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TLR2 Transmodulates Monocyte Adhesion and Transmigration via Rac1- and PI3K-Mediated Inside-Out Signaling in Response to Porphyromonas gingivalis This information is current as Fimbriae of September 25, 2021. Evlambia Harokopakis, Mohamad H. Albzreh, Michael H. Martin and George Hajishengallis J Immunol 2006; 176:7645-7656; ; doi: 10.4049/jimmunol.176.12.7645 Downloaded from http://www.jimmunol.org/content/176/12/7645

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

TLR2 Transmodulates Monocyte Adhesion and Transmigration via Rac1- and PI3K-Mediated Inside-Out Signaling in Response to Porphyromonas gingivalis Fimbriae

Evlambia Harokopakis,* Mohamad H. Albzreh,‡ Michael H. Martin,*† and George Hajishengallis2*†

We present evidence for a novel TLR2 function in transmodulating the adhesive activities of human monocytes in response to the fimbriae of Porphyromonas gingivalis, a pathogen implicated in chronic periodontitis and atherosclerosis. Monocyte recruitment into the subendothelium is a crucial step in atherosclerosis, and we investigated the role of P. gingivalis fimbriae in stimulating monocyte adhesion to endothelial cells and transendothelial migration. Fimbriae induced CD11b/CD18-dependent adhesion of human monocytes or mouse macrophages to endothelial receptor ICAM-1; these activities were inhibited by TLR2 blockade or Downloaded from deficiency or by pharmacological inhibitors of PI3K. Moreover, this inducible adhesive activity was sensitive to the action of Clostridium difficile toxin B, but was not affected by Clostridium botulinum C3 exoenzyme, pertussis toxin, or cholera toxin. Accordingly, we subsequently showed through the use of dominant negative signaling mutants of small GTPases, that Rac1 mediates the ability of fimbria-stimulated monocytes to bind ICAM-1. A dominant negative mutant of Rac1 also inhibited the lipid kinase activity of PI3K suggesting that Rac1 acts upstream of PI3K in this proadhesive pathway. Furthermore, fimbriae stimulated monocyte adhesion to HUVEC and transmigration across HUVEC monolayers; both activities required TLR2 and Rac1 signaling http://www.jimmunol.org/ and were dependent upon ICAM-1 and the high-affinity state of CD11b/CD18. P. gingivalis-stimulated monocytes displayed enhanced transendothelial migration compared with monocytes stimulated with nonfimbriated isogenic mutants. Thus, P. gingivalis fimbriae activate a novel proadhesive pathway in human monocytes, involving TLR2, Rac1, PI3K, and CD11b/CD18, which may constitute a mechanistic basis linking P. gingivalis to inflammatory atherosclerotic processes. The Journal of Immunology, 2006, 176: 7645–7656.

␤ he 2 integrin heterodimer CD11b/CD18 (Mac-1, CR3) is (1, 10). Inside-out signaling pathways for CD11b/CD18 activation a multifunctional receptor with significant and diverse can be induced upon stimulation of other surface receptors, such as by guest on September 25, 2021 T roles in immunity and inflammation (1, 2). The functional chemotactic receptors (1, 10) or TLRs (11, 12). versatility of this integrin is attributed, at least partly, to its ability The potential of CD11b/CD18 for vascular cell interactions by to recognize multiple and structurally unrelated molecules, includ- binding to ICAM-1 or to endothelial-associated matrix proteins, ing its endothelial counter-receptor ICAM-1, fibrinogen, iC3b, such as fibrinogen, may contribute to cardiovascular inflammation Factor X, and platelet glycoprotein Ib␣ (3–6). Abundantly ex- (5, 13). In this context, the adhesion of bloodborne leukocytes to pressed by neutrophils and monocytes, CD11b/CD18 plays a role the arterial endothelium, followed by their migration into the sub- in their migration to sites of extravascular inflammation (3, 7–9). endothelial area is a hallmark of early atherogenesis (14). The The interactions of CD11b/CD18 with fibrinogen and ICAM-1 transmigratory process is mediated by interacting sets of cell ad- mediate adhesion of neutrophils or monocytes to sites of fibrino- hesion molecules, including the CD11b/CD18-ICAM-1 pair, gen deposition and the endothelium, respectively (3, 9). These ad- which has been experimentally implicated in atherosclerosis and hesive interactions are tightly regulated. Although the default con- other inflammatory conditions (13, 15–17). It is thought that information of CD11b/CD18 in resting cells is of low affinity, a rapid fectious agents contribute to vascular inflammation and certain and transient shift to a high-affinity binding state (referred to as bacterial pathogens such as Chlamydia pneumoniae, Helicobacter CD11b/CD18 activation) can be triggered by inside-out signaling pylori, and Porphyromonas gingivalis have been implicated as ac- cessory factors in the development or acceleration of atherosclerosis (14, 18, 19). In this regard, infection-driven chronic inflam- † *Center for Oral Health and Systemic Disease, Department of Periodontics/End- matory diseases, including periodontitis, are associated with odondics and Department of Microbiology/Immunology, University of Louisville Health Sciences Center, Louisville, KY 40292; and ‡Center of Excellence in Oral and increased risk for cardiovascular disease (20–22). Craniofacial Biology, Louisiana State University Health Sciences Center, New Or- P. gingivalis is a Gram-negative oral bacterium that is strongly leans, LA 70119 associated with chronic periodontitis (23). This pathogen may dis- Received for publication January 26, 2006. Accepted for publication April 6, 2006. seminate from periodontal lesions into the systemic circulation and The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance P. gingivalis-specific DNA has been detected in human athero- with 18 U.S.C. Section 1734 solely to indicate this fact. sclerotic plaques (24). Studies in animal models of periodontitis or 1 This work was supported by U.S. Public Health Service Grant DE015254 from the atherosclerosis have established the P. gingivalis fimbriae (fila- National Institutes of Health. mentous appendages on the cell surface) as a major virulence fac- 2 Address correspondence and reprint requests to Dr. George Hajishengallis, Center tor of this pathogen (25, 26). Although both wild-type P. gingivalis for Oral Health and Systemic Disease, University of Louisville Health Sciences Cen- ter, 501 South Preston Street, Room 206, Louisville, KY 40292. E-mail address: and an isogenic non-fimbriated mutant are detected in the blood [email protected] and aortic arch tissue of orally infected hyperlipidemic mice, only

