HMGB1 Acts In Synergy With LPS In Activating Macrophages To Secret The Proinflammatory Cytokines

High mobility group box-1 protein (HMGB1), which was first discovered as a nuclear protein with rapid electrophoretic migration, is a 30 kDa, nonhistone, DNA-binding molecule that is highly conserved: it has a 99% sequence identity across mammalian species. HMGB1 has been recognized for many years as an abundant nuclear chromatin protein that binds to and distorts DNA; in doing so, it regulates many transcriptional events.HMGB1 is a bifunctional alarmin, extracellular it acts as a cytokine, helps to activate natural and adaptable immune, plays an important role in the pathophysiological process of sepsis, infection, and autoimmune disease.Human HMGB1 has 215 amino residues, contains two DNA binding domains: A box and B box, and an acidic tail. The interaction of HMGB1 with DNA has been determined by nuclear magnetic resonance spectroscopy, which showed that the A-box and B-box of HMGB1 bind to the minor groove of DNA. HMGB1 enhances the interaction of other proteins with DNA, and through its L-shaped A-box and B-box, which are each ~70 amino acids, it enhances transcriptional activation and, in this way, regulates several families of DNA-binding proteins. In its resting state, the long acidic tail of HMGB1 interacts with basic stretches in the A-box and B-box, shielding them from other interactions that might occur before HMGB1 binds DNA. Recently, many researchers used the indirect-ELISA to study the binding of HMGB1 and other molecules, and found that HMGB1 bound to many molecules such as ODN CpG, LPS, and IL-1.The putative cellular receptors that bind HMGB1 are at present controversial and may include TLR2 and TLR4 as well as RAGE (receptor for advanced glycation end-products). RAGE belongs to the immunoglobulin superfamily, express on the surface of many cells, and has high affinity with HMGB1. HMGB1 signal through RAGE to triggers a cascade of intracellular signals, including p21 Ras and other downstream targets, such as mitogenactivated protein kinase kinase, mitogen-activated protein kinase, and NF-κB.1999 Wang et al reported that HMGB1 was a late mediator in endotoxemia, and the extracellular functions of HMGB1 as a cytokine become a research highlight. HMGB1 induces the production of such as TNF-α, IL-1β, and IL-6 in monocytes/macrophages. And the released proinflammatory cytokines further activate the monocytes/macrophages to produce more proinflammatory cytokines. The extracellular functions of HMGB1 relays on the B Box and A Box peptide alone is an antagonist of B Box or HMGB1. HMGB1 not only participates in the acute inflammatory processes such as sepsis and acute lung injury, but also has an important role in the pathophysiological process of many autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus. Orlova VV et al found that In vitro, HMGB1 enhanced the interaction between Mac-1 and RAGE. Consistently, HMGB1 activated Mac-1 as well as Mac-1-mediated adhesive and migratory functions of neutrophils in a RAGE-dependent manner. Moreover, HMGB1-induced activation of NF-κB in neutrophils required both Mac-1 and RAGE. Together, they found a novel HMGB1-dependent pathway for inflammatory cell recruitment and activation that requires the functional interplay between Mac-1 and RAGE.Recent data have brought into question the proinflammatory properties of HMGB1. HMGB1 isolated and purified from calf thymus or recombinant in mammal-expression system did not appear to be capable of inducing macrophage or dendritic cells activation unless bound to DNA-containing immune complexes or to damage-associated pattern molecules such as IL-1βand LPS. Jane Tian et al found Thymus-derived HMGB1 synergizes with TLR7, TLR9, TLR2 and TLR4 ligands in the induction of cytokines in murine bone marrow cells, and the synergy occurs through a RAGEdependent mechanism. These data suggest that HMGB1 lacks a direct pro-inflammatory property but primes cells to augment the response through distinct PAMPs. RAGE is involved in the recognition of HMGB1- and DNA-containing immune complexes, and the DNA dependent association of TLR9 and RAGE. Along with the endosomal localization of RAGE, raise the possibility that RAGE determines the subcellular localization and/or retention of DNA-TLR9 complexes in the endosome. Recognition and/or activation of HMGB1- and DNAcontaining immune complexes by means of RAGE may therefore be important in the loss of tolerance to self antigens. Sha Y et al reported that IL-1βwas bound to HMGB1 isolated from cells cultured with this cytokine, and purified HMGB1 incubated with recombinant IL-1βacquired proinflammatory activity. Addition of anti-IL-1βAbs or the IL-1 receptor antagonist to cell cultures blocked the proinflammatory activity of HMGB1 purified from IL-1β-exposed cells, indicating that such activity was dependent on interaction with the IL-1 receptor. These results demonstrate that HMGB1 acquires proinflammatory activity through binding to proinflammatory mediators, such as IL-1β. Because of the dual functions of HMGB1, it has been shown playing a crucial role in many pathophysiological processes, such as rheumatoid arthritis, liver injury, and tumors. Our previous plan was to figure