Background/Purpose:  Granulocytic S100A12 is a member of the calgranulin-subgroup within the S100 family of calcium-binding proteins. Similar to other S100 proteins S100A12 can bind divalent metal ions, preferentially Ca²⁺ and Zn²⁺and subsequently arrange into different homo-multimeric complexes. Although S100A12 is highly overexpressed in auto-inflammatory diseases such as systemic juvenile idiopathic arthritis (sJIA) or familial mediterenean fever (FMF) the protein’s intracellular function is poorly defined. Yet, it is well accepted that once released from granulocytes S100A12 can operate as damage associated molecular pattern (DAMP) molecule or ‘alarmin’. In this function S100A12 was originally reported as ligand of the receptor for advanced glycation endproducts (RAGE), while we recently described human monocytes to respond to S100A12 stimulation in an exclusively toll like receptor 4 (TLR4)-dependent manner.

Methods:  Oligomers of recombinant as well as native S100A12 were isolated from serum samples and freshly isolated human granulocytes after cross-linking. Eluted S100A12 oligomers from patients’ serum were further applied to size exclusion chromatography. TLR4/MD2-binding assay and surface plasmon resonance assays were performed to investigate ligand-receptor interactions. Monocytic cells lines and primary human monocytes were used for stimulation experiments using defined S100A12 complexes.

Results:  Here we demonstrate that binding and signaling of S100A12 through TLR4 is dependent on the protein’s arrangement into a hexameric quarternary structure. Hexameric S100A12 triggers pro-inflammatory cytokine production by human monocytes and TLR4-expressing cell lines, which is sensitive to TLR4 and CD14 but not MD2 blockade. Importantly, the arrangement of S100A12 into its hexameric structure appears to depend on extracellular Ca²⁺ and Zn²⁺ion strengths. While S100A12-hexamers are not detectable inside human granulocytes, these protein complexes can be found in and isolated from human serum specimens, particularly sera obtained from patients with auto-inflammatory disease.

Conclusion:  Our data demonstrate that extracellular ion levels can serve as a molecular switch rendering a protein with non-inflammatory cell-intrinsic function into a pro-inflammatory DAMP. Hexameric S100A12 complexes are responsible for the pro-inflammatory functions of the protein. A detection system specifically quantifying its hexameric forms in human serum will improve the performance of S100A12 biomarker assays. Specific blockade of hexameric S100A12 may provide a novel therapeutic target.

Disclosures: The authors declare no conflict of interest. The described findings and principles are part of an international patent application (WO2016/178154A1).