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Cellular Microbiology (2006) 8(5), 738757 doi:10.1111/j.1462-5822.2005.00661.xFirst published online 8 December 2005

2005 The AuthorsJournal compilation 2005 Blackwell Publishing Ltd

Blackwell Science, LtdOxford, UKCMICellular Microbiology 1462-5814 2005 The Authors; Journal compilation 2005 Blackwell Publishing Ltd85738757Original ArticleP.gingivalis fimbria-dependent inflammatory responsesY.Takahashi et al.

Received 22 July, 2005; revised 27 September, 2005; accepted 3October, 2005. *For correspondence. E-mail [email protected]; Tel. (+1) 617 414 5305; Fax (+1) 617 414 5280.

Both authors contributed equally to this body of work.

Fimbria-dependent activation of pro-inflammatorymolecules in Porphyromonas gingivalisinfected humanaortic endothelial cells

Yusuke Takahashi,

1,2

Michael Davey,

3

Hiromichi Yumoto,

1,4

Frank C. Gibson III

1

and

Caroline Attardo Genco

1,3,5

*

1

Department of Medicine, Section of Infectious Diseases,

Boston University School of Medicine, Evans Biomedical

Research Center, 650 Albany Street, Boston, MA 02218,

USA.

2

Department of Oral Microbiology, Kanagawa Dental

College, 82 Inaokoa-cho, Yokosuka 238-8580, Japan.

3

Department of Periodontology and Oral Biology,

Goldman School of Dental Medicine, Boston University,100 East Newton Street, Boston, MA 02118, USA.

4

Department of Conservative Dentistry, Tokushima

University School of Dentistry, 3-18-15 Kuramoto-cho

Tokushima 770-8504, Japan.

5

Department of Microbiology, Boston University School of

Medicine, 715 Albany Street, L-504, Boston, MA 02118,

USA.

Summary

Epidemiological studies support that chronic peri-

odontal infections are associated with an increasedrisk of cardiovascular disease. Previously, we

reported that the periodontal pathogen Porphyromo-

nas gingivalisaccelerated atherosclerotic plaque for-

mation in hyperlipidemic apoE

/

mice, while an

isogenic fimbria-deficient (FimA-) mutant did not. In

this study, we utilized 41 kDa (major) and 67 kDa

(minor) fimbria mutants to demonstrate that major

fimbria are required for efficient P. gingivalisinvasion

of human aortic endothelial cells (HAEC). Enzyme-

linked immunosorbent assay (ELISA) revealed that

only invasive P. gingivalisstrains induced HAEC pro-

duction of pro-inflammatory molecules interleukin

(IL)-1, IL-8, monocyte chemoattractant protein

(MCP)-1, intracellular adhesion molecule (ICAM)-1,

vascular cellular adhesion molecule (VCAM)-1 and E-

selectin. The purified native forms of major and minor

fimbria induced chemokine and adhesion molecule

expression similar to invasive P. gingivalis, but failed

to elicit IL-1 production. In addition, the major and

minor fimbria-mediated production of MCP-1 and

IL-8 was inhibited in a dose-dependent manner

by P. gingivalis lipopolysaccharide (LPS). Both

P. gingivalis LPS and heat-killed organisms failed to

stimulate HAEC. Treatment of endothelial cells with

cytochalasin D abolished the observed pro-inflamma-

tory MCP-1 and IL-8 response to invasive P. gingivalis

and both purified fimbria, but did not affectP. gingivalisinduction of IL-1. These results suggest

that major and minor fimbria elicit chemokine produc-

tion in HAEC through actin cytoskeletal rearrange-

ments; however, induction of IL-1 appears to occur

via a separate mechanism. Collectively, these data

support that invasive P. gingivalisand fimbria stimu-

late endothelial cell activation, a necessary initial

event in the development of atherogenesis.

Introduction

Increasing evidence has focused attention on infectionwith specific microbial pathogens as a risk factor and

novel reservoir of agents that potentiate atherosclerosis

and its associated inflammatory changes (Ross, 1999).

