Helena Erlandsson Harris

Reversing established sepsis with antagonists of endogenous high-mobility group box 1

Despite significant advances in intensive care therapy and antibiotics, severe sepsis accounts for 9% of all deaths in the United States annually. The pathological sequelae of sepsis are characterized by a systemic inflammatory response, but experimental therapeutics that target specific early inflammatory mediators [tumor necrosis factor (TNF) and IL-1β] have not proven efficacious in the clinic. We recently identified high mobility group box 1 (HMGB1) as a late mediator of endotoxin-induced lethality that exhibits significantly delayed kinetics relative to TNF and IL-1β. Here, we report that serum HMGB1 levels are increased significantly in a standardized model of murine sepsis, beginning 18 h after surgical induction of peritonitis. Specific inhibition of HMGB1 activity [with either anti-HMGB1 antibody (600 μg per mouse) or the DNA-binding A box (600 μg per mouse)] beginning as late as 24 h after surgical induction of peritonitis significantly increased survival (nonimmune IgG-treated controls = 28% vs. anti-HMGB1 antibody group = 72%, P < 0.03; GST control protein = 28% vs. A box = 68%, P < 0.03). Animals treated with either HMGB1 antagonist were protected against the development of organ injury, as evidenced by improved levels of serum creatinine and blood urea nitrogen. These observations demonstrate that specific inhibition of endogenous HMGB1 therapeutically reverses lethality of established sepsis indicating that HMGB1 inhibitors can be administered in a clinically relevant time frame.

RAGE is the Major Receptor for the Proinflammatory Activity of HMGB1 in Rodent Macrophages

High‐mobility group box chromosomal protein 1 (HMGB1) is a protein with both intranuclear functions and extracellular cytokine‐like effects. In this report, we study possible candidate receptors for HMGB1 on macrophages (Mφ) and define pathways activated by HMGB1 binding. Bone marrow Mφ were prepared from Dark Agouti (DA) rats and stimulated in vitro with HMGB1. The kinetics of tumour necrosis factor (TNF) production, NO production, activation of p38 mitogen‐activated protein kinase (MAPK), p44/42 MAPK‐ and SAPK/JNK‐signalling pathways, nuclear translocation of nuclear factor kappa B (NF‐κB) and HMGB1‐induced upregulation of major histocompatibility complex (MHC) class II and CD86 were analysed. Mφ from interleukin (IL)‐1 receptor type I–/–, Toll‐like receptor 2 (TLR2–/–) and RAGE–/– mice were used to investigate the role of these receptors in HMGB1 signalling. HMGB1 induced TNF and NO production by Mφ, phosphorylation of all investigated MAP kinase pathways and NF‐κB translocation, and expression of MHC class II was increased. Mφ from RAGE–/– mice produced significantly lower amounts of TNF, IL‐1β and IL‐6, while IL‐1RI–/– and TLR2–/– Mφ produced cytokine levels comparable with wildtype controls in response to HMGB1 stimulation. We conclude that HMGB1 has the potential to induce a proinflammatory phenotype in Mφ, with RAGE as the major activation‐inducing receptor.

HMGB1: A multifunctional alarmin driving autoimmune and inflammatory disease

HMGB1 is a non-histone nuclear protein that can serve as an alarmin to drive the pathogenesis of inflammatory and autoimmune disease. Although primarily located in the cell nucleus, HMGB1 can translocate to the cytoplasm, as well as the extracellular space, during cell activation and cell death; during activation, HMGB1 can undergo post-translational modifications. The activity of HMGB1 varies with the redox states of the cysteine residues, which are required for binding to TLR4. In addition to stimulating cells directly, HMGB1 can form immunostimulatory complexes with cytokines and other endogenous and exogenous factors. In the synovia of patients with rheumatoid arthritis, as well as animal models of this disease, extranuclear expression of HMGB1 is increased and blockade of HMGB1 expression attenuates disease in animal models. In systemic lupus erythematosus, HMGB1 can be a component of immune complexes containing anti-DNA because of its interaction with DNA. In myositis, expression of HMGB1 is enhanced in inflamed muscle and can perturb muscle function. Together, these findings indicate that HMGB1 might be an important mediator and biomarker in rheumatic diseases as well as a target of new therapy.

