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Intracellular recognition of lipopolysaccharide by toll-like receptor 4 in intestinal epithelial cells.

Hornef MW, Normark BH, Vandewalle A, Normark S - J. Exp. Med. (2003)

Bottom Line: Here we demonstrate that disruption of the integrity of the Golgi apparatus significantly reduced LPS-mediated nuclear factor kappaB activation.In contrast to macrophages, prevention of ligand internalization by use of LPS-coated beads significantly impaired recognition by epithelial cells.Thus, our data provide evidence that in contrast to the situation in macrophages, LPS recognition in intestinal epithelial cells may occur in the Golgi apparatus and require LPS internalization.

View Article: PubMed Central - PubMed

Affiliation: Swedish Institute for Infectious Disease Control SMI, 17182 Solna, Sweden. mathias.hornef@smi.ki.se

ABSTRACT
Toll-like receptor (TLR)4 has recently been shown to reside in the Golgi apparatus of intestinal crypt epithelial m-ICcl2 cells, colocalizing with internalized lipopolysaccharide (LPS). Here we demonstrate that disruption of the integrity of the Golgi apparatus significantly reduced LPS-mediated nuclear factor kappaB activation. Also, the TLR4 adaptor protein MyD88 and the serine/threonine kinase IRAK-1 were rapidly recruited to the Golgi apparatus upon stimulation. LPS-mediated activation required lipid raft formation and intact clathrin-dependent internalization. In contrast to macrophages, prevention of ligand internalization by use of LPS-coated beads significantly impaired recognition by epithelial cells. The localization of TLR4 to the Golgi apparatus was abrogated by expression of a genetically modified form of the TLR4 binding chaperone gp96. Thus, our data provide evidence that in contrast to the situation in macrophages, LPS recognition in intestinal epithelial cells may occur in the Golgi apparatus and require LPS internalization.

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Recruitment of the TLR4 adaptor protein MyD88 and IRAK-1 to the Golgi apparatus upon LPS stimulation. (A) m-ICcl2 cells transfected with an MyD88-GFP fusion protein were left untreated or stimulated with 10 ng/ml LPS for the indicated time period. (B) m-ICcl2 cells transfected with an MyD88-GFP fusion protein were left untreated or stimulated with 10 ng/ml LPS or 100 ng/ml IL-1β. The cellular activation is illustrated by the nuclear translocation of the NF-κB subunit p65. (C) m-ICcl2 cells carrying the MyD88-GFP expression plasmid were left untreated or stimulated with LPS, fixed, and immunostained for TLR4 (red). (D) m-ICcl2 cells carrying the MyD88-GFP expression plasmid were incubated with 5 μM TR C5 ceramide to identify the Golgi apparatus. Subsequent stimulation with 10 ng/ml LPS for 15 min illustrates recruitment of the TLR4 adaptor protein MyD88 to the Golgi apparatus. (E) Double immunostaining of m-ICcl2 cells for MyD88 (red) and TLR4 (green) after stimulation with 10 ng/ml LPS or 100 ng/ml IL-1β for the indicated time. Note the transient change of the TLR4+ Golgi apparatus from green to yellow resulting from an overlay of the FITC-labeled TLR4 and the TxR-labeled MyD88 (merge images), indicating colocalization. (F) Double immunostaining of m-ICcl2 cells for IRAK-1 (red) and TLR4 (green) in unstimulated and LPS- or IL-1β–stimulated cells for 15 min. ×1,000.
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fig3: Recruitment of the TLR4 adaptor protein MyD88 and IRAK-1 to the Golgi apparatus upon LPS stimulation. (A) m-ICcl2 cells transfected with an MyD88-GFP fusion protein were left untreated or stimulated with 10 ng/ml LPS for the indicated time period. (B) m-ICcl2 cells transfected with an MyD88-GFP fusion protein were left untreated or stimulated with 10 ng/ml LPS or 100 ng/ml IL-1β. The cellular activation is illustrated by the nuclear translocation of the NF-κB subunit p65. (C) m-ICcl2 cells carrying the MyD88-GFP expression plasmid were left untreated or stimulated with LPS, fixed, and immunostained for TLR4 (red). (D) m-ICcl2 cells carrying the MyD88-GFP expression plasmid were incubated with 5 μM TR C5 ceramide to identify the Golgi apparatus. Subsequent stimulation with 10 ng/ml LPS for 15 min illustrates recruitment of the TLR4 adaptor protein MyD88 to the Golgi apparatus. (E) Double immunostaining of m-ICcl2 cells for MyD88 (red) and TLR4 (green) after stimulation with 10 ng/ml LPS or 100 ng/ml IL-1β for the indicated time. Note the transient change of the TLR4+ Golgi apparatus from green to yellow resulting from an overlay of the FITC-labeled TLR4 and the TxR-labeled MyD88 (merge images), indicating colocalization. (F) Double immunostaining of m-ICcl2 cells for IRAK-1 (red) and TLR4 (green) in unstimulated and LPS- or IL-1β–stimulated cells for 15 min. ×1,000.

