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C o p y r i g h t 8 Munksgaard 1997

Ieriodontology 2000,Vol. 14, 1997, 33-53

Printed in Denmark . AZl rights reserved

PERIODONTOLOGY 2000
ISSN 0906-6713

The host response to the microbial

challenge in periodontitis:
assembling the players
KENNETH S. KORNMAN,ROYC. PAGE& MAURIZIO
S. TONETTI
Introduction
The empty stage
A great amount of effort and preparation is required
prior to a theatrical production. Although the curtains may open to an apparently quiet and inanimate scene, all of the stage-hands, lighting staff,
technical crews, actors and musicians know their
places on the stage and respond to specific signals
during the play. Gingival health is a dynamic state
that may be viewed as one scene in a play. As certain
signals are given, specific players respond in a practiced manner and take their places in the scene. If
disease-initiating signals are given, the players take
positions on the stage that allow them to participate
in the disease scene.
This chapter describes how the stage changes
from health to one that is prepared for disease initiation and progression. Each change in the scenes
leading from health to disease is presented with successively finer granularity: the scene is described according to the histological characteristics of the
tissue, cellular activity and molecular changes.
Although the special interest of periodontologists
is to interpret everything that happens in the periodontal tissues as a factor in periodontal diseases,
the cellular and molecular events that occur are
dominated by routine host responses that are intended to protect the host from a bacterial challenge.
This chapter primarily focuses on the routine events
that occur to set the stage for the periodontal disease
process. There is reason to believe that specific bacterial products and the characteristics of specific individuals produce important local aberrations in the
routine response patterns. These factors may well be
the critical determinants of how the play evolves
once the stage is set and are discussed in detail in
other chapters.

General comments
Efforts to delineate the pathogenic processes of a
complex chronic disease have some inherent limitations and require many assumptions based on
cross-sectional observations from the specific disease and knowledge from general biological mechanisms.
Bacteria have generation times that allow an evolutionary adaptation to an inhospitable environment.
Thus, many of the bacteria in the gingival sulcus
have evolved mechanisms to avoid and manipulate
the host defense mechanisms. SimilarIy, the host has
developed countermeasures against these microorganisms. In an encounter between the host and a
single subgingival bacterial species, the sequence of
events in the host response may be reasonably well
defined. In a bacterial ecosystem as complex as the
gingival sulcus, however, the co-evolutionary processes between the diverse bacterial species and the
host have most likely produced a variety of host molecules to cope with the bacterial challenge. This
phenomenon is only one of the factors that makes it
extremely difficult to determine which host factors
are actually destructive or protective.
In addition, once periodontitis has established in
the tissues, it is reasonable to assume that substantial feedback mechanisms are at play to ultimately
regulate and control the immunoinflammatory responses. Such mechanisms are finely tuned, so that
the synthesis and inhibition of various cytokines and
other mediators will undergo repeated changes to
allow a response that properly limits the bacterial
challenge but is then modulated to limit tissue destruction and allow repair. Most importantly, the immunoinflammatory response in adult periodontal
disease should not be viewed as a one-dimensional
process that leads to destruction, but rather an iter-

33

Kornman et al.

Fig. 1. The epithelial and vascular response to early bacterial accumulations is shown diagrammatically in Scene
1. The bacteria release normal metabolic products, including fatty acids and the peptide N-formyl-methionylleucyl-phenylalanine(FMLP) and the lipopolysaccharides
(LPS) of gram-negative bacteria. These products activate
junctional epithelial cells to release various inflammatory
mediators, including interleukin-8 (IL-8), interleukin-la
(IL- la), prostaglandin E2 (PGE2), matrix metalloproteinases (MMP) and tumor necrosis factor a (TNFa). In addition, neural components originating in the epithelium
release neuropeptides that influence the local vascular response. The bacterial products and epithelial response activates perivascular mast cells to release histamine and
activates vascular endothelial cells to release IL-8 within
the vessels to assist in localizing neutrophils.

ative process that is constantly being adjusted to

meet multiple local and systemic needs. Variation in
tissue levels of these mediators certainly should be
expected.
Overview of the action in the play
Periodontitis is an infectious disease process. Bacteria and their products interact with the junctional
epithelium and penetrate into the underlying connective tissue. The small blood vessel plexus im-

34

mediately deep to the junctional epithelium becomes inflamed, leukocytes exit the post-capillary
venules and there is a very large increase in the
numbers of leukocytes, especially neutrophils, migrating through the junctional epithelium and into
the sulcus or pocket. The collagen and other components of the perivascular extracellular matrix are
destroyed. As supragingival plaque extends apically
into the gingival sulcus, the coronal cells of the junctional epithelium are stimulated to proliferate, and a
gingival pocket is formed. Later on, the apical cells
of the junctional epithelium are induced to proliferate and extend apically along the root surface, subsequently to be converted into an ulcerated pocked
epithelium. At an early stage, there is an enlarging
leukocyte infiltrate dominated by lymphocytes, including B cells and T cells with characteristics of
both T-helper 1 (Thl) and Th2 cells. Subsequently,
the lesion becomes dominated by B cells but also
present are T cells, macrophages and neutrophils, all
of which become activated. B and T cells are antigenically or mitogenically activated to replicate to
give rise to clones, and B cells are driven to differentiate into clones of antibody producing plasma cells.
As the disease worsens, periodontal pockets deepen,
the components of the extracellular matrix of the
gingiva and periodontal ligament are destroyed and
alveolar bone is resorbed.

Scene 1. Acute bacterial challenge

phase: the epithelial and vascular
elements respond to the bacterial
challenge (Fig. 1)
Several mechanisms operate in the vicinity of the
teeth to fend off microbial infection and prevent the
development of periodontitis. There is strong evidence that in some individuals and under some conditions, periodontal pathogens may be present but
clinical manifestations of disease do not develop
(76). The intact epithelial barrier of the gingival, sulcular and junctional epithelium normally prevents
bacterial invasion of the periodontal tissues and is
normally an effective barrier against penetration by
bacterial products and components. Salivary secretions provide a continuous flushing of the oral cavity
as well as providing a continuing supply of agglutinins and specific antibodies that can aggregate and
kill bacteria, and greatly influence the species and
numbers that can survive in the oral cavity. The gingival crevicular fluid continuously flushes the sulcus

The host response to the microbial challenge

or pocket and delivers all the components of blood

serum including complement proteins and specific
antibodies, which bathe the bacteria of the subgingival flora. A large population of B cells and plasma
cells that accumulate in the wall of the sulcus or
pocket produce antibodies that may be specific for
antigens of challenging bacteria and may tag them
for phagocytosis and killing. There is a very high
level of turnover of both the epithelium and the
components of the extracellular matrix, and this permits rapid replacement of cells and tissue components damaged by the microbial challenge.
The normal gingival connective tissue consists of
highly organized collagen fibers, proteoglycans and
serum-derived components such as albumin. Some
elastin fibers may also be present. Resident fibroblasts are uniformly distributed and a few macrophages and scattered infiltrating leukocytes, especially plasma cells may be observed but foci of inflammatory cells are not seen.
The epithelium in health is exposed to various
bacterial products
If the gingival condition is healthy, bacteria that are
early colonizers of the tooth surface are usually
found adjacent to the gingival margin. These bacteria include gram-positive facultative streptococci
and Actinomyces species, gram-negative capnophilic
Cupnocytophuga species, and the gram-negative anaerobes, Fusobucterium nucZeatum and Prevotellu
intermedia (75).
The microbial mass releases large quantities of
metabolites that may diffuse through the junctional
epithelium. These may include fatty acids such as
butyric and propionic that are toxic to the tissues
(74), peptides of the N-formyl-methionyl-leucylphenylalanine type which are potent chemoattractants for leukocytes, and the lipopolysaccharide of
gram-negative bacteria. Several other products from
periodontal bacteria have been shown to activate
various host mechanisms, but their relative importance in the disease process is unknown (151). These
and other proinflammatory mediators synthesized
by the junctional epithelium such as interleukin 1
(IL- 11, prostaglandin E2 and matrix metalloproteinases, can traverse the junctional epithelium and enter the connective tissues (1).It is via this mechanism that gingival vessels become inflamed and a
gradient of chemoattractant signals is established
that can guide the emigrating leukocytes to the location of the microbial plaque (Fig. 2 ) . Other bacterial
components such as lipopolysaccharide can activate

