Periodontology 2000, Vol.

64, 2014, 57–80 Printed

PERIODONTOLOGY 2000
in Singapore. All rights reserved
Ó 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Inflammatory and immune ​pathways

in the pathogenesis of periodontal

disease
A​LI ​C​EKICI​, A​LPDOGAN ​K​ANTARCI​, H​ATICE ​H​ASTURK ​& ​T​HOMAS ​E.

V​AN ​D​YKE
disease. Unlike many infectious diseases, periodontal diseases
ap- pear to be infections mediated by the overgrowth of
commensal organisms, rather than by the acquisition of an
exogenous pathogen. As microorganisms evolve more rapidly
Inflammation is the physiological response to a variety of
than their mammalian hosts, immune mechanisms that
injuries or insults, including heat, chemical agents or bacterial
determine the ecological balance of commensal organisms also
infection. In the acute phase of inflammation, the response is
need to change to pre- serve homeostasis (65).
rapid and of short duration. If the insult or injury is not resolved,
the response becomes chronic, which can be considered as Knowledge of how immune mechanisms and in-
nonphysiologic or pathologic. When inflammation becomes flammatory responses are regulated is critical for understanding
chronic, the adaptive immune response is activated with the pathogenesis of complex diseases, such as periodontitis. The
involvement of the cellular and non- cellular mechanisms of pathogenesis of perio- dontal diseases is mediated by the
acquired immunity. Immune mechanisms play further roles in inflammatory re- sponse to bacteria in the dental biofilm (Fig.
the resolution of inflammation and in the healing process, 1). However, identification of the true ​Ô​pathogens​Õ ​in
including the repair and the regeneration of lost or damaged periodontitis has been elusive. There is evidence that specific
tissues. Thus, innate (inflammatory) immunity and acquired microbes are associated with the progressive forms of the
immunity must be coordinated to return the injured tissue to disease; however, the presence of these microorganisms in
homeostasis (85). ndividuals with no evidence of disease progression suggests
hat the disease is the net effect of the immune response and the
The etiology of periodontal diseases is bacteria. The
nflammatory processes, not the mere presence of the bacteria.
human oral cavity harbors a substantial and continuously
Regulation of immune–inflammatory mechanisms governs
evolving load of microbial species. The ecological interactions
patient susceptibility and is modified by environmental factors
between the host and mi- crobes determine the severity of the
219, 220, 241). This review will address the pathways of
inflamma- tion in periodontal diseases by focusing on immu- The first is gingivitis, which is defined as inflammation of the
nologic mechanisms to elucidate sites of regulation. Clinical gingiva in which the connective tissue attachment to the tooth
features of the periodontal diseases are be- yond the scope of remains at its original level. The disease is limited to the
this work but are within the context of the pathogenic soft-tissue com- partment of the gingival epithelium and
mechanisms. Possible clinical outcomes will be discussed in connective tissue (12). The second is periodontitis, which is an
relation to the inflammatory–immunologic changes throughout nflammation of the supporting tissues of the teeth with
the disease process. progressive attachment loss and bone destruc- tion (55). Both
diseases and their symptoms are very common in populations
worldwide. In the USA, adolescents have gingivitis and signs of
gingival
Periodontal diseases: what do we ​know?

There are two common diseases affecting the peri- odontium.

Flagellin CpG DNA LPS Capsule Fimbria
TARC
IFN-γ I-309 MDC
IgG-G IgG-A IgG-M
IFN-γ IgG-G IgG-A IgG-M
Th2
Th17
Treg ​CTLA-4 TGF-β
Plasma cell ​Secretions of cells

Fig. 1. The immune inflammatory response in periodon- titis is complex and involves both innate and acquired immunity. This diagram
presents an overview of the effector molecules and effector cells in the pathogenesis of periodontitis based on our current understanding
of dis- ease pathways. BCA-1, B cell-attracting chemokine 1; CGRP, calcitonin gene-related peptide; CTLA-4, cytotoxic
T-lymphocyte-associated antigen 4; GM-CSF, granulocyte– macrophage colony-stimulating factor; IFN-c, interferon gamma; Ig-A,
immunglobulin A; Ig-G, immunglobulin G; Ig-M, immunglobulin M; IL-1b, interleukin-1beta; IL-2, interleukin-2; IL-3, interleukin-3;
IL-4, interleukin-4; IL-5, interleukin-5; IL-6, interleukin-6; IL-8, interleukin-8; IL-
MAC (C5b-C9)
IL-8 SDF-1α RANTES MCP-1 BCA-1
Mast cell
Histamin
MAC (C5b-C9)
IFN-γ
MMPs TGF-β PGE
RANTES IL-1β TNF-α IL-6
IL-2 IL-3 IFN-γ
Th1
Osteoblast Osteoclast
IgG-G
MMPs TIMP
IgG-A
IL-3 IgG-M
IL-4 IL-5
TNF-α GM-CSF
IL-13 GM-CSF
Substance P
M-CSF
CGRP PGE
IL-17
Th1
IL-22
Nerve cell
Collagen fibers
T Lymphocyte ​
Macrophage Fibroblast ​ Neutrophil B Lymphocyte Bacterial antigen

13, interleukin-13; IL-17, interleukin-17; IL-22, interleu- kin-22; LPS, lipopolysaccharide; M-CSF, macrophage col-
Complement protein ​

ony-stimulating factor; MAC, membrane attack complex; MCP-1, macrophage chemotactic protein-1; MDC, mac- rophage-derived

chemokine; MMPs, matrix metallopro- teinases; OPG, osteoprotegerin; PGE​2​, prostaglandin E​2​; RANTES,
​ regulated and normal T cell
expressed and se- creted; SDF-1a, stromal cell-derived factor-1alpha; TARC, thymus and activation-regulated chemokine; TGF-b,
transforming growth factor beta; Th1, T-helper 1 cell; Th2, T-helper 2 cell; Th17, T-helper 17 cell; TIMP, tissue inhibitor of matrix
metalloproteinases; TNF-a, tumor necrosis factor alpha; Treg T-regulatory cell.
bleeding, whereas 54​% ​of the adult population in the USA exhibits gingival bleeding (99). Thirty-seven per cent of the
adult population in the USA suffers from severe periodontitis (160). In both cases, the disease is associated with the
accumulation of bacteria at the dento–gingival margin, while the causal relationship of specific organisms is not fully clear.
The host re- sponds to microbial challenge by generating an inflammatory cell infiltrate in the tissue subjacent to the
 periodontal pocket (186).
58
Cekici et al.
The initial inflammation in the periodontal tissues should be considered a physiologic defense mecha- nism against the
microbial challenge, rather than pathology. The clinical findings of the disease at this stage include supragingival and
subgingival plaque formation, which are usually accompanied by cal- culus formation and gingival inflammation (154). If
plaque is removed, there is resolution with return to homeostasis; if the lesion persists, it becomes pathology. For
convenience, we will use the well-
known stages of gingivitis and periodontitis, de- scribed by Page & Schroeder, in 1976 (186), for descriptive purposes: the initial
lesion; the early le- sion; the established lesion; and the advanced lesion. The advanced lesion is also called the destructive phase,
because it represents the transition from gin- givitis to periodontitis. What makes inflammation and immunologic events underlying
periodontal diseases confusing is that the immunologic events overlap in different phases of disease. It should be emphasized that
division of the immune response into various systems, such as innate immunity and adaptive immunity, is a rather arbitrary
distinction imposed by immunologists (12). Although it is easier to describe the inflammation in compartments, the mechanisms
involved in inflammation, resolution and healing include all components of the immune system that interact cooperatively to
protect the periodontium (266). It is important to bear in mind that as the lesion progresses, the preceding pathways still function.
The initial lesion is the response of resident leu- kocytes and endothelial cells to the bacterial biofilm. At this stage, there are
no signs of clinical inflam- mation, but the changes in the tissues can be observed histologically. The metabolic products of bacteria
trigger junctional epithelium cells to pro- duce cytokines and stimulate neutrons to produce neuropeptides, which cause
vasodilatation of local blood vessels. Neutrophils leave the vessel and mi- grate toward the site of inflammation in response to
chemokines. The early lesion follows, with increased numbers of neutrophils in the connective tissue and the appearance of
macrophages, lymphocytes, plas- ma cells and mast cells. Complement proteins are activated. The epithelium proliferates to form
rete pegs, observed histologically, and clinical signs of gingival inflammation, such as bleeding, can be seen. Gingival crevice fluid
flow is increased.
The following stage is the established lesion. This can be considered as the period of transition from the innate immune
response to the acquired im- mune response. Macrophages, plasma cells, and T and B lymphocytes are dominant, with IgG1 and
IgG3 subclasses of B lymphocytes also present. Blood flow is impaired, and the collagenolytic activity is increased. There is also
increased collagen production by fibroblasts. Clinically, this stage is a moderate to severe gingivitis with gingival bleeding and
color and contour changes. The final stage is the transition to periodontitis: the advanced lesion. Irreversible attachment loss and
bone loss are ob- served histologically and clinically. The inflamma-
Inflammatory and immune pathways in periodontal disease

tory lesion extends deeper, affecting the alveolar bone (51).

