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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 equi-

librium and periodontal tissue homeostasis. Mounting evi-

dence 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 crevic-

ular 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 inflamma-

tion 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 abun-

dant 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 10

9

cells/kg per day from

common myeloid precursors. A combination of stem cell

factors, interleukin (IL)-3, and granulocyte macrophage

colony-stimulating factor (GM-CSF) is required for commit-

ment to the myeloid lineage [1]. Differentiated PMNs are

released into blood as band (incompletely matured) or seg-

mented (mature) neutrophils after a 12-day maturation period.

Classically, it was thought that PMNs spend 6–8hinthe

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 tis-

sue homeostasis. It is generally accepted that PMN recruit-

ment and microbicidal function are essential for the mainte-

nance of periodontal health. Research has shown that de-

viations 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 dis-

ruption 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 main-

taining homeostasis. It is estimated that 30,000 PMNs transit

through periodontal tissues every minute and that their pres-

ence 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

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

Curr Oral Health Rep (2014) 1:95–103

DOI 10.1007/s40496-014-0015-x

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-αor 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. Activat-

ed PMNs increase surface expression of chemokine receptors

(CXCR1 or CD181, CXCR2 or CD182), selectin ligands (P-

selectin glycoprotein ligand 1 or PSGL-1 or CD162 and

SLew

x

containing glycoproteins), and β2-integrins (lympho-

cyte function-associated antigen 1 [LFA-1] or CD11a/CD18).

The role of commensal periodontal bacteria in PMN recruit-

ment 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-leucyl-

phenylalanine; fMLP, C5a). It appears that PMNs preferen-

tially 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 en-

gagement can also mediate recruitment. Prior to transmigra-

tion 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 inter-

actions. Using a mouse model of acute gingivitis, we have

shown that the leukocyte rolling rate 2 h after proinflam-

matory stimulation with tumor necrosis factor-αshows a

dose-response relationship with the stimulus [9]. Several

physiological inhibitors of PMN integrins have been des-

cribed. Galectin-1, Lipoxin A4, and Resolvin D series

interfere with integrin activation by altered transcription,

decreased expression of endothelial ligands, and de-

creased upregulation. Pentraxin 3 (PTX-3), growth diffe-

rentiation 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 engage-

ment, respectively [10]. At the disease level, Lipoxin A4

was shown to prevent bone loss and mediate bone regene-

ration 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 IL-

17 expression [12].

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 composi-

tion 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 en-

hanced by opsonization through IgG and complement protein

C3b. Following recognition of opsonized pathogens, the PMN

forms phagocytic cups and then phagosomes, which ultimate-

ly fuse with lysosomes to form digestive vacuoles

(phagolysosomes) where oxidative and proteolytic antimicro-

bial molecules are released into an acidic milieu. These in-

clude 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 phagocy-

tose 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 produc-

tion 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 peri-

odontitis [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 poor-

ly understood. Recent evidence suggests that some periodon-

tal 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

96 Curr Oral Health Rep (2014) 1:95–103

bacteria ultimately leading to alveolar bone loss [19•]. There-

fore, 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’sweb.”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 character-

ized by nuclear swelling, chromatin degradation, and extra-

cellular 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 deiminase-

4 is activated. The latter is known to hypercitrullinate the

condensed nuclear chromatin promoting chromatin de-

condensation. 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 gingi-

vitis 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 peri-

odontal 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

may be associated with failure of resolution and onset

of periodontitis.

Resolution of Inflammation

Resolution of inflammation is a tightly regulated active pro-

cess 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 need-

ed 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 infil-

tration and activation to resolution and return to tissue homeo-

stasis [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 self-

destroying mechanisms in a non-inflammatory manner initi-

ating 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 car-

bohydrates 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 apopto-

tic PMNs by macrophages. PRM that have been correlated

with apoptotic cells include thrombospondin 1, C1q,

mannose-binding lectin, and surfactant proteins (SP-A, SP-

D) with affinity for carbohydrates, and pentraxins CRP, PTX3,

and serum amyloid (SAP) that probably bind chromatin re-

leased from dying cells. Specific scavenger receptors includ-

ing 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. Neverthe-

less, 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

Curr Oral Health Rep (2014) 1:95–103 97

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 circu-

lating PMN numbers to <1,500 cells/μL. Control of endoge-

nous 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 therapy-

associated) 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 neu-

tropenia 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 neu-

tropenia 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 neutro-

penia persisting for 3–10 days is consistently associated with

gingivitis and periodontitis [40]. Treatment includes human

recombinant granulocyte colony stimulating factor (hrG-

CSF) 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 compli-

cations 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 neutro-

penia and associated with periodontitis are Felty syndrome (an

uncommon complication of rheumatoid arthritis), lazy leuko-

cyte syndrome manifested by qualitative and quantitative

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 mole-

cules 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 T

h

17 lymphocytes producing IL-17

that further enhances G-CSF production. It is currently be-

lieved 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, hypo-

gammaglobulinemia, 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 in-

creased 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 periodon-

tium, and increased retention in periodontal microvasculature.