Fig. 2. Schematic drawing of the relationships of the gingival sulcus. Note the microbial plaque (P) on the tooth surface at the entrance to the gingival sulcus. Neutrophils are
leaving vessels in the connective tissue (CT) and emigrating chemotactically through the junctional epithelium
(JE) but not through the oral sulcular epithelium (OSE) of
the lateral wall of the sulcus. Source: Schroeder HE, Attstrom R. Pocket formation: an hypothesis. In: Lehner T, Cimasoni M, ed. Borderland between caries and periodontal
disease, Vol. 11. Orlando: Grune and Stratton, 1980).

the endothelial cells either directly or indirectly by

inducing the production and release of inflammatory mediators from various cells in the connective
tissue. These include histamine from perivascular
mast cells, prostaglandins and interleukins such as
IL-lp, and matrix metalloproteinases from resident
tissue macrophages, fibroblasts or keratinocytes
(84). Lipopolysaccharide can also activate the complement cascade via the indirect pathway as well as
induce the production of kinins, all of which can act
on the blood vessels and their endothelial cells.
The epithelium provides both signaling and
protective functions
Recent evidence (129) has indicated that mucosal
epithelia, besides providing protection due to their

35

Kornman et al.

barrier function and rapid cell turnover leading to

constant shedding of the mucosal surface exposed
to bacterial colonization, play a crucial role in intraepithelial recruitment of phagocytes and specific
lymphocyte subsets and thus in controlling bacterial
penetration through the mucosal integuments. The
discussed evidence also indicates that keratinocytes
may play an active role in this process. This is not
unexpected since keratinocytes represent the interface between the body and the external environment
exposed to bacterial challenges. Substantial evidence
indicates that keratinocytes can respond to a variety
of bacterial stimuli via the production of a wide array
of pro-inflammatory mediators, and of cytolunes in
particular (73, 124). Such ability to sense the external environment and to respond with the generation
of specific autocrine and paracrine messages has
been confirmed in a series of in vitro investigations on mucosal and cutaneous keratinocyte cultures. Emerging evidence indicates that interaction
between periodontal bacteria and keratinocytes
leads to keratinocyte activation to express a variety
of inflammatory mediators (40). These stimuli may
act locally to transmit the inflammatory message in
a centripetal direction towards the subepithelial
microvessels and thereby induce an inflammatory
reaction.
So far, evidence that these mechanisms are at play
in the marginal periodontium is sketchy. In particular, evidence of the elements conferring specificity to
the process is lacking. Is the ability of different bacterial components of dental plaque to induce these
centripetal stimuli essentially the same, or are specific bacteria and/or specific exo- and endocellular
toxins needed? Further, it is unclear whether or not
individual subjects may present differences in junctional epithelium keratinocyte responsiveness to
bacterial plaque stimuli.
Besides the role of epithelial keratinocytes, more
recent evidence has indicated that the presence of
specific leukocytes within the epithelium decreases
the chance that bacteria can gain access to the
underlying connective tissue in the gastrointestinal
tract. In fact, a significant increase in bacterial translocation to mesenteric lymph nodes was observed in
athymic nude mice (101). Furthermore, Gautreaux et
al. (50) have shown that experimental depletion of T
cells in otherwise irnmunocompetent mice resulted
in a significant increase in Escherichia coli translocation to the mesenteric lymph nodes. Maintenance
of an intact barrier function in mucosae, therefore,
seems to depend on complex interactions of colonizing or infecting microorganisms with keratinocytes

36

and intraepithelial leukocytes. Several investigations

have described the presence of specific intraepithelial leukocytes within the junctional epithelium including: CDla-positive antigen-presenting cells
(most likely Langerhans or dendritic cells), specific
lymphocyte subsets expressing the dELP7 integrin
(mucosal T cells), the cutaneous lymphocyte antigen
and the y6 T-cell receptor (a subpopulation of T cells
that has a repertoire of receptors enriched for bacterial antigens) (25, 52,79, 135, 138, 147).The density
of cells expressing these phenotypes is selectively increased in the junctional epithelium with respect to
the underlying infiltrated connective tissue, suggesting that specific mechanisms able to selectively
increase the probability of these cells to gain access
to the epithelium may be at play. The significance
of the presence of these accessory cells within the
junctional epithelium is unknown; cells bearing
these specific phenotypes, however, may control the
intraepithelial availability of proinflammatory stimuli and/or play an important role in antigen processing and presentation.

The bacterial challenge induces changes in the

epithelium to facilitate both vascular
permeability and the influx of neutrophils
Junctional epithelium is the structure initially most
directly challenged by bacteria. Junctional epithelium is a unique structure, differing in many respects
from all of the other oral and nonoral epithelia (117).
It manifests a uniform interface with the underlying
connective tissue without rete ridges and is roughly
15 to 18 cells thick at the sulcus bottom, tapering to
4 or 5 cells at the most apical termination. Junctional
epithelium consists of basal and suprabasal strata,
although all the cells are morphologically very similar. Though stratified, the cells do not undergo maturation; they manifest differentiation markers typical
of simple epithelia, including production of keratin
pair K 8 and K18 (47, 80, 81). Basal cells, which produce the basal lamina at the interface between the
junctional epithelium and connective tissue as well
as the interface with the tooth surface, have the capacity to synthesize DNA and divide. The sloughing
surface is at the sulcus bottom.
Cells along the tooth surface and near the sulcus
bottom contain acid phosphatase-positive lysosomes and manifest evidence of phagocytosis of
neutrophil granules and bacteria. Since the cells
have neither keratohyalin- nor membrane-coating
granules, the diffusion barrier found in most stratified epithelia is absent. The cells can make several