Mentions: Activation of TLR4 leads to recruitment of the adaptor protein MyD88 and the serine/threonine kinase IRAK-1, facilitating downstream signaling. Therefore, we analyzed the cellular localization of MyD88 and IRAK-1 in the presence or absence of LPS to determine the localization of the initiation of the TLR4-mediated signaling cascade. In a first attempt, we used an MyD88-GFP fusion construct recently described (14). Fluorescence microscopy revealed a diffuse cytoplasmic staining of MyD88-GFP in untreated cells. Subsequent stimulation with 10 ng/ml LPS induced rapid redistribution of MyD88-GFP and paranuclear condensation after 15 min exposure (Fig. 3 A). This paranuclear redistribution of MyD88-GFP was observed after stimulation with LPS but not with IL-1β (Fig. 3 B). It colocalized with intracellular TLR4 staining as well as the cellular distribution of the Golgi marker C5 ceramide, indicating that signaling occurred from this TLR4+ compartment (Fig. 3, C and D). In contrast, LPS stimulation of murine macrophage-like RAW 264.7 cells expressing MyD88-GFP induced recruitment to the cell membrane as recently described (unpublished data and 14). In addition, endogenous MyD88 was detected in m-ICcl2 cells using immunohistochemistry using an MyD88-specific antiserum. Similar to the MyD88-GFP fusion protein, endogenous MyD88 concentrated at a paranuclear localization 15 min after LPS stimulation at a concentration of 10 ng/ml and colocalized with TLR4 (Fig. 3 E). Also, IRAK-1 was redistributed toward the TLR4+ subcellular compartment 15 min after exposure to LPS (Fig. 3 F). These data demonstrate assembly of the TLR4-mediated cytoplasmic signaling cascade at the site of the Golgi apparatus in intestinal crypt epithelial m-ICcl2 cells and suggest an active role of Golgi-resident TLR4 in the process of LPS-mediated cell activation.


Intracellular recognition of lipopolysaccharide by toll-like receptor 4 in intestinal epithelial cells.

Hornef MW, Normark BH, Vandewalle A, Normark S - J. Exp. Med. (2003)

Recruitment of the TLR4 adaptor protein MyD88 and IRAK-1 to the Golgi apparatus upon LPS stimulation. (A) m-ICcl2 cells transfected with an MyD88-GFP fusion protein were left untreated or stimulated with 10 ng/ml LPS for the indicated time period. (B) m-ICcl2 cells transfected with an MyD88-GFP fusion protein were left untreated or stimulated with 10 ng/ml LPS or 100 ng/ml IL-1β. The cellular activation is illustrated by the nuclear translocation of the NF-κB subunit p65. (C) m-ICcl2 cells carrying the MyD88-GFP expression plasmid were left untreated or stimulated with LPS, fixed, and immunostained for TLR4 (red). (D) m-ICcl2 cells carrying the MyD88-GFP expression plasmid were incubated with 5 μM TR C5 ceramide to identify the Golgi apparatus. Subsequent stimulation with 10 ng/ml LPS for 15 min illustrates recruitment of the TLR4 adaptor protein MyD88 to the Golgi apparatus. (E) Double immunostaining of m-ICcl2 cells for MyD88 (red) and TLR4 (green) after stimulation with 10 ng/ml LPS or 100 ng/ml IL-1β for the indicated time. Note the transient change of the TLR4+ Golgi apparatus from green to yellow resulting from an overlay of the FITC-labeled TLR4 and the TxR-labeled MyD88 (merge images), indicating colocalization. (F) Double immunostaining of m-ICcl2 cells for IRAK-1 (red) and TLR4 (green) in unstimulated and LPS- or IL-1β–stimulated cells for 15 min. ×1,000.
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Related In: Results  -  Collection

