Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Jan 15;186(2):1119-30.
doi: 10.4049/jimmunol.1001647. Epub 2010 Dec 17.

Bacterial lipoprotein TLR2 agonists broadly modulate endothelial function and coagulation pathways in vitro and in vivo

Hae-Sook Shin  1 , Fengyun Xu, Aranya Bagchi, Elizabeth Herrup, Arun Prakash, Catherine Valentine, Hrishikesh Kulkarni, Kevin Wilhelmsen, Shaw Warren, Judith Hellman
Affiliations

Bacterial lipoprotein TLR2 agonists broadly modulate endothelial function and coagulation pathways in vitro and in vivo

Hae-Sook Shin et al. J Immunol. .

Abstract

TLR2 activation induces cellular and organ inflammation and affects lung function. Because deranged endothelial function and coagulation pathways contribute to sepsis-induced organ failure, we studied the effects of bacterial lipoprotein TLR2 agonists, including peptidoglycan-associated lipoprotein, Pam3Cys, and murein lipoprotein, on endothelial function and coagulation pathways in vitro and in vivo. TLR2 agonist treatment induced diverse human endothelial cells to produce IL-6 and IL-8 and to express E-selectin on their surface, including HUVEC, human lung microvascular endothelial cells, and human coronary artery endothelial cells. Treatment of HUVEC with TLR2 agonists caused increased monolayer permeability and had multiple coagulation effects, including increased production of plasminogen activator inhibitor-1 (PAI-1) and tissue factor, as well as decreased production of tissue plasminogen activator and tissue factor pathway inhibitor. TLR2 agonist treatment also increased HUVEC expression of TLR2 itself. Peptidoglycan-associated lipoprotein induced IL-6 production by endothelial cells from wild-type mice but not from TLR2 knockout mice, indicating TLR2 specificity. Mice were challenged with TLR2 agonists, and lungs and plasmas were assessed for markers of leukocyte trafficking and coagulopathy. Wild-type mice, but not TLR2 mice, that were challenged i.v. with TLR2 agonists had increased lung levels of myeloperoxidase and mRNAs for E-selectin, P-selectin, and MCP-1, and they had increased plasma PAI-1 and E-selectin levels. Intratracheally administered TLR2 agonist caused increased lung fibrin levels. These studies show that TLR2 activation by bacterial lipoproteins broadly affects endothelial function and coagulation pathways, suggesting that TLR2 activation contributes in multiple ways to endothelial activation, coagulopathy, and vascular leakage in sepsis.