m e host response to the microbial challenge

keratins with keratin 19 considered to be a marker
for junctional epithelium (2). The cells express intercellular attachment molecule and leukocyte function
antigen 3 on their surfaces even under healthy noninflammatory conditions (27, 871, and intercellular
attachment molecule 1 expression by keratinocytes
can be upregulated by pro-inflammatory cytokines
but not by lipopolysaccharide (140, 141). IL-8 messenger RNA was recently found (41, 134, 138) to be
present in gingival tissue and was localized primarily
to the junctional epithelium. This localization may
play a role in directing neutrophils to the gingival
sulcus area. The cells also express epidermal growth
factor, which is especially prominent in cells adjacent to the tooth surface (130), and epidermal
growth factor receptors (91) the functional role of
which is not understood.
Matrix metalloproteinases capable of degrading
the extracellular matrix of the periodontium have
been identified in the gingival epithelium (84), and
prostaglandin H synthase, which is capable of producing prostaglandin E2,has been identified in gingival epithelium (20).
A mediator derived from gingival epithelial cells
has also been shown to stimulate collagenase production by periodontal ligament fibroblasts. The action of the epithelial cell mediator was blocked by
IL-la-neutralizing antibody (97).
In addition, epithelial cells in general are known
to produce a broad range of cytokines, including ILla, IL- lp, granulocyte-macrophagecolony-stimulating factor, interferon p, tumor necrosis factor a,
transforming growth factor p, IL-3, IL-6, IL-7, IL-8,
IL-10, IL-11, and IL-12 (18, 33, 39, 42, 58, 70, 108,
124, 129). Mucosal epithelial cells exposed to bacterial products were recently shown to produce tumor necrosis factor a, IL-6 and IL-8 (38, 58).
In addition to inflammatory mediators, such as
cytokines, produced by activated epithelial cells,
neural components may be a key aspect of the early
tissue response to bacterial stimuli. Although not yet
defined in humans, the rat junctional epithelium has
been shown to include an unusually dense network
of unmyelinated nerve fibers, most likely c-fibers,
that produce the neuropeptides substance P and calcitonin gene-related peptide (15, 16, 82, 89, 131).
Many branching fibers formed a network around the
basal cells and extended through out the junctional
epithelium. These networks are the most dense yet
observed in oral tissues. These types of fibers routinely form localized loops that extend from the epithelium to innervate the local blood vessels and activate the mast cells that are routinely resident ad-

jacent to small vessels. The neuropeptides and mast

cell activation initiated by the c-fibers extending
from the junctional epithelium may be effector
mechanisms involved in the early vascular response
and cell replication. The cytokines produced by the
responding epithelial cells are also known to activate
adhesion molecules on endothelial cells and cytokine production by endothelial cells.
Scene 1 (Fig. 1) therefore involves epithelial, and
most likely local neural, responses to the bacterial
products. The result evident in scene 2 is enhanced
recruitment of neutrophils, proliferation of the epithelial ceUs and localized secretion of enzymes that
initiate destruction of the extracellular matrix.

Scene 2. Acute inflammatory

response phase: the tissues
respond to the early signals (Fig. 3)
Turnover of the junctional epithelium is unusually
high. Cells of the junctional epithelium are connected
by fewer than half the number of desmosomes seen
in other oral epithelia,and the extracellular spaces are
usually wide. Most of the vacuoles and vesicles associated with the junctional epithelium cells on electron
micrographs are, in fact, portions of the extracellular
compartment (119). In mild inflammation, the spaces
are widened further and filled with fluid, which can
serve as a medium for diffusion and through which
neutrophil migration occurs. Studies in the rat show
that antigen placed in the gingival sulcus readily traverses the junctional epithelium (1). As with all epithelial surfaces, an increased bacterial load in the gingival sulcus increases the cell turnover rate of the sulcular epithelium (17). Infiltrating cells occupy about
1-2% of the extracellular space in human junctional
epithelium and form a gradient with the greatest
number of cells found coronally. Independent of this
gradient, and in the absence of inflammation, large
numbers of leukocytes, estimated to total about
30,00O/min, migrate through the junctional epithelium (113). These are mostly neutrophils but also include monocytes and lymphocytes. Langerhans cells
and other HLA-DR-positive antigen-presenting cells
are also present.
The vascular leakage enhances the localized
response
Following the generation of proinflammatory afferent stimuli within the junctional epithelium, and

37

Kornman el al.

including IL- 1p or tumor necrosis factor a, the endothelial cells of the microcirculation become activated
(13) (Fig. 4, 5). The vessels of the microcirculation
become inflamed, dilated and engorged with blood,
and the blood flow slows. The endothelial cell junctions open and protein-rich fluid leaves the vessels
at the site of the post-capillary venules and accumu-

Fig. 3. Scene 2 diagrammatically shows the addition to

Scene 1 of serum protein systems and macrophage activation. The vascular leakage and activation of serum proteins, such as complement, begin to amplify the local inflammatory response and produce further endothelial cell
activation. Additional leukocytes and monocytes are recruited and activated macrophages produce many mediators of the immune and inflammatory responses, including interleukin 18 (IL-lp), interleukin 1 receptor antagonist (IL-ha),interleukins 6,lO and 12 (IL-6,IL-10 and
IL- 12), tumor necrosis factor a ("Fa),
prostaglandin E2
( P G k ) , matrix metalloproteinases (MMPs), interferon y
(IFNy) and a series of chemotactic substances: monocyte
chemoattractant protein (MCP), macrophage inflammatory protein (MIP) and RANTES (regulated on activation,
normal T-cell expressed and secreted). PMNS: polymorphonuclear lymphocytes.

early in the local inflammatory response, subepithelial venules are activated and display: an increase
in vascular permeability; the expression of leukocyte
cell adhesion molecules; and the release of specific
leukocyte-activating agents. The effects of these
phenomena are thought to be an increased leakage
of plasma components, including acute-phase proteins, into the gingival crevicular fluid and leukocyte
extravasation, leading to the formation of a perivascular connective tissue infiltrate.
In the presence of lipopolysaccharide or cytokines

38

Fig. 4. Schematic drawing illustrating the process of diapedesis. Endothelial cells of the venules become activated
by lipopolysaccharide (LPS) as well as by the proinflammatory cytokines IL-18 and tumor necrosis factor fl (not
shown) and are induced to express E-selectin. Neutrophils
constitutively express sialyl Lewis-X, which forms a loose
binding to E-selectin, resulting in slowing of the leukocyte
to a rolling motion along the endothelial surface. Leukocyte adhesion receptors (CD11118) form a tight binding
with the intercellular adhesion molecule (ICAM), which is
constitutively expressed by the endothelial cells, and initiates movement of the leukocyte between the endothelial cells into the extravascular compartment. (Figure
courtesy of Richard Darveau)

Fig. 5. Section through a small vessel located subjacent to

the junctional epithelium in a biopsy specimen taken
from a human 8 days after the cessation of plaque control.
Note the presence of neutrophlls (N) within the vessel and
possibly adherent to the endothelial cells. Original magnification ~ 1 2 5 0Source:
.
Payne WA, Page RC, Ogilvie AL,
Hall WB.Histopathological features of the initial and early
stages of experimental gingivitis in man. J Periodont Res
1975: 10: 51-64.

