrease
or absence of granulocytes and peripheral leukopenia [37•].
Altered Polymorphonuclear Neutrophil (PMN) Release
During Periodontal Inflammation
Release of PMNs from the bone marrow in response to
proinflammatory stimuli is a rate-limiting step in the host
response to pathogens. Under normal conditions, more than
90 % of all PMNs are in the bone marrow and roughly 50 % of
the circulating PMNs are marginating in postcapillary venules
throughout the body [1]. Systemic proinflammatory molecules mediate release of PMNs from the bone marrow through
increased G-CSF expression. The mechanisms underlying
increased G-CSF expression in periodontitis are incompletely
understood. However, persistent PMN recruitment at sites of
periodontal inflammation has been associated with activation
of the IL-23-IL-17 axis [42]. IL-23 stimulates naive CD4+ T
cells to differentiate into Th17 lymphocytes producing IL-17
that further enhances G-CSF production. It is currently believed that G-CSF interferes with the CXCR4-CXCL12
(SDF-1, stromal cell-derived factor 1) axis, a major regulator
of PMN retention in the bone marrow. CXCR4 mutations
associated with the WHIM syndrome (warts, hypogammaglobulinemia, infections, and myelokathexis) affect
the desensitization of CXCR4 upon stimulation with SDF-1,
leading to retention of PMN in the bone marrow [43]. In
children with WHIM syndrome, periodontitis is associated
with premature tooth loss and recurrent superficial infections.
Downstream of CXCR4, Rac GTPases play a crucial role in
cytoskeletal organization and genetic control of proliferation
and survival pathways. Rac2 plays critical roles in PMN
homing in the bone marrow and its absence is associated with
neutrophilia [44]. Using Rac null mice, we have recently
shown that PMN release from the bone marrow in response
to localized ligature-induced periodontitis is significantly increased 24 h after induction in the absence of Rac2 [45]. Rac2
null mice are more susceptible to alveolar bone loss, possibly
because of reduced recruitment and function in the periodontium, and increased retention in periodontal microvasculature.
Therefore, existing evidence suggests that increased or decreased PMN release from the bone marrow is associated with
higher susceptibility to periodontal inflammation.
Altered PMN Recruitment
Leukocyte adhesion deficiencies (LADs) represent a group of
inherited disorders associated with defects in the expression or
function of leukocyte adhesion molecules. Specifically in
LAD type 1 (LAD-I), there is a deficiency in β2 integrins,
in LAD type 2 (LAD-II), there is a deficiency in glycosylation
of selectin ligands, and in LAD type 3 (LAD-III), there is
dysfunction of signaling intermediates affecting integrin

Curr Oral Health Rep (2014) 1:95–103

activation. A fourth type of LAD (LAD-IV) was proposed for
Rac2 mutations affecting PMN chemotaxis and margination
[46]. All LADs are characterized by neutrophilia in the absence of infection and impaired PMN recruitment into tissues.
Other conditions similar to LADs include Chediak-Higashi
syndrome associated with mutations of the LYST gene, which
encodes for a protein involved in the regulation of lysosomal
trafficking and the Papillon-Lefèvre syndrome caused by deficiency in cathepsin C (dipeptidyl peptidase-I), a lysosomal
exo-cysteine protease also involved in pro-enzyme activation
(cathepsin G, elastase, and proteinase 3). All syndromes characterized by impaired PMN recruitment and function are
associated with increased susceptibility for early onset of
severe forms of periodontitis affecting both primary and permanent dentitions [10]. The roles of Rac GTPases in PMN
recruitment in periodontitis are less well characterized, possibly because Rac mutations (LAD-IV) are less common. However, observations of PMN dysfunction associated with severe
infections in patients with D57N RAC2 mutation similar to
Rac2 null PMN phenotype in mice suggests that Rac2 may
play critical roles in PMN function to control subgingival
biofilms. We have recently shown that the initial inflammatory
response to induced periodontitis was altered in mice with
Rac2 null PMNs. Further, the alveolar bone loss was significantly higher than healthy mice with periodontitis [45]. These
findings may be relevant for epigenetic changes that alter the
expression of Rac2 and therefore increase susceptibility to
periodontitis. Recent focus on epigenetic regulation of gene
expression in periodontal inflammation points to potentially
significant roles of opportunistic pathogens and the environment on PMN function in inflammatory responses [47].
Impaired Pathogen Clearance
The association between periodontal pathogens and periodontitis was only defined in cross-sectional studies; therefore
failing to demonstrate a direct cause-effect relationship. Human PMN from healthy individuals can efficiently phagocytose and kill the traditionally known “periodontal pathogens”
Aggregatibacter actinomycetemcomitans, P. gingivalis, Treponema denticolla, and Tannerella forsythia [48, 49]. However,
these so-called pathogens also possess immune evasion mechanisms that impair or delay their phagocytosis and killing by
PMN via leukotoxins, gingipains, and fimbriae that ultimately
leads to bacterial tissue and intracellular persistence. Lowabundance bacteria with community-wide effects such as
P. gingivalis are critical for altering host-biofilm symbiosis
by acting directly or indirectly on PMN. Porphyromonas
gingivalis can impair PMN recruitment by altering coordinated expression of chemokines (IL-8), tumor necrosis factor-α,
and adhesion molecules (E-selectin) [50••, 51]. These effects
are only transient in vivo suggesting that P. gingivalis does not
block the recruitment of PMN but rather can delay it.