Cells and mediators of periodontal ​inflammation

The innate immune system includes cells of nonhe- matopoietic origin, especially epithelial cells; myeloid cells of hematopoietic
origin (phagocytes); and the innate humoral defense, the complement cascade (266). Neuropeptides contribute to this initial,
immediate response to microbial challenge (236). The initial response, acute inflammation, is the physiologic response to the
microbial challenge to recruit adequate cells to sites of infection through the production of cytokines and chemokines (Fig. 1). If the
infection fails to clear, the chronic lesion is ini- tiated with transition to the early lesion. Still an in- nate immune response pathways
are stimulated that will activate the adaptive immune response. Innate immunity was formerly thought to be nonspecific,
characterized by the phagocytosis and digestion of microorganisms and foreign substances by macro- phages and neutrophils (97,
166). Phagocytes such as macrophages and neutrophils have surface receptors that recognize and bind surface molecules of bacteria
(266). These pattern recognition receptors, including the toll-like receptors, distinguish between the host and the bacteria (197).
After recognition of micro- organisms and foreign substances, chemokines are secreted to attract phagocytes. The complement
system also generates biologically active proteins, including the anaphylotoxins C3a, C4a and C5a that attract the different host
immune cells monocytes, lymphocytes and neutrophils, respectively. Comple- ment proteins can also directly kill certain bacteria.
Histamine-induced vasodilatation by mast cells in- creases blood flow and recruitment of phagocytes.

Complement ​The complement cascade can be activated through three pathways: the classical pathway; the lectin pathway; and
the alternative pathway.
The classical pathway is activated by immuno- globulin – IgG or IgM – which binds the first com- plement protein, C1q, to
a domain on its Fc tail. Bound C1q binds other C1 proteins to form a com- plex, C1qrs, which initiates a series of enzymatic
reactions, cleaving C4 to C4a and C4b, and C2 to C2a and C2b. C4b and C2a become part of the C1 com- plex, forming a C3

convertase that cleaves C3 to C3a​

59
systems, and C3b. C3b binds to the bacterial surface and, with
which may result in modulation of the several accessory proteins, forms a new enzyme to
inflammatory response (80, 165). Neuropeptides sig- cleave C5 to C5a and C5b. C5b interacts with the
nal nonneuronal cells through G protein-coupled terminal complement proteins, C6 to C9, to form the
receptors located on the cell membrane (150). Va- membrane attack complex that inserts C8 and C9
nilloid receptor-1, a neuropeptide receptor, is up- into the bacterial membrane, forming pores to dis-
regulated in inflammatory bowel disease, suggesting rupt the membrane.
a possible role of this receptor in chronic inflamma- The lectin pathway employs a mannose-binding
tion (262). lectin to bind carbohydrate on the bacterial cell-
The contribution of the nervous system to inflam- surface to form mannose-associated serine protease-
mation is not limited to vasodilatation and immune- 2. This molecule has the capacity to interact with the
cell recruitment. Cytokines and other products of complement proteins C4 and C2 to convert C3 to C3a
immune cells can modulate the action, differentia- and C3b, as in the classical pathway.
tion and survival of nerve cells, while neuropeptides The alternative pathway is activated by bacterial
released from neurons play pivotal roles in influ- polysaccharides, such as zymosan, lipopolysaccharide
encing the immune response. The interaction relies or aggregated IgA, through factor P (properdin) to
on the receptor-sensitizing characteristics of the cleave C3. C3b, and factors B and D convert C5 into
cytokines. When a cytokine engages a neuron C5a and C5b and the cascade continues to completion.
receptor, it initiates the release of neuropeptides Both gingivitis and chronic periodontitis have been
(184). The discovery of protease-activated receptors characterized primarily as activators of the alterna-
revealed that protease-activated receptor 2 has an tive pathway. This is of some interest because it
especially important role in chronic inflammation suggests that even though pathogen-specific anti-
(246). Protease-activated receptor 2 is co-expressed bodies are formed in chronic periodontitis, most of
with substance P and calcitonin gene-related peptide the complement activation in this disease is still via
on sensory nerves, where it is believed to mediate the alternative pathway (256). It is also known that
inflammation (53, 222) (Fig. 1). In addition, cytokines other than these very well-studied pathways, there
have been shown to regulate the expression of sub- are proteins that can interact directly with C3 and C5:
stance P and to facilitate the lipopolysaccharide-in- plasmin can cleave C3 into C3a and C3b; and
duced release of calcitonin gene-related peptide in thrombin has the ability to cleave C5.
sympathetic neurons (92, 113). Also, calcitonin gene- related peptide enhances interleukin-1-induced ​Neuropeptides
accumulation of neutrophils and induces T-cell During the innate immune response to periodontal
cytokine secretion (26, 137). Neural growth factor has pathogens, another element of the human defense
been shown to directly regulate the synthesis of cal- system is also activated. Neurons generate electric
citonin gene-related peptide by B-cells as it does in impulses in response to chemical or mechanical
sensory neurons (22). In the course of inflammation, stimuli, conduct the impulse and translate the elec-
kininogens are degraded to form kinins, including trical activity into a chemical signal. Alternatively,
bradykinin, which together are important mediators peptide neurotransmitters – neuropeptides – can be
 of inflammation. Kinins are known to be proinflam- secreted into the extracellular fluid, where they act
matory, leading to vasodilatation, plasma extravasa- locally through receptors on other neurons or im-
tion and the release of other inflammatory mediators, mune cells. Most neuropeptides act on nonneuronal
notably the neuropeptides substance P and calcito- targets (90), such as receptors for substance P and
nin gene-related peptide (67). calcitonin gene-related peptide, found on immune
With the identification of neuropeptides in gingival cells, suggesting that the paracrine action of neuro-
crevice fluid, it is becoming increasingly evident that peptides has important immunomodulatory roles
periodontitis and other orofacial inflammatory dis- (80, 150, 165) (Fig. 1). Recently, the nervous system
orders may be modulated by imbalances in certain has been identified as a critical regulator of inflam-
neuropeptides (78, 141, 142, 151, 152). In addition to mation in periodontal diseases (236). Furthermore,
the presence of neuropeptides in gingival crevice under pathological conditions some neuropeptides
fluid, fibers innervating the periodontal tissues in are synthesized and released from inflammatory cells
humans have been shown to be immunoreactive to a (168). Therefore, the identification of neuropeptide
number of neuropeptides, including substance P, receptors on immune cells suggests that communi-
calcitonin gene-related peptide, vasoactive intestinal cation exists between the immune and neurological
peptide and neuropeptide Y (153).
60
Cekici et al.
The major function of neuropeptides in inflam- mation is vasodilatation, vasoconstriction and the recruitment and regulation
of immune cells (6, 28, 126). Three major neuropeptides have modulatory effects in periodontal inflammation: substance P,
calcitonin gene-related peptide and vasoactive intestinal peptide.

Substance P

Substance P is a member of the tachykinin family of neuropeptides, also known as the neurokinins (150). Substance P evokes a
rapid response upon release and causes increased microvascular permeability, edema formation and subsequent plasma protein
extravasation. Vasodilatation caused by substance P occurs indirectly by stimulating histamine release from mast cells (29, 150).
Accordingly, edema in- duced by substance P is primarily caused by in- creased vascular permeability mediated through its action
on neurokinin 1 receptors on endothelial cells (136). Several studies have shown the presence of substance P in human gingival
tissues and in gingival crevice fluid (11, 151). The levels of substance P in the gingival crevice fluid are reduced after periodontal
treatment, supporting the view of a local source of tachykinins in the gingival tissues (151).
The actions of substance P on immune cells are also important. Substance P limits the production of transforming growth
factor beta by macrophages (161) and induces the synthesis of interleukin-6 by monocytes (140). Interestingly, substance P is also
synthesized by immune cells. Sources of substance P, in addition to neurons, include monocytes, dendritic cells, eosinophils,
T-lymphocytes and mast cells (130, 150). Mononuclear phagocytes and dendritic cells produce substance P when activated with
lipopoly- saccharide in vitro (131).