The hosr response to the microbial challenge

Fig. 6. Cross-section of an inflamed venule or capillary

with neutrophils (N)adherent to the vessel wall that are
moving between two endothelial cells. Portions of these
and additional neutrophils are seen in the connective
tissue. Magnification ~ 8 1 0 0Source:
.
Schluger S, Yuodelis
R, Page RC, Johnson RH. Periodontal diseases. 2nd edn.
Philadelphia:Lea & Febiger, 1990: 224.

gradient of chemoattractant through the connective

tissues and junctional epithelium, to form a barrier
between the subgingival microbial plaque and the
gingival tissue (Fig. 7). These cells are still viable and
capable of phagocytosis and killing of bacteria and
they serve to prevent apical and lateral extension of
the subgingival plaque. Diseases such as leukocyte
adhesion deficiency syndrome (103, 146) or procedures in experimental animals that alter neutrophi1 function (60, 117) result in rapid, severe periodontal destruction.
There is good evidence that the movement of
leukocytes from the vasculature to the tissue is regulated by different types of adhesion molecules that
are expressed on endothelial cells and the leukocytes
(100, 156). The specificity of the adhesion molecules
for leukocyte subpopulations determines which cells
migrate into the tissue. The local immunoinflammatory response is a function of which cells are in
the tissue and the mediator signals that influence
the behavior of the cells.
Leukocyte entry into tissues is a tightly regulated
process that has evolved to ensure optimal availabil-

lates in the extracellular matrix. Several studies of

early or experimental gingivitis show increases in inflammatory mediators in the gingiva or crevicular
fluid (57, 68).
Acute phase proteins such as a2-macroglobdin
have been shown to increase very early in the gingivitis process (3,671, indicating an increased vascular
leakage. The increased plasma in the tissue will produce a localized activation of complement and plasmin. These plasma-derived enzyme systems amplify
the inflammatory response and produce further endothelial cell activation.

Leukocytes selectively emigrate from the vessels to

alter the immunoinflammatory cell populations in
the gingival tissues
All the above mechanisms are important in fending
off microbial challenge and periodontal infection,
but the large numbers of emigrating leukocytes, especially neutrophils, comprise the most important
local defense. Neutrophils exit the inflamed vessels
of the microcirculation (Fig. 6) and migrate along a

Fig. 7. The most apically situated subgingival colony of

spirochetes attached to the tooth surface by a cuticular
substance and separated from the epithelium by a layer
of neutrophik (N)in the pocket of a beagle dog. Magnification ~9750.
Source: Theilade J, Attstrom R. Distribution
and ultrastructure of subgingival plaque in beagle dogs
with gingival inflammation. J Periodont Res 1985: 10: 131145.

39

Komman et al.

ity of inflammatory cells to injured tissues while

avoiding unnecessary damage. Leukocyte entry into
the periodontal stage requires an increase in the
stickiness of leukocytes in response to the intravascular release of proinflammatory agents, including IL-8, by activated endothelial cells (61, 123) (Fig.
4). This induces rolling of the leukocytes on the endoluminal aspect of the venules and increases the
probability of specific interactions among vascular
cell adhesion molecules and leukocyte integrins and
thus the chance of inducing leukocyte extravasation
by diapedesis into the exuavascular spaces. Specific
differences in the resulting inflammatory infiltrate in
various tissues indicates that this process possesses
a high degree of specificity, enabling the selective enrichment of specific cell types (123). The selectively
of the process seems to depend on the expression
on the endothelial cells of specific adhesins such as
endothelial cell adhesion molecule 1, vascular cell
adhesion molecule 1 and mucosal addressin cell adhesion molecule 1, whose complementary receptors
are expressed on specific leukocyte subpopulations.
As examples, endothelial cell adhesion molecule 1
is thought to be important for the extravasation of
neutrophils and some lymphocytes (92, 109, 122),
while expression of mucosal addressin cell adhesion
molecule 1 seems to be associated with the selective
enrichment of a&, lymphocytes at mucosal sites
(11). Intercellular attachment molecule 1 expression,
on the other hand seems to confer little specificity,
since its complementary receptor, the p2 integrin, is
expressed on all leukocytes. Intercellular attachment
molecule 1 expression, therefore, is considered to be
important for the common pathway leading to diapedesis, once rolling and margination have been induced by the more selective interactions. Endothelial
cells express both intercellular attachment molecule
1 and platelet endothelial cell attachment molecule
constitutively. Vascular cell adhesion molecule 1 is
thought to be more specific for mononuclear leukocytes.
Several investigations have described the expression of intercellular attachment molecule 1, vascular cell adhesion molecule 1, and endothelial cell
adhesion molecule 1 on gingival microvessels (2628, 51, 87, 136). Such expression has been shown to
be maximal in microvessels subjacent to the junctional epithelium. Interestingly, however, histopathological observations indicate that only a fraction of the microvessels express intercellular attachment molecule 1 (136). In a stable condition,
therefore, leukocyte entry into tissues is limited to a
few venules immediately subjacent to the junctional

40

epithelium. In most tissues, endothelial cell adhesion molecule 1 is not expressed until the cells are
activated by exposure to bacterial components such
as lipopolysaccharide or cytokines such as IL-1p.It
has been reported that gingival endothelial cells of
the high-endothelium microvessels express both endothelial cell adhesion molecule 1 and vascular cell
adhesion molecule 1 even in the absence of a detectable inflammatory stimulus (27, 87, 136). Approximately 23-28% of the microvessels that are positive
for intercellular attachmentmolecule 1 and platelet
endothelial cell attachment molecule 1 were also
positive for endothelial cell adhesion molecule 1 and
vascular cell adhesion molecule 1 (136). Production
of these adhesion molecules in normal gingiva may
be constitutive or alternatively, production may result from continuous low-level stimulation by bacterial substances. This may be a critical feature for
the continuous migration of leukocytes from the
small vessels into the junctional epithelium and sulcus for maintenance of normal host defense against
microbial challenge.
Fate of extravasated leukocytes
Following extravasation, leukocytes infiltrate the
perivascular connective tissue and/or migrate
through the junctional epithelium towards the gingival sulcus. Several classical investigations have observed that the leukocyte population retrieved from
the gingival sulcus is substantially different from the
one observed in the perivascular inflammatory infiltrate and within the junctional epithelium. Polymorphonuclear leukocytes represent the majority of the
cells gaining access to the bacteria present in the
gingival sulcus; mononuclear cells constitute the
majority of the tissue infiltrate. On the other hand,
both mononuclear and polymorphonuclear cells are
present within the junctional epithelium. These early
observations have recently been extended; studies of
the functional phenotype of the leukocytes present
in the inflammatory infiltrate, the junctional epithelium and the gingival sulcus have indicated that
selective enrichments of specific phenotypes occur
in specific topographic locations (138). A comparison of the density of cells expressing specific phenotypes in the inflammatory infiltrate and in the junctional epithelium found that the densities of neutrophils, memorylactivated lymphocytes (CD45RO+
cells), mucosal lymphocytes (aIELP7+
T cells), y6 T
cells and CD l a + antigen-presenting cells are selectively increased in the junctional epithelium as compared with the underlying perivascular inflamma-