Much of this interest has been centred on infection with

Chlamydia pneumoniae, a common respiratory patho-

gen, because of epidemiological and experimental

reports linking infection with this organism to atheroscle-

rosis (Kuo et al., 1993; Jackson et al., 1997; Moazed

et al., 1999). The association between human periodontal

disease, a chronic bacterial infection of the tissue that

supports the teeth, and cardiovascular disease (CVD)

has also been recently strengthened by both epidemio-

logical and in vitro studies (Beck et al., 1996; Loesche

et al., 1998; Arbes et al., 1999; Dorn et al., 1999; Harasz-

thy et al., 2000; Loos et al., 2000; Kolltveit and Eriksen,

2001; Glurich et al., 2002). Chiu (1999) reported that

42% of specimens obtained from human atherosclerotic

plaques reacted with antibody to the primary aetiological

agent of periodontal disease, Porphyromonas gingivalis.

In addition to a broad array of known virulence factors,

this organism expresses two distinct types of fimbria, the


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2005 The AuthorsJournal compilation 2005 Blackwell Publishing Ltd, Cellular Microbiology

, 8

, 738757

41 kDa and 67 kDa protein subunit types denoted as

major and minor respectively (Hamada et al., 1994). The

major fimbria has been shown to be required for adhe-

sion to gingival cells, gingival fibroblasts and endothelial

cells (Lamont et al., 1995; Deshpande et al., 1998;

Nakagawa et al., 2002). P. gingivalis major fimbria have

been reported to induce the expression of inflammatory

cytokines, such as tumour necrosis factor (TNF)- and

interleukin (IL)-1, in both human and murine monocytes/

macrophages or monocytic cell lines. These studies

indicated that the major fimbria of P. gingivalis plays a

crucial role as both a bacterial adhesin and a potent

stimulus capable of eliciting host inflammatory responses

(Ogawa et al., 1994; Saito et al., 1996; Hajishengallis

et al., 2002a; Graves et al., 2005). Recently, we reported

that infection of apolipoprotein E (ApoE) knockout mice

with invasive P. gingivalis plays a major role in acceler-

ated atheroma development. Results from these studies

showed that only wild-type P. gingivalis (possessing the

major fimbria), but not the non-invasive (FimA-) mutant,

could accelerate plaque formation in the aortic arch ofthese mice despite the observation of both wild-type and

FimA mutant DNA in the blood and aortic arch tissue of

infected animals (Gibson III., et al., 2004). As compared

with the major fimbria, very little is known about the func-

tion(s) of minor fimbria. In purified native form, the minor

fimbria of P. gingivalishas been reported to induce TNF-

, IL-1, or IL-6 production in human monocytic cell lines

and murine peritoneal macrophages (Hajishengallis

et al., 2002a; Hiramine et al., 2003). However, the precise

role of the minor fimbria in P. gingivalisvirulence remains

poorly defined.

The healthy vascular endothelium maintains an intact,anti-inflammatory state that inhibits thrombosis (Charo

et al., 1998). Disruption of this homeostatic state by a

mechanical breach of this barrier, or by exposure to bac-

terial or viral infection, can readily convert the endothelium

to a pro-thrombotic environment (Visser et al., 1988). Pro-

inflammatory cytokines, cell adhesion molecules (CAMs)

and Toll-like receptors (TLRs) are believed to be actively

involved in this infection-mediated activation of the endot-

helium (Collins et al., 1995; Faure et al., 2001; Zeuke

et al., 2002) and acceleration of atherosclerosis (Nageh

et al., 1997; Boring et al., 1998; Gu et al., 1998; Bjork-

backa et al., 2004; Michelsen et al., 2004). Numerous

studies have been reported on the interaction of endothe-

lial cells and Chlamydia pneumonia(Gaydos et al., 1996;

Fryer et al., 1997; Campbell et al., 1998; Dechend et al.,

1999; Gaydos, 2000). These studies demonstrated that

Chlamydial infection of these cells can induce expression

of many pro-inflammatory mediators associated with ath-

erosclerosis including cytokines, CAMs and chemokines,

as well as molecules associated with pro-coagulant activ-

ity and those promoting the oxidation of low density lipo-

protein (Kaukoranta-Tolvanen et al., 1996; Fryer et al.,

1997; Molestina et al., 1998; Krull et al., 1999; Summers-

gill et al., 2000; Dittrich et al., 2004). We recently demon-

strated that P. gingivalis invasion of human aortic

endothelial cells (HAEC) stimulates TLR expression, prim-

ing these cells to respond to interactions with TLR-specific

ligands (our unpubl. data). However, this response was

not observed with non-invasive P. gingivalis, heat-killed

organisms, purified native P. gingivalismajor or minor fim-

bria, or P. gingivalis lipopolysaccharide (LPS).