Mini-review : the nuclear protein HMGB1 as a proinflammatory mediator

The intranuclear architectural protein that is termed high mobility group box chromosomal protein 1 (HMGB1) was recently identified as a potent proinflammatory mediator when present extracellularly. HMGB1 has been demonstrated to be a long‐searched‐for nuclear danger signal passively released by necrotic, as opposed to apoptotic, cells that will induce inflammation. Furthermore, HMGB1 can also be actively secreted by stimulated macrophages or monocytes in a process requiring acetylation of the molecule, which enables translocation from the nucleus to secretory lysosomes. Subsequenttransport out of the cells depends on a secretion signal mediated by either extracellular lysophophatidyl‐choline or ATP. HMGB1 passively released from necrotic cells and HMGB1 actively secreted byinflammatory cells are thus molecularly different. Extracellular HMGB1 acts as a cytokine by signaling via the receptor for advanced glycated end‐products and via members of the Toll‐like receptor family. The initiated inflammatory responses include the production of multiple cytokines, chemoattraction of certain stem cells, induction of vascular adhesion molecules and impaired function of intestinalepithelial cells. Therapeutic administration of HMGB1 antagonists rescues mice from lethal sepsis, even when initial treatment is delayed for 24 h after the onset of infection, establishing a clinically relevant therapeutic window that is significantly wider than for other known cytokines.

Alarmin(g) news about danger: workshop on innate danger signals and HMGB1.

The EMBO Workshop on Innate Danger Signals and HMGB1 took place between 8 and 11 February 2006 in Milan, Italy, and was organized by M. Bianchi, K. Tracey and U. Andersson. ![][1] During evolution, multicellular organisms have developed mechanisms to counteract life‐threatening events, such as infections and tissue injury, as well as to restore tissue homeostasis. These mechanisms are called ‘the inflammatory response‘. To initiate an appropriate inflammatory response, organisms have developed ways to recognize potentially life‐threatening events. Danger signals—the molecules that alert the innate immune system and trigger defensive immune responses—have been classically defined as exogenous, pathogen‐associated molecular pattern (PAMP) molecules. PAMPs—for example, lipopolysacharide (LPS), viral RNA and bacterial petidoglycans—interact with dedicated receptors on immune cells, the so‐called pattern recognition receptors (PRRs). On ligation, PRRs transduce activation signals that lead to the production of proinflammatory molecules such as tumour necrosis factor (TNF). A well‐known family of PRRs is the toll‐like receptor (TLR) family in which each member recognizes a specific set of PAMPs. However, several endogenous molecules also initiate inflammatory responses by interacting with signalling receptors; such innate danger signals are known as endokines and/or alarmins. The term endokine reflects the potential of these molecules with intranuclear and/or intracellular functions also to act extracellularly, in this case to be immunostimulatory on their release from necrotic cells. The endokine family includes high‐mobility‐group box (HMGB) proteins, interleukins such as IL‐1α, cytosolic calcium‐binding proteins of the S100 family, heat‐shock proteins (HSPs) and nucleosomes. The term alarmin, coined by J. Oppenheim (Frederick, MD, USA) and co‐workers, denotes an array of structurally diverse multifunctional host proteins that are rapidly released during infection or tissue damage, and that have mobilizing and activating effects on receptor‐expressing cells engaged in host defence and tissue repair. Innate‐immune mediators that have alarmin function include defensins, eosinophil‐derived neurotoxin, cathelicidins and HMGB1 … [1]: /embed/graphic-1.gif

The alarmin HMGB1 acts in synergy with endogenous and exogenous danger signals to promote inflammation

