Toll-like receptor 9 can be expressed at the cell surface of distinct populations of tonsils and human peripheral blood mononuclear cells

Abstract

Unmethlylated CpG dinucleotides induce a strong T-helper-1-like inflammatory response, presumably mediated by Toll-like receptor 9 (TLR9). However, the nature and cellular localization of TLR9 in primary human cells remain controversial. Here we demonstrate, using flow cytometry and immunofluorescence microscopy techniques, that TLR9 can be expressed at the cell surface. The primary human cell subsets that were positive for TLR9 expression were distinct depending on the tissues analyzed. Specifically, in human peripheral blood mononuclear cells (PBMC) the majority of cell surface TLR9(+) cells were confined to the major histocompatibility complex (MHC) class II(+) CD19(-) populations that express CD11c and/or CD14, whereas in tonsils the same gated population contained primarily MHC class II(+) CD19(+) cells. Cells positive for surface expression represented a minor fraction of the total cell populations examined, varying between 2 and 10%. In addition, we found that TLR9 expression at the surface of PBMC was up-regulated approximately fourfold following stimulation with the gram-negative bacterial cell wall component lipopolysaccharide, suggesting a potential modulatory role of TLR4 agonists on TLR9 expression. Taken together, these data validate human TLR9 expression at the surface of primary cells, in addition to the previously described intracellular localization. Further, our results suggest that human antigen-presenting cells comprise the major cell populations expressing cell surface TLR9.

FIG. 1.

Cell surface TLR9 is expressed on human tonsil cells. (A) A representative dot plot displays forward and side scatters of the tonsil cells used for live-cell gating (R1). (B) Staining with the TLR9 MAb (dark open line) is shown overlaying staining observed with the isotype control MAb (gray filled line). R2 indicates the cell surface TLR9⁺ cells. Dot plots of HLA-DR (MHC class II) and CD19 expression are shown for (C) the total live R1 gated cells and (D) the TLR9⁺ cells (R2 gate). Dead cells were excluded by 7AAD staining.

FIG. 2.

Cell surface TLR9 is expressed on human PBMC. (A) A representative dot plot displays forward and side scatters of the PBMC used for live-cell gating (R1). (B) Staining with the TLR9 MAb (dark open line) is shown overlaying staining observed with the isotype control MAb (gray filled line). R2 indicates the cell surface TLR9⁺ cells. Dot plots of (C, D) MHC class II and CD19, (E, F) MHC class II and CD123, (G, H) MHC class II and CD11c expression are shown for (C, E, G) R1-gated cells and (D, F, H) R2 TLR9⁺ cells. Dead cells were excluded by 7AAD staining.

FIG. 3.

Forward and side scatter profiles of TLR9 positive cells in (A) tonsils and (B) human PBMC.

FIG. 4.

Specificity of TLR9 staining. The anti-human TLR9 MAb was made by immunizing mice with a 17-mer peptide of TLR9 (aa 268 to 284) located in the putative TLR9 extracellular domain. Specificity of the TLR9 staining was tested by preincubating the TLR9 MAb with the TLR9 peptide prior to the initiation of the flow cytometric staining protocol. PBMC preparations were stained with the mouse anti-human TLR9-APC that had been preincubated with either (A) a peptide from the extracellular domain of TLR9 or (B) a negative control peptide. The histograms shown are gated on live cells and show fluorescence for the mouse anti-human TLR9-APC MAb (gray histogram), the mouse anti-human TLR9-APC MAb preincubated with the TLR9 peptide (A) or the control peptide (B) (bold black line), and the APC-labeled isotype-matched MAb (thin stippled line). Preincubation of mouse anti-human TLR9 MAb with a TLR9 peptide reduced the fluorescence staining observed for TLR9 to near background levels observed with the isotype control (A). Preincubation of the mouse anti-human TLR9 MAb with the control peptide had no effect on TLR9 staining (B). The data shown are representative of those from three independent experiments.

FIG. 5.

Specificity of TLR9 staining. The specificity of the TLR9 staining was further tested by assessing cell surface TLR9 expression on RAW 264.7 cells that were mock transfected or transfected with a plasmid containing human TLR9. At 24 h posttransfection, cells were stained with mouse anti-human TLR9 antibody, which was detected with an FITC-goat anti-mouse IgG/IgM antibody. The histogram plot shown depicts mock-transfected cells stained with only the secondary reagent (gray dashed line), mock-transfected cells stained with the TLR9 antibody plus the secondary detection reagent (gray solid line), and TLR9-transfected RAW cells stained with the TLR9 antibody plus secondary detection reagent (black solid line). RAW cells transfected with human TLR9 display cell surface TLR9 staining distinct from that of the mock-transfected cells. The data shown are from one experiment and are representative of those from three independent experiments.

FIG. 6.

Cell surface TLR9 expression on human PBMC is up-regulated following LPS stimulation. Human PBMC were cultured for 24 h in 30, 10, or 1 μg of LPS per ml and then stained with the murine anti-human TLR9-APC MAb or the isotype control. The histogram plots shown are (A) PBMC cultured in medium alone stained with the TLR9 antibody (black line) overlaying the isotype control (gray histogram), (B) human PBMC cultured in LPS (10 μg/ml) stained with the TLR9 antibody (black line) overlaying the isotype control (gray histogram), and (C) human PBMC cultured in LPS (10 μg/ml) stained with the TLR9 antibody (black line) overlaying human PBMC cultured in medium alone stained with the TLR9 antibody (gray histogram). The MFIs for the TLR9⁺ cells are shown in histograms (A) and (B). No difference in the MFI of TLR9 up-regulation was observed among the various concentrations of LPS (data not shown). The data shown are representative of those from three independent experiments.

FIG. 7.

Cell surface TLR9 expression visualized by immunofluorescence staining. Double staining of LPS-stimulated human PBMC with (A) the APC-labeled isotype-matched MAbs and (B) FITC-labeled anti-CD19 MAbs, using the same microscopic field, and double staining of LPS-stimulated PBMC with (C) the APC-labeled anti-TLR9 MAbs and (D) FITC-labeled anti-CD19 MAbs, using the same microscopic field, are shown. Staining was observed under a fluorescence microscope at a magnification of ×40. Arrows indicate cell surface TLR9 staining (C), which did not colocalize with CD19 staining (arrows in panel D). (E) Staining observed with the anti-CD19 MAbs (Fig. 6D) overlaid by the staining observed with the TLR9 MAbs (Fig. 6C) shows distinct cells expressing TLR9 (red) and CD19 (green).