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Human variability in innateimmunity

DE N I S F. KI N A N E, D O N A L D R. D E M U T H , SV E N -UL R I K G O R R ,G E O R G E N. HA J I S H E N G A L L I S & M I C H A E L H. MA R T I N

Chronic inflammatory periodontal disease is initiated

by oral bacteria perturbing epithelial cells, which

trigger innate, inflammatory, and adaptive immune

responses. These processes result in the destruction

of the tissues surrounding and supporting the teeth

and eventually result in tissue, bone, and, finally,

tooth loss. This review will focus on individual human

variations in the innate immune responses and

variability in the oral bacteria challenging the innate

system and the signaling and subsequent innate

immune-generated host response relevant to the

periodontal diseases. It will address the initial bac-

teriumhost contact and the subsequent innate def-

ense afforded by the host cells and how epithelia and

phagocytes differ in their response. Recent work in

this area reports host variation in susceptibility to

simple gingival inflammation that might intriguinglyrelate to differences in the innate immune system of

subjects. It is feasible that variability exists in the Toll-

like receptors expressed by an individual, or in the

complex receptorligand recognition and signal

transduction pathways that result in the host cell re-

lease of cytokine and anti-microbial molecules. Thus

this review introduces variability in the innate im-

mune system and includes discussion of: (i) human

susceptibility to inflammation; (ii) human variation in

Toll-like receptors; (iii) variability in signaling; (iv)

variation in response to bacterial challenges and; (v)

the antimicrobial peptides and cytokines released.

Innate immunity andinflammation in the periodontaltissues

The primary etiologic agent in the initiation of the

periodontal diseases is the accumulation of bacterial

plaque in the gingival crevice. Irritation of the gingival

tissues induces an inflammatory response which

comprises physiologic responses that are the first

organized reaction to any injurious challenge to the

body, including bacterial infection. The rapid, gener-

alized inflammatory processes that occur in response

to challenges constitute an early step in the initiation

of the immune response, and are part of the innate

immunesystem, so called to reflect that they are part

of the inherent inborn biological responses that re-

quire no prior learning or experience. Inflammation is

a well-coordinated process that is discussed in detail

in this volume comprising increased vascular per-

meability, migration of polymorphonuclear leuko-

cytes, monocytes, and lymphocytes into the irritated

tissues, and activation of cells to secrete inflammatory

mediators that guide an amplifying cascade of bio-

chemical and cellular events (120, 176). Althoughinflammation was once considered a non-specific

arm of the immune response, current knowledge

leads to the conclusion that the inflammatory

response is a relatively specific event, and a wide-

ranging repertoire of receptors and corresponding

ligands are involved. The specific nature of inflam-

mation allows rapid identification and a response that

is better tailored to the infection (96) or to other

threatening external stimuli (134). For example, bac-

terial lipopolysaccharide, a common antigen of gram-

negative bacteria, is specifically recognized by host

receptors such as soluble lipopolysaccharide-bindingprotein, membrane-associated CD14, and Toll-like

receptors. The interactions between lipopolysaccha-

ride and these host proteins activate an intracellular

cascade of events that lead to the secretion of specific

inflammatory mediators and antimicrobial proteins.

These specific interactions may explain why the

inflammatory response to gram-positive bacteria is

less pronounced than the inflammatory response to

gram-negative bacteria during in vitro and in vivo

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Periodontology 2000, Vol. 45, 2007, 1434

Printed in Singapore. All rights reserved

2007 The Authors.Journal compilation 2007 Blackwell Munksgaard

PERIODONTOLOGY 2000

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inflammatory assays (39). In addition, the discovery

that there is a group of Toll-like receptors that can

recognize a wide but restricted set of pathogen-asso-

ciated molecular structures may explain how different

bacteria induce different responses. In fact, even,

lipopolysaccharide from different bacteria may acti-

vate different Toll-like receptors, and induce a dif-

ferent response (10, 129). These interactions enable

hosts to sample and sort their current environmentalcondition, to discriminate between pathogenic bac-

terial challenges, and to mount a selective and

appropriate response (39). Recent data indicate that

Toll-like receptors may respond to bacterial and non-

bacterial challenges, such as oxidized low-density

lipoprotein cholesterol (18, 19). Thus the host may

respond through inflammation to a range of chal-

lenges, from bacteria to cholesterol (39). However, the

nature of the response differs and its character de-

pends on the microbial triggering of specific recep-

tors, the signal transduction pathways and the way

cells and tissues respond to these signals in terms of

cytokine and defensive protein production.

Susceptibility

Not all individuals are equally affected by the accu-

mulation of bacterial plaque. Some susceptible indi-

viduals will develop aggressive forms of periodontitis

at a relatively young age, while others might never

develop periodontitis (111). Most of the population

lies between these extremes, and will develop somedegree of periodontitis if exposed to periodontal

pathogens for sufficient time. In some cases, the dis-

ease will progress slowly and the risk of tooth loss will

be minimal, while in others the rate will endanger the

dentition. The findings that high levels of inflamma-

tory mediators, such as interleukin-1, tumor necrosis

factor, and prostaglandin E2, correlate with perio-

dontal destruction (85, 153, 181) and that these

mediators aggravate the inflammatory response (61),

led to the hypothesis that some individuals may be

high-responders and respond to periodontal infec-

tion with high levels of inflammatory mediators,which results in attachment loss. Molvig et al. (146)

have shown that there are stable inter-individual dif-

ferences in the response of monocytes to lipopoly-

saccharide stimulation. They hypothesized that this

pattern of response to environmental stimuli is gen-

etically determined, and may be the basis for chronic

inflammatory disorders. Shapira et al. (174) and oth-

ers support this hypothesis, and suggest that inter-

individual differences in the monocyte macrophage

response can be found in periodontitis patients

compared to subjects with no periodontitis (58, 174).

Engebretson et al. (43) have shown that probing

depth and attachment levels are strongly correlated

with gingival crevice fluid interleukin-1b levels.

However, patients with severe disease had higher

gingival crevice fluid levels of interleukin-1b in each

probing depth category than those with mild mod-

erate disease. The differences between these two pa-tient groups were more pronounced (around twofold)

in shallow pockets, suggesting that high gingival

crevice fluid interleukin-1b expression is in part a host

trait, and not strictly a function of clinical parameters.

During experimental gingivitis studies, individual

variations in the rate of development of gingival

inflammation have been noted. Wiedemann et al.

(207) reported that in a group of 62 subjects who

withdrew from oral hygiene measures, eight were

found to be resistant and did not develop gingivitis

within 21 days, while another group of 25 subjects

were found to be susceptible and exhibited sub-

stantial gingival inflammation within 14 days. The

remaining subjects were an intermediate group and

developed gingival inflammation by day 21. In a later

study, a group of subjects that consistently exhibited

greater than average gingival inflammation and an-

other resistant group were described (198). The dif-

ference in gingivitis susceptibility between the two

groups could not be ascribed to plaque differences.