The nuclear protein HMGB1 has previously been demonstrated to act as an alarmin and to promote inflammation upon extracellular release, yet its mode of action is still not well defined. Access to highly purified HMGB1 preparations from prokaryotic and eukaryotic sources enabled studies of activation of human PBMC or synovial fibroblast cultures in response to HMGB1 alone or after binding to cofactors. HMGB1 on its own could not induce detectable IL‐6 production. However, strong enhancing effects on induction of proinflammatory cytokine production occurred when the protein associated with each of the separate proinflammatory molecules, rhIL‐1β, the TLR4 ligand LPS, the TLR9 ligand CpG‐ODN, or the TLR1‐TLR2 ligand Pam3CSK4. The bioactivities were recorded in cocultures with preformed HMGB1 complexes but not after sequential or simultaneous addition of HMGB1 and the individual ligands. Individual A‐box and B‐box domains of HMGB1 had the ability to bind LPS and enhance IL‐6 production. Heat denaturation of HMGB1 eliminated this enhancement. Cocultures with HMGB1 and other proinflammatory molecules such as TNF, RANKL, or IL‐18 did not induce enhancement. HMGB1 thus acts broadly with many but not all immunostimulatory molecules to amplify their activity in a synergistic manner.

Citrullinated proteins have increased immunogenicity and arthritogenicity and their presence in arthritic joints correlates with disease severity

Autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in patients with rheumatoid arthritis and have been suggested to be involved in the disease pathogenesis. The targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deiminase (PAD), which converts positively charged arginine to polar but uncharged citrulline. The aim of this study was to explore the effects of citrullination on the immunogenicity of autoantigens as well as on potential arthritogenicity. Thus, immune responses to citrullinated rat serum albumin (Cit-RSA) and to unmodified rat serum albumin (RSA) were examined as well as arthritis development induced by immunisation with citrullinated rat collagen type II (Cit-CII) or unmodified CII. In addition, to correlate the presence of citrullinated proteins and the enzyme PAD4 with different stages of arthritis, synovial tissues obtained at different time points from rats with collagen-induced arthritis were examined immunohistochemically. Our results demonstrate that citrullination of the endogenous antigen RSA broke immunological tolerance, as was evident by the generation of antibodies directed against the modified protein and cross-reacting with the native protein. Furthermore we could demonstrate that Cit-CII induced arthritis with higher incidence and earlier onset than did the native counterpart. Finally, this study reveals that clinical signs of arthritis precede the presence of citrullinated proteins and the enzyme PAD4. As disease progressed into a more severe and chronic state, products of citrullination appeared specifically in the joints. Citrullinated proteins were detected mainly in extracellular deposits but could also be found in infiltrating cells and on the cartilage surface. PAD4 was detected in the cytoplasm of infiltrating mononuclear cells, from day 21 after immunisation and onwards. In conclusion, our data reveal the potency of citrullination to break tolerance against the self antigen RSA and to increase the arthritogenic properties of the cartilage antigen CII. We also show that citrullinated proteins and the enzyme PAD4 are not detectable in healthy joints, and that the appearance and amounts in arthritic joints of experimental animals are correlated with the severity of inflammation.

MD-2 is required for disulfide HMGB1–dependent TLR4 signaling

Yang et al. show that a disulfide isoform of HMGB1, with a role in TLR4 signaling, physically interacts with and binds MD-2. MD-2 deficiency in macrophage cell lines or in primary mouse macrophages stimulated with HMGB1 implicates MD-2 in TLR4 signaling. They also identify an HGMB1 peptide inhibitor, P5779, which when administered in vivo can protect mice from acetaminophen-induced hepatoxicity, ischemia/reperfusion injury, and sepsis.

