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RESEARCH REPORT |
1 Department of Endodontics, School of Dentistry, Lyons Building, Rm. 441, 520 N. 12th Street, PO Box 980566, Richmond, VA 23298-0566, USA;
2 Department of Biostatistics and
3 Department of Microbiology and Immunology, School of Medicine, Virginia Commonwealth University, Richmond, VA, USA
* corresponding author, tew{at}hsc.vcu.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: streptococcus • monocytes • endothelium • tissue factor • IL-1ß
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Various virulence factors—such as exo-polysaccharide, fibronectin binding protein, and platelet aggregation association protein—have been implicated in the initial colonization of bacteria to the fibrin on the damaged cardiac valves (Herzberg, 1996; Chia et al., 2004). However, synthesis of exo-polysaccharides does not enhance infectivity by S. gordonii, as does S. mutans (Wells et al., 1993), and it even inhibits platelet binding of S. salivarius and S. mitis (Sullam et al., 1993). Moreover, there is no direct relationship between the ability of various viridans streptococci to adhere to platelet clots in vitro and their ability to cause endocarditis (Crawford and Russell, 1986). Furthermore, oral streptococci that do not possess these known virulence factors have been isolated from endocarditis lesions. The heterogeneity of oral streptococci recovered from endocarditis lesions prompted us to look for additional mechanisms to explain the initiation of the native valve endocarditis.
Endocardits studies in animals have indicated that tissue factor (TF) plays a key role in the development and maintenance of vegetations (Drake et al., 1984; Bancsi et al., 1996). TF is synthesized by monocytes and endothelial cells upon stimulation with lipopolysaccharides (LPS), inflammatory mediators, and lectins (Colucci et al., 1983; Furie and Furie, 1996), and it initiates the extrinsic and intrinsic clotting pathways (Edgington et al., 1991). TF from endothelial cells plays an important role early in vegetation formation (Drake et al., 1984), and TF from monocytes is important in perpetuating these lesions (Bancsi et al., 1996).
A close relationship between coagulation and pro-inflammatory cytokines, such as IL-1 and tumor necrosis factor-alpha (TNF-
), has been well-established (Nawroth et al., 1986a; Napoleone et al., 1997). Oral streptococci as a group can stimulate peripheral blood mononuclear monocytes (PBMC) to produce extraordinary amounts of pro-inflammatory cytokines quickly (Takada et al., 1993; Kjeldsen et al., 1995), prompting the hypothesis that rapid pro-inflammatory cytokine production during streptococcal bacteremia could up-regulate TF and contribute to the initial clot formation on activated endothelial cells.
Pro-inflammatory cytokines promote the expression of adhesion molecules such as E-selectin and intercellular adhesion molecule 1 (ICAM-1) on endothelial cells. E-selectin facilitates the initial tethering of leukocytes to endothelial cells, and up-regulates monocytic TF expression (Lo et al., 1995). ICAM-1 not only binds to integrins to affirm leukocyte adhesion, but also promotes clot formation and fibrin deposition at the vascular wall (Becker et al., 2000; Arefieva and Krasnikova, 2001). Thus, the ability of activated endothelial cells to recruit monocytes further amplifies coagulation (Collins et al., 1995). Moreover, we recently reported that streptococcus-infected monocytes differentiate into short-lived dendritic cells, which are known to express a high titer of TF (Broussas et al., 2000; Hahn et al., 2005). Thus, up-regulation of adhesion molecules on activated endothelial cells could facilitate the adherence of infected monocytes converting to dendritic cells, as well as deposition of fibrinogen that facilitates vegetation formation.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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PBMC and Monocyte-depleted Preparations
Venous blood from healthy donors was drawn after informed consent, according to a protocol approved by the Institutional Review Board. PBMC were prepared as previously described (Hahn et al., 2005), and the viable cell number was determined by trypan blue exclusion. For monocyte-depleted PBMC cultures (Mo-depleted), CD14-reactive microbeads were used according to the manufacturer’s instructions (Miltenyi Biotec, Auburn, CA, USA). PBMC or Mo-depleted cultures (106/mL) were challenged with live bacteria (107/mL) in enriched RPMI 1640 (0.01 M Hepes, Invitrogen, Carlsbad, CA) with 10% fetal calf serum (Hyclone, Logan, UT) for 4 hrs before the supernatant fluids were harvested. Supernatant fluids from Salmonella typhimurium LPS (Sigma, St. Louis, MO, USA) stimulated PBMC were used as the positive control. PBMCs alone served as negative controls. Supernatant fluids were stored at –20°C until the TF assay.