99

Porphyromonas gingivalis can escape immune clearance
through proactive manipulation of leukocyte receptors and
complement. The most documented such mechanism is the
P. gingivalis-induced C5aR-TLR2 crosstalk in macrophages
that impairs iNOS-dependent intracellular bacterial killing
[52]. Although these and other mechanisms through which
P. gingivalis and other periodontal pathogens can manipulate
the inflammatory response and bactericidal function of innate
immune cells are incompletely understood, a recently proposed model that accommodates these concepts is called the
polymicrobial synergy and dysbiosis (PSD) model [51, 53].
This model implies that physiologically compatible organisms
aggregate and coexist in a controlled immuno-inflammatory
state being proinflammatory and toxic for the tissues, but the
host response controls their overgrowth and overt pathogenicity. Consistent with the ecological plaque hypothesis proposed
in the early 1990s to explain the microbial etiology of periodontal diseases, the PSD model supports the concept that
non-specific accumulation of subgingival biofilms leads to
inflammation of gingival tissues but it is the latter that drives
the microbial changes associated with irreversible tissue destruction seen in periodontitis [26••, 54]. In fact, gingivitis is a
risk factor for periodontitis whereas only certain bacterial coaggregates and their quantitative changes can predict onset of
disease [55••, 56].
Diabetes Mellitus
Diabetes mellitus (DM) seems to modify periodontal tissues in
several ways including immunological dysfunction, microvascular alteration, and changes in extracellular matrix. The
relative contribution of these mechanisms to the increased
susceptibility for chronic periodontitis and rapid disease progression in diabetic patients is unknown. However, regardless
of dental plaque index, gingivitis is more prevalent in patients
with DM than in healthy individuals suggesting a direct impact of DM on the local immune response to the bacterial
biofilm [57–60]. It is well documented that DM, particularly
when uncontrolled, leads to impairment of PMN adherence,
chemotaxis, and pathogen clearance [61, 62]. Furthermore,
similar to LADs, a common finding in patients with DM is
neutrophilia [63]. Although PMNs from patients with DM
appear to be primed for hyper-responsive superoxide release,
their ability to kill bacteria is often paradoxically impaired [9,
64]. These alterations combined with increased expression of
leukocyte adhesion molecules may lead to ectopic inflammatory responses and tissue degradation though enzymatic and
oxidative mechanisms. Increased leukocyte adhesion molecule expression and gingival microvascular permeability in
DM in the absence of periodontitis suggests an immunevascular priming that predisposes to periodontitis [9]. Therefore, it seems that in DM, PMNs are released in higher
numbers from the bone marrow and are primed to respond

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Curr Oral Health Rep (2014) 1:95–103

Mounting evidence that a failure to resolve inflammation is
critical to the chronicity of periodontal tissue inflammation is
changing the current paradigm that treatment should primarily
focus on control of infectious agents. It is becoming evident that
innate immune alterations in response to overgrowing

subgingival biofilms result in failure to restore tissue homeostasis and host-microbial symbiosis. PMN apoptosis and expression of “eat me signals” lead to macrophage polarization to proresolution phenotypes in the periodontal environment, which is
essential for control of commensal biofilm ecology and tissue
integrity. Increased PMN survival and decreased apoptosis was
reported in GCF and tissue samples from chronic periodontitis
patients [54, 65]. In fact, one study has shown that GCF PMNs
from diseased sites show histone citrullination, a change indicative of PMN activation and initial stages of NET formation.
Furthermore, many crevicular PMNs are in advanced stages of
NET formation [66]. Similarly, in oral rinses from periodontitis
patients, there are more viable and fewer apoptotic PMNs,
suggesting increased cell survival in disease [67].