In this study, using defined P. gingivalisfimbrial mutants,

as well as purified native P. gingivalis major and minor

fimbria, we demonstrated that an invasive P. gingivalis

genotype and both the major and minor fimbria of

P. gingivaliscan stimulate potent inflammatory responses

consistent with expression of pro-inflammatory chemok-

ines and adhesion molecules in HAEC. In addition, we

demonstrate that only invasion by intact P. gingivalis

induces the more complex, temporally accelerated pro-

inflammatory response potentially seen in HAEC during

the initial events of a developing atherosclerotic lesion invivo.

Results

Inactivation of the 67 kDa minor fimbrillin gene (mfa1) of

P. gingivalis

Porphyromonas gingivalis attachment to host cells has

been suggested to be a bi-phasic process whereby the

major fimbria is responsible for initially tethering the bac-

teria to the host cell (Lamont and Jenkinson, 1988;

Njoroge et al., 1997). The exact mechanism by whichmore intimate attachment occurs is currently undefined.

As P. gingivalis possesses two types of fimbria, 41 kDa

and 67 kDa protein subunits, we generated several fim-

bria-deficient mutants in the wild-type strain 381 back-

ground. To create a P. gingivalis 67 kDa fimbrial mutant,

the truncated 67 kDa fimbrillin gene (mfa1) was amplified

by polymerase chain reaction (PCR) and cloned into

pBluescriptII KS+ and disrupted by the tetQ gene

(Fig. 1A). Transformation of the recombinant plasmid into

P. gingivalis strain 381 and previously constructed

P. gingivalisDPG3 (Malek et al., 1994) generated two new

strains, 381MF1 (a minor fimbria-deficient mutant) and

DPGMFB (a major and minor fimbria-deficient mutant)

respectively. Growth curves for all strains of P. gingivalis

demonstrated no differences in growth over a 36 h period

(data not shown). Both newly generated strains failed to

express the 67 kDa minor fimbria as demonstrated by

sodium dodecyl sulphate-polyacrylamide gel electro-

phoresis (SDS-PAGE) and by immuno-blotting with anti-

67-kDa fimbria antiserum (Fig. 1B). It should be noted that

immuno-blots of cell lysates from similar colony-forming


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Y. Takahashiet al.

2005 The AuthorsJournal compilation 2005 Blackwell Publishing Ltd, Cellular Microbiology, 8, 738757

units (cfu) counts of wild-type strain 381, DPG3 and

381MF1 verified that loss of Mfa1 or fimA does not result

in altered expression of the 41 kDa or 67 kDa fimbrial

proteins by 381MF1 and DPG3 respectively (data not

shown). As expected, SDS-PAGE and immuno-blot anal-

ysis of purified fimbria isolated from all strains revealed

that each fimbrillin protein appeared as a single band, with

the corresponding molecular weights of 41 kDa and

67 kDa, respectively, and reacted to their anti-41-kDa

(major) or anti-67-kDa (minor) fimbria-specific antiserum

(Fig. 1C). Electron microscopy revealed that strain

381MF1 expressed fimbria that were distinct from fimbria

of strain DPG3. As expected, no filamentous structures

were observed on the cell surface of DPGMFB (Fig. 1D).

These strains and purified native fimbrial proteins were

utilized for the remainder of our studies.