PubMed Disclaimer

Figures

Figure 1
Figure 1. TLR2 agonists activate human endothelial cells to secrete IL-6 and IL-8
(A, B) HUVEC monolayers were incubated with dilutions of Peptidoglycan-associated lipoprotein (PAL) for 18 hours (n = 4). Levels of IL-6 (A) and IL-8 (B) were quantified in the culture supernatants at 18 hours. (*p < 0.05, ** p < 0.01 PAL versus medium). (C) HUVEC monolayers were incubated with PAL (1 μg/ml) for intervals through 24 hours (n = 3). A time course of IL-6 and IL-8 levels induced by PAL was plotted. (* p < 0.05, ** p < 0.01, *** p < 0.001 IL-8, PAL versus medium; ## p < 0.01, ### p < 0.001, IL-6, PAL versus medium). (D) HUVEC monolayers were incubated for 18 hours with equal concentrations (1.5 μg/ml) of PAL or MLP, both of which are naturally occurring TLR2 agonists, or Pam3Cys, which is a synthetic TLR2 agonist (n = 3). IL-6 levels in supernatants (**p < 0.01, *** p < 0.001 for TLR2 agonist versus medium). (E and F) Monolayers of human lung microvascular endothelial cells (E) and human coronary artery endothelial cells (F) were treated with medium or PAL (1 μg/ml) for 18 hours (n = 4). Levels of IL-6 and IL-8 were quantified in culture supernatants. (**p < 0.01, *** p < 0.001 PAL versus medium). (G) Lung microvascular endothelial cells from wild-type and TLR2−/− mice were incubated overnight with PAL (1 μg/ml) or medium (n = 6). Levels of IL-6 were quantified in culture supernatants. LPS (1 μg/ml), a TLR4 agonist, was used as the positive control to verify intact intracellular TLR signaling. (*** p < 0.001 PAL versus medium in wild-type endothelial cells; ### p < 0.001, PAL-treated endothelial cells from wild-type versus TLR2−/− mice).
Figure 2
Figure 2. TLR2 agonists upregulate adhesion molecule expression by endothelial cells and facilitate neutrophil-endothelial adhesion in vitro.
(A) Surface expression of the adhesion molecule E-selectin was assessed using a cell-based ELISA in HUVEC, human lung microvascular endothelial cells and human coronary artery endothelial cells, after they were treated with Pam3Cys (1 μg/ml, 24 hours, shown for HUVEC) or PAL (1 μg/ml, 2 hours, shown for HUVEC, HMVEC-L and HCAEC) (n = 4). Data are expressed as the optical density (OD) of TLR2 agonist-treated cells relative to OD of medium-treated cells (Rel OD). (*** p < 0.001). (B) A time course of surface E-selectin expression was assessed in HUVEC monolayers treated with PAL (2 μg/ml) for increasing intervals through 18 hours (n = 4). Data are expressed as the optical density (OD) of TLR2 agonist-treated cells relative to OD of medium-treated cells at the same time point (Rel OD). (* p < 0.05, ** p < 0.01, *** p < 0.001). (C) Flow cytometry was performed to assess surface expression of E-selectin after 18 hours of treatment with Pam3Cys or PAL (1 μg/ml). (D) Neutrophil-endothelial adhesion was assessed using fluorescence intensity. HUVEC monolayers were incubated for 2 hours with the TLR2 agonists PAL, MLP or Pam3Cys, each at a concentration of 1.5 μg/ml (n = 3), and were then washed gently and calcein-labeled neutrophils were added for 1 hour. Plates were inverted and centrifuged gently to remove non-adherent neutrophils, and calcein fluorescence was measured in a fluorescent plate reader. (*p < 0.05, *** p < 0.001 TLR2 agonist versus medium; # p < 0.05 PAL versus MLP). A representative fluorescent micrograph is shown on the right.
Figure 3
Figure 3. TLR2 activation increases adhesion molecule and chemokine expression and increases lung myeloperoxidase levels in vivo
(A) Wild-type and TLR2−/− mice were challenged intravenously with PAL (25 μg/mouse) or carrier (n = 6). Soluble E-selectin was quantified in plasmas after 90 minutes. (** p < 0.01 PAL versus carrier). (B and C) Expression of adhesion molecule (E-selectin and P-selectin) and chemokine (MCP-1) mRNAs was measured using quantitative real-time PCR, (B) in lungs of wild-type mice (n = 4) over a time course of 30 min to 22 hours after intravenous challenge with Pam3Cys (400 μg/mouse) or carrier. (**p < 0.01, *** p < 0.001 Pam3Cys versus carrier), and (C) in lungs of wild-type and TLR2−/− mice (n = 4) 2 hours after intravenous challenge with PAL (25 μg/mouse) or carrier. (** p < 0.01 PAL-treated wild-type versus TLR2−/− mice). Changes in gene expression were normalized to 18S ribosomal RNA levels and are expressed as the fold-increase above baseline levels for carrier injected mice. (D) Myeloperoxidase (MPO) levels were used to assess neutrophil activity in the lungs of wild-type and TLR2−/− mice (n = 4) 4 hours after IV challenge with carrier or Pam3Cys (25 μg/mouse). LPS (100 ng/mouse), a TLR4 agonist, was used as a positive control and verified intact intracellular TLR signaling. (** p < 0.01, *** p < 0.001, Pam3Cys-treated wild-type versus TLR2−/− mice).