Calcitonin gene-related peptide

Calcitonin gene-related peptide has potent vasodila- tor activity and is frequently co-localized with sub- stance P, which has been
shown to regulate the vasodilator activity of calcitonin gene-related peptide (23, 38). In addition to its known vasodilator activity,
calcitonin gene-related peptide has immunosup- pressive actions that down-regulate the inflammatory response (232), such as
suppressing interleukin-2 production and the proliferation of murine T-cells (252). It inhibits hydrogen peroxide production by
Inflammatory and immune pathways in periodontal disease

macrophages in response to interferon gamma and presenting antigen (177, 185). Calcitonin gene-related peptide also impacts bone
metabolism, thus inhibit- ing osteoclastic bone resorption and stimulating osteogenesis (123).

Vasoactive intestinal peptide is an important im- mune-modulatory peptide that is capable of regu- lating the production of both
proinflammatory and anti-inflammatory mediators (15, 57, 193). The major nonneuronal sources of vasoactive intestinal peptide are
neutrophils and mast cells (36, 178). While vaso- active intestinal peptide inhibits lipopolysaccharide- induced production of tumor
necrosis factor alpha, interleukin-6 and interleukin-12 in activated macro- phages, it stimulates the production of the potent
anti-inflammatory cytokine, interleukin-10, and suppresses T-cell proliferation (57). The levels of vasoactive intestinal peptide are
significantly ele- vated in periodontitis sites compared with clinically healthy sites, and nonsurgical periodontal treatment results in
a clinical improvement along with a con- comitant reduction in the levels of vasoactive intes- tinal peptide (142).

Toll-like receptors ​In the oral epithelium, complementary defense mechanisms are present. Epithelial cells have tight
intercellular junctions that impede the entry of bacteria and their metabolites. Lipopolysaccharide is a cell-wall component of all
gram-negative micro- organisms (197). Once exposed to lipopolysaccha- ride, a series of complex mechanisms are triggered, which
lead to extracellular matrix degradation and the initiation of osteoclastogenesis. The main func- tion of dendritic cells is
presentation of antigen to other cells of the immune system. Recognition of innate immune signals by dendritic cells relies on a
limited number of pathogen-related receptors. These include toll-like receptors and related proteins that regulate apoptosis,
inflammation and immune responses (3, 95) (Fig. 1).
The toll-like receptors family of proteins is the best-characterized class of pattern-recognition receptors. Dendritic cells
express toll-like receptors, and different dendritic-cell subsets express distinct toll-like receptors that are associated with particular
functions in innate responses and the generation of distinct T-cell subsets (98, 103). Toll-like receptors
Vasoactive intestinal peptide
 61
Table 1. The source cell, location and associated bacteria for toll-like receptors.
Receptor Location Cells Bacteria

Toll-like receptor 1 Cell membrane Myeloid dendritic cells, monocytes Not specified
myeloid dendritic cells
Toll-like receptor 2 Cell membrane Monocytes, natural killer cells, myeloid
Escherichia coli
dendritic cells,mast cells, T-cells, epithelial cells
Porphyromonas gingivalis
Escherichia coli Tannerella Toll-like receptor 7 Intracellular Plasmacytoid dendritic cells,
forsythia Prevotella intermedia B-cells, eosinophils
Prevotella nigrescens Treponema Not specified
denticola

Toll-like receptor 3 Intracellular Myeloid dendritic cells, natural killer Toll-like receptor 8 Intracellular Natural killer cells, T-cells,
cells, epithelial cells myeloid cells, myeloid dendritic cells
Not specified Not specified

Toll-like receptor 9 Intracellular Plasmacytoid dendritic cells, B-cells,

Toll-like receptor 4 Cell membrane Monocytes, mast cells, neutrophils, T cells,
epithelial cells, endothelial cells natural killer cells
Aggregatibacter Porphyromonas gingivalis
actinomycetemcomitans, Veillonella parvula Aggregatibacter

Toll-like receptor 5 Cell membrane Monocytes, natural killer cells, myeloid

Toll-like receptor 10 Cell membrane B-cells, plasmacytoid dendritic cells,
dendritic cells epithelial cells
Not specified myeloid dendritic cells
Not specified

Toll-like receptor 6 Cell membrane Myeloid cells, mast cells, B-cells,

Toll-like receptor 11 Intracellular Macrophages, dendritic cells, epithelial cells Not specified

are also expressed on lymphocytes and osteoclast precursors, as 62

well as on macrophages, osteoblasts and stromal and epithelial Cekici et al.
cells, each of which has different toll-like-receptor expression
profiles (83, 96, 107, 228). Toll-like receptors are unique
receptors that recognize molecules that are broadly shared by
microorganisms, but are distinguishable from host molecules;
these are collectively referred to as ​Ô​pathogen-associated
molecular patterns​Õ​. Toll-like receptors detect multiple
pathogen-associated molecular patterns, including
lipopolysaccharide, bacterial lipoproteins and lipoteichoic acids,
flagellin, CpG DNA of bacteria and viruses, double-stranded
RNA and single-stranded viral RNA (96). To date, 11 different
toll-like-receptor molecules have been identified in human
periodontal tissues, and their expression, distribution and ligand
specificities have been characterized (125, 145, 195, 228) (Table
1).
When toll-like receptors bind pathogen-associated
molecular patterns, a series of intracellular events are initiated,
leading to the production of cytokines,
 toll-like receptor 4 predominate in periodontal tissues (82).
Interestingly, the same toll-like receptor can trigger different
responses depending upon the intracellular adapter protein. For
example, when lipopolysaccha- ride binds to toll-like receptor 4
and uses myeloid differentiation primary response protein
(MyD88) and toll ⁄ interleukin-1 receptor domain-containing
adapter protein as adapters, the result is the pro- duction of
tumor necrosis factor alpha, interleukin-6 and interleukin-12. If
the adapters toll ⁄ interleukin-1 receptor domain-containing
adapter-inducing inter- feron beta-related adapter molecule and
toll ⁄ interleukin-1 receptor domain-containing adapter- inducing
interferon beta with the same toll-like receptor type are used,
release of interferon al- pha ⁄ beta and activation of interferon
regulatory factor 3 follows (1, 35, 107, 125, 253).
Toll-like receptors 1, 2, 4, 5 and 6 recognize mainly
products that are unique to bacteria and not made by the host.
This gives them the specificity to differen- tiate microorganisms
from the host (96). Recognition by toll-like receptor pathways is
a crucial phase in inflammation. For a complete review of
toll-like receptors and their pathways, see Uehara & Takada
(239).

