Toll-like receptor 2 (TLR2) and TLR4 are present inside human dendritic cells, associated with microtubules and the Golgi apparatus but are not detectable on the cell surface: integrity of microtubules is required for interleukin-12 production in response to internalized bacteria

Abstract

The activation of dendritic cells (DCs) by microbes is mediated by pattern recognition receptors including the Toll-like receptors (TLR). Bacterial lipopolysaccharide acts via TLR4 whereas peptidoglycan and lipoprotein responses are mediated by TLR2. It is generally accepted that TLR binding to microbes occurs at the cell surface but this has not been directly demonstrated for human DCs. We show here that TLR2 and TLR4 are expressed inside DCs in an abundant tubulovesicular pattern with a focus of intense staining adjacent to the nucleus. In contrast, there was no detectable expression on the cell surface. TLR2 and TLR4 were readily found both intracellularly and on the surface of monocytes. They were shown to be closely associated with the Golgi complex and colocalized with alpha-tubulin, displaying a high focal concentration at the microtubule organizing centre. Alignment of TLR2 and TLR4 with microtubules was observed, suggesting that microtubules serve as transport tracks for TLR vesicles. Depolymerization of the microtubule network disrupted the intracellular expression of TLR2 and TLR4 and profoundly inhibited interleukin-12 (IL-12) production in response to Neisseria meningitidis but did not prevent phagocytosis. These data are consistent with the bacterial signalling through TLR2 and TLR4 required for IL-12 production occurring inside DCs after phagocytosis.

Figure 1

Surface and intracellular staining of TLR2 and TLR4 in DCs (a) and monocytes (b). For surface staining cells were incubated on ice with TLR antibodies followed by FITC-conjugated F(ab)₂ goat anti-rabbit IgG. For intracellullar staining DCs were fixed with 4% PFA, permeabilized in Saponin buffer and then stained. Representative FACS histograms of surface and intracellular staining and confocal images of intracellular staining from five experiments are shown. Arrows point to a highly concentrated signal in the perinuclear region (a,b) as well as surface staining on monocytes (b).

Figure 2

Association of TLR2 and TLR4 with Golgi apparatus. DCs were fixed with 4% PFA, permeabilized in Saponin buffer and stained with Alexa-568 Golgin-97 and TLR2 (a) and TLR4 (b) antbodies. Merged images show localization of TLR and Golgin at the centre of the Golgi apparatus. Representative images from three experiments are shown.

Figure 3

Co-localization of TLR2 and TLR4 with α-tubulin. Fixed and permeabilized DCs were stained for TLR2 (a) or TLR4 (b) and α-tubulin. Microtubules were depolymerized for 5 hr with 100 ng/ml of colcemid and then stained for intracellular TLR2 and TLR4 together with α-tubulin (c). Depolymerization of microtubules disrupted the intracellular TLR2 and TLR4 vesicles. Representative confocal images from three experiments are shown.

Figure 4

Depolymerization of microtubules disrupts IL-12 production in response to Neisseria meningitidis. DCs were pre-incuabated for 5 hr in the presence or absence of 100 ng/ml colcemid and then stimulated for 14 hr with N. meningitidis bacteria at a ratio of 1 : 100 in the presence of brefeldin A. DCs producing IL-12 and TNF-α were determined by intracellular staining. Mean and SEM of three different experiments with three different donors are shown(a). Phagocytosis after 14 hr with the bacteria is shown by confocal imaging (b).