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Collaborative induction of inflammatory responses by dectin-1 and Toll-like receptor 2.

Gantner BN, Simmons RM, Canavera SJ, Akira S, Underhill DM - J. Exp. Med. (2003)

Bottom Line: Dectin-1, which is expressed at low levels on macrophages and high levels on dendritic cells, contains an immunoreceptor tyrosine-based activation motif-like signaling motif that is tyrosine phosphorylated upon activation.The receptor is recruited to phagosomes containing zymosan particles but not to phagosomes containing immunoglobulin G-opsonized particles.Dectin-1 expression enhances TLR-mediated activation of nuclear factor kappa B by beta-glucan-containing particles, and in macrophages and dendritic cells dectin-1 and TLRs are synergistic in mediating production of cytokines such as interleukin 12 and tumor necrosis factor alpha.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, University of Washington, Seattle, 98103, USA.

ABSTRACT
Toll-like receptors (TLRs) mediate recognition of a wide range of microbial products including lipopolysaccharides, lipoproteins, flagellin, and bacterial DNA, and signaling through TLRs leads to the production of inflammatory mediators. In addition to TLRs, many other surface receptors have been proposed to participate in innate immunity and microbial recognition, and signaling through some of these receptors is likely to cooperate with TLR signaling in defining inflammatory responses. In this report we have examined how dectin-1, a lectin family receptor for beta-glucans, collaborates with TLRs in recognizing microbes. Dectin-1, which is expressed at low levels on macrophages and high levels on dendritic cells, contains an immunoreceptor tyrosine-based activation motif-like signaling motif that is tyrosine phosphorylated upon activation. The receptor is recruited to phagosomes containing zymosan particles but not to phagosomes containing immunoglobulin G-opsonized particles. Dectin-1 expression enhances TLR-mediated activation of nuclear factor kappa B by beta-glucan-containing particles, and in macrophages and dendritic cells dectin-1 and TLRs are synergistic in mediating production of cytokines such as interleukin 12 and tumor necrosis factor alpha. Additionally, dectin-1 triggers production of reactive oxygen species, an inflammatory response that is primed by TLR activation. The data demonstrate that collaborative recognition of distinct microbial components by different classes of innate immune receptors is crucial in orchestrating inflammatory responses.

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Zymosan triggers activation of dectin-1 and enhanced inflammatory responses in mouse macrophages. (a) V5 epitope–tagged dectin-1 expression was measured by flow cytometry in RAW cells stably overexpressing wild-type dectin-1 (green line) and in control RAW cells (shaded). (b) Control cells (black line) and cells overexpressing dectin-1 (green line) were incubated with tetramethylrhodamine isothiocyanate zymosan for 1 h and phagocytosis was measured by flow cytometry and compared with unfed control cells (shaded). (c–e) The cellular distribution of epitope-tagged dectin-1 (green) was examined by immunofluorescence microscopy in resting cells (c), cells fed zymosan (red) for 5 min (d), or in cells fed IgG-opsonized sheep red blood cells (red) for 5 min (e). (f) Cells expressing V5-tagged dectin-1 were fed 100 μg/ml zymosan for 15 min in the presence or absence of 50 μg/ml laminarin, total cell lysates were prepared, and the protein was detected in all lysates by immunoblot using an antibody for the tag (bottom). Tyrosine-phosphorylated proteins were immunoprecipitated from the lysates and recovery of dectin-1 was analyzed by immunoblot (top). (g and h) Induction of inflammatory responses was measured by ELISA in control RAW cells and cells overexpressing dectin-1 stimulated with 100 μg/ml zymosan (zym), 100 ng/ml PAM3CSK4 lipopeptide (Lip), or 100 ng/ml LPS for 4 (g, TNF-α) or 24 h (h, IL-12 p40) in the presence or absence of 500 μg/ml laminarin as indicated. N.D., none detected. (i) Induction of IL-12 p40 mRNA was measured by quantitative real-time PCR in cells stimulated for 4 h as in g.
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fig3: Zymosan triggers activation of dectin-1 and enhanced inflammatory responses in mouse macrophages. (a) V5 epitope–tagged dectin-1 expression was measured by flow cytometry in RAW cells stably overexpressing wild-type dectin-1 (green line) and in control RAW cells (shaded). (b) Control cells (black line) and cells overexpressing dectin-1 (green line) were incubated with tetramethylrhodamine isothiocyanate zymosan for 1 h and phagocytosis was measured by flow cytometry and compared with unfed control cells (shaded). (c–e) The cellular distribution of epitope-tagged dectin-1 (green) was examined by immunofluorescence microscopy in resting cells (c), cells fed zymosan (red) for 5 min (d), or in cells fed IgG-opsonized sheep red blood cells (red) for 5 min (e). (f) Cells expressing V5-tagged dectin-1 were fed 100 μg/ml zymosan for 15 min in the presence or absence of 50 μg/ml laminarin, total cell lysates were prepared, and the protein was detected in all lysates by immunoblot using an antibody for the tag (bottom). Tyrosine-phosphorylated proteins were immunoprecipitated from the lysates and recovery of dectin-1 was analyzed by immunoblot (top). (g and h) Induction of inflammatory responses was measured by ELISA in control RAW cells and cells overexpressing dectin-1 stimulated with 100 μg/ml zymosan (zym), 100 ng/ml PAM3CSK4 lipopeptide (Lip), or 100 ng/ml LPS for 4 (g, TNF-α) or 24 h (h, IL-12 p40) in the presence or absence of 500 μg/ml laminarin as indicated. N.D., none detected. (i) Induction of IL-12 p40 mRNA was measured by quantitative real-time PCR in cells stimulated for 4 h as in g.

