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Paired activating and inhibitory immunoglobulin-like receptors, MAIR-I and MAIR-II, regulate mast cell and macrophage activation.

Yotsumoto K, Okoshi Y, Shibuya K, Yamazaki S, Tahara-Hanaoka S, Honda S, Osawa M, Kuroiwa A, Matsuda Y, Tenen DG, Iwama A, Nakauchi H, Shibuya A - J. Exp. Med. (2003)

Bottom Line: Immune responses are regulated by opposing positive and negative signals triggered by the interaction of activating and inhibitory cell surface receptors with their ligands.Here, we describe novel paired activating and inhibitory immunoglobulin-like receptors, designated myeloid-associated immunoglobulin-like receptor (MAIR) I and MAIR-II, whose extracellular domains are highly conserved by each other.Thus, MAIR-I and MAIR-II play important regulatory roles in cell signaling and immune responses.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Immune Receptor, RIKEN Research Center for Allergy and Immunology, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan.

ABSTRACT
Immune responses are regulated by opposing positive and negative signals triggered by the interaction of activating and inhibitory cell surface receptors with their ligands. Here, we describe novel paired activating and inhibitory immunoglobulin-like receptors, designated myeloid-associated immunoglobulin-like receptor (MAIR) I and MAIR-II, whose extracellular domains are highly conserved by each other. MAIR-I, expressed on the majority of myeloid cells, including macrophages, granulocytes, mast cells, and dendritic cells, contains the tyrosine-based sorting motif and the immunoreceptor tyrosine-based inhibitory motif-like sequences in the cytoplasmic domain and mediates endocytosis of the receptor and inhibition of IgE-mediated degranulation from mast cells. On the other hand, MAIR-II, expressed on subsets of peritoneal macrophages and B cells, associates with the immunoreceptor tyrosine-based activation motif-bearing adaptor DAP12 and stimulates proinflammatory cytokines and chemokine secretions from macrophages. Thus, MAIR-I and MAIR-II play important regulatory roles in cell signaling and immune responses.

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Expression of DAP12 in B cells. (A) Spleen cells were stained with biotin-conjugated anti–MAIR-II and PE-conjugated B220, followed by allophycocyanin-conjugated streptavidin. MAIR-II+B220+ and MAIR-II−B220+ cells were purified by repeating sorting twice on flow cytometry (>99.8% purity). The RNA was extracted from the fractionated and total spleen cells and was subjected to semiquantitive RT-PCR (30 cycles) for DAP12 and HPRT, according to template dose by dilution. (B) B cells were purified from spleen cells by repeating positive selection twice with B220+ MACS-beads (>99.0% purity). 5 × 106 purified B cells were stimulated or not with 10 μg/ml LPS for 24 h, lysed in 1% NP-40 lysis buffer, and immunoprecipitated with control Ig or anti-DAP12. Immunoprecipitates or lysates of 106 raw cells were immunoblotted with anti-DAP12.
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fig8: Expression of DAP12 in B cells. (A) Spleen cells were stained with biotin-conjugated anti–MAIR-II and PE-conjugated B220, followed by allophycocyanin-conjugated streptavidin. MAIR-II+B220+ and MAIR-II−B220+ cells were purified by repeating sorting twice on flow cytometry (>99.8% purity). The RNA was extracted from the fractionated and total spleen cells and was subjected to semiquantitive RT-PCR (30 cycles) for DAP12 and HPRT, according to template dose by dilution. (B) B cells were purified from spleen cells by repeating positive selection twice with B220+ MACS-beads (>99.0% purity). 5 × 106 purified B cells were stimulated or not with 10 μg/ml LPS for 24 h, lysed in 1% NP-40 lysis buffer, and immunoprecipitated with control Ig or anti-DAP12. Immunoprecipitates or lysates of 106 raw cells were immunoblotted with anti-DAP12.

Mentions: Although human primary B cells and a mouse B cell line also express DAP12 transcript (35, 36), there has been no paper reporting that primary mouse B cells express DAP12. To investigate the functional role of MAIR-II on B cells, we examined whether spleen B cells express DAP12. B cells were highly fractionated from spleen cells stimulated with LPS according to MAIR-II expression by repeating sorting on flow cytometry (>99.8% purity, as determined by reanalysis by flow cytometry) and subjected to semiquantitative RT-PCR. As demonstrated in Fig. 8 A, both fractions of B cells express DAP12 transcript. To further examine whether B cells express DAP12 protein, DAP12 was immunoprecipitated from purified B cells before or after stimulation with LPS for 24 h and immunoblotted with anti-DAP12. Although DAP12 protein was undetectable in primary resting B cells, we observed a significant up-regulation of DAP12 protein expression in stimulated B cells (Fig. 8 B). These results suggest that MAIR-II associates with DAP12 (also on B cells, at least in the activated state with LPS), resulting in MAIR-II–mediated signaling in B cells.