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fig3: Recruitment of the TLR4 adaptor protein MyD88 and IRAK-1 to the Golgi apparatus upon LPS stimulation. (A) m-ICcl2 cells transfected with an MyD88-GFP fusion protein were left untreated or stimulated with 10 ng/ml LPS for the indicated time period. (B) m-ICcl2 cells transfected with an MyD88-GFP fusion protein were left untreated or stimulated with 10 ng/ml LPS or 100 ng/ml IL-1β. The cellular activation is illustrated by the nuclear translocation of the NF-κB subunit p65. (C) m-ICcl2 cells carrying the MyD88-GFP expression plasmid were left untreated or stimulated with LPS, fixed, and immunostained for TLR4 (red). (D) m-ICcl2 cells carrying the MyD88-GFP expression plasmid were incubated with 5 μM TR C5 ceramide to identify the Golgi apparatus. Subsequent stimulation with 10 ng/ml LPS for 15 min illustrates recruitment of the TLR4 adaptor protein MyD88 to the Golgi apparatus. (E) Double immunostaining of m-ICcl2 cells for MyD88 (red) and TLR4 (green) after stimulation with 10 ng/ml LPS or 100 ng/ml IL-1β for the indicated time. Note the transient change of the TLR4+ Golgi apparatus from green to yellow resulting from an overlay of the FITC-labeled TLR4 and the TxR-labeled MyD88 (merge images), indicating colocalization. (F) Double immunostaining of m-ICcl2 cells for IRAK-1 (red) and TLR4 (green) in unstimulated and LPS- or IL-1β–stimulated cells for 15 min. ×1,000.
Mentions: Activation of TLR4 leads to recruitment of the adaptor protein MyD88 and the serine/threonine kinase IRAK-1, facilitating downstream signaling. Therefore, we analyzed the cellular localization of MyD88 and IRAK-1 in the presence or absence of LPS to determine the localization of the initiation of the TLR4-mediated signaling cascade. In a first attempt, we used an MyD88-GFP fusion construct recently described (14). Fluorescence microscopy revealed a diffuse cytoplasmic staining of MyD88-GFP in untreated cells. Subsequent stimulation with 10 ng/ml LPS induced rapid redistribution of MyD88-GFP and paranuclear condensation after 15 min exposure (Fig. 3 A). This paranuclear redistribution of MyD88-GFP was observed after stimulation with LPS but not with IL-1β (Fig. 3 B). It colocalized with intracellular TLR4 staining as well as the cellular distribution of the Golgi marker C5 ceramide, indicating that signaling occurred from this TLR4+ compartment (Fig. 3, C and D). In contrast, LPS stimulation of murine macrophage-like RAW 264.7 cells expressing MyD88-GFP induced recruitment to the cell membrane as recently described (unpublished data and 14). In addition, endogenous MyD88 was detected in m-ICcl2 cells using immunohistochemistry using an MyD88-specific antiserum. Similar to the MyD88-GFP fusion protein, endogenous MyD88 concentrated at a paranuclear localization 15 min after LPS stimulation at a concentration of 10 ng/ml and colocalized with TLR4 (Fig. 3 E). Also, IRAK-1 was redistributed toward the TLR4+ subcellular compartment 15 min after exposure to LPS (Fig. 3 F). These data demonstrate assembly of the TLR4-mediated cytoplasmic signaling cascade at the site of the Golgi apparatus in intestinal crypt epithelial m-ICcl2 cells and suggest an active role of Golgi-resident TLR4 in the process of LPS-mediated cell activation.

Bottom Line: Here we demonstrate that disruption of the integrity of the Golgi apparatus significantly reduced LPS-mediated nuclear factor kappaB activation.In contrast to macrophages, prevention of ligand internalization by use of LPS-coated beads significantly impaired recognition by epithelial cells.Thus, our data provide evidence that in contrast to the situation in macrophages, LPS recognition in intestinal epithelial cells may occur in the Golgi apparatus and require LPS internalization.

View Article: PubMed Central - PubMed

Affiliation: Swedish Institute for Infectious Disease Control SMI, 17182 Solna, Sweden. mathias.hornef@smi.ki.se

ABSTRACT
Toll-like receptor (TLR)4 has recently been shown to reside in the Golgi apparatus of intestinal crypt epithelial m-ICcl2 cells, colocalizing with internalized lipopolysaccharide (LPS). Here we demonstrate that disruption of the integrity of the Golgi apparatus significantly reduced LPS-mediated nuclear factor kappaB activation. Also, the TLR4 adaptor protein MyD88 and the serine/threonine kinase IRAK-1 were rapidly recruited to the Golgi apparatus upon stimulation. LPS-mediated activation required lipid raft formation and intact clathrin-dependent internalization. In contrast to macrophages, prevention of ligand internalization by use of LPS-coated beads significantly impaired recognition by epithelial cells. The localization of TLR4 to the Golgi apparatus was abrogated by expression of a genetically modified form of the TLR4 binding chaperone gp96. Thus, our data provide evidence that in contrast to the situation in macrophages, LPS recognition in intestinal epithelial cells may occur in the Golgi apparatus and require LPS internalization.

Show MeSH
Related in: MedlinePlus