From these experimental gingivitis studies, it appears

that approximately 13% of subjects are resistant

(190). Trombelli and associates (190, 194) have alsoshown that while all individuals will develop some

degree of inflammation, there are inter-individual

differences in response to dental plaque. These dif-

ferences may be explained by genetics or environ-

ment. Using the twin study approach, Michalowicz

et al. (142) could not demonstrate an association be-

tween gingival inflammation and genetics, perhaps

because of the cross-sectional approach of the study.

However, their data support the major role of genetics

in the development of periodontitis, in which gingival

inflammation is considered as a major part of the

pathogenesis. In summary, all of the above studies areconsistent with the hypothesis of genetically based

host modulation of gingival inflammation.

Variation in microbial modulationof the innate immune response

Innate immunity represents the inherited resistance

to microbial infection, which is detected by pattern-

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recognition receptors. These are strategically located

at the interface between the mammalian host and the

microbes, and have evolved to recognize conserved

microbial motifs, known as microbe-associated

molecular patterns (4, 137). Toll-like receptors con-

stitute an evolutionarily ancient pattern-recognition

receptor family, which plays a central role in the

induction of innate immune and inflammatory re-

sponses (5, 17). Not surprisingly, Toll-like receptorsare expressed predominantly in cells that mediate

first-line defense, such as neutrophils, mono-

cytes macrophages, and dendritic cells, as well as on

epithelial cells. Distinct members of the Toll-like

receptor family respond to different types of mi-

crobe-associated molecular patterns, endowing the

innate response with a relative specificity (5, 17). For

example, Toll-like receptor 2 responds to lipoteichoic

acid and microbial lipoproteins, Toll-like receptor 4

responds to lipopolysaccharide, Toll-like receptor 5

responds to flagellin, and Toll-like receptor 9 re-

sponds to bacterial central pattern generator DNA

islands (5, 17).

The discovery of Toll-like receptors and the iden-

tification of their ligand repertoire have prompted the

bar code hypothesis of innate recognition of mi-

crobes. According to this concept, Toll-like receptors

read a bar code on microbes, which is then decoded

intracellularly to tailor the appropriate type of innate

response (1). For instance, simultaneous activation of

Toll-like receptor 5 and Toll-like receptor 4 would be

interpreted as infection with a flagellated gram-neg-

ative bacterium, whereas activation of Toll-likereceptor 2 together with Toll-like receptor 5 would

likely indicate the presence of a flagellated gram-

positive bacterium. However, this bar code detec-

tion system would not readily distinguish between

pathogens and commensals, because they both share

similar invariant structures (e.g. lipoteichoic acid,

lipopolysaccharide, or flagellae). Yet, the immune

system generally elicits a vigorous inflammatory re-

sponse against pathogens aimed at eliminating them,

whereas it normally tolerates commensals.

Signal transduction featuresrelevant to host sensing ofmicrobes

Agonist induced activation of the Toll-like receptor

complex sets forth a diverse array of intracellular

signaling pathways that can dictate both qualitative

and quantitative aspects of the host inflammatory

response. The fundamental basis of this initial Toll-

like receptor-mediated signal transduction depends

upon the association with, as well as the recruitment

of, various adapter molecules that contain the

structurally conserved Toll interleukin-1 receptor

(TIR) domain. To date, the best-described Toll

interleukin-1 receptor-containing adapter molecules

that impart specificity to a given Toll-like receptor

signal transduction pathway include myeloid

differentiation factor 88 (MyD88), Toll interleukin-1receptor-containing adaptor protein (TIRAP), Toll

interleukin-1 receptor domain-containing adaptor

inducing interferon-b (TRIF), and the Toll inter-

leukin-1 receptor domain-containing adaptor indu-

cing interferon-b-related adapter molecule (TRAM)

(48, 83, 104, 138, 214). In turn, these adaptor

molecules provide the necessary framework to recruit

and activate downstream kinases and transcription

factors that subsequently dictate the nature, magni-

tude, and duration of myeloid differentiation factor

88-dependent and myeloid differentiation factor 88-

independent responses (see Fig. 1) (48, 83, 104, 138,

214). While much attention and extraordinary studies

have been focused on discerning the molecular dif-

ferences between the myeloid differentiation factor

88-dependent and myeloid differentiation factor

88-independent pathways, it appears that a major

mechanism modulating the nature and magnitude of

the inflammatory response to a variety of Toll-like

receptor-agonists involves the recruitment and acti-

vation of specific kinase pathways. In this regard,

most studies identifying and characterizing the

regulatory processes that govern the inflammatoryresponse to Porphyromonas gingivalis or associated

virulence factors have highlighted the importance of

the mitogen-activated protein kinase (MAPK) and

phosphatidylinositol-3 kinase (PI3 K) pathways.

Therefore, the aim of this section is to summarize

how these signal transduction pathways regulate

innate immune responses to P. gingivalis and may

be a source of intersubject variability in disease

susceptibility.

Mitogen-activated protein kinasepathways

One of the most highly conserved signaling cascades

activated in both the innate and adaptive immune

systems are a family of serine-threonine kinases

collectively referred to as the mitogen-activated

protein kinases. The best-described mitogen-activa-

ted protein kinases include the extracellular signal-

related kinase 1 2 (ERK1 2), p38 mitogen-activated

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protein kinase, and the c-Jun N-terminal kinase

(JNK1 2) (41). Although mitogen-activated protein

kinases were initially identified as being critical kin-

ases involved in the regulation of G-protein-coupled

receptor signaling pathways, subsequent studies have

clearly demonstrated that mitogen-activated protein

kinases play an integral role in Toll-like receptor

signaling (46). Mitogen-activated protein kinases

are sequentially activated by upstream kinases via

phosphorylation of a Thr-X-Tyr tripeptide (see Fig. 1)

(93, 97, 107, 133, 171, 191). Mitogen-activated protein

kinases can be located within both the cytosolic and

nuclear compartments of innate immune cells and,

upon phosphorylation, can regulate the activity of

many different downstream transcription factors via

post-translational modification. Alternatively, mito-

MAPKSignaling

MyD88/MyD88independentSignaling PI3K Signaling

TLR TLR4 TLR1/6 TLR2

PDK1PDK2

PI3K

PIP3

Akt

GSK-3

IFN-IRF3

JNK 1/2

ERK 1/2

MEK 1/2

MEKK 1/4

Pak1Raf

Rac1Ras

MKK 4/7

MKK 3/6

p38

TBK1/IKKa

TRAM TRIFTIRAP

MyD88TAK1Ask1

NF-B

NF-B

Late NF-Bactivation (Type IIFNs)

IL-10

IL-8

IL-12 p40/p70

Early NF-Bactivation(Proinflammatorycytokines)

Anti-inflammatorycytokines

CREB

Invasion

P. gingivalis

CBP

Attenuated Pro-inflammatory

cytokines/p65-CBP

Fig. 1. The Toll-like receptor (TLR) and Toll-like receptor

4 (TLR4) signal transduction pathways and their differ-

ences in terms of mitogen-activated protein kinase

(MAPK), myeloid differentiation factor 88 (MyD88) and

phosphatidylinositol-3 kinase (PI3 K) signaling. CBP,

cyclic adenosine monophosphate response-element-

binding protein binding protein; CREB, cyclic adenosine

monophosphate response-element-binding protein;