Figure 4
Figure 4. TLR2 agonists alter coagulation pathway factor expression in vitro
(A) HUVEC monolayers were treated with medium or increasing concentrations of Pam3Cys as indicated in the figure (n = 4), and PAI-1 levels were quantified at 24 hours. (* p < 0.05, ** p < 0.01 Pam3Cys versus medium). (B) HUVEC monolayers were treated with medium or increasing concentrations of Pam3Cys as indicated in the figure (n = 4), and tPA levels were quantified at 24 hours. (* p < 0.05, ** p < 0.01 Pam3Cys versus medium). (C) HUVEC monolayers were treated with PAL (20 μg/ml) or medium (n = 3) and TFPI levels were quantified in the cultures supernatants after 24 hours. (*** p < 0.001 Pam3Cys versus medium). (D) A representative immunoblot showing expression of tissue factor (TF, upper panel) in HUVEC lysates after 1 hour, 6 hours and 24 hours of exposure to PAL (20 μg/ml). The actin blot (lower panel) indicates equal loading of different lanes. 250 μg of protein were loaded per lane.
Figure 5
Figure 5. TLR2 agonists induce systemic PAI-1 production and increases lung fibrin levels in mice
(A) PAI-1 levels were quantified in the plasmas of wild-type mice (n = 6) 24 hours after IV challenge with PAL (25 μg/mouse) or carrier. (*** p < 0.001 PAL versus carrier). (B) PAI-1 levels were quantified in the plasmas of wild-type and TLR2−/− mice (n = 5) 24 hours after IV challenge with carrier or Pam3Cys (50 μg/mouse). (** p < 0.01 Pam3Cys-treated wild-type versus TLR2−/− mice). (C) Immunoblots were used to assess fibrin levels in the lungs 20 hours after intratracheal administration of Pam3Cys (200 μg/mouse) versus carrier. Right panel – density analysis of immunoblot bands (n = 4). (**p < 0.01 Pam3Cys versus carrier).
Figure 6
Figure 6. TLR2 activation increases HUVEC permeability to albumin
HUVEC were grown to confluence in the upper (luminal) chamber of 0.4 μM transwells, and were treated with dilutions of Pam3Cys (n = 3, 5–15μg/ml). FITC-Albumin was added to the luminal chamber after 24 hours, and the flux of FITC-albumin across the membrane was quantified as described in the methods and is expressed as the permeability coefficient for albumin (Pa) of the experimental condition versus control (medium alone). Cytomix (a mixture of IL-1β, TNFα, and IFN-γ, each at a concentration of 10 ng/ml) was used as a positive control for the assay. (** p < 0.01 Pam3Cys versus medium).
Figure 7
Figure 7. Effects of TLR2 agonist on HUVEC viability and apoptosis
HUVEC monolayers were treated with dilutions of Pam3Cys for 24 hours (n = 3, 1.67–40 μg/ml) to assess the effects of TLR2 activation on (A) endothelial cell viability, using the MTT assay and (B) endothelial cell apoptosis, using the TUNEL assay. (** p < 0.01 Pam3Cys versus medium).
Figure 8
Figure 8. Pam3Cys upregulates TLR2 expression by HUVEC
(A) Immunoblot analyses of lysates of HUVEC monolayers that were incubated with Pam3Cys at different concentrations (Left Panel) and for intervals up to 24 hours (Right Panel, Pam3Cys concentration 20 μg/ml). Fifteen μg of protein were loaded per lane. Primary antibodies for the immunoblot were goat anti-human TLR2 IgG and goat anti-actin IgG. Molecular weight markers are indicated at the left. (B) Cell-based ELISA for surface expression of TLR2 after 20 hours of treatment with Pam3Cys (1 and 10 μg/ml). (* p < 0.05 TLR2 agonist versus medium) There was no increase in binding of control IgG to the cell surface at either concentration of Pam3Cys. (C) Flow cytometry for surface expression of TLR2 after 20 hours of treatment with PAL (1 and 10 μg/ml). Similar results were obtained using Pam3Cys (not shown).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Rittirsch D, Flierl MA, Ward PA. Harmful molecular mechanisms in sepsis. Nat Rev Immunol. 2008;8:776–787. - PMC - PubMed
    1. Schouten M, Wiersinga WJ, Levi M, van der Poll T. Inflammation, endothelium, and coagulation in sepsis. J Leukoc Biol. 2008;83:536–545. - PubMed
    1. Aird WC. The role of the endothelium in severe sepsis and multiple organ dysfunction syndrome. Blood. 2003;101:3765–3777. - PubMed
    1. Reinhart K, Bayer O, Brunkhorst F, Meisner M. Markers of endothelial damage in organ dysfunction and sepsis. Crit Care Med. 2002;30:S302–S312. - PubMed
    1. McGill SN, Ahmed NA, Christou NV. Endothelial cells: role in infection and inflammation. World J Surg. 1998;22:171–178. - PubMed

Publication types

MeSH terms

-