Antigen presentation and ​activation of

acquired immunity

If the early lesion persists without resolution, bacte- rial antigens

are processed and presented by lym- phocytes, macrophages and
dendritic cells. Broadly, two different subsets of lymphocytes
have evolved to recognize extracellular and intracellular
chemokines and antimicrobial peptides (104). The pathogens after being presented with antigens by the innate
toll-like-receptor domain can bind four different adapter proteins immune cells: T-lymphocytes and B-lymphocytes. B-
and has the potential to induce various cytokines through lymphocytes bear immunoglobulin molecules on their surface,
nuclear factor of kappa light polypeptide gene enhancer in which function as antigen receptors. Antibody, which is a
B-cells pathways in the nucleus of the cell. Known adapter soluble form of immunoglobu- lin, is secreted following
proteins of toll-like receptors are myeloid differentiation activation of B-cells to bind pathogens and foreign material in
primary response protein (Myd88), toll ⁄ interleukin-1 recep- tor the extracellular spaces (humoral immunity). T-cells are the
domain-containing adapter protein, toll ⁄ inter- leukin-1 receptor effectors of cell-mediated immunity (delayed hypersensitiv- ity).
domain containing adapter-induc- ing interferon beta and toll ⁄ The T-cell antigen receptor is a membrane- bound molecule,
interleukin-1 receptor domain containing adapter-inducing similar to immunoglobulin, which recognizes peptide fragments
interferon beta- related adapter molecule. Different toll-like of pathogens. Activa- tion of the T-cell receptor requires the
receptors induce different responses. For example, in dendritic major histo- compatibility complex, which is also a member of
cells, the interaction of toll-like receptor 4 and lipo- the immunoglobulin superfamily. Two classes of major
polysaccharide results in the production of proin- flammatory histocompatibility complex molecules are required for the
cytokines such as interleukin-12, and the interaction of toll-like activation of distinct subsets of T-cells. Various T-cell subsets
receptor 3 with lipopolysac- charide results in the production of kill infected target cells and activate macrophages, B-cells and
type-I interferon. It is known that toll-like receptor 2 and other T-cells.
 cells, respectively
Inflammatory (4, 207,
and immune 254).inT-helper
pathways 17 disease
periodontal cells are named for
heir unique production of interleukin-17. T-helper 17 cells also
Thus, T-cells are essential for the regulation of both humoral produce interleukin-22. T-helper 17 lymphocytes, like T-helper
and cell-mediated responses. 1 cells, are also noted for their stimulatory role in
osteoclastogenesis (258). T-helper 17 cells are observed in
Classically, T-lymphocytes have been classified into
chronic periodontitis sites, and T-helper 17-related cytokines are
subsets based on the cell-surface expression of CD4 or CD8
produced in periodontal le- sions (180, 226, 247).
molecules. CD4​+ ​T-cells (T-helper cells) were initially
subdivided into two subsets, designated T-helper 1 and T-helper T-regulatory cells have a protective role in peri- odontal
2, on the basis of their pat- tern of cytokine production (172). issue damage. Natural T-regulatory cells are CD4- and
T-helper 1 cells secrete interleukin-2 and interferon gamma, CD25-expressing T-cells that specifically regulate the
whereas T-helper 2 cells produce interleukins 4, 5, 6, 10 and 13. activation, proliferation and effector functions of activated
Both cell types produce interleukin-3, tumor necrosis factor conventional T-cells (4, 14, 207). T-regulatory cells are found in
alpha and granulocyte–macrophage colony-stimulating factor periodontal disease sites (30, 175). The cytokines produced by
(112, 266). The major role of the T-helper 1 cytokines T-regulatory cells are transforming growth factor beta and
interleukin-2 and inter- feron gamma is to enhance cell-mediated T-lympho- cyte-associated molecule 4 (cytotoxic T-lymphocyte
responses, whereas the T-helper 2 signature cytokine, interleu- antigen 4), which down-regulate inflammation. Inter- leukin-10,
kin-4, suppresses cell-mediated responses (170). T-cell subsets ransforming growth factor beta and cyto- toxic
are also important in the behavior of B-cells. For example, T-lymphocyte-associated antigen 4 are reported to decrease
T-helper 1 cells direct B-cell secretion of IgG2, whereas periodontal disease progression (30).
T-helper 2 cells up-regu- late IgG1 secretion. CD8 T-cells New data suggest the existence of an antigen-pre-
(cytotoxic T-cells) are immune effector cells that also secrete senting cell type from the follicular Th-cell lineage that
cytokines which are characteristic of either T-helper 1 or produces interleukin-21 (118). This type of antigen-presenting
T-helper 2 cells (266).
More recent studies have described two new cell is characterized by the​ 63
well-defined CD4 T-cell subsets, T-helper 17 and T-regulatory
T-cells, which play antagonistic roles as effector and suppressor
components, expression of the chemokine receptor, chemokine
reduced oxygen metabolites, nitric (C-X-C motif) receptor 5 (24, 50, 211).
oxide and antibodies. This is also important because The other important antigen-presenting cell is the
in severe periodontal lesions, B-cells are the macrophage. Macrophages, which are phagocytic
predominant antigen-presenting cells, suggesting cells from the myeloid lineage, efficiently ingest
that B-cell antigen presentation may allow further particulate antigen and express the major histocom-
activation and clonal expansion of already activated patibility complex class II molecules to induce
T-cells (63, 266). costimulatory activity on T-cells. Macrophages are
The role of antibody and cell-mediated immunity widely distributed cells that play an indispensible
in the pathogenesis of periodontal diseases is beyond role in homeostasis and defense. Macrophages can
the scope of this review. For a recent detailed review be phenotypically polarized by the microenviron-
of acquired immune mechanisms in periodontitis, ment. The classic inflammatory macrophage (M1) is
see Berglundh & Donati (17). The role of cytokines activated by interferon gamma and lipopolysaccha-
and other mediators from cells of the acquired im- ride. Alternatively activated macrophages (M2) are
mune network is discussed in subsequent sections of important cells in the resolution of inflammation;
this review. they have reduced capacity to produce proinflam- matory cytokines (18).
The transition from the established lesion, domi- nated by T- and B-cells, to the advanced lesion
Cytokines and chemokine networks ​(progressive periodontitis) is not well understood. We know that
dendritic cells also express the major his-
Cytokines and chemokines (chemotactic cytokines) tocompatibility complex class II molecules and have
are the messages between cells. The immune response costimulatory activity. The unique ability of B-cells to
to infection is regulated by cytokine and chemokine bind and internalize antigens via their immunoglobulin
signals. Cytokines are low-molecular-weight proteins receptors may be important in activating T-lympho-
 involved in the initiation and further stages of cytes, pointing out that costimulatory molecules are
inflammation, in which they regulate the amplitude present on B-cells. Costimulation can be thought of
and the duration of the response. The genetic regula- as the mechanism by which antigen-presenting cells
tion leading to the secretion of proinflammatory inform T-cells that the antigen requires a proliferative
cytokines from a variety of cells is generally dependent response preventing T-cell apoptosis or anergy (266).
on the activation of nuclear factor kappa-B transcrip- It has become clear that CD4 T-cells and certain in-
tion (8, 75). The nuclear factor kappa-B regulated nate immune cells, such as dendritic cells, monocytes
pathways are activated by pathogen-associated and neutrophils, are in perfect communication
molecular patterns, such as lipopolysaccharide, through cytokine networks (7, 66, 108, 220, 231).
through the toll-like receptor pathway (75). It is evident that innate and adaptive systems are
Cytokines are produced by resident cells, such as coordinately involved in the inflammatory response
epithelial cells and fibroblasts, and by phagocytes and tissue destruction, although we lack a complete
(neutrophils and macrophages) in the acute and early understanding of the mechanism. In the case of peri-
chronic phases of inflammation, and by immune odontal disease, where all elements of the immune
cells (lymphocytes) in established and advanced le- system are involved, inflammatory mechanisms and
sions (5). After recognition and presentation of signals are dysregulated. For instance, T-cells ex-
microbes to the appropriate cells, cytokines of the tracted from diseased periodontal tissues exhibit a
innate response, including tumor necrosis factor al- reduced response to stimuli, which suggests that the
pha, interleukin-1beta and interleukin-6, are the first cell-mediated response is suppressed in patients with
to appear in the periodontal disease pathogenesis periodontal disease (34); following periodontal ther-
pathways (58). Interleukin-1beta and interleukin-6 apy, lymphocyte reactivity has been reported to return
are signature innate cytokines and have been char- to normal (225).
acteristically associated with inflammatory cell Activation of B-cells is an important step in the
migration and osteoclastogenesis (56, 73). Tumor maturation of the antibody response. This event is
necrosis factor alpha is a multi-effect cytokine that mediated mainly by the tumor necrosis factor family
has many functions, from cell migration to tissue of proteins and their receptors. In addition to their
destruction. Tumor necrosis factor alpha impacts cell role in presenting antigen, B-cells also function as
migration by inducing the up-regulation of adhesion effectors, through cytokine secretion, lysosomal
molecules to promote rolling and adhesion of neu-
64
Cekici et al.
trophils to the vessel wall, leading to extravasation. It also stimulates the production of chemokines in- volved in cell migration to
infected and inflamed sites (42, 117, 190, 251). Tumor necrosis factor alpha up- regulates the production of interleukin-1beta and
interleukin-6 (42, 59, 73, 128, 173, 182, 251). Tumor necrosis factor alpha is also correlated with extra- cellular matrix degradation
and bone resorption through actions promoting the secretion of matrix metalloproteinases and RANKL (62, 72, 73) and cou- pled
bone formation (13). Accordingly, experimental periodontitis in tumor necrosis factor alpha p55 receptor-deficient mice was
characterized by a significant decrease in matrix metalloproteinase and RANKL expression and resistance to periodontitis (59).
Chemokines are chemotactic cytokines that play a very important role in the migration of phagocytic cells to the site of infection.
Once blood leukocytes exit a vessel, they are attracted, by functional gradi- ents of chemotactic factors, to the site of infection (200,
267). Chemokines are synthesized by a variety of cells including endothelial, epithelial and stromal cells, as well as leukocytes.
Functionally, chemokines can be grouped as homeostatic or inflammatory (171). In addition to their cell-trafficking role,
chemokines provide messages leading to other bio- logical processes, such as angiogenesis, cell prolifer- ation, apoptosis, tumor
metastasis and host defense (48, 171, 200, 201, 267). Bacterial peptides are also chemotactic for inflammatory cells, but the discus-
sion herein will focus upon host-derived chemokines. Chemokines are small, heparin-binding proteins ranging from 7 to 15 kDa.
They are classified into four subfamilies according to the configuration of cysteine residues near the N-terminus. The nomenclature
is as follows: chemokines are designated CXC and CX3C if two cysteines are separated and as CC and C if they are not. Their
receptors are named by adding ​Ô​R​Õ ​to the end of the particular chemokine, for example, ​Ô​CXCR​Õ ​or ​Ô​CCR​Õ​. Detailed
information on the classi- fication codes, the designated names of the chemo- kines, their receptors and the target cells they are
affecting is presented in Table 2 (21, 37, 64, 74, 101, 110, 146, 147, 200, 208, 209, 249, 264, 267). Binding of a chemokine to its
respective receptor initiates the cell-migration process, beginning with integrin- dependent adhesion and diapedesis. Chemokines
 target leukocytes of the innate immune system, as well as lymphocytes of the adaptive immune system (233).
The first cytokine identified to have chemotactic activity was interleukin-8 ⁄ chemokine (C-X-C motif) ligand 8. In the
periodontium, this cytokine is pro-
Inflammatory and immune pathways in periodontal disease

