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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 engages receptors on macrophages other than TLRs. (a and b) Bone marrow–derived macrophages from wild-type, TLR2−/−, or MyD88−/− mice were incubated with tetramethylrhodamine isothiocyanate–labeled zymosan for 15 min. Phagocytosis was visualized by immunofluorescence microscopy (a) and quantified by flow cytometry (b). (c) Bone marrow macrophages from MyD88−/− or wild-type mice were stimulated with 100 μg/ml zymosan or 100 ng/ml PAM3CSK4 lipopeptide for 4 h and TNF-α mRNA production was measured by quantitative real-time PCR. (d and e) Production of ROS was assayed by luminol-enhanced chemiluminescence in peritoneal macrophages from wild-type, TLR2−/−, or MyD88−/− mice (d) stimulated with 200 μg/ml zymosan or in RAW 264.7 macrophages (e) stimulated with 100 μg/ml zymosan, 100 ng/ml LPS, or 100 ng/ml PAM3CSK4 lipopeptide.
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fig1: Zymosan engages receptors on macrophages other than TLRs. (a and b) Bone marrow–derived macrophages from wild-type, TLR2−/−, or MyD88−/− mice were incubated with tetramethylrhodamine isothiocyanate–labeled zymosan for 15 min. Phagocytosis was visualized by immunofluorescence microscopy (a) and quantified by flow cytometry (b). (c) Bone marrow macrophages from MyD88−/− or wild-type mice were stimulated with 100 μg/ml zymosan or 100 ng/ml PAM3CSK4 lipopeptide for 4 h and TNF-α mRNA production was measured by quantitative real-time PCR. (d and e) Production of ROS was assayed by luminol-enhanced chemiluminescence in peritoneal macrophages from wild-type, TLR2−/−, or MyD88−/− mice (d) stimulated with 200 μg/ml zymosan or in RAW 264.7 macrophages (e) stimulated with 100 μg/ml zymosan, 100 ng/ml LPS, or 100 ng/ml PAM3CSK4 lipopeptide.

Mentions: We have previously observed that expression of inhibitory forms of TLR2, TLR6, and MyD88 (an adaptor molecule required for TLR signaling; references 4, 5, and 28) do not inhibit recognition and phagocytosis of zymosan by macrophages (11, 13). We have extended these observations using macrophages from TLR2– and MyD88-deficient mice and examining the production of ROS. Phagocytosis of unopsonized zymosan is unaffected in bone marrow–derived macrophages from mice deficient in TLR2 or MyD88, indicating that additional receptor(s) recognize the particle and trigger intracellular signaling for phagocytosis (Fig. 1 , a and b). Macrophages from mice lacking MyD88 fail to produce inflammatory cytokines such as TNF-α in response to the pure TLR2 stimulus, PAM3CSK4 lipopeptide (a synthetic tri-palmitoylated bacterial lipopeptide; reference 13), or in response to the more complex TLR2 stimulus zymosan (Fig. 1 c). Zymosan particles trigger the production of ROS in murine macrophages and although TLR activation is required for cytokine production, TLR2−/− and MyD88−/− macrophages still produce ROS in response to zymosan (Fig. 1 d). TLR stimulation alone is not sufficient to induce ROS because PAM3CSK4 lipopeptide and LPS fail to trigger ROS production in a mouse macrophage cell line (Fig. 1 e) or in primary peritoneal macrophages (unpublished data). Thus, recognition through receptors independent of TLRs mediates ROS production and phagocytosis.


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 engages receptors on macrophages other than TLRs. (a and b) Bone marrow–derived macrophages from wild-type, TLR2−/−, or MyD88−/− mice were incubated with tetramethylrhodamine isothiocyanate–labeled zymosan for 15 min. Phagocytosis was visualized by immunofluorescence microscopy (a) and quantified by flow cytometry (b). (c) Bone marrow macrophages from MyD88−/− or wild-type mice were stimulated with 100 μg/ml zymosan or 100 ng/ml PAM3CSK4 lipopeptide for 4 h and TNF-α mRNA production was measured by quantitative real-time PCR. (d and e) Production of ROS was assayed by luminol-enhanced chemiluminescence in peritoneal macrophages from wild-type, TLR2−/−, or MyD88−/− mice (d) stimulated with 200 μg/ml zymosan or in RAW 264.7 macrophages (e) stimulated with 100 μg/ml zymosan, 100 ng/ml LPS, or 100 ng/ml PAM3CSK4 lipopeptide.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2193968&req=5

fig1: Zymosan engages receptors on macrophages other than TLRs. (a and b) Bone marrow–derived macrophages from wild-type, TLR2−/−, or MyD88−/− mice were incubated with tetramethylrhodamine isothiocyanate–labeled zymosan for 15 min. Phagocytosis was visualized by immunofluorescence microscopy (a) and quantified by flow cytometry (b). (c) Bone marrow macrophages from MyD88−/− or wild-type mice were stimulated with 100 μg/ml zymosan or 100 ng/ml PAM3CSK4 lipopeptide for 4 h and TNF-α mRNA production was measured by quantitative real-time PCR. (d and e) Production of ROS was assayed by luminol-enhanced chemiluminescence in peritoneal macrophages from wild-type, TLR2−/−, or MyD88−/− mice (d) stimulated with 200 μg/ml zymosan or in RAW 264.7 macrophages (e) stimulated with 100 μg/ml zymosan, 100 ng/ml LPS, or 100 ng/ml PAM3CSK4 lipopeptide.
Mentions: We have previously observed that expression of inhibitory forms of TLR2, TLR6, and MyD88 (an adaptor molecule required for TLR signaling; references 4, 5, and 28) do not inhibit recognition and phagocytosis of zymosan by macrophages (11, 13). We have extended these observations using macrophages from TLR2– and MyD88-deficient mice and examining the production of ROS. Phagocytosis of unopsonized zymosan is unaffected in bone marrow–derived macrophages from mice deficient in TLR2 or MyD88, indicating that additional receptor(s) recognize the particle and trigger intracellular signaling for phagocytosis (Fig. 1 , a and b). Macrophages from mice lacking MyD88 fail to produce inflammatory cytokines such as TNF-α in response to the pure TLR2 stimulus, PAM3CSK4 lipopeptide (a synthetic tri-palmitoylated bacterial lipopeptide; reference 13), or in response to the more complex TLR2 stimulus zymosan (Fig. 1 c). Zymosan particles trigger the production of ROS in murine macrophages and although TLR activation is required for cytokine production, TLR2−/− and MyD88−/− macrophages still produce ROS in response to zymosan (Fig. 1 d). TLR stimulation alone is not sufficient to induce ROS because PAM3CSK4 lipopeptide and LPS fail to trigger ROS production in a mouse macrophage cell line (Fig. 1 e) or in primary peritoneal macrophages (unpublished data). Thus, recognition through receptors independent of TLRs mediates ROS production and phagocytosis.

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