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3156 Sho Yamasaki Eur. J. Immunol. 2013. 43: 3156–3158DOI: 10.1002/eji.201344131

Signaling while eating: MCL is coupled with Mincle

Sho Yamasaki

Division of Molecular Immunology, Research Center for Infectious Diseases, Medical Instituteof Bioregulation, Kyushu University, Fukuoka, Japan

Recently, C-type lectin receptors (CLRs) have been identified as a newly emerging familyof pattern-recognition receptors for pathogen-associated molecular patterns. The ligandand function of many “individual” CLRs have been clarified in the past few years. A newreport by Lobato-Pascual et al. in this issue of the European Journal of Immunology [Eur. J.Immunol. 2013. 43: 3167–3174] shows that a rat CLR, named macrophage C-type lectin(also called Clec4d), forms a heterodimer with another CLR, macrophage inducible C-typelectin (also called Clec4e). This finding sheds light on a possible synergistic regulation ofdifferent C-type lectin receptors by forming heterodimers: such heterodimers may amplifysignaling, expand ligand specificity, or confer multiple functions.

Keywords: C-type lectin � Mincle � Myeloid cells � Signaling adaptor

See accompanying article by Lobato-Pascual et al.

C-type lectin receptors (CLRs) have recently been identified asa fourth family of pattern-recognition receptors for pathogen-associated molecular patterns, following TLRs, Nod-like recep-tors, and RIG-I-like receptors [1]. Among the CLRs is macrophageC-type lectin (MCL), which is also called Clec4d or Clecsf8. MCLwas first described in 1998 as a C-type lectin receptor with expres-sion restricted to murine macrophage cell lines, such as J774.2and RAW 264.7 cells [2]. MCL is mapped to the natural killergene complex on murine chromosome 6 [3]. This locus forms agene cluster including related C-type lectin receptor genes such asmacrophage-inducible C-type lectin (Mincle, also called Clec4e),dendritic cell immunoreceptor (also called Clec4a2), and dendriticcell-associated lectin-2 (Dectin-2, also called Clec4n). Arce et al.reported that human MCL is also located in the human naturalkiller gene complex on chromosome 12p13, and potentially actsas an endocytic receptor [4]. However, the ligand and function ofMCL have not been identified for the past decade.

The past two years however has brought tremendous progressin understanding the function of MCL. MCL is now found to

Correspondence: Dr. Sho Yamasakie-mail: yamasaki@bioreg.kyushu-u.ac.jp

be also expressed in dendritic cells and neutrophils as well asmacrophages [5–7]. In human PBMCs, MCL is highly expressedon CD16+CD14+ monocyte subsets [5, 8]. Graham et al. demon-strated that MCL can mediate phagocytosis, the respiratory burst,and inflammatory cytokine production, and activates Syk kinasethrough unknown adaptors [5]. Lobato-Pascual et al. [6] andour group [7] have reported that MCL delivers activating signalsthrough the ITAM-containing FcRγ chain.

The physiological functions of MCL have been previouslydemonstrated using MCL-deficient mice. MCL-deficient mice havesubstantial defects in the immune response against mycobacte-ria and Klebsiella pneumonia infection [7, 10]. No defects wereobserved however in the ability of MCL-deficient mice to resistinfection with other pathogens, such as fungi (Candida albi-cans), extracellular bacteria (Staphylococcus aureus), intracellularbacteria (Listeria monocytogenes), or nematodes (Nippostrongylusbrasiliensis) [5]. In contrast, Zhu et al. reported that MCL-deficientmice are highly susceptible to Candida albicans infection [11]. MCLcould recognize mycobacterial TDM (trehalose-6,6′-dimycolate)with low affinity [7, 13], whereas the ligand in Gram-negativepneumonia (Klebsiella pneumoniae) remains unknown. It is stillpossible that MCL recognizes other unknown ligand(s) derivedfrom pathogens (pathogen-associated molecular patterns) and/ordamaged-self (damage-associated molecular patterns).

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Eur. J. Immunol. 2013. 43: 3156–3158 HIGHLIGHTS 3157

Figure 1. Proposed model for a heteromeric complex comprising MCL,Mincle, and FcRγ. MCL may form a functional heteromeric complexwith Mincle and FcRγ. Upon ligand recognition, FcRγ delivers an acti-vating signal through the Syk-CARD9 axis to produce proinflamma-tory cytokines. Simultaneously, the MCL-Mincle-FcRγ complex may beinvolved in the efficient phagocytosis of pathogens. Thus, the MCL-Mincle interaction may confer an additional function to each of theindividual receptors: MCL could acquire signaling potential by couplingwith the Mincle-FcRγ complex, whereas Mincle could acquire endocyticreceptor function by coupling with MCL. It is also possible that addi-tional ligands may be recognized by the Mincle/MCL heterodimer thatare not recognized by Mincle or MCL alone.

In this issue of the European Journal of Immunology, Lobato-Pascual et al. [9] propose a novel heteromeric complex of MCLand Mincle. As MCL lacks a positively charged residue in itstransmembrane region, Lobato-Pascual et al. speculate that MCLmight require other bridging molecules to interact with FcRγ. Theauthors had previously established a rat myeloid cell line, RMW,which expresses high levels of MCL [6]. By using this cell line, theyfound that MCL and Mincle show tight colinear expression. Theauthors thus hypothesized that MCL and Mincle may be expressedtogether in a molecular complex on the cell surface.