Mentions: To examine the role of β-glucan recognition in zymosan-induced inflammatory signaling in macrophages, we established a model system using the RAW 264.7 (RAW) mouse macrophage cell line. RAW cells express surface CD14, low levels of mRNA for dectin-1, and bind and internalize zymosan. We generated a stable population of cells overexpressing an epitope-tagged dectin-1. By real-time PCR, these cells express 34-fold (±3) more dectin-1 mRNA than the parental cells. As demonstrated by flow cytometry, the population uniformly expresses epitope-tagged dectin-1 (Fig. 3 a) and immunofluorescence microscopy revealed that the receptor is expressed evenly at the cell surface and is also found in some intracellular compartments (Fig. 3 c).


Collaborative induction of inflammatory responses by dectin-1 and Toll-like receptor 2.

Gantner BN, Simmons RM, Canavera SJ, Akira S, Underhill DM - J. Exp. Med. (2003)

Zymosan triggers activation of dectin-1 and enhanced inflammatory responses in mouse macrophages. (a) V5 epitope–tagged dectin-1 expression was measured by flow cytometry in RAW cells stably overexpressing wild-type dectin-1 (green line) and in control RAW cells (shaded). (b) Control cells (black line) and cells overexpressing dectin-1 (green line) were incubated with tetramethylrhodamine isothiocyanate zymosan for 1 h and phagocytosis was measured by flow cytometry and compared with unfed control cells (shaded). (c–e) The cellular distribution of epitope-tagged dectin-1 (green) was examined by immunofluorescence microscopy in resting cells (c), cells fed zymosan (red) for 5 min (d), or in cells fed IgG-opsonized sheep red blood cells (red) for 5 min (e). (f) Cells expressing V5-tagged dectin-1 were fed 100 μg/ml zymosan for 15 min in the presence or absence of 50 μg/ml laminarin, total cell lysates were prepared, and the protein was detected in all lysates by immunoblot using an antibody for the tag (bottom). Tyrosine-phosphorylated proteins were immunoprecipitated from the lysates and recovery of dectin-1 was analyzed by immunoblot (top). (g and h) Induction of inflammatory responses was measured by ELISA in control RAW cells and cells overexpressing dectin-1 stimulated with 100 μg/ml zymosan (zym), 100 ng/ml PAM3CSK4 lipopeptide (Lip), or 100 ng/ml LPS for 4 (g, TNF-α) or 24 h (h, IL-12 p40) in the presence or absence of 500 μg/ml laminarin as indicated. N.D., none detected. (i) Induction of IL-12 p40 mRNA was measured by quantitative real-time PCR in cells stimulated for 4 h as in g.
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fig3: Zymosan triggers activation of dectin-1 and enhanced inflammatory responses in mouse macrophages. (a) V5 epitope–tagged dectin-1 expression was measured by flow cytometry in RAW cells stably overexpressing wild-type dectin-1 (green line) and in control RAW cells (shaded). (b) Control cells (black line) and cells overexpressing dectin-1 (green line) were incubated with tetramethylrhodamine isothiocyanate zymosan for 1 h and phagocytosis was measured by flow cytometry and compared with unfed control cells (shaded). (c–e) The cellular distribution of epitope-tagged dectin-1 (green) was examined by immunofluorescence microscopy in resting cells (c), cells fed zymosan (red) for 5 min (d), or in cells fed IgG-opsonized sheep red blood cells (red) for 5 min (e). (f) Cells expressing V5-tagged dectin-1 were fed 100 μg/ml zymosan for 15 min in the presence or absence of 50 μg/ml laminarin, total cell lysates were prepared, and the protein was detected in all lysates by immunoblot using an antibody for the tag (bottom). Tyrosine-phosphorylated proteins were immunoprecipitated from the lysates and recovery of dectin-1 was analyzed by immunoblot (top). (g and h) Induction of inflammatory responses was measured by ELISA in control RAW cells and cells overexpressing dectin-1 stimulated with 100 μg/ml zymosan (zym), 100 ng/ml PAM3CSK4 lipopeptide (Lip), or 100 ng/ml LPS for 4 (g, TNF-α) or 24 h (h, IL-12 p40) in the presence or absence of 500 μg/ml laminarin as indicated. N.D., none detected. (i) Induction of IL-12 p40 mRNA was measured by quantitative real-time PCR in cells stimulated for 4 h as in g.
