The Toll-like receptor 2 (TLR2) ligand FSL-1 is internalized via the clathrin-dependent endocytic pathway triggered by CD14 and CD36 but not by TLR2

Abstract

Little is known of how Toll-like receptor (TLR) ligands are processed after recognition by TLRs. This study was therefore designed to investigate how the TLR2 ligand FSL-1 is processed in macrophages after recognition by TLR2. FSL-1 was internalized into the murine macrophage cell line, RAW264.7. Both chlorpromazine and methyl-beta-cyclodextrin, which inhibit clathrin-dependent endocytosis, reduced FSL-1 uptake by RAW264.7 cells in a dose-dependent manner but nystatin, which inhibits caveolae- and lipid raft-dependent endocytosis, did not. FSL-1 was co-localized with clathrin but not with TLR2 in the cytosol of RAW264.7 cells. These results suggest that internalization of FSL-1 is clathrin dependent. In addition, FSL-1 was internalized by peritoneal macrophages from TLR2-deficient mice. FSL-1 was internalized by human embryonic kidney 293 cells transfected with CD14 or CD36 but not by the non-transfected cells. Also, knockdown of CD14 or CD36 in the transfectants reduced FSL-1 uptake. In this study, we suggest that (i) FSL-1 is internalized into macrophages via a clathrin-dependent endocytic pathway, (ii) the FSL-1 uptake by macrophages occurs irrespective of the presence of TLR2, and (iii) CD14 and CD36 are responsible for the internalization of FSL-1.

Figure 4

Effects of chemicals on FSL-1 uptake by RAW264.7 cells. RAW264.7 cells were incubated for 30 min without (control) or with various concentrations of nystatin (Nys), and (a–c), chlorpromazine (CPZ) (d–f), and methyl-β-cyclodextrin (MbCD) (g–i) and then incubated for 2 hr with 100 μg/ml fluorescein isothiocyanate-conjugated (FITC-) FSL-1. Results are shown as images obtained by CLSM (FITC-FSL-1, green; Alexa-Concanvalin A, red) and as histograms (b, e, h) and mean fluorescence intensity (MFI) obtained from the histograms by flow cytometry.

Figure 3

Inhibition of fluorescein isothiocyanate-conjugated (FITC-) FSL-1 uptake by unlabelled FSL-1. FITC-FSL-1 uptake by RAW264.7 cells was measured in the presence of 9 or 35 μg/ml FSL-1 by flow cytometry. The results shown are a histogram and their relative mean fluorescence intensity (MFI) for A, cell only; B, cell + FITC-FSL-1; C, cell + FSL-1 (9 μg/ml) + FITC-FSL-1 (100 μg/ml); D, cell + FSL-1 (35 μg/ml) + FITC-FSL-1 (100 μg/ml). Relative MFIs were calculated as [(MFI of cells incubated with FITC-FSL-1in the absence or the presence of unlabelled FSL-1)/(MFI of cells incubated without FSL-1).

Figure 2

Temperature dependency of FSL-1 internalization into RAW264.7 cells. RAW264.7 cells were incubated with 100 μg/ml fluorescein isothiocyanate-conjugated (FITC-) FSL-1 at 4° (on ice) or 37° for 2 hr. The uptake level of FITC-FSL-1 was observed by using confocal laser scanning microscopy at (a) 4° and (b) 37° and flow cytometry at (c) 4° and (d) 37°.

Figure 1

Induction of tumour necrosis factor-α (TNF-α) production by RAW264.7 cells stimulated with FSL-1 or fluorescein isothiocyanate-conjugated (FITC-) FSL-1. RAW264.7 cells in 96-well plates were cultured and then stimulated at 37° for 15 hr with various concentrations of FSL-1 or FITC-FSL-1 in RPMI-1640 base medium. The culture supernatants were collected and examined for the amount of TNF-α, which was determined by enzyme-linked immunosorbent assay. Results are expressed as the means ± SD of three determinations.