ERK1 2, extracellular signal-related kinase 1 2; GSK-3b,

glycogen synthase kinase 3b; IFN-b, interferon-b; IL-10,

interleukin-10; IRF, interferon regulatory factor; JNK,

c-Jun N-terminal kinase; MEK, mitogen-activated protein

kinase extracellular signal-related kinase 1 2 kinase;

MKK, mitogen-activated protein kinase kinase-4 7; NF-

jB, nuclear factor-jB; PDK1, phosphoinositide-dependent

kinase 1; PDK2, phosphoinositide-dependent kinase 2;

PI3 K, phosphatidyl inositol-3 kinase; PIP3, phosphatidy-

linositol-3,4,5-trisphosphate; TAK1, transforming growth

factor-b-activated kinase 1; TBK, tank-binding kinase 1;

TIRAP, Toll interleukin-1 receptor-containing adaptor

protein; TRAM, Toll interleukin-1 receptor domain-con-

taining adaptor inducing interferon-b-related adapter

molecule; TRIF, Toll interleukin-1 receptor domain-

containing adaptor inducing interferon-b.

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gen-activated protein kinases can act as intermediate

kinases that are involved in activating phosphory-

lating other kinases. Although mitogen-activated

protein kinase pathways have been shown to contain

many conserved upstream signaling molecules, i.e.

transforming growth factor-b-activated kinase 1

(TAK1) (Fig. 1), that can mutually interact with p38,

c-Jun N-terminal kinase 1 2, and extracellular sig-

nal-related kinase 1 2 pathways, it is also welldocumented that the upstream kinases leading to

mitogen-activated protein kinase activation can also

be exquisitely specific for the respective mitogen-

activated protein kinases (41, 69).

Studies utilizing small molecule inhibitors that

selectively target p38, extracellular signal-related

kinase 1 2 (mitogen-activated protein kinase kinase

1 or 1 2), and c-Jun N-terminal kinase 1 2, have

begun to elucidate their functional roles in regulating

innate immune responses to P. gingivalis. The inva-

sion process of human gingival epithelial cells by

P. gingivalis was shown to result in the down-regu-

lation of extracellular signal-related kinase 1 2

activity whereas phospho-c-Jun N-terminal kinase

1 2 levels were enhanced (203). Moreover, these

studies suggested that c-Jun N-terminal kinase 1 2,

but not extracellular signal-related kinase 1 2, was

associated with P. gingivalisinvasion. The regulation

of specific pro-inflammatory cytokines produced by

gingival epithelial cells stimulated with P. gingivalis

has also highlighted the differential involvement of

mitogen-activated protein kinases. In this regard,

Huang et al. (89) showed that the production ofinterleukin-8 by P. gingivalis-stimulated gingival

epithelial cells involved both p38 and extracellular

signal-related kinase 1 2. In contrast, the down-

regulation of interleukin-8 from gingival epithelial

cells stimulated with P. gingivalis was selectively

mediated by extracellular signal-related kinase 1 2

(89). Other studies characterizing how mitogen-acti-

vated protein kinases are involved in P. gingivalis-

mediated apoptosis have provided evidence that

extracellular signal-related kinase 1 2 and p38

exhibit additive effects on the promotion of cell

death, whereas c-Jun N-terminal kinase 1 2 activityexerted cytoprotective properties (123). Several

laboratories have further defined how mitogen-acti-

vated protein kinases are involved in the regulation of

inflammatory responses by assessing the production

of pro- and anti-inflammatory cytokines by innate

immune cells stimulated with P. gingivalis or asso-

ciated virulence factors. Our laboratory identified

that inhibition of the extracellular signal-related

kinase 1 2 pathway differentially regulated inter-

leukin-10 and interleukin-12 synthesis by P. gingi-

valis-stimulated monocytes in that interleukin-10

production was suppressed by approximately 60%,

while interleukin-12 levels were enhanced by more

than twofold (131). Interestingly, inhibition of extra-

cellular signal-related kinase 1 2 resulted in the

ability of P. gingivalis lipopolysaccharide to induce

bioactive interleukin-12 p70 from innate immune

cells, whereas no detectable levels of interleukin-12p70 were evident when monocytes were stimulated

with P. gingivalis lipopolysaccharide alone (131).

Studies by Darveau et al. (33) have identified an

additional level of complexity in the regulation of

mitogen-activated protein kinases by demonstrating

that P. gingivalis lipopolysaccharide-mediated p38

activation can be largely affected by cell type and

thus act as a p38 agonist or antagonist. Collectively,

these studies demonstrate the multitude of cellular

effects members of the mitogen-activated protein

kinase family can exert and further highlight their

differential activation and functional involvement in

a variety of cellular responses to P. gingivalis.

Phosphatidylinositol-3 kinasepathway

Phosphatidylinositol-3 kinase is a heterodimeric en-

zyme that consists of a regulatory subunit (p85) and a

catalytic subunit (p110) (28). Activation of phospha-

tidylinositol-3 kinase occurs via its regulatory subunit

(p85) binding to phosphotyrosine residues (YXXM)present within activated cellular receptors located

on the plasma membrane (28, 192). Once activated,

phosphatidylinositol-3 kinase catalyzes the pro-

duction of phosphatidylinositol-3,4,5-trisphosphate

(PIP3). This allows for recruitment and co-localization

of phosphoinositide-dependent kinase 1 (PDK1) and

the serine-threonine kinase Akt via their pleckstrin

homology domains (50, 183). This co-localization

allows activation of Akt via the phosphorylation at

threonine 308 by phosphoinositide-dependent kinase

1 and phosphorylation at serine 473 by a still uniden-

tified kinase called phosphoinositide-dependent kin-ase2 (PDK2;50, 121,183).Upon dual phosphorylation,

Akt phosphorylates several downstream targets

including the serine phosphorylation and subsequent

inhibition of the constitutively active serine thre-

onine kinase glycogen synthase kinase 3 (GSK3) (31).

The identification of intracellular signaling path-

ways that regulate the production of both pro- and

anti-inflammatory cytokines upon Toll-like receptor-

activation has been an area of intense research. An

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elegant study by Arbibe et al. (7) has elucidated direct

evidence for the involvement of phosphatidylinositol-

3 kinase in the Toll-like receptor-signaling pathway.

Analysis of the cytoplasmic domain of Toll-like

receptor 2 demonstrated that it contains a YXXM

motif that is required for the recruitment of the p85

subunit of phosphatidylinositol-3 kinase and site-

directed mutations within the cytoplasmic domain to

disrupt the YXXM motif found in Toll-like receptor 2resulted in a loss of the ability of p85 to associate with

Toll-like receptor 2 and blunted Toll-like receptor 2-

mediated activation of the nuclear factor-jB (NF-jB)

p65 transcription factor (7). While it appears that

phosphatidylinositol-3 kinase activation can occur

via direct recruitment to Toll-like receptor 2, studies

analyzing Toll-like receptor 5-signaling have shown

that phosphatidylinositol-3 kinase activity is abro-

gated in the absence of the adaptor protein myeloid

differentiation factor 88 (167). From a functional

standpoint, direct inhibition of phosphatidylinositol-

3 kinase has been shown to augment the production

of tumor necrosis factor and interleukin-12 p40 p70

(52, 69, 131). Moreover, our laboratory further iden-

tified that phosphatidylinositol-3 kinase activity

negatively regulated the levels of interleukin-12

p40 p70 while concurrently enhancing the levels of

the anti-inflammatory cytokine interleukin-10 (131).