the presence of wild-type P. gingivalis is associated with periodon- mosphere in PromoCell Endothelial Cell Growth medium (2% FCS, 0.1 tal disease and increased atherosclerotic plaque formation (26). ng/ml epidermal growth factor, 1.0 ng/ml basic fibroblast growth factor, ␮ In this study, we have identified a plausible inflammatory mech- 1.0 g/ml hydrocortisone, 0.4% endothelial cell growth supplement/hep- arin, 50 ␮g/ml gentamicin, 50 ng/ml amphotericin B), according to the anism whereby P. gingivalis fimbriae may contribute to the ath- supplier’s recommendations. Thioglycolate-elicited macrophages were iso- erosclerotic process. Based on earlier findings that P. gingivalis lated from the peritoneal cavity of mice deficient in CD14, TLR2, TLR4, fimbriae bind CD14 and stimulate TLR2/PI3K-mediated inside- both TLR2 and TLR4, or from wild-type control mice, as previously de- out signaling for CD11b/CD18 activation (11, 27), we now show scribed (27, 32). The mice deficient in CD14, TLR2, or TLR4 were of C57BL/6 genetic background, whereas mice harboring homozygous TLR2 that activation of this pathway leads to increased monocyte adhe- and TLR4 mutations were 9-fold backcrossed toward the C3H genetic sion to fibrinogen, ICAM-1, and endothelial cells. This inducible background (kindly donated by Dr. C. Kirschning, Technical University of proadhesive pathway is distinct from other CD11b/CD18 activa- Munich, Munich, Germany). Mouse macrophages were cultured in com- tion pathways stimulated by FMLP or PMA, as shown by differ- plete RPMI as described. The use of animals was reviewed and approved ential toxin sensitivity. On the basis of toxin sensitivity data and by the Institutional Animal Care and Use Committee. Human or mouse cell 3 viability was monitored using the CellTiter-Blue Cell Viability assay kit additional experiments using dominant negative (DN) signaling (Promega). The use of fimbriae and other agonists as well as treatments mutants of small GTPases, we found that Rac1 acts upstream of with blocking mAbs or other antagonists did not affect cell viability as PI3K and is essential for the ability of fimbria-stimulated mono- compared with medium-only control treatments. cytes to bind CD11b/CD18 ligands, endothelial cells, and transmigrate across endothelial monolayers. The property of P. gingi- THP-1/CD14 cell transfections valis fimbriae to induce CD11b/CD18-dependent adhesive Transfections of THP-1/CD14 cells were performed using the FuGene 6 interactions may contribute to the role of monocytes in the process transfection reagent (Roche Applied Science) at a reagent to DNA ratio of Downloaded from of atherosclerosis or other inflammatory conditions. 3:1, according to the manufacturer’s instructions. Plasmids expressing DN versions of the human Rac1, Cdc42, and RhoA genes (Rac1/T17N, Cdc42/ T17N, and RhoA/T19N, respectively) as well as the empty control vector Materials and Methods pUSEampϩ were obtained from Upstate Biotechnology. A plasmid ex- Reagents pressing a DN mutant of human TLR2 (pZERO-hTLR2tirless) and empty mAbs to human TLR2 (clone TL2.1), TLR4 (HTA125), CD11b vector control (pZERO-mcs) were obtained from InVivogen. The cells (CBRM1/5, FITC-labeled), MHC class I (W6/32), and Ig isotype controls were used in functional assays 48 h posttransfection. Transient transfection http://www.jimmunol.org/ (IgG1, IgG2a) were purchased from eBioscience. FITC-labeled mAb to efficiency was 40–45% as determined by GFP reporter plasmid transfec- human CD11b (Bear-1) and mAbs to human CD14 (MEM-18), mouse tion and fluorescence microscopy to detect cells presenting green CD11b (M1/70), and its IgG2b isotype control were from Caltag Labora- fluorescence. tories. mAb to human CD11b (2LPM19c) was from DakoCytomation. mAb to ICAM-1 (BBIG-I1) and human rICAM-1 plus rIL-1␤ were from Monocyte adhesion to immobilized ligands or endothelial cells R&D Systems. FMLP, PMA, wortmannin, LY294002, LY30351, GF109203X, and cell culture-grade BSA were purchased from Sigma- The 96-well microtiter plates were coated with 10 ␮g/ml human fibrinogen Aldrich. Human fibrinogen (depleted of plasminogen, von Willebrand fac- or rICAM-1. Following overnight incubation at 4°C, remaining uncoated tor, and fibronectin) was obtained from Enzyme Research Laboratories. sites on the plates were blocked with 5 mg/ml BSA for1hatroom tem- ␤ XVA143, a 2 integrin allosteric antagonist (28), was generously provided perature. Monocytes were labeled with the fluorescent dye calcein AM (2.5 by guest on September 25, 2021 by Dr. N. Fotouhi (Roche, Nutley, NJ). P. gingivalis was grown anaero- ␮M; Molecular Probes) for 30 min, washed, and resuspended in assay ϩ ϩ bically at 37°C in brain-heart infusion broth supplemented with hemin (5 buffer (HBSS, 10 mM HEPES (pH 7.4), 1 mM Mg2 , and 0.42 mM Ca2 ). ␮g/ml) and menadione (l ␮g/ml). P. gingivalis strains used included wild- Labeled cells were added to the ligand-coated wells (5 ϫ 104 cells per well) type strains 381 and A7436 (donated by Dr. H. Kuramitsu, University of at 37°C in the absence or presence of 30-min stimulation with native fim- Ϫ Buffalo, Buffalo, NY) and their respective isogenic fimbria-deficient mu- briae or rFimA (1 ␮g/ml) or positive control agonists (FMLP, 10 7 M; tants (25, 29). Fimbriae were purified from P. gingivalis strain 381 as PMA, 0.1 ␮g/ml). In certain experiments, the cells were pretreated for 30 previously described (11). Recombinant fimbrillin (rFimA) was purified min with blocking mAbs or pharmacological inhibitors before stimulation. from Escherichia coli BL21 (DE3) transformed with the fimA gene of P. At the end of the 30-min binding time, nonadherent cells were removed by gingivalis 381 as previously described (30) with an additional step involv- careful washing repeated four times. Cell adhesion was quantified using a ing chromatography through agarose-immobilized polymyxin B to remove fluorescence microplate reader (FL600, Bio-Tek Instruments) with excita- residual endotoxin. The final fimbrial preparations were free of any con- tion/emission wavelength settings of 485/530 nm, and was expressed as a taminating substances on silver-stained SDS-PAGE and tested negative for percentage of total cells added using formula (bound fluorescence/total endotoxin (Ͻ6 EU/mg of protein) according to a quantitative Limulus ame- fluorescence added ϫ 100). HUVEC (between passages 2 and 5) were bocyte lysate assay (BioWhittaker). seeded on 96-well plates at 5 ϫ 104 cells/well and cultured for 2 days to form a confluent monolayer. Calcein AM-labeled monocytes were added at Cell culture 2 ϫ 105 cells per well of confluent HUVEC monolayer and incubated for 30 min at 37°C and 5% CO2 atmosphere. Nonadherent monocytes were Monocytes were purified from the peripheral blood of healthy human vol- gently washed off and monocyte adhesion was assessed as described using unteers as previously described (11). Briefly, monocytes were separated a Bio-Tek fluorescence microplate reader. Wells containing only HUVEC from lymphocytes upon centrifugation of peripheral blood over NycoPrep were used to determine background fluorescence, which was minimal and 1.068 (Axis-Shield). Incidental nonmonocytes were removed by magnetic subtracted from each experimental value. depletion using a mixture of biotin-conjugated mAbs and magnetic mi- crobeads coupled to anti-biotin mAb (Monocyte isolation kit II; Miltenyi

