Studies of high mobility group chromosomal protein 1 as a pro-inflammatory cytokine

High mobility group box chromosomal protein 1 (HMGB 1) is a nuclear protein recently recognised as a pro-inflammatory cytokine with implications both in acute and in chronic inflammation. HMGB1 has been reported to act as a late mediator of septic shock in mice. Patients with severe sepsis or septic shock have persistently elevated circulating levels of HMGB1. HMGB1 can be secreted by activated macrophages as well as by cells undergoing necrosis. Here we have studied the pro-inflammatory activity of HMGB1 on endothelial cells and the role of its receptor, receptor for advanced glycation end-products (PAGE).

Both the full-length protein and the B-box domain of HMGB1 induced NF-kappaB mediated activation of endothelial cells with the translocation of its subunits p65 and c-rel. This led to a release of interleukin (IL)-8 and G-CSF as well as an increased expression of ICAM-1 and VCAM-1. This HMGB1-induced pro-inflammatory activity appeared to signal partially through R-AGE, and included phosphorylation of the signal transduction factor Elk-1.

Furthermore, we demonstrated that endothelial cells also released HMGB1 in response to endotoxin or TNF-alpha exposure suggesting that the endothelium may be another important source of secreted HMGB 1. Furthermore, the corticosteroid dexamethasone inhibited this pro-inflammatory activity in endothelial cells of HMGB1. Meanwhile, CNI-1493 a tetravalent guanylhydrazone, inhibited HMGB1 pro-inflammatory activity in primary monocytes but not in endothelial cells.

Macrophages from IL-1 receptor type I-/-' (IL-1R1-/-), Toll-like receptor 2 (TLR2-/-) and RAGE -/- mice were used to investigate the role of these receptors in HMGB1 signalling. HMGB 1 led to TNF-alpha and NO production, the phosphorylation of several MAP kinase pathways, NF-kappaB subunit translocation and the increased expression of MHC class II molecules. Macrophages from PAGE--/- mice produced significantly lower amounts of TNF-alpha, IL-1beta and IL-6, while IL1RI-/- and TLR2-/- macrophages produced cytokine levels comparable with wildtype controls in response to HMGB1 stimulation, suggesting that RAGE is a central receptor for HMGB1-induced cytokine production.

HMGB1 was injected intraperitoneal ly into mice of PAGE-/- phenotype or wild type controls. The RAGE -/- mice showed a reduced inflammatory response in the lungs compared to wild type controls. To further understand the role of PAGE in humans, we investigated the response of soluble RAGE (sRAGE) to inflammation. Healthy volunteers subjected to endotoxin showed an immediate increase of circulating sRAGE. Sera obtained from patients with severe sepsis or septic shock were analyzed for sRAGE. Levels of sRAGE were elevated upon admittance compared to healthy controls and correlated with IL-6.

These results suggest that secreted HMGB1 may be an important pro-inflammatory cytokine that is also released from endothelial cells; and that RAGE is a central receptor for HMGB 1 induced inflammation.

List of scientific papers

I. Treutiger CJ, Mullins GE, Johansson AS, Rouhiainen A, Rauvala HM, Erlandsson-Harris H, Andersson U, Yang H, Tracey KJ, Andersson J, Palmblad JE (2003). High mobility group 1 B-box mediates activation of human endothelium. J Intern Med. 254(4): 375-85.
https://doi.org/10.1046/j.1365-2796.2003.01204.x

II. Mullins GE, Sunden-Cullberg J, Johansson AS, Rouhiainen A, Erlandsson-Harris H, Yang H, Tracey KJ, Rauvala H, Palmblad J, Andersson J, Treutiger CJ (2004). Activation of human umbilical vein endothelial cells leads to relocation and release of high-mobility group box chromosomal protein 1. Scand J Immunol. 60(6): 566-73.
https://doi.org/10.1111/j.0300-9475.2004.01518.x

III. Kokkola R, Andersson A, Mullins G, Ostberg T, Treutiger CJ, Arnold B, Nawroth P, Andersson U, Harris RA, Harris HE (2005). RAGE is the major receptor for the pro-inflammatory activity of HMGB1 in rodent macrophages. Scand J Immunol. 61(1): 1-9.
https://doi.org/10.1111/j.0300-9475.2005.01534.x

IV. Mullins GE, Sunden-Cullberg J, Tokics L, Soop A, Sollevi A, Harris HE, Arnold B, Andersson J, Treutiger CJ (2005). Increase of soluble RAGE in patients with severe sepsis/septic shock. [Manuscript]