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Mast cell-expressed orphan receptor CCRL2 binds chemerin and is required for optimal induction of IgE-mediated passive cutaneous anaphylaxis.

Zabel BA, Nakae S, Zúñiga L, Kim JY, Ohyama T, Alt C, Pan J, Suto H, Soler D, Allen SJ, Handel TM, Song CH, Galli SJ, Butcher EC - J. Exp. Med. (2008)

Bottom Line: Mast cells contribute importantly to both protective and pathological IgE-dependent immune responses.In contrast to other "silent" or professional chemokine interreceptors, chemerin binding does not trigger ligand internalization.Rather, CCRL2 is able to bind the chemoattractant and increase local concentrations of bioactive chemerin, thus providing a link between CCRL2 expression and inflammation via the cell-signaling chemerin receptor CMKLR1.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Immunology and Vascular Biology, Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA. bazabel@alum.mit.edu

ABSTRACT
Mast cells contribute importantly to both protective and pathological IgE-dependent immune responses. We show that the mast cell-expressed orphan serpentine receptor mCCRL2 is not required for expression of IgE-mediated mast cell-dependent passive cutaneous anaphylaxis but can enhance the tissue swelling and leukocyte infiltrates associated with such reactions in mice. We further identify chemerin as a natural nonsignaling protein ligand for both human and mouse CCRL2. In contrast to other "silent" or professional chemokine interreceptors, chemerin binding does not trigger ligand internalization. Rather, CCRL2 is able to bind the chemoattractant and increase local concentrations of bioactive chemerin, thus providing a link between CCRL2 expression and inflammation via the cell-signaling chemerin receptor CMKLR1.

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CCRL2 binds chemerin. (A) Chemerin blocks anti-CCRL2 mAb binding. Various concentrations of human chemerin or CCL2 were incubated with total peritoneal mast cells on ice for 5 min, followed by incubation with CCRL2-specific mAb BZ2E3 or anti-IgE and detected with secondary anti–rat PE or anti–mouse IgE PE. (B and C) Radiolabeled chemerin binding. (B) Displacement of iodinated chemerin (residues 21–148) binding to mCMKLR1, huCCRL2, and mCCRL2 by full-length chemerin. (C) Saturation binding of 125I-chemerin21-148 to mCCRL2-transfected cells. (D) Immunofluorescence-based chemerin binding. Various concentrations of untagged serum form chemerin were incubated with mCCRL2-HA, huCCRL2-HA, mCRTH2-HA, or mCMKLR1-HA L1.2 transfectants in the presence of 10 nM His8-tagged serum form chemerin. Samples were incubated on ice for 30 min. Secondary anti-His6 PE was added to detect levels of bound His8-tagged chemerin, and mean fluorescence intensity values are displayed. Mean fluorescence intensity ± range of duplicate staining wells are shown. (E) Mast cell binding. 1,000 nM of untagged chemerin isoforms were incubated with total peritoneal cells from either WT or CCRL2 KO mice in the presence or absence of 10 nM His8-tagged chemerin isoforms. Secondary anti-His6 PE was added to detect levels of bound His8-tagged chemerin. SSChigh F4/80− c-kit+ mast cells were analyzed. For B and C, the mean of quadruplicate wells ±SD is shown for individual experiments. A representative dataset of the three (for B, D, and E) or two experiments (for A and C) performed, each of which gave similar results, is shown.
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fig6: CCRL2 binds chemerin. (A) Chemerin blocks anti-CCRL2 mAb binding. Various concentrations of human chemerin or CCL2 were incubated with total peritoneal mast cells on ice for 5 min, followed by incubation with CCRL2-specific mAb BZ2E3 or anti-IgE and detected with secondary anti–rat PE or anti–mouse IgE PE. (B and C) Radiolabeled chemerin binding. (B) Displacement of iodinated chemerin (residues 21–148) binding to mCMKLR1, huCCRL2, and mCCRL2 by full-length chemerin. (C) Saturation binding of 125I-chemerin21-148 to mCCRL2-transfected cells. (D) Immunofluorescence-based chemerin binding. Various concentrations of untagged serum form chemerin were incubated with mCCRL2-HA, huCCRL2-HA, mCRTH2-HA, or mCMKLR1-HA L1.2 transfectants in the presence of 10 nM His8-tagged serum form chemerin. Samples were incubated on ice for 30 min. Secondary anti-His6 PE was added to detect levels of bound His8-tagged chemerin, and mean fluorescence intensity values are displayed. Mean fluorescence intensity ± range of duplicate staining wells are shown. (E) Mast cell binding. 1,000 nM of untagged chemerin isoforms were incubated with total peritoneal cells from either WT or CCRL2 KO mice in the presence or absence of 10 nM His8-tagged chemerin isoforms. Secondary anti-His6 PE was added to detect levels of bound His8-tagged chemerin. SSChigh F4/80− c-kit+ mast cells were analyzed. For B and C, the mean of quadruplicate wells ±SD is shown for individual experiments. A representative dataset of the three (for B, D, and E) or two experiments (for A and C) performed, each of which gave similar results, is shown.

