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Pivotal Advance: Inhibition of HMGB1 nuclear translocation as a mechanism for the anti-rheumatic effects of gold sodium thiomalate

“Gold compounds such as gold sodium thiomalate (GST) can reduce the symptoms of rheumatoid arthritis (RA)”, as has been demonstrated in large clinical trials. It has been shown in in-vitro models that GST has “diverse anti-inflammatory and immunosuppressive effects on macrophages and monocytes”. Since the proinflammatory mediator HMGB1 (high mobility group box chromosomal protein 1) may play a key role in the pathogenesis of RA, the authors investigated whether GST inhibits the release of HMGB1 as a basis of the mode of action.

Macrophage cells from mice (RAW 264.7) and human macrophage cells (TCP-1) were stimulated (activated) in cultures using agents which cause extracellular release of HMGB1 (LPS, IFN-, poly(I:C) (polyinosinic:polycytidylic acid), IFN-, NO). External secretion as well as intracellular localisation of HMGB1 were examined (in the cell HMGB1 regulates DNA transcription, and it “can translocate from the nucleus to the cytoplasm to enter endolysosomes for secretion”).

As shown in the study (by means of ELISPOT assay), “GST (10–250 M) caused a dose-dependent inhibition of the extracellular HMGB1 translocation” from unstimulated as well as stimulated RAW-264.7 cells and from stimulated TCP-1 cells. The former was confirmed by Western blotting. Visual inspection did not show apoptosis.

GST did not affect spontaneous or LPS + IFN--induced TNF- secretion from RAW-264.7 cells. “Cell viability was more than 95%”.

Unstimulated RAW-264.7 cells showed (using immunohistochemical staining) stronger nuclear HMGB1 signals with GST than without. Cells activated by LPS + IFN- showed low or no nuclear HMGB1 signals and strong HMGB1 accumulation in the cytoplasm without GST, while with GST, conversely, they produced intense nuclear and weak cytoplasmic HMGB1 signals.

Without and with GST the intracellular TNF- production was low in unstimulated RAW-246.7 cells; however, in the case of stimulation by LPS and IFN- it was strong in most cells. These cells could be recognised through the TNF accumulation in the Golgi apparatus – in contrast to HMGB1 “which does not traverse the endoplasmic reticulum-Golgi system”.

GST can block the response of the RAW-264.7 cells to the mediators NO and IFN-, which induce the release of HMGB1. This indicates “that GST affects HMGB1 release in more than one step”.

RAW-264.7 cells activated by LPS or poly(I:C) showed a release of HMGB1. With GST this was reduced significantly; not so, however, with sodium thiomalate (without gold). This points to the role of the gold component in GST in the blocking of HMGB1 release.

Also with AuCl₃ as an example of another gold salt instead of GST, the release of HMGB1 from RAW-264.7 cells (activated by poly(I:C) or LPS) was inhibited, which is a confirmation of “the role of the gold component in blocking HMGB1 release from activated macrophages”.

The study provides new insights into the mechanism of how gold salts work and shows “that a DMARD [disease-modifying anti-rheumatic drug] can decrease the release of the proinflammatory mediator HMGB1 from activated macrophages”. The fact that GST did not block TNF- production is an argument “against GST-mediated cell toxicity or death as the basis for decreased HMGB1 secretion”. GST affects the intracellular traffic of HMGB1 as well as the retention of this proinflammatory mediator in the nucleus.

The “extracellular transport of the HMGB1 occurs by a nonconventional pathway”. In the cell, “HMGB1 can shuttle between the nucleus and cytoplasm”. By activation it is relocated to the cytosol. MRP1 (multidrug resistance-related protein 1) then translocates it “into the secretory lysosomes for extracellular exocytosis”.

LPS and “other TLR ligands as well as endogenous mediators such as cytokines and NO can all induce HMGB1 translocation and release”. GST can inhibit the release of the key mediators NO and IFN-, likewise AuCl₃ can downregulate NO release. Previous studies have shown that the gold compound auranofin can “downregulate NO synthesis in activated rat macrophages”. Altogether this indicates “that this effect may be a general characteristic of therapeutic gold compounds”.

“Neither NO nor IFN- was able to induce HMGB1 release from RAW-264.7 cells treated with GST.” Hence, GST is not only able to block “the production of the pivotal mediators NO and IFN-, but also to “counteract a biological response” to them. Thus, “GST can interdict HMGB1 release at more than one step”. The study also indicates “that the inhibitory effects of GST relate to the gold component of this compound rather than the thiomalate moiety”.

The study shows “that GST inhibits cytoplasmic and extracellular HMGB1 translocation in cultures of myeloid cells at pharmacologically relevant concentrations”. The “mechanism could explain the important anti-rheumatic effects of gold therapy in RA, as decreased levels of HMGB1 should ameliorate synovial inflammation and tissue destruction”. Although gold exerts “systemic, anti-inflammatory effects”, it “may act locally”. “As gold affects a number of intracellular signalling systems (e.g., PKC and haem oxygenase), studies are in progress to identify the key system responsible for the effects on HMGB1 release.”