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Toll-like receptor 4 resides in the Golgi apparatus and colocalizes with internalized lipopolysaccharide in intestinal epithelial cells.

Hornef MW, Frisan T, Vandewalle A, Normark S, Richter-Dahlfors A - J. Exp. Med. (2002)

Bottom Line: Toll-like receptor (TLR) 4 is mainly found on cells of the myelopoietic lineage.LPS added to the supernatant was internalized by m-IC(cl2) cells and colocalized with TLR4.The cytoplasmic location of TLR4, which is identical to the final location of internalized LPS, further indicates an important role of cellular internalization and cytoplasmic traffic in the process of innate immune recognition.

View Article: PubMed Central - PubMed

Affiliation: Microbiology and Tumor Biology Center, Karolinska Institutet, 17177 Stockholm, Sweden. mathias.hornef@mtc.ki.se

ABSTRACT
Toll-like receptor (TLR) 4 is mainly found on cells of the myelopoietic lineage. It recognizes lipopolysaccharide (LPS) and mediates cellular activation and production of proinflammatory cytokines. Less is known about the distribution and role of TLR4 in epithelial cells that are continuously exposed to microbes and microbial products. Here we show that the murine small intestinal epithelial cell line m-IC(cl2) is highly responsive to LPS and expresses both CD14 and TLR4. Transcription and surface membrane staining for CD14 were up-regulated upon LPS exposure. Surprisingly, TLR4 immunostaining revealed a strictly cytoplasmic paranuclear distribution. This paranuclear compartment could be identified as the Golgi apparatus. LPS added to the supernatant was internalized by m-IC(cl2) cells and colocalized with TLR4. Continuous exposure to LPS led to a tolerant phenotype but did not alter TLR4 expression nor cellular distribution. Thus, intestinal epithelial cells might be able to provide the initial proinflammatory signal to attract professional immune cells to the side of infection. The cytoplasmic location of TLR4, which is identical to the final location of internalized LPS, further indicates an important role of cellular internalization and cytoplasmic traffic in the process of innate immune recognition.

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Analysis of TLR4 expression in m-ICcl2 cells and isolated intestinal crypts. (A) Ribonuclease protection assay using radioactively labeled antisense RNA probes specifically hybridizing with murine tlr4, md-2, tlr2, l32, and gapdh mRNA. (B) Immunostaining for murine TLR4 on RAW 264.7 cells and m-ICcl2 cells. As control, the primary antibody was incubated with 10 μg/ml of the peptide before use for immunization. ×1,000. (C) Immunostaining of peritoneal macrophages and bone marrow–derived macrophages isolated from wild-type C57BL/10ScSn and TLR4-deficient C57BL/10ScN mice. ×1,000. (D) Western blot of m-ICcl2 cell lysate revealing a band of the predicted protein size of TLR4 at ∼96 kD (arrow). In addition, a larger band at ∼120 kD was found representing the glycosylated form of TLR4 (arrowhead). Overlay with Con A showing binding to the larger band (arrowhead). (E) The 120-kD band of m-ICcl2 cell lysate stained with TLR4 after treatment with Endo H or PNGase F. (F) Comparison of TLR4 staining in m-ICcl2 cells using our TLR4 antibody and the TLR4 sc-12511 antibody. As control, the peptide-blocked primary antibody, or only the secondary antibody, was used for TLR4 and sc-12511, respectively. (G) TLR4 staining of isolated murine small intestinal crypts. Crypts were isolated from the small intestine of C57BL/10ScSn and TLR4-deficient C57BL/10ScN mice. ×400.
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fig3: Analysis of TLR4 expression in m-ICcl2 cells and isolated intestinal crypts. (A) Ribonuclease protection assay using radioactively labeled antisense RNA probes specifically hybridizing with murine tlr4, md-2, tlr2, l32, and gapdh mRNA. (B) Immunostaining for murine TLR4 on RAW 264.7 cells and m-ICcl2 cells. As control, the primary antibody was incubated with 10 μg/ml of the peptide before use for immunization. ×1,000. (C) Immunostaining of peritoneal macrophages and bone marrow–derived macrophages isolated from wild-type C57BL/10ScSn and TLR4-deficient C57BL/10ScN mice. ×1,000. (D) Western blot of m-ICcl2 cell lysate revealing a band of the predicted protein size of TLR4 at ∼96 kD (arrow). In addition, a larger band at ∼120 kD was found representing the glycosylated form of TLR4 (arrowhead). Overlay with Con A showing binding to the larger band (arrowhead). (E) The 120-kD band of m-ICcl2 cell lysate stained with TLR4 after treatment with Endo H or PNGase F. (F) Comparison of TLR4 staining in m-ICcl2 cells using our TLR4 antibody and the TLR4 sc-12511 antibody. As control, the peptide-blocked primary antibody, or only the secondary antibody, was used for TLR4 and sc-12511, respectively. (G) TLR4 staining of isolated murine small intestinal crypts. Crypts were isolated from the small intestine of C57BL/10ScSn and TLR4-deficient C57BL/10ScN mice. ×400.

Mentions: Although TLR4 might not directly bind LPS, it represents the sensor portion of the LPS recognition complex indicated by chimeric transfection experiments (20, 21). Expression of TLR4 as well as the accessory protein MD-2 was detected in epithelial m-ICcl2 cells by ribonuclease protection assay (Fig. 3 A). Interestingly, the transcriptional levels of TLR4 and MD-2 did not significantly differ from those found in the macrophage RAW 264.7 cells. TLR2 transcription could also be demonstrated in m-ICcl2 cells, suggesting that these cells recognize a broader spectrum of microbial pattern molecules.