duced primarily by gingival fibroblasts, gingival epi- thelial cells and endothelial cells (227, 229, 265). Interleukin-8 is a
polymorphonuclear leukocyte chemoattractant. It is detectable in healthy and dis- eased periodontal tissues and has been associated
with subclinical inflammation of the initial lesion, which is comprised of polymorphonuclear neutroph- ils (163, 188, 263).
Interleukin-8 ⁄ chemokine (C-X-C motif) ligand also has an important role in bone metabolism. It has direct actions on osteoclast
differ- entiation and activity by signaling through the specific receptor, chemokine (C-X-C motif) receptor 1 (16).
Another crucial chemokine of innate immunity is macrophage chemotactic protein-1 ⁄ chemokine (C-C motif) ligand 2.
Macrophage chemotactic protein-1 mediates the recruitment of monocytes ⁄ macro- phages, the second wave of the innate response
to bacteria (76, 183). Macrophage inflammatory protein 1 alpha ⁄ chemokine (C-C motif) ligand 3 is the most abundantly expressed
chemokine in periodontitis tissues, with its expression localized in the connective tissue subjacent to the pocket epithelium of
inflamed gingival tissues. Together with regulated and normal T cell expressed and secreted ⁄ chemokine (C-C mo- tif) ligand 5,
macrophage inflammatory protein 1 alpha ⁄ chemokine (C-C motif) ligand 3 may also be involved in the migration of macrophages
to peri- odontal tissues (64, 102). Chemokine (C-X-C motif) receptor 3 and its ligand, interferon gamma-induced protein 10 ⁄
chemokine (C-X-C motif) ligand 10, are also expressed in diseased periodontal tissues, (61, 102) and are associated with higher
levels of inter- feron gamma in inflammation. Chemokine (C-C motif) receptor 4 is expressed at higher levels in chronic
periodontitis and it is associated with higher levels of interleukin-4 and interleukin-10 in the periodontium (61, 62).
It has become increasingly clear that chemokines are multipurpose ligands in mediating repair and angiogenesis. The time
and the place of secretion are of utmost importance. In a study by DiPietro et al., macrophage chemotactic protein-1 ⁄ chemokine
(C-C motif) ligand 2-deficient mice demonstrated delayed wound re-epithelialization (43, 149). Chemokines are equally crucial in
guiding adaptive immunity and also play a critical role in bone metabolism. For in- stance, chemokines such as
macrophage-derived chemokine ⁄ chemokine (C-C motif) ligand 22, thy- mus and activation-regulated chemokine ⁄ chemoki- ne
(C-C motif) ligand 17 and I-309 ⁄ chemokine (C-C motif) ligand 1 attract T-helper 2 and T-regulatory cells binding to chemokine

(C-X-C motif) receptor 4 and chemokine (C-X-C motif) receptor 8, respectively​

65
Table 2. Chemokine classification codes, designated names and affected cell types.
Receptor (classification code) Cell type affected
Ligand
Ligand (classification code)
(designated name)
CCR1 Monocytes ⁄ macrophages CCL3 MIP-1a
Neutrophils CXCL8 CXCL6 CXCL1 CCL23
IL-8 GCP-2 GROa CKb8
Osteoclast precursors and
mature osteoclasts
RANTES MCP-3 MIP-1c
CCR2 Monocytes ⁄ macrophages and
osteoclast precursors
CCL5 CCL7 CCL9
CCL2 MCP-1
CCR3 Th1 lymphocytes CXCL9 MIG
Th2 lymphocytes and osteoblasts CXCL10 CXCL11 CCL7 CCL11 CCL13 CCL15
IP-10 I-TAC MCP-3 Eotaxin MCP-4 HCC-2
CCR4 Th2 lymphocytes and osteoblasts CCL22 CCL17 CXCL12
MDC TARC SDF-1
CCR5 Monocytes ⁄ macrophages, Th1
lymphocytes and osteoblasts
 CCL5 RANTES
B lymphocytes CXCL13 BCA-1
CCR8 Th2 lymphocytes CCL1 I-309
CXCR1 Osteoclast precursors and osteoblasts CXCL8 IL-8
CXCR3 Osteoclast precursors and Osteoblasts CXCL9 MIG
CXCR4 Osteoclast precursors,
mature osteoclasts and osteoblasts
CXCL12 SDF-1
CXCR5 Osteoblasts CCL5
CXCL13
RANTES BCA-1
BCA-1, B cell-attracting chemokine 1; CCL, chemokine (C-C motif) ligand; CCR, chemokine (C-C motif) receptor; CKb8, transcript variant CKb8; CXCL, chemokine (C-X-C
motif) ligand; CXCR, chemokine (C-X-C motif) receptor; GCP-2, granulocyte chemotactic protein 2; GROa, melanoma growth stimulatory factor; HCC-2, hemofiltrate
CC-chemokine-2; IL-8, interleukin-8; I-TAC, interferon-inducible T-cell chemoattractant; MCP-3, macrophage chemotactic protein-3; MCP-4, mac- rophage chemotactic
protein-4; MDC, macrophage-derived chemokine; MIG, monokine induced by gamma interferon; MIP-1a, macrophage inflammatory protein 1alpha; MIP-1c, macrophage
inflammatory protein 1gamma; RANTES, regulated and normal T cell expressed and secreted; SDF-1, stromal cell-derived factor-1; TARC, thymus and activation-regulated
chemokine; Th1, T-helper 1; Th2, T-helper 2.
(37, 74, 208). It has been suggested that the expression of T-helper 2 and T-regulatory cell chemoattractants, such as
macrophage-derived chemokine ⁄ chemokine (C-C motif) ligand 22, thymus and activation-regu- lated chemokine ⁄
chemokine (C-C motif) ligand 17 and I-309 ⁄ chemokine (C-C motif) ligand 1, reduce periodontal disease severity (175). B
cell-attracting chemokine 1 ⁄ chemokine (C-X-C motif) ligand 13, an important B-cell chemoattractant, is expressed in
diseased tissues, suggesting a role for the accumula- tion of these cells in the periodontium. The expres- sion of B
cell-attracting chemokine 1 ⁄ chemokine (C-X-C motif) ligand 13 may be important in the
66
Cekici et al.
local humoral response to periodontal pathogens (119).
Chemokines are involved in both the physiology and the pathology of bone metabolism. They are essential signals for the
trafficking of osteoblast and osteoclast precursors, and consequently as potential modulators of bone homeostasis (16,
257). The chemokines implicated in regulating bone metabo- lism are identified through expression of receptors including
chemokine (C-C motif) receptors 1 and 2, and chemokine (C-X-C motif) receptors 3 and 4; these receptors are expressed
on osteoclast precur- sors, mature osteoclasts and osteoblasts. The poten-
tial chemokine ligands include stromal cell-derived factor-1 ⁄ chemokine (C-X-C motif) ligand 12, mac- rophage inflammatory
protein 1 alpha ⁄ chemokine (C-C motif) ligand 3, regulated and normal T cell expressed and secreted ⁄ chemokine (C-C motif)
ligand 5, macrophage inflammatory protein 1 gamma ⁄ chemokine (C-C motif) ligand 9, macro- phage chemotactic protein-1 beta ⁄
chemokine (C-C motif) ligand 2, macrophage chemotactic protein-3 ⁄ chemokine (C-C motif) ligand 7, monokine induced by
gamma interferon ⁄ chemokine (C-X-C motif) ligand 9 and transcript variant CKb8 ⁄ chemokine (C-C motif) ligand 23 (115, 116,
127, 134, 183, 249, 257, 259, 264). Interferon gamma-induced protein 10 ⁄ chemokine (C-X-C motif) ligand 10 induces osteo- blast
proliferation through chemokine (C-C motif) receptor 3 (71, 143), while stromal cell-derived factor- 1 alpha ⁄ chemokine (C-X-C
motif) ligand 12 and B cell-attracting chemokine 1 ⁄ chemokine (C-X-C mo- tif) ligand 13 induce both proliferation and collagen
type I mRNA expression in osteoblasts through chemokine (C-C motif) receptors 4 and 5 (144). In addition to a role in
osteoclastogenesis, chemokines also impact osteoclast functions. Stromal cell-derived factor-1 alpha ⁄ chemokine (C-X-C motif)
ligand 12 increases the activity of matrix metalloproteinase 9 in human osteoclasts, resulting in increased bone resorption (70).
There is some evidence that regulated and normal T cell expressed and secreted ⁄ chemokine (C-C motif) ligand 5 acts on
osteoblasts, resulting in chemotaxis and promoting cell survival (260). Inter- estingly, RANKL also induces the production of
macrophage chemotactic protein-1 ⁄ chemokine (C-C motif) ligand 2, macrophage inflammatory protein 1 ⁄ chemokine (C-C motif)
ligand 3, regulated and normal T cell expressed and secreted ⁄ chemokine (C-C motif) ligand 5 and monokine induced by gamma
interferon ⁄ chemokine (C-X-C motif) ligand 9 by osteoclasts, suggesting a coupling contribution to bone resorption (115). Taken
together, these studies suggest that chemokines can effectively con- tribute to bone remodeling by driving osteoblast migration and
activation.
Interferon gamma is a signature cytokine of the adaptive immune response. Its main function is to promote
antigen-presenting cell binding of antigen by up-regulating major histocompatibility complex class I and class II expression (199,
238). Interferon gamma also plays a major role in B-cell maturation, and, accordingly, in immunoglobulin secretion (179). In
 periodontal disease, interferon gamma is present at high levels in periodontal lesions, and is associated
Inflammatory and immune pathways in periodontal disease