Biotec). Purified monocytes were cultured at 37°C and 5% CO2 atmo- Transmigration assay sphere, in RPMI 1640 (Invitrogen Life Technologies) supplemented with To assess monocyte transmigration across a HUVEC monolayer, we used 10% heat-inactivated FBS, 2 mM L-glutamine, 10 mM HEPES, 100 U/ml ␮ penicillin G, 100 ␮g/ml streptomycin, and 0.05 mM 2-ME (complete the Transwell plate system (6.5-mm Transwell inserts with 8.0- m pores; RPMI). Human blood collections were conducted in compliance with es- Corning Costar) and a modification of a previously described protocol (33). ϫ 4 tablished guidelines approved by the institutional review board. THP-1 cell Briefly, HUVEC (5 10 ) were seeded in the upper chamber of each lines stably transfected with human CD14 (THP-1/CD14) or with empty transwell and grown for 2 days to form a confluent monolayer. Confluency vector (THP-1/RSV) (31) were cultured in complete RPMI. Both cell lines was confirmed by microscopic inspection. Calcein AM-labeled primary ϫ 5 were provided by Dr. P. S. Tobias (The Scripps Research Institute, La monocytes or THP-1 cells were added to the upper chamber (2 10 cells) Jolla, CA). HUVEC (PromoCell) were cultured at 37°C and 5% CO at- and were allowed to migrate through the HUVEC monolayer into the lower 2 chamber at 37°C for 3 or 4 h, respectively. Subsequently, the upper chamber was removed to stop transmigration. The fluorescence intensity in the 3 Abbreviations used in this paper: DN, dominant negative; PRR, pattern-recognition lower compartment was measured using a Bio-Tek fluorescence microplate receptor; PTx, pertussis toxin; CTx, cholera toxin; CdTxB, C. difficile toxin B; PIP3, reader and was expressed as a percentage of total cell-associated fluores- phosphatidylinositol-3,4,5-trisphosphate; PIP2, phosphatidylinositol 4,5-bisphosphate. cence added in the upper compartment (percentage of transmigrated cells). The Journal of Immunology 7647

PI3K activation assay Results PI3K activity was measured as enzymatic production of phosphatidylino- P. gingivalis fimbriae induce CD11b/CD18-dependent cell sitol-3,4,5-trisphosphate (PIP3) from phosphatidylinositol 4,5-bisphosphate adhesion to immobilized ICAM-1 or fibrinogen via TLR2- (PIP2) substrate by means of a PI3K ELISA kit (Echelon Biosciences) as mediated inside-out signaling previously described (27). Briefly, PI3K was immunoprecipitated from cell lysates using anti-PI3K Ab and protein A-agarose beads, and the bead- We have previously shown that P. gingivalis fimbriae induce an activation-specific neoepitope (CBRM1/5) on CD11b, via a novel bound enzyme was subsequently incubated with PIP2 substrate in kinase reaction buffer for2hatroom temperature. The generation of PIP3 product inside-out signaling pathway involving CD14, TLR2, and PI3K was determined by competitive ELISA. (11). We now investigated the functional significance of this signaling pathway. First, we examined the ability of fimbria-stimulated monocytes to bind well-characterized ligands, such as CD11b/CD18 activation assay ICAM-1 and fibrinogen, which are bound by CD11b/CD18 only The CBRM1/5 epitope induction assay was used to monitor the activation when this integrin is activated (6). Upon 30-min stimulation at state of CD11b/CD18, as we have previously described (11). The assay is 37°C with 1 ␮g/ml native fimbriae or rFimA, human monocytes based on the property of the CBRM1/5 mAb to detect a conformational bound efficiently to ICAM-1- or fibrinogen-coated microtiter change on CD11b that signifies the high-affinity binding state of CD11b/ wells, in contrast to medium-only-treated monocytes that bound CD18 (34). poorly (6–16% of the binding activity of stimulated cells; Fig. 1, A and B). When monocytes were pretreated with anti-CD11b mAb (2LPM19c, 10 ␮g/ml) or with an allosteric antagonist of CD11b/ Statistical analysis Downloaded from CD18 (XVA143, 1 ␮M) before stimulation with fimbriae, their Data were evaluated by ANOVA and the Dunnett multiple-comparison test ability to bind immobilized ICAM-1 (Fig. 1A) or fibrinogen (Fig. using the InStat program (GraphPad). Where appropriate (comparison of Ͻ two groups only), two-tailed t tests were also performed. Statistical differ- 1B) was significantly ( p 0.05) diminished. Pretreatment with ences were considered significant at the level of p Ͻ 0.05. Experiments IgG1 isotype control or a mAb to an unrelated surface Ag (MHC were performed using triplicate samples and were performed twice or more class I) had no effect in this regard (Fig. 1, A and B). These data to verify the results. indicate that P. gingivalis fimbriae (in native or recombinant form) http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 1. P. gingivalis fimbriae stimulate CD11b/CD18-dependent monocyte adhesion via TLR2 inside-out signaling. Fluorescently labeled human monocytes were added to 96-well plates coated with ICAM-1 (A, C, and E) or fibrinogen (B, D, and F). The cells were allowed to bind for 30 min at 37°C in the absence or presence of stimulation with 1 ␮g/ml P. gingivalis native fimbriae or rFimA, or additionally with 0.1 ␮g/ml PMA (E and F). Before the assays, monocytes were pretreated for 30 min with the indicated inhibitors or control molecules. All mAbs and isotype controls were used at 10 ␮g/ml; XVA143 (allosteric antagonist of CD11b/CD18) at 1 ␮M; wortmannin (WTM) at 50 nM; LY294002 and LY30351 (inactive analog) at 20 ␮M; and GF109203X at 10 ␮M. After washing to remove nonadherent monocytes, cell adhesion was measured on a microplate fluorescence reader and was expressed as a percentage of total cells added. Results are presented as the mean Ϯ SD of triplicate determinations, from one of two independent sets of .p Ͻ 0.05) inhibition of cell adhesion due to various treatments is indicated ,ء) experiments that yielded similar findings. Statistically significant 7648 TRANSENDOTHELIAL MIGRATION DEPENDENCE ON TLR2, Rac1, PI3K stimulate monocyte adhesion to immobilized ICAM-1 or fibrino- mouse CD11b or with XVA143 (Fig. 2, C and D). The data from gen in a CD11b/CD18-dependent way. Figs. 1 and 2 collectively indicate that fimbriae interact with CD14 However, the data described do not necessarily show that the and TLR2 leading to activation of PI3K-mediated intracellular sig- observed fimbria-induced monocyte adhesion is mediated via the naling for enhanced CD11b/CD18-dependent cell adhesion. CD14-TLR2-PI3K inside-out signaling pathway (11). We thus pretreated monocytes with mAbs to CD14 or TLR2, and deter- Toxin sensitivity of fimbria-stimulated monocyte adhesion mined their ability to bind immobilized ICAM-1 or fibrinogen The ability of P. gingivalis fimbriae to induce the CD11b activa- upon activation with native fimbriae or rFimA. In contrast to iso- tion-specific CBRM1/5 neoepitope is not affected by the G protein type controls or mAbs with irrelevant specificities (TLR4 or MHC inhibitor, pertussis toxin (PTx), in contrast with PTx-sensitive in- class I), anti-CD14 or anti-TLR2 significantly inhibited adhesion duction of CBRM1/5 by FMLP (11). Similarly, PTx had no effect ( p Ͻ 0.05; Fig. 1, C and D). The effect of combined anti-TLR2 and on the ability of fimbriae to stimulate monocyte adhesion to im- anti-TLR4 treatment was not significantly different from the use of mobilized ICAM-1 or fibrinogen, although the same toxin signif- anti-TLR2 alone (Fig. 1, C and D). We next determined the effect icantly inhibited ( p Ͻ 0.05) FMLP-stimulated cell adhesion (Figs. of PI3K inhibitors on fimbria-stimulated monocyte adhesion. Spe- 3A and 4A). To further characterize the fimbria-induced proadhe- cifically, pretreatment with wortmannin or LY294002 (but not sive pathway, we have investigated three additional toxins that with its inactive analog, LY303511) resulted in significantly ( p Ͻ inhibit integrin activation by interfering with activation signals, 0.05) reduced cell adhesion to immobilized ICAM-1 or fibrinogen namely cholera toxin (CTx), Clostridium difficile toxin B (CdTxB), (Fig. 1, E and F, respectively). The inhibitory action of wortman- and Clostridium botulinum C3 transferase exoenzyme (C3 exoen- nin or LY294002 could not be attributed to nonspecific toxic ef- zyme) (35–37). Following pretreatment with various doses of the Downloaded from fects, because both compounds had no influence on PMA-stimu- toxins, human monocytes were allowed to bind immobilized lated cell adhesion, which was however inhibitable by ICAM-1 (Fig. 3) or fibrinogen (Fig. 4) for 30 min at 37°C in the GF109203X, a PKC inhibitor (Fig. 1, E and F). absence or presence of stimulation with P. gingivalis native fim- The involvement of CD14 and TLR2, but not of TLR4, in P. briae, FMLP, or PMA. CTx did not significantly influence mono- gingivalis fimbria-induced cell adhesion was conclusively shown cyte adhesion to ICAM-1 (Fig. 3B) or fibrinogen (Fig. 4B) regard-

