Glucosyltransferases of Viridans Group Streptococci Modulate Interleukin-6 and Adhesion Molecule Expression in Endothelial Cells and Augment Monocytic Cell Adherence

Abstract

Recruitment of monocytes plays important roles during vegetation formation and endocardial inflammation in the pathogenesis of infective endocarditis (IE). Bacterial antigens or modulins can activate endothelial cells through the expression of cytokines or adhesion molecules and modulate the recruitment of leukocytes. We hypothesized that glucosyltransferases (GTFs), modulins of viridans group streptococci, may act directly to up-regulate the expression of adhesion molecules and also interleukin-6 (IL-6) to augment monocyte attachment to endothelial cells. Using primary cultured human umbilical vein endothelial cells (HUVECs) as an in vitro model, we demonstrated that GTFs (in the cell-bound or free form) could specifically modulate the expression of IL-6, and also adhesion molecules, in a dose-and time-dependent manner. Results of inhibition assays suggested that enhanced expression of adhesion molecules was dependent on the activation of nuclear factor B (NF-B) and extracellular signal-regulated kinase and that p38 mitogen-activated protein kinase pathways also contributed to the release of IL-6. Streptococcus-infected HUVECs or treatment with purified IL-6 plus soluble IL-6 receptor ␣ enhanced the expression of ICAM-1 and the adherence of the monocytic cell line U937. These results suggest that streptococcal GTFs might play an important role in recruiting monocytic cells during inflammation in IE through induction of adhesion molecules and IL-6, a cytokine involved in transition from neutrophil to monocyte recruitment.

C-Terminal Repeats ofClostridium difficileToxin A Induce Production of Chemokine and Adhesion Molecules in Endothelial Cells and Promote Migration of Leukocytes

“…All experiments were performed between the second and fifth passage before the cells undergo senescence that may result in a higher level of cytokine production. The identity and purity of the HUVECs were characterized by flow cytometry using anti-CD31 or von Willebrand factor monoclonal antibody (MAb) (50).…”

“…Purified His-tagged rARU was kindly provided by David M. Lyerly and Limin Zheng (Techlab, Inc., Blacksburg, VA) (31), and His-tagged rGTFC was purified as described previously (50). These two recombinant proteins were purified to homogeneity by affinity chromatography, and the protein purity was determined by silver staining of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels.…”

“…Preparations with detectable amounts of LPS were further processed by polymyxin B agarose column chromatography (Sigma) to remove the contaminating LPS. For Western blot analysis, a monoclonal anti-ARU was also provided by David M. Lyerly and Limin Zheng, and polyclonal anti-GTFC was prepared as described previously (50).…”

“…Primers specific for IL-6, IL-8, monocyte chemoattractant protein 1 (MCP-1), and GAPDH were selected based on the published human cDNA sequences. The oligonucleotide primers used and reverse transcription (RT)-PCR procedures were described previously (50).…”

“…GTFC binds to glucan through its C-terminal repeated domain that is homologous to the CRD of TcdA (43,44). Our previous study demonstrated that GTFC is a potent modulin for human endothelial cells (50). In this work, it was tested in parallel as a positive control for endothelial activation and also for the purpose of comparison in a carbohydrate-binding screening assay.…”