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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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Transcriptional expression and immunostaining of m-ICcl2 cells for CD14. (A) Ribonuclease protection assay using specific antisense probes for murine mRNA encoding CD14 and GAPDH. Before RNA isolation, m-ICcl2 cells were exposed to 0.0, 0.1, or 10.0 μg/ml LPS for 12 h. RAW 264.7 cells were kept unstimulated. (B) Immunostaining for murine CD14 on m-ICcl2 cells. Cells were incubated for 12 h in the presence of various concentrations of LPS before fixation, as indicated. As control, immunostaining was performed on untreated cells by omitting the primary antibody. Immunostaining was detected using the horseradish peroxidase reaction and cells were counterstained with Mayer's hematoxylin. ×1,000. (C) Comparison of MIP-2 secretion in response to LPS stimulation by m-ICcl2 cells cultured in the presence or absence of serum. As control, 2 μg/ml recombinant CD14 was added before LPS stimulation.
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fig2: Transcriptional expression and immunostaining of m-ICcl2 cells for CD14. (A) Ribonuclease protection assay using specific antisense probes for murine mRNA encoding CD14 and GAPDH. Before RNA isolation, m-ICcl2 cells were exposed to 0.0, 0.1, or 10.0 μg/ml LPS for 12 h. RAW 264.7 cells were kept unstimulated. (B) Immunostaining for murine CD14 on m-ICcl2 cells. Cells were incubated for 12 h in the presence of various concentrations of LPS before fixation, as indicated. As control, immunostaining was performed on untreated cells by omitting the primary antibody. Immunostaining was detected using the horseradish peroxidase reaction and cells were counterstained with Mayer's hematoxylin. ×1,000. (C) Comparison of MIP-2 secretion in response to LPS stimulation by m-ICcl2 cells cultured in the presence or absence of serum. As control, 2 μg/ml recombinant CD14 was added before LPS stimulation.

Mentions: The absence of CD14 expression in gastrointestinal tissue was recently suggested to explain the unresponsive phenotype in respect to the normal intestinal flora (12, 13). Surprisingly, CD14 expression was detected by ribonuclease protection assay in m-ICcl2 cells, although at a significantly lower level as RAW 264.7 cells (Fig. 2 A). Furthermore, preincubation of epithelial cells with increasing amounts of LPS significantly enhanced the level of CD14 mRNA expression. The relative amount of CD14 mRNA in m-ICcl2 cells compared with RAW 264.7 cells was <1% in untreated cells, but increased to 14% at 100 ng/ml LPS and 21% at 10 μg/ml LPS for 24 h. CD14 expression was confirmed by immunohistochemistry, demonstrating a weak surface staining on untreated m-ICcl2 cells and an increasingly intense staining signal after LPS exposure (Fig. 2 B). The functional relevance of this autonomous CD14 production was demonstrated by the preservation of the highly LPS-susceptible phenotype of m-ICcl2 cells grown in the absence of serum (Fig. 2 C). Thus, m-ICcl2 cells synthesize CD14 and seem to enhance rather than diminish their LPS-binding capacity in response to LPS exposure.


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)

Transcriptional expression and immunostaining of m-ICcl2 cells for CD14. (A) Ribonuclease protection assay using specific antisense probes for murine mRNA encoding CD14 and GAPDH. Before RNA isolation, m-ICcl2 cells were exposed to 0.0, 0.1, or 10.0 μg/ml LPS for 12 h. RAW 264.7 cells were kept unstimulated. (B) Immunostaining for murine CD14 on m-ICcl2 cells. Cells were incubated for 12 h in the presence of various concentrations of LPS before fixation, as indicated. As control, immunostaining was performed on untreated cells by omitting the primary antibody. Immunostaining was detected using the horseradish peroxidase reaction and cells were counterstained with Mayer's hematoxylin. ×1,000. (C) Comparison of MIP-2 secretion in response to LPS stimulation by m-ICcl2 cells cultured in the presence or absence of serum. As control, 2 μg/ml recombinant CD14 was added before LPS stimulation.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2193765&req=5

fig2: Transcriptional expression and immunostaining of m-ICcl2 cells for CD14. (A) Ribonuclease protection assay using specific antisense probes for murine mRNA encoding CD14 and GAPDH. Before RNA isolation, m-ICcl2 cells were exposed to 0.0, 0.1, or 10.0 μg/ml LPS for 12 h. RAW 264.7 cells were kept unstimulated. (B) Immunostaining for murine CD14 on m-ICcl2 cells. Cells were incubated for 12 h in the presence of various concentrations of LPS before fixation, as indicated. As control, immunostaining was performed on untreated cells by omitting the primary antibody. Immunostaining was detected using the horseradish peroxidase reaction and cells were counterstained with Mayer's hematoxylin. ×1,000. (C) Comparison of MIP-2 secretion in response to LPS stimulation by m-ICcl2 cells cultured in the presence or absence of serum. As control, 2 μg/ml recombinant CD14 was added before LPS stimulation.
Mentions: The absence of CD14 expression in gastrointestinal tissue was recently suggested to explain the unresponsive phenotype in respect to the normal intestinal flora (12, 13). Surprisingly, CD14 expression was detected by ribonuclease protection assay in m-ICcl2 cells, although at a significantly lower level as RAW 264.7 cells (Fig. 2 A). Furthermore, preincubation of epithelial cells with increasing amounts of LPS significantly enhanced the level of CD14 mRNA expression. The relative amount of CD14 mRNA in m-ICcl2 cells compared with RAW 264.7 cells was <1% in untreated cells, but increased to 14% at 100 ng/ml LPS and 21% at 10 μg/ml LPS for 24 h. CD14 expression was confirmed by immunohistochemistry, demonstrating a weak surface staining on untreated m-ICcl2 cells and an increasingly intense staining signal after LPS exposure (Fig. 2 B). The functional relevance of this autonomous CD14 production was demonstrated by the preservation of the highly LPS-susceptible phenotype of m-ICcl2 cells grown in the absence of serum (Fig. 2 C). Thus, m-ICcl2 cells synthesize CD14 and seem to enhance rather than diminish their LPS-binding capacity in response to LPS exposure.

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