Fig. 1 Polymorphonuclear neutrophil (PMN) recruitment and function in periodontium. In health, persistent low-abundance commensal
bacteria in the subgingival biofilm trigger PMN recruitment to the periodontium and GCF gingival crevicular fluid. Recruitment is mediated
predominantly by CXCL2 chemokine (C-X-C motif) ligand 2. In the
gingival crevice, PMN form a “wall” between host tissues and the biofilm.
Most oral PMNs are short lived or apoptotic and exhibit a downregulation
of pro-survival gene expression. In gingivitis, CXCL8-mediated PMN
recruitment is predominant; PMN accumulate in higher numbers in the
tissue and GCF, particularly in early stages of gingivitis, and have decreased survival and increased apoptosis. In both health and gingivitis
inflammation, resolution leads to restoration of homeostasis once PMNs
and tissue debris have been cleared by pro-resolution macrophages. In both
entities, there is no clinical or histological evidence of alveolar bone loss. In

periodontitis (chronic or aggressive forms), local and systemic proinflammatory markers stimulate release of PMNs from the bone marrow. Conditions that impact the release, recruitment, and function of PMNs (e.g.,
diabetes mellitus, neutropenias, LADs) are associated with different forms
of periodontitis. Microbial chemotactic agents such as fMLP, particularly in
advanced disease, likely mediate recruitment into periodontal tissues. GCF
and oral PMNs have increased survival and decreased apoptosis. CB
commensal bacteria, PB pathogenic bacteria, APC antigen-presenting cell,
fMLP formylmethionyl-leucyl-phenylalanine, G-CSF granulocyte colonystimulating factor, TNF-α tumor necrosis factor-α, MIP2-α macrophage
inflammatory protein 2-α, PSGL-1 P-selectin glycoprotein ligand 1, ICAM1 intercellular adhesion molecule 1, IL interleukin, WHIM warts, hypogammaglobulinemia, infections, and myelokathexis, LADs leukocyte adhesion deficiencies

to inflammatory stimuli before reaching the sites of periodontal inflammation. In addition, PMN and microvascular changes associated with DM seem to predispose to altered PMN
margination with subsequent degranulation and oxidative
stress in the vasculature that contribute to increased susceptibility, severity, and progression of periodontitis.
Impaired Initiation of Resolution

Curr Oral Health Rep (2014) 1:95–103

The study of oral PMNs offers several advantages in the
case of periodontitis because the subgingival biofilm is located at the interface between periodontal tissues and the oral
environment. Therefore, most oral PMN are likely to have
encountered the bacterial biofilm, thus offering information
on the result of this interaction. The collecting of oral PMNs
that extravasate into the mouth through the periodontal crevice
was carried out initially by Klinkhammer in the 1960s [68].
Recent work using this approach has demonstrated that PMNs
from sites with periodontal disease display increased numbers
of gene changes compared with oral neutrophils that enter the
healthy periodontium [67]. Genes associated with PMN survival, delayed apoptosis, and PMN recruitment to sites of
inflammation were all found to be upregulated in PMNs,
which have located to the infected periodontium. This approach has also allowed for the phenotypic characterization
of tissue-recruited PMNs. With the identification of novel
PMN phenotypes found in tumors, it has been suggested that
novel neutrophil subtypes may be associated with some inflammatory diseases [69, 70]. A recent paper has identified a
novel oral PMN subset in healthy patients that expresses the Tcell receptor CD3 [71]. While previous reports suggested that
some circulating neutrophils express CD3 receptors, this work
is the first to identify tissue neutrophils expressing T-cell
receptors. This finding is significant as it supports the view
that PMNs act as a critical bridge between the innate and
adaptive immune responses in the context of altered resolution
programs. This notion is supported by a well-designed study
by Pelletier et al., which demonstrated that activated PMN
communicate directly with Th17 cells by mutual recruitment.
PMN also release chemokines, such as CCL20 and CCL2,
which have chemotactic activity, and Th17 cells release C-XC motif chemokine 8 (CXCL8), which attracts PMN. Using
the same approach, they also demonstrated that PMN release
CXCL10 and CCL2, which promote chemotaxis of Th1 cells
[72]. It therefore seems that increased PMN survival and
activity that initiates adaptive immune responses, two indicators of impaired inflammation resolution, are characteristics of
active periodontitis.