using pattern-recognition receptor (PRR) knockout mouse macro- less of the agonists used for cell stimulation. However, there was http://www.jimmunol.org/ phages. We found that wild-type or TLR4-deficient macrophages a statistically significant linear trend ( p Ͻ 0.05) regarding the ef- could readily bind to immobilized ICAM-1 or fibrinogen upon fect of CTx on cell adhesion induced by fimbriae or FMLP, but not stimulation with native fimbriae or rFimA (Fig. 2, A and B). In with PMA. Specifically, CTx appeared to have a modest enhancing stark contrast, similarly stimulated macrophages deficient in CD14 effect on fimbria-induced cell adhesion, whereas the opposite or TLR2 or macrophages with combined TLR2 and TLR4 defi- trend was observed for FMLP-induced cell adhesion (Figs. 3B ciencies failed to display enhanced binding to the same molecules and 4B). Interestingly, within the same dose range we tested, (Fig. 2, A and B). FMLP and PMA were used as controls and their CTx acts as a potent inhibitor of P. gingivalis fimbria-induced stimulatory effect on macrophage adhesion was not influenced by cytokine release (38). any PRR deficiency tested (Fig. 2, A and B). The observed cell In contrast to CTx, CdTxB had a profound inhibitory effect by guest on September 25, 2021 adhesion to ICAM-1 or fibrinogen was dependent upon CD11b/ ( p Ͻ 0.05) on both fimbria- and FMLP-stimulated monocyte ad- CD18 as shown by the inhibitory effect of treatments with anti- hesion to immobilized ICAM-1 or fibrinogen (Figs. 3C or 4C,

FIGURE 2. Adhesion of P. gingivalis fimbria-stimulated mouse macrophages to ICAM-1 or fibrinogen is inhibited by CD14 or TLR2 deficiency. Fluorescently labeled macrophages from wild-type mice or mice deficient in CD14, TLR2, TLR4, or both TLR2 and TLR4 (TLR2/4) were added to 96-well plates coated with ICAM-1 (A and C) or fibrinogen (B and D). The cells were allowed to bind for 30 min at 37°C in the absence or presence of stimulation with P. gingivalis native fimbriae or rFimA (both at 1 ␮g/ml), or with control agonists (FMLP, 10Ϫ7 M; PMA, 0.1 ␮g/ml). Before the assays (C and D), wild-type macrophages were pretreated for 30 min with 10 ␮g/ml IgG2b isotype control or anti-CD11b, or with 1 ␮M XVA143. Cell adhesion was assessed fluorometrically as outlined in the legend to Fig. 1. Data are presented as the mean Ϯ SD (n ϭ 3) from a typical set of experiments that were repeated p Ͻ 0.05) due to receptor deficiency (A and B) or due to CD11b/CD18 blockade ,ء) yielding similar results. Significantly reduced macrophage adhesion (C and D) is shown. The Journal of Immunology 7649 Downloaded from http://www.jimmunol.org/