Figure 5

Involvement of clathrin in FSL-1 uptake by RAW264.7 cells. RAW264.7 cells were incubated for 2 hr with100 μg/ml of fluorescein isothiocyanate-conjugated (FITC-) FSL-1 (green) and then stained with anti-clathrin monoclonal antibody and second antibody (red). White arrows show co-localization of FSL-1-containing compartments with clathrin-coated compartments (a). Down-regulation of clathrin messenger RNA (mRNA) by transfection of clathrin heavy-chain-specific small interfering (siRNA) was determined by using Real-time TaqMan polymerase chain reaction (b). Effect of knockdown of clathrin mRNA on FSL-1 uptake by RAW264.7 cells was determined by flow cytometry. Black line, cell only; grey area, cells with FITC-FSL-1; red line, negative control siRNA-transfected cells with FITC-FSL-1; and blue line, clathrin siRNA-transfected cells with FITC-FSL-1 (c) and mean fluorescence intensity (MFI) obtained from the histogram (d, N, cells with FITC-FSL-1; SC, clathrin siRNA-transfected cells with FITC-FSL-1; and NC, negative control siRNA-transfected cells with FITC-FSL-1).

Figure 6

Maturation of FSL-1-containing endosome. The maturation was determined by staining RAW264.7 cells with Lysotracker, which had been incubated with 100 μg/ml of fluorescein isothiocyanate-conjugated (FITC-) FSL-1. White arrows show co-localization of FSL-1-containing endosomes with Lysotracker-containing endosomes.

Figure 8

The time–course of cell surface Toll-like receptor 2 (TLR2) expression after FSL-1 stimulation. RAW264.7 cells were stimulated with FSL-1 (10 nm), and cell surface expression level of TLR2 was determined by flow cytometry. The results were shown as both histograms (a, black line, cell only; grey line, cells with isotype control antibody; faint green area, TLR2 expression on unstimulated cells; blue line, TLR2 expression on 1-hr stimulated cells; red line, TLR2 expression on 2-hr stimulated cells) and mean fluorescence intensity (MFI) obtained from the histogram (b).

Figure 7

Involvement of Toll-like receptor 2 (TLR2) in FSL-1 uptake. Co-localization of fluorescein isothiocyanate-conjugated FITC-) FSL-1 (green) and TLR2 (red) in RAW264.7 cells was observed by confocal laser scanning microscopy (CLSM) (a). Peritoneal macrophages (PMφs) from TLR2^+/+ mice and TLR2^−/− mice were incubated for 2 hr with 100 μg/ml FITC-FSL-1 and the uptake was determined by flow cytometry (b, c) and CLSM (d, e). Small histograms in (b) and (c) show the surface expression level of TLR2 in each PMφ.

Figure 9

Involvement of CD14 or CD36 in FSL-1 uptake. HEK293WT (a), HEK293/CD14 (b), HEK293/CD36 (c), HEK293/TLR2 (d), HEK293/CD14/TLR2 (e) or HEK293/CD36/TLR2 (f) were incubated with 100 μg/ml of fluorescein isothiocyanate-conjugated (FITC-) FSL-1 for 2 hr. Results are shown as images obtained by confocal laser scanning microscopy (FITC-FSL-1, green; Alexa-Concanavalin A, red) and as histograms obtained by flow cytometry (faint grey area, cell only; black line, cell + FITC-FSL-1).

Figure 11

Effect of co-transfection of CD14 and CD36 on the uptake of FSL-1. HEK293 cells were transiently transfected with CD14 and/or CD36. The level of CD14 and CD36 expression was confirmed by flow cytometry (not shown). FSL-1 uptake by the transfectants was measured in the presence of 100 μg/ml of fluorescein isothiocyanate-conjugated (FITC-) FSL-1. Relative mean fluorescence intensities (MFIs) were calculated as [(MFI of transfectants incubated with FITC-FSL-1)/(MFI of transfectants)].

Figure 10

Effects of knockdown of CD14 in HEK293/CD14 and knockdown of CD36 in HEK293/CD36 on the uptake of FSL-1. HEK293/CD14 and HEK293/CD36 cells were transiently transfected with small interfering RNA (siRNA) against CD14 and CD36, respectively. The cell surface expression levels of CD14 (a) and CD36 (b) were confirmed by using flow cytometry. The cells transfected with siRNA against CD14 and CD36 were incubated with 100 μg/ml of fluorescein isothiocyanate-conjugated (FITC-) FSL-1 and the uptake was shown as both relative mean fluorescence intensity (MFI) (c, d) and the histograms (e, f). Relative MFIs were calculated as [(MFI obtained by specific antibody)/(MFI obtained by isotype control antibody)] (a, b) or [(MFI of transfectants with FITC-FSL-1)/(MFI of HEK293WT)] (c, d).