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opportunistic microbial pathogenicity and disease onset
(Fig. 1). Genetic and epigenetic factors may contribute to
various degrees to altered PMN function associated with loss
of periodontal clinical attachment and tooth loss. The heterogeneity in disease expression can be in part explained by the
multiple rate-limiting steps in PMN function in relation to
periodontal biofilms. Oral PMN characterization throughout
the entire spectrum of disease states will further our understanding of inflammation resolution in the periodontium and
provide diagnostic tools for monitoring disease activity and
response to therapy.

Compliance with Ethics Guidelines
Conflict of Interest Dr. Corneliu Sima and Dr. Michael Glogauer each
declare no potential conflicts of interest relevant to this article.
Human and Animal Rights and Informed Consent This article does
not contain any studies with human or animal subjects performed by any
of the authors.

References
Papers of particular interest, published recently, have been
highlighted as:
• Of importance
•• Of major importance
1.

2.

3.
4.•

5.

Conclusions
The function of PMN from bone marrow to the oral cavity is
critical to host-microbial homeostasis and maintenance of
periodontal health. Unbalanced (increased or decreased) release, recruitment and function of PMN in the periodontium
are likely critical to inflammatory tissue damage, failure of
inflammation resolution, and progression from gingivitis to
periodontitis. Increasing evidence validates the ecological
plaque hypothesis and polymicrobial synergy and dysbiosis
theory that account for an altered inflammatory response to
accumulating commensal periodontal biofilms as the cause for

6.

7.

8.•

9.

Summers C, Rankin SM, Condliffe AM, Singh N, Peters AM,
Chilvers ER. Neutrophil kinetics in health and disease. Trends
Immunol. 2010;31:318–24.
Pillay J, den Braber I, Vrisekoop N, Kwast LM, de Boer RJ,
Borghans JAM, et al. In vivo labeling with 2H2O reveals a
human neutrophil lifespan of 5.4 days. Blood. 2010;116:
625–7.
Bekkering S, Torensma R. Another look at the life of a neutrophil.
World J Hematol. 2013;2:44–58.
Hajishengallis G. Immunomicrobial pathogenesis of periodontitis:
keystones, pathobionts, and host response. Trends Immunol.
2013;35:3–11. This review addresses the host biofilm interations
in pathogenesis of periodontitis with focus on inflammation.
Delima AJ, Van Dyke TE. Origin and function of the cellular components in gingival crevice fluid. Periodontol 2000.
2003;31:55–7.
Tonetti MS, Imboden MA. Neutrophil migration into the gingival
sulcus is associated with transepithelial gradients of interleukin-8
and ICAM-1. J Periodontol. 1998;69:1139–47.
Zenobia C, Luo XL, Hashim A, Abe T, Jin L, Chang Y, et al.
Commensal bacteria-dependent select expression of CXCL2 contributes to periodontal tissue homeostasis. Cell Microbiol. 2013;15:
1419–26.
Kolaczkowska E, Kubes P. Neutrophil recruitment and function in
health and inflammation. Nat Rev Immunol. 2013;13:159–75. This
review addresses the mechanisms of PMN recruitment and their
bactericidal function, including the formation of NETs.
Sima C, Rhourida K, Van Dyke TE, Gyurko R. Type 1
diabetes predisposes to enhanced gingival leukocyte