Paired activating and inhibitory immunoglobulin-like receptors, MAIR-I and MAIR-II, regulate mast cell and macrophage activation.

Yotsumoto K, Okoshi Y, Shibuya K, Yamazaki S, Tahara-Hanaoka S, Honda S, Osawa M, Kuroiwa A, Matsuda Y, Tenen DG, Iwama A, Nakauchi H, Shibuya A - J. Exp. Med. (2003)

Expression of DAP12 in B cells. (A) Spleen cells were stained with biotin-conjugated anti–MAIR-II and PE-conjugated B220, followed by allophycocyanin-conjugated streptavidin. MAIR-II+B220+ and MAIR-II−B220+ cells were purified by repeating sorting twice on flow cytometry (>99.8% purity). The RNA was extracted from the fractionated and total spleen cells and was subjected to semiquantitive RT-PCR (30 cycles) for DAP12 and HPRT, according to template dose by dilution. (B) B cells were purified from spleen cells by repeating positive selection twice with B220+ MACS-beads (>99.0% purity). 5 × 106 purified B cells were stimulated or not with 10 μg/ml LPS for 24 h, lysed in 1% NP-40 lysis buffer, and immunoprecipitated with control Ig or anti-DAP12. Immunoprecipitates or lysates of 106 raw cells were immunoblotted with anti-DAP12.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2194075&req=5

fig8: Expression of DAP12 in B cells. (A) Spleen cells were stained with biotin-conjugated anti–MAIR-II and PE-conjugated B220, followed by allophycocyanin-conjugated streptavidin. MAIR-II+B220+ and MAIR-II−B220+ cells were purified by repeating sorting twice on flow cytometry (>99.8% purity). The RNA was extracted from the fractionated and total spleen cells and was subjected to semiquantitive RT-PCR (30 cycles) for DAP12 and HPRT, according to template dose by dilution. (B) B cells were purified from spleen cells by repeating positive selection twice with B220+ MACS-beads (>99.0% purity). 5 × 106 purified B cells were stimulated or not with 10 μg/ml LPS for 24 h, lysed in 1% NP-40 lysis buffer, and immunoprecipitated with control Ig or anti-DAP12. Immunoprecipitates or lysates of 106 raw cells were immunoblotted with anti-DAP12.
Mentions: Although human primary B cells and a mouse B cell line also express DAP12 transcript (35, 36), there has been no paper reporting that primary mouse B cells express DAP12. To investigate the functional role of MAIR-II on B cells, we examined whether spleen B cells express DAP12. B cells were highly fractionated from spleen cells stimulated with LPS according to MAIR-II expression by repeating sorting on flow cytometry (>99.8% purity, as determined by reanalysis by flow cytometry) and subjected to semiquantitative RT-PCR. As demonstrated in Fig. 8 A, both fractions of B cells express DAP12 transcript. To further examine whether B cells express DAP12 protein, DAP12 was immunoprecipitated from purified B cells before or after stimulation with LPS for 24 h and immunoblotted with anti-DAP12. Although DAP12 protein was undetectable in primary resting B cells, we observed a significant up-regulation of DAP12 protein expression in stimulated B cells (Fig. 8 B). These results suggest that MAIR-II associates with DAP12 (also on B cells, at least in the activated state with LPS), resulting in MAIR-II–mediated signaling in B cells.

Bottom Line: Immune responses are regulated by opposing positive and negative signals triggered by the interaction of activating and inhibitory cell surface receptors with their ligands.Here, we describe novel paired activating and inhibitory immunoglobulin-like receptors, designated myeloid-associated immunoglobulin-like receptor (MAIR) I and MAIR-II, whose extracellular domains are highly conserved by each other.Thus, MAIR-I and MAIR-II play important regulatory roles in cell signaling and immune responses.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Immune Receptor, RIKEN Research Center for Allergy and Immunology, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan.

ABSTRACT
Immune responses are regulated by opposing positive and negative signals triggered by the interaction of activating and inhibitory cell surface receptors with their ligands. Here, we describe novel paired activating and inhibitory immunoglobulin-like receptors, designated myeloid-associated immunoglobulin-like receptor (MAIR) I and MAIR-II, whose extracellular domains are highly conserved by each other. MAIR-I, expressed on the majority of myeloid cells, including macrophages, granulocytes, mast cells, and dendritic cells, contains the tyrosine-based sorting motif and the immunoreceptor tyrosine-based inhibitory motif-like sequences in the cytoplasmic domain and mediates endocytosis of the receptor and inhibition of IgE-mediated degranulation from mast cells. On the other hand, MAIR-II, expressed on subsets of peritoneal macrophages and B cells, associates with the immunoreceptor tyrosine-based activation motif-bearing adaptor DAP12 and stimulates proinflammatory cytokines and chemokine secretions from macrophages. Thus, MAIR-I and MAIR-II play important regulatory roles in cell signaling and immune responses.

Show MeSH
Related in: MedlinePlus