Furthermore, these studies identified significant

cross-talk between the phosphatidylinositol-3 kinase

and mitogen-activated protein kinase pathways be-

cause inhibition of phosphatidylinositol-3 kinase

attenuated P. gingivalislipopolysaccharide-mediatedextracellular signal-related kinase 1 2 activity in

human monocytes (131). These studies were the first

to identify that the phosphatidylinositol-3 kinase

pathway differentially controlled the production of

two critical immunoregulatory cytokines and sug-

gested that the ability of phosphatidylinositol-3 kin-

ase to regulate the activity of a downstream effector

molecule was responsible for this pathway to differ-

entially regulate pro- and anti-inflammatory cytokine

levels. Indeed, using a variety of molecular and

pharmacological techniques we demonstrated that

phosphatidylinositol-3 kinase activity was necessaryto indirectly mediate the phosphorylation activation

of Akt (Ser473 Thr308) (99). In addition, inhibition

of Akt resulted in similar effects on the levels of

interleukin-10 and interleukin-12, as compared to

phosphatidylinositol-3 kinase inhibition; thus

suggesting that preventing the inactivation phos-

phorylation of a downstream kinase within the phos-

phatidylinositol-3 kinase pathway was responsible for

enhancing pro-inflammatory and suppressing anti-

inflammatory cytokine production. Analysis of the

phosphorylation status of downstream kinases within

the phosphatidylinositol-3 kinase-Akt pathway iden-

tified that Toll-like receptor-stimulation resulted in

the phosphorylation (Ser9) and inactivation of the

serine threonine kinase glycogen synthase kinase 3-

b and that this process was dependent upon the

kinase activity of both phosphatidylinositol-3 kinase

and Akt (99). Using a panel of glycogen synthasekinase 3-specific pharmacological inhibitors, small

interfering RNA targeting glycogen synthase kinase

3b and glycogen synthase kinase 3-b-deficient mouse

embryonic fibroblasts, it was subsequently shown

that inactivation of glycogen synthase kinase 3-b was

responsible for the ability of the phosphatidylinosi-

tol-3 kinase-Akt pathway to augment interleukin-10

and suppress interleukin-12 production. Additional

experiments utilizing selective agonists for Toll-like

receptor 2, Toll-like receptor 4, Toll-like receptor 5,

and Toll-like receptor 9 showed that inhibition of

glycogen synthase kinase 3 resulted in a selective

reduction of 50)90% in the production of all proin-

flammatory cytokines tested [interleukin-1, IFN-c,

interleukin-12p40 and interleukin-6 (99)]. In contrast,

the production of the anti-inflammatory cytokine

interleukin-10 was increased three- to eightfold

regardless of which Toll-like receptor agonist was

used. These studies highlight the fundamental

importance of the phosphatidylinositol-3 kinase

pathway in regulating both qualitative and quantita-

tive aspects of the host inflammatory response and

provide a mechanistic framework by which Toll-likereceptor-mediated pro- and anti-inflammatory cy-

tokines are regulated.

Glycogen synthase kinase 3 has been linked to the

regulation of the main eukaryotic transcription factor

nuclear factor-jB, which regulates many diverse

cellular processes, including proinflammatory cyto-

kine responses (36, 84, 172). Nuclear factor-jB

activity can be regulated at multiple steps, including

degradation of IjB inhibitory molecules, processing

of the nuclear factor-jB p105 and p100 molecules,

and nuclear factor-jB p65 phosphorylation-depend-

ent association with cellular coactivators (65). How-ever, subsequent experiments showed that the ability

of glycogen synthase kinase 3 to modulate host

inflammatory responses was not associated with any

discernible down-regulation of nuclear factor-jB

post-translational processing or nuclear import (99).

In contrast, glycogen synthase kinase 3 inhibition

exerted a potent augmentation in the nuclear levels

of the cyclic adenosine monophosphate response-

element-binding protein (CREB) (Ser133). These

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findings were of major significance in our under-

standing of how glycogen synthase kinase 3 was

regulating host inflammatory responses because past

studies characterizing the transcriptional properties

of cyclic adenosine monophosphate response-ele-

ment-binding protein demonstrated that, as nuclear

levels of phospho-cyclic adenosine monophosphate

response-element-binding protein increased, nuclear

factor-jB p65 was displaced from the coactivatorof transcription cyclic adenosine monophosphate

response-element-binding-protein-binding-protein

(CBP) and resulted in a reduction in nuclear factor-

jB p65-dependent transcription (161, 221). Further-

more, studies by Platzer et al. (163) characterizing the

transcriptional regulation of interleukin-10 produc-

tion in lipopolysaccharide-stimulated monocytes

showed that cyclic adenosine monophosphate re-

sponse-element-binding protein was critical for

optimal interleukin-10 levels. A direct role for cyclic

adenosine monophosphate response-element-bind-

ing protein in regulating pro- and anti-inflammatory

cytokine production in glycogen synthase kinase-3-

inhibited monocytes was shown by the use of small

interfering RNA experiments targeting cyclic adeno-

sine monophosphate response-element-binding

protein (99). In this regard, it was shown that glyco-

gen synthase kinase 3 inhibition was unable to

differentially regulate the levels of pro- and anti-

inflammatory cytokines in lipopolysaccharide-

stimulated monocytes exhibiting more than 70%

knockdown in cellular cyclic adenosine monophos-

phate response-element-binding protein levels.Taken together, these studies have identified and

characterized a central mechanism by which glyco-

gen synthase kinase 3 differentially affects the nature

and magnitude of the inflammatory response.

Variation in microbial challenge

The apparent inability of Toll-like receptors (or pat-

tern-recognition receptors in general) to distinguish

microbe-associated molecular patterns from patho-

genic and commensal organisms suggests that amajor distinction between pathogens and commen-

sals could be made by considering what pathogens

can uniquely do in terms of modulating the innate

response. Pathogens could either exploit pattern-

recognition receptors, or modify the subsequent

signaling and or the response outcome. These pro-

cesses would require the presence of specific viru-

lence factors, which commensals lack. In this regard,

virulence factors (e.g. protein adhesins invasins)

and microbe-associated molecular patterns (e.g.

lipopolysaccharide or lipoteichoic acid) are not

equivalent terms and generally represent distinct

molecular entities. Microbe-associated molecular

patterns are essential for performing microbial

physiologic functions and are thus relatively con-

served within a certain microbial class (130). Since

most, if not all, microbe-associated molecular pat-

terns existed long before mammalian pattern-recog-nition receptors, it becomes obvious that from

the microbes point of view, microbe-associated

molecular patterns evolved to perform functions

unrelated to hostpathogen interactions. From the

host viewpoint, however, the conserved nature of

microbe-associated molecular patterns rendered

them ideal targets in the course of evolution for

detection by pattern-recognition receptors. In stark

contrast to microbe-associated molecular patterns,

virulence factors are responsible for adaptive fitness

within a particular host environment (130) and are

thus characterized by mutability and lack of conser-

vation. This strongly suggests that virulence factors

are unlikely to have been selected in the course of

evolution as targets of pattern recognition. However,

the converse notion that virulence factors, such as

protein adhesins, may have evolved to interact with

and possibly exploit the pattern recognition system

cannot be ruled out and will be discussed. In fact, it is

not only non-proteinaceous, invariant structures that

display Toll-like receptor reactivity as was originally

assumed; it is now firmly established that microbial

virulence proteins do interact with Toll-like receptors(44, 72, 73, 132, 160, 179, 195).