out the crucial role of HMGB1 in the pathogenesis of rheumatoid arthritis, especially which receptor and pathway are involved, and the feasibility of HMGB1 as a therapy target of rheumatoid arthritis. We first studied the activation of macrophages by HMGB1 in vitro, and found that different Lots of HMGB1 had different proinflammatory activity. So we adjusted our study plan to figure out the molecular nature of the extracellular function of HMGB1 proteins.This study has three parts: Part one, Results from different labs showed different proinflammatory activity of HMGB1, and the HMGB1 used in many experiments are recombined in E. coli, and even after strict purification procedures, we still cannot completely abolish the influence of endotoxin. We first found that HMGB1 proteins from different companies have different proinflammatory activities and were concordant with the endotoxin contamination. Recombinant HMGB1 proteins have little endotoxins and show marginal proinflammatory activity, while HMGB1 proteins contain over 3000EU/mg endotoxin (Lot 077K4002) can induce macrophages to secrete amount of TNF-α. Real-time PCR showed the same results that HMGB1 contains endotoxin can up-regulate the expression of many proinflammatory cytokines such as IL-1, IL-6, TNF-α, and Cxcl2. Our results support that HMGB1 itself barely has proinflammatory activity; the proinflammatory activity of HMGB1 depends on the endotoxin it contains. Except endotoxins, other components of E. coli such as CpG DNA may also contribute to the activity of HMGB1.Part two: Although Lot 077K4002 contained significantly more amounts of endotoxin than others, this does not fully explain the different activities. We next hypothesized that HMGB1 could enhance LPS-induced proinflammatory cytokines secretion. To confirm this hypothesis, we cultured macrophages with LPS, HMGB1 (Lot 018K4157), and both of them. Results showed that recombinant HMGB1 enhanced the LPS-induced production of proinflammatory cytokines, such as IL-6 and TNF-αin vitro. To further confirm that HMGB1 acted in synergy with LPS in vivo, we used an intraarticular injection of HMGB1-induced experimental arthritis model. After intraarticular injection with 5μg HMGB1 alone for 7 days, one out of eight DBA/1 mice developed mild synovitis, and seven out of eight mice developed arthritis in the group that was injected with 5μg HMGB1 plus 2ng LPS. Given LPS alone, three out of eight mice developed mild arthritis. The control group injected with PBS or that remained untreated showed no signs of arthritis. All evidence showed that HMGB1 actually acts in synergy with LPS to induce the inflammatory response.Part three, In order to elucidate the mechanism of the synergetic effect between HMGB1 and LPS, we first added blocking antibodies (Anti-TLR2, Anti-TLR4, and RAGE-Fc) 30 minutes before LPS and /or HMGB1 were added to the medium and measured the secretion and expression of proinflammatory cytokines, and results showed that both TLR4 and RAGE were involved. Recently, HMGB1 was reported to bind to IL-1βand then acquire the proinflammatory activity. IL-1 receptors and TLR4 have similar intracellular Toll-IL-1 receptor (TIR) domains, and initiate innate immunity via NF-κB activation leading to production of proinflammatory cytokines, so we next added blocking antibodies (Anti-TLR2, Anti-TLR4, and RAGE-Fc) 30 minutes before IL-1βand /or HMGB1 were added to the medium and measured the secretion and expression of proinflammatory cytokines. Results showed that HMGB1 signaled through RAGE to enhance the proinflammatory activity of LPS. We then used the protein inhibitors to inhabit the activation of MAPK proteins and NF-κB. Results showed that blockading the activation of NF-κB abolished the production of TNF-αor IL-6, and blockading the activation of MAPK P38 partly inhibited the production of TNF-αor IL-6, whereas blockading the activation of ERK or JNK had no significant effect on the production of proinflammatory cytokines. Finally, we tested the phosphorylation level and the activiation of NF-κB with western blot.The TIR superfamily initiates innate immunity via NF-κB activation, leading to the production of proinflammatory cytokines. TLRs share common intracellular MyD88-dependent signal pathways to recruit the TRAF6, which leads to activation of four protein kinase cascades and culminates in the activation of NF-κB and the MAP kinases p38, JNK and ERK1/2. The crosstalk between TLRs and other signal pathway is ubiquitous in macrophage activation, and it had been reported that HMGB1 can signal through both RAGE and TLR4 to activate the NF-κB. We think maybe it is the LPS that HMGB1 contains that signals through the TLR4 to activate the macrophages, and that the crosstalk between TLR4 and RAGE leads to the enhanced phosphorylation of MAPK p38.In conclusion, our data indicate that the proinflammatory activity of HMGB1 recombined from E. coli is accordant with the endotoxin levels it contains. HMGB1 acts in synergy with LPS in activating macrophages to secrete proinflammatory cytokines, may signal through RAGE, and enhances the phosphorylation of MAPK p38 and the activation of NF-κB. The crosstalk between TLR4 and RAGE may lead to the enhancement of inflammatory responses in macrophages.