EfficientP. gingivalis invasion of HAEC requires 41 kDa

(major) fimbria

Previously we reported that human umbilical vein endot-

helial cells (HUVEC) express chemokines (Nassar et al.,

2002) and adhesion molecules (Khlgatian et al., 2002) in

response to invasive P. gingivalis. Since publication of the

aforementioned manuscripts, several groups have dem-

onstrated diversity in the biochemical composition

Fig. 1. Establishment of P. gingivalis41 kDa (major) and 67 kDa (minor) fimbria mutants.A. Construction of the recombinant plasmid for inactivation of the 67 kDa (minor) fimbrillin gene. The truncated 67 kDa fimbrillin gene (mfa1, socalled minor fimbria gene) was cloned into a pBluescriptII KS+ plasmid and disrupted by the tetracycline resistance gene tetQ. The resultingplasmid was transformed into strain 381 and DPG3 following linearization with SacI. Tetracycline resistant transformants were recovered after

23 weeks of anaerobic culture.B. SDS-PAGE and immuno-blot analysis of the fimbrial mutants (wild-type strain 381 expresses 41 kDa major and 67 kDa minor fimbria, mutantstrain DPG3 expresses only the 67 kDa minor fimbria (Malek et al., 1994), mutant strain 381MF1 expresses only the 41 kDa major fimbria,and mutant strain DPGMFB does not express either fimbria).

C. SDS-PAGE and immuno-blot analysis of purified native fimbria isolated from strains 381, DPG3 and 381MF1. Whole cell samples of thebacterial cells or purified native fimbria were separated using 12% gels stained by Coomassie brilliant blue R-250 or transferred to PVDFmembranes and incubated with rabbit anti-major or anti-minor fimbria-specific antiserum for detection of fimbria as indicated (B and C).D. Electron microscopy analysis of fimbria mutants. P. gingivaliscells were fixed and applied to collodion coated copper grids, negatively stainedby 2% uranyl acetate and visualized with a JEM1220 transmitting electron microscope. Bars indicate 200 nm.


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(Ghitescu and Robert, 2002), as well as differences in

gene expression patterns (Chi et al., 2003), between arte-

rial and venous endothelial cells. As the aorta is a princi-

pal site of atherosclerotic plaque accumulation during

CVD, we obtained primary HAEC from multiple donors

and challenged these cells with live P. gingivalis. To exam-

ine the role of the major and minor fimbria in the invasion

efficiency of P. gingivalis into HAEC, we first carried out

infection experiments with our constructed fimbria

mutants. As shown in Fig. 2A, the invasion efficiency of

P. gingivalis into HAEC was dependent upon expression

of the major fimbria. Internalization of the bacteria

occurred predominantly within the first hour of infection for

strains possessing the major fimbria. P. gingivalis strain

381MF1, possessing only the major fimbria, exhibited

invasion efficiencies comparable to wild-type P. gingivalis

strain 381; while the P. gingivalisstrains failing to express

the major fimbria (DPG3 and DPGMFB) displayed 100- to

1000-fold lower invasion efficiencies, respectively, when

compared with wild-type P. gingivalisafter 6 h infection of

HAEC (Fig. 2A). To ensure that observed differences ininvasion efficiency were not the result of altered suscep-

tibility to antibiotic treatment, existing strains 381 and

DPG3, as well as newly constructed mutant strains

381MF1 and DPGMFB were tested for susceptibility to

metronidazole killing. No differences were observed in

efficiency of metronidazole killing for any of the strains

tested (data not shown).

As mentioned previously, the interaction of P. gingivalis

with host cells has been described as a two-stage process

of initial attachment mediated by the 41 kDa major fimbria,

followed by more intimate attachment facilitating endocy-

tosis of the bacteria (Lamont and Jenkinson, 1988;Njoroge et al., 1997). In addition, it has been demon-

strated that expression of Fim A is not sufficient for inva-

sion and that other surface molecules may be involved

(Dorn et al., 2000). As a minimal percentage of the initial

DPG3 (0.0023%) and DPGMFB (0.00041%) inoculums,

which are devoid of the major or both fimbria, respectively,

were able to invade the endothelial cells, we next deter-

mined the contribution of the minor fimbria and other

surface components in P. gingivalis invasion of HAEC. To

better define the initial process of major fimbria-mediated

attachment, wild-type P. gingivalis and the fimbrial

mutants were centrifuged onto HAEC, as described pre-

viously (Walter et al., 2004), to enhance the rate of infec-

tion and potentially alleviate the requirement for major

fimbria in achieving efficient invasion. Centrifugation of the

bacteria resulted in a two-log-unit increase in invasion for

both the DPG3 and DPGMFB strains when compared with

non-centrifuged bacteria (Fig. 2B). In addition, the

enhancement of DPG3 attachment to the endothelial sur-

face resulted in invasion percentages that were similar to

non-centrifuged wild-type 381 and mutant strain 381MF1

which possess the major fimbria. Collectively, these

results stress the importance of major fimbria for initiation

of attachment and provide novel information regarding the

role of the minor fimbria in endothelial invasion.

Chemokine and cytokine responses of HAEC infected

withP. gingivalis

To functionally assess the invasive capabilities of the

Fig. 2. P. gingivaliswild-type and fimbrial mutant invasion of HAEC.A. Non-centrifuged invasion efficiency of P. gingivaliswild-type and

mutant strains into HAEC. Endothelial cells were infected for 1, 2 and6 h. Internalized bacterial cells were recovered by lysis of HAEC with

water. Cell lysates were plated onto blood agar plates and incubatedanaerobically at 37C for 7 days prior to cfu count. Percent invasionwas expressed as the percentage of the initial inoculums.B. Centrifuge-enhanced invasion of the endothelium. Bacteria wereeither added to medium (black bars) or centrifuged onto HAEC mono-

layers (350 gfor 5 min) (open bars). After 1 h, internalized bacteriawere recovered as described above and percent invasion wasexpressed as the percentage of the initial inoculums. All assays wereperformed in triplicate. Values represent the mean of triplicate sam-

ples and are indicative of a typical experiment standard error.


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P. gingivalisfimbria mutants, we next examined chemok-

ine and cytokine expression by HAEC in response to

invasive P. gingivalis infection utilizing enzyme-linked

immunosorbent assay (ELISA) and reverse transcriptase

polymerase chain reaction (RT-PCR). Infection with inva-

sive P. gingivalisstrains 381 and 381MF1 (expressing the

major fimbria) stimulated production of monocyte

chemoattractant protein (MCP)-1, IL-8 and IL-1 by HAEC

(Fig. 3A, C and E respectively). It should be noted that

infection with P. gingivalis381MF1 stimulated IL-8 mRNA

expression at 6 h (196 relative densitometry units, RDU)

(Fig. 3C) that remained elevated for 24 h (209 RDU), while

infection with wild-type strain 381 resulted in an initial

induction of IL-8 mRNA at 6 h (167 RDU) that appeared

to decrease over time (104 RDU). In addition, infection

with invasive strains of P. gingivalis resulted in protein

levels of IL-1 (Fig. 3E) which increased markedly after

2 h, followed by decreased levels observed at the 6 and

24 h time points. This trend was not observed with either

MCP-1 (Fig. 3A) or IL-8 production (Fig. 3C). As

expected, neither the major fimbria mutant DPG3 nor thefimbrial-null mutant DPGMFB stimulated expression of

MCP-1, IL-8, or IL-1 (Fig. 3A, C and E respectively). To

confirm that active invasion of P. gingivalisinto HAEC was

required for the observed chemokine and cytokine

responses, cells were treated with 1 g ml1 of cytochala-

sin D prior to the addition of P. gingivalis. Trypan blue

staining (Singh et al., 1985) revealed that cells remained

viable after cytochalasin D treatment. However, we

observed that cytochalasin D treatment did inhibit

P. gingivalis invasion (data not shown). In addition, pro-

duction of MCP-1 and IL-8 was significantly decreased

(P< 0.05) by cytochalasin D to 5% and 30%, respectively,when compared with non-inhibited, P. gingivalis chal-

lenged, HAEC control (Fig. 3B and D). In contrast, we

observed no significant inhibition of IL-1 production by

HAEC treated with cytochalasin D following stimulation

with wild-type P. gingivalis or the fimbrial mutants

(Fig. 3F). These data suggest that while the observed

chemokine responses appear to be dependent on cytosk-

eletal rearrangements induced by P. gingivalisinvasion of

HAEC, induction of the cytokine IL-1 seems to occur via

a separate mechanism.

Invasion-induced expression of adhesion molecules

in HAEC

In addition to chemokines, other markers of vascular acti-

vation including CAMs, have been indicated as contribu-

tors to the pro-inflammatory state associated with CVD

(Davies et al., 1993; OBrien et al., 1993; 1996; Johnson-

Tidey et al., 1994; DeGraba et al., 1998). To assess CAM

expression on endothelial cells infected with P. gingivalis,HAEC were cultured with wild-type P. gingivalisand fim-

brial mutants for 1, 6 and 24 h. CAM expression was

determined by flow cytometry and RT-PCR. Following 6 h

of infection, we observed increased levels of mRNA for

ICAM-1, VCAM-1 and E-selectin in HAEC cultured with

invasive strains 381 and 381MF1 (Fig. 4A). No differences

in P-selectin mRNA expression were observed at 6 or

24 h for any of the strains tested; however, total surface

expression of P-selectin by HAEC appeared to decrease

from 6 to 24 h. Protein levels of all CAMs stimulated by

invasive strains of P. gingivaliswere maximally expressed

on HAEC by 6 h of infection with a gradual return to

baseline expression levels following 24 h of infection

(Fig. 4C). The major fimbria-deficient mutants, DPG3 and

DPGMFB, did not induce similar CAM responses. In addi-

tion, heat-killed P. gingivalis 381 did not induce surface

expression of CAMs (data not shown). Taken together,

these results suggest that P. gingivalis viability and an

invasion phenotype is required to stimulate increased

expression of adhesion molecules on HAEC.

Stimulation of HAEC with purified native fimbria induces

MCP-1 and IL-8, but not an IL-1 response

To better define the role of fimbria in invasion-induced

expression of inflammatory molecules, we examined pro-

tein production and gene expression of MCP-1 (Fig. 5A)

and IL-8 (Fig. 5B) following incubation of HAEC with puri-

fied native major or minor fimbria. Following 6 h incubation

with either the major or minor fimbria, we observed a

dose- and time-dependent increase in MCP-1 protein pro-

duction and gene expression by HAEC (Fig. 5A). Incuba-

tion of HAEC with major or minor fimbria (10 g ml1)

stimulated 25 and 40 ng ml1 of MCP-1, respectively, as

Fig. 3. Expression of MCP-1, IL-8 and IL-1 by HAEC infected with P. gingivalis. HAEC were incubated with P. gingivalisstrains at an moi of 100for 1 h, 6 h and 24 h as indicated below each panel.A, C and E. Vertical stripes, black bars, open bars, grey bars and diagonal stripes represent uninfected control, 381, DPG3, 381MF1 and DPGMFB

respectively.B, D and F. For inhibition of invasion, HAEC were treated with 1 g ml1 (open bars) of cytochalasin D for 30 min prior to infection. Cells werethen infected with P. gingivalisstrains at moi of 100 for 6 h. Control samples not treated with cytochalasin D are indicated by black bars.Supernatant and total RNA from infected HAEC were analysed by ELISA and semi-quantitative RT-PCR. Production of MCP-1 (A and B), IL-8(C and D) and IL-1 (E and F) were expressed as mean SDs. Corresponding gene expression, as determined by RT-PCR, is displayed beloweach graph (AF). Semi-quantitative densiometric analyses have been performed for all mRNA data as indicated below each panel and are listedas relative densitometry units (RDUs). The results shown are representative of three independent experiments. *P< 0.05 versus uninfectedcontrol. #P< 0.05 control versus cytochalasin D treated HAEC.


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744 Y. Takahashiet al.


observed at 24 h post infection. Protein levels of IL-8 on

HAEC appeared to follow a similar trend as that observed

for MCP-1 (Fig. 5B); however, the doseresponse of

mRNA induced by either fimbria was no longer observed

at 24 h. Interestingly, we observed that unlike infection

with live bacteria, IL-1 was not produced following stim-

ulation of HAEC with either type of fimbria (data not

shown). These results indicate that purified native minor

fimbria of P. gingivalis is as capable as the major fimbria

in stimulating chemokine secretion by HAEC. Additionally,

the lack of detectable IL-1 expression in response to

either purified antigen suggests a complex, and potentially

interactive role for major and minor fimbria in mediating

the stimulation of HAEC by live P. gingivalis.