The host resvonse to the microbial challenae

tory infiltrate (25,79, 135, 138, 147). These observations have indicated that leukocyte infiltration and
migration into the gingival sulcus are not random
diffusion processes and suggested that selective
mechanisms are likely to be important (138).
Neutrophil migration into the gingival sdcus
Following extravasation, neutrophils seem to gain
access to the more coronal portion of the junctional
epithelium and to selectively migrate through this
multilayered epithelium to gain access to the bacterial flora. New developments in immunobiology
have described at least two mechanisms of possible
importance in the regulation of neutrophil migration
towards the gingival sulcus or the periodontal pocket
following neutrophil extravasation: 1) the expression
of leukocyte adhesion molecules, such as the intercellular adhesion molecule 1, in epithelial cells; and
2) the discovery of a new family of low-molecularweight cytokines with potent and, to a great extent,
cell type-specific leukocyte chemotactic properties:
the chemokines (88,991, and the neutrophil-selective interleukin 8, in particular (14).
Intercellular attachment molecule 1 expression
can mediate heterotypic interactions between leukocytes and keratinocytes via interaction with receptors of the p2 integrin subfamily present on leukocytes and may therefore be able to direct leukocyte
infiltration along specific haptotactic gradients (34,
72, 142). The intercellular attachment molecule
1/p2integrin interaction has also been shown to be
a necessary step in neutrophil migration across mucosal membranes (106).In fact, neutrophil adhesion
to and migration through epithelial monolayers is
sharply inhibited by antibodies against intercellular
attachment molecule 1 (70% inhibition) and against
its p2 integrin counter-receptor (complete inhibition) present on the neutrophil surface (106,142).
Further, a variety of mucosal pathogens whose infections are associated with neutrophil recruitment at
mucosal sites have been shown to enhance intercellular attachment molecule 1 expression on mucosal keratinocytes in uitro (129).
The chemokines, on the other hand, are thought
to selectively recruit and activate specific leukocytes
to the sites of inflammation. In this respect, emerging evidence on medically relevant mucosal infections involving gram-negative bacteria indicates the
importance of both the release of neutrophil chemoattractants and the induction of specific adhesion
molecules for neutrophil recruitment at mucosal
sites of infection (129). For instance, in E. coli uri-

nary tract infections, neutrophil urinary counts have

been shown to be associated with the concentrations
of the neutrophil chemotactic cytokine IL-8 during
the course of naturally occurring infections or deliberate colonization in humans (4). Similar results
have been observed ex uiuo and in uitro in the gastrointestinal mucosa in response to Salmonella spp.,
Listeria monocytogenes and Helicobacter pylori infection (29,37).Both in the urinary and gastrointestinal
tracts, an important source of IL-8has been shown
to be the mucosal epithelium.
These facts show that mucosal keratinocytes play
a crucial active role in neutrophil recruitment at mucosal sites of infection and that IL-8and intercellular
attachment molecule 1 expression represent key
steps in this process. Recent evidence of intercellular
attachment molecule 1 and IL-8 expression in the
gingiva supports this concept. Junctional epithelium
keratinocytes have been shown to express high levels
of intercellular attachment molecule 1 and IL-8(27,
87, 136, 137).The intercellular attachment molecule
1 staining intensities increase within the junctional
epithelium in an apico-coronal direction: the superficial keratinocytes, possibly exposed to bacterial
plaque accumulation, expressing higher staining intensities (140). Further, intercellular attachment
molecule 1 expression in the gingival epithelia is
limited to the junctional epithelium: the only epithelium associated with significant neutrophil transmigration.
Substantial evidence indicates that IL-8is present
and expressed locally in the gingiva. High concentrations of IL-8 have been detected in the gingival
fluid (107).Investigations focused on the detection
of specific IL-8messenger RNA in the gingiva have
indicated the presence of the message and its source
within the junctional epithelium (134,137).The intensity of the in situ signal appeared to be maximal
in the most superficial layers of the epithelium,
possibly because they are exposed to bacterial
plaque. In a subsequent quantitative analysis, the
surface density of IL-8messenger RNA-positive cells
was significantly higher than the surface density of
leukocytes, indicating that at least part of the message originated from junctional epithelium keratinocytes (139).The spatial polarization of IL-8messenger RNA-positive cells appears to be consistent with
the source of a chemotactic stimulus attracting neutrophils towards the superficial layers of the epithelium. The biological effects of IL-8 on neutrophils
have been shown to be dose dependent: lower concentrations stimulate cell migration, and higher ones
lead to activation of neutrophil antibacterial mech-

41

Kornman et al.

cated that neutrophils that have gained access to the

gingival sulcus show an activated phenotype (149).
The exudate observed initially is predominated by
neutrophils and has all of the manifestations of an
acute inflammatory response. The important functional role of the neutrophil-dominated exudate cannot be overemphasized. In both rats (60) and dogs
(7), large reduction in numbers of circulating neutrophils by administration of anti-neutrophil antiserum
or cyclophosphamide results in the rapid invasion of
the periodontal tissues by pathogenic bacteria (Fig.
8). Suppression of lymphocytes in rats by administration of cyclosporin A does not enhance invasion
(60).It is paradoxical that accompanying the formation of the acute inflammatory exudate are alterations in the perivascular connective tissue components, specifically destruction of collagen. This destruction is most likely brought about by release of
a preformed collagenase (117) or activation of procollagenase stored in the connective tissue (144).
The inflammatory infiltrate within the tissues

Fig. 8. Scene 3 depicts the increase in hfhmrnatory cellular activity in the tissues and the epithelial changes associated with a gingival pocket. Soon after inflammation
starts, the exudate from the vessels becomes predominated by mononuclear cells. In addition to the activation
of macrophages and serum proteins, as in Scene 2, T cells,
B cells and plasma cells become evident in the tissue. Activated T cells produce cytokines that help to shape the immune response, including interleukins 2 , 3 , 4, 5, 6, 10 and
13 (IL-2, IL-3, IL-4, IL-5, IL-6, IL-10 and IL-13), tumor necrosis factor a (TNFa), transforming growth factor b
(TGFB), interferon-y (IFNy) and a series of chemotactic
substances: monocyte chemoattractant protein (MCP),
macrophage inflammatory protein (MIP) and RANTES
(regulated on activation, normal T-cell expressed and secreted). Plasma cells become prominent in the tissues and
produce immunoglobulins, such as IgG, and cytokines interleukin-6 (IL-6) and tumor necrosis factor a. Some of
the polymorphonuclear leukocytes (PMNS) become activated in the tissue and produce IL-la, IL-la, IL-6 and IL8, tumor necrosis factor a, leukotrienes (LT) and matrix
metalloproteinases (MMP). Fibroblasts are shown producing collagen, matrix metalloproteinases and tissue inhibitors of matrix metdoproteinases (TIMP). FMLP: N-formyl-methionyl-leucyl-phenylalanine; PGE,:
prostaglandin E2.

anisms (8). The observed polarization of the IL-8

source may also lead to higher IL-8 concentrations
on the epithelial surface and thus lead to neutrophil
activation. In fact, a previous investigation has indi-

42

Although most neutrophils recruited into the gingival

tissues emigrate out into the gingival sulcus, the majority of the mononuclear cells persist in the perivascular connective tissue and form the local inflammatory infiltrate. As mentioned above, specific rnononuclear cell phenotypes are selectively recruited to
gain access to the overlying epithelium; the majority
of the mononuclear phenotypes, however, is present
at higher surface densities within the inflarnmatoq
infiltrate than in the junctional epithelium. Presence
of these phenotypes within the epithelium can therefore be interpreted as the result of random migration
Specific chemokines, produced within the inflammatory infiltrate, such as the monocyte chemoattractant protein 1, have been suggested to be partlj
responsible for the spatial demarcation of the area 01
leukocyte infiltration (153).Recent evidence has indi.
cated that, among the immunocompetent cells pres.
ent in the inflammatory infiltrate, some specificall1
recognize antigenic determinants of periodontal bac
teria (95). Initial evidence seems to indicate that spe
cific lymphocyte clones may selectivelyhome into tht
gingiva (35). At the moment, the molecular basis o
this selectivity is largely unknown; locally processec
antigens presented with class I1 molecules on the en
doluminal aspect of endothelial cells may be a mech
anisrn able to selectively enrich for cells committed tc
periodontal bacteria.
Another interesting aspect of the inflammatory in
filtrate physiology is that, in clinically healthy con

The host response to the microbial challenge

ditions, its size seems to remain fairly constant over

time .of exposure to bacterial plaque. ?tvo observations suggest that specific mechanisms regulate
the life span of the terminally differentiated mononuclear cells of the gingival infiltrate: 1) in clinically
normal gingiva, a fraction of the vessels of the local
microcirculation is activated to express a variety of
vascular adhesion molecules and therefore allows
leukocyte transendothelial migration, and 2) the size
of the infiltrate actually seems to decrease when exceptional oral hygiene efforts are instituted. One
such potential mechanism in the regulation of gingival inflammatory homeostasis is programmed cell
death or apoptosis. Several investigations have implicated apoptotic cell death in the inflammatory
process by its limiting effect on the lifetime of terminally differentiated inflammatory cells (23, 56). In
this respect a recent investigation has described the
presence of apoptosis-associatedDNA breaks in cells
of the gingival inflammatory infiltrate, accompanied
by expression of the apoptosis-inducing p53 tumorsuppressing gene (24). This initial observation indicates that programmed cell death may be a physiological mechanism controlling the life span of leukocytes in the infiltrated gingival connective tissue and
possibly the size of the inflammatory infiltrate.
Scene 2 (Fig. 3) therefore involves a reactive defensive response to the bacterial products. This response is evidenced by the activation of adhesion
molecules to enhance neutrophil migration, flow
and activation of serum proteins into the tissues,
movement of neutrophils out of the vessels and into
the sulcus, epithelial cell proliferation and selective
accumulation of mononuclear cells in the tissues.

Scene 3. Immune response phase:

activation of mononuclear cells
shapes the local and systemic
immune response (Fig. 8)
The bacterial products and epithelially derived
cytokines also activate the local tissue mononuclear
cells that shape the local immune response
Macrophages have been reported to be few in
healthy gingiva. Although they increase in gingivitis
and periodontitis, macrophage density in the tissue
does not become great, and they remain in low proportions relative to other cell types.
Soon after inflammation starts, however, the exudate from the vessels becomes predominated by
mononuclear cells. In addition to endothelial cell ad-

Fig. 9. Infiltrated area of connective tissue immediately

subjacent to the junctional epithelium (JE) in a human biopsy specimen illustratingcharacteristic features of the inflammatory infiltrate. Medium lymphocytes (ML), fibroblasts (FI), s m d lymphocytes (SL),macrophages (MC) and
collagen fibers (CO) are shown. Note the predominance of
lymphoid cells, vacuoliation of the fibroblasts and paucity
of collagen fibers. In many areas (arrows),lymphoid cells
intimately contact pathologically altered fibroblasts. Original magnification X9000. Source: Schroeder HE, MiinzelPedrazzoli S,Page RC. Correlated morphometric and biochemical analysis of gingival tissue in early chronic gingivitis in man. Arch Oral Bioll973: 18:899.

hesion molecule 1, activated endothelial cells express vascular cell adhesion molecule 1, which selectively binds mononuclear cells, allowing them to exit
the small blood vessels and become a part of the
extravascular exudate. Very soon after the initiation
of the acute inflammatory response, small lymphocytes consisting of both T cells and B cells predominate in the tissue infiltrate. Subsequently, in the
presence of antigen and various cytokines, these
lymphoid cells begin to enlarge and replicate to form
clones of CD4' and CD8+ T cells, and the B cells
are driven to differentiate into clones of antibodyproducing plasma cells. In studies of gingival specimens obtained from patients with adult periodontitis, Meikle et al. (84) observed that CD4+ cells
were present in larger numbers than CD8+ cells. The

43

Kornman et al.

Fig. 10. Features of the connective tissue of the gingiva in

chimpanzees with periodontitis. A. Connective tissue zone
subjacent to the pocket epithelium. Note the blood vessels
(V) engorged with white blood cells, numerous plasma
cells (PI, lymphocytes (L)and strands of pocket epithelium PE).Collagen fibers are absent. Hematoxylin & eosin,
original magnification ~ 8 0 0 B.
. Features of the deeper
connective tissues showing the inflammatory cell infiltrate and parts of two blood vessels. There are few collagen fibers (Co), and the predominant inflammatory cell is
the plasma cell (P). Epon-embedded, original magnification ~ 8 0 0Source:
.
Page RC, Simpson DM, Ammons WE
Host tissue response in chronic inflammatory periodontal
disease. W. The periodontal and dental status of a group
of aged great apes. J Periodontol 1975: 46: 144-155.

numbers of B cells varied greatly and were entirely

absent from some specimens, although plasma cells
were prominent (Fig. 9, 10).
With a bacterial challenge, the macrophage is activated through antigen nonspecific mechanisms and
serves to enhance the inflammatory response as well
as initiate the immune response. Macrophages thus
become effector cells that, depending on the nature of
the challenge, secrete a restricted set of cytokines and
express surface receptors that influence the antigenspecific immune response that directly targets the
pathogen. Macrophages exposed to lipopolysaccharide produce several cytokines (83), including interferon y (461, tumor necrosis factor a (191, trans-

44

forming growth factor @ (661, IL-la and p (12,33),IL6 (132, 145), IL-10 (9, 86), IL-12 (59, 152), IL-15 f53),
the chemokines monocyte chemoattractant protein,
macrophage inflammatory protein and RANTES
(regulated on activation, normal T-cell expressed and
secreted), matrix metalloproteinases and prostaglandin E2. Some of the factors from monocytes, in
particular IL-lp, tumor necrosis factor a and prostaglandin E2 are prominent components of the periodontitis lesion and have been strongly implicated in
the pathogenesis of periodontal diseases (94).
The macrophage products substantially alter the
local environment in several ways. First, the macrophages in the gingival area have been shown to produce chemokines (155) that would recruit additional
monocytes and lymphocytes into the local area.
Second, the factors produced by lipopolysaccharide-activated macrophages in vitro, such as prostaglandin E2, matrix metalloproteinases and various
cytokines, are certainly capable of altering the environment to favor collagen degradation. The gingival macrophages, when stimulated, are known to
produce matrix metalloproteinases (82). Matrix
metalloproteinases and tissue inhibitors of matrix
metalloproteinases play a major role in determining
the progress of destruction of the components of the
extracellular matrix. In addition, the macrophage
products may alter collagen metabolism of the local
fibroblasts. For example, recent studies have demonstrated that IL- 1@ increased collagenase production
by both periodontal ligament and gingival fibroblasts (5, 96), decreased collagen synthesis (64) and
increased fibroblast cell-associated products that
were capable of increasing collagenase production
and activating bone resorption in experimental test
systems (98). Prostaglandin E2 also decreased collagen synthesis by gingival fibroblasts (6).
Third, antigen-specific CD4+ T-lymphocytes
would be activated by the macrophage activity and
differentiate to cytokine producing T cells that are
capable of providing help for B-cell differentiation
and antibody production. IgG is detectable in the
gingival crevicular fluid of gingivitis sites, and increases are associated with the development of gingivitis (36, 54).