Epithelial cells in the oral cavity or other mucosal

sites express Toll-like receptors and can thus recog-

nize and respond to microbe-associated molecular

patterns from pathogens or commensals (53, 112,

186). A certain degree of basal or constitutive acti-

vation of the innate immune response is required to

maintain health. In this regard, the interactions of

commensals with host Toll-like receptors or pattern-

recognition receptors normally lead to a state of

physiological inflammation, which helps to contain

the commensals at the mucosal surfaces in a more-or-less harmonious hostmicrobe coexistence (39).

The inability of commensal organisms to actively

penetrate or disrupt the integrity of the mucosal

surface may also contribute to this balance and

accidentally trespassing commensals (e.g. as a result

of trauma) would most likely stimulate a strong host

response and be eliminated (86, 139). However, this

balance may be disrupted by pathogens because

virulence factor-mediated manipulation of normal

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pattern recognition mechanisms may result in

perturbation of the otherwise homeostatic Toll-

like receptormicrobe-associated molecular pattern

interactions, leading to pathogen persistence and to

non-productive inflammation detrimental to the host

(166). Metaphorically, these divergent states for host

commensal and hostpathogen interactions have

been characterized as armed peace and open war,

respectively (170). Modulation of the host innateresponse and the concept of pathogen exploitation of

pattern recognition will be considered in the context

of periodontal disease and the oral cavity with its

network of hostmicrobial interactions. P. gingivalis

and Actinobacillus actinomycetemcomitans will be

used as model virulent organisms which exemplify

these concepts.

Pathogenic bacteria are equipped with virulence

protein adhesins that enable them to cross epithelial

barriers (117, 152, 169). P. gingivalis, for instance,

traverses the epithelia via a fimbria-dependent

invasion mechanism (117, 152) or by degrading

the epithelial barrier junctions (102). Trespassing

P. gingivalis would subsequently be detected in the

underlying connective tissue by macrophages and

other inflammatory cells, as is the case with other

invasive organisms (101, 148, 170). This could lead to

the clearance of P. gingivalis, unless it has developed

effective counter-immune strategies. This is a realis-

tic possibility because P. gingivalis is a successful

pathogen that can also be found in systemic tissues

(66, 115, 119). However, the mechanism(s) whereby

P. gingivalis resists immune elimination are poorlyunderstood. The ability of this oral pathogen to act-

ively inhibit production of the interleukin-8 chemo-

kine by gingival epithelial cells (34) or to degrade

secreted cytokines through the action of cysteine

proteinases (13, 27) has been suggested to suppress

host defense. More recent evidence from our group

suggests that P. gingivalis has co-opted a pro-adhe-

sive signaling pathway, normally involved in leuko-

cyteendothelial cell interactions, for subverting the

innate immune response (77, 78). Specifically,

P. gingivalis fimbriae interact with the CD14 Toll-

like receptor 2 recognition complex and inducephosphatidylinositol 3-kinase-mediated inside-out

signaling for activating the ligand-binding capacity of

CR3 (CD11b CD18) (77, 78) a multifunctional b2integrin with pattern-recognition receptor capabilit-

ies (42, 212). While CR3 activation may contribute to

the mobilization of inflammatory cells to sites of

infection (77, 126) this pattern-recognition receptor

may be exploited by P. gingivalis. Specifically,

P. gingivalisfimbriae not only activate, but also bind

CR3 resulting in CR3-mediated inhibition of biolo-

gically active interleukin-12 (interleukin-12 p70) (72),

a major cytokine involved in intracellular bacterial

clearance (193). Interestingly, the phagocytosis of

apoptotic cells by macrophages is heavily dependent

on CR3 and is associated with inhibition of interleu-

kin-12 p70, because apoptotic cells are not normally

recognized as danger (108, 140). It thus appears that

P. gingivalis has co-opted a natural anti-inflamma-tory CR3-dependent mechanism to suppress innate

immunity. This mechanism may not be unique to

this pathogen. Indeed, the interaction of Bordetella

pertussis filamentous hemagglutinin with CR3 also

leads to the inhibition of interleukin-12 p70 (136),

although the filamentous hemagglutinin uses a dif-

ferent mechanism for inducing the adhesive activity

of CR3 (94). Strikingly, the ability of P. gingivalis

fimbriae to stimulate Toll-like receptor 2 inside-out

signaling for CR3 activation (78) is shared by the

lipoarabinomannan of mycobacteria, which exploit

this pathway for promoting CR3-dependent inter-

nalization by monocytes macrophages (173).

Additional molecular mechanisms employed by

P. gingivalis to manipulate the host innate response

are diverse and may involve the structural modifica-

tion of microbe-associated molecular patterns which

alters their interaction with host pattern-recognition

receptors. For example, it has been shown that

P. gingivalis expresses a heterogeneous mixture of

lipid A species that can induce cell activation through

Toll-like receptor 2 or Toll-like receptor 4 or even

antagonize Toll-like receptor 4-induced cell activa-tion (35, 40). The heterogeneity that characterizes

P. gingivalis lipid A moieties is environmentally

regulated and the resulting changes in P. gingivalis

lipopolysaccharide structure may determine the type

of host response (35, 40). These findings suggest that

by altering the proportions of its different lipid A

moieties, P. gingivalis may increase its virulence

through manipulation of the innate response in ways

that may favor its survival.

In contrast, A. actinomycetemcomitans takes a

quite different approach in avoiding and modulating

the innate immune response. While both organismsare capable of invading and thriving in gingival epi-

thelial cells, which presumably shields them from the

host response, their mechanisms of internalization,

intracellular growth, and cell-to-cell spreading are

organism specific (118, 141). Interestingly, the

epithelial cell response to interaction with A.

actinomycetemcomitans and P. gingivalisalso differs

significantly. For example, microarray studies of

infected gingival epithelial cells showed that

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A. actinomycetemcomitans influences the expression

of many genes involved in cellular metabolism and

transcriptional regulation, and also activates pro-

apoptotic molecules such as GADD45A, BL3, ATM

and E2 F1 while repressing cMYC (74). In contrast,

P. gingivalis modulates the expression of genes in-

volved in cell proliferation, the regulation of the cell

cycle, and activates the transcription factors cMYC,

Bcl-2, and Bfl1 (74). This is consistent with its anti-apoptotic phenotype in the early stages of infection

(197). P. gingivalisinvasion of gingival epithelial cells

also results in inhibition of interleukin-8 (34) and

intracellular adhesion molecule-1 (90) production via

a process that has been designated local chemokine

paralysis (34). A non-fimbriated mutant that was

unable to invade gingival epithelial cells did not

antagonize interleukin-8 accumulation. These studies

suggest that the invasion process may retard the

recruitment of neutrophils to the site of P. gingivalis

infection. On the other hand, invasion of gingival

epithelial cells by A. actinomycetemcomitans increa-

ses interleukin-8 production (89), which is mediated

at least in part through the activity of outer mem-

brane protein 100 (9).