Therefore, existing evidence suggests that increased or de-

creased 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 β2integrins,

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

98 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 ab-

sence 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 de-

ficiency 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 char-

acterized by impaired PMN recruitment and function are

associated with increased susceptibility for early onset of

severe forms of periodontitis affecting both primary and per-

manent dentitions [10]. The roles of Rac GTPases in PMN

recruitment in periodontitis are less well characterized, possi-

bly because Rac mutations (LAD-IV) are less common. How-

ever, 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 signifi-

cantly 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 environ-

ment on PMN function in inflammatory responses [47].

Impaired Pathogen Clearance

The association between periodontal pathogens and periodon-

titis was only defined in cross-sectional studies; therefore

failing to demonstrate a direct cause-effect relationship. Hu-

man PMN from healthy individuals can efficiently phagocy-

tose and kill the traditionally known “periodontal pathogens”

Aggregatibacter actinomycetemcomitans,P. gingivalis,Trepo-

nema denticolla,andTannerella forsythia [48,49]. However,

these so-called pathogens also possess immune evasion mech-

anisms that impair or delay their phagocytosis and killing by

PMN via leukotoxins, gingipains, and fimbriae that ultimately

leads to bacterial tissue and intracellular persistence. Low-

abundance 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 coordinat-

ed 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.

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 pro-

posed 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 pathogenic-

ity. Consistent with the ecological plaque hypothesis proposed

in the early 1990s to explain the microbial etiology of peri-

odontal 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 de-

struction seen in periodontitis [26••,54]. In fact, gingivitis is a

risk factor for periodontitis whereas only certain bacterial co-

aggregates 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, micro-

vascular alteration, and changes in extracellular matrix. The

relative contribution of these mechanisms to the increased

susceptibility for chronic periodontitis and rapid disease pro-

gression 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 im-

pact 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 inflamma-

tory responses and tissue degradation though enzymatic and

oxidative mechanisms. Increased leukocyte adhesion mole-

cule expression and gingival microvascular permeability in

DM in the absence of periodontitis suggests an immune-

vascular priming that predisposes to periodontitis [9]. There-

fore, it seems that in DM, PMNs are released in higher

numbers from the bone marrow and are primed to respond

Curr Oral Health Rep (2014) 1:95–103 99

to inflammatory stimuli before reaching the sites of periodon-

tal inflammation. In addition, PMN and microvascular chang-

es associated with DM seem to predispose to altered PMN

margination with subsequent degranulation and oxidative

stress in the vasculature that contribute to increased suscepti-

bility, severity, and progression of periodontitis.

Impaired Initiation of Resolution

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 homeosta-

sis and host-microbial symbiosis. PMN apoptosis and expres-

sion of “eat me signals”lead to macrophage polarization to pro-

resolution 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 indic-

ative 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 func-

tion in periodontium. In health, persistent low-abundance commensal

bacteria in the subgingival biofilm trigger PMN recruitment to the peri-

odontium 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 de-

creased 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 proinflam-

matory markers stimulate release of PMNs from the bone marrow. Condi-

tions 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 colony-

stimulating factor, TNF-αtumor necrosis factor-α,MIP2-αmacrophage

inflammatory protein 2-α,PSGL-1 P-selectin glycoprotein ligand 1, ICAM-

1intercellular adhesion molecule 1, IL interleukin, WHIM warts, hypo-

gammaglobulinemia, infections, and myelokathexis, LADs leukocyte ad-

hesion deficiencies

100 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 locat-

ed 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 sur-

vival, 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 ap-

proach 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 in-

flammatory diseases [69,70]. A recent paper has identified a

novel oral PMN subset in healthy patients that expresses the T-

cell 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 T

h

17 cells by mutual recruitment.

PMN also release chemokines, such as CCL20 and CCL2,

which have chemotactic activity, and T

h

17 cells release C-X-

C motif chemokine 8 (CXCL8), which attracts PMN. Using

the same approach, they also demonstrated that PMN release

CXCL10 and CCL2, which promote chemotaxis of T

h

1cells

[72]. It therefore seems that increased PMN survival and

activity that initiates adaptive immune responses, two indica-

tors of impaired inflammation resolution, are characteristics of

active periodontitis.

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) re-

lease, 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

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 hetero-

geneity 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 under-

standing 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.

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