102
margination and macromolecule extravasation in vivo. J
Periodontol Res. 2010;45:748–56.
10. Hajishengallis E, Hajishengallis G. Neutrophil homeostasis and
periodontal health in children and adults. J Dent Res.
2014;93(3):231–7.
11. Serhan C, Jain A, Marleau S, Clish C, Kantarci A, Behbehani B,
et al. Reduced inflammation and tissue damage in transgenic rabbits
overexpressing 15-lipoxygenase and endogenous antiinflammatory lipid mediators. J Immunol. 2003;171(12):6856–65.
12. Eskan MA, Jotwani R, Abe T, Chmelar J, Lim JH, Liang S, et al.
The leukocyte integrin antagonist Del-1 inhibits IL-17-mediated
inflammatory bone loss. Nat Immunol. 2012;13(5):465–73.
13. Martire B, Rondelli R, Soresina A, Pignata C, Broccoletti T,
Finocchi A, et al. Clinical features, long-term follow-up and outcome of a large cohort of patients with chronic granulomatous
disease: an Italian multicenter study. Clin Immunol.
2008;126:155–64.
14. Cohen MS, Leong PA, Simpson DM. Phagocytic cells in periodontal defense: periodontal status of patients with chronic granulomatous disease of childhood. J Periodontol. 1985;56(10):611–7.
15. Johnstone AM, Koh A, Goldberg MB, Glogauer M. A hyperactive
neutrophil phenotype in patients with refractory periodontitis. J
Periodontol. 2007;78:1788–94.
16. Aboodi GM, Goldberg MB, Glogauer M. Refractory periodontitis
population characterized by a hyperactive oral neutrophil phenotype. J Periodontol. 2011;82:726–33.
17. Feng Z, Weinberg A. Role of bacteria in health and disease of
periodontal tissues. Periodontol 2000. 2006;40:50–76.
18. Bostanci N, Thurnheer T, Aduse-Opoku J, Curtis MA, Zinkernagel
AS, Belibasakis GN. Porphyromonas gingivalis regulates TREM-1
in human polymorphonuclear neutrophils via its gingipains. PLoS
ONE. 2013;8:e75784.
19.• Hajishengallis G, Liang S, Payne MA, Hashim A, Jotwani R, Eskan
MA, et al. Low-abundance biofilm species orchestrates inflammatory periodontal disease through the commensal microbiota and
complement. Cell Host Microbe. 2011;10:497–506. This study
demonstrated that an inflammtory disease such as periodontitis
can be caused by dysregulation of host-polymicrobial interactions
instigated by a single species (Porphyromonas gingivalis) that
appears to act as keystone pathogen.
20. Brinkmann V. Neutrophil extracellular traps kill bacteria. Science.
2004;303:1532–5.
21. Cooper PR, Palmer LJ, Chapple ILC. Neutrophil extracellular traps
as a new paradigm in innate immunity: friend or foe? Periodontol
2000. 2013;63:165–97.
22. Brinkmann V, Zychlinsky A. Beneficial suicide: why neutrophils
die to make NETs. Nat Rev Microbiol. 2007;5:577–82.
23. Menegazzi R, Decleva E, Pietro D. Killing by neutrophil extracellular traps: fact or folklore? Blood. 2012;119:1214–6.
24. Bianchi M, Hakkim A, Brinkmann V, Siler U, Seger RA,
Zychlinsky A, et al. Restoration of NET formation by gene therapy
in CGD controls aspergillosis. Blood. 2009;114:2619–22.
25. Papayannopoulos V, Metzler KD, Hakkim A, Zychlinsky A.
Neutrophil elastase and myeloperoxidase regulate the formation
of neutrophil extracellular traps. J Cell Biol. 2010;191:677–91.
26.•• Bartold PM, Van Dyke TE. Periodontitis: a host-mediated disruption of microbial homeostasis: unlearning learned concepts.
Periodontol 2000. 2013;62:203–17. This paper overviews new concepts in periodontal disease pathogenesis that challenge the
existing paradigm of biofilm-initiated tissue destruction.
27.•• Serhan CN, Chiang N, Van Dyke TE. Resolving inflammation: dual
anti-inflammatory and pro-resolution lipid mediators. Nat Rev
Immunol. 2008;8:349–61. This paper is a landmark synthesis of
concepts used in analysis of inflammation resolution. It also overviews
a new class of endogenously produced pro-resolution lipid mediators.

Curr Oral Health Rep (2014) 1:95–103
28.
29.
30.

31.

32.

33.

34.

35.
36.
37.•

38.

39.

40.
41.

42.

43.

44.

45.

46.
47.
48.
49.