A. actinomycetemcomitans does not appear to

modify its lipid A to the same extent as P. gingivalis

(discussed above). Furthermore, A. actinomycetem-

comitanslipopolysaccharide is an activator of Toll-like

receptor 4 (124). Thus, P. gingivalis lipopolysaccha-

ride and invasion of gingival epithelial cells limit the

initiation of the inflammatory response whereas

A. actinomycetemcomitans lipopolysaccharide andinvasion of gingival epithelial cells are stimulators

of pro-inflammatory cytokines. These activities of

A. actinomycetemcomitanswould appear to be coun-

terproductive for establishing an infection. How then

might A. actinomycetemcomitans overcome the

inflammatory response of the host to establish infec-

tion and persist? A. actinomycetemcomitans appears

to counteract these processes by expressing potent

toxins, e.g. leukotoxin (114), cytolethal distending

(immunosuppressive) toxin (177, 185) and the heat-

shock protein GroEL (150) that target immune cells

responding to the site of infection. Theorganism is alsoresistant to neutrophil-mediated phagocytosis (162)

and expresses enzymes, e.g. superoxide dismutase and

catalase, that inactivate reactive oxygen components

and may confer resistance to killing by the respiratory

burst. Thus, A. actinomycetemcomitans is capable of

modulating the innate response by targeting the cells

and other components of the inflammatory response

rather than by inhibiting the initiation of inflamma-

tion. A. actinomycetemcomitansalso expresses a toxin

that is related to the cytolethal distending toxin. Cy-

tolethal distending toxin was initially identified in

enteric organisms (157) and the A. actinomycetem-

comitanstoxin is most similar to cytolethal distending

toxin expressed by Haemophilus ducreyi (177). The

toxin is active against a variety of cells including HeLa

cells (185), epithelial cells (3, 100), and human gingival

fibroblasts (16). However, Shenker et al. (177) showed

that the specific activity of cytolethal distending toxinwas higher for human lymphocytes than for HeLa cells

and suggested that the toxin may be an immunosup-

pressive factor. Expressing both theleukotoxin and the

cytolethal distending toxin may allow A. actinomyce-

temcomitans to influence multiple facets of the host

immune response.

Periodontal disease arises from a polymicrobial

infection within the gingival crevice or periodontal

pocket by facultative and obligate anaerobic bacteria.

The interactions of different organisms in this biofilm

likely influence the virulence of this microbial com-

munity. Indeed, synergistic effects on virulence in

murine model systems has been documented for

several periodontal pathogens, e.g. P. gingivalis and

Fusobacterium nucleatum (47), Streptococcus con-

stellatus and F. nucleatum (116), Peptostreptococcus

micros and Prevotella intermedia (6), and P. gingi-

valis and Tannerella forsythia (217, 218). Thus, it is

possible that the diverse mechanisms utilized

by P. gingivalis and A. actinomycetemcomitans to

modulate the host innate response may function sy-

nergistically to facilitate colonization, persistence

and virulence of multi-species microbial populationsin the gingival pocket. For example, the exploitation

of the Toll-like receptor2 CR3 pathway by P. gingi-

valis results in down-regulation of interleukin-12,

which may reduce or delay the local host inflamma-

tory response. While this may allowP. gingivalistime

to establish an infection and or to invade host cells,

it would also benefit co-habiting organisms occupy-

ing the same niche as P. gingivalis. The reduced

induction of interleukin-12 may also affect the acti-

vation of cytotoxic T cells or natural killer cells

resulting in reduced clearance of host cells that have

been invaded by P. gingivalis or A. actinomycetem-comitans. In addition, the antagonism of Toll-like

receptor 4-mediated responses by specific isoforms

of P. gingivalis lipopolysaccharide may function sy-

nergistically to reduce the inflammatory response

induced by organisms such as A. actinomycetem-

comitans, whose lipopolysaccharide is a strong

agonist of Toll-like receptor 4-mediated signaling.

Conversely, the killing of neutrophils, macrophages,

and lymphocytes by the potent toxins secreted by

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A. actinomycetemcomitansmight benefit P. gingivalis

and other organisms that do not possess mechanisms

to target these immune cells directly. As we learn

more about the molecular mechanisms that govern

how pathogens modulate the innate immune

response, it will be possible to test directly the

hypothesis that the pathogenesis of polymicrobial

infections is driven at least in part by a cooperative

assault on the host response by different microbialspecies. Thus variability in the microbial plaque will

translate to variation in disease among subjects.

Genetic variation in the innatesystem

The recognition of lipopolysaccharide by inflamma-

tory cells and the transduction of the lipopolysac-

charide signal involve the Toll-like receptors and

several additional molecules, particularly the CD14

receptor and lipopolysaccharide-binding protein (29,

174). Recently, base-pair changes in the genes for

both CD14 and Toll-like receptor 4 have been des-

cribed in humans, in particular two co-segregating

polymorphisms in the extracellular domain of the

receptor of the human Toll-like receptor 4 gene (8).

These mutations were found to be associated with

decreased airway responsiveness after lipopoly-

saccharide stimulation, and support functional

variability that may affect the host response to a

gram-negative bacterial infection. A polymorphism in

the promoter region of the CD14 gene has also beendescribed (11). The homozygote genotype of this

polymorphism has been associated with increased

circulating soluble CD14 levels and a higher density

of CD14 receptors on monocytes (11, 92). Agnese

et al. (2) have found a significantly higher incidence

of gram-negative infections among patients with the

Toll-like receptor 4 polymorphism, but no associ-

ation between CD14 polymorphism(s) and the inci-

dence of infection or outcome. Two polymorphisms

have been identified in the Toll-like receptor 2 gene,

and have been found to diminish the ability of Toll-

like receptor 2 to mediate a response to bacterialcomponents (23). Its unique ability to respond to

P. gingivalis antigens means that the polymorphic

changes in the Toll-like receptor 2 structure may af-

fect the course of periodontitis associated with

P. gingivalis infection. Periodontal infection is dom-

inated by gram-negative bacterial pathogens, and it is

reasonable to hypothesize that any functional poly-

morphism in lipopolysaccharide receptors may affect

the inflammatory process and the clinical outcomes.

Antimicrobial peptides

An outcome of innate immune system triggering is the

production of cytokines and anti-microbial peptides

by stimulated cells. To maintain health in the upper

respiratory tract and oral cavity, a major entry point

for bacteria and other microorganisms (54), the mu-

cosal secretions of oral and airway epithelia containmultiple anti-microbial proteins. These proteins are

early responses to microbial challenges. The antimi-

crobial proteins can confer rapid protection from

pathogens, either before or during the development of

an acquired immune response. Over 800 eukaryotic

antimicrobial peptides have been identified and

are accessible in antimicrobial peptide databases

(http://aps.unmc.edu/AP/main.php, http://www.

bbcm.univ.trieste.it/~tossi/antimic.html). Many anti-

microbial peptides are organized in multigene famil-

ies although antimicrobial peptides within individual

families exhibit remarkable sequence diversity. As anexample, defensins typically share a few key amino

acids that are needed for overall structure but other-

wise vary greatly between the different family mem-

bers (56, 127). It is thought that this diversity of

antimicrobial peptides allows the innate immune

system to respond effectively to a wide range of

microorganisms. Moreover, a host response that

involves multiple antimicrobial proteins to a single

pathogen may be less likely to be met with antimi-

crobial resistance. Hence a broad understanding of

the antimicrobial peptides that act in oral and airway

mucosa is likely to be a prerequisite for the rational

development of antibiotics based on these peptides.

Antimicrobial peptides in the oralcavity

Recent surveys of human saliva indicate that many

known antimicrobial proteins are found in the oral

cavity, including neutrophil defensins, beta defensins,

lysozyme, bactericidal permeability-increasing pro-

tein, bactericidal permeability-increasing protein-like proteins, histatins, proline-rich proteins, salivary

agglutinin (GP-340), cathelicidin LL-37, cystatins,

lactoferrin, salivary peroxidase, mucins, and secretory

leukoproteinase inhibitor (63, 70, 76, 87, 88, 201, 202,

208, 211, 216). The different antimicrobial protein

families represent a variety of antimicrobial functions

including membrane permeabilization, cell wall deg-

radation, intracellular inhibitors, bacterial aggrega-

tion, and oxidation (32, 188).

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Cationic proteins constitute a large group of anti-

microbial proteins that represent different antimi-

crobial activities. The importance of these proteins is

illustrated by the finding that broad depletion of

cationic proteins from human airway fluid also re-

moves its antibacterial activity (30). The different

antimicrobial functions of cationic peptide are rep-

resented by (i) defensins, cathelicidin, and the palate,

lung, and nasal epithelium carcinoma-associatedprotein (PLUNC) that can interact directly with the

bacterial cell membrane leading to its permeabiliza-

tion (57); (ii) lysozyme, which cleaves the peptidog-

lycans of bacterial cell walls leading to membrane

rupture; and (iii) the antifungal histatins that bind to

a fungal cell membrane receptor to enter the cells

and block mitochondrial function (103).

Both the overexpression and the lack of antimi-

crobial peptides have been linked with the develop-

ment of oral diseases. Defensin expression is induced

by oral bacteria (199) and the levels of defensin-1 are

significantly higher in patients with oral inflamma-

tion than in normal controls (145). Morbus Kostmann

is a congenital neutropenia that is associated with

recurrent infections and periodontal disease and

deficiencies in LL-37 and the a-defensins human

neutrophil peptide (HNP) 13 (164). The finding that

one patient who had undergone a bone marrow

transplant had normal levels of LL-37 and no perio-

dontal disease, underscores the potential role of

antimicrobial peptides in host defense of the oral

cavity (164). Evaluating the levels of antimicrobial

peptides may have diagnostic value. Thus, low levels

of HNP13 were correlated with high caries incidence

in children. Other antimicrobial (hBD-3, LL-37) pep-

tides did not correlate with caries incidence in this

study (189). While these single protein deficiencies

are linked to specific conditions, they are unlike the

rampant oral infections and dental decay that occur

when all salivary proteins are lacking in dry mouth

conditions such as Sjogrens syndrome. This differ-

ence underscores the importance of complementarity

in the mucosal innate immune defense (188). With a

more comprehensive understanding of the antimi-

crobial proteins that act in the oral cavity, it is poss-

ible that protein expression signatures (fingerprints)

can be developed for the diagnosis of individual oral

diseases or the identification of at-risk individuals,

before development of the disease. Several families of

antimicrobial proteins have been extensively studied

(12, 25, 32, 55, 103). This section of the review will

focus on the expression and function of a new family

of bactericidal permeability-increasing protein-like

antimicrobial proteins that have recently been iden-

tified in the oral cavity and airway epithelia.

Bactericidal permeability-increasing protein-like proteins

The completion of the sequence of human chro-

mosome 20 led to the identification of a novel

family of nine secretory proteins that are expressed

Table 1. The human bactericidal permeability-increasing protein-like protein family

HGNC symbol Aliases Tissue localization

PLUNC SPLUNC1, LUNX, SPURT, NASG, bA49 G10.5 Lung, nasal, trachea, saliva

BPIL1 RYSR, LPLUNC2, C20orf184, dJ726C3.2 Nasal, SMG, tonsil

BPIL2 dJ149A16.7 Skin

BPIL3 LPLUNC6 Trachea, tonsil

C20orf114 VEMSGP, LPLUNC1, MGC14597, bA49 G10.6,

dJ1187 J4.1

Von Ebners minor salivary gland,

lung, trachea, saliva

C20orf185 RYA3, LPLUNC3, dJ726C3.4 Nasal

C20orf186 RY2 G5, dJ726C3.5, LPLUNC4 Nasal

C20orf70 SPLUNC2, PSP, bA49 G10.1 SMG, parotid, saliva

C20orf71 SPLUNC3, bA49 G10.4,

N A BASE, RP11-49 G10.8 Breast cancer, salivary gland

The currently recognized members of the human bactericidal permeability-increasing protein-like protein family are listed with their HGNC approved genenames (when available), aliases and sites of expression. All genes are located on chromosome 20q11.21 with the exception of BPIL2, which is located on 22q12.3.

BPI, bactericidal permeability-increasing protein; PLUNC, palate, lung, and nasal epithelium carcinoma-associated protein; PSP, parotid secretory protein;SMG, submandibular gland; SPLUNC, short PLUNC; LPLUNC, long PLUNC; SPURT, secretory protein in upper respiratory tracts; VEMSGP, von Ebner s minorsalivary gland protein.

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in oral and airway epithelia (20, 21) (Table 1). Some

of these proteins had also been identified inde-

pendently (14) (GenBank no. AF432917) (37, 95) and

a related gene that is expressed in skin was identi-

fied on chromosome 22 (149). Sequencesequence

comparison and sequencestructure analysis using

the 3D-PSSM program (106) revealed that these

proteins are related to a family of mammalian lipid-

binding proteins that includes bactericidal per-

meability-increasing protein, lipopolysaccharide-

binding protein, cholesteryl ester transport protein

(CETP) and phospholipid transport protein (PLTP)

(20, 149, 206) (Fig. 2).

Several of the human bactericidal permeability-

increasing protein-like proteins correspond to previ-

ously identified animal proteins including parotid

secretory protein (PSP) (128), palate, lung, and nasal

epithelium carcinoma-associated protein (205),

bovine salivary protein 30 KD (BSP30) (165) and von

Ebners minor salivary gland protein (VEMSGP;

GenBank no. U46068). The human proteins have

been termed the palate, lung, and nasal epithelium

carcinoma-associated protein family (20) or bacteri-cidal permeability-increasing protein-like proteins

(149). We prefer the latter name because it is linked to

the protein structure that characterizes the family

rather than to the identification of a single protein

or some of its expression sites (palate, lung, nasal

epithelium). Bactericidal permeability-increasing

protein is an antibacterial protein with selectivity

for gram-negative bacteria. In addition, bacteri-

cidal permeability-increasing protein binds lipo-

polysaccharides and exhibits anti-inflammatory

activity by inhibiting the binding of lipopolysaccha-

ride to lipopolysaccharide-binding protein (204). TheX-ray structure has been elucidated (PDB ID: 1EWF)

and consists of two bactericidal permeability-

increasing protein domains, BPI1 and BPI2 (15). This

structure formed the basis for the identification of the

bactericidal permeability-increasing protein-like

proteins.

The bactericidal permeability-increasing protein-

like proteins are either about 250 amino acids in

length or more than 450 amino acids. The predicted

structure of these proteins is related to the bacteri-

cidal permeability-increasing protein structure,

containing one or two of the bactericidal permeab-

ility-increasing protein domains. As an example,

parotid secretory protein and palate, lung, and nasal

epithelium carcinoma-associated protein are similar

to the N-terminal of domain 1 of bactericidal per-

meability-increasing protein while the longer von

Ebners minor salivary gland protein and bactericidal

permeability-increasing protein-like 2 (BPIL2) con-

tain both domains 1 and 2 (Fig. 2). We have specu-

lated that the shorter proteins arose by loss of the

C-terminal domain from a progenitor gene (21). The

functional consequences of this model remain to be

determined.

As is the case in other families of antimicrobial

proteins (see above) the sequence conservation

among bactericidal permeability-increasing protein-

like proteins is poor, with the exception of two con-

served Cys residues. These residues are also found in

bactericidal permeability-increasing protein where

they form a disulfide bridge. The disulfide bridge has

not been verified in the bactericidal

permeability-increasing protein-like proteins, but a parotid secre-

tory protein mutant lacking the Cys residues is not

secreted, suggesting that its structure is significantly

altered (Geetha & Gorr, unpublished observation).

Expression of bactericidal permeability-increasingprotein-like proteins

Several of the bactericidal permeability-increasing

protein-like proteins are expressed in human gingivalkeratinocytes where their expression is regulated by

the oral bacterium P. gingivalis and the pro-inflam-

matory cytokine tumor necrosis factor-a (178, and

Gorr et al., manuscript in preparation). In the air-

ways, palate, lung, and nasal epithelium carcinoma-

associated protein expression is up-regulated by

retinoic acid (37) and expression has been linked to

epithelial injury, irritation, and cancers (22, 144, 187).

In contrast, palate, lung, and nasal epithelium carci-

PLUNC

VEMSGP

BPIL2

Fig. 2. Domain structure of selected

bactericidal permeability-increasing

protein (BPI) -like proteins. Data

obtained from the NCBI Conserved

Domain Search service (98).

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noma-associated protein expression is down-regula-

ted in nasopharyngeal carcinoma (220), some smok-

ers (63), and seasonal allergic rhinitis (62). Palate,

lung, and nasal epithelium carcinoma-associated

protein gene polymorphisms have also been linked to

nasopharyngeal carcinoma (82). However, it is not

yet clear if palate, lung, and nasal epithelium carci-

noma-associated protein plays a direct functional

role in these cancers.

Function of bactericidal permeability-increasingprotein-like proteins

The differential expression of palate, lung, and nasal

epithelium carcinoma-associated protein in epithelial

injury and irritation suggests that either this protein or

the other bactericidal permeability-increasing pro-

tein-like proteins could play a role in inflammation.

Consistent with this suggestion, palate, lung, andnasal

epithelium carcinoma-associated protein binds to

lipopolysaccharide (64). Indeed, we have found that

peptides based on the predicted parotid secretory

protein structure block the binding of lipopolysac-

charide to lipopolysaccharide-binding protein and

block the lipopolysaccharide-stimulated secretion of

tumor necrosis factor-a from macrophages (59). In

addition, intact parotid secretory protein is antibac-

terial to Pseudomonas aeruginosa (60) and preliminary

data suggest that parotid secretory protein peptides

are antibacterial to gram-negative but not gram-pos-itive bacteria (unpublished observation). Antibacterial

and anti-inflammatory activity has also been reported

for human palate, lung, and nasal epithelium carci-

noma-associated protein (135). The functional

characterization of the bactericidal permeability-

increasing protein-like proteins is still in the early

stages and functional data are lacking for the other

members of this gene family. Their elucidation will

provide a more complete picture of the antimicrobial

activities that exist in theoral cavityand upper airways.

Antimicrobial peptides are attractive candidates for

novel antibiotics. The need for such drugs to combatthe ever-increasing resistance of bacterial infections

is undisputed. It is predicted that antimicrobial

peptides naturally occurring in humans will cause

little toxicity and that their co-evolution with differ-

ent bacteria will lead to fewer problems with resist-

ance. However, caution has been urged in this field

because a broad resistance to these naturally occur-

ring antibiotics could render patients defenseless

against pathogens. Indeed, to date the study of anti-

microbial peptides has not led to new clinically

important antibiotics. Individual peptides including

magainin and recombinant bactericidal permeabil-

ity-increasing protein have been tested in clinical

trials but are not approved for clinical use. However,

the use of single peptides may not adequately mimic

how these peptides act in vivo. Since antimicrobial

peptides act in concert in mucosal surfaces, it is

possible that combination therapies must be de-signed to maximize clinical efficacy and minimize the

development of resistance.

Variability and the innate system

Irritation of the gingival tissues by periodontal plaque

biofilm bacteria induces an inflammatory response

that varies depending on the subjects genetics, their

Toll-like receptors, the niche and biofilm bacteria

making the challenge, and potential variations in

signaling and subsequent cytokine and antimicrobial

responses. Furthermore, downstream variations

should also be considered, particularly in the inflam-

matory response, the adaptive immune response and

the healing responses, all of which can be affected by

genetic and environmental factors. Identifying causes

of variability in the innate system may indicate diag-

nostic tools and therapeutic modalities to address the

differences between subjects in terms of susceptibility

to gingivitis, periodontitis, and other chronic micro-

bially induced diseases. Our knowledge of host sus-

ceptibility will be enhanced by a fuller understandingof the nature of the challenge, variation in specific

receptors, the signal transduction pathways and the

ways in which cells and tissues respond to these signals

in terms of cytokine and defense molecule production.

Acknowledgements

This work was supported by the following grants:

R01DE015254, R01DE018292 (G. Hajishengallis);

R01DE14605,R01DE10729(D. Demuth);R01DE017384

(D.F. Kinane); R01DE017680 (M.H. Martin).

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