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 kappaB (NF-kappaB) 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 alpha 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.

FIG. 1.

Adherence of S. mutans and purified rGTFC to HUVECs. (A) FACS analysis of trypsinized HUVECs revealed positive staining of CD31 without CD45-positive leukocytes. (B) SDS-PAGE analysis of purified rGTFC by silver staining (a) or Western blotting using rabbit antibodies specific for GTFB/C (b). (C) Detection of rGTFC bound to HUVECs by Western blotting. HUVECs were treated separately with the amounts of rGTFC indicated at 37°C for 1 h, and unbound protein was washed away. Total cell lysates were harvested and Western blotting of rGTFC was performed as described in Materials and Methods. (D) Detection of S. mutans or rGTFC binding to HUVECs by fluorescence. Confluent HUVECs, untreated (a) or stimulated with purified rGTFC (b), S. mutans GS-5 (c), or GTF-null mutant NHS1DD (d), were fixed and stained with an anti-S. mutans antibody for panels c and d or anti-GTFB/C antibody for panel b. The results were observed by fluorescence microscopy after reaction with a secondary FITC-conjugated anti-rabbit IgG antibody.

FIG. 2.

Induction of cytokines and chemokine by S. mutans wild type or mutants. The confluent monolayer of HUVECs was treated with wild-type strain GS-5 (hatched bars), GTFB-expressing strain NHR1DD (dark gray bars), GTFB/C-expressing strain GS-5DD (black bars), or the GTF-null mutant NHS1DD (light gray bars) and compared with the untreated control (spotted bars) for the time indicated. Culture supernatants or cell lysates were harvested for detection of IL-6, IL-8, and IL-1β levels by a sandwich ELISA. IL-6 (A) and IL-8 (B) were released by HUVECs, and IL-1β (C) was detected in the cell lysates but not in the supernatants. Data are expressed as means for triplicate experiments from three independent assays. Error bars indicate standard deviations. * and **, P ≤ 0.05 and P ≤ 0.01, respectively, relative to uninfected cells; # and ##, P ≤ 0.05 and P ≤ 0.01, respectively, relative to NHS1DD. (D) Upregulation of cytokine and chemokine mRNA expression. HUVECs (1 × 10⁶ cells) were stimulated with 5 × 10⁸ CFU of S. mutans or NHS1DD. Total cellular RNA was extracted, reverse transcribed, and analyzed by RT-PCR using synthetic oligonucleotides. PCR products were separated on 2% agarose gel and stained with ethidium bromide. Relative amounts of RNA were quantified by a densitometry analysis as described in Materials and Methods; GAPDH mRNA expression was used as an internal control.

FIG. 3.

Dose-dependent induction of IL-6 release and up-regulation of cytokine mRNA in HUVECs induced by rGTFC. (A) Up-regulation of cytokine and chemokine mRNA expression in rGTFC-stimulated HUVECs. TNFR1, TNF receptor 1. (B) Dose-dependent induction of IL-6. A confluent monolayer of HUVECs (1 × 10⁵) was stimulated by crude extracts of cell wall-associated (CA) or secreted (CF) proteins from wild-type GS-5 or from GTF-null mutant NHS1DD, purified rGTFC (1.56 to 50 μg ml⁻¹), or recombinant immunodominant glycoprotein 60 (idg60; 25 μg ml⁻¹) for 24 h. (C) Dose-dependent inhibition of LPS-stimulated IL-6 production in HUVECs stimulated by polymyxin B. rGTFC-induced IL-6 release was not affected by polymyxin B. HUVECs were stimulated in the presence or absence of 20 or 40 μg ml⁻¹ polymyxin B for 24 h. IL-6 in supernatants was determined by sandwich ELISA. Data are expressed as means from triplicate experiments. Error bars indicated standard deviations. *, P ≤ 0.05; **, P ≤ 0.01.

FIG. 4.

Time-dependent expression of adhesion molecules on stimulated HUVECs. A confluent monolayer of HUVECs (1 × 10⁵ cells) was treated with culture medium (solid line) or rGTFC (25 μg ml⁻¹; heavy solid line) for 6 h or 16 h. The cells stained with secondary antibody served as a negative control (dot line). Cell surface expression of adhesion molecules was determined by indirect immunofluorescence and FACS analysis. Representative histograms are shown. The x axis indicates relative fluorescence intensity on a logarithmic scale; the y axis shows the number of cells on a linear scale. A total of 10⁴ cells were analyzed for each histogram.

FIG. 5.

Inhibitor-mediated inhibition of IL-6 or IL-8 and NF-κB translocation in rGTFC-stimulated HUVECs. (A) Inhibitor-mediated inhibition of IL-6 and IL-8 in stimulated HUVECs. HUVECs were pretreated with different inhibitors for 1 h and then stimulated with purified rGTFC (25 μg ml⁻¹) for 24 h at 37°C. Levels of IL-6 (white bars) and IL-8 (dark bars) in the cell supernatants were determined by sandwich ELISA. The results are expressed as percentages of inhibition of IL-6 and IL-8 release and are means ± standard deviation for triplicate determinations from three independent assays. *, P ≤ 0.05; **, P ≤ 0.01. (B) Immunofluorescence detection of p65 translocation and PDTC-mediated inhibition in HUVECs. HUVECs were left unstimulated (A, B, and C) or were stimulated with purified rGTFC (D, E, and F) for 1 h, and then cells were fixed, permeabilized, and stained with an anti-NF-κB p65 antibody (visually by the FITC-labeled secondary antibody). For inhibition assays, HUVECs were pretreated with PKC inhibitor Ro-31028 (B and E) or with NF-κB inhibitor PDTC (C and F) and then stimulated with rGTFC. Original magnification, ×400.