FIGURE 3. Toxin sensitivity of stimulated monocyte adhesion to ICAM-1. Fluorescently labeled human monocytes were added to 96-well plates coated by guest on September 25, 2021 with ICAM-1. The cells were allowed to bind for 30 min at 37°C in the absence or presence of stimulation with 1 ␮g/ml P. gingivalis fimbriae, 10Ϫ7 M FMLP, or 0.1 ␮g/ml PMA. Before the assay, monocytes were pretreated with PTx for2h(A);CTxfor2h(B); CdTxB for2h(C); and C3 exoenzyme for 24 h (D) at the indicated doses. After washing to remove nonadherent monocytes, cell adhesion was assessed fluorometrically and was expressed as a percentage of total cells added. Results are presented as the mean Ϯ SD of triplicate determinations, from one of two independent sets of experiments .p Ͻ 0.05) inhibition of cell adhesion due to toxin treatment is indicated ,ء) that yielded similar findings. Statistically significant respectively). However, suppression of PMA-induced cell adhe- human monocytic THP-1/CD14 cells to bind immobilized sion by CdTxB did not reach statistical significance, despite a sig- ICAM-1 or fibrinogen, upon cell transfection with empty vector nificant linear trend with increasing toxin dose (Figs. 3C and 4C). control or with DN inhibitors of Rac1, Cdc42, or RhoA. We found The C3 exoenzyme had no effect, whatsoever, on fimbria-stimu- that THP-1/CD14 cells transfected with Rac1-DN, but not with lated cell adhesion, although the same toxin significantly inhibited Cdc42-DN or RhoA-DN, displayed significantly reduced adhesion ( p Ͻ 0.05) the activity of FMLP and PMA (Figs. 3D and 4D). The to ICAM-1 in response to fimbriae ( p Ͻ 0.05, compared with toxins did not influence basal cell adhesion, which was Յ8% of empty vector-transfected cells) (Fig. 5A). Similar results were that seen in the presence of agonists (data not shown). These data obtained when adhesion was tested on fibrinogen-coated plates suggest that the signaling pathway involved in P. gingivalis fim- (data not shown). In a parallel experiment using similarly trans- bria-stimulated monocyte adhesion to a ligand (fibrinogen) or a fected but FMLP-stimulated THP-1/CD14 cells, cell adhesion to counterreceptor (ICAM-1) of CD11b/CD18 is distinct from those ICAM-1 was significantly inhibited ( p Ͻ 0.05) by RhoA-DN but stimulated by FMLP or PMA. The fimbria-stimulated pathway is not by Rac1-DN or Cdc42-DN (Fig. 5A). Therefore, Rac1 appears sensitive to the action of CdTxB but is not affected by PTx, CTx, to be a second signaling intermediate, in addition to PI3K, in- or C3 exoenzyme. volved in P. gingivalis fimbria-stimulated cell adhesion to ICAM-1. These data further support that P. gingivalis fimbriae and Rac1 is involved in P. gingivalis fimbria-induced cell adhesion FMLP activate distinct intracellular signaling pathways, involving and is acting upstream of PI3K Rac1 and RhoA, respectively, for CD11b/CD18 activation. CdTxB is known to inhibit the small molecular weight GTPases, Rac1 and PI3K regulate cellular function through various, often Rho, Rac, and Cdc42, whereas C3 exoenzyme specifically inhibits overlapping, signaling pathways and either of the two intracellular Rho (RhoA, B, and C), but not Rac or Cdc42 (39). Therefore, on enzymes can activate the other, depending on the specific pathway the basis of the toxin sensitivity data (Figs. 3 and 4), we hypoth- involved (40). We examined whether Rac1-DN could additionally esized that the proadhesive pathway activated by P. gingivalis inhibit fimbria-induced PI3K activation to determine whether Rac1 fimbriae involves participation of Rac or Cdc42, but not of Rho acts upstream of PI3K. Specifically, we examined whether GTPase. To test this hypothesis, we determined the ability of Rac1-DN inhibits the lipid kinase activity of PI3K, monitored 7650 TRANSENDOTHELIAL MIGRATION DEPENDENCE ON TLR2, Rac1, PI3K Downloaded from http://www.jimmunol.org/

FIGURE 4. Toxin sensitivity of stimulated monocyte adhesion to ﬁbrinogen. Fluorescently labeled human monocytes were added to 96-well plates

coated with fibrinogen. The cells were allowed to bind for 30 min at 37°C in the absence or presence of stimulation with 1 ␮g/ml P. gingivalis fimbriae, by guest on September 25, 2021 10Ϫ7 M FMLP, or 0.1 ␮g/ml PMA. Before the assay, monocytes were pretreated with PTx for2h(A);CTxfor2h(B); CdTxB for2h(C); and C3 exoenzyme for 24 h (D) at the indicated doses. Cell adhesion was assessed as shown in Fig. 3. Data are presented as the mean Ϯ SD (n ϭ 3), from one p Ͻ 0.05) inhibition of cell adhesion due to toxin treatment ,ء) of two independent sets of experiments that yielded similar results. Statistically significant is indicated.

through the generation of PIP3 from PIP2 substrate. TLR2-DN and unstimulated HUVECs (Fig. 6A). When monocytes were preincu- RhoA-DN were used as positive and negative controls, respec- bated with anti-CD11b mAb or the XVA143 allosteric antagonist tively. We found that Rac1-DN and TLR2-DN (but not RhoA-DN) of CD11b/CD18 before being exposed to fimbriae and added to the could significantly inhibit ( p Ͻ 0.05) the ability of fimbriae to HUVEC monolayer, their adhesive activity was significantly re- activate PI3K (Fig. 5B). Therefore, Rac1 appears to be a signaling duced ( p Ͻ 0.05; Fig. 6B). In contrast, an IgG1 isotype control or intermediate, acting between TLR2 and PI3K, in the P. gingivalis a mAb to an unrelated surface Ag (MHC class I) were without fimbria-stimulated pathway for CD11b/CD18-dependent cell effect in this regard (Fig. 6B). Furthermore, when unstimulated or adhesion. IL-1␤-stimulated HUVECs were preincubated with anti-ICAM-1 mAb before addition of monocytes and fimbriae, their ability to P. gingivalis fimbriae promote monocyte adhesion to HUVEC support monocyte adhesion was significantly decreased ( p Ͻ 0.05; monolayer Fig. 6C). In contrast, control treatments (isotype control or irrel- Our findings that P. gingivalis fimbriae stimulate monocyte adhe- evant mAb) had no effect (Fig. 6C). These data collectively sug- sion to ICAM-1, a major endothelial receptor, suggested that fim- gest that P. gingivalis fimbriae promote monocyte adhesion to briae may similarly up-regulate monocyte adhesion to HUVEC. To HUVEC in a CD11b/CD18- and ICAM-1-dependent way. investigate this possibility, monocytes were added with or without fimbriae (1 ␮g/ml) to a HUVEC monolayer that was previously P. gingivalis fimbriae promote monocyte transmigration through either activated (by 1 ng/ml IL-1␤ for 16 h) or maintained un- HUVEC monolayer stimulated (medium only). Unstimulated or IL-1␤-stimulated We next examined the ability of fimbria-stimulated monocytes for HUVEC were washed before addition of monocytes to remove transmigration across HUVEC monolayers. For this purpose, IL-1␤ from the coculture system. We found that the ability of monocytes were added with or without fimbriae (1 ␮g/ml) to the monocytes to adhere to the HUVEC was significantly ( p Ͻ 0.05) upper chamber of Transwells containing HUVEC monolayers, and higher in the presence of fimbriae than in the presence of medium the incubation was conducted for 3 h. We found that the ability of only, regardless of whether the HUVECs were prestimulated with monocytes for transmigration was significantly enhanced ( p Ͻ IL-1␤ (Fig. 6A). However, monocyte adhesion to IL-1␤-stimulated 0.05) in the presence of fimbriae than in the presence of medium HUVECs was significantly enhanced ( p Ͻ 0.05) compared with only (Fig. 7A). However, when monocytes were preincubated with The Journal of Immunology 7651

FIGURE 5. Rac1 is involved in P. gingivalis fimbria-induced cell adhesion to ICAM-1 (A) and stimulation of the lipid kinase activity of PI3K (B). A, THP-1/CD14 cells transfected with empty vector control or with DN mutants of Rac1, Cdc42, or RhoA (at the indicated microgram amounts of plasmid DNA per 2 ϫ 105 cells) were fluorescently labeled and added to 96- well plates coated with ICAM-1. The cells were allowed to bind for 30 min at 37°C in the absence or presence of stimulation with 1 ␮g/ml P. gingivalis fimbriae or 10Ϫ7 M FMLP. After washing to remove nonadherent monocytes, cell adhesion was assessed fluorometrically and was expressed as a percentage of total cells Downloaded from added. B, THP-1/CD14 cells transfected with DN mutants of TLR2, Rac1, or RhoA were stimulated for 30 min with 1 ␮g/ml fimbriae. Subse- quently, PI3K was immunoprecipitated from cell lysates and its enzymatic activity was assessed as http://www.jimmunol.org/ described in Materials and Methods. Data are presented as the mean Ϯ SD (n ϭ 3), from one of two (A) or three (B) independent sets of experiments that yielded similar results. Statisti- -p Ͻ 0.05) inhibi ,ء) cally significant tion of cell adhesion (A)orofPIP3 production (B) due to transfection

with DN mutants is indicated. by guest on September 25, 2021

anti-CD11b mAb or the XVA143 antagonist before being exposed activation-specific CBRM1/5 neoepitope (Fig. 8A). Moreover, the to fimbriae and added to HUVEC, their ability for transendothelial fimbria-induced transendothelial migration activity of THP-1/ migration was significantly inhibited ( p Ͻ 0.05; Fig. 7B). On the CD14 cells was significantly higher ( p Ͻ 0.05) compared with that contrary, an IgG1 isotype control or mAb with irrelevant specific- of THP-1/RSV cells (Fig. 8B), confirming that CD11b/CD18 ac- ity (MHC class I) did not affect monocyte transmigration (Fig. 7B). tivation plays an important role in the transmigration process. The Furthermore, pretreatment of HUVEC with anti-ICAM-1 mAb be- relative inability of THP-1/RSV cells to respond to fimbriae was fore addition of monocytes and fimbriae resulted in significantly re- not due to any inherent defects in this cell line. Indeed, when PMA duced ( p Ͻ 0.05) monocyte transmigration, whereas control treat- was used instead as an agonist, both cell lines could equally well ments had no effect in this regard (Fig. 7C). These data jointly suggest induce the CBRM1/5 neoepitope (Fig. 8A) and stimulate transmi- that P. gingivalis fimbriae promote transendothelial migration of gration (Fig. 8B). monocytes in a CD11b/CD18- and ICAM-1-dependent mode. Under the experimental conditions used, it was possible that Rac1 is involved in fimbria-stimulated monocytic cell adhesion fimbriae also activated the HUVEC. Indeed, the incubation time (3 to HUVEC and transendothelial migration h) appeared adequate for fimbria-induced up-regulation of Rac1, but not RhoA, is required for enhanced THP-1/CD14 adhe- ICAM-1 expression in HUVEC (41). We therefore set out to con- sion to ICAM-1 in response to P. gingivalis fimbriae (Fig. 5). We firm that fimbria-induced transendothelial migration of monocytes now determined whether Rac-1 is similarly involved in the ability was mediated, at least in part, by direct effects of fimbriae on of fimbria-stimulated THP-1/CD14 cells to adhere to HUVEC and monocytes (i.e., through induction of CD11b/CD18 activation). To transmigrate across the HUVEC monolayer, and moreover, exam- this end, we compared the transmigration activity of monocytic ined whether these activities correlate with CD11b/CD18 activa- THP-1/CD14 cells to that of CD14-nonexpressing THP-1/RSV tion. For this purpose, we used THP-1/CD14 cells transiently cells. The rationale was that fimbriae would not effectively induce transfected with Rac1-DN. Cells transfected with TLR2-DN or CD11b/CD18 activation in the latter cell line (due to diminished RhoA-DN were used as positive or negative controls, respectively. CD14 expression, required for fimbria-induced inside-out signal- We found that cells transfected with Rac1-DN or TLR2-DN dis- ing (11)), resulting in reduced transmigration activity of THP-1/ played significantly reduced ability ( p Ͻ 0.05) for CD11b/CD18 RSV cells compared with THP-1/CD14 cells. Indeed, fimbriae activation (CBRM1/5 epitope induction), adhesion to HUVEC, readily induced CD11b/CD18 activation in THP-1/CD14 cells (but and transendothelial migration in response to P. gingivalis fim- not in THP-1/RSV cells), as evidenced by induction of the CD11b briae, as compared with untransfected cells or cells transfected 7652 TRANSENDOTHELIAL MIGRATION DEPENDENCE ON TLR2, Rac1, PI3K Downloaded from http://www.jimmunol.org/

FIGURE 6. P. gingivalis fimbriae promote monocyte adhesion to HUVEC in a CD11b/CD18- and ICAM-1-dependent way. Confluent HUVEC monolayers were stimulated with IL-1␤ (1 ng/ml; 16 h) or not and by guest on September 25, 2021 washed before addition of fluorescently labeled monocytes (2 ϫ 105 per well) with or without P. gingivalis fimbriae (1 ␮g/ml). The monocytes were allowed to bind for 30 min at 37°C. B, Monocytes were preincubated for 30 min with IgG1 isotype control, anti-CD11b mAb, or anti-MHC class I (all at 10 ␮g/ml) or an allosteric antagonist of CD11b/CD18 (XVA413, 1 ␮M) before exposure to fimbriae and addition to the HUVEC monolayer. C, HUVEC were preincubated for 30 min with IgG1 isotype control, anti- ICAM-1 mAb, or anti-MHC class I mAb (all at 10 ␮g/ml) before addition of monocytes and fimbriae. After a 30-min incubation of monocytes with HUVEC, nonadherent monocytes were gently washed off and monocyte adhesion was measured fluorometrically and expressed as a percentage of total cells added. Results are presented as the mean Ϯ SD (n ϭ 3), from one of three (A) or two (B and C) independent experiments that yielded p Ͻ 0.05) differences in cell FIGURE 7. P. gingivalis fimbriae promote monocyte transmigration ,ء) similar findings. Statistically significant adhesion due to differential stimulation treatments (A) or due to the use of through HUVEC monolayers in a CD11b/CD18- and ICAM-1-dependent ϫ 5 inhibitory treatments (B and C) are indicated. way. A, Fluorescently labeled monocytes (2 10 ) were added with or without P. gingivalis fimbriae (1 ␮g/ml) to confluent HUVEC monolayers in the upper chamber of Transwells. B, Monocytes were preincubated for 30 min with IgG1 isotype control, anti-CD11b mAb, or anti-MHC class I with empty vector control (Fig. 9). Transfection with Rac1-DN or (all at 10 ␮g/ml) or an allosteric antagonist of CD11b/CD18 (XVA413, 1 TLR2-DN had no effect on surface expression of CD11b/CD18, as ␮M) before exposure to fimbriae and addition to the HUVEC monolayer. shown by staining with Bear-1, a mAb that detects CD11b regard- C, HUVECs were preincubated for 30 min with IgG1 isotype control, anti-ICAM-1 mAb, or anti-MHC class I (all at 10 ␮g/ml) before addition less of its activation state (data not shown). In contrast to THP-1/ of monocytes and fimbriae. The monocytes were allowed to migrate into CD14 cells transfected with Rac1-DN or TLR2-DN, transfection the lower chamber at 37°C for 3 h. Subsequently, the fluorescence intensity with RhoA-DN did not influence their ability for CD11b/CD18 in the lower chamber was measured and expressed as a percentage of total activation and adhesion to HUVEC (Fig. 9). However, the trans- cell-associated fluorescence added in the upper chamber (percentage of migrating activity of RhoA-DN-transfected cells was similarly af- transmigrated cells). Results are presented as the mean Ϯ SD of triplicate fected and was significantly diminished ( p Ͻ 0.05) relative to determinations from one of three (A) or two (B and C) independent exper- (p Ͻ 0.05 ,ء) untransfected or empty vector-transfected cells (Fig. 9). Therefore, iments that yielded similar findings. Statistically significant RhoA may be involved in fimbria-induced monocyte transmigra- differences in monocyte transmigration due to differential stimulation treat- tion by acting on a process that is independent of CD11b/CD18 ments (A) or due to the use of inhibitory treatments (B and C) are indicated. activation and adhesion to HUVEC. These data demonstrate that The Journal of Immunology 7653

potential threats; in contrast, these wandering cells should hold fast to sites of infection they encounter and thus need to be rapidly reprogrammable. In this context, we consider that TLRs are appropriate transmodulators of the adhesive activities of leukocytes because these PRRs can both detect infection and transduce acti- vating intracellular signals (43). Our identification of a novel, TLR2-mediated inside-out signaling pathway (11) and, most im- portantly, the current demonstration that this pathway regulates the adhesive and transmigrating activities of human monocytes clearly ␤ support this notion. CD11b/CD18 and 2 integrins in general are appropriate effectors of such proadhesive pathways due to their ability to engage, once activated, diverse ligands or counterrecep- tors implicated in the inflammatory response (1, 2, 4, 6). The significance of inside-out signaling in the regulation of leukocyte adhesion was emphasized by the discovery of an alternative form of ␤ leukocyte adhesion deficiency (44). In this type of deficiency, 1, ␤ ␤ 2, and 3 integrins are normally expressed on the cell surface but fail to be activated by intracellular signaling pathways to bind

ligands (44). Downloaded from This study showed that P. gingivalis ﬁmbriae stimulate mono- ␤ cyte adhesion to 2 integrin ligands (Fig. 1) or to endothelial cells (Fig. 6) and activate monocyte transendothelial migration (Figs. 7 and 8B) through induction of inside-out signaling. On the basis of previous (11) and current data, this proadhesive signaling pathway

involves the sequential participation of CD14, TLR2, Rac1, PI3K, http://www.jimmunol.org/ and the CD11b/CD18 (Fig. 11). Initial evidence that this mechanism of CD11b/CD18 activation involves a distinct intracellular signaling pathway from those induced by FMLP or PMA came FIGURE 8. CD11b/CD18 activation is important for transendothelial migration of THP-1 cells in response to P. gingivalis fimbriae. A, THP-1/ from data of differential toxin sensitivity (Figs. 3 and 4). The novel CD14 or CD14-nonexpressing THP-1/RSV cells were incubated with me- pathway activated by P. gingivalis fimbriae was sensitive to the dium only, fimbriae (1 ␮g/ml), or PMA (0.1 ␮g/ml). After 30 min, the cells action of CdTxB, but was not influenced by C3 exoenzyme, PTx, were assessed for induction of a CD11b/CD18 activation-specific neo- or CTx. These findings pointed to a possible participation of Rac1 epitope (CBRM1/5) by staining with FITC-labeled CBRM1/5 mAb. Cell- in this pathway because Rac1 is inhibitable by CdTxB but not by associated fluorescence was measured and expressed in relative fluores- C3 exoenzyme (39). At the same time, these data suggested that by guest on September 25, 2021 cence units (RFU). B, Fluorescently labeled THP-1/CD14 and THP-1/RSV RhoA was not a likely signaling molecule candidate in the fimbria- ␮ ␮ cells were added with or without fimbriae (1 g/ml) or PMA (0.1 g/ml) induced pathway because RhoA is inhibitable by both CdTxB and to the upper chamber of Transwells containing HUVEC monolayers, and C3 exoenzyme (39). Experiments examining the effects of DN the incubation was conducted for 4 h. The percentage of transmigrated cells signaling mutants on the ability of fimbriae to induce CD11b/ was determined as outlined in the legend to Fig. 7. Results are presented as the mean Ϯ SD (n ϭ 3) from one of two (A) or three (B) independent set CD18-dependent monocyte adhesive activities confirmed the im- of experiments that yielded similar findings. Significantly enhanced trans- portance of Rac1 and the irrelevance of RhoA, although the latter p Ͻ 0.05) compared with indicated control is shown. was important for the FMLP-induced proadhesive pathway. An ,ء) migration additional difference regarding the mechanisms whereby fimbriae and FMLP stimulate monocyte adhesion involves the lack of PI3K Rac1 is an essential component of the P. gingivalis fimbria-in- requirement in the case of FMLP. Specifically, although FMLP duced inside-out signaling pathway that leads to enhanced mono- activates PI3K in our experimental system, the use of specific cyte adhesion to HUVEC and transendothelial migration. PI3K inhibitors (wortmannin or LY294002) does not inhibit the ability of FMLP to stimulate monocyte adhesion (our unpublished Fimbriated P. gingivalis stimulate monocyte adhesion and observation). Therefore, PI3K is not a point of convergence in the transendothelial migration proadhesive pathways activated by fimbriae or FMLP. FMLP-ac- We then determined whether fimbriae could activate monocyte trans- tivated PI3K may mediate other effector functions in monocytes, endothelial migration in cell-associated form. This examination used such as induction of NF-␬B activation (45). The ability of fimbriae whole cells of P. gingivalis from wild-type strains 381 and A7436 or to stimulate monocyte transendothelial migration was inhibited not their respective isogenic non-fimbriated mutants. We found that the only by the Rac1-DN but also by the RhoA-DN mutant (Fig. 9C). wild-type strains were significantly more potent ( p Ͻ 0.05) than their Because RhoA is not involved in fimbria-induced activation of non-fimbriated mutants in stimulating monocyte adhesion to HUVEC CD11b/CD18 and monocyte adhesion to endothelial cells, this and transmigration across HUVEC monolayers (Fig. 10). These data finding appeared somewhat unexpected. However, RhoA was pre- suggest that P. gingivalis has the potential to contribute to the inflam- viously found to be essential for the retraction of the tail of the matory processes in atherosclerosis by stimulating monocyte recruit- migrating monocyte to complete diapedesis (33) and this may ac- ment into subendothelial areas. count for the inhibitory effect of the RhoA-DN mutant in our transmigration model. Discussion Rac1 and other small GTPases of the Rho family are regulated It has been speculated that inside-out signaling mechanisms by GTP/GDP exchange and function as molecular switches that evolved to resolve two competing objectives (42). On the one control signaling pathways involved in kinase regulation, gene hand, there is need for highly mobile leukocytes to roam and detect transcription, cytoskeleton organization, cell motility, and other 7654 TRANSENDOTHELIAL MIGRATION DEPENDENCE ON TLR2, Rac1, PI3K

