HMGB1-secreting capacity of multiple cell lineages revealed by a novel HMGB1 ELISPOT assay.

High mobility group box protein 1 (HMGB1) exerts different biological functions dependent on its cellular localization. Nuclear HMGB1 maintains chromatin architecture and is required for undisturbed transcription activity, and extracellularly released HMGB1 mediates inflammation and tissue regeneration. A present paucity of readily accessible methods to quantify released HMGB1 represents a problem concerning the exploration of HMGB1 biology. We have now developed a HMGB1-specific ELISPOT assay enabling enumeration of individual HMGB1-releasing cells. The method also allows automated, semiquantitative assessment of released HMGB1 by evaluating areas of single HMGB1 spots. Actively secreted HMGB1 as well as cells passively releasing the protein following necrotic cell death are visualized distinctly using this ELISPOT assay. Kinetics of HMGB1 secretion after different stimuli was studied using cell lines of various lineages. IFN-gamma already induced substantial HMGB1 secretion from the monocytic cell line RAW 264.7 within 24 h and even more so after 48 h. LPS only stimulated a modest HMGB1 release within 24 h, but this increased considerably by 48 h. TNF-induced HMGB1 release was unexpectedly low. Mast cells, which share the secretory, lysosomal pathway with macrophages/monocytes, did not secrete HMGB1 in response to any studied mode of activation. Most transformed cells overexpress HMGB1, but the ELISPOT assay revealed that all transformed cell lines will not actively secrete the protein. We believe the ELISPOT method provides a novel tool to study pathways promoting or inhibiting HMGB1 secretion.