Mentions: Although we failed to identify evidence of signaling effects of any of the tested chemoattractants, we were able to identify a high-affinity ligand for the receptor: in independent studies in which we were using our anti-CCRL2 mAbs as controls for staining, we serendipitously discovered that chemerin, a protein ligand for signaling receptor CMKLR1 (for review see reference 28), inhibited the binding of mCCRL2-specific mAbs to mouse peritoneal mast cells. In Fig. 6 A, we illustrate the potent ability of chemerin to inhibit anti-CCRL2 staining of mouse peritoneal mast cells. Increasing concentrations of chemerin blocked the binding of anti-mCCRL2 BZ5B8 (Fig. 6 A) and BZ2E3 (not depicted) in a dose-dependent manner (EC50 = 21 nM). The effect was specific to anti-mCCRL2:mCCRL2 interactions because binding of IgE to FcRεI was unaffected by 1,000 nM chemerin (Fig. 6 A), and 1,000 nM CCL2 had no effect on CCRL2 staining (Fig. 6 A).


Mast cell-expressed orphan receptor CCRL2 binds chemerin and is required for optimal induction of IgE-mediated passive cutaneous anaphylaxis.

Zabel BA, Nakae S, Zúñiga L, Kim JY, Ohyama T, Alt C, Pan J, Suto H, Soler D, Allen SJ, Handel TM, Song CH, Galli SJ, Butcher EC - J. Exp. Med. (2008)

CCRL2 binds chemerin. (A) Chemerin blocks anti-CCRL2 mAb binding. Various concentrations of human chemerin or CCL2 were incubated with total peritoneal mast cells on ice for 5 min, followed by incubation with CCRL2-specific mAb BZ2E3 or anti-IgE and detected with secondary anti–rat PE or anti–mouse IgE PE. (B and C) Radiolabeled chemerin binding. (B) Displacement of iodinated chemerin (residues 21–148) binding to mCMKLR1, huCCRL2, and mCCRL2 by full-length chemerin. (C) Saturation binding of 125I-chemerin21-148 to mCCRL2-transfected cells. (D) Immunofluorescence-based chemerin binding. Various concentrations of untagged serum form chemerin were incubated with mCCRL2-HA, huCCRL2-HA, mCRTH2-HA, or mCMKLR1-HA L1.2 transfectants in the presence of 10 nM His8-tagged serum form chemerin. Samples were incubated on ice for 30 min. Secondary anti-His6 PE was added to detect levels of bound His8-tagged chemerin, and mean fluorescence intensity values are displayed. Mean fluorescence intensity ± range of duplicate staining wells are shown. (E) Mast cell binding. 1,000 nM of untagged chemerin isoforms were incubated with total peritoneal cells from either WT or CCRL2 KO mice in the presence or absence of 10 nM His8-tagged chemerin isoforms. Secondary anti-His6 PE was added to detect levels of bound His8-tagged chemerin. SSChigh F4/80− c-kit+ mast cells were analyzed. For B and C, the mean of quadruplicate wells ±SD is shown for individual experiments. A representative dataset of the three (for B, D, and E) or two experiments (for A and C) performed, each of which gave similar results, is shown.
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fig6: CCRL2 binds chemerin. (A) Chemerin blocks anti-CCRL2 mAb binding. Various concentrations of human chemerin or CCL2 were incubated with total peritoneal mast cells on ice for 5 min, followed by incubation with CCRL2-specific mAb BZ2E3 or anti-IgE and detected with secondary anti–rat PE or anti–mouse IgE PE. (B and C) Radiolabeled chemerin binding. (B) Displacement of iodinated chemerin (residues 21–148) binding to mCMKLR1, huCCRL2, and mCCRL2 by full-length chemerin. (C) Saturation binding of 125I-chemerin21-148 to mCCRL2-transfected cells. (D) Immunofluorescence-based chemerin binding. Various concentrations of untagged serum form chemerin were incubated with mCCRL2-HA, huCCRL2-HA, mCRTH2-HA, or mCMKLR1-HA L1.2 transfectants in the presence of 10 nM His8-tagged serum form chemerin. Samples were incubated on ice for 30 min. Secondary anti-His6 PE was added to detect levels of bound His8-tagged chemerin, and mean fluorescence intensity values are displayed. Mean fluorescence intensity ± range of duplicate staining wells are shown. (E) Mast cell binding. 1,000 nM of untagged chemerin isoforms were incubated with total peritoneal cells from either WT or CCRL2 KO mice in the presence or absence of 10 nM His8-tagged chemerin isoforms. Secondary anti-His6 PE was added to detect levels of bound His8-tagged chemerin. SSChigh F4/80− c-kit+ mast cells were analyzed. For B and C, the mean of quadruplicate wells ±SD is shown for individual experiments. A representative dataset of the three (for B, D, and E) or two experiments (for A and C) performed, each of which gave similar results, is shown.
Mentions: Although we failed to identify evidence of signaling effects of any of the tested chemoattractants, we were able to identify a high-affinity ligand for the receptor: in independent studies in which we were using our anti-CCRL2 mAbs as controls for staining, we serendipitously discovered that chemerin, a protein ligand for signaling receptor CMKLR1 (for review see reference 28), inhibited the binding of mCCRL2-specific mAbs to mouse peritoneal mast cells. In Fig. 6 A, we illustrate the potent ability of chemerin to inhibit anti-CCRL2 staining of mouse peritoneal mast cells. Increasing concentrations of chemerin blocked the binding of anti-mCCRL2 BZ5B8 (Fig. 6 A) and BZ2E3 (not depicted) in a dose-dependent manner (EC50 = 21 nM). The effect was specific to anti-mCCRL2:mCCRL2 interactions because binding of IgE to FcRεI was unaffected by 1,000 nM chemerin (Fig. 6 A), and 1,000 nM CCL2 had no effect on CCRL2 staining (Fig. 6 A).

Bottom Line: Mast cells contribute importantly to both protective and pathological IgE-dependent immune responses.In contrast to other "silent" or professional chemokine interreceptors, chemerin binding does not trigger ligand internalization.Rather, CCRL2 is able to bind the chemoattractant and increase local concentrations of bioactive chemerin, thus providing a link between CCRL2 expression and inflammation via the cell-signaling chemerin receptor CMKLR1.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Immunology and Vascular Biology, Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA. bazabel@alum.mit.edu

ABSTRACT
Mast cells contribute importantly to both protective and pathological IgE-dependent immune responses. We show that the mast cell-expressed orphan serpentine receptor mCCRL2 is not required for expression of IgE-mediated mast cell-dependent passive cutaneous anaphylaxis but can enhance the tissue swelling and leukocyte infiltrates associated with such reactions in mice. We further identify chemerin as a natural nonsignaling protein ligand for both human and mouse CCRL2. In contrast to other "silent" or professional chemokine interreceptors, chemerin binding does not trigger ligand internalization. Rather, CCRL2 is able to bind the chemoattractant and increase local concentrations of bioactive chemerin, thus providing a link between CCRL2 expression and inflammation via the cell-signaling chemerin receptor CMKLR1.

Show MeSH
Related in: MedlinePlus