Human Umbilical Vein Endothelial Cell Preparation
Confluent human umbilical vein endothelial cells (HUVEC) were grown in 24-well plates with Medium 200 supplemented with low serum and antibiotics, according to the manufacturer’s instructions (Cascade Biologics, Seattle, WA, USA). One day before experimentation, HUVEC were washed twice to remove antibiotics, and serum-supplemented Medium 200 (without antibiotics) was used in the experiments.
Transwell Studies
Freshly prepared PBMCs (106) were added to transwell inserts (0.4 µ, Costar, Corning Inc., Corning, NY, USA) and stimulated with live oral streptococci (107) in 200 µL enriched RPMI without antibiotics. The bottom wells contained confluent HUVEC cells in supplemented Medium 200 without antibiotics (500 µL/well), and induction of endothelial TF activity (FXa assay) was measured after 6 hrs. Transwell inserts containing PBMC without bacteria and HUVEC directly stimulated with PBMC or bacteria (without inserts) were included as negative controls.
Neutralizing Antibodies
Neutralizing antibodies were added to endothelial cell wells 30 min before the addition of 10-fold-diluted supernatant fluids from PBMC cultures. Anti-IL-1 (10 µg/mL, BD Pharmingen, San Diego, CA, USA), anti-IL-1ß (10 µg/mL, BD Pharmingen), anti-IL-1ra (20 µg/mL, R & D Systems, Minneapolis, MN, USA), or isotype IgG1 antibody for rIL-1 and rIL-1ß (10 µg/mL, eBioscience, San Diego, CA, USA) and rabbit anti-TNF- (20 µg/mL, Biosource, Camarillo, CA, USA) were used.
Endothelial Surface TF Activity Assay
Endothelial cell surface TF activity of confluent HUVEC was measured in triplicate with a modified factor Xa assay, described by Veltrop et al.(1999). After 6 hrs of co-culture with stimulant, HUVEC were washed with warm PBS twice and incubated with 150 µL of buffer containing 0.125 pmole of purified factor VII (Calbiochem, San Diego, CA, USA) and 0.125 nmole of CaCl2 for 20 min at 37°C, to allow for formation of a TF-factor VII-Ca complex. After another 5 min of incubation with 20 µL of factor X (10 U/mL, Calbiochem) at 37°C, 100 µL of the mixture from each HUVEC well were transferred to a 96-well ELISA plate. Ice-cold buffer B (100 µL/well) was added, followed by room-temperature buffer C (100 µL/well). Pefachrom (10 mg/mL, Centerchem, Norwalk, CT, USA) was then added to the sample mixture (5 µL/well) and incubated for 20 min at 37°C. HUVEC wells treated with rIL-1 (10 ng/mL) or rIL-1ß (10 ng/mL) were included as positive controls. A factor Xa calibration curve was generated from purified factor X (1 U/100 µL) activated by Russell’s Viper Venom (25 µL of 50 U/mg, Centerchem, CT, USA). The conversion of the substrate was determined by means of an ELISA reader (OD405) (UV MAX Kinetic Microplate Reader, Biostad, Saint-Julie, Quebec, Canada).
Flow Cytometry
Up-regulation of adhesion molecules on endothelial cells after being challenged with PBMC supernatant fluids was examined by flow cytometry. HUVECs in triplicate were stimulated for 6 hrs with PBMC supernatant fluids that were harvested 4 hrs after challenge with oral streptococci or LPS. HUVECs were removed from the wells with non-enzymatic Cellstripper (Cellgro, Mediatech Inc., Herndon, VA, USA), washed with PBS, and labeled with ICAM-1 (CD54-APC, BD Pharmingen) and E-selectin (CD62E-PE, BD Pharmingen), according to the manufacturer’s instructions. The percentage of ICAM-1 and E-selectin expression was analyzed with Cytomics software (Beckman Coulter, Miami, FL, USA). HUVEC wells treated with rIL-1 (10 ng/mL) or rIL-1ß (10 ng/mL) were used for positive controls.
Statistical Analysis
Increases in endothelial TF activity and adhesion molecule (CD54, CD62E) were analyzed with a mixed-model ANOVA and Tukey’s HSD. The differences between PBMC and Mo-depleted preparations were examined with two-way ANOVA and post hoc comparisons.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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). There were no significant differences in TF induction among streptococci that were originally isolated from endocarditis lesions or oral cavity. HUVEC directly challenged with PBMC or bacteria alone did not induce measurable surface TF activity (data not shown).
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did not. Anti-TNF- treatment did not result in significant change from the control (Fig. 2). A similar inhibition pattern was observed with S. sanguinis 10556: a dramatic TF inhibition by IL-1ß, but not by IL-1 or TNF- (data not shown). Thus, IL-1ß appeared to be the major cytokine necessary for early TF activity.
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). Supernatant fluids from PBMC medium control or Mo-depleted PBMC induced no detectable TF activity (data not shown).