FIGURE 9. Rac1 is involved in induction of CD11b/CD18 activation, adhesion to HUVEC, and transendothelial migration of fimbria-stimulated monocytic cells. THP-1/CD14 cells, transfected with empty vector control (EVC) or with DN mutants of TLR2, Rac1, or RhoA (at the indicated microgram 5 amounts of plasmid DNA per 2 ϫ 10 cells), were stimulated with 1 ␮g/ml P. gingivalis fimbriae and tested for induction of CD11b/CD18 activation, Downloaded from adhesion to HUVECs, and transendothelial migration. Results were normalized to the activity of untransfected THP-1/CD14 cells, and are presented as the p Ͻ 0.05) of cell ,ء) mean Ϯ SD (n ϭ 3) from one of two independent sets of experiments that yielded similar findings. Statistically significant inhibition activity due to transfection with DN mutants is indicated. cellular processes (46, 47). The property of Rac1 to function as a cate at the level of PI3K; downstream activation of Akt induces molecular on-off switch is consistent with its involvement in P. NF-␬B-dependent transcription (49), whereas stimulation of alter- http://www.jimmunol.org/ gingivalis fimbria-induced inside-out signaling pathway, which native PI3K effectors may result in activation of CD11b/CD18- needs to be rapidly and transiently activated. PI3K is also a com- dependent adhesion as seen in the present study. ponent of this signaling pathway and appears to act downstream of The ability of CD14/TLR2 to detect P. gingivalis fimbriae and Rac1 (Fig. 11). In this regard, it was shown that Rac1, but not initiate inside-out signaling for CD11b/CD18 activation is a po- RhoA, can bind PI3K and augment its activity (48). These findings tentially protective mechanism, which can contribute to monocyte are in line with our observations that a DN inhibitor of Rac1, but recruitment to sites of P. gingivalis infection. Fimbriae could stim- not of RhoA, inhibits PI3K activity (Fig. 5B). Two PI3K binding ulate this proadhesive pathway in bacterial cell-associated form or

motifs are present on the TLR2 cytoplasmic tail and PI3K is re- more effectively as free molecules shed from the bacterial cell by guest on September 25, 2021 cruited to TLR2 upon activation with heat-killed Staphylococcus surface or as components of released outer membrane vesicles that aureus (49). This pathway proceeds downstream of PI3K through can readily infiltrate tissues (50). However, the ability of P. gin- the Ser/Thr kinase Akt and results in NF-␬B activation (49). Rac1 givalis fimbria-stimulated TLR2 to transmodulate the adhesive ac- is also essential for NF-␬B activation in the S. aureus-stimulated tivity of CD11b/CD18 may also constitute a potentially harmful TLR2 pathway and it similarly acts upstream of PI3K (49). There- mechanism contributing to destructive inflammation in conditions fore, it appears that P. gingivalis fimbriae and heat-killed S. aureus associated with P. gingivalis. Evidence from biopsy studies on may both activate a TLR2-Rac1-PI3K pathway, which may bifur- human carotid endarterectomy specimens or from experimental

FIGURE 10. The ability of P. gingivalis to promote monocyte adhesion (A) and transmigration through HUVEC monolayers (B) correlates with its fimbriation state. Fluorescently labeled monocytes (2 ϫ 105) were added to confluent HUVEC monolayers and assayed for adhesion or transmigration. The monocytes were added in the presence or absence of wild-type (Wt) P. gingivalis strains 381 or A7436 or their non-fimbriated isogenic mutants (Mt) at the indicated multiplicity of infection. Results are presented as the mean Ϯ SD (n ϭ 3) from one of two independent experiments that yielded similar -p Ͻ 0.05) of monocyte adhesion or transmigration by wild-type P. gingivalis compared with corre ,ء) findings. Statistically significant enhancement sponding mutant is shown. The Journal of Immunology 7655

process is augmented when macrophages are exposed to wild-type P. gingivalis rather than a non-fimbriated mutant (59). The paral- lels between P. gingivalis and C. pneumoniae suggest that similar pathogenic mechanisms induced by different infectious agents may link chronic inflammatory conditions with the development of cardiovascular disease. However, this investigation is the first time that pathogen-induced TLR-dependent inside-out signaling has been demonstrated and suggested as a possible mechanism for infection-driven monocyte accumulation into the subendothelium. In conclusion, innate recognition of P. gingivalis fimbriae results in activation of TLR2, which transmodulates the adhesive activity of CD11b/CD18 via Rac1 and PI3K (Fig. 11). Activated CD11b/CD18 renders the monocytes capable of binding endothelial ICAM-1 and transmigrating across endothelial cells. Because monocyte recruitment into subendothelial areas plays an important role in the early steps of atherogenesis, the inducible proadhesive pathway described in this study may form a mechanistic basis FIGURE 11. P. gingivalis fimbria-activated TLR2 transmodulates the linking P. gingivalis to inflammatory atherosclerotic processes. adhesive activity of monocytes. P. gingivalis fimbriae interact with CD14 Downloaded from and TLR2 and induce Rac1- and PI3K-mediated inside-out signaling lead- Acknowledgment ing to activation of the ligand-binding capacity of CD11b/CD18. Activated We thank Dr. Sarah Gaffen (University at Buffalo, Buffalo, NY) for critical CD11b/CD18 can thereby bind endothelial ICAM-1, thus promoting review of the manuscript. monocyte-endothelial cell interactions. Disclosures The authors have no financial conflict of interest. http://www.jimmunol.org/ atherosclerosis in a mouse model suggests that P. gingivalis can localize to sites of atheroma development (24, 26). Moreover, ep- idemiological and experimental evidence suggests that periodontal References 1. Shimaoka, M., J. Takagi, and T. A. Springer. 2002. Conformational regulation of disease and P. gingivalis may be risk factors contributing to the integrin structure and function. Annu. Rev. Biophys. Biomol. Struct. 31: 485–516. pathogenesis of atherosclerosis (20–22, 26, 51, 52). Because 2. Ehlers, M. R. W. 2000. CR3: a general purpose adhesion-recognition receptor monocyte recruitment into the subendothelium is a crucial step in essential for innate immunity. Microbes Infect. 2: 289–294. 3. Carlos, T. M., and J. M. Harlan. 1994. Leukocyte-endothelial adhesion mole- atherosclerosis (14), our findings that P. gingivalis stimulates cules. Blood 84: 2068–2101. monocyte transendothelial migration suggest a plausible mecha- 4. Yakubenko, V. P., V. K. Lishko, S. C. Lam, and T. P. Ugarova. 2002. A mo-

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