with progressive lesions or severe forms of peri- odontitis (46, 61, 88).
Interleukin-4 is another important adaptive- immunity cytokine that induces proliferation of T-cells and regulates B-cell
immunoglobulin secre- tion. It is considered an anti-inflammatory cytokine. Interleukin-4 has known antitumor actions. Inter-
leukin-4 inhibits the activity of proinflammatory cytokines, such as the interleukin-2-induced generation of natural killer cells and
the activation of macro- phages (179). Interleukin-4 can also block nitric oxide generation by macrophages (170). Studies also sug-
gest that interleukin-4 down-regulates the production of other cytokines, including interleukin-1beta, tumor necrosis factor alpha
and interleukin-6, by human peripheral blood monocytes and T-helper 1 cells (44, 49), inhibiting the transcription of these
proinflam- matory cytokines and interferon gamma. Additionally, interleukin-4 inhibits the production of matrix metalloproteinases
and RANKL, and concomitantly induces the up-regulation of tissue inhibitor of metalloproteinases and osteoprotegerin (93, 204),
reinforcing its potential protective role in periodontal diseases (68). Interleukin-4 also induces the produc- tion of interleukin-10,
another anti-inflammatory cytokine (191). Interleukin-10 plays a major role in suppressing immune responses by inhibiting the
antigen-presenting capacity of macrophages (40, 52). Interleukin-10 is a potent effector for activated human B-cells (202). It is
widely expressed in inflamed peri- odontal tissues, where it is thought to limit disease severity (60, 62, 133). Interleukin-10
interferes directly with the production of interferon gamma and inter- leukin-17 by T-helper 17 cells (100, 176). Interleukin- 10
plays a direct protective role in tissue destruction by down-regulating both matrix metalloproteinases and RANKL. Interleukin-10
characteristically induces the up-regulation of tissue inhibitor of metallopro- teinases, which inhibit the matrix metalloproteinase
family of proteins (31, 33, 62).
Interleukin-12 was originally described as a factor that promotes the activity of natural killer cells and CD8 T-cells (132). It
has the capacity to enhance T-cell and natural killer cell proliferation after acti- vation by other stimuli (121, 189). Natural killer
cells appear to be most effective at preventing early infection, but T- and B-cells and their products are required to resolve the
infection (9). Interleukin-4 and interleukin-10 are powerful inhibitors of the production of interleukin-12. It has been suggested that
these two cytokines may determine the balance between T-helper 1 and T-helper 2 cells in the peri- odontal lesion (237).

