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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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Rat MCL is expressed on monocytes, macrophages and granulocytes. A,mAb WEN42 is specific for MCL. The specificity of WEN42 was tested in flow cytometry analysis of BWN3G cells stably expressing rat MCL or Mincle. B, Flow cytometry analysis of rat MCL surface expression on different cell types isolated from different organs, as indicated. Histograms display cell subsets gated as described in the materials and methods section. Black line histograms: MCL. Filled grey histograms: isotype control.
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pone-0057406-g001: Rat MCL is expressed on monocytes, macrophages and granulocytes. A,mAb WEN42 is specific for MCL. The specificity of WEN42 was tested in flow cytometry analysis of BWN3G cells stably expressing rat MCL or Mincle. B, Flow cytometry analysis of rat MCL surface expression on different cell types isolated from different organs, as indicated. Histograms display cell subsets gated as described in the materials and methods section. Black line histograms: MCL. Filled grey histograms: isotype control.

Mentions: As no antibody against rat MCL was commercially available, we produced our own in-house monoclonal anti-MCL antibody. BWZ cells virally-transduced with a chimeric protein consisting of the extracellular sequence of MCL, the transmembrane domain from killer cell lectin receptor H1 (KLRH1) and the cytoplasmic segment of the T-cell surface glycoprotein CD3 zeta chain were used for both immunization and screening as previously described [20]. Among the selected clones that showed high and consistent antibody production, we obtained three antibodies that bound specifically to rat MCL. One of these, WEN42, gave consistently better staining in flow cytometry, worked for immunoprecipitation and Western blot, and was used for all studies described herein. This antibody bound specifically to BWN3G cells expressing MCL (BWN3G.MCL), but not to the parent cells, or to cells expressing Mincle (BWN3G.Mincle) (Figure 1A). In addition, WEN42 did not bind to cells transfected with human MCL, or with other APLEC receptors (Figure S1).


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)

Rat MCL is expressed on monocytes, macrophages and granulocytes. A,mAb WEN42 is specific for MCL. The specificity of WEN42 was tested in flow cytometry analysis of BWN3G cells stably expressing rat MCL or Mincle. B, Flow cytometry analysis of rat MCL surface expression on different cell types isolated from different organs, as indicated. Histograms display cell subsets gated as described in the materials and methods section. Black line histograms: MCL. Filled grey histograms: isotype control.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585393&req=5

pone-0057406-g001: Rat MCL is expressed on monocytes, macrophages and granulocytes. A,mAb WEN42 is specific for MCL. The specificity of WEN42 was tested in flow cytometry analysis of BWN3G cells stably expressing rat MCL or Mincle. B, Flow cytometry analysis of rat MCL surface expression on different cell types isolated from different organs, as indicated. Histograms display cell subsets gated as described in the materials and methods section. Black line histograms: MCL. Filled grey histograms: isotype control.
Mentions: As no antibody against rat MCL was commercially available, we produced our own in-house monoclonal anti-MCL antibody. BWZ cells virally-transduced with a chimeric protein consisting of the extracellular sequence of MCL, the transmembrane domain from killer cell lectin receptor H1 (KLRH1) and the cytoplasmic segment of the T-cell surface glycoprotein CD3 zeta chain were used for both immunization and screening as previously described [20]. Among the selected clones that showed high and consistent antibody production, we obtained three antibodies that bound specifically to rat MCL. One of these, WEN42, gave consistently better staining in flow cytometry, worked for immunoprecipitation and Western blot, and was used for all studies described herein. This antibody bound specifically to BWN3G cells expressing MCL (BWN3G.MCL), but not to the parent cells, or to cells expressing Mincle (BWN3G.Mincle) (Figure 1A). In addition, WEN42 did not bind to cells transfected with human MCL, or with other APLEC receptors (Figure S1).

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.

Show MeSH
Related in: MedlinePlus