Mentions: To examine the role of β-glucan recognition in zymosan-induced inflammatory signaling in macrophages, we established a model system using the RAW 264.7 (RAW) mouse macrophage cell line. RAW cells express surface CD14, low levels of mRNA for dectin-1, and bind and internalize zymosan. We generated a stable population of cells overexpressing an epitope-tagged dectin-1. By real-time PCR, these cells express 34-fold (±3) more dectin-1 mRNA than the parental cells. As demonstrated by flow cytometry, the population uniformly expresses epitope-tagged dectin-1 (Fig. 3 a) and immunofluorescence microscopy revealed that the receptor is expressed evenly at the cell surface and is also found in some intracellular compartments (Fig. 3 c).

Bottom Line: Dectin-1, which is expressed at low levels on macrophages and high levels on dendritic cells, contains an immunoreceptor tyrosine-based activation motif-like signaling motif that is tyrosine phosphorylated upon activation.The receptor is recruited to phagosomes containing zymosan particles but not to phagosomes containing immunoglobulin G-opsonized particles.Dectin-1 expression enhances TLR-mediated activation of nuclear factor kappa B by beta-glucan-containing particles, and in macrophages and dendritic cells dectin-1 and TLRs are synergistic in mediating production of cytokines such as interleukin 12 and tumor necrosis factor alpha.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, University of Washington, Seattle, 98103, USA.

ABSTRACT
Toll-like receptors (TLRs) mediate recognition of a wide range of microbial products including lipopolysaccharides, lipoproteins, flagellin, and bacterial DNA, and signaling through TLRs leads to the production of inflammatory mediators. In addition to TLRs, many other surface receptors have been proposed to participate in innate immunity and microbial recognition, and signaling through some of these receptors is likely to cooperate with TLR signaling in defining inflammatory responses. In this report we have examined how dectin-1, a lectin family receptor for beta-glucans, collaborates with TLRs in recognizing microbes. Dectin-1, which is expressed at low levels on macrophages and high levels on dendritic cells, contains an immunoreceptor tyrosine-based activation motif-like signaling motif that is tyrosine phosphorylated upon activation. The receptor is recruited to phagosomes containing zymosan particles but not to phagosomes containing immunoglobulin G-opsonized particles. Dectin-1 expression enhances TLR-mediated activation of nuclear factor kappa B by beta-glucan-containing particles, and in macrophages and dendritic cells dectin-1 and TLRs are synergistic in mediating production of cytokines such as interleukin 12 and tumor necrosis factor alpha. Additionally, dectin-1 triggers production of reactive oxygen species, an inflammatory response that is primed by TLR activation. The data demonstrate that collaborative recognition of distinct microbial components by different classes of innate immune receptors is crucial in orchestrating inflammatory responses.

Show MeSH
Related in: MedlinePlus