The moiecdes that mediate the

immunoinflammatory response become
prominent in the gingival tissues
The events that comprise the pathogenesis of periodontitis are initiated, driven and regulated by mediators of the inflammatory process. They are known

~-

to be major participants in acute and chronic inflammation regardless of its location, and there is
strong evidence for participation of these mediators
in periodontitis. They are produced by activated resident gingival cells and infiltrating leukocytes and the
complement cascade and kinin system in blood
plasma. Monocytes from individuals susceptible to
or having severe periodontitis produce elevated
amounts of mediators (481, and mediators are present in inflamed gingiva and gingival crevicular fluid
from diseased sites at elevated concentrations (93).
Concentrations decrease following successful
therapy.
IL-1 is a major mediator in periodontitis (104, 125,
126). IL-lp comes mostly from activated macrophages and fibroblasts and IL-la from keratinocytes
of the junctional or pocket epithelium. Production
is induced by lipopolysaccharide and other bacterial
components and by IL- 1 which is autostimulatory.
Production is suppressed by bacterial metabolites
such as butyric and propionic acid and by IL-1 receptor antagonist, which is also produced by macrophages.
IL- 1 upregulates complement and Fc receptors on
neutrophils and monocytic cells, and adhesion molecules on fibroblasts and leukocytes. It induces homing receptors for lymphoid cells in the extracellular
matrix and induces osteoclast formation and bone resorption. It enhances production of itself, matrix
metalloproteinases and prostaglandins by macrophages, fibroblasts and neutrophils. IL- 1 upregulates
major histocompatibility complex expression by B
and T cells to facilitate their activation, clonal expansion and immunoglobulin production. In conjunction with tumor necrosis factor a and IL-6, 1L-1 induces production of acute-phase proteins by the liver.
IL-2, IL-3, IL-4 and IL-5 are all involved, among
other things, in lymphocyte clonal expansion, differentiation of B cells into antibody-producing plasma
cells and isotype switching. IL-2 is produced by T
cells and antigen-presenting cells and, in the presence of antigen, induces expression of clones of specific T cells and secretion of IL-3 and IL-4. IL-4 regulates IgGl and IgE production and suppresses activated macrophages and causes their apoptosis. IL-6
is produced by macrophages, fibroblasts, lymphocytes and endothelial cells. Production is induced by
IL- 1 and lipopolysaccharide and suppressed by estrogens and progesterone. It may be through IL-6
that these hormones exert their effects on gingiva.
IL-6 causes fusion of monocytes to form multinuclear cells that resorb bone.
IL-8 is a member of the chemokine superfamily,

The host response to the microbial challenae

all of which are chemoattractants. It is produced by

lipopolysaccharide-activated macrophages, synovial
cells, endothelium and junctional epithelial cells.
Neutrophils, the primary target cell, have high-affinity receptors that can become occupied at low concentrations of chemoattractant and cause the cells
to undergo directed migration, and low-affinity receptors, which become occupied at high concentrations of chemoattractant and cause the cells to
undergo the metabolic burst and to degranulate on
arrival at the site of challenge. Monocyte chemoattractant protein 1 is a potent attractant for monocytes. Monocyte chemoattractant protein l-producing cells are commonly found in inflamed gingiva
(1551, and there is a good correlation between cells
that produce IL-8 and monocyte chemoattractant
protein 1 and neutrophil and macrophage accumulation and location in gingivitis and periodontitis
(137).
Transforming growth factor p is produced by activated T cells. It is a chemoattractant for monocytes
and it suppresses their activation. Transforming
growth factor p induces production of IgA and
IgG2b. Transforming growth factor a is produced by
macrophages and it serves as a mitogen for fibroblasts and for epithelial and endothelial cells. In contrast, interferon y, which is produced by CD8+ cytotoxic T cells, recruits and activates macrophages and
upregulates the major histocompatibility complex
on virally infected cells and targets them for killing.
The major source of prostaglandins and leukotrienes in inflamed periodontal tissues is the activated macrophage, although they can also be produced by other cells such as fibroblasts. Prostaglandins, especially prostaglandin EZ, comprise the
primary pathway of alveolar bone destruction in
periodontitis. Leukotrienes, especially leukotriene
B,, are potent chemoattractants for neutrophils. Endothelial cells activated by cytokines or lipopolysaccharide secrete another bioactive lipid known as
platelet-activating factor, which induces vasodilation
and platelet aggregation and degradation. The
cells also release prostacycline, which is a potent
vasodilator and inhibitor of platelet aggregation, and
thromboxane, which causes vasoconstriction and
platelet aggregation.
Serum proteins, such as the kinin and complement systems, provide a mechanism for rapid expansion of the inflammatory response. Bradykinin
enhances permeability and inflammation of vessels
of the microcirculation. It is an 8-amino acid peptide
cleaved from a plasma protein precursor. Activation
of the complement cascade either by the classic or

45

Kornman et al.

alternative pathways results in production of C3a

and C5a which are chemotactic peptides and C3b
which is an anaphylatoxin.
The matrix metalloproteinases comprise a large
family of Zn++-dependent enzymes produced by
macrophages, fibroblasts and keratinocytes activated by lipopolysaccharide or cytokines. Matrix
metalloproteinases collectively can digest all of the
components of the extracellular matrix. Production
of these enzymes is tightly controlled, complex and
not well understood. Transcription is upregulated by
IL-1, transforming growth factor a, epidermal
growth factor and transforming growth factor a.
With some exceptions, transcription is down regulated by transforming growth factor p and interferon
y. Matrix metalloproteinase activity is suppressed by
tissue inhibitors of metalloproteinases, which are
also produced by macrophages.
The local antibody response is activated to assist
in controlling the bacterial challenge

As the bacterial challenge increases, the host tissues

are protected by neutrophil activity in the sdcus, facilitated by specific antibody that is produced systemically and in the local tissues. Circulating antibody may be far greater in amount and importance
than locally produced antibody. Most, although not
all, early-onset periodontitis and adult periodontitis
patients mount a systemic humoral immune response to antigens of the infecting bacteria (65).This
response likely results from the access of subgingival
plaque bacteria and bacteria cell wall components
to local lymph nodes and through the circulation to
the spleen. Antibody titers can be remarkably high,
although biological activity is often low, as measured
by antibody avidity and capacity to opsonize and enhance phagocytosis and killing. Specific antibody
from the blood comprises a major portion of the
specific antibody in gingival crevicular fluid (69,
128).
The local antibody response is directed by the
cytokine profile within the tissues and the presentation of antigens by professional antigen-presenting
cells such as the macrophage. In addition to the
macrophage influence in shaping the T-cell and Bcell responses within the tissues, other cells participate in essential ways in preparing the early response. Production of IL-4 through antigen nonspecific mechanisms appears to be an important aspect of the early immune response to various
pathogens (49). Although the initial source of local
IL-4 in the gingival tissues is unclear, mast cells and

46

basophils, which are routinely associated with small

blood vessels, have been shown to produce IL-4
upon activation (101, and natural killer cells, capable
of producing IL-4,are found near the gingival epithelium and increase in numbers in periodontitis
(22, 45, 71).
Studies (150) of the antibody response in subjects
without periodontitis found that individuals with
low antibody titers tended to have hjgh antibody
avidity and opsonic ability, suggesting that the antibody response in subjects with health and gingivitis
is capable of providing protective functions. This is
also consistent with the finding that antibody from
gingivitis subjects recognized fewer specific I! gingivulis epitopes than antibody from periodontitis subjects (110).
The net outcome of Scene 3 (Fig. 8) is an increase
in tissue lymphocytes, plasma cells and macrophages shifts in the metabolism of the local fibroblasts to favor a reduction in collagen synthesis, and
activation of the local and systemic specific immune
response, with production of antibody directed
against highly immunogenic bacterial epitopes.