Toll-like receptor 4 resides in the Golgi apparatus and colocalizes with internalized lipopolysaccharide in intestinal epithelial cells.

Hornef MW, Frisan T, Vandewalle A, Normark S, Richter-Dahlfors A - J. Exp. Med. (2002)

Analysis of TLR4 expression in m-ICcl2 cells and isolated intestinal crypts. (A) Ribonuclease protection assay using radioactively labeled antisense RNA probes specifically hybridizing with murine tlr4, md-2, tlr2, l32, and gapdh mRNA. (B) Immunostaining for murine TLR4 on RAW 264.7 cells and m-ICcl2 cells. As control, the primary antibody was incubated with 10 μg/ml of the peptide before use for immunization. ×1,000. (C) Immunostaining of peritoneal macrophages and bone marrow–derived macrophages isolated from wild-type C57BL/10ScSn and TLR4-deficient C57BL/10ScN mice. ×1,000. (D) Western blot of m-ICcl2 cell lysate revealing a band of the predicted protein size of TLR4 at ∼96 kD (arrow). In addition, a larger band at ∼120 kD was found representing the glycosylated form of TLR4 (arrowhead). Overlay with Con A showing binding to the larger band (arrowhead). (E) The 120-kD band of m-ICcl2 cell lysate stained with TLR4 after treatment with Endo H or PNGase F. (F) Comparison of TLR4 staining in m-ICcl2 cells using our TLR4 antibody and the TLR4 sc-12511 antibody. As control, the peptide-blocked primary antibody, or only the secondary antibody, was used for TLR4 and sc-12511, respectively. (G) TLR4 staining of isolated murine small intestinal crypts. Crypts were isolated from the small intestine of C57BL/10ScSn and TLR4-deficient C57BL/10ScN mice. ×400.
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Related In: Results  -  Collection

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fig3: Analysis of TLR4 expression in m-ICcl2 cells and isolated intestinal crypts. (A) Ribonuclease protection assay using radioactively labeled antisense RNA probes specifically hybridizing with murine tlr4, md-2, tlr2, l32, and gapdh mRNA. (B) Immunostaining for murine TLR4 on RAW 264.7 cells and m-ICcl2 cells. As control, the primary antibody was incubated with 10 μg/ml of the peptide before use for immunization. ×1,000. (C) Immunostaining of peritoneal macrophages and bone marrow–derived macrophages isolated from wild-type C57BL/10ScSn and TLR4-deficient C57BL/10ScN mice. ×1,000. (D) Western blot of m-ICcl2 cell lysate revealing a band of the predicted protein size of TLR4 at ∼96 kD (arrow). In addition, a larger band at ∼120 kD was found representing the glycosylated form of TLR4 (arrowhead). Overlay with Con A showing binding to the larger band (arrowhead). (E) The 120-kD band of m-ICcl2 cell lysate stained with TLR4 after treatment with Endo H or PNGase F. (F) Comparison of TLR4 staining in m-ICcl2 cells using our TLR4 antibody and the TLR4 sc-12511 antibody. As control, the peptide-blocked primary antibody, or only the secondary antibody, was used for TLR4 and sc-12511, respectively. (G) TLR4 staining of isolated murine small intestinal crypts. Crypts were isolated from the small intestine of C57BL/10ScSn and TLR4-deficient C57BL/10ScN mice. ×400.
Mentions: Although TLR4 might not directly bind LPS, it represents the sensor portion of the LPS recognition complex indicated by chimeric transfection experiments (20, 21). Expression of TLR4 as well as the accessory protein MD-2 was detected in epithelial m-ICcl2 cells by ribonuclease protection assay (Fig. 3 A). Interestingly, the transcriptional levels of TLR4 and MD-2 did not significantly differ from those found in the macrophage RAW 264.7 cells. TLR2 transcription could also be demonstrated in m-ICcl2 cells, suggesting that these cells recognize a broader spectrum of microbial pattern molecules.

Bottom Line: Toll-like receptor (TLR) 4 is mainly found on cells of the myelopoietic lineage.LPS added to the supernatant was internalized by m-IC(cl2) cells and colocalized with TLR4.The cytoplasmic location of TLR4, which is identical to the final location of internalized LPS, further indicates an important role of cellular internalization and cytoplasmic traffic in the process of innate immune recognition.

View Article: PubMed Central - PubMed

Affiliation: Microbiology and Tumor Biology Center, Karolinska Institutet, 17177 Stockholm, Sweden. mathias.hornef@mtc.ki.se

ABSTRACT
Toll-like receptor (TLR) 4 is mainly found on cells of the myelopoietic lineage. It recognizes lipopolysaccharide (LPS) and mediates cellular activation and production of proinflammatory cytokines. Less is known about the distribution and role of TLR4 in epithelial cells that are continuously exposed to microbes and microbial products. Here we show that the murine small intestinal epithelial cell line m-IC(cl2) is highly responsive to LPS and expresses both CD14 and TLR4. Transcription and surface membrane staining for CD14 were up-regulated upon LPS exposure. Surprisingly, TLR4 immunostaining revealed a strictly cytoplasmic paranuclear distribution. This paranuclear compartment could be identified as the Golgi apparatus. LPS added to the supernatant was internalized by m-IC(cl2) cells and colocalized with TLR4. Continuous exposure to LPS led to a tolerant phenotype but did not alter TLR4 expression nor cellular distribution. Thus, intestinal epithelial cells might be able to provide the initial proinflammatory signal to attract professional immune cells to the side of infection. The cytoplasmic location of TLR4, which is identical to the final location of internalized LPS, further indicates an important role of cellular internalization and cytoplasmic traffic in the process of innate immune recognition.

Show MeSH
Related in: MedlinePlus