Jesenberger V, Jentsch S. Deadly encounter: ubiquitin meets apoptosis. Nat Rev Mol Cell Biol. 2002;3:112–21.
Tait SWG, Green DR. Caspase-independent cell death: leaving the
set without the final cut. Oncogene. 2008;27:6452–61.
Roos A, Xu W, Castellano G, Nauta AJ, Garred P, Daha MR, et al.
Mini-review: a pivotal role for innate immunity in the clearance of
apoptotic cells. Eur J Immunol. 2004;34:921–9.
Nauta AJ, Daha MR, van Kooten C, Roos A. Recognition and
clearance of apoptotic cells: a role for complement and pentraxins.
Trends Immunol. 2003;24:148–54.
Hébert MJ, Takano T, Holthöfer H, Brady HR. Sequential morphologic events during apoptosis of human neutrophils: modulation by
lipoxygenase-derived eicosanoids. J Immunol. 1996;157:3105–15.
Bellingan GJ, Caldwell H, Howie SE, Dransfield I, Haslett C. In
vivo fate of the inflammatory macrophage during the resolution of
inflammation: inflammatory macrophages do not die locally,
but emigrate to the draining lymph nodes. J Immunol.
1996;157:2577–85.
Savill JS, Wyllie AH, Henson JE, Walport MJ, Henson PM, Haslett
C. Macrophage phagocytosis of aging neutrophils in inflammation:
programmed cell death in the neutrophil leads to its recognition by
macrophages. J Clin Invest. 1989;83:865–75.
Serhan CN, Savill J. Resolution of inflammation: the beginning
programs the end. Nat Immunol. 2005;6:1191–7.
Hart TC, Atkinson JC. Mendelian forms of periodontitis.
Periodontol 2000. 2007;45:95–112.
Deas DE, Mackey SA, McDonnell HT. Systemic disease and periodontitis: manifestations of neutrophil dysfunction. Periodontol
2000. 2003;32:82–104.
Zaromb A, Chamberlain D, Schoor R, Almas K, Blei F.
Periodontitis as a manifestation of chronic benign neutropenia. J
Periodontol. 2006;77:1921–6.
Armitage GC, Cullinan MP. Comparison of the clinical features of chronic and aggressive periodontitis. Periodontol
2000. 2010;53:12–27.
Morley AA, Carew JP, Baikie AG. Familial cyclical neutropenia.
Br J Haematol. 1967;13:719–38.
Ye Y, Carlsson G, Wondimu B, Fahlén A, Karlsson-Sjöberg J,
Andersson M, et al. Mutations in the ELANE gene are associated
with development of periodontitis in patients with severe congenital
neutropenia. J Clin Immunol. 2011;31:936–45.
Gaffen SL, Hajishengallis G. A new inflammatory cytokine on the
block: re-thinking periodontal disease and the Th1/Th2 paradigm in the context of Th17 cells and IL-17. J Dent Res.
2008;87(9):817–28.
Lagane B, Chow KYC, Balabanian K, Levoye A, Harriague J,
Planchenault T, et al. CXCR4 dimerization and beta-arrestinmediated signaling account for the enhanced chemotaxis to
CXCL12 in WHIM syndrome. Blood. 2008;112:34–44.
Cancelas JA, Jansen M, Williams DA. The role of chemokine
activation of Rac GTPases in hematopoietic stem cell marrow
homing, retention, and peripheral mobilization. Exp Hematol.
2006;34:976–85.
Sima C, Gastfreund S, Sun C, Glogauer M. Rac-null leukocytes are
associated with increased inflammation-mediated alveolar bone
loss. Am J Pathol. 2013;184:472–82.
Pai S-Y, Kim C, Williams DA. Rac GTPases in human diseases. Dis
Markers. 2010;29:177–87.
Barros SP, Offenbacher S. Modifiable risk factors in periodontal
disease. Periodontol 2000. 2013;64:95–110.
Ryder MI. Comparison of neutrophil functions in aggressive and
chronic periodontitis. Periodontol 2000. 2010;53:124–37.
Eick S, Pfister W, Sigusch B, Straube E. Phagocytosis of
periodontopathogenic bacteria by crevicular granulocytes is depressed in progressive periodontitis. Infection. 2000;28:301–4.