67
but Interleukin-13 is another potent modulator of
at low doses it has the potential to increase IgG. human monocyte ⁄ macrophage and B-cell function.
When combined with interleukin-4, low doses of Monocyte ⁄ macrophage cell-surface major histo-
prostaglandin E​2 ​induce a synergistic rise in IgG compatibility complex class II and several integrin
production, suggesting an immune-regulatory role molecules are up-regulated by interleukin-13 (39).
for prostaglandin E​2 (79).
​ The monocyte ⁄ macrophage-related production of interleukin-1alpha, interleukin-1beta,
interleukin-6, interleukin-8 and tumor necrosis factor alpha is
Destruction of periodontal tissues ​inhibited by interleukin-13, and interleukin-1 recep- tor antagonist
secretion is enhanced (39, 269)
Destruction of bone ​suggesting an anti-inflammatory role along with
It is now generally accepted that disruption of the interleukin-4 and interleukin-10 (269).
balance between osteoblast and osteoclast activities Transforming growth factor beta is a growth factor
by bacterial products and inflammatory cytokines that regulates cell growth, differentiation and matrix
constitutes the main underlying causes of inflam- production, and is also a potent immunosuppressive
mation-induced bone loss (145). Lipopolysaccharide factor that down-regulates the transcription of proin-
directly stimulates bone resorption when added to flammatory factors (such as interleukin-1beta and
osteoclast precursor cultures containing osteoblasts tumor necrosis factor alpha) and matrix metallopro-
and ⁄ or stromal cells (94). A toll-like receptor and teinases (181, 223). In active periodontal lesions, the
inflammation-induced osteoclastogenesis pathway is levels of transforming growth factor beta are negatively
implicated in the initiation of bone loss (187, 192). correlated with the levels of RANKL, reinforcing its
 Inflammation-induced bone loss in response to protective role against tissue destruction (45, 46, 223).
periodontal infection has been well studied. Complex inflammatory signals and cytokine networks regulate
Lipid mediators of inflammation
osteoclastogenesis through RANKL, interleukin- 1beta, interleukin-6, tumor necrosis factor alpha and
Prostaglandins are derived from the hydrolysis of
prostaglandin E​2 ​(84) (Fig. 1).
Before the discovery of RANK, its ligand (RANKL) membrane phospholipids. Phospholipase A​2 cleaves ​
and its antagonist (osteoprotegerin), the develop- the sn-2 position of membrane phospholipids to
ment and the formation of osteoclasts were thought generate arachidonic acid, a precursor of a group of
to be controlled by factors produced by osteoblasts small lipids known as eicosanoids (139). Arachidonic
and bone marrow stromal cells (162, 198). It is now acid is metabolized by two major enzyme pathways:
clear that RANKL and osteoprotegerin are the key (i) lipoxygenases, which catalyze the formation of
regulators of bone remodeling and are directly in- hydroxyeicosatetraenoic acids, leading to the forma-
volved in the differentiation, activation and survival tion of leukotrienes; and (ii) cyclooxygenases 1 and 2,
of osteoclasts and osteoclast precursors (2, 129, 261). which catalyze the conversion of arachidonic acid
RANKL is expressed by osteoblasts, stromal cells, into prostaglandins, prostacyclins and thrombox-
chondrocytes and other mesenchymal cells. In addi- anes. Prostaglandins have 10 subclasses, of which D,
tion, activated T-cells and B-cells can also express E, F G, H and I are the most important (65). Inflamed
RANKL (111, 158, 235). RANK is expressed by osteo- gingiva synthesizes significantly larger amounts of
clast progenitors, mature osteoclasts, chondrocytes, prostaglandins when incubated with arachidonic
monocytes ⁄ macrophages and dendritic cells (2, 91). acid than does healthy gingiva (167). Prostaglandin E​2
The decoy receptor, osteoprotegerin, is known to be is a potent stimulator of alveolar bone resorption (41,
expressed by periodontal tissue cells, including 69). Within gingival lesions, prostaglandin E​2 ​is
fibroblasts and periodontal ligament cells (145). mainly localized to macrophage-like cells and is
Blocking RANKL activity with osteoprotegerin signif- secreted when stimulated with bacterial lipopoly-
icantly inhibits bone loss in rheumatoid arthritis, saccharide (148). Periodontal ligament cells also
osteoporosis, cancer-related bone metastasis and produce prostaglandin E​2​, even when unstimulated.
diabetes-associated alveolar bone destruction (25, 87, This secretion is enhanced by interleukin-1beta,
89, 122, 158, 169), confirming the critical role of the tumor necrosis factor alpha and parathyroid hor-
RANKL ⁄ RANK ⁄ osteoprotegerin triad in osteoclas- mone (196, 205, 206). It is important to note that
togenesis. However, it is not that simple. Macrophage prostaglandin E​2 ​has biphasic actions on immune
colony-stimulating factor is also required, which is function. In high doses, it decreases the levels of IgG,
produced by osteoblasts ⁄ stromal cells (155, 230).
68
Cekici et al.
The periodontal implication of the macrophage through a myeloid differentiation primary response protein
colony-stimulating factor requirement is that the pathogen, stress (MyD88)- dependent mechanism (210). Also, osteoclasts and
or pathology that influences the production of macrophage their precursors have been shown to express toll-like receptors,
colony-stimulating factor via proinflammatory cytokines will especially toll-like receptors 2, 4 and 9 (83, 86). Moreover, in
have a significant influence on subsequent osteoclast activity. mouse calvarial osteoblasts, expression of toll-like receptors 4
For example, toll-like receptor 2 activation in human gingival and 9 results in the activation of nuclear factor of kappa light
fibroblasts up-regulates the expression of macrophage polypeptide gene enhancer in B-cells and increased secretion of
colony-stimulating factor (27). tumor necrosis factor alpha and macrophage colony- stimulating
It is known that lipopolysaccharide from different factor (268). Lipopolysaccharide-induced production of
pathogens stimulates bone resorption in vitro and in animal interleukin-1beta through toll-like- receptor pathways can
models, as in primary mouse calvarial osteoblasts. The up-regulate RANKL and can also inhibit the expression of
activation of toll-like receptor 2 and toll-like receptor 6 by osteoprotegerin by osteoblasts, resulting in osteoclast formation
lipopolysaccharide causes enhanced expression of RANKL
 periodontal inflam- mation (241). Matrix metalloproteinase
in a prostaglandin E​2​-dependent manner (224). The crucial
​ role
activation involves tissue and plasma proteinases and bacterial
of toll-like receptor 2 is that it substan- tially decreases the proteinases, together with oxidative stress (174, 242).
responses to lipopolysaccharide (27). These findings point out
The expression and pathologic release of matrix
that lipopolysaccha- ride, directly via toll-like receptor
metalloproteinases was originally thought to be limited to
pathways, induces osteoclast development and activity. Thus, it
neutrophils (241), but it is now clear that a broad range of cell
is believed that toll-like receptors influence the inflammatory
types present in normal and diseased human periodontium
response in the bone microenviron- ment and may play a critical
(including gingival epithelial cells, fibroblasts, endothelial cells,
role in modulating inflammation-induced osteoclastogenesis and
mono- cytes ⁄ macrophages and plasma cells) express distinct
bone loss. It is also interesting that recent evidence also points to
matrix metalloproteinases (77, 114, 234, 250). Transcription of
important roles for resident cells in periodontal bone loss
matrix metalloproteinase genes is very low in healthy
because periodontal ligament fibroblasts and osteoclast
periodontal tissue. In periodontal disease, secretion of specific
precursors synergistically increase the expression of genes
matrix metalloprotein- ases is stimulated or down-regulated by
related to osteocl- astogenesis (20).
various cytokines. The main stimulatory cytokines for matrix
metalloproteinases are tumor necrosis factor alpha, interleukin-1
and interleukin-6. It is also known that active matrix
Destruction of extracellular matrix ​There is significant metalloproteinases are capable of activating other matrix
evidence showing that collagen- ases, along with other matrix metalloproteinases in a mutual activation cascade (248). Certain
metalloproteinases, play an important role in periodontal tissue cytokines are specifically related to particular matrix metallo-
destruction. Matrix metalloproteinases are a family of proteinases. For example, interleukin-1beta and tu- mor necrosis
structurally related, but genetically distinct, enzymes factor alpha can stimulate the secretion of matrix
Inflammatory
metalloproteinases 3, pathways
and immune in periodontal
8 and 9 from gingivaldisease
fibroblasts and the
secretion of matrix metallopro- teinase-13 from osteoblasts.
that degrade extracellular matrix and basement membrane Transforming growth factor beta suppresses the transcription of
components. This group of 23 human enzymes is classified into matrix metalloproteinase-1, -3 and -8 genes, but induces matrix
collagenases, gelatinases, stromelysins, membrane-type matrix metalloproteinase-2 and matrix metallopro- teinase-13, mainly
metallopro- teinases and other matrix metalloproteinases, mainly in keratinocytes (19, 106, 124).
based on the substrate specificity and the molecular structure. The involvement of matrix metalloproteinases in
Matrix metalloproteinases are involved in physiological nflammation is an active area of investigation. A good example
processes such as tissue development, remodeling and wound of the interaction is interleukin-8 secretion in response to
healing. Matrix metallo- proteinase activity is controlled by bacterial biofilm. Interleu- kin-8 recruits neutrophils to the site
changes in the delicate balance between the expression and syn- containing bio- film. The neutrophils will secrete cytokines, as
thesis of matrix metalloproteinases and their major endogenous well as
inhibitors, tissue inhibitor of matrix metalloproteinases. It is
clear that matrix metallo- proteinases are up-regulated in