High mobility group box protein 1 in complex with lipopolysaccharide or IL-1 promotes an increased inflammatory phenotype in synovial fibroblasts

IntroductionIn addition to its direct proinflammatory activity, extracellular high mobility group box protein 1 (HMGB1) can strongly enhance the cytokine response evoked by other proinflammatory molecules, such as lipopolysaccharide (LPS), CpG-DNA and IL-1β, through the formation of complexes. Extracellular HMGB1 is abundant in arthritic joint tissue where it is suggested to promote inflammation as intra-articular injections of HMGB1 induce synovitis in mice and HMGB1 neutralizing therapy suppresses development of experimental arthritis. The aim of this study was to determine whether HMGB1 in complex with LPS, interleukin (IL)-1α or IL-1β has enhancing effects on the production of proinflammatory mediators by rheumatoid arthritis synovial fibroblasts (RASF) and osteoarthritis synovial fibroblasts (OASF). Furthermore, we examined the toll-like receptor (TLR) 4 and IL-1RI requirement for the cytokine-enhancing effects of the investigated HMGB1-ligand complexes.MethodsSynovial fibroblasts obtained from rheumatoid arthritis (RA) and osteoarthritis (OA) patients were stimulated with HMGB1 alone or in complex with LPS, IL-1α or IL-1β. Tumour necrosis factor (TNF) production was determined by enzyme-linked immunospot assay (ELISPOT) assessment. Levels of IL-10, IL-1-β, IL-6 and IL-8 were measured using Cytokine Bead Array and matrix metalloproteinase (MMP) 3 production was determined by ELISA.ResultsStimulation with HMGB1 in complex with LPS, IL-1α or IL-1β enhanced production of TNF, IL-6 and IL-8. HMGB1 in complex with IL-1β increased MMP production from both RASF and OASF. The cytokine production was inhibited by specific receptor blockade using detoxified LPS or IL-1 receptor antagonist, indicating that the synergistic effects were mediated through the partner ligand-reciprocal receptors TLR4 and IL-1RI, respectively.ConclusionsHMGB1 in complex with LPS, IL-1α or IL-1β boosted proinflammatory cytokine- and MMP production in synovial fibroblasts from RA and OA patients. A mechanism for the pathogenic role of HMGB1 in arthritis could thus be through enhancement of inflammatory and destructive mechanisms induced by other proinflammatory mediators present in the arthritic joint.

The role of HMGB1 in the pathogenesis of rheumatic disease.

HMGB1 is a ubiquitous nuclear protein that can be released by any damaged cell or by activated macrophages and certain other cell types. HMGB1 has been successfully therapeutically targeted in multiple preclinical models of infectious and sterile diseases including arthritis. Extracellular HMGB1 mediates inflammation via induction of cytokine and metalloproteinase production and recruitment and activation of dendritic cells needed for priming of naïve T helper type 1 lymphocytes. HMGB1 can bind endogenous molecules such as IL-1beta and nucleosomes and exogenous agents like endotoxin and microbial DNA. These complexes synergistically increase the capacity for activation of adaptive and innate immunity. HMGB1-nucleosome complexes induce autoantibody formation against double-stranded DNA and nucleosomes, which does not occur if HMGB1 is absent. These antibodies are central in the pathogenesis of systemic lupus erythematosus and patients with active disease have both increased HMGB1 and HMGB1-nucleosome levels in circulation. Furthermore, HMGB1 is strongly bipolar charged, enabling cell membrane passage and intracellular transport of complexed molecules including DNA. Rheumatoid arthritis patients have excessive extracellular HMGB1 levels in joints and serum. The HMGB1 release is caused by cytokines, activated complement and hypoxia. The most prominent HMGB1 protein and mRNA expression arthritis is present in pannus regions, where synovial tissue invades articular cartilage and bone. HMGB1 promotes the activity of proteolytic enzymes, and osteoclasts need HMGB1 for functional maturation. Neutralizing HMGB1 therapy in preclinical models of arthritis confers striking protection against structural damage. This review summarizes selected aspects of HMGB1 biology relevant for induction and propagation of some autoimmune conditions.