To further address this coassociation, Lobato-Pascual et al. [9]transiently transfected 293T cells with a combination of rat Min-cle, MCL, and FcRγ. Cotransfection of rat Mincle and FcRγ leads toan increase in Mincle expression on the cell surface, as previouslydescribed for mouse Mincle [12]. Intriguingly, cotransfection ofMincle and MCL leads to a significant increase in Mincle levels.Finally, the highest expression levels of Mincle are upon cotrans-fection of all three molecules together: MCL, Mincle, and FcRγ.

Lobato-Pascual et al. [9] further analyzed rat peritonealmacrophages to confirm that these two receptors are alsoexpressed together as a heterodimer in primary cells. Flow cyto-metric analysis of the LPS-stimulated macrophages also demon-strated colinear increased expression of MCL and Mincle. Thesedata implicate the presence of MCL/Mincle heterodimers on thesurface of primary rat myeloid cells.

To further assess the formation of heterodimers between MCLand Mincle, Lobato-Pascual et al. [9] next analyzed the com-

plex by immunoprecipitation using 293T cells. Under nonreduc-ing conditions, anti-MCL precipitation from MCL-transfected cellsreveals bands of 30 and 60 kDa, which may correspond to MCLmonomers and homodimers, respectively. An additional band of55 kDa could be detected under nonreducing conditions whencells were cotransfected with Mincle and MCL. The authors spec-ulate that a band of 55 kDa would correspond to a Mincle/MCLheterodimer, as the band was shifted to a lower mobility underreducing conditions. Immunoprecipitation with anti-MCL resultsin the coprecipitation of FcRγ when MCL is transfected togetherwith Mincle. The authors conclude that rat Mincle and rat MCLform covalent disulfide-linked heterodimers at the cell surface,thus allowing MCL to indirectly associate with FcRγ [9].

Miyake et al. [7] have reported that mouse MCL coimmunopre-cipitates with mouse FcRγ in the absence of Mincle. However, thisdoes not exclude the current idea of Lobato-Pascual et al. [9] thatMincle may promote the efficient coupling of MCL with FcRγ. Theindividual structures of monomeric MCL and Mincle respectivelyhave also been recently solved [13]. Further understanding of thenature of the complex will be achieved by the structural analysesof the crystallized heteromeric MCL-Mincle complex.

As Lobato-Pascual et al. did not generate myeloid cells defi-cient in MCL or Mincle, the functional consequence of theMincle/MCL/FcRγ complex formation was assessed in 293T cells.Coexpression of MCL together with Mincle and FcRγ leads to astrong synergistic increase in the efficiency of phagocytosis of anti-Mincle beads, although the authors did not test TDM-coated beadsor mycobacteria.

It is still uncertain whether endogenous MCL and Minclecould form complexes or not. Is the complex formed induciblyor constitutively? Precise analyses of MCL or Mincle on primarycells from wild-type, MCL- and Mincle-deficient mice will answerthese questions. Previous papers have suggested that transfectedMCL tends to form disulfide-linked homodimers [2, 4]. A recentreport demonstrated that MCL is capable of forming heterodimerswith dendritic cell-associated lectin-2 but not with Mincle [11].However, Lobato-Pascual et al. show that the heterodimerformation between MCL and Mincle is specific, as MCL does notinteract with other CLRs. The molecular mechanism underlyingthe formation of specific heterodimers still requires furtherclarification. We have demonstrated, using ligand-stimulatedDCs from MCL-deficient mice, that MCL is critical for inducibleMincle expression [7]. It is also interesting to speculate thatthe reduced expression of Mincle protein in MCL-deficient cellsmay be partly due to the lack of MCL-Mincle complex on thecell surface, which might stabilize the Mincle protein on the cellsurface.

The current findings of Lobato-Pascual et al. prompt us toconsider the analogy between CLR and TLR, as TLR2 forms het-erodimers with other members of the TLR family. Namely, TLR1-TLR2 and TLR2-TLR6 heterodimers recognize different ligands,such as triacylated lipopeptide and diacylated lipopeptide, respec-tively [14]. In a similar manner, some C-type lectin receptors mighthave altered ligand specificity by forming heterodimer with othermembers of the C-type lectin receptors.

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3158 Sho Yamasaki Eur. J. Immunol. 2013. 43: 3156–3158

The evidence for the heteromeric conformation of C-type lectinreceptors provides some implications in this field. First, het-eromeric receptors may be able to respond to a wide variety ofligands (expand the ligand specificity); second, they may con-fer additional functions to a single receptor, i.e. add internaliza-tion potential to an activating (signaling) receptor (Fig. 1). Theprecise regulatory mechanisms that govern a balance betweenchaotic complexity and sophisticated organization generated byheterodimeric C-type lectin receptors is an intriguing issue thatneeds to be addressed in future studies.

Acknowledgments: This work was supported by Funding Pro-gram for Next Generation World-Leading Researchers (NEXTProgram).

Conflict of interest: The author declares no financial or commer-cial conflict of interest.

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Abbreviations: CLR: C-type lectin receptor · MCL: macrophage C-type

lectin · Mincle: macrophage-inducible C-type lectin

Full correspondence: Dr. Sho Yamasaki, Division of MolecularImmunology, Research Center for Infectious Diseases, MedicalInstitute of Bioregulation, Kyushu University, 3-1-1 MaidashiHigashiku, Fukuoka 812-8582, JapanFax: +81-92-642-4614e-mail: yamasaki@bioreg.kyushu-u.ac.jp

See accompanying article:http://dx.doi.org/10.1002/eji.201343752

Received: 30/9/2013Revised: 30/9/2013Accepted: 17/10/2013

C© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu