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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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Phagocytosis of anti-MCL-coated beads. A,Comparative surface expression of the receptors, numbers represent median fluorescence intensity. B. Imaging flow cytometry analysis of a RMW cell showing a cell interacting with two beads. Channel 1 shows visible light image; bead-fluorescence is shown in channel 10, with DyLight-594 counterstaining in channel 4. Black arrowhead shows an internalized bead (green fluorescence) C. Percentage of cells with internalized beads, or with only surface-bound beads. D. Efficiency of phagocytosis expressed as a ratio of cells phagocytosing beads to cells with only surface-bound beads. 10,000 cells events were collected.
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pone-0057406-g006: Phagocytosis of anti-MCL-coated beads. A,Comparative surface expression of the receptors, numbers represent median fluorescence intensity. B. Imaging flow cytometry analysis of a RMW cell showing a cell interacting with two beads. Channel 1 shows visible light image; bead-fluorescence is shown in channel 10, with DyLight-594 counterstaining in channel 4. Black arrowhead shows an internalized bead (green fluorescence) C. Percentage of cells with internalized beads, or with only surface-bound beads. D. Efficiency of phagocytosis expressed as a ratio of cells phagocytosing beads to cells with only surface-bound beads. 10,000 cells events were collected.

Mentions: To determine whether MCL could play a role in phagocytosis, we coated fluorescent beads with antibodies to MCL or to CD45RC. Analysis of flow cytometry data showed that these antigens were expressed at similar levels on the surface of RMW cells (Figure 6A). After incubating beads with RMW cells for 1 h, we counterstained to differentiate internalized and phagocytosed beads, and analyzed using imaging flow cytometry. Figure 6B shows a sample image demonstrating an internalized bead and a surface bead on the same cell. We collected 10,000 cells and determined the percentage of cells that had internalized beads, and the percentage that only had cell surface beads. As shown in Figure 6C, the majority of cells that bound anti-MCL beads were able to phagocytose these beads, while the majority of cells that bound anti-CD45RC-beads did not. To correct for the fact that more cells bound to anti-CD45RC-beads than to anti-MCL-beads (Figure 6D), the data are expressed as a ratio of cells phagocytosing beads to cells with only surface-bound beads. These data indicate that MCL activates phagocytosis.


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)

Phagocytosis of anti-MCL-coated beads. A,Comparative surface expression of the receptors, numbers represent median fluorescence intensity. B. Imaging flow cytometry analysis of a RMW cell showing a cell interacting with two beads. Channel 1 shows visible light image; bead-fluorescence is shown in channel 10, with DyLight-594 counterstaining in channel 4. Black arrowhead shows an internalized bead (green fluorescence) C. Percentage of cells with internalized beads, or with only surface-bound beads. D. Efficiency of phagocytosis expressed as a ratio of cells phagocytosing beads to cells with only surface-bound beads. 10,000 cells events were collected.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0057406-g006: Phagocytosis of anti-MCL-coated beads. A,Comparative surface expression of the receptors, numbers represent median fluorescence intensity. B. Imaging flow cytometry analysis of a RMW cell showing a cell interacting with two beads. Channel 1 shows visible light image; bead-fluorescence is shown in channel 10, with DyLight-594 counterstaining in channel 4. Black arrowhead shows an internalized bead (green fluorescence) C. Percentage of cells with internalized beads, or with only surface-bound beads. D. Efficiency of phagocytosis expressed as a ratio of cells phagocytosing beads to cells with only surface-bound beads. 10,000 cells events were collected.
Mentions: To determine whether MCL could play a role in phagocytosis, we coated fluorescent beads with antibodies to MCL or to CD45RC. Analysis of flow cytometry data showed that these antigens were expressed at similar levels on the surface of RMW cells (Figure 6A). After incubating beads with RMW cells for 1 h, we counterstained to differentiate internalized and phagocytosed beads, and analyzed using imaging flow cytometry. Figure 6B shows a sample image demonstrating an internalized bead and a surface bead on the same cell. We collected 10,000 cells and determined the percentage of cells that had internalized beads, and the percentage that only had cell surface beads. As shown in Figure 6C, the majority of cells that bound anti-MCL beads were able to phagocytose these beads, while the majority of cells that bound anti-CD45RC-beads did not. To correct for the fact that more cells bound to anti-CD45RC-beads than to anti-MCL-beads (Figure 6D), the data are expressed as a ratio of cells phagocytosing beads to cells with only surface-bound beads. These data indicate that MCL activates phagocytosis.

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