Figure 9.

Expression and regulation of Toll-like receptors (TLRs) in glomerular endothelial cells (GENCs). GENCs were cultured as described in the Materials and methods. (a) RNA was isolated from GENCs kept in medium (black bars) or medium plus 500 units/ml tumour necrosis factor (TNF) and 200 units/ml interferon (IFN)-γ (grey bars) for 3 hr, and TLR mRNA expression levels were determined by real-time polymerase chain reaction (PCR) and expressed as a ratio relative to the respective 18S rRNA levels. Data represent the mean ± standard error of the mean (SEM) of three independent experiments. (b, c) GENCs were stimulated as above for 24 hr and flow cytometry was performed for TLR2 (b) and TLR4 (c). Lipopolysaccharide (LPS) (1 μg/ml) and pam3cys (P₃C) (1 μg/ml) were used for stimulation. Data are expressed as the percentage of gated events and represent the mean ± SEM of three independent experiments. Med-Iso, medium-treated cells stained with the isotype antibody; Med-TLR2, medium-treated cells stained with the TLR2 antibody; Med-TLR4, medium-treated cells stained with the TLR4 antibody; +TNF-α/IFN-γ, TNF-α/IFN-γ-treated cells; medium, medium-treated cells. (d, e) GENCs were stimulated as before and with increasing doses of P₃C or LPS as indicated, and interleukin (IL)-6 and chemokine (C-C motif) ligand 2 (CCL2) were measured in cell culture supernatants by enzyme-linked immunosorbent assay (ELISA). Data represent the mean ± SEM of three independent experiments. (f) Albumin permeability through GENC monolayers was determined as described in Fig. 6e. LPS (10 μg/ml) and P₃C (10 μg/ml) were used for stimulation. Data illustrate the fold change in fluorescence optical density (OD) of the filterate of P₃C- or LPS-treated groups versus the medium-treated group at different time-points as indicated. Data represent the mean ± SEM of four experiments, each performed in duplicate. *P < 0·05 versus medium; $P < 0·05 versus medium + TNF-α/IFN-γ; @P < 0·05 versus the respective ligand group with medium.