Scene 4. Regulation and resolution

phase: determinants of protective
components in the sulcus and
collagen balance in the tissues
(Fig. 11)
The local cellular and humoral responses described
above appear to be sufficiently competent in most
individuals to manage a limited bacterial challenge.
There appear to be at least two conditions in which
the routine host response becomes more destructive
at the local level. The first appears to involve a specific bacterial biomass that directly inhibits key components of the host defense mechanism. As discussed by Darveau et al. in this volume, some subgingival biomasses and the specific bacteria that
accumulate in those ecosystems are capable of interfering with neutrophil function in multiple ways, including the presence of leukocyte killing toxins (1431,
the production of short-chain fatty acids and polyamines that are toxic to neutrophils (90, 148), and
the inhibition of E-selectin upregulation on endothelial cells (30). There is also reason to believe that
certain bacterial challenges are capable of shifting
the T-cell and B-cell responses to result in less effective antibody. Selected periodontal bacteria produce
proteases that cleave the Fc regions of IgG or de-

The host response to the microbial challenge

grade the C3 component of the complement, thereby

interfering with phagocytosis and killing (55, 112,
127). In addition, antibody avidity to E gingiualis is
higher in periodontally healthy individuals and
chronic adult periodontitis patients than in rapidly
progressive cases and improved following therapy
(21, 77, 85, 105). It is reasonable to expect the above
factors to substantially alter the protective balance
within the sulcus.
The second type of factor that appears to result in
a more destructive response to the bacterial challenge includes the host response modifiers, such as
smoking, systemic disease and genetic variation. Salvi et al. and Hart & Kornman discuss the influences
of these factors in this volume. In general, they appear to predispose the individual to a more destructive response.
The net result of the unrelenting bacterial challenge provides the scene for the destructive phase of
periodontitis. Large numbers of T-lymphocytes and
plasma cells are evident in the tissues. Although
large numbers of neutrophils are migrating into the
sulcus, many are also activated within the tissues.
Macrophages are producing products, such as matrix metalloproteinases, prostaglandin E2,tumor necrosis factor a and IL-lp that are known to enhance
both bone and connective tissue destruction. In addition, in the chronic inflammation state, fibroblasts
may be poised for a net loss of collagen.
Fibroblasts shift to a state that favors destruction
of the extracellular matrix (Fig. 10)
Histomorphometric studies of health and gingivitis
have clearly demonstrated that as gingival inflammation develops the volume of tissue occupied by
fibroblasts decreases (102, 114-1 16). In addition,
there are good reasons to expect connective tissue
metabolism to be substantially altered in tissues
dominated by a chronic inflammatory state (32). The
net collagen balance is a function of matrix metalloproteinase activity and collagen synthesis, both of
which are regulated by various cytokines, growth factors, and prostanoids (see Reynolds & Meikle in this
volume). Using immune localization on biopsy
specimens obtained from patients with adult periodontitis, Meikle et al. (84) observed enormous variation in numbers and distribution of cells staining
positively for matrix metalloproteinase messenger
RNA among and within specimens. Matrix metalloproteinases were produced by macrophages, fibroblasts and epithelial cells. The extent of matrix
metalloproteinase expression correlated with the ex-

Fig. 11. Scene 4 represents the initid loss of attachment

and the increased mononuclear cell activity in the tissues.
The inflammatory mediator load in the tissue increases
and includes contributions by the fibroblasts of interleukin-1s (IL-lfl), IL-6, IL-8, prostaglandin $!I (PGE,), tumor
necrosis factor a (TNFa), as well as collagen, matrix
metalloproteinases (MMP) and tissue inhibitors of matrix
metalloproteinases (TIMP). Plasma cells are prominent.
MCP monocyte chemoattractant protein; MIP: macrophage inflammatory protein; RANTES: regulated on activation, normal T-cell expressed and secreted;TGFP: transforming growth factor & IFNy: interferon y; PMNS: polymorphonuclear leukocytes; LT: leukotrienes; IgG:
immunoglobulin G; IL-lra: interleukin 1 receptor antagonist.

tent of inflammation in some specimens but not in

others. In insulin-dependent diabetes, most of the
matrix metalloproteinases are derived from neutrophils, whereas in localized juvenile periodontitis
most matrix metalloproteinases are of the fibroblast
type (121). Collagenase production by gingival
fibroblasts has been observed in biopsies from adult
periodontitis and appears to be localized primarily
to the interface between the epithelium and the connective tissue (84). Increased neutrophil-type (matrix metalloproteinase 8 ) and fibroblast-type (matrix
metalloproteinase 1) collagenases may be detected
in tissue from periodontitis as compared with
healthy sites (63, 133). In addition, fibroblasts isolated form ligature-induced periodontitis sites in

47

Kornman et al.

monkeys produce less total protein and collagen

than cells isolated from noninflamed sites ( 6 2 ) .Van
der Zee et al. (144) have shown that the extracellular
matrix of the connective tissues surrounding bone
accumulate substantial quantities of procollagenase
when exposed to IL-lor alone or IL-lor plus epidermal growth factor. At a later time the stored procollagenase may be released in an active form by the
addition of plasmin. Proteases from periodontopathic bacteria have also been shown to activate latent
procollagenases (120). Of perhaps major importance
is the cellular differentiation and production of inflammatory mediators by fibroblasts that have been
exposed to bacterial products or an inflammatory
environment (31, 32, 118).
Selective cytokine expression molds the immune
response
Recent studies (44) have found that mononuclear
cells isolated from inflamed gingival tissues produced predominately interferon y, IL-6, IL-10 and
IL-13, but IL-2, IL-4 and IL-5 were not detected.
Others, however, have reported increased IL-4-producing cells in periodontitis lesions (154) and the
presence of IL-5-specific message in gingival mononuclear cells (43, 111, 154). Of perhaps greatest importance is the clear agreement among several investigators that the local cytokine profiles in the inflamed gingival tissues are only a subset of those
produced in peripheral blood of the same patients.
If the reduced local expression of IL-4 and IL-5 is
confirmed, one would expect a more prominent Thl
response in the gingivitis and periodontitis lesions,
resulting in less effective antibody. In periodontitis
subjects, low avidity antibody and IgG2 antibody,
which lacks strong complement fixation and opsonization properties, appear to predominate (78, 150),
most likely reducing the effectiveness of the humoral
response in clearing pathogens. It is certainly
reasonable to expect that a chronically inflamed
condition establishes a local cytokine environment
that influences the immune response.
The scene is set for the initiation and progression
of periodontitis (Fig. 11)
With a chronic bacterial challenge, the periodontal
tissues are continuously exposed to specific bacterial
components that can alter many local cell functions.
The tissues are populated with T lymphocytes and
macrophages that are producing selective subsets of
cytokines and prostanoids that favor net loss of col-

48

lagen and bone, and less effective antibody production. The efficiency of neutrophil migration is reduced, and it is likely that more neutrophils are activated within the tissue. The total impact of the above
changes is to subtly shift the scene from one in
which the host is controlling the bacterial challenge
to one in which the challenge is less well controlled
and the tissue-destructive phase is dominant. Such
factors as smoking and genetic influences on cytokine expression that are capable of modifymg critical
aspects of the neutrophil-antibody protection and/
or fibroblast function would be expected to alter the
balance of the systems between protection and destruction.

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