CLEC‐2 signaling via Syk in myeloid cells can regulate inflammatory responses

Abstract

Myeloid cells express a plethora of C-type lectin receptors (CLRs) that can regulate immune responses. CLEC-2 belongs to the Dectin-1 sub-family of CLRs that possess an extracellular C-type lectin-like domain and a single intracellular hemITAM motif. CLEC-2 is highly expressed on mouse and human platelets where it signals via Syk to promote aggregation. We generated a monoclonal antibody (mAb) against mouse CLEC-2 and found that CLEC-2 is additionally widely expressed on leukocytes and that its expression is upregulated during inflammation. MAb-mediated crosslinking of CLEC-2 leads to hemI-TAM-dependent signaling via Syk, Ca 21 and NFAT and, in myeloid cells, modulates the effect of toll-like receptor (TLR) agonists to selectively potentiate production of IL-10. A macrophage/dendritic cell-dependent increase in IL-10 is also observed in mice given anti-CLEC-2 mAb together with LPS. Collectively, these data indicate that CLEC-2 is expressed in myeloid cells and acts as a Syk-coupled CLR able to modulate TLR signaling and inflammatory responses.Key words: CLEC-2 . C-type lectin . HemITAM . IL-10 . Syk Supporting Information available online IntroductionSignaling by some members of the C-type lectin receptor (CLR) superfamily plays a key role in innate immunity and in regulation of myeloid cell function [1,2]. A classical example is Dectin-1, which is expressed by macrophages (MØs), neutrophils and dendritic cells (DCs) and acts as a pattern recognition receptor for b-glucans present on fungal and some bacterial cell walls. Dectin-1 signals via a phospho-tyrosine-based hemITAM motif in its intracellular domain that recruits spleen tyrosine kinase (Syk) [3,4]. Dectin-1 signaling through Syk in myeloid cells can variably lead to phagocytosis, production of reactive oxygen species (ROS) and/or induction of innate response genes, including those encoding pro-inflammatory cytokines [5,6]. 3040Pro-inflammatory Dectin-1 signaling requires CARD9-dependent activation of NF-kB [7] although Dectin-1 can also signal to NF-kB via a Syk-independent pathway [8]. Although many DC types are potently activated by Dectin-1 agonists, in other myeloid cells Dectin-1 signaling only weakly activates the proinflammatory gene program [9]. In those cells, Dectin-1 acts primarily in synergy with other innate immune receptors such as toll-like receptors (TLRs) [10,11]. Notably, both Dectin-1 and CARD9 are important for resistance to fungal infection in both mice [7,12,13] and humans [14,15], indicating the importance of the CLR/Syk signaling pathway in immunity.The features of Dectin-1, including type II membrane topology, single Dectin-1-like C-type lectin-like domain and intracellular hemITAM are shared by two other CLRs, DNGR-1 (CLEC9A) and CLEC-2 (CLEC1B). DNGR-1 is selectively expressed by DCs and acts as a receptor for dead cells, facilitating cross-priming to cell-associated antigens [16]. CLEC-2 was first identified in a screen for natural killer (NK) cell receptors and its mRNA was found in myeloid cells and in some NK-cell clones [...

HemITAM: A single tyrosine motif that packs a punch

“…In mice, Clec-2 was found not only on platelets but also on neutrophils and activated monocytes (36) and, in a subsequent study, also on macrophages, DCs, and a subset of NK cells (44). The latter study also reported accumulation of Clec-2 on myeloid cells and NK cells upon lipopolysaccharide (LPS) challenge together with a de novo appearance of Clec-2 on B cells (44). A more recent report, however, attributed Clec-2 on mouse B cells to the acquisition from exogenous sources, for example, by trogocytosis, and showed that Clec-2 on mouse myeloid cells is lost during the entrance of these cells into secondary lymphoid organs (45).…”

“…However, CLEC-2 glycoproteins turned out to be mostly restricted to platelets and were also detected at low levels on liverresident Kupffer cells (24,42,43). In mice, Clec-2 was found not only on platelets but also on neutrophils and activated monocytes (36) and, in a subsequent study, also on macrophages, DCs, and a subset of NK cells (44). The latter study also reported accumulation of Clec-2 on myeloid cells and NK cells upon lipopolysaccharide (LPS) challenge together with a de novo appearance of Clec-2 on B cells (44).…”

Tetraspanin Tspan9 regulates platelet collagen receptor GPVI lateral diffusion and activation

“…Fluorescently labelled monoclonal antibodies used for flow cytometry and single particle tracking were from Emfret, apart from anti-mouse integrin α6 (AbD Serotec), anti-mouse CLEC-2 17D9 [30] and anti-ADAM10 and anti-CD9 (R&D Systems). Atto 647N and 565 fluorescent dyes were from Sigma, and 3,3′-dihexyloxacarbocyanine iodide (DiOC6) membrane dye was from Molecular Probes.…”

Podoplanin negatively regulates CD4+ effector T cell responses

“…PDPN has 3 known ligands: CLEC-2 (22), galectin-8 (23), and the chemokine CCL21 (24). Expression of CLEC-2 is largely restricted to hematopoietic cells, including platelets, DCs (25), and B cells (26). We at the peak of disease by quantitative PCR (Figure 6C).…”