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Rat macrophage C-type lectin is an activating receptor expressed by phagocytic cells.

Lobato-Pascual A, Saether PC, Dahle MK, Gaustad P, Dissen E, Fossum S, Daws MR - PLoS ONE (2013)

Bottom Line: We found that MCL co-immunoprecipitated with the activating adaptor protein FcεRIγ in these cells.Moreover, beads coated with anti-MCL antibody increased phagocytosis in the RMW cells.Together, these observations indicate that rat MCL is a receptor that activates phagocytosis in myeloid cells under inflammatory conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway. a.l.pascual@medisin.uio.no

ABSTRACT
Macrophage C-type lectin (MCL) is a membrane surface receptor encoded by the Antigen Presenting Lectin-like gene Complex (APLEC). We generated a mouse monoclonal antibody for the study of this receptor in the rat. We demonstrate that rat MCL is expressed on blood monocytes and neutrophils, as well as on several tissue macrophage populations, including alveolar and peritoneal cavity macrophages. We also demonstrate MCL expression on a subset of resident spleen macrophages. Immunohistochemistry analysis of the spleen showed staining specifically in the marginal zone and red pulp. Exposure to pro-inflammatory mediators or to yeast cell wall extract (zymosan) increased surface MCL expression on peritoneal macrophages. We characterized a rat myeloid cell line, RMW, which expresses high levels of MCL. We found that MCL co-immunoprecipitated with the activating adaptor protein FcεRIγ in these cells. Moreover, beads coated with anti-MCL antibody increased phagocytosis in the RMW cells. Together, these observations indicate that rat MCL is a receptor that activates phagocytosis in myeloid cells under inflammatory conditions.

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Biochemical analysis of rat MCL. A,Western blot analysis of anti-FLAG immunoprecipitate from CHO cells stably transfected with FLAG-tagged rat MCL. Lysate from parental CHO cells was used as control. Anti-FLAG mAb M2 was used for immunoprecipitation; blotting was performed with a rabbit polyclonal anti-FLAG antibody. B, Western blot analysis of anti-MCL immunoprecipitate from non-transfected RMW cells. Biotinylated anti-MCL mAb WEN42 was used for immunoblotting. C, Rat MCL co-precipitates with tyrosine phosphorylated molecules of different molecular weight. RMW cells were untreated (−) or treated with pervanadate (PV) before lysis, immunoprecipitated with anti-MCL mAb and subjected to Western blot analysis with an anti-phosphotyrosine mAb. D, Anti-FcεRIγ Western blot analysis of anti-MCL immunoprecipitate from unstimulated RMW cells. Iso: isotype control mAb. IP: immunoprecipitation. NR: non-reducing conditions. R: reducing conditions. Apparent molecular weight in kDa is indicated.
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pone-0057406-g005: Biochemical analysis of rat MCL. A,Western blot analysis of anti-FLAG immunoprecipitate from CHO cells stably transfected with FLAG-tagged rat MCL. Lysate from parental CHO cells was used as control. Anti-FLAG mAb M2 was used for immunoprecipitation; blotting was performed with a rabbit polyclonal anti-FLAG antibody. B, Western blot analysis of anti-MCL immunoprecipitate from non-transfected RMW cells. Biotinylated anti-MCL mAb WEN42 was used for immunoblotting. C, Rat MCL co-precipitates with tyrosine phosphorylated molecules of different molecular weight. RMW cells were untreated (−) or treated with pervanadate (PV) before lysis, immunoprecipitated with anti-MCL mAb and subjected to Western blot analysis with an anti-phosphotyrosine mAb. D, Anti-FcεRIγ Western blot analysis of anti-MCL immunoprecipitate from unstimulated RMW cells. Iso: isotype control mAb. IP: immunoprecipitation. NR: non-reducing conditions. R: reducing conditions. Apparent molecular weight in kDa is indicated.

Mentions: Rat MCL is a protein of 218 amino acid residues with predicted molecular weight of 25.3 kDa. It contains two putative N-linked glycosylation sites and two disulphide bonds involved in the stabilization of the C-type lectin-like domain. Stably transfected CHO cells were examined for possible formation of MCL multimers by Western blot analysis. In accordance with previously published results about human and mouse MCL [9], [11], the anti-rat MCL mAb reacted with bands of approximately 30 and 35 kDa in SDS-PAGE under reducing conditions. The ∼10 kDa increase over the predicted weight of the naïve MCL polypeptide most likely reflects glycosylation, and the occurrence of two bands is likely due to the presence of incompletely processed intracellular protein forms. Under non-reducing conditions, MCL migrated as a band of approximately 70 kDa, indicating that MCL exists as a disulfide-linked homodimer (Figure 5A). However, MCL was also found to migrate as a monomer under non-reducing conditions. Bands of similar size were also detected in RMW cells, suggesting that this receptor also exists as a single chain or forms non-covalently linked multimers. (Figure 5A, B). Following pervanadate treatment, a number of tyrosine phosphorylated proteins co-precipitated with MCL from RMW cells (Figure 5C), including a band of approximately 12 kDa, consistent with the expected size of FcεRIγ. A band of 12 kDa was also detected with specific antibody towards FcεRIγ in anti-MCL immunoprecipitation experiments, using non-stimulated RMW cells. Together, these data indicate that MCL associates with the activating transmembrane adaptor protein FcεRIγ, suggesting an activating function for MCL (Figure 5D).