69
epithelial matrix metalloproteinases 8 and 9 (221), resulting in
cells (241). Overall, the role of matrix me- the degradation of the extracellular matrix and the
talloproteinase-7 in periodontal disease is not clear. signaling other effector cells to produce matrix me-
Matrix metalloproteinase-3 (stromelysin-1) does talloproteinases. The major collagen-degrading en-
not digest interstitial collagen. The main substrates of zyme in periodontitis is matrix metalloproteinase-8,
matrix metalloproteinase-3 are basement membrane which is mainly produced by neutrophils. This en-
components such as laminins and type IV collagen. zyme is found in gingival crevice fluid and saliva in
Matrix metalloproteinase-3 is found in gingival cre- diseased periodontal tissue. The main function of
vice fluid and gingival tissue during periodontal matrix metalloproteinase-8 is the degradation of
inflammation (47, 164, 248). interstitial collagens (221).
Regulation of matrix metalloproteinase activity is a Matrix metalloproteinase-1 (collagenase-1) from
function of tissue inhibitor of metalloproteinases. mononuclear phagocytes, fibroblasts and epithelial
 The tissue inhibitor of metalloproteinases class of cells has a wide range of substrates. It digests inter-
enzymes function in the regulation of extracellular stitial collagen, extracellular matrix components and
matrix metabolism. Four members of the family of soluble nonmatrix mediators (221). Matrix metallo-
tissue inhibitor of matrix metalloproteinases (tissue proteinase-9 (gelatinase B) is a gelatinolytic enzyme
inhibitor of matrix metalloproteinases 1–4) have been that degrades several types of extracellular matrix,
identified to date. Although the main function of including basement membrane type IV collagen
tissue inhibitor of matrix metalloproteinases is to (135). Matrix metalloproteinase-9 is expressed by
inhibit matrix metalloproteinases, they also regulate neutrophils, but also by cultured epithelial cells. The
matrix metalloproteinase transportation, stabiliza- production of matrix metalloproteinase-9 is stimu-
tion and localization in the extracellular matrix. Tis- lated by several cytokines, especially tumor necrosis
sue inhibitor of matrix metalloproteinases 1, 2 and 4 factor alpha, epidermal growth factor and by some
are secreted extracellular proteins, whereas tissue bacterial products such as lipopolysaccharide and
inhibitor of matrix metalloproteinase 3 is an extra- phospholipase C (54, 194).
cellular matrix-bound molecule (248). Matrix metalloproteinase-2 (gelatinase A) has been shown to be strongly expressed
in inflamed pocket epithelium and to be important in epithelial cell
Resolution of inflammation ​migration (159). Matrix metalloproteinase-13 (colla- genase 3) is expressed by
the basal cells of the gin-
Periodontal inflammation begins as a protective re- gival pocket epithelium (240); it degrades type I, type
sponse to bacterial biofilm. In susceptible individu- III and type IV collagens, as well as fibronectin,
als, periodontal inflammation fails to resolve and tenascin and some proteoglycans (105, 120). Matrix
chronic inflammation becomes the periodontal metalloproteinase-13 plays an important role in the
pathology. Periodontal disease results from excess growth of pocket epithelium into periodontal con-
inflammation and may be considered a failure of nective tissue. Some oral bacterial species, especially
resolution pathways. An essential goal of interven- Fusobacterium nucleatum, induce matrix metallo-
tions in inflammatory disease is the return of tissue to proteinase-13 (241). Matrix metalloproteinase-7
homeostasis, defined as an absence of inflammation. (matrilysin) is another epithelial matrix metallopro-
Hence, the rapid and complete elimination of teinase with a broad spectrum of substrates. It de-
invading leukocytes from a lesion is the ideal out- grades fibronectin, laminin, type IV collagen, gelatin,
come following an inflammatory event (243). elastin, entactin, tenascin and proteoglycans. The
Accordingly, inadequate resolution and failure to re- enzyme is not commonly secreted by the gingival
turn tissue to homeostasis results in neutrophil- tissues and has not been reported in human gingival
mediated destruction and chronic inflammation epithelium or in the pocket epithelium of periodon-
(245), with destruction of both extracellular matrix titis patients. It is expressed constitutively in many
and bone, and scarring and fibrosis (244). Scarring adult epithelial cells, most notably in the salivary
and fibrosis in periodontitis prevent the return to glands, and its secretion is observed in suprabasal
homeostasis (243). epithelial cells (203, 255). Some periodontal patho-
To date, the efforts to control inflammation have gens, including F. nucleatum, Fusobacterium necro-
been focused on the use of pharmacologic agents phorum, Porphyromonas endodontalis and Prevotella
that inhibit proinflammatory mediator pathways denticola, were found to induce the expression of
(e.g. nonsteroidal anti-inflammatory drugs) (214). matrix metalloproteinase-7 in porcine gingival
Nonsteroidal anti-inflammatory drugs target cyclo-
70
Cekici et al.
oxygenase 1- and cyclooxygenase 2-dependent pathways, More recent discoveries have uncovered the natural
inhibiting the generation of prostanoids. Newer classes of proresolving pathways, which are an extension of the same
inhibitors target lipoxygenase pathways and leukotriene eicosanoid pathways that produce proinflam- matory mediators.
production, or tumor necrosis factor alpha. The side-effect The physiologic end of the acute inflammatory phase occurs
profiles of these agents prohibit their extended use in when there is a ​ÔÔ​class switch​ÕÕ ​of eicosanoid pathways in
periodontal therapy. neutrophils (138, 243). This class switch is mediated by the
 up-regula- tion of 15-lipoxygenase by neutrophils late in both anti-inflammatory and proresolution activities, reinforcing
inflammation. Neutrophils in the early acute phase produce only he active nature of the resolution process (216). In an animal
5-lipoxygenase for the production of leukotrienes. model of periodontitis, treatment with resolvin-E1 completely
15-lipoxygenase catalyzes a second reaction with eliminated the signs of inflammation, enabling the regeneration
hydroxyeicosatetraenoic acid products generated earlier by of lost tissues (81).
neutrophils or other cells (213). The series of enzymatic
reactions starts with the oxi- dation of arachidonic acid by a
lipoxygenase (5-, 12- or 15-lipoxygenase, depending on the cell Conclusion
of origin). A 5-, 12- or 15-S-hydroxy-(p)-eicosatetraenoic acid
intermediate is produced, which is then further acted on by
15-lipoxygenase to induce the synthesis of doubly substituted Periodontal diseases are inflammatory diseases in which
intermediates (5, 15 hydroxy-(p)- eicosatetraenoic acids, for microbial etiologic factors induce a series of host responses that
example) that are further metabolized into lipoxins, such as mediate inflammatory events (Fig. 1). In susceptible individuals,
lipoxins A​4 and B​4 (109, 245). Lipoxins are receptor agonistsdysregulation of inflammatory and immune pathways leads to
​ ​
that stimu- late the resolution of inflammation and promote the chronic inflammation, tissue destruction and disease.
restoration of tissue homeostasis through a number of Physiologic inflammation is a well-orchestrated net- work of
mechanisms. These include limiting the migration of cells, mediators and tissues. It is very important to consider the
polymorphonuclear neutrophils into sites of inflammation and nflammatory ⁄ immune response as a whole, rather than many
modulating the phenotype of macrophages to stimulate the different modules working separately. As disease appears to be
uptake of apoptotic polymorphonuclear neutrophils without he result of loss of regulation and a failure to return to
secreting proinflammatory cytokines (156, 157, 217). homeostasis, it is important to achieve a more complete
understanding of the molecular and cellu- lar events in this
Unlike other nonsteroidal anti-inflammatory drugs, complex system.
aspirin has unique characteristics. Aspirin acetylates
cyclooxygenase 2 to inhibit further pro- duction of prostanoids The paradigm shift in our understanding of
from arachidonic acid metabolism, but the acetylated nflammatory disease, such as periodontitis, is that resolution of
cyclooxygenase 2 has new enzyme activity as a nflammation is an active, rather than a passive, process that
15-epi-lipoxygenase. This alternative pathway leads to the activates specific biochemical programs of resolution. Precursor
synthesis of 15-R- hydroxy-(p)-eicosatetraenoic acid. This fatty-acid sub- strates from cells (arachidonic acid) and dietary
molecule is transformed into 5(6)-epoxytetraene with the help of sources (omega-3 fatty acids) yield lipid mediators (lipoxins and
5-lipoxygenase, and the product is 15-epi-LXs or resolvins, respectively) that counter- regulate proinflammatory
aspirin-triggered lipoxins (245). Aspirin-triggered lipoxin, the signals. It is increasingly evident that future care of periodontal
epimer of native lipoxin, possesses more powerful proresolving nfections and periodontal surgical patients will rely on
properties (32, 218, 245). clinicians having a detailed map and molecular appreciation of
he resolution programs for inflammation and tissue injury.
Lipoxins are the natural proresolving molecules derived
from endogenous fatty acids (arachidonic
Systematic temporal study of infection​
71
Inflammatory and immune pathways in periodontal disease
and resolution in human tissues is of paramount importance in
acid). Dietary fatty acids of the omega-3 class are also the treatment of bacterially initiated disease. Studies of models
metabolized by similar pathways, and the products (resolvins of disease suggest that the shift to chronicity of the infection and
and their aspirin-triggered derivatives) have similar biologic the persistence of the pathogen results from increased
activity to lipoxins (215, 243). Resolvins stimulate the resolution inflammation and a failure of innate mucosal antibacterial
of inflammation through multiple mechanisms, including systems. Susceptibility to chronic inflammatory disorders may
preventing neutrophil penetration, the phagocytosis of apoptotic therefore result from uncontrolled resolution of the inflammatory
neutrophils to clear the lesion and enhancing the clearance of process. Considering the limited and semi-successful treatment
inflammation within the lesion to pro- mote tissue regeneration options for periodontitis, research on the orchestration of this
(10, 81, 212). Interestingly, the classic inflammatory eicosanoids complex system can bring us one step closer for better treatment
(i.e. prosta- glandins and leukotrienes), in addition to activating opportunities. As many current and widely used drugs have been
and amplifying the cardinal signs of inflammation, are developed without knowledge of their impact in resolution
responsible for inducing the production of mediators that have circuits, some agents, such as selective cyclooxygenase 2
inhibitors and certain lipoxygenase inhibitors, have proven to be
toxic to the resolution programs (216). It will be important to metalloproteinases in human periodontal diseases.J Periodontol 1993:64:
learn whether resolution pharmacology leads to new treatments 474–484. 20. Bloemen V, Schoenmaker T, de Vries TJ, Everts V. Direct
cell-cell contact between periodontal ligament fibroblasts and osteoclast
for human disease.
precursors synergistically increases the expression of genes related to
osteoclastogenesis. J Cell Physiol 2009: 222: 565–573. 21. Bonecchi R,
Bianchi G, Bordignon PP, D​Õ​Ambrosio D, Lang R, Borsatti A, Sozzani S,
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