Inflammation: Dealing with excessive cell death

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Macrophage-inducible C-type lectin (Mincle) recognizes an endogenous ligand that is released from dead cells, as well as triggering the production of cytokines and chemokines that attract neutrophils to damaged tissue.

Macrophage-inducible C-type lectin (Mincle; also known as CLEC4E) controls a new pathway by which the immune system can detect excessive cell death in the absence of infection — for example, as a result of tissue injury or irradiation. According to new research, an endogenous ligand that is released from dead cells is recognized by Mincle expressed by macrophages, triggering the production of cytokines and chemokines that attract neutrophils to the damaged tissue.

Expression of Mincle is known to be upregulated by macrophages in response to cell stress, but until now its ligand, signalling pathway and function have been unknown. Mincle was shown to associate selectively with the immunoreceptor tyrosine-based activation motif (ITAM)-containing adaptor FcR gamma (Fc receptor gamma -chain) through a conserved arginine (Arg42) in the transmembrane domain of Mincle, and the signalling capacity of Mincle was Arg42 dependent. Antibody-mediated crosslinking of Mincle on peritoneal macrophages induced the production of several pro-inflammatory mediators, including CXC-chemokine ligand 2 (CXCL2; also known as MIP2), through a phosphorylation cascade that was triggered by spleen tyrosine kinase (SYK). This pathway was markedly abrogated in FcR gamma -deficient cells. In line with the known role of CARD9 (caspase-recruitment domain protein 9) in linking SYK activation to inflammatory responses, the production of CXCL2 in response to Mincle activation was also abrogated in CARD9-deficient macrophages. So, the authors conclude that Mincle activates macrophages through a FcR gamma –SYK–CARD9 signalling pathway.

But what is the physiological ligand that triggers signalling through this pathway? The authors established a T-cell hybridoma that expresses both Mincle and FcR gamma , as well as a reporter construct that produces green fluorescent protein (GFP) in response to ITAM-mediated signalling. When these cells were cultured alone without changing the medium, an increase in cell death was associated with an increase in the number of GFP⁺ cells. This effect was almost completely inhibited by Mincle-specific blocking antibodies, which indicates that dead cells can activate ITAM signalling through Mincle. A soluble form of Mincle was produced and used to screen lysates of dead cells; spliceosome-associated protein 130 (SAP130; also known as SF3B3), which is a component of a small nuclear ribonucleoprotein, was shown to selectively bind Mincle, and recombinant SAP130 activated Mincle-expressing cells. SAP130 from living cells and dead cells bound to Mincle with equal efficiency, which indicates that it is the release of SAP130 from its normal nuclear localization, rather than protein modification, during cell death that activates Mincle.

Whole-body irradiation of mice induces extensive death of thymocytes and the transient infiltration of neutrophils into the thymus; in this model, the expression of Mincle by thymic macrophages was rapidly upregulated after irradiation. A Mincle-specific blocking antibody inhibited the production of CXCL2 by thymic macrophages and markedly decreased neutrophil infiltration of the thymus after irradiation.

So, the authors conclude that Mincle is a sensor of non-physiological cell death that transduces an inflammatory signal. Importantly, control experiments were carried out to prevent signalling through Toll-like receptors (TLRs), which also induce inflammation in response to endogenous ligands from dead cells, to exclude the possibility of TLR-ligand contaminants.

Kirsty Minton

References

Yamasaki, S. et al. Mincle is an ITAM-coupled activating receptor that senses damaged cells. Nature Immunol. 9, 1179–1188 (2008) | Article | PubMed |