Expression of adhesion molecules following stimulation

of HAEC with purified native fimbria

After observing similar chemokine responses by endothe-

lial cells incubated with live bacteria or purified fimbrial

antigen, we next assessed CAM expression of HAECcultured with fimbria by RT-PCR and FACS respectively

(Fig. 6A and C). As previously reported with other endot-

helial cells (McEver et al., 1989; Khlgatian et al., 2002),

we observed that P-selectin gene expression in HAEC

was constitutive and that differences between samples

were not observed at 1 and 6 h time points. Interestingly,

a slight decrease in P-selectin mRNA expression, relative

to unstimulated HAEC control (185 RDU) was observed

with the highest concentrations of major and minor fimbria

stimulation after 24 h (121 and 134 RDU respectively). It

should be noted that a similar decrease in 24 h mRNA

expression of P-selectin was also observed with liveP. gingivalis (Fig. 4A). P-selectin is one of the first cell

surface molecules to be expressed on endothelial cells in

response to inflammatory stimuli and triggers initial neu-

trophil rolling along the vascular endothelium (Jones et al.,

1993; Mayadas et al., 1993; Smith, 1993; Nolte et al.,

1994; Kanwar et al., 1997; Burns et al., 1999; Akgur et al.,

2000; Takano et al., 2002). Its expression on the luminal

surface of the endothelial cell has been shown to peak

within minutes following activation by circulating mediators

(Akgur et al., 2000). This rapid surface expression reflects

the release of preformed P-selectin from Weibel-Palade

(WP) bodies located inside the cell membrane (Weibel

and Palade, 1964; Bonfanti et al., 1989; Hattori et al.,

1989a,b; McEver et al., 1989; Burns et al., 1999). How-

ever, the mechanisms by which P-gingivalistriggers this

rapid P-selectin expression remains to be elucidated. In

Fig. 4B, it is clear that P-selectin is expressed in response

to invasive strains 381 and 381MF1 which possess the

major fimbria. As purified major fimbria failed to induce

surface expression of P-selectin (Fig. 6C), it appears that

strains 381 and 381MF1 activate P-selectin expression in

a manner that is independent of the major fimbria and that

this expression may instead be dependent upon invasion.

Following a 6 h stimulation of HAEC with major or minor

fimbria, we observed increased transcription of ICAM-1,

VCAM-1 and E-selectin in a dose-dependent manner.

Analysis of cell surface expression of these adhesion mol-

ecules by FACS demonstrated a similar increase for ICAM-

1, VCAM-1 and E-selectin for the same 6 h period. Fol-

lowing 24 h incubation with major or minor fimbria, surface

expression of VCAM-1 had returned to preincubation levels

while surface expression of ICAM-1 remained at signifi-

cantly elevated levels (P< 0.001) (Fig. 6C). These results

indicate that both the P. gingivalis major and the minor

fimbria are capable of inducing CAM expression on HAEC.

Stimulation of HAEC with purified native fimbria induces

chemokine production through regulation of actin

cytoskeleton dynamics

Previous studies have reported that fimbria-mediated

adhesin of P. gingivalis to gingival epithelial cells inducesthe formation of integrin-associated focal adhesions with

subsequent remodelling of the actin cytoskeleton (Yilmaz

et al., 2002; 2003). Reports by Okada et al. (1998) sug-

gested that a mechanical breach of the vascular cell wall

potentially promotes atherogenesis. This study demon-

strated that cyclic stretch in human endothelial cells

resulted in alteration of actin cytoskeletal integrity and

resultant enhancement of MCP-1 and IL-8 secretion. To

determine whether rearrangement of the actin cytoskele-

ton plays a role in fimbria-mediated induction of chemok-

ines by HAEC, endothelial cells were treated with 1 g ml

1

of cytochalasin D for 30 min prior to incubation withpurified major or minor fimbria. We observed that incuba-

tion with purified native major or minor fimbria, cultured

with cytochalasin D treatment, significantly inhibited (P

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