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). Untreated HUVECs were negative, and supernatant fluids from PBMC medium control induced a low background of CD54 and CD62E. Interestingly, LPS were significantly less potent in E-selectin-inducing activity than were the oral streptococci.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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, has been reported as important in inducing endothelial TF activity in streptococci-infected endothelial cell in vitro (Veltrop et al., 2001), and our results were consistent with this pattern. Endothelial cells are about 10 times more sensitive to IL-1ß than IL-1 in TF induction, and monocyte-IL-1ß in endothelial cell co-cultures is important in the up-regulation of endothelial TF (Wharram et al., 1991; Napoleone et al., 1997). Our neutralizing antibody study also demonstrated that monocyte-IL-ß induced by oral streptococci was the main cytokine contributing to early TF induction. TNF- may play a subsequent role in endothelial TF induction, since the TF activity of 20-hour PBMC culture supernatants was modestly inhibited by anti-TNF- (data not shown). IL-1ß is the major IL-1 cytokine secreted by monocytes upon stimulation (Wharram et al., 1991; Muller-Alouf et al., 1994). Previous work demonstrated that a similar amount of IL-1ß was induced from monocytes by both Gram-negative and Gram-positive bacteria (Hessle et al., 2005). Their results could explain the comparable endothelial TF activity induced by LPS-stimulated (100 ng/mL) and S. mutans-stimulated PBMC supernatant fluids (Fig. 2
). IL-1ra is released concurrently with IL-1 and can inhibit the effect of IL-1 by blocking the IL-1 receptor (Waage and Steinshamn, 1993). However, IL-1ra was not released by PBMC, within the first 4 hrs of bacterial challenge, in an amount adequate to alter results.
E-selectin is an early indicator of endothelial dysfunction in sites of inflammation. E-selectin molecules are de novo synthesized upon stimulation by endotoxin or cytokines in 6 hrs (Kuhns et al., 1995). Increased E-selectin levels in persons with infective endocarditis correlate with subsequent embolizations (Korkmaz et al., 2001). ICAM-1 on endothelial cells enhances interaction with monocytes and can lead to higher pro-coagulant activity (Collins et al., 1995). In addition, ICAM-1 can bind to fibrinogen and promote clot formation and fibrin deposition at the vascular wall (Becker et al., 2000; Arefieva and Krasnikova, 2001). Cross-linking of E-selectin and ICAM-1 on endothelial cells can induce autocrine secretion of platelet-activating factor and TNF-, which further increase endothelial TF (Schmid et al., 1995). We previously reported that mature dendritic cells could be generated from monocytes within a single day in vitro upon encounter with oral streptococci (Hahn et al., 2005). Mature dendritic cells have remarkable ability to produce TF and adhesion molecules (D’Amico et al., 1998; Broussas et al., 2000). Thus, endothelial cells expressing ICAM-1 and E-selectin in the heart valves could attract infected monocytes/dendritic cells, and the adherent streptococci-infected monocytes/dendritic cells could contribute to further coagulation. Moreover, dendritic cells are short-lived and, upon death, leave viable streptococci, which could infect the site (Hahn et al., 2005).
It is important to understand the initiation of cardiac vegetations in native valve endocarditis. Herzberg proposed that perturbed or modestly denuded endothelium would bind platelets and initiate septic vegetations (Herzberg, 1996). We reasoned that the rapid IL-1ß-inducing ability of oral streptococci during streptococcal bacteremia could serve as a virulence factor by promoting coagulation in vivo. This could help explain the heterogeneity of viridans isolated from endocarditis lesions. Interestingly, a recent rabbit endocarditis study demonstrated that infective endocarditis developed in pre-exiting sterile vegetations only when IL-1 was given 3 hrs before bacterial challenge (Dankert et al., 2006). The authors reasoned that 3 hrs were required for IL-1 to induce maximal endothelial TF and subsequent thrombin generation in rabbits (Nawroth et al., 1986b), and concluded that a pro-inflammatory stimulus was a risk factor for the development of infective endocarditis.
In conclusion, interactions between streptococci and PBMC up-regulated TF activity and adhesion molecules’ expression on endothelial cells. Monocyte-derived IL-1ß appeared to be the main cytokine in early TF activity. Vegetations and/or activated endothelial cells in the valvular area could attract streptococci or streptococci-infected monocytes, which differentiate into mature dendritic cells that produce TF and die, leaving viable streptococci to initiate local infection. We reasoned that the right combination of bacteremia to induce monocyte-IL-1ß and a subsequent bacteremia of oral streptococci could result in infective endocarditis. Thus, this rapid induction of monocyte-IL-1ß could serve as a virulence factor, leading to streptococcal endocarditis.
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ACKNOWLEDGMENTS |
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Received May 10, 2006; Last revision October 31, 2006; Accepted November 7, 2006
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REFERENCES |
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