Upon receptor activation, the myeloid C-type lectin receptor Mincle signals via the Syk-CARD9-Bcl10-MALT1 pathway. It does so by recruiting the ITAM-bearing FcεRI-γ. The related receptor macrophage C-type Lectin (MCL) has also been shown to be associated with Syk and to be dependent upon this signaling axis. We have previously shown that MCL co-precipitates with FcεRI-γ, but were unable to show a direct association, suggesting that MCL associates with FcεRI-γ via another molecule. Here, we have used rat primary cells and cell lines to investigate this missing link. A combination of flow cytometric and biochemical analysis showed that Mincle and MCL form heteromers on the cell surface. Furthermore, association with MCL and FcεRI-γ increased Mincle expression and enhanced phagocytosis of Ab-coated beads. The results presented in this paper suggest that the Mincle/MCL/FcεRI-γ complex is the functionally optimal form for these C-type lectin receptors on the surface of myeloid cells.Keywords: C-type lectin r Macrophage C-type Lectin (MCL) r Mincle r Myeloid cells r Signaling adaptor See accompanying Commentary by YamasakiAdditional supporting information may be found in the online version of this article at the publisher's web-site IntroductionMacrophage inducible C-type lectin (Mincle) (also called CLEC4E) and macrophage C-type lectin (MCL) (also called CLEC4D) are single-pass transmembrane proteins that belong to the C-type lectin-like domain superfamily, and their genes lie adjacent to each other in the APLEC (antigen-presenting lectin-like complex) gene complex [1] in all species thus far examined. Mincle and MCL are expressed on cells of myeloid origin [2][3][4][5][6][7][8]. Mincle is normally expressed at low levels, but receptor levels are increased by exposure to different inflammatory signals [6,7,9]. Mincle has been shown to recognize the mycobacterial glycolipid trehalose-6,6-dimycolate (TDM, also called cord factor), present in the cell wall of some Mycobacterium species and considered as a virulence Correspondence: Dr. Michael R. Daws e-mail: m.r.daws@medisin.uio.no factor [10,11]. Moreover, Mincle-deficient mice show increased mycobacterial burden following challenge with Bacillus CalmetteGuérin (BCG), suggesting that Mincle has an important in vivo role in the immune response to mycobacteria [12]. In addition, Mincle recognizes a number of pathogenic fungi, particularly Malassezia spp. [7,8], and the endogenous ligand spliceosome-associated protein 130 released during cell necrosis [9]. We have been unable to identify a ligand for MCL, but it has recently been reported that MCL can also recognize TDM [13].The majority of activating C-type lectin receptors signal via associated adaptor proteins. Mincle has been shown to be associated with . MCL carries no known signaling motifs in its cytoplasmic region, and has no charged residues in its transmembrane domain, but it has been shown to activate spleen tyrosine kinase (Syk) [4]. We have recently shown that immunoprecipitation of MCL from a rat myeloid cel...
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.
Dendritic cell activating receptor-1 (DCAR1) is a cell-surface receptor encoded by the Antigen Presenting Lectin-like gene Complex (APLEC). We generated a mouse monoclonal antibody against rat DCAR1, and used this to characterize receptor expression and function. Rat DCAR1 was expressed on minor subsets of myeloid cells in lymphoid tissue, but was uniformly expressed at a high level by eosinophils, and at a low level by neutrophils. It was expressed by eosinophils in the peritoneal cavity and the lamina propria of the gut, and by subsets of macrophages or dendritic cells at these sites. Polarization of peritoneal macrophages showed that DCAR1 expression was absent on M1 macrophages, and increased on M2 macrophages. DCAR1 could be expressed as a homodimer and its associated with the activating adaptor protein FcεRIγ. This association allowed efficient phagocytosis of antibody-coated beads. Additionally, cross-linking of DCAR1 on the surface of rat eosinophils lead to production of reactive oxygen species. These data show that DCAR1 is an activating receptor. Its expression on M2 macrophages and eosinophils suggests that it may play a role in the immune response to parasites.
MCL, Mincle and Dectin‐2 are C‐type lectin receptors expressed by subsets of myeloid cells, and their genes cluster together in the APLEC/Dectin‐2 gene complex. We have previously shown that MCL and Mincle form a heterodimer in the rat, and others have shown that MCL and Dectin‐2 form a heterodimer in the mouse. In the rat, Dectin‐2 is a pseudogene, but here, we examine the association of the three receptors in human. In co‐transfection experiments analyzed with flow cytometry and immunoprecipitation, we here show that human MCL and Mincle form a disulphide‐linked heterodimer that associates with the signalling adaptor molecule FcεRIγ, in accordance with our previous findings in the rat. In contrast to previous findings in the rat, data in this paper indicate a direct association of MCL with FcεRIγ, as previously shown for mouse MCL. We were unable to demonstrate the formation of a heterodimer between human MCL and Dectin‐2. Thus, despite similarities, there may be important differences in the conformation of these receptors between rat, mouse and human, and this may have functional consequences.
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