Rat macrophage C-type lectin is an activating receptor expressed by phagocytic cells.

Lobato-Pascual A, Saether PC, Dahle MK, Gaustad P, Dissen E, Fossum S, Daws MR - PLoS ONE (2013)

Biochemical analysis of rat MCL. A,Western blot analysis of anti-FLAG immunoprecipitate from CHO cells stably transfected with FLAG-tagged rat MCL. Lysate from parental CHO cells was used as control. Anti-FLAG mAb M2 was used for immunoprecipitation; blotting was performed with a rabbit polyclonal anti-FLAG antibody. B, Western blot analysis of anti-MCL immunoprecipitate from non-transfected RMW cells. Biotinylated anti-MCL mAb WEN42 was used for immunoblotting. C, Rat MCL co-precipitates with tyrosine phosphorylated molecules of different molecular weight. RMW cells were untreated (−) or treated with pervanadate (PV) before lysis, immunoprecipitated with anti-MCL mAb and subjected to Western blot analysis with an anti-phosphotyrosine mAb. D, Anti-FcεRIγ Western blot analysis of anti-MCL immunoprecipitate from unstimulated RMW cells. Iso: isotype control mAb. IP: immunoprecipitation. NR: non-reducing conditions. R: reducing conditions. Apparent molecular weight in kDa is indicated.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585393&req=5

pone-0057406-g005: Biochemical analysis of rat MCL. A,Western blot analysis of anti-FLAG immunoprecipitate from CHO cells stably transfected with FLAG-tagged rat MCL. Lysate from parental CHO cells was used as control. Anti-FLAG mAb M2 was used for immunoprecipitation; blotting was performed with a rabbit polyclonal anti-FLAG antibody. B, Western blot analysis of anti-MCL immunoprecipitate from non-transfected RMW cells. Biotinylated anti-MCL mAb WEN42 was used for immunoblotting. C, Rat MCL co-precipitates with tyrosine phosphorylated molecules of different molecular weight. RMW cells were untreated (−) or treated with pervanadate (PV) before lysis, immunoprecipitated with anti-MCL mAb and subjected to Western blot analysis with an anti-phosphotyrosine mAb. D, Anti-FcεRIγ Western blot analysis of anti-MCL immunoprecipitate from unstimulated RMW cells. Iso: isotype control mAb. IP: immunoprecipitation. NR: non-reducing conditions. R: reducing conditions. Apparent molecular weight in kDa is indicated.
Mentions: Rat MCL is a protein of 218 amino acid residues with predicted molecular weight of 25.3 kDa. It contains two putative N-linked glycosylation sites and two disulphide bonds involved in the stabilization of the C-type lectin-like domain. Stably transfected CHO cells were examined for possible formation of MCL multimers by Western blot analysis. In accordance with previously published results about human and mouse MCL [9], [11], the anti-rat MCL mAb reacted with bands of approximately 30 and 35 kDa in SDS-PAGE under reducing conditions. The ∼10 kDa increase over the predicted weight of the naïve MCL polypeptide most likely reflects glycosylation, and the occurrence of two bands is likely due to the presence of incompletely processed intracellular protein forms. Under non-reducing conditions, MCL migrated as a band of approximately 70 kDa, indicating that MCL exists as a disulfide-linked homodimer (Figure 5A). However, MCL was also found to migrate as a monomer under non-reducing conditions. Bands of similar size were also detected in RMW cells, suggesting that this receptor also exists as a single chain or forms non-covalently linked multimers. (Figure 5A, B). Following pervanadate treatment, a number of tyrosine phosphorylated proteins co-precipitated with MCL from RMW cells (Figure 5C), including a band of approximately 12 kDa, consistent with the expected size of FcεRIγ. A band of 12 kDa was also detected with specific antibody towards FcεRIγ in anti-MCL immunoprecipitation experiments, using non-stimulated RMW cells. Together, these data indicate that MCL associates with the activating transmembrane adaptor protein FcεRIγ, suggesting an activating function for MCL (Figure 5D).

Bottom Line: We found that MCL co-immunoprecipitated with the activating adaptor protein FcεRIγ in these cells.Moreover, beads coated with anti-MCL antibody increased phagocytosis in the RMW cells.Together, these observations indicate that rat MCL is a receptor that activates phagocytosis in myeloid cells under inflammatory conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway. a.l.pascual@medisin.uio.no

ABSTRACT
Macrophage C-type lectin (MCL) is a membrane surface receptor encoded by the Antigen Presenting Lectin-like gene Complex (APLEC). We generated a mouse monoclonal antibody for the study of this receptor in the rat. We demonstrate that rat MCL is expressed on blood monocytes and neutrophils, as well as on several tissue macrophage populations, including alveolar and peritoneal cavity macrophages. We also demonstrate MCL expression on a subset of resident spleen macrophages. Immunohistochemistry analysis of the spleen showed staining specifically in the marginal zone and red pulp. Exposure to pro-inflammatory mediators or to yeast cell wall extract (zymosan) increased surface MCL expression on peritoneal macrophages. We characterized a rat myeloid cell line, RMW, which expresses high levels of MCL. We found that MCL co-immunoprecipitated with the activating adaptor protein FcεRIγ in these cells. Moreover, beads coated with anti-MCL antibody increased phagocytosis in the RMW cells. Together, these observations indicate that rat MCL is a receptor that activates phagocytosis in myeloid cells under inflammatory conditions.

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Related in: MedlinePlus