Curr Oral Health Rep (2014) 1:95–103
50.•• Darveau RP. Periodontitis: a polymicrobial disruption of host homeostasis. Nat Rev Microbiol. 2010;8:481–90. This review integrates recent findings in periodontal microbiology and immunology
into contemporary views of periodontitis pathogenesis.
51. Hajishengallis G, Lamont RJ. Beyond the red complex and into
more complexity: the polymicrobial synergy and dysbiosis (PSD)
model of periodontal disease etiology. Mol Oral Microbiol.
2012;27:409–19.
52. Wang M, Krauss JL, Domon H, Hosur KB, Liang S, Magotti P,
et al. Microbial hijacking of complement-toll-like receptor
crosstalk. Sci Signal. 2010;3:ra11.
53. Hajishengallis G, Lamont RJ. Breaking bad: manipulation of the
host response by Porphyromonas gingivalis. Eur J Immunol.
2014;44(2):328–38.
54. Lucas H, Bartold PM, Dharmapatni AASSK, Holding CA, Haynes
DR. Inhibition of apoptosis in periodontitis. J Dent Res. 2009;89:
29–33.
55.•• Lang NP, Schätzle MA, Löe H. Gingivitis as a risk factor in
periodontal disease. J Clin Periodontol. 2009;36:3–8. This work
provides proof-of-concept evidence that gingival inflammation is a
risk factor for periodontitis.
56. Charalampakis G, Dahlén G, Carlén A, Leonhardt Å. Bacterial
markers vs. clinical markers to predict progression of chronic
periodontitis: a 2-yr prospective observational study. Eur J Oral
Sci. 2013;121:394–402.
57. Lalla E, Cheng B, Lal S, Kaplan S, Softness B, Greenberg E, et al.
Diabetes mellitus promotes periodontal destruction in children. J
Clin Periodontol. 2007;34:294–8.
58. Mattout C, Bourgeois D, Bouchard P. Type 2 diabetes and periodontal indicators: epidemiology in France 2002-2003. J
Periodontol Res. 2006;41:253–8.
59. Novak KF, Taylor GW, Dawson DR, Ferguson JE, Novak MJ.
Periodontitis and gestational diabetes mellitus: exploring the link
in NHANES III. J Public Health Dent. 2006;66:163–8.
60. Sarelius IH. Macromolecule permeability of in situ and excised
rodent skeletal muscle arterioles and venules. Am J Physiol Heart
Circ Physiol. 2005;290:H474–80.

103
61.

Manouchehr-Pour M, Spagnuolo PJ, Rodman HM, Bissada NF.
Impaired neutrophil chemotaxis in diabetic patients with severe
periodontitis. J Dent Res. 1981;60:729–30.
62. McMullen JA, Van Dyke TE, Horoszewicz HU, Genco RJ.
Neutrophil chemotaxis in individuals with advanced periodontal
disease and a genetic predisposition to diabetes mellitus. J
Periodontol. 1981;52:167–73.
63. Gkrania-Klotsas E, Ye Z, Cooper AJ, Sharp SJ, Luben R, Biggs
ML, et al. Differential white blood cell count and type 2 diabetes:
systematic review and meta-analysis of cross-sectional and prospective studies. PLoS ONE. 2010;5:e13405.
64. Gyurko R, Siqueira CC, Caldon N, Gao L, Kantarci A, Van Dyke
TE. Chronic hyperglycemia predisposes to exaggerated inflammatory response and leukocyte dysfunction in Akita mice. J Immunol.
2006;177:7250–6.
65. Gamonal J, Sanz M, O'Connor A, Acevedo A, Suarez I, Sanz A,
et al. Delayed neutrophil apoptosis in chronic periodontitis patients.
J Clin Periodontol. 2003;30:616–23.
66. Vitkov L, Klappacher M, Hannig M, Krautgartner WD. Neutrophil
fate in gingival crevicular fluid. Ultrastruct Pathol. 2010;34:25–30.
67. Lakschevitz FS, Aboodi GM, Glogauer M. Oral neutrophil transcriptome changes result in a pro-survival phenotype in periodontal
diseases. PLoS ONE. 2013;8:e68983.
68. Klinkhamer JM. Quantitative evaluation of gingivitis and periodontal disease: I. The orogranulocytic migratory rate. Periodontics.
1968;6:207–11.
69. Bird L. Tumour immunology: neutrophil plasticity. Nat Rev
Immunol. 2009;9:672–3.
70. Fridlender ZG, Sun J, Kim S, Kapoor V, Cheng G, Ling L, et al.
Polarization of tumor-associated neutrophil phenotype by TGF-β:
“N1” versus ‘N2’ TAN. Cancer Cell. 2009;16:183–94.
71. Lakschevitz FS, Aboodi GM, Glogauer M. Oral neutrophils display
a site-specific phenotype characterized by expression of T-cell
receptors. J Periodontol. 2013;84:1493–503.
72. Pelletier M, Maggi L, Micheletti A, Lazzeri E, Tamassia N,
Costantini C, et al. Evidence for a cross-talk between human
neutrophils and Th17 cells. Blood. 2010;115:335–43.



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